Small molecules for mouse satellite cell proliferation

Abstract

To provide compositions and methods for inducing proliferation of satellite cells.SOLUTION: The invention provides a method of inducing, enhancing or increasing satellite cell proliferation. The method comprises contacting a satellite cell with a compound selected from a group consisting of kinase inhibitors, G protein coupled receptor (GPCR) modulators, epigenic modifiers, histone deacetylase (HDAC) modulators, hedgehog signaling pathway modulators, neuropeptides, dopamine receptor modulators, serotonin receptor modulators, histamine receptor modulators, adenosine receptor agonists etc. In another aspect, there is provided a method for repairing or regenerating a damaged muscle tissue of a subject. In still another aspect, a method for screening candidate compounds for inducing, enhancing or increasing satellite cell proliferation is provided.SELECTED DRAWING: Figure 50

Description

[Technical field]

[0001] Related Applications This application is a continuation-in-part of, and claims priority to, U.S. Patent Application No. 15 / 012,656, filed February 1, 2016. U.S. Patent Application No. 15 / 012,656 is a continuation-in-part of, and claims priority to, U.S. Patent Application No. 14 / 126,716, filed June 13, 2014 (now U.S. Patent No. 9,248,185, issued February 2, 2016), which is a national stage application under U.S. Patent Act 371 of International Application No. PCT / US2012 / 042964, filed June 18, 2012. International Application No. PCT / US2012 / 042964 claims the benefit under U.S. Patent Act § 119(e) of U.S. Provisional Application No. 61 / 497,708, filed June 16, 2011. The contents of the above applications are incorporated herein by reference in their entirety.

[0002] The present disclosure relates generally to compositions and methods for promoting the proliferation of satellite cells. [Background technology]

[0003] Satellite cells are a population of skeletal muscle stem cells that are located under the basal lamina surrounding muscle fibers and are required for muscle growth and muscle repair after injury or exercise. The number and function of satellite cells are affected by normal aging and in some diseases, resulting in progressive muscle wasting or inefficient recovery after injury. Examples include Duchenne muscular dystrophy, where satellite cells are depleted by constant use, and sarcopenia, where satellite cells are not only depleted but also their proliferation capacity can be adversely affected by changes in their environment (Jejurikar and Kuzon, Apoptosis, vol. 8 (2003), pp. 573-578). Finding a treatment that expands the endogenous population of satellite cells could be of great help in the treatment of these debilitating diseases. Thus, there is a need in the art for compositions and methods that induce the proliferation of satellite cells. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Jejurikar and Kuzon, Apoptosis (2003), 8, 573-578. Summary of the Invention [Means for solving the problem]

[0005] We performed an image-based screen of selected small molecules for their ability to increase proliferation in satellite cells isolated from adult mouse muscle tissue. Satellite cells were isolated using cell surface markers (Sherwood et al., Cell, vol. 119 (2004), pp. 543-554), cultured in the presence of small molecules for 4 days, and then analyzed using automated confocal microscopy to determine cell number. Using this procedure, we discovered several compounds that act through defined signaling pathways and can enhance proliferation in cultures of primary mouse satellite cells. The myogenic capacity of treated cells is currently being tested by marker analysis and differentiation assays, as well as in vivo muscle transplantation. The compounds can proliferate cells without adversely affecting differentiation potential and can be used to treat diseases that have skeletal muscle defects as one of their components. These compounds are also referred to herein as proliferation enhancers.

[0006] Therefore, the present specification provides a method for inducing, enhancing or increasing the proliferation of satellite cells.The method includes contacting satellite cells with a compound selected from the group consisting of kinase inhibitors, G protein-coupled receptor (GPCR) modulators, epigenic modifiers, histone deacetylase (HDAC) modulators, hedgehog signaling pathway modulators, neuropeptides, dopamine receptor modulators, serotonin receptor modulators, histamine receptor modulators, adenosine receptor agonists, ionophores, ion channel modulators, gamma-secretase modulators, corticosteroids, and any combination thereof.The satellite cells that are contacted can be in vitro, ex vivo or in vivo.

[0007] In another aspect, provided herein is a method for repairing or regenerating damaged muscle tissue in a subject, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of a kinase inhibitor, a G protein-coupled receptor (GPCR) modulator, an epigenetic modifier, a histone deacetylase (HDAC) modulator, a hedgehog signaling pathway modulator, a neuropeptide, a dopamine receptor modulator, a serotonin receptor modulator, a histamine receptor modulator, an ionophore, an ion channel modulator, a gamma-secretase modulator, and any combination thereof.

[0008] In yet another aspect, provided herein is a method of screening candidate compounds for inducing, enhancing, or increasing satellite cell proliferation, comprising the steps of (a) contacting a population of satellite cells with a test compound; (b) evaluating satellite cell proliferation; and (c) selecting a compound that induces, increases, or enhances satellite cell proliferation.

[0009] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Department upon request and payment of the necessary fee. [Brief description of the drawings]

[0010] [Figure 1AB] Figures 1A-1C show Opera analysis of satellite cell numbers. Images were captured using a Perkin Elmer Opera automated confocal imaging system. Since all cells isolated from CAG-EGFP animals were fluorescent, we were able to image the cells directly (Figure 1A). Images were analyzed using Acapella software to count cells with signals within set thresholds for each parameter (Figure 1B). Cells or debris that were too dim (arrows) or too small (arrowheads) were excluded. Cells and debris that were highly fluorescent or too large in size were also excluded. Cells could then be further grouped according to set criteria such as roundness (Figure 1C). [Figure 1C] Same as above.

[0011] [Figure 2A] Figures 2A-2D show the analysis of positive hit compounds. Mature mouse satellite cells were isolated by FACS based on the procedure outlined in Sherwood et al. (Cell, vol. 119 (2004), pp. 543-554). Cells were then treated with compounds, grown for 4 days, fixed, and imaged. Compound-induced proliferation was compared to that found with DMSO vehicle control (Figure 2A) or bFGF (Figure 2B). Two example images from positive hit compounds are shown, an Flt3 kinase inhibitor (Figure 2C) and an adenosine receptor agonist (Figure 2D). [Figure 2BC] Same as above. [Figure 2D] Same as above.

[0012] [Figure 3A] Figures 3A and 3B show validation of some exemplary hit compounds. Compounds identified as hits in the primary screen were then tested in a dose-response assay. The level of proliferation is measured as a fold change above DMSO vehicle control values ​​of (A.1 ± 0.3 (Figure 3A) and 1 ± 0.4 (Figure 3B). For comparison, bFGF positive control values ​​were 3.4 ± 0.9 (Figure 3A) and 5.6 ± 1.7 (Figure 3B). [Figure 3B] Same as above.

[0013] [Figure 4] Figure 4 shows the optimization of culture conditions. Culture conditions were optimized by testing the effect of the compound in the presence or absence of bFGF at different exposure times. For all time points, the medium was replaced with standard growth medium on the indicated days. For this compound, the inclusion of bFGF had an additive effect on proliferation. Also, the compound itself produced similar proliferation to the bFGF positive control at all time points, but was found to be more effective at shorter exposure times.

[0014] [Figure 5AB]Figures 5A-5C show differentiation of treated cultures. CAG-EGFP satellite cells were grown under our standard growth conditions and exposed to compounds for 4 days. Under our standard growth conditions, cells were cultured on laminin-coated plates in Ham's F-10 medium supplemented with 10% heat-inactivated horse serum, 100 units / mL penicillin / 100ug / mL streptomycin, and 2mM L-glutamine. Compounds were added the day after plating. Compounds and medium were refreshed daily or after 3 days. To differentiate cells and form myotubes, after 4 days under growth conditions, medium was switched to high glucose DMEM supplemented with 10% heat-inactivated horse serum, 10% fetal bovine serum, 0.5% chicken embryo extract, 100 units / mL penicillin / 100ug / mL streptomycin, and 2mM L-glutamine. Cultures were grown in differentiation medium for 3-5 days until myotube formation was observed and then fixed. Cultures were then switched to differentiation medium and cultured for an additional 4 days. They were then fixed and stained with anti-myosin heavy chain (red channel) and Hoechst (blue channel). Cells grown with only the DMSO vehicle control did not form myotubes because they were not dense enough in the culture (data not shown). Cells grown in the presence of the bFGF positive control (Figure 5A) were able to form large myotubes. Cultures grown in the presence of the Flt3 kinase inhibitor (Figure 5B) and adenosine agonist (Figure 5C) were also able to form myotubes. [Figure 5C] Same as above.

[0015] [Figure 6] Figure 6 shows the formation of myogenic colonies in single SMPs treated with CEP / DMSO for 5 days. Treatment interval d1-d3.5 and n=3.

[0016] [Figure 7] Figure 7 shows compound exposure from d2 to d4.5. Initial cell number was 250 and n=3.

[0017] [Figure 8A] 8A and 8B show the expression of CXCR4 and beta-1 integrin on cultured SMPs after 5 days: CEP (FIG. 8A) and DMSO (FIG. 8B). [Figure 8B] Same as above.

[0018] [Figure 9] Figures 9A-13 show dose-response curves for some exemplary hit compounds: sunitinib (SU11248, Figures 9A and 9B), Jak3 inhibitor VI (Figures 10A and 10B), lestaurtinib (CEP701, Figures 11A and 11B), bosutinib (Figure 12), and SU11652 (Figure 13). [Figure 10] Same as above. [Figure 11] Same as above. [Figure 12] Same as above. [Figure 13] Same as above.

[0019] [Figure 14] 14-17 are bar graphs showing the synergistic effect of bFGF with CEP701 (FIGS. 14 and 15) and the Jak3 inhibitor VI (FIG. 16), and sunitinib (FIG. 17) on satellite cell proliferation. [Figure 15] Same as above. [Figure 16] Same as above. [Figure 17] Same as above.

[0020] [Figure 18] 18-20 are photographs showing satellite cell differentiation following treatment with CEP701 (FIG. 18), sunitinib (FIG. 19), and the Jak3 inhibitor VI (FIG. 20). [Figure 19] Same as above. [Figure 20] Same as above.

[0021] [Figure 21] FIG. 21 is a bar graph showing the synergistic effect of CEP701 with a TGF-beta inhibitor (Alk5 inhibitor II).

[0022] [Figure 22] Figure 22 shows the specificity of CEP701 for satellite cell proliferation. Left panel: CEP701; right panel: DMSO.

[0023] [Figure 23] FIG. 23 is a bar graph showing that CEP701 has no effect on primary fibroblasts.

[0024] [Figure 24] Figures 24 and 25 are bar graphs showing that CEP701 is effective in both old (Figure 24) and young (Figure 25) tissues. [Diagram 25] Same as above.

[0025] [Figure 26] Figures 26 and 27 are line graphs showing dose response curves for N6-cyclopentyladenosine (Figure 26) and budesonide (Figure 27). [Figure 27] Same as above.

[0026] [Figure 28] Figures 28 and 29 are bar graphs showing the synergistic effect of bFGF with N6-cyclopentyladenosine (Figure 28) and budesonide (Figure 29). [Figure 29] Same as above.

[0027] [Diagram 30] Figures 30 and 31 are photographs showing satellite cell differentiation following treatment with N6-cyclopentyladenosine (Figure 30) and budesonide (Figure 31). [Diagram 31] Same as above.

[0028] [Diagram 32] FIG. 32 is a bar graph showing the synergistic effect of N6-cyclopentyladenosine and a TGF-beta inhibitor (Alk5 inhibitor II).

[0029] [Diagram 33] FIG. 33 illustrates screening assays performed to identify primary and secondary compounds shown to increase satellite cell proliferation in vitro.

[0030] [Diagram 34] FIG. 34 illustrates the customer screening library that we used to screen a set of approximately 400 compounds to identify compounds that increase satellite cell proliferation.

[0031] [Diagram 35] Figure 35 shows the results of the assays performed, demonstrating that lestaurtinib (CEP701), sunitinib (SU11248), JAK3 inhibitor VI, and N6-cyclopentyladenosine (CPA) were found to increase satellite cell proliferation in vitro. Lestaurtinib (CEP701) was identified as the top hit, was effective at nanomolar doses, and had target overlap with several other hit compounds.

[0032] [Diagram 36] FIG. 36 shows the results of the assays performed, demonstrating that lestaurtinib (CEP701) increases the proliferation of aged satellite cells in vitro.

[0033] [Figure 37] Figures 37A-37B show the dose response assay (Figure 37A) and response curves (Figure 37B) for lestaurtinib (CEP701), sunitinib (SU11248), JAK3 inhibitor VI, and N6-cyclopentyladenosine (CPA), respectively.

[0034] [Figure 38]FIG. 38 demonstrates that both CEP-701 and AC220 increase human satellite cells by more than 2-fold at a concentration of 1 nM relative to the control (DMSO).

[0035] [Figure 39] FIG. 39 shows the results of the assays performed, confirming that the compounds identified as hits (e.g., CEP701, SU11248, JAK3 inhibitor VI, CPA and Tyr AG490) drive myoblast differentiation.

[0036] [Diagram 40] FIG. 40 demonstrates that CEP701 enhances myoblast differentiation in differentiation medium relative to DMSO controls, as evidenced by the observed increases in both myoblast area and length.

[0037] [Diagram 41] Figures 41A-41B show the experimental protocol (Figure 41A) and the results of that experiment (Figure 41B), which show that CEP701 treatment of cells increases the number of GFP+ fibers per section relative to the control (DMSO).

[0038] [Fig. 42AB] Figures 42A-42D show the experimental protocol (Figure 42A) and the observed results. As shown in Figures 42B-42D, treatment with CEP701 increased both the size of regenerating fibers and the number of satellite cells in vivo in both adult and aged mice. [Fig.42CD] Same as above.

[0039] [Diagram 43]Figures 43A-43B show the effect that the identified compounds have on RTK (Figure 43A) and compare the fold change in phospho-RET in both intact contralateral tibial anterior (TA) muscles with that observed 2 days after cardiotoxin injury, as shown in Figure 43B. As shown in Figure 43B, an approximately 8-fold increase in phospho-RET was observed by ELISA relative to intact contralateral TA muscles 2 days after cardiotoxin injury.

[0040] [Diagram 44] Figures 44A-44B show that satellite cells express RET in vitro.

[0041] [Figure 45A] Figures 45A-45D show that CEP701 treatment inhibits RET phosphorylation in vitro. [Figure 45BCD] Same as above

[0042] [Diagram 46] FIG. 46 shows the results of a study evaluating the effect of in vitro deletion of RET.

[0043] [Figure 47] FIG. 47 shows that by using a conditional RET mutant and a reporter, the inventors were able to determine that the RET promoter was active in at least 25% of satellite cells.

[0044] [Figure 48] FIG. 48 shows that RET knockout cells grow better in vitro than wild-type cells (n=6; p=0.0004).

[0045] [Figure 49] FIG. 49 demonstrates the fold change relative to controls in untreated FLT3 and RET knockout cells.

[0046] [Figure 50] Figure 50 identifies small molecules that promote satellite cell proliferation. (A) Chemical screening schematic outlining FACS isolation and compound library treatment of satellite cells. (B) Representative dose-response curves for four of the top 10 compounds. The top 10 compounds were selected based on the highest fold change in cell proliferation relative to vehicle control. Proliferation was assessed by high content imaging using Hoechst33342 as a cell marker. (C) Representative fluorescent images of FACS sorted satellite cells from Tg:Pax7-nGFP mice on 96w plates cultured for 4 days and treated with vehicle, compound or positive control (Jak3 ​​inhibitor 6). The optimal treatment concentration for each compound was determined by dose response; 3uM for XMD8-92, 5uM for SB23906, 800nM for XMD11-50, and 400nM for vorinostat. Hoechst33342 was used as a cell marker. Scale bar indicates 100um. (D) Fold change over vehicle control for several compounds that promote satellite cell expansion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Provided herein is a method of increasing satellite cell proliferation, comprising contacting satellite cells with a compound selected from the group consisting of a kinase inhibitor, a G protein-coupled receptor (GPCR) modulator, an epigenetic modifier, a histone deacetylase (HDAC) modulator, a hedgehog signaling pathway modulator, a neuropeptide, a dopamine receptor modulator, a serotonin receptor modulator, a histamine receptor modulator, an adenosine receptor agonist, an ionophore, an ion channel modulator, an adenosine receptor modulator, a gamma-secretase modulator, a corticosteroid, or any combination thereof.

[0048] As used herein, the term "proliferation" refers to the growth and division of cells. In some embodiments, the term "proliferation" as used herein with respect to cells refers to a group of cells that can increase in number over a period of time.

[0049] As used herein, "inducing," "enhancing," or "increasing" satellite cell proliferation means that satellite cells replicate at a more rapid rate and / or at a higher frequency. In some embodiments of this and other aspects described herein, satellite cell proliferation is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more, relative to a non-treated control. The percentage or fold increase in satellite cell proliferation can be determined by measuring the number of replicating satellite cells while in contact with a compound described herein, relative to a control in which the satellite cells are not in contact with the compound. The increase in proliferation can also be based on the ratio of replicating cells to the total number of cells in the respective treated and non-treated controls. In some embodiments, the total number of cells in the treated and non-treated controls is used to determine proliferation. Satellite cell proliferation can be determined using the BrdU incorporation method described in US Patent Application Publication No. 2009 / 0136481, the contents of which are incorporated herein by reference.

[0050] Muscle satellite cells or satellite cells are small mononuclear precursor cells found in mature muscle that contain virtually no cytoplasm. They are found sandwiched between the basement membrane and the sarcolemma (cell membrane) of individual muscle fibers and can be difficult to distinguish from the subsarcolemmal nuclei of the fibers. Satellite cells can differentiate and fuse to augment existing muscle fibers and form new fibers. These cells represent the oldest known adult stem cell niche and are involved in the normal growth of muscle as well as regeneration after injury or disease.

[0051] In uninjured muscle, the majority of satellite cells are quiescent; they do not differentiate or divide. In response to mechanical strain, satellite cells become activated. Activated satellite cells initially proliferate as skeletal myoblasts and then undergo muscle differentiation.

[0052] Markers characteristic of satellite cells include expression of cell surface proteins or coding genes, expression of intracellular proteins or coding genes, cell morphological characteristics, etc. Those skilled in the art will recognize that the presence and absence of satellite cell specific characteristics can be readily ascertained by known immunofluorescence, immunochemistry, polymerase chain reaction, in situ hybridization, Northern blot analysis, chemical or radiochemical or biological methods.

[0053] If desired, the cell type(s) in the population of satellite cells can be determined using techniques well known in the art. For example, using cell type specific stains. Alternatively, immunofluorescence staining can be performed using antibodies directed against various satellite cell specific proteins. In addition, cell type can be determined by its morphology using techniques such as, for example, light microscopy or electron microscopy.

[0054] Satellite cells express several distinct genetic markers. For example, the current consensus is that all satellite cells express PAX7 and PAX3 (F. Rlaix et al., Nature, 2005, 435(7044):898-899). Activated satellite cells express myogenic transcription factors such as Myf5 and MyoD. They also begin to express muscle-specific fibrous proteins such as desmin as they differentiate.

[0055] Little is known about the regulation of satellite cells. Although PAX3 and PAX7 together now form the definitive satellite markers, the Pax genes may be poor transcriptional activators. The dynamics of activation and quiescence and the induction of the myogenic program by the myogenic regulators, Myf5, MyoD, myogenin, and MRF4, remain to be determined. Several studies have shown that satellite cells are negatively regulated by a protein called myostatin. Elevated levels of myostatin upregulate a cyclin-dependent kinase inhibitor called p21, thereby inducing satellite cell differentiation.

[0056] In some embodiments, satellite cells are in a stable state, e.g., cells are taken from a subject and processed so that they can be stored for a period of time.For example, cells can be frozen, e.g., using methods known in the art for freezing primary cells, so that the cells are viable when thawed.For example, methods known in the art for freezing and thawing embryos to generate live mammals can be adapted for use in the present method.Such methods can include, for example, using liquid nitrogen with one or more cryoprotectants, e.g., agents that prevent freeze-thaw damage to cells.

[0057] Kinase inhibitors As used herein, the term "kinase" refers to any phosphotransferase enzyme that transfers phosphate groups. In some embodiments, the kinase is a protein kinase. Protein kinases are a family of enzymes that catalyze the phosphorylation of specific residues in proteins. In general, protein kinases are classified into several groups; those that preferentially phosphorylate serine and / or threonine residues, those that preferentially phosphorylate tyrosine residues, and those that phosphorylate both tyrosine and Ser / Thr residues.

[0058] Protein kinases include, for example, but are not limited to, members of the protein tyrosine kinase family (PTK), which can then be divided into cytoplasmic PTKs and receptor PTKs (RTKs).Cytoplasmic PTKSs include the SRC family (including BLK; FOR; FYN; HCK; LCK; LYN; SRC; YES and YRK); the BRK family (including BRK; FRK, SAD; and SRM); the CSK family (including CSK and CTK); the BTK family (including BTK; ITK; TEC; MKK2 and TXK), the Janus kinase family (including JAKI, JAK2, JAK3 and Tyk2), the FAK family (including FAK and PYK2); the Fes family (including FES and FER), the ZAP70 family (including ZAP70 and SYK); the ACK family (including ACK1 and ACK2); and the Abl family (including ABL and ARG).RTK families include the EGF-receptor family (including EGFR, HER2, HER3, and HER4); the insulin receptor family (including INS-R and IGF1-R); the PDGF-receptor family (including PDGFRa, PDGFR|3, CSF1R, KIT, FLK2); the VEGF-receptor family (including FLT1, FLK1, and FLT4); the FGF-receptor family (including FGFR1, FGFR2, FGFR3, and FGFR4); the CCK4 family (including CCK4); the MET family (including MET and RON); the TRK family (including TRKA, TRKB, and TRKC); the AXL family (including AXL, MER, and SKY); the TIE / TEK family (including T IE and TIE2 / TEK); the EPH family (including EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6); the RYK family (including RYK); the MCK family (including MCK and TYRO10); the ROS family (including ROS); the RET family (including RET); the LTK family (including LTK and ALK); the ROR family (including ROR1 and ROR2); the Musk family (including Musk); the LMR family (including LMR1, LMR2 and LMR3); and the SuRTK106 family (including SuRTK106).

[0059] Representative, non-limiting examples of kinases include Abl, Abl(T315I), ALK, ALK4, AMPK, Arg, Arg, ARKS, ASK1, Aurora-A, Axl, Blk, Bmx, BRK, BrSK1, BrSK2, BTK, CaMKI, CaMKII, CaMKIV, CDK1 / cyclin B, CDK2 / cyclin A, CDK2 / cyclin E, CDK3 / cyclin E, CDK5 / p25, CDK5 / p35, CDK6 / cyclin D3, CDK7 / cyclin H / MAT1, CDK9 / cyclin T1, CHK1, CHK2, CK1( y), CK18, CK2, CK2a2, cKit(D816V), cKit, c-RAF, CSK, cSRC, DAPK1, DAPK2, DDR2, DMPK, DRAK1, DYRK2, EGFR, EGFR(L858R), EGFR(L861Q), EphA1, EphA2 , EphA3, EphA4, EphAS, EphAV, EphAS, EphB1, EphB2, EphB3, EphB4, ErbB4, Per, Fes, FGFR1, FGFR2, FGFR3, FGFR4, Fgr, Fill, Flt3(D835Y), Flt3, Flt4, F ms, Fyn, GSK3(3, GSK3a, Hck, HIPK1, HIPK2, HIPK3, IGF-1R, IKK(3, IKKa, IR, IRAKI, IRAK4, IRR, ITK, JAK2, JAK3, JNK1a1, JNK2a2, JNK3, KDR, Lck, LIMK 1, LKB1, LOK, Lyn, Lyn, MAPK1, MAPK2, MAPK2, MAPKAP-K2, MAPKAP-K3, MARK1, MEK1, MELK, Met, MINK, MKK4, MKK6, MKK7(3, MLCK, MLK1, Mnk2, MRCK-beta, MR CKa, MSK1, MSK2, MSSK1, MST1, MST2, MST3, MuSK, NEK2, NEKS, NEK6, NEK7, NLK, p70S6K, PAK2, PAK3, PAK4, PAK6, PAR-lBa, PDGFR(3, PDGFRa, PDK1, PI3K beta, PI3K delta, PI3K gamma, Pim-1, Pim-2, PKA(b), PKA, PKB(3, PKBa, PKBy, PKC^i, PKC(3I, PKC(3II, PKCa, PKCy, PKC8, PKCe, PKC^, PKCr|, PKC9, PKCi, PKD2,PKG1(3, PKGla, Plk3, PRAK, PRK2, PrKX, PTK5, Pyk2, Ret, RIPK2, ROCK-I, ROCKII, ROCK-II, Ron, Ros, Rse, Rskl, Rskl, Rsk2, Rsk3, SAPK2a, SAPK2a(T106M), SAP Includes K2b, SAPK3, SAPK4, SGK, SGK2, SGK3, SIK, Snk, SRPK1, SRPK2, STK33, Syk, TAK1, TBK1, Tie2, TrkA, TrkB, TSSK1, TSSK2, WNK2, WNK3, Yes, ZAP-70, ZIPK. In embodiments, the kinase may be ALK, Aurora-A, Axl, CDK9 / cyclinTl, DAPK1, DAPK2, Per, FGFR4, GSK3(3, GSK3a, Hck, JNK2a2, MSK2, p70S6K, PAK3, PI3K delta, PI3K gamma, PKA, PKB(3, PKBa, Rse, Rsk2, Syk, TrkA, and TSSK1. In other embodiments, the kinase may be ABL, AKT, AURORA, CDK, DBF2 / 20, EGFR, EPH / ELK / ECK, ERK / MAPKFGFR, GSK3, IKKB, INSR, JAK, Selected from the group consisting of DOM 1 / 2, MARK / PRKAA, MEK / STE7, MEKK / STE11, MLK, mTOR, PAK / STE20, PDGFR, PI3K, PKC, POLO, SRC, TEC / ATK, and ZAP / SYK.

[0060] Similarly, the serine / threonine-specific kinases comprise several distinct subfamilies, including extracellular signal-regulated kinases, (p42 / ERK2 and p44 / ERKI); c-Jun NH2-terminal kinase (JNK); cAMP response element-binding protein kinase (CREBK); cAMP-dependent kinase (CAPK); mitogen-activated protein kinase-activated protein kinase (MAPK and its relatives); stress-activated protein kinase-p38 / SAPK2; mitogen- and stress-activated kinase (MSK); protein kinases, PKA, PKB and PKC, among others.

[0061] In some embodiments, the kinase is an FMS-like tyrosine kinase 3 (Flt3), PDGFR / EGFR, Bcr-abl, Jak3, or SRC kinase inhibitor. Flt3 is also known as FLK2 (fetal liver kinase-2) and STK1 (human stem cell kinase-1).

[0062] As used herein, the term "kinase inhibitor" refers to any compound, molecule or composition that inhibits or reduces the activity of kinase, for example, phosphotransferase activity.Without limitation, kinase inhibitor can be selected from the group consisting of small or large organic or inorganic molecules; monosaccharides; disaccharides; trisaccharides; oligosaccharides; polysaccharides; biological macromolecules, such as proteins, peptides, peptide analogs and their derivatives, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, enzymes, antibodies, antibody parts or fragments; extracts made from biological materials, such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions; and any combination thereof.

[0063] A wide variety of kinase inhibitors are known in the art and can be used in the compositions and methods described herein. Kinase inhibitors include FMS-like tyrosine kinase 3 inhibitors; Aurora kinase inhibitors; Aurora-B kinase inhibitors; Aurora-C kinase inhibitors; beta-adrenergic receptor kinase inhibitors; checkpoint kinase inhibitors; cyclin-dependent kinase 1 inhibitors; cyclin-dependent kinase 2 inhibitors; cyclin-dependent kinase 4 inhibitors; cyclin-dependent kinase inhibitors; EphB2 kinase inhibitors; epidermal growth factor receptor kinase inhibitors; N-acylmannosamine kinase inhibitors; MAP kinase inhibitors; Opheline kinase inhibitors; phosphatidylinositol 3-kinase beta inhibitors; phosphatidylinositol 3-kinase gamma inhibitors; protein kinase (CK1) inhibitors; protein kinase B inhibitors; protein kinase C The inhibitors may be selected from the group consisting of eta inhibitors; protein-serine-threonine kinase inhibitors; proto-oncogene tyrosine-protein kinase Fyn inhibitors; proto-oncogene tyrosine-protein kinase Kit inhibitors; pyridoxal kinase inhibitors; Raf kinase B inhibitors; Raf kinase inhibitors; Rho-associated kinase inhibitors; ribosomal protein S6 kinase inhibitors; and any combination thereof.

[0064] In certain embodiments, the compounds disclosed herein are RET kinase inhibitors.In certain embodiments, the compounds disclosed herein (for example, CEP-701 and / or AC220) are or comprise RET inhibitors or otherwise inhibit RET phosphorylation.In certain embodiments, the compounds disclosed herein (for example, CEP-701 and / or AC220) are or comprise C-RET inhibitors or otherwise inhibit C-RET phosphorylation.

[0065] Also contemplated are compounds and compositions that reduce RET kinase ligand.For example, in certain embodiments, the disclosed compounds and compositions include antibodies or agents that interfere with RET kinase activation, such as antibodies and agents that bind to C-RET or otherwise interfere with the binding of C-RET ligand (e.g., GDNF) to it.

[0066] In certain embodiments, the kinase inhibitor is a B-Raf inhibitor, a JAK3 inhibitor, a p38MAPK inhibitor, a C-Raf1 inhibitor, an Akt inhibitor, a BMK1 / ERK5 inhibitor, a p38MAPK inhibitor, a RTK inhibitor, an ERK5 inhibitor, a Bcr-Abl inhibitor, a RhoK inhibitor, a p38 inhibitor, a p110 inhibitor, a FAK inhibitor, an ATP-competitive JNK inhibitor, or a MELK inhibitor. In some embodiments, the kinase inhibitor inhibits a pathway identified in Table 5. In some embodiments, the kinase inhibitor is one identified in Table 5.

[0067] Kinase inhibitors suitable for the compositions and methods described herein also can be found in, for example, U.S. Patent Nos. 5,674,998; 5,795,977; 5,864,033; 6,194,939; 6,239,133; 6,346,625; 6,391,894; 6,448,277; 6,492,409; 6,498,165; 670,6711; 6,723,726; 6,825,190; 6,825,355; 6,943,161; 6,951,859; 6,982,266, the entire contents of which are incorporated herein by reference. ; Same No. 6982266; Same No. 7056925; Same No. 7101884; Same No. 7105531; Same No. 7105531; Same No. 7115 No. 597; No. 7153856; No. 7183307; No. 7196090; No. 7199137; No. 7199147; No. 7 No. 223757; No. 7232826; No. 7262199; No. 7265134; No. 7309787; No. 7314940 ; Same No. 7326713; Same No. 7326713; Same No. 7449488; Same No. 7456169; Same No. 7459554; Same No. 74706 No. 93; No. 7470713; No. 7488826; No. 7504429; No. 7511040; No. 7514435; No. 7 No. 517882; No. 7521460; No. 7528132; No. 7550478; No. 7550598; No. 7572914 ; Same No. 7582652; Same No. 7598272; Same No. 7601852; Same No. 7618982; Same No. 7635703; Same No. 7648 No. 987; No. 7662977; No. 7683060; No. 7687506; No. 7732613; No. 7749994; No. 7 No. 767674; No. 7790739; No. 7812166; No. 7820662; No. 7855211; No. 7872031 ; Same No. 7893064; Same No. 7893081; Same No. 7901894; Same No. 7915443; Same No. 7943629; Same No. 79685 No. 46; No. 7994159; No. 7998507; No. 8022057; No. 8024821; No. 8026234; No. 8 No. 026246; No. 8026247; No. 8044221; No. 8093239; No. 8093383; No. 8143410;Nos. 8,148,361; and 8,152,630, and U.S. Patent Application Publication Nos. 20070161673; 20090181940; 20090215785; 20100097654; 20100234404; 20110008211; 20030044203; 20030065180; 20030087919; 20030119839; 20030139462; 20030187001; 20030199511; 20030199525; 20030199525; 0030216446; 20040034038; 20040034075; 20040082581; 200 No. 40180897; No. 20040192725; No. 20050043347; No. 20050096324; No. 20050 No. 131022; No. 20050153990; No. 20050171076; No. 20050187247; No. 2005019 No. 2304; No. 20050203114; No. 20050215556; No. 20050239794; No. 2005023981 No. 5; No. 20050261318; No. 20050267133; No. 20050277642; No. 20050277642 ; Same No. 20050288290; Same No. 20050288321; Same No. 20050288321; Same No. 20060019958; Same No. No. 20060058304; No. 20060058341; No. 20060079563; No. 20060122389; No. 2 No. 0060122389; No. 20060148824; No. 20060178388; No. 20060217369; No. 2006 No. 0264438; No. 20060270694; No. 20060276490; No. 20060281789; No. 200602 No. 87370; No. 20060287381; No. 20070049600; No. 20070054906; No. 20070060 No. 619; No. 20070078140; No. 20070099856; No. 20070099935; No. 2007012353 No. 4; No. 20070173516; No. 20070173525; No. 20070185139; No. 20070191420;Same as No. 20070191420; Same as No. 20070203143; Same as No. 20070213386; Same as No. 20070254896; Same as No. 20070259869; Same as No. 20070270425; Same as No. 20070280928; Same as No. 20080027063; Same as No. 2 No. 0080108611; No. 20080153869; No. 20080161297; No. 20080167330; No. 20080207613; No. 20080207613; No. 20080207632; No. 20080255155; No. 20080 No. 255184; same as No. 20080269244; same as No. 20080293714; same as No. 20080293785; same as No. 20080312307; same as No. 20090054425; same as No. 20090054436; same as No. 20090105209; same as No. 200901246 No. 02; same as No. 20090131407; same as No. 20090131437; same as No. 20090131506; same as No. 20090149389; same as No. 20090162376; same as No. 20090175852; same as No. 20090197862; same as No. 20090215750; Same as No. 20090221616;Same as No. 20090233960;Same as No. 20090264446;Same as No. 20090286779;Same as No. 20090298855;Same as No. 20090318440;Same as No. 20100004234;Same as No. 20100041645;Same as No. 2 No. 0100041684; No. 20100041684; No. 20100048599; No. 20100081662; No. 20100093767; No. 20100099710; No. 20100113454; No. 20100120772; No. 20100 No. 120801; same as No. 20100144732; same as No. 20100144745; same as No. 20100160303; same as No. 20100168102; same as No. 20100179134; same as No. 20100179146; same as No. 20100190816; same as No. 201002042 No. 21; same as No. 20100222342; same as No. 20100234386; same as No. 20100298301; same as No. 20100317643; same as No. 20100324041; same as No. 20100331314; same as No. 20110009410; same as No. 20110070317;Same No. 20110077237; Same No. 20110118285; Same No. 20110124623; Same No. 20110136789; Same No. 20110190280; Same No. 20110195980; Same No. 20110257238 No. 20110269739; No. 20110269772; No. 20110275630; No. 20110281857; No. 20110281866; No. 20110288097; No. 2011029374 No. 5; No. 20110294812; No. 20120015937; No. 20120041024; No. 20120053187; No. 20120065213; No. 20120071490; No. 20120071494; No. 20120077851; No. 20120095014; No. 20120095233; No. 20120212961; No. 20100267774 and No. 20100324074.;

[0068] In some embodiments, the kinase inhibitor is: [ka] and any combination thereof.

[0069] In some embodiments, the kinase inhibitor is or comprises quizartinib (AC220), or any salt, ester, or chelate thereof. In certain embodiments, the kinase inhibitor is [ka] It is.

[0070] In certain embodiments, the kinase inhibitor is selected from the group consisting of BAY-439006 (i.e., sorafenib; HMSL10008-101-1); HG-6-64-01 (i.e., HMSL10017-101-1); HKI-272 (i.e., neratinib; HMSL10018-101-1); KIN001-055 (i.e., HY-11067; HMSL10033-101-1); SB239063 (i.e., HMSL10036-101-1); KIN001-242 (i.e., HMSL10044-104-1); SB590885 (i.e., G SK2118436; HMSL10046-101-1; AZ-628 (i.e., HMSL10050-101-1); MK2206 (i.e., HMSL10057-102-1); XMD11-50 (i.e., LRRK2-in-1; HMSL10086-101-1); XMD8-92 (i.e., HMSL10094-101-1); BIRB796; Doramapimod (i.e., HMSL10169-101-1); Sunitinib Malate (i.e., SU11248; Stent; HMSL10175-10 6-1); GDC-0879 (i.e., HMSL10181-101-1); XMD8-85 (i.e., HMSL10093-101-1); AMN-107 (i.e., nilotinib; HMSL10099-101-1); Y39983 (i.e., HMSL10149-102-1); SB203580 (i.e., RWJ64809; PB203580; HMSL10167-101-1); VX-745 (i.e., HMSL10168-101-1); pseudoXL765 (i.e., HMSL10173-101-1); Y-276 32 (i.e., HMSL10176-101-1); PH-797804 (i.e., HMSL10439-101); VX-702 (i.e., HMSL10440-101); NG25 (i.e., HMSL10419-101); SB202190 (i.e., HMSL10441-101); BI-D1870 (i.e., HMSL10423-101); BIX02565 (i.e., HMSL10434-101); URMC-099 (i.e., HMSL10453-101); staurosporine aglycone (i.e., K252C;HMSL10454-101); ralimetinib (i.e., LY2228820; HMSL10438-103); BMX-IN-1 (i.e., HMSL10427-101); PF3644022 (i.e., HMSL10476-101); NVP-BHG712 (i.e., KIN001-265; HMSL10200-101); bosutinib (i.e., SKI-606; HMSL10189-101); NVP-TAE226 (i.e., CHIR-265; H MSL10207-101; RAD001 (i.e., everolimus; HMSL10235-101); CC-401 (i.e., HMSL10185-101); CGP74514A (i.e., HMSL10355-101); KIN001-269 (i.e., HMSL10195-101); RAF265 (i.e., HMSL10206-101); OTSSP167 (i.e., HMSL10337-102); Dorsomorphin (i.e., Compound C; BML275; HMSL10399-102; losmapimod (i.e., GSK-AHAB; SB856553; GW856553X; HMSL10402-101); AZD5363 (i.e., HMSL10370-101); RO31-8220 (i.e., bisindolylmaleimide IX; HMSL10407-103); sotrastaurin (i.e., AEB0 71; HMSL10408-101); TAK-632 (i.e., HMSL10409-101); FRAX597 (i.e., HMSL10400-101); GW2580 (i.e., HMSL10401-101); Alisertib (i.e., MLN8237; HMSL10391-101) or derivatives, salts, metabolites, prodrugs, and stereoisomers thereof. In some embodiments, the compound is XMD8-92, SB239063, XMD11-50, or derivatives, salts, metabolites, prodrugs, and stereoisomers thereof.

[0071] In some embodiments of this and other aspects described herein, the activity of the kinase is inhibited or reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a non-inhibited control. Without wishing to be bound by theory, the activity of the kinase can be determined using any assay known in the art for measuring the activity of a kinase, for example, by measuring phosphorylation.

[0072] Hedgehog signaling pathway modulators As used herein, the term "modulate" with respect to the Hedgehog signaling pathway means to regulate, either positively or negatively, the normal function of a component of the Hedgehog signaling pathway. Thus, the term modulate can be used to refer to increasing, decreasing, masking, altering, neutralizing, or restoring the normal function of a component of the Hedgehog signaling pathway.

[0073] In some embodiments of the aspects described herein, the modulator modulates at least one activity of the Hedgehog signaling pathway by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0074] The terms "hedgehog signaling pathway", "hedgehog pathway" and "hedgehog signaling pathway" are all used to refer to a series of events that are typically mediated by hedgehog, smoothened, Ptch1 and Gli, among others, and that lead to gene expression changes and other phenotypic changes typical of hedgehog activity. Activation of downstream components can activate the hedgehog pathway even in the absence of hedgehog protein. For example, overexpression of smoothened activates the pathway in the absence of hedgehog, and Gli and Ptch1 gene expression are indicators of an active hedgehog-signaling pathway. Thus, the compounds described herein can be used to overcome inappropriate increases in Hedgehog signaling, regardless of whether said increases in signaling are the result of a mutation / lesion in a component of the Hedgehog signaling pathway (e.g., Ptch1, Glil, Gli3, smoothened, etc.) or whether said increases in signaling occur in the context of a cell that does not contain a mutation / lesion in a component of the Hedgehog signaling pathway (e.g., a wild-type cell with respect to the component of the Hedgehog signaling pathway). Thus, in some embodiments, the cells have a phenotype of smoothened gain-of-function, hedgehog gain-of-function, patched (Ptc) loss-of-function, Gli gain-of-function, and / or overexpression of a hedgehog ligand.

[0075] The term "smoothened gain of function" refers to abnormal modification or mutation of the smo gene or increased expression level of the gene, which results in a phenotype similar to contact of cells with hedgehog protein, e.g., abnormal activation of the hedgehog pathway. Without wishing to be bound by any particular theory, it is noted that Ptch1 does not directly signal cells, but rather modulates the activity of smoothened, another membrane-bound protein that is downstream of Ptch1 in hedgehog signaling (Marigo et al. (1996) Nature, 384:177-179; Taipale et al. (2002) Nature, 418, 892-896). The smo gene is a segment polarity gene required for the correct patterning of each segment in Drosophila (Alcedo et al. (1996) Cell 86:221-232). The human homologue of smo has been identified. See, e.g., Stone et al. (1996) Nature 384:129-134, and GenBank accession U84401. The smoothened gene encodes a heterotrimeric G protein-coupled receptor; i.e., an integral membrane protein with the characteristics of seven transmembrane domains. The protein shows homology to the Drosophila Frizzled (Fz) protein, a member of the wingless pathway. Ptc is an Hh receptor. Cells expressing Smo are unable to bind Hh, indicating that smo does not directly interact with Hh (Nusse, (1996) Nature 384:119-120). Rather, it is believed that Sonic Hedgehog (SHH) binds to its receptor, and PTCH prevents the normal inhibition of smoothened by PTCH. Activation of smoothened mutations is known to occur in sporadic basal cell carcinomas (Xie et al., Nature, 1998, vol. 391:90-92) and in primitive neuroectodermal tumors of the central nervous system (Reifenberger et al., Cancer Res., 1998, vol. 58:1798-1803).

[0076] The term "hedgehog gain of function" refers to a phenotype similar to contact of cells with hedgehog protein, for example, abnormal modification or mutation of Ptch1, hedgehog, or smoothened genes, or reduced (or lost) expression levels of such genes, resulting in abnormal activation of the hedgehog pathway. Gain of function may include loss of ability of Ptch1 gene product to regulate the expression levels of Ci homolog genes, for example, Gli1, Gli2, and Gli3. The term "hedgehog gain of function" is also used herein to refer to any similar cellular phenotype (e.g., exhibiting hyperproliferation) caused by changes in any of the hedgehog signaling pathway, including but not limited to modification or mutation of hedgehog itself. For example, tumor cells with abnormally high proliferation rates due to activation of the hedgehog signaling pathway will have a "hedgehog gain of function" phenotype, even if hedgehog is not mutated in the cell.

[0077] The term "patched loss of function" refers to the abnormal modification or mutation of Ptch1 gene, or the decreased expression level of the gene, which results in a phenotype similar to the contact of cells with hedgehog protein, for example, the abnormal activation of hedgehog pathway. Loss of function can include the loss of ability of Ptch1 gene product to regulate the expression level or activity of Ci homolog genes, for example, Gli1, Gli2, and Gli3. The term "Ptch1 loss of function" is also used herein to refer to any similar cellular phenotype (e.g., hyperproliferation) caused by any change in the hedgehog signaling pathway, including but not limited to the modification or mutation of Ptch1 itself. For example, tumor cells with abnormally high proliferation rate due to activation of hedgehog signaling pathway will have a "Ptch1 loss of function" phenotype, even if Ptch1 is not mutated in the cell.

[0078] The term "Gli gain of function" refers to an abnormal modification or mutation of a Gli gene, or increased expression levels of the gene, that results in a phenotype similar to a cellular response to a hedgehog protein, e.g., abnormal activation of the hedgehog pathway.

[0079] The vertebrate family of Hedgehog genes includes three members present in mammals known as Desert (Dhh), Sonic (Shh) and Indian (Ihh) Hedgehog, all of which code for secreted proteins. These various Hedgehog proteins consist of a signal peptide, a highly conserved N-terminal region and a more divergent C-terminal domain. Biochemical studies have shown that autoproteolytic cleavage of the Hh precursor protein proceeds through an internal thioester intermediate, which is subsequently cleaved in a nucleophilic displacement. Presumably, the nucleophile is a small lipophilic molecule that covalently binds to the C-terminus of the N-peptide, tethering it to the cell surface. The biological consequences are profound. As a result of that tethering, a high local concentration of the N-terminal Hedgehog peptide occurs at the surface of Hedgehog-producing cells. It is this N-terminal peptide that is necessary and sufficient for short-term and long-term Hedgehog signaling activity.

[0080] In an inactive Hedgehog signaling pathway, the transmembrane protein receptor Patched (Ptc) inhibits the activity of the seven-transmembrane protein Smoothened (Smo). The transcription factor Gli, a downstream component of Hh signaling, is processed into a repressor form, and nuclear accumulation of the activator form is prevented through interactions with cytoplasmic proteins including Fused and Suppressor of fused (Sufu). As a result, transcriptional activation of Hedgehog target genes is suppressed. Activation of the pathway is initiated through binding of any of three mammalian ligands (Dhh, Shh or Ihh) to Ptc. Ligand binding results in the reversal of repression of Smo and thus activation of a cascade that results in the translocation of the active form of the transcription factor Gli to the nucleus. Nuclear Gli activates target gene expression, including Ptc and Gli itself. Elevated levels of Hedgehog signaling are sufficient to initiate cancer formation and are required for tumor survival.

[0081] Hedgehog signaling pathway modulators can be agonists or antagonists of the Hedgehog signaling pathway.

[0082] The term "hedgehog agonist" refers to an agent that antagonizes or blocks the biological activity of patched to increase the transcription of target genes. Hedgehog antagonists can be used to overcome ptc gain-of-function and / or smoothened loss-of-function, where the latter are also referred to as "smoothened agonists." The term "hedgehog antagonist" also refers to any agent that can act by directly inhibiting the normal function of hedgehog protein, as well as any agent that inhibits the hedgehog signaling pathway to recapitulate the function of ptc.

[0083] Exemplary hedgehog signaling pathway modulators include, but are not limited to, AY9944, triparanol, jervine, cyclopamine, tomatidine, and the like.

[0084] GPCR Modulators As used herein, the term "modulate" in reference to a GPCR means to regulate, positively or negatively, the normal function of a GPCR signaling pathway. Thus, the term modulate can be used to refer to increasing, decreasing, masking, altering, neutralizing, or restoring the normal function of a GPCR. A GPCR modulator can be a GPCR agonist or a GPCR antagonist.

[0085] In some embodiments of the aspects described herein, the modulator modulates at least one activity of the GPCR by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0086] G protein-coupled receptors (GPCRs) form a widespread superfamily of cell surface receptors characterized by an amino-terminal extracellular domain, a carboxyl-terminal intracellular domain, and a serpentine structure that spans the cell membrane seven times. Thus, such receptors are sometimes also referred to as seven-transmembrane (7TM) receptors. These seven transmembrane domains define three extracellular and three intracellular loops in addition to the amino- and carboxy-terminal domains. The extracellular portion of the receptor is responsible for recognizing and binding one or more extracellular binding partners (e.g., ligands), while the intracellular portion is responsible for recognizing and communicating with downstream molecules in a signaling cascade.

[0087] Overall, GPCRs can be divided into six classes based on sequence homology and functional similarity: class A (or 1) (rhodopsin-like); class B (or 2) (secretin receptor family); class C (or 3) (metabotropic glutamate / pheromone); class D (or 4) (fungal mating pheromone receptors); class E (or 5) (cyclic AMP receptors); and class F (or 6) (Frizzled / Smoothened). The very large rhodopsin A group has been further subdivided into 19 subgroups (A1–A19). More recently, an alternative classification system called GRAFS (glutamate, rhodopsin, adhesion, Frizzled / Taste2, secretin) has been proposed.

[0088] As used herein, the term "GPCR ligand" refers to a molecule that binds to a GPCR. G protein-coupled receptors bind a variety of ligands, including calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants, and even photons, and are important for the normal (and sometimes abnormal) function of many cell types [see generally Strosberg, Eur. J. Biochem. 196:1-10 (1991) and Bohm et al., Biochem J. 322:1-18 (1997)]. When a specific ligand binds to its corresponding receptor, the ligand typically stimulates the receptor to activate a specific heterotrimeric guanine-nucleotide-binding regulatory protein (G protein) that is coupled to the intracellular portion of the receptor. The G protein then transmits a signal to an effector molecule within the cell by stimulating or inhibiting the activity of the effector molecule. These effector molecules include adenylate cyclase, phospholipases, and ion channels. Adenylyl cyclase and phospholipase are enzymes involved in the production of the second messenger molecules cAMP, inositol triphosphate, and diacylglycerol. Through this sequence of events, extracellular ligand stimulation leads to intracellular changes via G protein-coupled receptors. Each such receptor has its own characteristic primary structure, expression pattern, ligand binding profile, and intracellular effector system.

[0089] GPCRs include those that mediate sensory signals (e.g., light and olfactory stimuli); adenosine, bombesin, bradykinin, endothelin, gamma-aminobutyric acid (GABA), hepatocyte growth factor (HGF), melanocortins, neuropeptide Y, opioid peptides, opsin, somatostatin, GH, tachykinins, members of the vasoactive intestinal peptide family, and vasopressin; biogenic amines (e.g., dopamine, epinephrine, norepinephrine, histamine, glutamate (metabotropic), glucagon, and ketones). GPCRs include receptors for: acetylcholine (muscarinic action), acetylcholine (muscarinic action), and serotonin; chemokines; lipid mediators of inflammation (e.g., prostaglandins, prostanoids, platelet-activating factor, and leukotrienes); and peptide hormones (e.g., calcitonin, C5a anaphylatoxin, follicle-stimulating hormone (FSH), gonadotropin-releasing hormone (GnRH), neurokinins, thyrotropin-releasing hormone (TRH), cannabinoids, and oxytocin). GPCRs that act as receptors for as yet unidentified stimuli are known as orphan receptors.

[0090] However, in other types of receptors that have been studied, where the ligand is externally bound to the membrane, the ligand of the GPCR typically binds within the transmembrane domain, whereas protease-activated receptors are activated by cleavage of part of their extracellular domain.

[0091] Types of GPCR ligands include, but are not limited to: agonists, which shift the equilibrium in favor of the active state; inverse agonists, which shift the equilibrium in favor of the inactive state; and neutral antagonists, which do not affect the equilibrium. When an active GPCR encounters a G protein, it can activate the G protein. GPCRs are the target of approximately 40% of all prescription drugs on the market. (Filmore, Modern Drug Discovery, November 2004, p. 11). Examples of commonly prescribed GPCR-based drugs include atenolol (TENORMIN®), albuterol (VENTOLIN®), ranitidine (ZANTAC®), loratadine (CLARITIN®), hydrocodone (VICODIN®), theophylline (THEODUR®), and fluoxetine (PROZAC®).

[0092] Exemplary GPCR modulators include, but are not limited to, corticotropin releasing factor (CRF), urocortin 1, urocortin 2, usorcortin 3, parathyroid hormone, PTH-related hormone, TIP39, calcitonin, amylin, CGRP (CALCA and CALCB), adrenomedullin, secretin, VIP, PACAP, glucagon, GHRH, GLP-1, GLP-2, dynorphin A, dynorphin A amide. , dynorphin A(1-6), dynorphin A(1-13), dynorphin A(2-13), dynorphin A(2-17), MetEnk, Met-Enk-RF-amide, Met-Enk-Arg-Phe, Met-Enk-Glyleu, [D-pGlul, D-Phe2, D-Trp3,6]-LH-RH, gl-MSH amide, g2-MSH, [N-MePhel, D-Pro4]-morphiceptin (PL017), ACTH (human), Leu-Enk, Adrenomedullin(22-52), Adrenomedullin(26-52)(human)(ADM antagonist), Agouti 1-40 amide, Agouti-related protein(87-132)-amide, alpha-MSH, alpha-Neo-endorphin, Amylinamide, BAM(1-20), BAM(1-22), BAM(2-22), BAM(6-22), BAM(1-20), ANP (atrial natriuretic peptide), Anti-inflammatory peptide 1, Anti-inflammatory peptide 2, (3- Endorphin, Benzylureido-Met-Leu-Phe, beta-ANP, beta-endorphin, beta-MSH, Big Endothelin-1, Big Gastrin-1, BNP (Brain Natriuretic Peptide-32), BNP-45 (Cardiac Natriuretic Peptide, Bombesin, BAM(8-25), BAM(8-20), FLRF, Calcitonin Gene-Related Peptide, NPFF, Calcitonin, Calcitonin Gene-Related Peptide(8-37), CART(55-1,02), CART(55102) [Met(O)67, CART(61-102), CGRP(8-37), CGRP II, cholecystokinin octapeptide [CCK(26-33)], cholecystokinin-33, CNP-22 (C-type natriuretic peptide), corticotropin releasing factor, cortistatin-14, NPAF, SST, NPY, FMRFamide, Orpanin FQFMRFamide related peptide, YMRFamide, YLPLRFamide, YFMRFamide, LPLRFamide, dFMRFamide, W-Nle-RF-amide, and ACEP.

[0093] Polypeptide modulators of GPCRs include, but are not limited to, vasopressin, oxytocin, somatostatin, neuropeptide Y, GnRH, luteinizing hormone, follicle-stimulating hormone, parathyroid hormone, orexin, urotensin II, endorphins, enkephalins, etc. A list of GPCR modulators is compiled online at pharminfo.pharm.kyoto-u.ac.jp / services / glida / ligand_classification.php.

[0094] In some embodiments, the GPCR modulator inhibits binding of the ligand by at least about any one of or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to a control. Binding of a ligand to a GPCR can be determined by any method known to one of skill in the art.

[0095] In some embodiments, a GPCR modulator reduces activity of a GPCR by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a non-inhibited control.

[0096] In some embodiments, a GPCR modulator enhances activity of a GPCR by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative to a non-activated control.

[0097] In some embodiments, a GPCR modulator is capable of binding to the active site of a GPCR (eg, the binding site for a ligand).

[0098] In some embodiments, the serotonin receptor modulator is capable of binding to an allosteric site of a GPCR.

[0099] In some embodiments of this and other aspects described herein, the GPCR antagonist has an IC50 of less than or equal to 500 nM, less than or equal to 250 nM, less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equal to 0.001 nM.

[0100] In some embodiments of this and other aspects of the invention, the GPCR agonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM.

[0101] In some embodiments, the GPCR modulator comprises: [ka] and any combination thereof.

[0102] Dopamine Receptor Modulators As used herein, the term "dopamine receptor modulator" refers to a compound that modulates one or more dopamine receptors. A dopamine receptor modulator can be a dopamine agonist or a dopamine antagonist. As used herein, the term "dopamine agonist" refers to a compound that activates and / or stimulates one or more dopamine receptors, and / or increases the level of dopamine (such as L-dopa or drugs that inhibit dopamine metabolism), and / or stimulates the dopamine signaling pathway, and / or decreases the level of norepinephrine, and / or inhibits the norepinephrine signaling pathway. The term "dopamine agonist" also includes analogs of dopamine molecules that exhibit at least some biological activity in common with natural human dopamine receptors. Thus, the term "dopamine agonist" encompasses dopaminergic agents. As used herein, the term "dopaminergic agents" refers to compounds that mimic the action of dopamine. Thus, the term dopaminergic agent is intended to encompass dopamine, derivatives of dopamine, and compounds that have dopamine-like actions on dopamine receptors. Exemplary analogs of dopamine include ergolines and aporphines, such as apomorphine, pergolide, bromocriptine, and lisuride.

[0103] In some embodiments of the aspects described herein, the modulator modulates at least one activity of a dopamine receptor by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0104] Without wishing to be bound by theory, dopamine agonist may act through one of several pathways.For example, dopamine agonist may activate or enhance D1 dopamine receptor and / or Dj-like receptor, for example, D1 and D5 dopamine receptor and / or D2 dopamine receptor (for example, D2, D2 short and D2 long receptor, D4 and D4 dopamine receptor) and / or D3 dopamine receptor and / or D4 dopamine receptor.Dopamine agonist may act by inhibiting one or more enzymes involved in the biosynthesis and / or conversion and / or degradation of dopamine.

[0105] Exemplary dopamine agonists include, but are not limited to, (-)-7-{[2-(4-phenylpiperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol; (+)-4-propyl-9-hydroxynaphthoxazine ((+)PHNO); (E)-1-aryl-3-(4-pyridinepiperazin-1-yl)propanone oxime; (R)-3-(4-propylmorpholin-2-yl)phenol (PF-219,061); (R,R)-S32504; 2-(N-phenylethyl-N-propylamino)- 5,6,7,8-Tetrahydro-6-(2-propen-1-yl)-4H-thiazolo[4,5-d]azepin-2-amine (BHT-920);5-HT uptake inhibitors;5-HT-1A agonists (e.g. roxindole);6-Br-APB;6-Methyl-8-a-(N-acyl)amino-9-ergoline;6-Methyl-8-a-(N-phenyl-acetyl)amino-9-ergoline;6-Methyl-8β-carbobenzyloxy-a Aminoethyl-10-a-ergoline;7,8-dihydroxy-5-phenyl-octahydrobenzo[h]isoquinoline;8-acylaminoergolines;9,10-dihydroergocomine;a2-adrenergic antagonists (e.g., terguride);A-412,997;A-68,930;A-77,636;A-86,929;ABT-670;ABT-724;AF-14;alaptide;amisulpride;Any D-2-halo-6-alkyl-8-substituted ergoline;Aplindore;Apomorphine;A Lipiprazole (known as Abilify in the USA); benzazepine analogues; BP-897; bromocriptine; bromocriptine mesylate; cabergoline; cis-8-hydroxy-3-(n-propyl)-1,2,3a,4,5,9b-hexahydro-1H- and trans-N-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]cyclohexyl}-3-methoxybenzamide; clozapine; COMT inhibitors (e.g. CGP-28014, entacapone and tolcapone); CP-226,269; CP-96,345; CY-208,243;D-2-Bromo-6-methyl-8-cyanomethylergoline;Dihydrexidine;Dihydro-alpha-ergocryptine;Dihydro-alpha-ergotoxine;Dihydroergocryptine;Dihydroergocryptine;Dihydroergotoxine (Hydergine);Dinapsoline;Dinoxyline;Domperidone;Dopamine;Dopamine D1 receptor agonist;Dopamine D2 receptor agonist;Dopamine D3 receptor agonist;Dopamine D4 receptor agonist;Dopamine D5 receptor agonist;Dopamine uptake inhibitors (GBR-1290 9, GBR-13069, GYKI-52895, and NS-2141, etc.); doprexin; doxanthrine; ER-230; erfotoxine; ergocornine; ergoline derivatives; ergot alkaloid derivatives; eticlopride; etisulergine; FAUC299; FAUC316; fenoldopam; flibanserin; haloperidol; iloperidone; L-dopa; levodopa; lisuride; lisuride; LSD; L U111995; Mazapertine; Methylphenidate; Monoamine oxidase-B inhibitors (such as selegiline, N-(2-butyl)-N-methylpropargylamine, N-methyl-N-(2-pentyl)propargylamine, AGN-1133, ergot derivatives, lazabemide, LU-53439, MD-280040 and mofegiline); N-0434; Naxagolide; Olanzapine; Opiate receptor agonists (such as NIH-10494); PD-118,440; PD-168,077; Pergolide (A-68939, A-77636, Dihydrexidine, etc.) , and SKF-38393;PIP3EA;Piribedil;Primipexole;Quinagolide;Quinelorane;Quinpirole;Racemic trans-10,11-dihydroxy5,6,6a,7,8,12b-hexahydro and related benzazepine analogues;Raclopride;Remoxipride;Risperidone;Ro10-5824;Ropinirole;Rotigotine;Salvinorin A;SDZ-HDC-912;Sertindole;SKF-38,393;SKF-75,670;SKF-81,297;SKF-82,526 (Fenoldopam);SKF-82,598;SKF-82,957;SKF-82,958;SKF-38,393;SKF-77,434;SKF-81,297;SKF-82,958;SKF-89,145;SKF-89,626;spiperone;spiroperidol;sulpride;sumanilol;talipexole;terguride;tropapride;WAY-100635;YM09151-2;zetidoline;β-adrenergic receptor agonists;and analogs, derivatives, enantiomers, metabolites, prodrugs, and pharma- ceutically acceptable salts thereof.;

[0106] Exemplary beta-3 adrenergic receptor agonists include, but are not limited to, DPDMS; dopexamine; AJ-9677; AZ-40140; BMS187413; BMS-194449; BMS-210285; BRL-26830A; BRL-28410; BRL-35135; BRL-37344; CGP 12177;CL-316243;CP-114271;CP-331648;CP-331679;D-7114;FR-149175;GW-2696;GW-427353;ICI-198157;L-750355;L-796568;LY-377604;N-5984;SB-226552;SR-58611A;SR-59062A;SWR0342SA;ZD-2079; and analogues, derivatives, enantiomers, metabolites, prodrugs, and pharma- ceutically acceptable salts thereof.

[0107] In some embodiments, the dopamine agonist enhances activity of a dopamine receptor by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative to a non-activated control.

[0108] In some embodiments, the dopamine agonist inhibits binding of a ligand to its receptor by at least about any one of or by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to a control.

[0109] In some embodiments, the dopamine receptor modulator is capable of binding to the active site of a dopamine receptor (eg, the binding site for a ligand).

[0110] In some embodiments, the dopamine receptor modulator is capable of binding to an allosteric site of a dopamine receptor.

[0111] In some embodiments of this and other aspects described herein, the dopamine agonist has an IC50 of less than or equal to 500 nM, less than or equal to 250 nM, less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equal to 0.001 nM.

[0112] In some embodiments of this and other aspects of the invention, the dopamine agonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

[0113] In some embodiments, the dopamine agonist inhibits dopamine beta-hydroxylase, which converts dopamine to norepinephrine. Thus, by inhibiting dopamine beta-hydroxylase, intracellular dopamine increases and norepinephrine decreases.

[0114] Exemplary inhibitors of DBH include, but are not limited to, fusaric acid; 1,1',1",1"'-[disulfanediylbis-(carbonothioylnitrilo)]tetraethane (disulfuram); 2-hydroxy-2,4,6-cycloheptatrien-1-one (also called tropolone, 2-hydroxytropone, or purprocatechol); 5-(aminomethyl)-1-[(2S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-1,3-dihydro-2H-imidazole-2-thione (Nepicastat, INN, or SYN117); 1-(4-hydroxybenzyl)imidazole-2-thiol; FLA-63; diethyldithiocarbamate (diethyidi thiocarbamate); beta-chlorophenethylamine; 4-hydroxybenzyl cyanide; 2-halo-3(p-hydroxyphenyl)-1-propene; 1-phenyl-1-propyne; 2-phenylallylamine; 2-(2-thienyl)allylamine; 2-thiophene-2(2-thienyl)allylamine; 3-phenylpropargylamine; 1-phenyl-1(aminoethyl)ethane; N-(trifluoroacetyl)phenyl(aminoethyl)ethane; 5-picolinic acid substituted with alkyl groups containing up to 6 carbon atoms; 5-picolinic acid substituted with haloalkyl groups containing up to 6 carbon atoms; and analogs, derivatives, enantiomers, metabolites, prodrugs, and pharma- ceutically acceptable salts thereof.

[0115] Other inhibitors of dopamine beta-hydroxylase include, but are not limited to, those described in U.S. Pat. Nos. 4,487,761; 4,634,711; 4,719,223; 4,743,613; 4,749,717; 4,761,415; 4,762,850; 4,798,843; 4,810,800; 4,835,154, the contents of all of which are incorporated herein by reference in their entirety. These include Nos. 4,839,371; 4,859,779; 4,876,266; 4,882,348; 4,906,668; 4,935,438; 4,963,568; 4,992,459; 5,100,912; 5,189,052; 5,597,832; 6,407,137; 6,559,186; 7,125,904; and 7,576,081.

[0116] In some embodiments of this and other aspects of the invention, activity of dopamine beta-hydroxylase is inhibited or reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a non-inhibited control.

[0117] In some embodiments, the dopamine beta-hydroxylase inhibitor has a desired activity at a concentration lower than the concentration of the inhibitor required to produce another unrelated biological effect. In some exemplary embodiments, the concentration of the inhibitor required for dopamine beta-hydroxylase inhibitory activity is at most about 2-fold lower, at most about 5-fold lower, at most about 10-fold lower, or at most about 20-fold lower than the concentration required to produce the unrelated biological effect.

[0118] In some embodiments of this and other aspects described herein, the dopamine beta-hydroxylase inhibitor has an IC50 of less than or equal to 500 nM, less than or equal to 250 nM, less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equal to 0.001 nM.

[0119] In some embodiments, the dopamine receptor modulator inhibits binding of the ligand by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to a control, or by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. Binding of a ligand to a dopamine receptor can be determined by any method known to one of skill in the art.

[0120] In some embodiments, the dopamine receptor modulator reduces the activity of the dopamine receptor by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a non-inhibited control.

[0121] In some embodiments, the dopamine receptor modulator enhances activity of a dopamine receptor by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative to a non-activated control.

[0122] In some embodiments, the dopamine receptor modulator is capable of binding to the active site of a dopamine receptor (eg, the binding site for a ligand).

[0123] In some embodiments, the serotonin receptor modulator is capable of binding to an allosteric site of a dopamine receptor.

[0124] In some embodiments of this and other aspects described herein, the GPCR antagonist has an IC50 of less than or equal to 500 nM, less than or equal to 250 nM, less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equal to 0.001 nM.

[0125] In some embodiments of this and other aspects of the invention, the GPCR agonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM.

[0126] Serotonin Receptor Modulators As used herein, the term "modulate" in relation to serotonin receptor means to regulate the normal function of serotonin receptor positively or negatively.Thus, the term modulate can be used to refer to increasing, decreasing, masking, altering, neutralizing, or restoring the normal function of serotonin receptor.Serotonin receptor modulators can be agonists or antagonists of serotonin receptor.

[0127] In some embodiments of the aspects described herein, the modulator modulates at least one activity of a serotonin receptor by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0128] Serotonin (5-hydroxytryptamine, 5-HT) is a major neurotransmitter that elicits its effects through a number of receptors. To date, at least 15 different 5-HT receptors have been identified, mostly as a result of cDNA cloning, and these receptors have been classified into seven families (5-HT 1 ~5-HT 7 ). See, e.g., Hoyer et al., Pharmacol. Biochem. Behav., 2002, 71:533-554. Fourteen of the fifteen cloned 5-HT receptors are expressed in the brain. 5-HT has been implicated in many pathologies, particularly conditions of the central nervous system, including depression, anxiety, schizophrenia, eating disorders, obsessive-compulsive disorder, learning and memory dysfunction, migraine headaches, chronic pain, sensory perception, motor activity, thermoregulation, nociception, sexual behavior, hormone secretion, and cognition.

[0129] As used herein, the term "serotonin receptor modulator" intends and includes compounds that bind to serotonin receptors or inhibit the binding of ligands thereto, or reduce, eliminate, increase, enhance, or mimic the activity of serotonin receptors.Thus, "serotonin receptor modulator" includes both serotonin receptor antagonists and serotonin receptor agonists. In some embodiments, the serotonin receptor modulator binds to or inhibits binding of a ligand to a 5-HT1A and / or 5-HT1B and / or 5-HT2A and / or 5-HT2B and / or 5-HT2C and / or 5-HT3 and / or 5-HT4 and / or 5-HT6 and / or 5-HT7 receptor, or reversibly or irreversibly reduces, eliminates, increases, enhances, or mimics the activity of a 5-HT1A and / or 5-HT1B and / or 5-HT2A and / or 5-HT2B and / or 5-HT2C and / or 5-HT3 and / or 5-HT4 and / or 5-HT6 and / or 5-HT7 receptor.

[0130] In some embodiments, the serotonin modulator is a serotonin receptor antagonist.

[0131] Exemplary serotonin modulators include, but are not limited to, (-) cisapride; (-) norcisapride; (-) venlafaxine; (+) cisapride; (+) norcisapride; (+) venlafaxine; 1-(2-fluorophenyl)-3-(4-hy)-prop-2-en-1-one-O-(2-dimethylaminoethyl)-oxime; [5[3-(4-methylsulfonylamino)-benzyl-1,2,4-oxadiazol-5yl]-1H-indol-3-yl]ethanamine (L694247). ;2-Hydroxymethylolanzapine;2-Methyl-5-HT;2C-B;3-Tropanyl-indole-3-carboxylate;3-Tropanyl-indole-3-carboxylate methiodide;311C90;5-CT;5-MeO-DMT;5-MT;8-OH-DPAT;A-372,159;Agomelatine;AL-38022A;Almotriptan;Alnitidan;Alosetron;Alosetron;Alpha-Me-5-HT;Alprenolol;Amitriptyline;AR-A000002;Aripiprazole;AS-19;A Senapine; BIMU-8; BMY7378; BRL-15572; Bufotenin; Buspirone; BVT-933 (Biovitrum); BW-723C86; BZP; Cannabidiol; Chlorpromazine; Cilansetron; Cinitapride; Cisapride; Citalopram; Clomipramine; Clozapine; Cnanserin; CP-93,129; CP-94,253; Cyanopindolol; Dazopride; Demethylcitalopram; Demethylsertraline; Desipramine; Desmethylolanzapine; Dihydroergotamine; Dimebolin; DMT; Dolasetron; DOM; EGIS-12233; Eletriptan; Eletriptan; EMD-386,088; EMDT; Eplivanserin; Etoperidone; Fenfluramine; Flusinoxan; Flibanserin; Fluoxetine; Fluphenazine; Fluvoxamine; Frovatriptan; Gepirone; GR127935; Granisetron; Haloperidol; Homochlorcyclizine; Hydrodolasetron; Iloperidone; Imipramine; Iodocyanopindolol; Ipsapirone; Ketanserin;1-[5(2-thienylmethoxy)-1H-3-indolyl[propan-2-amine hydrochloride (BW723C86);L-lysine;Lecozotan;Lisuride;Lorcaserin;Loxapine;LSD;LY-278,584;LY-53,857;m-chlorophenylpiperazine (MCPP);MDL11939;MDMA;Mefway;Memantine;Mescaline;Metergoline;Methiothepin;Methiothepin;Methysergide;Metoclopramide;Mianserin;Mirtazapine;Mosapride;MS-245;Myristicin;N AN-190;naratriptan;nefazodone;norcisapride;norfenfluramine;norfluoxetine;nortriptaline;olanzapine;ondansetron;oxetron;oxprenolol;p-NPPL;paroxetine;perlapine;pivocerod;pimavanserin;pindolol;piperazine;pizotifen;propanolol;prucalopride;psilocin;psilocybin;quetiapine;quetiapine;risperidone;quipazine;R-hydroxynefazodone;r (-)Fluoxetine;r(+)Ondansetron;Rauwolscine;Renzapride;Renzapride;Risatriptan;Risperidone;Ritanserin;Rizatriptan;Ro04-6790;Robalzotan;RS-56812;RS-67333;RU24969;RU24969;s(+)Fluoxetine;S15535;SB206553;SB216641;SB242084;SB-258,585;SB-269,970;SB-271,046;SB-357,134;SB-399,885;S B-699,551;SDZ-205,557;Sertraline;Sibutramine;Spiperone;Sumatriptan;Tandospiroe;Tegaserod;TFMPP;Trazodone;Tropisetron;Tryptamine;UH-301;Urapidil;Valerenic acid;Venlafaxine;WAY-100,135;WAY-100,635;Zaliproden;YM-348;;Yohimbine;Zacopride;;Zalospirone;Zatosetron;Ziprasidone;Zolmitriptan;and analogs, derivatives, enantiomers, metabolites, prodrugs, and pharma- ceutically acceptable salts thereof.

[0132] Additional serotonin modulators include those described in U.S. Pat. Nos. 4,737,496; 4,782,063; 4,788,290; 4,789,673; 4,797,406; 4,903,691; 5,001,133; 5,017,582; 5,130,313; 5,143,916; 5,202,318; 5,232,924; 5,260,303; 5,320,317; 5,320,319; 5,420,320; 5,420,321; 5,420,322; 5,420,323; 5,420,324; 5,420,325; 5,420,326; 5,420,327 ... ,319,085; 5,356,934; 5,399,557; 5,434,161; 5,516,782; 5,591,749; 5,604,239; 5,612,366; 5,705,509 No. 5,728,835; No. 5,736,544; No. 5,874,429; No. 5,962,448; No. 6,187,772; No. 6,255,306; No. 6,235,745; No. 6,271,223; No. 6,288 , No. 6,316,468; No. 6,353,008; No. 6,436,964; No. 6,638,934; No. 6,686,374; No. 6,743,913; No. 6,828,330; No. 6,911,452; No. No. 7,109,339; No. 7,244,722; No. 7,297,711; No. 7,351,707; No. 7,375,114; No. 7,592,355; No. 7,655,691; No. 7,772,239; No. 7,781,47 Nos. 6, 7,851,474, and compounds described in U.S. Patent Application Publication Nos. 2003 / 0153576; 2005 / 0215555; 2006 / 0003990; 2006 / 0025601; 2006 / 0079567; 2006 / 0100266; 2006 / 0178366; 2007 / 0032481; 2007 / 0244086; 2010 / 0004264; and 2010 / 0069356.

[0133] In some embodiments, the serotonin receptor modulator inhibits the binding of the ligand by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the control, or by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%.The binding of the ligand to the serotonin receptor can be determined, for example, by the assay described in US Patent Application Publication No. 2009 / 0239854, the contents of which are incorporated herein by reference in their entirety.

[0134] In some embodiments, the serotonin receptor modulator reduces the activity of the serotonin receptor by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a non-inhibited control.

[0135] In some embodiments, a serotonin receptor modulator enhances activity of a serotonin receptor by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative to a non-activated control.

[0136] In some embodiments, a serotonin receptor modulator can bind to the active site of a serotonin receptor (eg, the binding site for a ligand).

[0137] In some embodiments, the serotonin receptor modulator is capable of binding to an allosteric site of a serotonin receptor.

[0138] In some embodiments of this and other aspects of the invention, the serotonin antagonist has an IC50 of less than or equal to 500 nM, less than or equal to 250 nM, less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equal to 0.001 nM.

[0139] In some embodiments of this and other aspects of the invention, the serotonin agonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

[0140] Histamine Receptor Modulators As used herein, the term "modulate" in reference to a histamine receptor means to regulate, positively or negatively, the normal function of the histamine receptor. Thus, the term modulate can be used to refer to increasing, decreasing, masking, altering, neutralizing, or restoring the normal function of the histamine receptor. A histamine receptor modulator may be an agonist or antagonist of the histamine receptor.

[0141] In some embodiments of the aspects described herein, the modulator modulates at least one activity of a histamine receptor by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0142] Histamine receptors belong to the superfamily of G protein-coupled seven-transmembrane proteins that have histamine as their endogenous ligand. G protein-coupled receptors constitute one of the major signal transduction systems in eukaryotic cells. The coding sequences for these receptors, within which regions thought to contribute to the agonist-antagonist binding sites, are fairly conserved across mammalian species. Histamine receptors are found in many peripheral tissues and within the central nervous system. There are four known histamine receptors, H 1 , H 2 , H 3 and H 4 exists.

[0143] As used herein, the term "histamine receptor modulator" intends and includes a compound that binds to histamine receptor or inhibits the binding of ligand thereto, or reduces or eliminates or increases or enhances or mimics the activity of histamine receptor.Thus, "histamine receptor modulator" includes both histamine receptor antagonists and histamine receptor agonists.In some embodiments, histamine receptor modulators bind to histamine H1 and / or H2 and / or H3 and / or H4 receptor or inhibit the binding of ligand thereto, or reversibly or irreversibly reduces or eliminates or increases or enhances or mimics the activity of histamine H1 and / or H2 and / or H3 and / or H4 receptor.

[0144] In some embodiments, the histamine modulator is a histamine receptor antagonist. Exemplary histamine modulators include, but are not limited to, A-349,821; ABT-239; Acrivastine; Alimemazine (Trimeprazine); Antazoline; Astemizole; Azatadine; Azelastine; Bepotastine; Bilastine; Bisfentidine; BL-6341A; BL-6548; BMY-25271; BMY-25405; BMY-52368; Brompheniramine; Carbinoxamine; Cetirizine; Chlorcyclizine. chlorphenamine (chlorpheniramine);chlorpromazine;cimetidine;ciproxifan;clemastine;clobenpropit;clozapine;cyclizine;cyproheptadine;D-16637;DA-4634;desloratadine;dexchlorpheniramine;dimebon;dimenhydrinate;dimethindene;diphenhydramine;donetidine;ebastine;ebrotidine;embramine;etintidine;famotidine;famotidine;fexofenadine;FRG-87 01;FRG-8813;Haloperidol;HB-408;HE-30-256;Hydroxyzine;ICI-162846;ICIA-5165;Impromidine;JNJ7777120;Ketotifen;L-643728;Lafutidine;Lantidine;Levocabastine;Levocetirizine;Loratadine;Locstidine;Lupitidine;Meclozine;Mepyramine;Mifentidine;Mizolastine;Nizatidine;Nizatidine;Olonzapine;Olopatadine;ORF- 17578; pheniramine; pifatidine; promethazine; quetiapine; quifenadine; ramixotidine; ranitidine; ritanserin; roxatidine; SKF-94482; SR-58042; sufotidine; terfenadine; thioperamide; tiotidine; VUF-6002; Wy-45727; zartidine; and analogs, derivatives, enantiomers, metabolites, prodrugs, and pharma- ceutically acceptable salts thereof.

[0145] Additional histamine modulators include those described in U.S. Pat. Nos. 3,932,644; 3,980,781; 4,060,621; 4,112,234; 4,117,131; 4,145,546; 4,153,793; 4,154,834; 4,159,329; 4,159,329; 4,218,452; 4,227,000; 4,234,588; 4,250,316; 4,255,248; No. 4,307,104; No. 4,309,433; No. 4,309,433; No. 4,318,913; No. 4,337,256 No. 4,338,328; No. 4,374,248; No. 4,374,248; No. 4,375,341; No. 4,380, No. 639; No. 4,385,058; No. 4,385,058; No. 4,399,294; No. 4,432,983; No. 4,4 No. 39,437; No. 4,442,110; No. 4,447,611; No. 4,481,199; No. 4,485,104; No. 4 , No. 496,567; No. 4,507,296; No. 4,520,025; No. 4,521,418; No. 4,522,943; Same No. 4,524,071; Same No. 4,526,973; Same No. 4,529,723; Same No. 4,543,352; Same No. 4,551,46 No. 6; No. 4,608,380; No. 4,638,001; No. 4,645,110; No. 4,670,487; No. 4,681 , No. 4,694,008; No. 4,738,969; No. 4,745,110; No. 4,764,612; No. 4,7 Nos. 77,179; 4,812,451; 4,812,452; 4,952,589; 5,273,984; 5,486,526; 5,541,343; 5,639,775; 5,753,671; 6,420,560; 6,552,047; 6,936,627; 7,115,600; 7,205,316; 7,256,205, and U.S. Patent Application Publication Nos. 2002 / 0086859; 2004 / 0138234; 2005 / 0070525;These include compounds described in US Pat. Nos. 2006 / 0047114; 2006 / 0069087; 2007 / 0238771; 2008 / 0015200; 2009 / 0239854; 2009 / 0325927; and 2010 / 0022580.

[0146] In some embodiments, the histamine receptor modulator inhibits ligand binding by at least about any one of or by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to a control.

[0147] In some embodiments, the histamine receptor modulator reduces the activity of the histamine receptor by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a non-inhibited control.

[0148] In some embodiments, the histamine receptor modulator enhances the activity of a histamine receptor by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative to a non-activated control.

[0149] In some embodiments, the histamine receptor modulator can bind to the active site of the histamine receptor (eg, the binding site for a ligand).

[0150] In some embodiments, the histamine receptor modulator is capable of binding to an allosteric site of the histamine receptor.

[0151] In some embodiments of this and other aspects of the invention, the histamine antagonist has an IC50 of less than or equal to 500 nM, less than or equal to 250 nM, less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equal to 0.001 nM.

[0152] In some embodiments of this and other aspects of the invention, the histamine agonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

[0153] In some embodiments, the histamine receptor modulator is methylhistamine dihydrochloride, i.e., histamine R(-)-alpha-methyl-dihydrochloride ( [ka] ) may be.

[0154] HDAC Modulator As used herein, the term "modulate" with respect to HDAC means to regulate the normal function of HDAC positively or negatively.Therefore, the term modulate can be used to refer to the increase, decrease, masking, alteration, neutralization or restoration of the normal function of HDAC.HDAC modulator can be an agonist or antagonist of serotonin receptor.

[0155] In some embodiments of the aspects described herein, the modulator modulates the activity of at least one HDAC by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0156] HDACs are a family that includes at least 18 enzymes that are divided into three classes (class I, II, and III). Class I HDACs include, but are not limited to, HDAC1, 2, 3, and 8. Class I HDACs can be found in the nucleus and are thought to be involved in transcriptional repressors. Class II HDACs include, but are not limited to, HDAC4, 5, 6, 7, and 9, and can be found in both the cytoplasm and even the nucleus. Class III HDACs are thought to be NAD-dependent proteins, including, but are not limited to, members of the sirtuin family of proteins. Non-limiting examples of sirtuin proteins include SIRT1-7.

[0157] The term "HDAC modulator," as used herein, refers to a compound that has the ability to modulate transcriptional activity.

[0158] In some embodiments, HDAC modulator is HDAC inhibitor.The term "HDAC inhibitor" as used herein refers to a compound that has the ability to inhibit histone deacetylase activity.This therapeutic class can block angiogenesis and cell cycle, and promote apoptosis and differentiation.HDAC inhibitors not only show targeted anticancer activity by themselves, but also improve the efficacy of existing drugs, and even other new targeted therapies.

[0159] As used herein, the term "selective HDAC inhibitor" refers to an HDAC inhibitor that does not significantly interact with all three HDAC classes.As used herein, "class I selective HDAC" refers to an HDAC inhibitor that interacts with one or more of HDAC1, 2, 3 or 8, but does not significantly interact with class II HDAC (i.e., HDAC4, 5, 6, 7 and 9).

[0160] Several compounds having HDAC inhibitory activity are known in the art (see, for example, Marks et al., J. Natl. Cancer Inst., vol. 92; pp. 1210-1216 (2000) and Miller et al., J. Med. Chem, vol. 46(24); pp. 5097-5115 (2003), which are incorporated herein by reference) and can be used as HDAC inhibitors in the present disclosure. The HDAC inhibitor can be, by way of non-limiting example, a short chain fatty acid, such as butyric acid, phenylbutyrate (PB), 4-phenylbutyrate (4-PBA), pivaloyloxymethylbutyrate (Pivanex, AN-9), isovalerate, valerate, valproate, valproic acid, propionate, butyramide, isobutyramide, phenylacetate, 3-bromopropionate, or tributyrin. Short chain fatty acid compounds having HDac inhibitory activity are described in U.S. Pat. Nos. 4,988,731, 5,212,326, 4,913,906, 6,124,495, 6,110,970, 6,419,953, 6,110,955, 6,043,389, 5,939455, 6,511,678, 6,528,090, 6,528,091, 6,713,086, and 6,720,004, U.S. Patent Application Publication No. 20040087652, and International Publication WO 02 / 007722, as well as in Phiel et al., J. Biol Chem 276(39):36734-41 (2001); Rephaeli et al., Int J Cancer 116(2):226-35 (2005); Reid et al., Lung Cancer 45(3):381-6 (2004); Gottlicher et al., 2001, EMBO J 22(13):3411-20 (2003); and Vaisburg et al., Bioorg Med Chem Lett 14(1):283-7 (2004). HDac inhibitors include, by way of non-limiting example, compounds having a hydroxyamic acid group, such as suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA), trichostatin C (TSC), salicylhydroxamic acid, oxamflatin, suberic bishydroxamic acid (SBHA), m-carboxycinnamic bishydroxamic acid (CBHA), pyroxamide (CAS RN 382180-17-8), diethyl bis-(pentamethylene-N,N dimethylcarboxamide) malonate (EMBA), azelaic bishydroxamic acid (AZA), benzoyl bishydroxamic acid (BS ... The compound may be 6-(3-chlorophenylureido)carpoic hydroxamic acid (ABHA), azelaic-l-hydroxamate-9-anilide (AAHA), 6-(3-chlorophenylureido)carpoic hydroxamic acid, or A-161906.

[0161] Hydroxamic acid compounds having HDac inhibitory activity are disclosed in U.S. Patent Nos. 6,800,638, 6,784,173, 6,531,472, 6,495,719, 6,512,123, and 6,511,990, U.S. Patent Application Publication Nos. 20060004041, 20050227976, 20050187261, 20050107348, 20050131018, 20050124679, 20050085507, 20050124679 ... Nos. 20040266818, 20040122079, 20040024067, and 20030018062, International Publication No. EP 1174438, WO / 2004092115, WO / 2005019174, WO0052033, WO018045, WO018171, WO0138322, WO0170675, WO9735990, W09911659, WO0226703, WO0230879, and WO0226696, as well as Butler et al., Clin Cancer Res. 7:962-970 (2001); Richon et al., Proc. Natl. Acad. Sci. USA:95;3003-3007 (1998); Kim et al., Oncogene:18(15);24612470 (1999); Klan et al., Biol Chem. 384(5):777-85 (2003); Yoshida et al., J Biol Chem. 265(28):17174-9 (1990); Suzuli et al., Bioorg Med Chem Lett. 15(2):331-5 (2005); Kelly et al., J Clin Oncol. 23(17):3923-31 (2005); Kelly et al., Clin Cancer Res. 9(10, part 1):3578-88 (2003); Sonoda et al., Oncogene 13(1):143-9 (1996); Richon et al., Proc Natl Acad Sci USA. 93(12):5705-8 (1996); Jung et al., J. Med. Chem. 42;4669-4679 (1999); Jung et al., Bioorg. Med. Chem.Lett, 7(13);1655-1658 (1997), Lavoie et al., Bioorg. Med. Chem. Letters, 11, 2847-2850 (2001), Remiszewski et al., J. Med. Chem. 45, 4, 753-757 (2002), Sternson et al., Org. Lett. 3, 26, 4239-4242 (2001), Bouchain et al., J Med Chem. 46(5):820-30 (2003), and Woo et al., J Med Chem. 45(13):2877-85 (2002).

[0162] The HDAC inhibitor may be, by way of non-limiting example, a cyclic tetrapeptide, such as depsipeptide (FK228), FR225497, trapoxin A, apicidin, chlamydocin, or HC-toxin. Cyclic tetrapeptides having HDAC inhibitory activity are described in U.S. Pat. Nos. 5,922,837, 6,403,555, 6,656,905, 6,399,568, 6,825,317, 6,831,061, U.S. Patent Application Publication Nos. 20050209134, 20040014647, 20030078369, and 20020120099, as well as in Kijima et al., J Biol Chem 268(30):22429-35 (1993); Jose et al., Bioorg Med Chem Ze# 14(21):5343-6 (2004); Xiao et al., Rapid Commun. Mass Spectrom., vol. 17(8): pp. 757-66 (2003); Furumai et al., Cancer Res., vol. 62(17): pp. 4916-21 (2002); Nakajima et al., Exp. Cell Res., vol. 241; pp. 126-133 (1998); Sandor et al., Clin Cancer Res., vol. 8(3): pp. 718-28 (2002); Jung et al., J. Med. Chem., vol. 42; pp. 4669-4679 (1999); and Jung et al., Bioorg. Med. Chem. Lett., vol. 7(13); pp. 1655-1658 (1997).

[0163] The HDAC inhibitor may be a benzamide, such as MS-275. Benzamides with HDAC inhibitory activity are described in U.S. Patent Nos. 6,174,905 and 6,638,530, U.S. Patent Application Publication Nos. 2004005513, 20050171103, 20050131018, and 20040224991, International Publication Nos. WO / 2004082638, WO / 2005066151, WO / 2005065681, European Patent No. 0847992, and Japanese Patent No. 258863 / 96, as well as Saito et al., Proc. Natl. Acad. Sci. USA, 96, 45924597 (1999); Suzuki et al., J. Med. Chem., 42, 3001-3003 (1999); Ryan et al., J Clin Oncol, 23(17):391222 (2005); Pauer et al., Cancer Invest., 22(6):886-96 (2004); and Undevia et al., Ann Oncol, 15(11):1705-11 (2004).

[0164] The HDAC inhibitor may be depudecin, a sulfonamide anilide (e.g., diallyl sulfide), BL1521, curcumin (diferuloylmethane), CI-994 (N-acetyldinaline), spiruchostatin A, Scriptaid, carbamazepine (CBZ), or a related compound. These and related compounds having HDac inhibitory activity are described in U.S. Pat. No. 6,544,957, as well as in Lea et al., Int. J. Oncol 15:347-352 (1999); Ouwehand et al., FEBS Lett. 579(6):1523-8 (2005); Kraker et al., Mol Cancer Ther. 2(4):401-8 (2003); de Ruijter et al., Biochem Pharmacol 68(7):1279-88 (2004); Liu et al., Acta Pharmacol Sin. 26(5):603-9 (2005); Fournel et al., Cancer Res. 62:4325-4330 (2002); Yurek-George et al., J Am Chem Soc, 126(4):1030-1 (2004); Su et al., Cancer Res., 60(12):3137-42 (2000); Beutler et al., Life Sci., 76(26):3107-15 (2005); and Kwon et al., Proc. Natl. Acad. Sci. USA, 95, 3356-3361 (1998).

[0165] The HDAC inhibitor may be a compound that comprises a cyclic tetrapeptide group and a hydroxamic acid group.Examples of such compounds are described in U.S. Patent Nos. 6,833,384 and 6,552,065, as well as in Nishino et al., Bioorg Med Chem., vol. 12(22):5777-84 (2004); Nishino et al., Org Lett, vol. 5(26):5079-82 (2003); Komatsu et al., Cancer Res., 61(11):4459-66 (2001); Furumai et al., Proc Natl Acad Sci USA., 98(1):87-92 (2001); Yoshida et al., Cancer Chemotherapy and Pharmacology, 48, Suppl 1;S20-S26 (2001); and Remiszeski et al., J Med Chem., 46(21):4609-24 (2003).

[0166] The HDAC inhibitor may be a compound that contains a benzamide group and a hydroxamic acid group. Examples of such compounds are described in Ryu et al., Cancer Lett. Jul., 9, 2005 (electronic publication); Plumb et al., Mol Cancer Ther, 2(8):721-8 (2003); Ragno et al., J Med Chem., 47(6):1351-9 (2004); Mai et al., J Med Chem., 47(5):1098-109 (2004); Mai et al., J Med Chem., 46(4):512-24 (2003); Mai et al., J Med Chem., 45(9):1778-84 (2002); Massa et al., J Med Chem., 44(13):2069-72 (2001); Mai et al., J Med Chem., 48(9):3344-53 (2005), and Mai et al., J Med Chem., 46(23):4826-9 (2003).

[0167] HDAC inhibitors may be disclosed in U.S. Pat. Nos. 6,897,220, 6,888,027, 5,369,108, 6,541,661, 6,720,445, 6,562,995, 6,777,217, or 6,387,673, 6,693,132, or U.S. Patent Application Publication Nos. 20060020131, 20060058553, 20060058298, 20060058282, 20060052599, 2006004712, 20060030554, 20060030555, 2006004712, 20060030554, 2006004712 ... 20060030543, 20050288282, 20050245518, 20050148613, 20 No. 050107348, No. 20050026907, No. 20040214880, No. 20040214862, No. 2004 No. 0162317, No. 20040157924, No. 20040157841, No. 20040138270, No. 200400 No. 72849, No. 20040029922, No. 20040029903, No. 20040023944, No. 200301253 No. 06, No. 20030083521, No. 20020143052, No. 20020143037, No. 20050197336 No. 20050222414, No. 20050176686, No. 20050277583, No. 20050250784, Same No. 20050234033, Same No. 20050222410, Same No. 20050176764, Same No. 20050107290, Same No. 20040043470, 20050171347, 20050165016, 20050159470, 200 No. 50143385, No. 20050137234, No. 20050137232, No. 20050119250, No. 20050 No. 113373, No. 20050107445, No. 20050107384, No. 20050096468, No. 20050085 No. 515, No. 20050032831, No. 20050014839, No. 20040266769, No. 2004025422 No. 0, No. 20040229889, No. 20040198830, No. 20040142953, No. 20040106599,The compound may be a compound described in No. 20040092598, No. 20040077726, No. 20040077698, No. 20040053960, No. 20040002506, No. 20030187027, No. 20020177594, No. 20020161045, No. 20020119996, No. 20020115826, No. 20020103192, or No. 20020065282.

[0168] The HDAC inhibitor may be an inhibitor selected from the group consisting of FK228, AN-9, MS-275, CI-994, LAQ-824, SAHA, G2M-777, PXD-101, LBH-589, MGCD-0103, MK0683, pyroxamide, sodium phenylbutyrate, CRA-024781, belinostat; (i.e., PXD101), MS-275 (i.e., entinostat; MS-27-275), vorinostat (i.e., suberoylanilide hydroxamic acid (SAHA); Zolinza), mocetinostat (i.e., MGCD0103), SB939 (i.e., pracinostat), rosilinostat (i.e., ACY-1215) and derivatives, salts, metabolites, prodrugs, and stereoisomers thereof.

[0169] Additional non-limiting examples include reported HDAC inhibitors selected from ONO-2506 or arundic acid (CAS RN 185517-21-9); MGCD0103 (Gelmon et al., "Phase I trials of the oral histone deacetylase (HDac) inhibitor MGCD0103 given either daily or 3x weekly for 14 days"). days every 3 weeks in patients (pts) with advanced solid tumors,” Journal of Clinical Oncology, 2005, ASCO Annual Meeting Proceedings. Vol. 23(16S, June 1, Suppl.), 2005:3147; and Kalita et al., “Pharmacodynamic effect of MGCD0103, an oral isotype-selective histone deacetylase (HDac) inhibitor, on HDac enzyme inhibition and histone acetylation induction in Phase I clinical trials in patients (pts) with advanced solid tumors or non-Hodgkin's lymphoma (NHL)”, Journal of Clinical Oncology, 2005, ASCO Annual Meeting Proceedings, Vol. 23 (16S, Part II, Part I, June 1, Supplement), 2005:9631, poster entitled "Enhanced Isotype-Selectivity and Antiproliferative Activity of Thiophenyl Derivatives of BenzamideHDac Inhibitors reported thiophenyl derivatives of benzamide HDac inhibitors as shown in "In Human Cancer Cells" (abstract #4725), and reported HDac inhibitors as described in U.S. Pat. No. 6,541,661; SAHA or Vorinostat (CAS RN 149647-78-9); PXD101 or PXD101 or PX105684 (CAS RN 414864-00-9), CI-994 or Tacedinaline (CAS RN 112522-64-2), MS-275 (CAS RN 209783-80-2), or inhibitors reported in WO2005 / 108367.

[0170] HDAC inhibitors may be novel HDac inhibitors identified using structure-activity relationships and teachings known in the art and described, for example, in Miller et al., J. Med. Chem., 46(24);5097-5115 (2003) and Klan et al., Biol Chem., 384(5):777-85 (2003), all of which are incorporated herein by reference in their entireties. Methods for assessing histone deacetylase activity are known in the art and described, for example, in Richon et al., Methods Enzymol 376:199-205 (2004); Wegener et al., Mol Genet Metab. 80(1-2):138-47 (2003); U.S. Pat. No. 6,110,697; and U.S. Patent Application Publication Nos. 20050118596, 20050227300, 20030161830, 20030224473, 20030082668, 20030013176, and 20040091951, all of which are incorporated by reference in their entireties.

[0171] Antisense oligonucleotides and ribozymes that inhibit transcription and / or translation of one or more HDacs are disclosed in U.S. Pat. No. 6,953,783, and U.S. Patent Application Publication Nos. 20050171042, 20040266718, 20040204373, 20040077578, 20040077084, 20040077 083, 20040072770, 20030236204, 20030216345, 20030152557, 20030148970, 20030078216, 20020137162, 20020164752, 20020115177, and 20020061860.

[0172] Some exemplary inhibitors of HDAC include low molecular weight carboxylates (e.g., less than about 250 amu), hydroxamic acids, benzamides, epoxyketones, cyclic peptides, and hybrid molecules (see, e.g., Drummond DC et al., Annu. Rev. Pharmacol. Toxicol., (2005) 45:495-528, including specific examples therein, which are incorporated herein by reference in their entirety). Non-limiting example HDAC inhibitors include, but are not limited to, suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, Nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyrate (e.g., sodium phenylbutyrate) and valproic acid ((VPA) and other short chain fatty acids), scriptaid, suramin sodium, trichostatin A (TSA), APHA Compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (i.e., N-acetyldinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (i.e., 6-(3-chlorophenylureido)caproic hydroxamic acid HDAC inhibitors include, for example, dominant negative forms (e.g., catalytically inactive forms) of HDACs, siRNA inhibitors of HDACs, and antibodies that specifically bind to HDACs.HDAC inhibitors are commercially available, for example, from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich. Additional HDAC inhibitors suitable for the present invention include, but are not limited to, those described in U.S. Patent Nos. 7,183,298; 6,512,123; 6,541,661; 6,531472; 6,960,685; 6,897,220; 6,905,669; 6,888, Nos. 207; 6,800,638 and 7,169,801, and U.S. Patent Application Publication Nos. 10 / 811,332; 12 / 286,769; 11 / 365,268; 11 / 581,570; 10 / 509,732; 10 / 546,153; 10 / 381,791 and 11 / 516,620.

[0173] In some embodiments, the HDAC modulator inhibits binding of the ligand by at least about any one of or by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to a control.

[0174] In some embodiments, the HDAC modulator reduces the activity of HDAC by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a non-inhibited control.

[0175] In some embodiments, an HDAC modulator enhances activity of HDAC by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative to a non-activated control.

[0176] In some embodiments, the HDAC modulator can bind to the active site of an HDAC (eg, a binding site for a ligand).

[0177] In some embodiments, the HDAC modulator can bind to an allosteric site of an HDAC.

[0178] In some embodiments of this and other aspects of the invention, the HDAC antagonist has an IC50 of less than or equal to 500 nM, less than or equal to 250 nM, less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equal to 0.001 nM.

[0179] In some embodiments of this and other aspects of the invention, the HDAC agonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

[0180] Epigenetic modifiers "Epigenetic modifier" refers to an agent that modifies the epigenetic state of a cell, i.e., the phenotype or gene expression in a cell, due to mechanisms other than changes in DNA sequence. The epigenetic state of a cell includes, for example, DNA methylation, histone modification(s) and RNA-associated silencing.

[0181] In certain embodiments, the epigenetic modifier alters (e.g., increases or decreases) the epigenetic state of the cell by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% compared to a control cell. In certain embodiments, the epigenetic modifier alters (e.g., increases or decreases) the epigenetic state of the cell by 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more relative to a control cell.

[0182] In some embodiments, the epigenetic modifier modulates histone modifications (e.g., HDAC modulators). In some embodiments, the epigenetic modifier modulates pathways involving BRD2, BRD4, or EGLN1. In some embodiments, the epigenetic modifier is (+)-JQ1; S)-JQ1; belinostat (i.e., PXD101); MS-275 (i.e., entinostat; MS-27-275); vorinostat (i.e., suberoylanilide hydroxamic acid (SAHA); Zolinza); mocetinostat (i.e., MGCD0103); I-BET (i.e., GSK525762A); SB939 (i.e., prasinostat; PFI-1); rosilinostat (i.e., ACY-1215); I-BET151 (i.e., GSK1210151A); IOX2; or derivatives, salts, metabolites, prodrugs, and stereoisomers thereof. In some embodiments, the epigenetic modifier is an epigenetic modifier shown in Table 5.

[0183] In some aspects, the epigenetic modifier is vorinostat.

[0184] Neuropeptides Neuropeptides are small protein-like molecules that are distinct from the larger neurotransmitters that neurons use to communicate with each other. They are neuronal signaling molecules that affect brain activity in specific ways and are therefore involved in brain functions such as pain analgesia, reward, food intake, learning and memory, among others. Neuropeptides are expressed and released by neurons and mediate or modulate neuronal signaling by acting on cell surface receptors. The human genome contains about 90 genes that code for precursors of neuropeptides. Currently, about 100 different peptides are known to be released by various populations of neurons in the mammalian brain.

[0185] Exemplary neuropeptides include, but are not limited to, hypothalamic hormones, such as oxytocin and vasopressin; hypothalamic releasing and inhibiting hormones, such as corticotropin releasing hormone (CRH), growth hormone releasing hormone (GHRH), luteinizing hormone releasing hormone (LHRH), somatostatin growth hormone releasing inhibiting hormone and thyrotropin releasing hormone; tachykinins, such as neurokinin a (substance K), neurokinin b, neuropeptide K and substance P; opioid peptides, such as b-endorphin, dynorphin and met- and leu-enkephalins; NPY and related peptides, such as neuropeptide tyrosine (NPY), pancreatic polypeptide and peptide tyrosine-tyrosine (PYY); VIP-glucagon family members, such as glucogen-like peptide-1 (GLP-1), peptide histidine isoleucine (PHI), pituitary adenylate cyclase activating peptide (PYY), and the like. PACAP) and vasoactive intestinal polypeptide (VIP); in addition, many other peptides, such as brain natriuretic peptide), calcitonin gene-related peptide (CGRP) (a- and b-types), cholecystokinin (CCK) and other forms, galanin, islet amyloid polypeptide (LAPP) or amylin, melanin-concentrating hormone (MCH), melanocortins (ACTH, a-MSH, etc.), neuropeptide FF (F8Fa), neurotensin, parathyroid hormone-related These include proteins, agouti gene-related protein (AGRP), cocaine- and amphetamine-regulated transcript (CART) / peptides, endomorphin-1 and -2, 5-HT-moduline, hypocretin / orexin, nociceptin / orphanin FQ, nocistatin, prolactin-releasing peptide, secretoneurin and urocortin; neurotensin; neuropeptide Y; neurotensin; substance P; TRH; enkephalin; and others.

[0186] In some embodiments, the neuropeptide is PD160170 ( [ka] ) may be.

[0187] Ionophores As used herein, the term "ionophore" includes molecules that can form complexes with certain ions, in some instances substantially to the exclusion of others. In general, ionophores facilitate the permeation of ions across lipid barriers by binding to the ions or by increasing the permeability of the barrier to them.

[0188] Without limitation, the ionophore may be selected from the group consisting of small or large organic or inorganic molecules; monosaccharides; disaccharides; trisaccharides; oligosaccharides; polysaccharides; biological macromolecules, such as proteins, peptides, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, enzymes, antibodies, portions or fragments of antibodies; extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions; and any combination thereof.

[0189] Exemplary potassium ion ionophores include, but are not limited to, valinomycin, crown ethers such as dimethyldibenzo-30-crown-10, dicyclohexyl-18-crown, dimethyldicyclohexyl-18-crown-6, tetraphenyl borate, tetrakis(chlorophenyl)borate. Sodium ion ionophores include, for example, methyl monensin, N,N',N"-triheptyl-N,N',N"trimethyl-4,4',4"-propylidinetris-(3-oxabutyramide), N,N,N,N'-tetracyclohexyl-1,2-phenylenedioxydiacetamide, 4-octadecanoyloxymethyl-N,N,N',N'-tetracyclohexyl-1,2-phenylenedioxydiacetamide, bis[(12-crown-4)methyl]dodecylmethylmalonate. Exemplary Exemplary calcium ion ionophores include, but are not limited to, bis(didecyl phosphate), bis(4-octylphenyl phosphate), bis(4-(1,1,3,3-tetramethylbutyl)phenyl phosphate tetracosamethylcyclododecasiloxane, N,N'-di(1,1,3,3 ethoxycarbonyl)undecyl)-N,N',4,5-tetramethyl-3,6 dioxaoctanediamide, calcium ionophore A23187 (also known as C-7522), calcium ionophore II 21193, and calcium ionophore IV 21198. Exemplary barium ionophores include, but are not limited to, calcium di(2-ethylhexyl)phosphate plus decan-1-ol, a barium complex of nonylphenoxypoly(ethyleneoxy)ethanol in ortho-nitrodiphenyl ether.Exemplary chloride ionophores include, but are not limited to, {u-[4,5-dimethyl-3,6-bis(octyloxy)-1,2-phenylene]}bis(trifluoroacetato-0)dimercuri (ETH9009), {(x-[4,5-dimethyl-3,6-bis(dodecyloxy)-1,2-phenylene]}bis(mercuric chloride) (ETH9033), 5,10,15,20-tetraphenyl 21H,23H-porphine manganese(III) chloride (MnTPPCl), tributyltin chloride (TBTC1), and trioctyltin chloride (TOTC1). Bicarbonate ionophores include, for example, quaternary ammonium ions. Ion exchangers include p-octodecyloxy-meta-chlorophenyl-hydrazone-methoxalonitrile. Ammonium ionophores include, for example, nonactin and monactin. Nitrate ionophores include, for example, tridodecylhexadecylammonium nitrate + n-octyl ortho-nitrophenyl, 1:10 phenanthroline nickel (II) nitrate + para-nitrocymene. Lithium ionophores include, for example, N,N'-diheptyl-N,N',5,5-tetramethyl-3,7-dioxononanediamide), 12-crown-4,6,6-dibenzyl-14-crown-4.Another non-limiting exemplary list of ionophores includes: for potassium, valinomycin, dicyclohexano-18-crown-6, dibenzo-18-crown-6, tetraphenyl borate, tetrakis(chlorophenyl)borate; for calcium, bis(didecyl phosphate), bis(4-octylphenyl phosphate), bis(4-(1,1,3,3-tetramethylbutyl)phenyl phosphate, tetracosamethylcyclododecasiloxane (tetra cosamefhylcyclododecasiloxane), N,N'-di(11-ethoxycarbonyl)undecyl)-N,N',4,5-tetramethyl-3,6-dioxaoctanediamide; with hydrogen, tridodecylamine, N-methyl-N-octadecyl(1-methyl, 2-hydroxy, 2-phenyl)ethylamine, N-octadecyl 3-hydroxy-n-propylamine, N,N'-bis(octadecylethyleneamine), p-octadecyloxy ci-m-chlorophenylhydrazone meso-oxalonitrile; sodium, monensin, N,N',N"-triheptyl-N,N,N"-trimethyl-4,4',4"-propylidinetris-(3-oxabutyramide), N,N,N',N'-tetracyclohexyl-1,2-phenylenedioxydiacetamide, 4-octadecanoyloxymethyl-N,N,N',N'-tetracyclohexyl-1,2-phenylenedioxydiacetamide, bis [(12-crown-4) methyl]dodecyl methylmalonate; for lithium, N,N'-diheptyl-N,N,5,5-tetramethyl-3,7-dioxononanediamide), 12-crown-4,6,6 dibenzyl-14 crown-4; for chloride, quaternary ammonium chloride, tributyltin chloride. Other suitable ionophores include ionomycin, monensin, lasalocid, laidlomycin, and the like.

[0190] Ion Channel Modulators As used herein, the term "ion channel modulator" refers to a compound that modulates at least one activity of an ion channel. As used herein, the term "ion channel modulator" is intended to include agents that can act as allosteric modulators of a channel by interacting with the channel pore itself or with a site on the channel complex. As used herein, the term "ion channel modulator" is also intended to include agents that indirectly modulate the activity of an ion channel. "Indirectly," when used in reference to a modulator interaction with an ion channel, means that the ion channel modulator does not directly interact with the ion channel itself, i.e., the ion channel modulator interacts with the ion channel through an intermediary. Thus, the term "indirectly" also encompasses the situation where the ion channel modulator requires another molecule to bind or interact with the ion channel.

[0191] Without limitation, the ion channel modulator may be selected from the group consisting of small or large organic or inorganic molecules; monosaccharides; disaccharides; trisaccharides; oligosaccharides; polysaccharides; biological macromolecules, such as proteins, peptides, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, enzymes, antibodies, portions or fragments of antibodies; extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions; and any combination thereof.

[0192] In some embodiments of the aspects described herein, the ion channel modulator modulates the passage of ions through an ion channel.

[0193] In some embodiments of the aspects described herein, the modulator is an inhibitor or antagonist of an ion channel. As used herein, the term "inhibitor" refers to a compound that blocks or reduces the flow of ions through an ion channel.

[0194] In some embodiments of the aspects described herein, the modulator is an ion channel agonist. As used herein, the term "agonist" refers to a compound that increases the flow of ions through an ion channel.

[0195] In some embodiments of the aspects described herein, the modulator modulates at least one activity of an ion channel by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0196] In some embodiments of the aspects described herein, the activity of at least one of the ion channels is inhibited or reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a control without the modulator.

[0197] In some embodiments of the aspects described herein, the ion channel modulator has an IC of less than or equal to 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM. 50 has.

[0198] In some embodiments of the aspects described herein, the ion channel modulator inhibits the flow of ions through the ion channel by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., a complete cessation of ion flow through the channel) relative to a control without the modulator.

[0199] In some embodiments of the aspects described herein, the ion channel modulator increases the flow of ions through the ion channel by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold or more relative to a control without the modulator.

[0200] In some embodiments of the aspects described herein, the ion channel modulator increases the concentration of an ion, e.g., sodium, in a cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold or more relative to a control without the modulator.

[0201] Without wishing to be bound by theory, ion channel modulators can modulate the activity of ion channels by several different mechanisms. For example, the modulator can bind to an ion channel and physically block ions from passing through the channel. Upon binding, the ion channel modulator can cause a conformational change in the ion channel, which can increase or decrease the interaction between ions and the channel, or increase or decrease the opening of the channel.

[0202] The modulator can modulate the energy-utilizing activity, e.g., the ATPase activity, of the ion channel. In some embodiments of the aspects described herein, the ion channel modulator inhibits the ATPase activity of the ion channel.

[0203] In some embodiments of the aspects described herein, the ion channel modulator is Na + / K + -Inhibit the ATPase activity of the ATPase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% (complete inhibition) relative to a control without the modulator. Without wishing to be bound by theory, ATPase activity can be measured by measuring the dephosphorylation of adenosine-triphosphate by utilizing methods well known to those skilled in the art for measuring such dephosphorylation reactions.

[0204] In some embodiments of the aspects described herein, the ion channel modulator inhibits RIG-I activation by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% (complete inhibition) relative to a control without the modulator.

[0205] In some embodiments of the aspects described herein, the ion channel modulator inhibits the ATPase activity of RIG-I by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% (complete inhibition) relative to a control without the modulator.

[0206] Without limitation, ion channel modulators may be small organic molecules, small inorganic molecules, polysaccharides, peptides, proteins, nucleic acids, extracts made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combination thereof.

[0207] In some embodiments of the aspects described herein, the ion channel modulator is a cardiac glycoside. As used herein, the term "cardiac glycoside" refers to a category of compounds that have a positive inotropic effect on the heart. Cardiac glycosides are also referred to in the art as cardiac steroids. They are used in the treatment of cardiac diseases, including cardiac arrhythmias, and have a rate-dependent effect on atrioventricular nodal conduction. As a general group of compounds, cardiac glycosides contain a steroid nucleus that contains a pyrone or butenolide substituent at C17 ("pyrone-type" and "butenolide-type"). In addition, cardiac glycosides may be optionally glycosylated at C3. The form of cardiac glycoside without glycosylation is also known as an "aglycone." Most cardiac glycosides contain one to four sugars attached to the 3β-OH group. The most commonly used sugars include L-rhamnose, D-glucose, D-digitoxose, D-digitalose, D-digginose, D-sarmentose, L-balalose, and D-fructose. In general, sugars affect the pharmacokinetics of cardiac glycosides with little other effect on biological activity. For this reason, aglycone forms of cardiac glycosides are available and are intended to be encompassed by the term "cardiac glycoside" as used herein. The pharmacokinetics of cardiac glycosides can be adjusted by adjusting the hydrophobicity of the molecule, with increasing hydrophobicity resulting in greater absorption and increased half-life. The sugar moiety may be modified with one or more groups, such as an acetyl group.

[0208] Numerous cardiac glycosides are known in the art. Exemplary cardiac glycosides include, but are not limited to, bufalin, ouabain, digitoxigenin, digoxin, lanatoside C, strophanthin K, usaligenin, desacetyllanatoside A, digitoxin, actyl digitoxin, desacetyllanatoside C, strophantoside, scillarenin, scillaren A, proscillaridin, proscillaridin A, BNC-1, BNC-4, digitoxose, gitoxin, strophanthidiol, oleandrin, acovenoside A, strophanthidin digiranovioside ... digilanobioside, strophanthidin-d-cimarroside, digitoxigenin-L-rhamnoside, digitoxigenin theretoside, strophanthidin, strophanthidin, strophanthidin digilanovioside, strophanthidin-d-cimarroside, digoxigenin, digoxigenin 3,12-diacetate, gitoxigenin, gitoxigenin 3-acetate, gitoxigenin 3,16-diacetate, 16-acetylgitoxigenin, acetylstrophanthidin, uabagenin, 3-epigoxigenin, neriifolin, acetylneriifolin cerverin cerberin, theventin, somalin, odoroside, honghelin, desacetyl digilanide, calotropin, calotoxin, lanatoside A, uzarin, strophanthidin-3β-digitoxoside, strophanthidin aL-rhamnopyranoside, and analogs, derivatives, pharmaceutically acceptable salts, and / or prodrugs thereof.

[0209] More than 100 cardiotonic glycosides have been identified as secondary metabolites in plants, mostly belonging to the angiosperm family. See, for example, Melero, CP, Medardea, M. & Feliciano, AS, A short review on cardiotonic steroids and their aminoguanidine analogues. Molecules, vol. 5, pp. 51-81 (2000), the contents of which are incorporated herein by reference. In general, cardiotonic glycosides are found in Digitalis purpurea and Digitalis lanata (foxglove), Nerium oleander (oleander), Thevetia peruviana (yellow oleander), Convallaria majalis (lily of the valley), Urginea maritima and Urginea Bufadienolides are found in a diverse group of plants, including Strophanthus indica (arrow), and Strophanthus gratus (ouabain). Recently, however, cardiac glycosides of the bufadienolide group have been identified in the skin and carotid glands of animals, primarily in the venom of several toad species. See Steyn, PS & van Heerden, FR, Bufadienolides of plant and animal origin. Nat. Prod. Rep. 15, 397-413 (1998), the contents of which are incorporated herein by reference.

[0210] In some embodiments of the aspects described herein, the ion channel modulator is a sodium pump blocker. As used herein, the terms "sodium pump blocker," "sodium pump inhibitor," and "sodium pump antagonist" refer to compounds that inhibit or block the flow of sodium and / or potassium ions across a cell membrane.

[0211] In some embodiments of the aspects described herein, the ion channel modulator is a calcium channel blocker. As used herein, the terms "calcium channel blocker", "calcium channel inhibitor" and "calcium channel antagonist" refer to compounds that inhibit or block the flow of calcium ions across a cell membrane. Calcium channel blockers include calcium ion influx inhibitors, slow channel blockers, and the like. Also known as calcium ion antagonists, calcium channel antagonist drugs, and as Class IV antiarrhythmic drugs. Exemplary calcium channel blockers include, but are not limited to, amiloride, amlodipine, bepridil, diltiazem, felodipine, isradipine, mibefradil, nicardipine, nifedipine (dihydropyridine), nickel, nimodimpine, nisoldipine, nitric oxide (NO), norverapamil, verapamil, and analogs, derivatives, pharma- ceutical acceptable salts, and / or prodrugs thereof.

[0212] In some embodiments of the aspects described herein, the calcium channel blocker is a beta blocker.Exemplary beta blockers include, but are not limited to, alprenolol, bucindolol, carteolol, carvedilol (with additional alpha-blocking activity), labetalol, nadolol, penbutolol, pindolol, propranolol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, esmolol, metoprolol, nebivolol, butaxamine, and ICI-118,551 (3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol), and analogs, derivatives, pharmaceutically acceptable salts, and / or prodrugs thereof.

[0213] Exemplary K +Ion channel modulators include, but are not limited to, 2,3-butanedione monoxime; 3-benzidino-6-(4-chlorophenyl)pyridazine; 4-aminopyridine; 5-(4-phenoxybutoxy)psoralen; 5-hydroxydecanoic acid sodium salt; L-α-phosphatidyl-D-myo-inositol; 4,5-diphosphate, dioctanoyl; Aa1; adenosine 5'-(β,γ-imido)triphosphate tetralithium salt hydrate; Agitoxin-1; Agitoxin-2; Agitoxin-3; Alinidine. ;Apamin;Aprindine hydrochloride;BDS-I;BDS-II;BL-1249;BeKm-1;CP-339818;Chrybdotoxin;Chrybdotoxin;Chlorzoxazone;Chromanol 293B;Cibenzoline succinate;Clofilium tosylate;Clotrimazole;Cromakalim;CyPPA;DK-AH269;Dendrotoxin-I;Dendrotoxin-K;Dequalinium chloride hydrate;DPO-1 needles;Diazoxide;Dofetilide;E-4031;Ergutoxin;Glimepiride;Glipizide;Gliben Chlamydide;Heteropodatoxin-2;Hongotoxin-1;ICA-105574;IMID-4F hydrochloride;Iberiotoxin;Ibutilide hemifumarate;Isopimaric acid;Kaliotoxin-1;Levcromakalim;Lq2;Margatoxin;Mast cell degranulating peptide;Maurotoxin;Mefeethyltetrazole;Mepivacaine hydrochloride;Minoxidil;Minoxidil sulfate;N-Acetylprocainamide hydrochloride ;N-Salicyloyltryptamine;NS1619;NS1643;NS309;NS8593 hydrochloride;Nicorandil;Noxiustoxin;Omeprazole;PD-118057;PNU-37883A;Pandinotoxin-Kα;Paxilline;Penitrem A;Phrixotoxin-2;Pinacidil monohydrate;Psora-4;Quinine;Quinine hemisulfate monohydrate;Quinine hydrobromide;Quinine hydrochloride dehydrate;Repaglinide;Rutaecarpine;S(+)-Niguldipine hydrochloride;SG-209;Scyllatoxin;Sematilide monohydrochloride monohydrate;Throtoxin;Stromatoxin-1;TRAM-34;Tamapine;Tertiapin;Tertiapin-Q trifluoroacetate;Tetracaine;Tetracaine hydrochloride;Tetraethylammonium chloride;Tityustoxin-Kα;Tolazamide;UCL1684;UCL- 1848 trifluoroacetate; UK-78282 monohydrochloride; VU590 dihydrochloride hydrate; XE-991; ZD7288 hydrate; Zatebradine hydrochloride; α-dendrotoxin; β-dendrotoxin; δ-dendrotoxin; γ-dendrotoxin; β-bungarotoxin; and analogs, derivatives, pharma- ceutically acceptable salts, and / or prodrugs thereof.

[0214] In some embodiments of the aspects described herein, the ion channel modulator is a potassium channel agonist. As used herein, a "potassium channel agonist" refers to an ion channel modulator that inhibits potassium channel activation. + K, which aids in the permeation of ions through ion channels + Ion channel modulators. Exemplary potassium channel agonists include, but are not limited to, diazoxide, minoxidil, nicorandil, pinacidil, retigabine, flupirtine, lemakalim, L-735534, and analogs, derivatives, pharma- ceutically acceptable salts, and / or prodrugs thereof.

[0215] In some embodiments of the aspects described herein, the ion channel modulator is bufalin; digoxin; ouabain; nimodipine; diazoxide; digitoxigenin; ranolazine; lanatoside C; strophanthin K; usaligenin; desacetyllanatoside A; actyldigitoxin; desacetyllanatoside C; strophanthoside; scillaren A; proscillaridin A; digitoxose; gitoxin; strophanthidiol; oleandrin; acobenoside A; strophanthi gindigiranovioside;strophanthidin-d-cimarroside;digitoxigenin-L-rhamnoside;digitoxigenin-teretoside;strophanthidin;digoxigenin-3,12-diacetate;gitoxigenin;gitoxigenin-3-acetate;gitoxigenin-3,16-diacetate;16-acetylgitoxigenin;acetylstrophanthidin;uabagenin;3-epigoxigenin;nerifolin;acetyhieriifolincerverin cerberin;ceventine;somalin;odroside;hongerin;desacetyldigylanide;carotropin;carotoxin;convallatoxin;oleandrigenin;periplocyrnarin;strophanthidin oxime;strophanthidin semicarbazone;strophanthidic acid lactone acetate;ernicyrnarin;sannentoside D;sarberogenin;sarmentoside A;sarmentogenin;proscillariditi ;Marinobufagenin;Amiodaron;Dofetilide;Sotalol;Ibutilide;Azimilide;Bretylium;Clofilium;N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide (E-4031);Nifekalant;Tedisamil;Sematilide;Ampyra;Apamin;Chrybdotoxin;1-Ethyl-2-benzimidazolinone (1-EBIO);3-Oxime-6,7-dichloro-1H-indole-2,3-dione (NS309);Cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA);GPCR antagonists;Ifenprodil;Glibenclamide;Tolbutamide;Diazoxide;Pinacidil;Halothane;Tetraethylammonium;4-Aminopyridine;Dendrotoxin;Retigabine;4-Aminopyridine;3,4-Diaminopyridine;Diazoxide;Minoxidil;Nicolandi;Retigabine;Fulpirtine;Quinidine;Procainamide;Disopyramide;Lidocaine ;Phenytoin;Mexiletine;Flecainide;Propafenone;Moricizine;Atenolol;Lopranolol;Esmolol;Timolol;Metoprolol;Atenolol;Bisoprolol;Amiodaron;Sotalol;Ibutilide;Dofetilide;Adenosine;Nifedipine;δ-Conotoxins;κ-Conotoxins;μ-Conotoxins;ω-Conotoxins;ω-Conotoxins GVIA;ω-Conotoxins CNVIIA;ω-Conotoxins CVIID;ω-Conotoxins AM336;Cilnidipine;L-Cysteine 2A;ω-Agatoxin IVA;N,N-Dialkyl-dipeptidyl-amines;SNX-111 (ziconotide);Caffeine;Lamotrigine;202W92 (Structural analogue of lamotrigine);Phenytoin;Carbamazepine;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]-pyridin-3-yl]-3-pyridinecarboxylic acid, 1-phenylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]-pyridin-3-yl]-3-pyridinecarboxlic acid, 1-phenylethyl ester 1,4-Dihydro-2,6-dimethyl-5-nitro-4-[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, cyclopropyl methyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno(3,2-c)pyridin-3-yl]-3-pyridinecarboxylic acid, butyl ester;(S)-1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-methylpropyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, methyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-methylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 2-propynyl ... Nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-methyl-2-propynyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 2-butynyl ester (este);1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-methyl-2-butynyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-methyl-2-butynyl ester 1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 2,2-dimethylpropyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 3-butynyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1,1-dimethyl-2prop ... R(+)-1,4-dihydro-2,6-dimethyl-5-nitro-4[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid (2A methyl-1-phenylpropyl) ester;S-(-)-1,4-dihydro-2,6-dimethyl(dimerhyl)-5-nitro-4[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 2-methyl-1-phenylpropyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-methylphenylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-phenylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, (1-phenylpropyl) ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2 ... Nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylic acid, (4-methoxyphenyl)methyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylic acid, 1-methyl-2-phenylethyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylic acid, 2-phenylpropyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c ]-pyridin-3-yl]-3-pyridinecarboxylic acid, phenylmethyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylic acid, 2-phenoxyethyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-thieno3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 3-phenyl-2 propynyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylic acid, 2-Methoxy 2-phenylethyl ester;(S)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-phenylethyl ester;(R)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-phenylethyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, cyclopropylmethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic acid, 1-cyclopropylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]pyridin-3-yl]-3-pyridinecarboxylic acid, 2-cyanoethyl ester;1,4-Dihydro-4-(2-{5-[4-(2-methoxyphenyl)-1-piperazinyl]pentyl}-3-furanyl)-2,6-dimethyl-5-nitro-3-pyridinecarboxylic acid , methyl ester;4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylic acid, {4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl} ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-(3-pyridinyl)-3-pyridinecarboxylic acid, {4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl} ester;4-(3-furanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylic acid, {2-[4- (2-Methoxyphenyl)-1-piperazinyl]ethyl} ester;4-(3-furanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylic acid, {2-[4-(2-pyrimidinyl)-1-piperazinyl]ethyl} ester;1,4-Dihydro-2,6-dimethyl-4-(1-methyl-1H-pyrrol-2-yl)-5-nitro-3-pyridinecarboxylic acid, {4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl} ester;1,4-Dihydro-2,6-dimethyl-4-(1-methyl -1H-Pyrrol-2yl)-5-nitro-3-pyridinecarboxylic acid, {4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl} ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-(3-thienyl)-3-pyridinecarboxylic acid, {2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl} ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-(3-thienyl)-3-pyridinecarboxylic acid, {2-[4-(2-pyrimidinyl)-1-piperazinyl]ethyl} ester;4-(3-furanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylic acid, {4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl} ester;(4-(2-furanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylic acid, {4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl} ester;1,4-dihydro-2,6-dimethyl-5-nitro-4- (2-Thienyl)-3-pyridinecarboxylic acid, {2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl} ester;1,4-Dihydro-2,6-dimethyl-4-(1-methyl-1H-pyrrol-2-yl)-5-nitro-3-pyridinecarboxylic acid, {2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl} ester;1,4-Dihydro-2,6-dimethyl-4-(1-methyl-1H-pyrrol-2-yl; 5-(4-chlorophenyl)-N-(3,5-dimethoxyphenyl)-2-furancarboxamide (A-803467); and analogs, derivatives, pharma- ceutically acceptable salts, and / or prodrugs thereof.

[0216] In some embodiments of the aspects described herein, the ion channel modulator is bufalin or an analog, derivative, pharma- ceutically acceptable salt, and / or prodrug thereof. Exemplary bufalin analogs and derivatives include, but are not limited to, 7β-hydroxylubufalin; 3-epi-7β-hydroxylubufalin; 1β-hydroxylubufalin; 15α-hydroxylubufalin; 15β-hydroxylubufalin; terocinobufagin (5-hydroxylubufalin); 3-epi-terocinobufagin; 3-epi-bufalin-3-O-β-d-glucoside; 11β-hydroxylubufalin; 12β-hydroxylubufalin; 1β,7β-dihydroxylubufalin; 16α-hydroxylubufalin; 7β,16α-dihybrid Bufalin analogs and derivatives include, without limitation, those that can cross the blood-brain barrier. Bufadienolides and their analogs and derivatives are also considered to be bufalin analogs or derivatives thereof.Additional bufalin or bufadienolide analogs and derivatives suitable for the present invention include those described in U.S. Patent Nos. 3,080,362; 3,136,753; 3,470,240; 3,560,487; 3,585,187; 3,639,392; 3,642,770; 3,661,941; 3,682,891; 3,682,895; 3,687,944; 3,706,72 No. 7; No. 3,726,857; No. 3,732,203; No. 3,80,6502; No. 3,812,106; No. 3,838,146; No. 4,001,401; No. 4,102,884; No. 4,175,078; No. 4,242,33; No. 4,380,624; No. 5,314,932; No. 5,874,423; and No. 7,087,590, as well as those described in Min et al., J. Steroid. Biochem. Mol. Biol. 91(1-2):87-98 (2004); Kamano, Y. & Pettit, GR, J. Org. Chem. 38(12):2202-2204 (1973); Watabe et al., Cell Growth Differ 8(8):871 (1997); and Mahringer et al., Cancer Genomics and Proteomics 7(4):191-205 (2010). The contents of all of the patents and references listed in the above paragraphs are incorporated herein by reference.

[0217] Adenosine Receptor Modulators As used herein, the term "adenosine receptor modulator" refers to a compound that modulates at least one activity of an adenosine receptor. As used herein, the term "adenosine receptor modulator" is intended to include agents that can act as allosteric modulators of the receptor by interacting with the adenosine receptor itself or with a site on the channel complex. As used herein, the term "adenosine receptor modulator" is also intended to include agents that indirectly modulate the activity of the adenosine receptor. When used in reference to the interaction of a modulator with an adenosine receptor, "indirectly" means that the modulator does not directly interact with the receptor itself, i.e., the modulator interacts with the ion channel through an intermediary. Thus, the term "indirectly" also encompasses situations in which the modulator requires another molecule to bind or interact with the receptor.

[0218] Adenosine receptors are proteins found in animals and humans that can bind the ligand, adenosine, and result in physiological responses. Adenosine receptors are located in a variety of tissues and cells, including the hippocampus, adipocytes, atrioventricular node, striatum, platelets, neutrophils, coronary vasculature, and olfactory tubercule.

[0219] The four adenosine receptors are generally referred to as A1, A2A, A2B, and A3. Among them, the stimulation of A1 receptors can inhibit nerve cells, reduce heart rate, slow down atrioventricular node conduction, and promote vasoconstriction. The stimulation of A2A receptors is generally anti-inflammatory, and can be used to sense excessive tissue inflammation and promote coronary vasodilation. The stimulation of A2B generally promotes vasodilation. Among them, the stimulation of A3 receptors can stimulate as well as inhibit cell proliferation, and promote tumor growth and angiogenesis. A large number of literature documents the current knowledge about adenosine receptors. These include Bioorganic & Medicinal Chemistry, vol. 6, (1998), pp. 619-641; Bioorganic & Medicinal Chemistry, vol. 6, (1998), pp. 707-719; J. Med. Chem., vol. 41, (1998), pp. 2835-2845; J. Med. Chem., vol. 41, (1998), pp. 3186-3201; J. Med. Chem., vol. 41, (1998), pp. 2126-2133; J. Med. Chem., vol. 42, (1999), pp. 706-721; J. Med. Chem., vol. 39, (1996), pp. 1164-1171; Arch. Pharm. Med. Chem., vol. 332, pp. 39^1, (1999); Am. J. Physiol., vol. 276, pp. H1113-1116, (1999); and Naunyn Schmied, Arch. Pharmacol., 362:375-381, (2000).

[0220] As used herein, the term "modulate" refers to the change or alteration of at least one biological activity of adenosine receptor. Modulation can be the increase or decrease of activity, the change of binding characteristics, or any other change of the biological, functional, or immunological properties of receptor. The ligand that binds to adenosine receptor and causes the inhibition of adenosine receptor physiological response is called adenosine receptor antagonist. Similarly, the ligand that binds to adenosine receptor and thereby produces a physiological response that mimics the response caused by adenosine receptor binding adenosine is called adenosine receptor agonist.

[0221] In some embodiments of the aspects described herein, the modulator is an agonist of the adenosine receptor. It will be appreciated that an adenosine receptor agonist includes compounds that act either directly or indirectly on the receptor, resulting in activation of the receptor or mimicking the action of the receptor with the same net effect.

[0222] In some embodiments of the aspects described herein, the modulator modulates at least one activity of the receptor by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0223] In some embodiments of the aspects described herein, the activity of at least one of the receptors is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% relative to a control without the modulator.

[0224] Exemplary adenosine receptor modulators include, but are not limited to, 2-(1-hexynyl)-N-methyladenosine; 2-Cl-IB-MECA; 2'-MeCCPA; 5'-N-ethylcarboxamidoadenosine; 8-cyclopentyl-1,3-dimethylxanthine (CPX); 8-cyclopentyl-1,3-dipropylxanthine (DPCPX); 8-phenyl-1,3-dipropylxanthine; ATL-146e; BAY60-6583; caffeine; CCPA; CF-101 (IB-MECA); CGS-2 1680;CP-532,903;CVT-6883;GR79236;Istradefylline;LUF-5835;LUF-5845;MRE3008F20;MRS-1191;MRS-1220;MRS-1334;MRS-1523;MRS-1706;MRS-1754;MRS-3558;MRS-3777;N6-Cyclopentyladenosine;PSB36;PSB-0788;PSB-10;PSB-11;PSB-1115;PSB-603;Regadenoson;SCH-442,416;SCH-58261;SDZ WAG994;Theophylline;VUF-5574;ZM-241,385 and others.

[0225] Exemplary adenosine receptor agonists include, but are not limited to, GR79236; SDZ WAG994; ATL-146e; CGS-21680; regadenoson; 5'-N-ethylcarboxamidoadenosine; BAY 60-6583; LUF-5835; LUF-5845; 2-(1-hexynyl)-N-methyladenosine; CF-101 (IB-MECA); 2-Cl-IB-MECA; CP-532,903; MRS-3558; N6-cyclopentyladenosine (CPA), N-ethylcarboxamidoadenosine (NECA), 2-[p-(2-carboxyethyl)phenethyl-amino-5'-N-ethyl]ade ... R-phenylisopropyl adenosine (CGS-21680); 2-chloroadenosine; N6-[2-(3,5-demethoxyphenyl)-2-(2-methoxyphenyl]ethyl adenosine; 2-chloro-N6-cyclopentyladenosine (CCPA); 2'-MeCCPA; N-(4-aminobenzyl)-9-[5-(methylcarbonyl)-beta-D-lobofuranosyl]adenine (AB-MECA); ([IS-[1a,2b,3b,4a(S*)]]-4-[7-[[2-(3chloro-2-thienyl)-1-methyl-propyl]amino]-3H-imidazole[4,5-b]pyridyl-3-yl]cyclopentanecarboxamide (AMP579); N6-(R)-phenylisopropyl adenosine (R-PLA); aminophenylethyl adenosine 9AP N-[3-(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT510); CVT-2759; allosteric enhancers such as PD81723; N-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl) adenosine (TCPA); 2-amino-3-naphthoylthiophene 78, and the like.

[0226] In some embodiments, the adenosine receptor agonist is N6-cyclopentyladenosine ( [ka] ) may be.

[0227] Gamma Secretase Ligand As used herein, the term "modulate" with respect to gamma secretase means to regulate the normal function of gamma secretase positively or negatively.Therefore, the term modulate can be used to refer to the increase, decrease, masking, alteration, neutralization, or restoration of the normal function of gamma secretase.Gamma secretase modulators can be gamma secretase agonists or gamma secretase antagonists.

[0228] In some embodiments of the aspects described herein, the modulator modulates at least one activity of gamma secretase by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, at least 98% or more relative to a control without modulation.

[0229] As used herein, the terms "gamma secretase protein" and "gamma secretase" refer to a protein that exhibits gamma secretase activity, which includes recognizing a polypeptide substrate having a gamma secretase cleavage sequence; and catalyzing cleavage of the gamma secretase cleavage sequence at a gamma secretase cleavage site to produce a substrate cleavage product.

[0230] Gamma-secretase is a macromolecular proteolytic complex composed of at least four proteins: presenilin (PS), nicastrin (NCT), PEN-2, and APH-1 (De Strooper, 2003, Neuron 38:9-12). Recently, CD147 and TMP21 have been found to associate with the gamma-secretase complex (Chen et al., 2006, Nature 440:1208-1212; Zhou et al., 2005, Proc. Natl. Acad. Sci. USA, 102:7499-7504). Of these known components, PS is believed to contain the active site of gamma secretase, which is recognized as an aspartyl protease (Esler et al., 2000, Nat. Cell. Biol., 2:428:434; Li et al., 2000, Nature 405:689-694; Wolfe et al., 1999, Nature 398:513-517). Considerable efforts have been made to understand the process of gamma secretase substrate recognition and its catalytic mechanism. PS-dependent proteases can process any single-pass transmembrane (TM) protein, regardless of its primary sequence, as long as the TM protein extracellular domain is smaller than 300 amino acids. Furthermore, the size of the extracellular domain appears to determine the efficiency of substrate cleavage (Struhl and Adachi, 2000, Mol. Cell 6:625-636).

[0231] Exemplary gamma secretase inhibitors include, but are not limited to, those described in U.S. Patent Application Publication Nos. 2003 / 0216380; 2006 / 0009467; 2004 / 0048848; 2004 / 0171614; 2005 / 0085506; 2006 / 0021621; and 2005 / 00216380, the entire contents of which are incorporated herein by reference. 6 / 0100427; 2005 / 0261495; 2007 / 0299053; 2006 / 0264417; 2006 / 0258638; Same No. 2005 / 0245501; Same No. 2003 / 0134841; Same No. 2008 / 004,5533; Same No. 2007 / 0213329; Same No. 2006 / 0041 Nos. 2004 / 0116404; and 2003 / 0114496, U.S. Patent Nos. 7,122,675; 6,683,091; 7,208,602; 7,256,186; 6,967,196; 7,304,056; 7,304,055; 7,101,870; 6, Nos. 962,913; 6,794,381; 7,304,094; and 6,984,663, as well as those described in PCT Publication Nos. WO03 / 013527; WO03 / 066592; WO00 / 247671; WO00 / 050391; WO00 / 007995; and WO03 / 018543. Additional exemplary gamma secretase modulators include certain nonsteroidal anti-inflammatory drugs (NSAIDs) and their analogs, as described in U.S. Patent Application Publication No. 2002 / 0128319, PCT Publication No. WO01 / 78721, and Weggen et al., Nature, 414 (2001), 212-16; Morihara et al., J. Neurochem., 83 (2002), 1009-12; and Takahashi et al., J. Biol. Chem., 278 (2003), 18644-70, all of which are incorporated herein by reference.

[0232] In some embodiments, the gamma secretase modulator inhibits binding of the substrate by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to a control, or by about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%. Binding of the substrate to gamma secretase can be determined by any method known to those skilled in the art.

[0233] In some embodiments, the gamma secretase modulator reduces the activity of gamma secretase by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g., complete loss of activity) relative to a non-inhibited control.

[0234] In some embodiments, the gamma secretase modulator enhances the activity of gamma secretase by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative to a non-activated control.

[0235] In some embodiments, the gamma secretase modulator can bind to the active site of a GPCR (eg, the binding site for a substrate).

[0236] In some embodiments, the serotonin receptor modulator is capable of binding to an allosteric site of gamma secretase.

[0237] In some embodiments of this and other aspects described herein, the GPCR antagonist has an IC50 of less than or equal to 500 nM, less than or equal to 250 nM, less than or equal to 100 nM, less than or equal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equal to 0.001 nM.

[0238] In some embodiments of this and other aspects of the invention, the GPCR agonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM.

[0239] Corticosteroids As used herein, the term "corticosteroids" refers to a group of steroid hormones produced in the adrenal cortex or synthetically produced. Corticosteroids are involved in a wide range of physiological systems, such as stress response, immune response and regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. Corticosteroids are generally classified into four groups based on chemical structure. Group A corticosteroids (short- to intermediate-acting glucocorticoids) include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, prednisolone, methylprednisolone, and prednisone. Group B corticosteroids include triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, and halcinonide. Group C corticosteroids include betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, and fluocortolone. Group D corticosteroids include hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate.

[0240] Without limitation, the corticosteroid may be selected from the group consisting of small or large organic or inorganic molecules; monosaccharides; disaccharides; trisaccharides; oligosaccharides; polysaccharides; biological macromolecules, such as proteins, peptides, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, enzymes, antibodies, portions or fragments of antibodies; extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions; and any combination thereof.

[0241] Exemplary corticosteroids include, but are not limited to, aldosternone, beclomethasone, beclomethasone dipropionate, betamethasone, betamethasone-21-phosphate disodium, betamethasone valerate, budesonide (also referred to herein as Bud), clobetasol, clobetasol propionate, clobetasone butyrate, clocortolone pivalate, cortisol, cortisterone, cortisone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, diflorasone diacetate, flucinonide, fludrocortisones acetate, flumethasone, flunisolide, flucinolone acetonide, fluticasone furoate, fluticasone valer ... furate), fluticasone propionate, halcinonide, halpmetasone, hydrocortisone, hydrocortisone acetate, hydrocortisone succinate, 16α-hydroxyprednisolone, isoflupredone acetate, medrysone, methylprednisolone, prednacinolone, predricarbate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisone, triamcinolone, triamcinolone, and triamcinolone diacetate.

[0242] As used herein, the term corticosteroids is intended to include the following generic and trade name corticosteroids: cortisone (CORTONE ACETATE, ADRESON, ALTESONA, CORTELAN, CORTISTAB, CORTISYL, CORTOGEN, CORTONE, SCHEROSON); oral dexamethasone (DECADRON-ORAL, DEXAMETH, DEXONE, HEXADROL-ORAL, DEXAMETHASONE INTENSOL, DEXONE 0.5, DEXONE 0.75, DEXONE 1.5, DEXONE 4); oral hydrocortisone (CORTEF, HYDROCORTONE); hydrocortisone cypionate (CORTEF ORAL SUSPENSION;ORAL METHYLPREDNISOLONE (MEDROL-ORAL);ORAL PREDNISOLONE (PRELONE, DELTA-CORTEF, PEDIAPRED, ADNISOLONE, CORTALONE, DELTACORTRIL, DELTASOLONE, DELTASTAB, DI-ADRESON F, ENCORTOLONE, HYDROCORTANCYL, MEDISOLONE, METICORTELONE, OPREDSONE, PANAAFCORTELONE, PRECORTISYL, PRENISOLONA, SCHERISOLONA, SCHERISOLONE);PREDNISONE (DELTASONE, LIQUID PRED, METICORTEN, ORASONE 1, ORASONE 5, ORASONE 10, ORASONE 20, ORASONE 50, PREDNICEN-M, PREDNISONE INTENSOL, STERAPRED, STERAPRED DS, ADASONE, CARTANCYL, COLISONE, CORDROL, CORTAN, DACORTIN, DECORTIN, DECORTISYL, DELCORTIN, DELLACORT, DELTADOME, DELTACORTENE, DELTISONA, DIADRESON, ECONOSONE, ENCORTON, FERNISONE, NISONA, NOVOPRED NISONE, PANAFCORT, PANASOL, PARACORT, PARMENISON, PEHACORT, PREDELTIN, PREDNICORT, PREDNICOT, PREDNIDIB, PREDNIMENT, RECTODELT, ULTRACORTEN, WINPRED); oral triamcinolone (KENACORT, ARISTOCORT, ATOLONE, SHOLOG) A, TRAMACORT-D, TRI-MED, TRIAMCOT, TRISTOPLEX, TRYLONE D, U-TRI-LONE).

[0243] Other exemplary corticosteroid drugs include cortisone, cortisol, hydrocortisone (11β, 17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxycortisone, dexamethasone (21-(acetyloxy)-9fluoro-β, 17-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione), and highly derivatized steroid drugs. Examples of corticosteroids include, for example, beconase (beclomethasone dipropionate, which is 9-chloro-11 (3,17,21, trihydroxy-16β-methylpregna-1,4-diene-3,20-dione 17,21-dipropionate). Other examples of corticosteroids include flunisolide, prednisone, prednisolone, methylprednisolone, triamcinolone, deflazacort, and betamethasone.

[0244] In some embodiments, the corticosteroid is budesonide ( [ka] ) may be.

[0245] Synergy The inventors have also found that many of the hit compounds have a synergistic effect on satellite cell proliferation when given together with a growth factor. Thus, in some embodiments of the aspects described herein, the compounds described herein are contacted with satellite cells together with a growth factor. Exemplary growth factors include, but are not limited to, basic epidermal growth factor (bEGF), fibroblast growth factor (FGF), FGF-1, FGF-2 (bFGF), FGF-4, thymosin, platelet-derived growth factor (PDGF), insulin-binding growth factor (IGF), IGF-1, IGF-2, epidermal growth factor (EGF), transforming growth factor (TGF), TGF-alpha, TGF-beta, chondroinductive factor-A and -B, osteoid inductive factor, osteogenin, bone morphogenetic proteins, and other bone growth factors, collagen growth factors, heparin-binding growth factor-1 or -2, and biologically active derivatives thereof.

[0246] The term "synergistic" as used herein is defined to mean a combination of components in which the activity of the combination is greater than the sum of the individual activities of each component of the combination. In some embodiments, the activity of the combination is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold or more greater than the sum of the individual activities of each component of the combination.

[0247] The compound and growth factor can be contacted with the satellite cells in any ratio. The ratio can be a molar:molar ratio or a weight:weight ratio, for example, the ratio can range from 100:1 to 1:100. In some embodiments, the proliferation enhancer and growth factor are in a ratio of 20:1 to 1:20. In some embodiments, the proliferation enhancer and growth factor are in a ratio of 10:1 to 1:10. In some embodiments, the proliferation enhancer and growth factor are in a ratio of 5:1 to 1:5. In some embodiments, the proliferation enhancer and growth factor are in a ratio of 15:1 to 1:5. In some embodiments, the proliferation enhancer and growth factor are in a ratio of 10:1 to 1:1. In one embodiment, the proliferation enhancer and growth factor are used in a ratio of 1:1.

[0248] In some embodiments, the growth factor may be selected from the group consisting of basic epidermal growth factor (bEGF), fibroblast growth factor (FGF), FGF-1, FGF-2 (bFGF), FGF-4, thymosin, platelet-derived growth factor (PDGF), insulin-binding growth factor (IGF), IGF-1, IGF-2, epidermal growth factor (EGF), transforming growth factor (TGF), TGF-alpha, TGF-beta, chondroinductive factor-A and -B, osteoid induced growth factor, osteogenin, bone morphogenetic proteins, and other bone growth factors, collagen growth factors, heparin-binding growth factor-1 or -2, and biologically active derivatives thereof.

[0249] Synergistic Compositions As discussed herein, the inventors have found that some of the proliferation enhancing agents, especially when used together with growth factors, exhibit synergistic effects on the proliferation of satellite cells.Therefore, the present disclosure also provides a synergistic composition comprising a proliferation enhancing agent and a growth factor.The growth factor can be selected.

[0250] Without limitation, the proliferation enhancer and the growth factor may be present in the synergistic composition in any ratio, which may be mole:mole or weight:weight. For example, the proliferation enhancer and the growth factor may be in a ratio of 100:1 to 1:100. In some embodiments, the proliferation enhancer and the growth factor are in a ratio of 20:1 to 1:20. In some embodiments, the proliferation enhancer and the growth factor are in a ratio of 10:1 to 1:10. In some embodiments, the proliferation enhancer and the growth factor are in a ratio of 5:1 to 1:5. In some embodiments, the proliferation enhancer and the growth factor are in a ratio of 15:1 to 1:5. In some embodiments, the proliferation enhancer and the growth factor are in a ratio of 10:1 to 1:1. In one embodiment, the proliferation enhancer and the growth factor are used in a ratio of 1:1. In some embodiments, the proliferation enhancer and growth factor may be in a ratio of 50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1.75, 1.5:1, or 1.25:1 to 1:1.25, 1:1.5, 1.75, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:20, 1:30, 1:40, or 1:50.

[0251] Contact of satellite cells with compounds The satellite cell population can be contacted with the proliferation-enhancing agent in cell culture, e.g., in vitro or ex vivo, or the proliferation-enhancing agent can be administered to a subject, e.g., in vivo. In some embodiments of the present invention, the proliferation enhancing agents described herein can be administered to a subject to repair or regenerate damaged muscle tissue.

[0252] As used herein with respect to contacting a population of satellite cells, the term "contacting" or "contacting" includes subjecting the satellite cells to an appropriate culture medium containing the indicated compound or agent. If the satellite cell population is in vivo, "contacting" or "contacting" includes administering the proliferation-enhancing agent or agent in a pharmaceutical composition to a subject via an appropriate route of administration such that the proliferation-enhancing agent or agent contacts the satellite cell population in vivo.

[0253] In the in vivo method, a therapeutically effective amount of the compound described herein can be administered to a subject.The method of administering the compound to a subject is known in the art and can be easily used by those skilled in the art.Promoting the proliferation of satellite cells in a subject can result in the treatment, prevention or amelioration of some diseases, disorders or conditions caused by damaged muscle tissue.

[0254] Satellite cells suitable for use in ex vivo methods can be obtained from a subject by methods well known to those of skill in the art.

[0255] The term "ex vivo" refers to cells that are removed from a living organism and cultured outside the organism (e.g., in a test tube). In ex vivo methods, the satellite cells may include autologous satellite cells, i.e., one or more cells taken from a subject in need of treatment for muscle injury or repair. Autologous satellite cells have the advantage of avoiding any immune-based rejection of the cells. Alternatively, the cells may be xenogeneic, e.g., taken from a donor. The second subject may be allogeneic or xenogeneic. Typically, when the cells are derived from a donor, they are from a donor that is sufficiently immunologically compatible with the recipient, i.e., not subject to transplant rejection, reducing or eliminating the need for immunosuppression. In some embodiments, the cells are taken from a xenogeneic source, i.e., the recipient, or a non-human mammal that has been genetically engineered to be sufficiently immunologically compatible with the recipient's species. Methods for determining immune compatibility are known in the art and include tissue typing to assess donor-recipient compatibility for HLA and ABO determinants. See, for example, Transplantation Immunology, edited by Bach and Auchincloss (Wiley, John & Sons, Incorporated, 1994).

[0256] Without wishing to be bound by theory, any suitable cell culture medium can be used in the ex vivo method of the present invention. For example, the inventors have discovered that the cells can survive in a minimum of 10% serum medium. The cells can survive in suspension cultures containing at least 30% serum, but they require a medium containing 10% serum to adhere when used in the absence of bFGF. The cells are optimal in culture when they are adhered on laminin. After ex vivo contact with the compounds described herein, the satellite cells can grow to a desired population number or density, for example, about 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2×10 7 When the number of cells reaches 100 or more, the cells can be transplanted into a subject in need of muscle repair or treatment of injury. The cells can be transplanted into the subject from which the cells were originally obtained or into another subject.

[0257] When satellite cells come into contact with a proliferation-enhancing agent, the proliferation-enhancing agent may have a direct or indirect effect on the satellite cells. As used herein, "direct effect" means that the proliferation-enhancing agent interacts directly with the satellite cells, e.g., binds to a cell surface receptor on the satellite cells and is taken up by the satellite cells. As used herein, "indirect effect" means that the proliferation-enhancing agent does not interact directly with the satellite cells. For example, the proliferation-enhancing agent may interact with non-satellite cells and indirectly affect the proliferation of the satellite cells. Without wishing to be bound by theory, the proliferation-enhancing agent may indirectly affect the satellite cells by inducing the expression and / or secretion of a molecule from the non-satellite cells, which then directly or indirectly affect the proliferation of the satellite cells.

[0258] As used herein, the term "damaged muscle tissue" refers to muscle tissue, such as skeletal or cardiac muscle, that has been altered, for example, by physical injury or accident, disease, infection, abuse, loss of blood circulation, or by genetic or environmental factors. Damaged muscle tissue may be dystrophic or aging muscle. Exemplary symptoms of muscle injury include, but are not limited to, swelling, bruising or redness, open cuts as a result of injury, pain at rest, pain when the particular muscle or joint associated with the muscle is used, muscle or tendon weakness, and inability to use the muscle at all.

[0259] In some embodiments of this and other aspects described herein, the damaged muscle tissue results from muscle atrophy / wasting.

[0260] In some embodiments of this aspect and other aspects described herein, the damaged muscle tissue results from sarcopenia.As used herein, the term "sarcopenia" refers to the loss of muscle mass and function that inevitably occurs with aging.Sarcopenia is the cause of a decrease in physical activity level, which can then lead to increased body fat and further loss of muscle.The loss of muscle mass results from a negative net balance between muscle protein synthesis and muscle protein degradation.The pathogenesis of this loss of skeletal muscle mass and function is not believed to be clear.A decrease in physical activity level, a decrease in motor units secondary to changes in the central nervous system, and inadequate protein intake are all implicated.

[0261] In some embodiments of this and other aspects described herein, the damaged muscle tissue results from a physical injury.

[0262] In some embodiments of this and other aspects of the invention, the damaged muscle is a skeletal muscle.

[0263] In some embodiments, the subject has or is otherwise affected by a muscle injury, insult, or disease.

[0264] In some embodiments of this aspect and other aspects described herein, the disease that causes damaged muscle tissue is a myopathy. Without limitation, the myopathy can be a congenital or acquired myopathy. Exemplary myopathy includes, but is not limited to, dystrophies, myotonia (neuromytonia), congenital myopathy (e.g., nemaline myopathy, multi / minicore myopathy, centronuclear myopathy (or myotubular myopathy)), mitochondrial myopathy, familial periodic paralysis, inflammatory myopathy, metabolic myopathy (e.g., glycogen storage disease and lipid storage disorder), dermatomyositis, polymyositis inclusion body myositis, myositis ossificans, rhabdomyolysis, and myoglobinurias.

[0265] In some embodiments of this and other aspects described herein, the muscle disorder is a dystrophy selected from the group consisting of muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, reflex sympathetic dystrophy, retinal dystrophy, Conal dystrophy, myotonic dystrophy, corneal dystrophy, and any combination thereof.

[0266] Congenital myopathy is a term sometimes applied to a group of rare inherited primary muscle disorders that may be present at birth, but usually result in hypotonia and weakness at birth or during the neonatal period, and in some cases later in childhood, delayed motor development. Patients may have a range of conditions from mild (onset in late childhood and ambulatory through adulthood) to severe (respiratory failure). Patients suffer from weakness ranging from pulmonary insufficiency (pulmonary insufficiency) to death within the first year of life.

[0267] The most common types of congenital myopathies are nemaline myopathies, myotubular myopathies, central core myopathies, congenital fiber type disproportion, and multicore myopathies. They are primarily differentiated by their histological features, symptoms, and prognosis. Diagnosis is indicated by characteristic clinical findings and confirmed by muscle biopsy.

[0268] In certain embodiments, a therapeutically effective amount of a compound described herein can be administered to a subject to treat any disease in which small muscles (e.g., sphincter muscles) are affected. In certain embodiments, a therapeutically effective amount of a compound described herein can be administered to a subject to treat esophageal diseases (e.g., esophageal reflux disease and other diseases caused by reduced esophageal muscle control, tone or motility). In certain embodiments, a therapeutically effective amount of a compound described herein can be administered to a subject to treat urinary incontinence. In certain embodiments, a therapeutically effective amount of a compound described herein can be administered to a subject to treat fecal incontinence. In certain aspects, a therapeutically effective amount of a compound contemplated herein can be administered to a subject to increase or improve sphincter tone.

[0269] X-linked myotubular myopathy (XLMTM): Myotubular myopathy is autosomal or X-linked. The more common autosomal alterations result in mild weakness and hypotonia in both sexes. X-linked alterations affect males, resulting in severe skeletal muscle weakness and hypotonia, facial weakness, dysphagia, and respiratory muscle weakness and failure. However, female carriers rarely develop significant clinical symptoms. Many patients die within the first year of life due to respiratory failure. Some patients may survive for several years and show spontaneous improvement of respiratory function after birth. XLMTM is also referred to in the art as CNM, MTMX, X-linked centronuclear myopathy, and XMTM.

[0270] Characteristic muscle histopathology consists of small, round muscle fibers with centrally located nuclei containing mitochondria, surrounded by halo devoid of contractile elements. The MTM1 gene is mutated in the majority of XLMTM patients. The gene is ubiquitously expressed and displays muscle-specific alternative transcripts due to the use of different polyadenylation signals. Over 133 different disease-associated mutations have been described in the MTM1 gene. A list of MTM1 mutations is maintained on the web at the Human Gene Mutation Database, under the XLMTM entry, and can be accessed at the following address: www.uwcm.ac.uk / uwcm / mg / search / 119439.html. Mutations in the MTM1 gene are spread throughout the gene, but compared to the ratio of the number of mutations to nucleotide length for each exon, more are found in exons 4, 12, 3, 8, 9, and 11, in that order. For a review of MTM1 mutations in X-linked myotubular myopathies, see J. Laporte et al., 2000, 15:393-409.

[0271] Mutations in the MTM1 gene include missense, nonsense, small insertions or deletions, large deletions, and splice site mutations. Most point mutations are truncating; however, approximately 25% of mutations are missense. Most truncating and splice mutations are associated with severe phenotypes, whereas some missense mutations are associated with milder or less severe phenotypes and longer survival.

[0272] Nemaline myopathy: Nemaline myopathy can be autosomal dominant or recessive and results from a variety of mutations in various chromosomes. Nemaline myopathy can be severe, moderate, or mild in newborns. Severely affected patients can experience weakness of the respiratory muscles and respiratory failure. Moderate disease results in progressive weakness of the muscles of the face, neck, trunk, and legs, but life expectancy can be near normal. Mild disease is non-progressive and life expectancy is normal.

[0273] Central core myopathy: Inheritance is autosomal dominant. Most affected patients develop hypotonia and mild proximal muscle weakness during the newborn period. Many also have facial weakness. The weakness is nonprogressive and life expectancy is normal. However, patients are at high risk of developing malignant hyperthermia (the genes associated with central core myopathy are also associated with increased susceptibility to malignant hyperthermia).

[0274] Congenital fiber inequality: Congenital fiber inequality is inherited, but the pattern is poorly understood. Hypotonia and weakness of the face, neck, trunk, and extremities are often associated with skeletal abnormalities and dysmorphic features. Most affected children improve with age, but a small percentage develop respiratory failure.

[0275] Multicore myopathy: Multicore myopathy is usually autosomal recessive but can be autosomal dominant. Infants typically present with proximal weakness, but some children later present with generalized weakness. Progression is highly variable.

[0276] In some embodiments, the methods described herein also include diagnosing the subject for a congenital muscular disorder prior to initiating administration of a compound described herein. The subject can be diagnosed for a congenital muscular disorder based on symptoms exhibited by the subject.

[0277] In general, symptoms of congenital myopathies include, but are not limited to, weakened reflexes, muscle swelling, difficulty relaxing muscles after contraction, stiff muscles, and muscle rigidity. Specific symptoms are described below.

[0278] Central core disease is characterized by mild, non-progressive muscle weakness. Signs of central core disease usually appear in infancy or early childhood, and may be present even earlier. Reduced fetal movement in utero and breech presentation may also be present. The main hallmarks of CCD are insufficient muscle tone (hypotonia), muscle weakness, and skeletal problems, including congenital hip dislocation, scoliosis (curvature of the spine), pes cavus (high-arched feet), and clubbed feet. Children with CCD experience delayed achievement of motor milestones and tend to sit and walk much later than children without the disorder. Children with the disease usually cannot run easily, and jumping and other physical activities may be found to be impossible in many cases. Although central core disease can be disabling, this does not usually affect intelligence or life expectancy.

[0279] People with central core disease are sometimes vulnerable to malignant hyperthermia (MH), a condition caused by anesthesia during surgery that can result in a rapid and sometimes fatal rise in body temperature and muscle stiffness.

[0280] There is variability in the age of onset, presence of symptoms, and severity of symptoms of nemaline myopathy (NM). Most commonly, NM presents in infancy or early childhood with weakness and poor muscle tone. In some cases, pregnancy complications such as polyhydramnios (excess amniotic fluid) and reduced fetal movement may be present. Children affected by NM tend to have delays in motor milestones such as rolling over, sitting, and walking. Muscle weakness occurs commonly in the face, neck, and upper limbs. Over time, a characteristic myopathic face (long, expressionless face) develops. Skeletal problems including chest deformity, scoliosis, and foot deformities may develop. In the most severe cases of NM, feeding difficulties and potentially fatal breathing problems may also occur. In children who survive the first two years of life, muscle weakness tends to progress slowly or not at all.

[0281] Typically, the X-linked form of MTM (XLMTM) is the most severe of the three types (X-linked, autosomal recessive, and autosomal dominant). XLMTM usually presents as a newborn male with poor muscle tone and respiratory distress. Pregnancy may be complicated by polyhydramnios and reduced fetal movement. Of children who survive the neonatal period, many are at least partially dependent on a ventilator for breathing. Many also have a gastrostomy tube (G-tube) due to the risk of aspiration. Boys with XLMTM may experience significant delays in achieving motor milestones and may never walk independently. They tend to be tall with characteristic facial features (long, narrow faces with highly curved palate and crowding teeth). Intelligence is generally unaffected. Medical complications that may develop include scoliosis, eye problems (ophthalmoplegia and droopy eyelids), and dental malocclusion (severe crowding). Other problems may also occur with XLMTM, including cryptorchidism, spherocytosis, purpura, elevated liver enzymes, and gallstones.

[0282] Autosomal recessive and autosomal dominant forms of MTM tend to have a milder course than the X-linked form. The autosomal recessive form may present in infancy, childhood, or early adulthood. Common hallmarks include generalized muscle weakness with or without facial weakness and ophthalmoplegia (paralysis of the eye muscles). Affected individuals usually survive infancy, although feeding and breathing problems may occur. Onset of the autosomal dominant form ranges from late childhood to early adulthood. It tends to be the mildest of the three forms of MTM. Unlike the X-linked condition, problems with other organs (such as the liver, kidneys, and gallbladder) have not been reported with the autosomal recessive and autosomal dominant forms of MTM.

[0283] Diagnosis of congenital myopathies generally involves evaluation of the subject's personal and family history, physical and neurological examinations testing reflexes and strength, and specialty testing. Because there is overlap between symptoms of congenital myopathies and other neuromuscular disorders, several tests can be performed to narrow the diagnosis. Serum CK (creatine kinase) analysis, EMG (electromyogram), nerve conduction studies, and muscle ultrasound tend to be of limited value in making this diagnosis. Definitive diagnosis of congenital myopathies usually relies on genetic testing and / or muscle biopsy. Similarly, muscle biopsy can be used to determine a patient's susceptibility to malignant hyperthermia.

[0284] X-Linked Myotubular Myopathy: The diagnosis of X-linked MTM is usually made on muscle biopsy. Findings include centrally located nuclei within muscle fibers that look like myotubes, the absence of structures known as myofibrils, and possibly the persistence of certain proteins normally found in fetal muscle cells. Genetic testing detects mutations (genetic changes that result in disease) in up to 97-98% of people with the X-linked form. Genetic testing may include (i) complete genetic sequencing of the MTM1 gene; (ii) mutation screening (scanning) by methods such as single-strand conformation polymorphism analysis (SSCP) or denaturing gradient high performance liquid chromatography (DHPLC), followed by sequencing of abnormal fragments; and (iii) deletion testing. X-linked MTM can also be diagnosed by measuring levels of myotubularin. Patients with known MTM1 mutations usually show abnormal myotubularin levels.

[0285] Central Core Disease: Muscle biopsies from individuals with CCD typically show a metabolically inactive "core" or central region that appears blank when stained (tested) for certain metabolic enzymes (proteins) that should be present there. These central regions also lack mitochondria, the energy producing "factories" of the cells. Genetic testing for RYR1 mutations is available on a research basis. The same genetic testing can be used to determine the presence of genetic changes in family members who may have or be at risk for the disease. In families in which RYR1 mutations have been found, prenatal diagnosis may be possible using DNA from fetal cells obtained from chorionic villus sampling (CVS) or amniocentesis.

[0286] Nemaline myopathy: The clinical diagnosis of NM is suspected in infants under 1 year of age who have muscle weakness and hypotonia (poor muscle tone). Definitive diagnosis of nemaline myopathy is made by demonstration of nemaline bodies, rod-shaped structures characteristic of the disease, in a muscle biopsy sample using a specific stain known as "Gomori's trichrome". Muscle biopsies may also show a predominance of structures known as type I fibers. Genetic testing is available on a clinical basis for one gene, the ACTA1 gene, located on the long arm of chromosome 1. Approximately 15% of NM cases are due to mutations in this gene. Prenatal diagnosis is possible in families with a known ACTA1 mutation. Fetal DNA can be tested using cells obtained from chorionic villus sampling (CVS) or amniocentesis.

[0287] Pharmaceutical Compositions For administration to a subject, the proliferation-enhancing agents can be provided in pharma- ceutically acceptable compositions that comprise a therapeutically effective amount of one or more proliferation-enhancing agents formulated together with one or more pharma- ceutically acceptable carriers (excipients) and / or diluents. As described in detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), gavage, lozenges, dragees, capsules, pills, tablets (e.g., buccal, sublingual, and those targeted for systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or as a sustained release formulation; (3) topical application, e.g., as a cream, ointment, or controlled release patch or spray applied to the skin; (4) vaginally or rectally, e.g., as a vaginal suppository, cream, or foam; (5) sublingually; (6) ophthalmic; (7) transdermal; (8) transmucosal; or (9) nasal. In addition, compound can be implanted into patient or injected using drug delivery system.See, for example, Urquhart et al., Ann. Rev. Pharmacol. Toxicol., vol. 24:199-236 (1984); Lewis, ed., "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Patent No. 3,773,919; and U.S. Patent No. 353,270,960, the entire contents of which are incorporated herein by reference.

[0288] As used herein, the term "pharmacologically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, and within the scope of sound medical judgment.

[0289] As used herein, the term "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricants, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, involved in carrying or transporting the compound from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of substances which serve as pharma- ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; and (9) oils, such as peanut oil, cottonseed oil, safflower oil, and the like. oils, such as sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL; (22) C 2 ~C 12Alcohols, e.g., ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation. The terms "excipient", "carrier", "pharmaceutical acceptable carrier" and the like are used interchangeably herein.

[0290] The phrase "therapeutically effective amount," as used herein, means an amount of a compound, substance, or composition containing the compound, described herein, that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment, e.g., the amount of a compound administered to a subject sufficient to produce statistically significant and measurable satellite cell proliferation or muscle repair or regeneration.

[0291] As used herein, the term "repair" refers to the process of reducing or completely eliminating muscle tissue damage. In some embodiments, at least one symptom of muscle tissue damage is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0292] Determination of a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art. Generally, a therapeutically effective amount may vary with the subject's history, age, condition, sex, as well as the severity and type of the subject's medical condition, and administration of other pharmacologic active agents.

[0293] As used herein, the term "administer" refers to placing a composition in a subject by a method or route that results in at least partial localization of the composition at a desired site so that a desired effect occurs. Suitable administration routes for the method of the present invention include both local and systemic administration. In general, local administration results in a large amount of the administered proliferation-enhancing agent (or satellite cells treated with the proliferation-enhancing agent) being delivered to a specific site compared to the whole body of the subject, whereas systemic administration results in the proliferation-enhancing agent (or satellite cells treated with the proliferation-enhancing agent) being delivered essentially to the whole body of the subject. One method of local administration is by intramuscular injection.

[0294] In the context of administering cells treated with a compound, the term "administering" also includes transplanting such cells into a subject. As used herein, the term "transplantation" refers to the process of implanting or transferring at least one cell into a subject. The term "transplantation" includes, for example, autotransplantation (removing cell(s) from one location in a patient and transferring them to the same or another location in the same patient), allotransplantation (transplantation between members of the same species), and xenotransplantation (transplantation between members of different species). A skilled artisan is well aware of the methods for implanting or transferring cells for muscle repair and regeneration suitable for the present invention. See, for example, U.S. Patent No. 7,592,174 and U.S. Patent Application Publication No. 2005 / 0249731, the contents of both of which are incorporated herein by reference.

[0295] Furthermore, the proliferation enhancer can be formulated in the form of ointment, cream, powder or other formulation suitable for topical preparation.Without wishing to be bound by theory, these formulations can deliver the proliferation enhancer through the skin to deeper muscle tissue.Therefore, such formulations can include one or more agents that enhance the penetration of active ingredients through the skin.For topical application, the proliferation enhancer can be included in wound dressing and / or skin covering composition.

[0296] The proliferation-enhancing agent or a composition comprising same can be administered by any suitable route known in the art, including oral or parenteral routes, including but not limited to intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

[0297] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In a preferred embodiment of the aspects described herein, the composition is administered by intravenous infusion or injection.

[0298] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a livestock or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. A patient or subject includes any subset of the above, such as all of the above, but excluding one or more groups or species, such as humans, primates, or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate, such as a human. The terms "patient" and "subject" are used interchangeably herein. The terms "patient" and "subject" are used interchangeably herein. A subject may be male or female.

[0299] In certain embodiments, the subject does not have or is otherwise not afflicted with acute myeloid leukemia (AML). In certain embodiments, the subject does not have or is otherwise not afflicted with cancer.

[0300] Preferably, the subject is a mammal.The mammal can be, but is not limited to, human, non-human primate, mouse, rat, dog, cat, horse, or cow.Non-human mammals can be advantageously used as subjects that represent the animal model of autoimmune disease or inflammation-related disorders.In addition, the methods and compositions described herein can be used to treat domesticated animals and / or pets.

[0301] The subject may be one who has previously been diagnosed with, identified as suffering from, or has a disorder characterized by muscle damage or muscle atrophy / wasting.

[0302] The subject can be a subject not currently being treated with a compound described herein.

[0303] The subject may be one who has previously been diagnosed with a disease being treated with a therapeutic regimen including a compound described herein, where the disease is not a disease characterized by muscle damage or muscle atrophy / wasting.

[0304] Thus, in some embodiments, the method of treatment includes adjusting the subject's therapeutic regimen such that at least one symptom of muscle damage is alleviated. Without limitation, the therapeutic regimen can be adjusted by modulating the frequency of administration of the proliferation-enhancing agent and / or by changing the site or mode of administration.

[0305] In some embodiments of the aspects described herein, the methods further include diagnosing the subject for muscle damage or muscle atrophy / wasting prior to treating the subject for muscle damage or muscle atrophy / wasting.

[0306] In some embodiments of the aspects described herein, the method further includes selecting a subject with muscle damage or muscle atrophy / wasting prior to treating the subject for muscle damage or muscle atrophy / wasting.

[0307] The compounds described herein can be co-administered to subjects in combination with pharmacoactive agents.Exemplary pharmacoactive compounds include, but are not limited to, those found in Harrison's Principles of Internal Medicine, 13th edition, edited by TR Harrison et al., McGraw-Hill NY, NY; Physicians' Desk Reference, 50th edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990; Goodman and Oilman's The Pharmacological Basis of Therapeutics, current edition; and The Merck Index, current edition, all of which are incorporated herein by reference in their entirety.

[0308] In some embodiments of the aspects described herein, the pharmacoactive agent is a growth factor.Exemplary growth factors include, but are not limited to, basic epidermal growth factor (bEGF), fibroblast growth factor (FGF), FGF-1, FGF-2 (bFGF), FGF-4, thymosin, platelet-derived growth factor (PDGF), insulin-binding growth factor (IGF), IGF-1, IGF-2, epidermal growth factor (EGF), transforming growth factor (TGF), TGF-alpha, TGF-beta, chondroinductive factor-A and -B, osteoid induced growth factor, osteogenin, bone morphogenetic protein, and other bone growth factors, collagen growth factor, heparin-binding growth factor-1 or -2, and their biologically active derivatives.

[0309] The proliferation enhancer and the pharmacoactive agent can be administered to a subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times).When administered at different times, the proliferation enhancer and the pharmacoactive agent can be administered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 24 hours of administration of the other.When the proliferation enhancer and the pharmacoactive agent are administered in different pharmaceutical compositions, the administration route can be different.

[0310] The amount of proliferation enhancing agent that can be combined with a carrier material to produce a single dosage form is generally that amount of proliferation enhancing agent that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 0.01% to 99% of the compound, preferably from about 5% to about 70%, and most preferably from 10% to about 30%.

[0311] Toxicity and therapeutic efficacy can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, for example, to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are preferred.

[0312] As used herein, the term ED refers to the effective dose and is used in relation to animal models, and the term EC refers to the effective concentration and is used in relation to in vitro models.

[0313] The data obtained from cell culture assay and animal research can be used to formulate the dosage range for use in humans.The dosage of such compounds is preferably within the circulating concentration range that includes the ED50 with little or no toxicity.Dosage can vary within this range depending on the dosage form used and the route of administration used.

[0314] Therapeutically effective doses can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the therapeutic agent that achieves half-maximal inhibition of symptoms) as determined in cell culture. Plasma levels can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by a suitable bioassay.

[0315] Dosage will be determined by the physician and may be adjusted as necessary to meet the observed effects of treatment. Generally, the composition is administered such that the proliferation enhancer is given at a dose of 1 μg / kg to 150 mg / kg, 1 μg / kg to 100 mg / kg, 1 μg / kg to 50 mg / kg, 1 μg / kg to 20 mg / kg, 1 μg / kg to 10 mg / kg, 1 μg / kg to 1 mg / kg, 100 μg / kg to 100 mg / kg, 100 μg / kg to 50 mg / kg, 100 μg / kg to 20 mg / kg, 100 μg / kg to 10 mg / kg, 100 μg / kg to 1 mg / kg, 1 mg / kg to 100 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 10 mg / kg, 10 mg / kg to 100 mg / kg, 10 mg / kg to 50 mg / kg, or 10 mg / kg to 20 mg / kg. It should be understood that the ranges set forth herein include all intermediate ranges, for example, a range of 1 mg / kg to 10 mg / kg includes 1 mg / kg to 2 mg / kg, 1 mg / kg to 3 mg / kg, 1 mg / kg to 4 mg / kg, 1 mg / kg to 5 mg / kg, 1 mg / kg to 6 mg / kg, 1 mg / kg to 7 mg / kg, 1 mg / kg to 8 mg / kg, 1 mg / kg to 9 mg / kg, 2 mg / kg to 10 mg / kg, 3 mg / kg to 10 mg / kg, 4 mg / kg to 10 mg / kg, 5 mg / kg to 10 mg / kg, 6 mg / kg to 10 mg / kg, 7 mg / kg to 10 mg / kg, 8 mg / kg to 10 mg / kg, 9 mg / kg to 10 mg / kg, etc. Furthermore, it should be understood that within the above intermediate ranges, for example, the range of 1 mg / kg to 10 mg / kg, dosage ranges such as 2 mg / kg to 8 mg / kg, 3 mg / kg to 7 mg / kg, and 4 mg / kg to 6 mg / kg are also within the scope of the present invention.

[0316] In some embodiments, the composition is administered to a subject in need of treatment with an agent that inhibits proliferation, such that the proliferation-enhancing agent or a metabolite thereof is less than 500 μM, less than 400 μM, less than 300 μM, less than 250 μM, less than 200 μM, less than 150 μM, less than 100 μM, less than 50 μM, less than 25 μM, less than 20 μM, less than 300 μM, less than 300 μM, less than 400 μM, less than 500 μM, less than 500 μM, less than 600 μM, less than 700 μM, less than 800 μM, less than 900 μM, less than 1000 μM, less than 1000 μM, less than 15 ... The pharmaceutical composition is administered at a dosage such that the pharmaceutical composition has an in vivo concentration of less than 10 μM, less than 5 μM, less than 1 μM, less than 0.5 μM, less than 0.1 μM, less than 500 nM, less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, less than 0.01 nM, less than 0.005 nM, less than 0.001 nM.

[0317] In some embodiments, XMD8-92 is administered at a dosage that provides an in vivo concentration of about 1-10 μM, preferably about 3 μM. In some embodiments, SB-23906 is administered at a dosage that provides an in vivo concentration of about 1-10 μM, preferably about 5 μM. In some embodiments, XMD11-50 is administered at a dosage that provides an in vivo concentration of about 500-1000 nM, preferably about 800 nM. In some embodiments, vorinostat is administered at a dosage that provides an in vivo concentration of about 100-500 nM, preferably about 400 nM.

[0318] With regard to duration and frequency of treatment, it is typical for a skilled physician to monitor the subject to determine when the treatment is showing therapeutic benefit and to determine whether to increase or decrease dosage, increase or decrease frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. Administration schedules may vary from once a week to daily, depending on several clinical factors, such as the subject's sensitivity to the polypeptide. The desired dose can be administered daily or every 3, 4, 5, or 6 days. The desired dose can be administered once or divided into partial doses, e.g., 2-4 partial doses, administered over a period of time, e.g., at suitable intervals throughout the day or at other suitable schedules. Such partial doses can be administered as unit dosage forms. In some embodiments of the aspects described herein, administration is chronic, e.g., over a period of weeks or months, in one or more doses daily. Examples of dosing schedules are daily, twice daily, three times daily, or four times daily or more for one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, or six months or more.

[0319] Screening assays In yet another aspect, the invention provides a method of screening a candidate compound for stimulating or increasing proliferation in a satellite cell population, comprising: (a) contacting a population of satellite cells with a test compound; (b) assessing satellite cell proliferation; (c) selecting a compound that induces, increases, or enhances satellite cell proliferation; The present invention provides a method comprising:

[0320] As used herein, the term "test compound" refers to the compound and / or composition to be screened for their ability to induce, stimulate, enhance or increase the proliferation of satellite cells.Test compound can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, such as small organic or inorganic molecules; saccharin; oligosaccharides; polysaccharides; biological macromolecules, such as peptides, proteins, and peptide analogs and derivatives; antibodies, antibody fragments, peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; extracts made from biological materials, such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combination thereof.

[0321] In some embodiments, the test compound is a small molecule.

[0322] The number of possible test compounds can reach millions. Methods for developing small molecule, polymer and genome-based libraries are described, for example, in Ding et al., J Am. Chem. Soc., 124:1594-1596 (2002) and Lynn et al., J. Am. Chem. Soc., 123:8155-8156 (2001). Commercially available compound libraries can be obtained, for example, from ArQule, Pharmacopia, graffinity, Panvera, Vitas-M Lab, Biomol International and Oxford. These libraries can be screened using the screening devices and methods described herein. NIH Roadmap, Molecular Libraries Chemical compound libraries such as those from the Screening Centers Network (MLSCN) can also be used. A comprehensive list of compound libraries can be found at www.broad.harvard.edu / chembio / platform / screening / compound_libraries / index.htm. A chemical library or compound library is a collection of stored chemicals, usually for eventual use in high-throughput screening or industrial manufacturing. A chemical library may exist in simple terms as a set of stored chemicals. Each chemical has associated information stored in some type of database, along with information such as the chemical structure, purity, quantity, and physiochemical characteristics of the compound.

[0323] Depending on the particular embodiment carried out, the test compound can be provided free in solution or attached to a carrier or solid support, such as beads. A number of suitable solid supports can be used to immobilize the test compound. Examples of suitable solid supports include agarose, cellulose, dextran (commercially available, i.e., Sephadex, Sepharose), carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resins, plastic film, polyamine methyl vinyl ether maleic acid copolymer, glass beads, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. In addition, in the methods described herein, the test compounds can be screened individually or in groups. Group screening is particularly useful when the hit rate of effective test compounds is expected to be low and no more than one positive result is expected for a given group.

[0324] In some embodiments, the test compound induces, enhances or increases satellite cell proliferation by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more compared to an untreated control.

[0325] In some embodiments, assessing satellite cell proliferation comprises detecting a satellite cell marker.

[0326] In some embodiments, the step of evaluating the proliferation of satellite cells includes detecting a satellite cell marker and a cell replication marker. The selected test compounds can be further limited to those compounds in which the satellite cell marker and the cell replication marker co-localize in the same cells.

[0327] Increased or enhanced satellite cell proliferation can be assessed by (i) an increase in the total number of cells in the culture compared to an untreated control; (ii) an increase in the total number of cells expressing at least one satellite cell marker in the culture compared to an untreated control; (iii) an increase in the ratio of cells expressing at least one satellite cell marker to the total number of cells in the culture compared to an untreated control; (iv) an increase in the number of cells expressing at least one cell replication marker compared to an untreated control; (v) an increase in the ratio of cells expressing at least one cell replication marker compared to an untreated control; or (vi) a combination thereof.

[0328] In some embodiments, satellite cell proliferation is evaluated by automated image acquisition and analysis using Cellomics ArrayScan VTI. Acquisition thresholds / parameters are established so that computer-based calls of replication events are consistent with human-based calls. Such automated image acquisition and analysis allows high-throughput screening of compounds.

[0329] Generally, plating density may range from about 10k cells / well to about 100k cells / well. In some embodiments, cell plating density ranges from about 25k cells / well to about 75k cells / well. In one embodiment, cell plating density is about 60k cells / well. Generally, at least 75%, 80%, 85%, 90%, 95% or more of the cells are viable at the time of plating.

[0330] After plating, the satellite cells are allowed to adhere to the surface for a sufficient time, for example, at least 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours or more, before contacting with the test compound. In some embodiments, the cells are allowed to adhere for 48 hours before compound treatment. After the cells have been allowed to adhere for a sufficient time, the medium can be changed before treatment with the compound of interest.

[0331] Generally, compounds can be tested at any concentration that can enhance β-cell replication over a control for an appropriate period of time. In some embodiments, compounds are tested at concentrations ranging from about 0.1 nM to about 1000 mM. Preferably, compounds are tested at concentrations ranging from about 0.1 μM to about 20 μM, about 0.1 μM to about 10 μM, or about 0.1 μM to about 5 μM. In one embodiment, compounds are tested at 1 μM.

[0332] The satellite cell population can be maintained at any temperature suitable for culturing satellite cells. In one embodiment, the satellite cells are maintained at a temperature ranging from about 15° C. to about 55° C. In one embodiment, the pancreatic cells are maintained at 37° C.

[0333] Generally, satellite cells can be counted in culture after being contacted with test compound for a sufficient time, for example, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks or longer.Cells can be counted manually or by an automated system.Using an automated system allows high-throughput screening of compounds.

[0334] The inventors have found that in some cases, long-term treatment with proliferation enhancer does not result in higher satellite cell proliferation compared to shorter-term treatment.Therefore, the number of satellite cells in culture can be counted after contacting with test compound for 1 hour to 7 days.For example, the number of satellite cells in culture can be counted after contacting with test compound for 1 day, 2 days, 3 days, 4 days, 5 days or 6 days.

[0335] After contacting satellite cells with the test compound for a sufficient time, for example, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, or longer, detection of satellite cell and replicating cell markers can be performed. After detection of markers, cell replicating and / or the number of cells expressing satellite cell markers can be counted. Detection of markers can include a step of preparing cells for a suitable assay, for example, fixing and / or staining cells.

[0336] In some embodiments, satellite cells can be contacted with a test compound for between 1 hour and about 7 days prior to detection of satellite cells and cell replication markers.

[0337] In some embodiments, detection of satellite cells and cell replication markers can be performed after contacting the satellite cells with a test compound for 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days.

[0338] In some embodiments, the method further comprises selecting a compound that increases the ratio of satellite cells to the total number of cells compared to an untreated control.

[0339] The term "satellite cell marker" refers, without limitation, to proteins, peptides, nucleic acids, polymorphisms of proteins and nucleic acids, splice variants, fragments of proteins or nucleic acids, elements, and other analytes that are specifically expressed or present in satellite cells. Exemplary satellite cell markers include, but are not limited to, PAX7, PAX3, Myf5, MyoD, and desmin. Other markers that can be used include, but are not limited to, beta-integrin 1 and CXCR4. Methods for identifying satellite cells are also described in Sherwood et al. (Cell, 2004, vol. 119:543-554), the contents of which are incorporated herein by reference.

[0340] The term "cell replication marker" refers to, without limitation, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically associated with cell proliferation. In addition, "cell replication marker" includes enzyme activity, where a change, e.g., an increase or decrease, in enzyme activity is specifically associated with cell proliferation. Exemplary cell replication markers include, but are not limited to, phosphorylated histone H3 (PH3), Ki-67 protein, phosphorylated MPM-2 antigen, proliferating cell nuclear antigen (PCNA, a protein expressed in the nucleus of cells during the DNA synthesis phase of the cell cycle), phospho-S780-Rb epitope (Jacobberger, JW et al., Cytometry A (2007), 73A:5-15), Cenp-F (mitocin), class III β-tubulin, spindle checkpoint protein hMad2, phosphorylated myosin light chain kinase, topoisomerase II, checkpoint kinase 1 (Chk1), vesicular monoamine transporter 2 (VMAT2), and reduced cyclin-dependent kinase 1 (Cdk1) kinase activity. Histone H3 may be phosphorylated at Ser28 or Ser10.

[0341] Cell replication markers and satellite cell markers can be detected by methods known in the art and readily available to those skilled in the art, for example, suitable ELISA, immunofluorescence, or immunohistochemistry assays can be used for detection. MIB-1 is a commonly used monoclonal antibody that detects Ki-67 protein. It is used in clinical applications to determine Ki-67 labeling index. The Ki-67 ELISA is described in Klein, CL et al., J. Mater. Sci. Mater. Med. (2000), 11:125-132; Frahm, SO et al., J. Immunol. Methods (199*0, 211:43-50; and Key G et al., J. Immunol. Methods (1994), 177:113-117. Phospho-histone H3 antibodies for detecting phosphorylated histone H3 are commercially available from Cell Signaling Technology and Millipore. Antibodies against PCNA are commercially available from Sigma Aldrich. Antibodies against the MPM-2 antigen are specific for cells undergoing mitosis and recognize a family of proteins sharing a common phosphorylated epitope.

[0342] In some embodiments of this and other aspects described herein, satellite cells can be isolated from a mammal. Methods for isolating cells from a subject are described, for example, in Sherwood et al. (Cell, 2004, 119:543-554), the contents of which are incorporated herein by reference.

[0343] In some embodiments of this and other aspects described herein, satellite cells can be isolated from a mouse.

[0344] Satellite cells may be transformed cells. As used herein, the term "transformed cells" is recognized in the art and refers to cells that have been transformed into a state of unlimited proliferation, i.e., they have acquired the ability to grow in culture by unlimited division. Transformed cells may be characterized by terms such as neoplasia, dedifferentiation and / or hyperplasia with respect to their loss of growth control. In general, the term "transformed satellite cells" refers to satellite cells that exhibit an increased ability to survive serial subculture or an increased growth rate in vitro.

[0345] In some embodiments, the screening method is high-throughput screening.High-throughput screening (HTS) is a method for scientific experiments that uses robotics, data processing and control software, liquid handling devices, and sensing detectors.High-throughput screening or HTS allows researchers to rapidly carry out millions of biochemical, genetic or pharmacological tests.High-throughput screening is well known to those skilled in the art, and is described in, for example, U.S. Patent Nos. 5,976,813; 6,472,144; 6,692,856; 6,824,982; and 7,091,048, the contents of each of which are incorporated herein by reference in their entirety.

[0346] HTS uses automation to perform screening assays against a library of candidate compounds. The assays are tests for specific activity: usually the inhibition or stimulation of a biochemical or biological mechanism. A typical HTS screening library or "deck" may contain over 100,000 to 2,000,000 compounds.

[0347] The key laboratory instrument or test vessel for HTS is the microtiter plate: a small, usually disposable, plastic vessel that features a grid of small open divots called wells. Modern microplates for HTS generally have either 384, 1536, or 3456 wells; all of these are multiples of 96, reflecting the original 96-well microplate, which had 8 × 12 wells spaced 9 mm apart.

[0348] To prepare an assay, a researcher fills each well of a plate with the appropriate reagents on which they wish to conduct the experiment, such as satellite cell populations. After some incubation time to allow the reagents to absorb, bind, or otherwise react (or fail to react) with the compounds in the wells, measurements are made, either manually or by instrument, across all the wells of the plate. Manual measurements are often necessary when (for example) the researcher is using microscopy to look for changes that a computer cannot easily determine on its own. Alternatively, specialized automated analytical instruments can run several experiments on the wells, such as colorimetric measurements, radioactivity counts, etc. In this case, the instrument outputs the results of each experiment as a grid of numbers, mapping each number to the value obtained from a single well. High-volume analytical instruments can thus measure dozens of plates within a period of minutes, very quickly yielding thousands of experimental data points.

[0349] By way of example, in one embodiment, the assay was performed as follows: Satellite cells were harvested from CAG-B-actin-GFP mice, 2-4 months old, by FACS isolation methods outlined in Sherwood et al., 2004 and Cerletti et al., 2008. Briefly, all limb and abdominal muscles were harvested from the animals and digested first in 0.2% collagenase solution, then in 0.0125% collagenase / 0.05% dispase solution. The filtered solution was stained for cell surface markers and subjected to FACS. Cells that were negative for Mac1, Sca1 and CD45, and positive for beta-integrin 1 and CXCR4 were used for the screening assay. Cells were plated directly from the FACS machine at 50 cells / well into 96-well plates coated with 10ug / mL laminin (4-6 hours at 37°C, then partially removed). The medium for screening was Ham's F-10 supplemented with 10% heat-inactivated horse serum, 1x penicillin / streptomycin and 1x L-glutamine. Basic FGF (bFGF) was added only to the positive control wells; all other wells received medium only. The day of plating was designated day 0. Compounds were added the day after plating, day 1. All compounds were dissolved in DMSO and initially tested in duplicate at 10uM, 1uM or 0.1uM. The negative control for each plate was DMSO at the same concentration (no compound dissolved). Compounds were incubated with cells until day 4 without changing the medium. bFGF was added daily to the positive control wells. On day 4, plates were fixed in 4% paraformaldehyde and washed with phosphate-buffered saline. Plates were directly imaged on an Opera Confocal imager. The number of cells in each well was counted using Acapella script. Wells were scored for proliferation based on cell number (DMSO-treated wells usually had about 50 cells at the end of the assay, bFGF-treated wells usually had 150-250 cells at the end).We selected compounds as hits if they had cell counts greater than or equal to one (or, in the second session of screening, three) standard deviation(s) from the DMSO control. Compounds so selected were then tested in an initial dose curve, usually at concentrations between 20 nM and 30 uM, corresponding to the concentrations that flagged them as hits. If the compound was able to grow to a cell count of at least two standard deviations relative to the DMSO negative control, it was active. Any compounds found to be active in the dose curve were resupplied from the original supplier. The resupplied compounds were tested again in the dose curve for growth potential. Compounds still found to be active were then queued for optimization and characterization.

[0350] In another aspect, the present invention provides compounds selected by the screening assays described herein. It should be understood that analogs, derivatives, and isomers of compounds selected by the screening assays described herein are also claimed herein.

[0351] kit In another aspect, the present invention provides a kit for muscle repair or regeneration.In some embodiments, the kit comprises a compound as described herein, for example, a compound selected from the group consisting of kinase inhibitors, G protein-coupled receptor (GPCR) modulators, histone deacetylase (HDAC) modulators, hedgehog signaling pathway modulators, neuropeptides, dopamine receptor modulators, serotonin receptor modulators, histamine receptor modulators, ionophores, ion channel modulators, gamma-secretase modulators, and any combination thereof.The compound can be pre-formulated into pharmaceutical formulations for administration, or the kit can provide the components for formulating into pharmaceutical formulations.

[0352] In some embodiments, the kit comprises a compound described herein, wherein the compound is formulated for topical application.

[0353] In some embodiments, the kit comprises a population of satellite cells, wherein at least one cell in the population has been pre-treated by contacting the cell with a compound described herein.

[0354] In addition to the above-mentioned components, the kit may include informational material. The informational material may be descriptive, instructional, marketing or other material regarding the methods described herein and / or the use of the compounds for the methods described herein. For example, the informational material describes a method for administering the formulation to a subject. The kit may also include a delivery device.

[0355] In one embodiment, the informational material may include instructions for administering the formulation in an appropriate manner, e.g., in an appropriate dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material may include instructions for identifying an appropriate subject, e.g., a human, e.g., an adult. The informational material of the kit is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., printed text, drawings, and / or photographs, e.g., a label or printed sheet. However, the informational material may be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or phone number, where a user of the kit can obtain substantial information about the formulation and / or its use in the methods described herein. Of course, the informational material may be provided in any combination of formats.

[0356] In some embodiments, each component of the formulation can be provided in one container.Alternatively, it may be desirable to provide the components of the formulation separately in two or more containers, for example, one container for oligonucleotide formulation and at least another container for carrier compound.For example, different components can be combined according to the instructions provided with the kit.The components can be combined according to the methods described herein, for example, to prepare and administer pharmaceutical compositions.

[0357] In addition to the formulation, the composition of the kit can include other components such as a solvent or buffer, a stabilizer or preservative, and / or a second agent for treating a condition or disorder described herein. Alternatively, the other components can be included in the kit, but in a composition or container separate from the formulation. In such an embodiment, the kit can include instructions for mixing the formulation and other components, or for using the oligonucleotide together with the other components.

[0358] The compound can be provided in any form, for example, liquid, dry or lyophilized form.Preferably, the formulation is substantially pure and / or sterile.When the formulation is provided in liquid solution, the liquid solution is preferably an aqueous solution, preferably a sterile aqueous solution.When the formulation is provided in dry form, reconstitution is generally by adding suitable solvent.Solvent, for example, sterile water or buffer, can be provided in the kit as an option.

[0359] In some embodiments, the kit contains separate containers, dividers or compartments for the formulation and the informational material. For example, the formulation may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained in a single, undivided container. For example, the formulation is contained in a bottle, vial, or syringe to which the informational material is attached in the form of a label.

[0360] In some embodiments, the kit comprises a plurality of individual containers, e.g., a pack, each of which contains one or more unit dosage forms of the formulation.For example, the kit comprises a plurality of syringes, ampoules, foil packets, or blister packs, each of which contains a single unit dose of the formulation.The containers of the kit can be airtight and / or waterproof.

[0361] Exemplary embodiments may also be described by one or more of the following numbered paragraphs. 1. A method for increasing satellite cell proliferation, comprising contacting satellite cells with a compound selected from the group consisting of a kinase inhibitor, a G protein-coupled receptor (GPCR) modulator, a histone deacetylase (HDAC) modulator, an epigenetic modifier, a hedgehog signaling pathway modulator, a neuropeptide, a dopamine receptor modulator, a serotonin receptor modulator, a histamine receptor modulator, an adenosine receptor agonist, an ionophore, an ion channel modulator, a gamma-secretase modulator, a corticosteroid, and any combination thereof. 2. The method of paragraph 1, wherein the compound is selected from the group consisting of small organic or inorganic molecules; saccharin; oligosaccharides; polysaccharides; peptides, proteins, peptide analogs and derivatives; antibodies, antibody fragments, peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; extracts made from biological materials; naturally occurring or synthetic compositions; and any combination thereof. 3. The method of any of paragraphs 1-2, wherein the compound is an Flt3 kinase, PDGFR / EGFR, Bcr-abl, Jak3, or SRC kinase inhibitor. 4. The method of any of paragraphs 1-3, wherein the compound is selected from the group consisting of a protein kinase inhibitor and a receptor kinase inhibitor. In some embodiments, the kinase inhibitor is lestaurtinib (CEP701, [ka] [ka] [ka] and any combination thereof. 5. The method of any of paragraphs 1-4, wherein the compound is contacted with the satellite cells at a concentration of about 0.01 nM to about 100 μM. 6. The method of any of paragraphs 1-5, wherein the contacting step is for at least 1 hour. 7. The method of any of paragraphs 1-6, wherein the contacting step lasts for 1 to 7 days. 8. The method of any of paragraphs 1-7, wherein the contacting is in vitro. 9. The method of any of paragraphs 1-8, wherein the contacting is ex vivo. 10. The method of any of paragraphs 1-9, wherein the contacting is in vivo. 11. The method of paragraph 10, wherein the in vivo contacting is in a mammal. 12. The method of paragraphs 10 or 11, wherein the in vivo contacting is in a human. 13. The method of any of paragraphs 10 to 12, wherein the in vivo contacting is in a subject, and the subject is in need of treatment for damaged muscle tissue. 14. The method of paragraph 13, wherein the damaged muscle tissue is the result of physical injury or accident, disease, infection, abuse, loss of blood circulation, or muscle atrophy or wasting. 15. The method of paragraphs 13 or 14, wherein the damaged muscle tissue is dystrophic muscle or aging muscle. 16. The method of any of paragraphs 13-15, wherein the damaged muscle tissue is the result of muscle atrophy / wasting. 17. A method for repairing or regenerating muscle in a subject, comprising administering to the subject a therapeutically effective amount of a compound, wherein the subject has damaged muscle tissue, and the compound is selected from the group consisting of kinase inhibitors, G protein-coupled receptor (GPCR) modulators, histone deacetylase (HDAC) modulators, epigenetic modifiers, hedgehog signaling pathway modulators, neuropeptides, dopamine receptor modulators, serotonin receptor modulators, histamine receptor modulators, ionophores, ion channel modulators, gamma-secretase modulators, and any combination thereof. 18. The method of paragraph 17, wherein the damaged muscle tissue is the result of physical injury or accident, disease, infection, abuse, loss of blood circulation, or muscle atrophy or wasting. 19. The method of any of paragraphs 17-18, wherein the damaged muscle tissue is dystrophic muscle or aging muscle. 20. The method of any of paragraphs 17-19, wherein the damaged muscle tissue is the result of muscle atrophy / wasting. 21. The method of any of paragraphs 17 to 20, wherein the subject is a mammal. 22. The method of any of paragraphs 17-21, wherein the subject is a human. 23. The method of any of paragraphs 17-22, wherein the compound is co-administered with a therapeutic agent. 24. The method of paragraph 23, wherein the compound and the therapeutic agent are administered in the same formulation. 25. The method of any of paragraphs 17-24, wherein the compound is administered at a dosage of 1 μg / kg to 150 mg / kg. 26. The method of any of paragraphs 17-25, wherein the administering step is by injection, infusion, instillation, inhalation, or ingestion. 27. The method of any of paragraphs 17-26, wherein the administering step is once a day. 28. The method of any of paragraphs 17-27, further comprising the step of diagnosing the subject for muscle damage or muscle atrophy / wasting prior to treating the subject for muscle repair or regeneration. 29. A compound: [ka] [ka] 29. The method according to any of paragraphs 17 to 28, selected from the group consisting of: and any combination thereof. 30. A high throughput assay for screening compounds that induce, stimulate, enhance, or increase satellite cell proliferation, comprising: (a) contacting satellite cells with a test compound; (b) assessing satellite cell proliferation; (c) selecting a compound that induces, stimulates, enhances, or increases satellite cell replication or growth; A high throughput assay comprising: 31. The assay of paragraph 30, wherein the step of assessing satellite cell proliferation comprises detecting a cell marker. 32. The assay of paragraph 31, wherein the cellular marker is selected from the group consisting of CXCR4, β1-integrin, Sca-1, Mac-1, CD45, PAX7, PAX3, Myf5, MyoD, desmin, and any combination thereof. 33. The assay of any of paragraphs 30-32, wherein the test compound has a concentration in the range of 0.1 nM to 1000 mM. 34. The assay of any of paragraphs 30 to 33, wherein the assay is carried out at a temperature in the range of about 15°C to about 55°C. 35. The assay of any of paragraphs 30 to 34, wherein the test compound is contacted with the pancreatic cells for a period of from 1 hour to 7 days. 36. The assay of any of paragraphs 30 to 35, wherein the test compound increases satellite cell proliferation by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more relative to an untreated control. 37. The assay of any of paragraphs 30 to 36, wherein satellite cells are isolated from a mammal. 38. The assay of any of paragraphs 30 to 37, wherein satellite cells are isolated from a subject, the subject being in need of treatment for damaged muscle tissue. 39. The method of paragraph 38, wherein the damaged muscle tissue is the result of physical injury or accident, disease, infection, abuse, loss of blood circulation, or muscle atrophy or wasting. 40. The method of paragraphs 38 or 39, wherein the damaged muscle tissue is dystrophic muscle or aging muscle. 41. The method of any of paragraphs 38-40, wherein the damaged muscle tissue is the result of muscle atrophy / wasting. 42. In some embodiments of any of paragraphs 1-41 above, the kinase inhibitor is: [ka] or a salt, ester or chelate thereof. 44. A method for increasing satellite cell proliferation, comprising contacting satellite cells with a compound, wherein the compound is a kinase inhibitor. 45. The method of paragraph 44, wherein the kinase is a protein kinase. 46. ​​The method of paragraph 44, wherein the protein kinase is a protein tyrosine kinase. 47. The method of paragraph 46, wherein the protein tyrosine kinase is a receptor protein tyrosine kinase. 48. The method of paragraph 47, wherein the receptor protein tyrosine kinase is a member of the RET family. 49. The method of paragraph 47, wherein the receptor protein tyrosine kinase is RET. 50. The method of paragraph 47, wherein the compound interferes with the binding of a ligand to a receptor protein tyrosine kinase. 51. The method of paragraph 44, wherein the compound is selected from the group consisting of small organic or inorganic molecules, saccharin, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives, antibodies, antibody fragments, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, naturally occurring or synthetic compositions, and any combination thereof. 52. The method of paragraph 51, wherein the compound is an antibody. 53. The method of paragraph 44, wherein the compound is contacted with the satellite cells at a concentration of about 0.01 nM to about 100 μM. 54. The method of paragraph 44, wherein the contacting is in vivo. 55. The method of paragraph 54, wherein the in vivo contacting is in a mammal. 56. The method of paragraph 55, wherein the in vivo contacting is in a mammal, and the mammal is in need of treatment of damaged muscle tissue. 57. The method of paragraph 56, wherein the damaged muscle tissue is the result of physical injury or accident, disease, infection, abuse, loss of blood circulation, muscle atrophy, muscle wasting, dystrophic muscle or aging muscle. 58. The method of any of paragraphs 44 to 57, wherein the compound comprises quizartinib (AC220), or any salt, ester, or chelate thereof. 59. A method for repairing or regenerating muscle in a subject having damaged muscle tissue, comprising administering to the subject a therapeutically effective amount of a compound, wherein the compound is a kinase inhibitor. 60. The method of paragraph 59, wherein the kinase is a protein kinase. 61. The method of paragraph 60, wherein the protein kinase is a protein tyrosine kinase. 62. The method of paragraph 61, wherein the protein tyrosine kinase is a receptor protein tyrosine kinase. 63. The method of paragraph 62, wherein the receptor protein tyrosine kinase is a member of the RET family. 64. The method of paragraph 61, wherein the receptor protein tyrosine kinase is RET. 65. The method of paragraph 61, wherein the compound interferes with the binding of a ligand to a receptor protein tyrosine kinase. 66. The method of paragraph 59, wherein the compound is selected from the group consisting of small organic or inorganic molecules, saccharin, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives, antibodies, antibody fragments, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, naturally occurring or synthetic compositions, and any combination thereof. 67. The method of paragraph 59, wherein the damaged muscle tissue is the result of physical injury or accident, disease, infection, abuse, loss of blood circulation, muscle atrophy, muscle wasting, dystrophic muscle or aging muscle. 68. The method of paragraph 59, wherein the subject is a mammal. 69. The method of any of paragraphs 59-68, wherein the compound comprises quizartinib (AC220), or any salt, ester, or chelate thereof. 70. A compound: [ka] [ka] 70. The method of any of paragraphs 44 to 69, selected from the group consisting of: and any combination thereof. 71. In some embodiments of any of paragraphs 1-41, 44, 51-57, 59-60, 66, and 67-68, supra, the kinase inhibitor comprises one or more B-Raf inhibitors, JAK3 inhibitors, p38MAPK inhibitors, C-Raf1 inhibitors, Akt inhibitors, ERK inhibitors, BMK1 / ERK5 inhibitors, p38MAPK inhibitors, RTK inhibitors, ERK5 inhibitors, Bcr-Abl inhibitors, RhoK inhibitors, p38 inhibitors, p110 inhibitors, FAK inhibitors, ATP-competitive JNK inhibitors, MELK inhibitors, or inhibitors of a pathway identified in Table 5, or a salt, ester, or chelate thereof. 72. In some embodiments of any of paragraphs 1-41, 44, 51-57, 59-60, 66, and 67-68, above, the kinase inhibitor is selected from the group consisting of BAY-439006 (i.e., sorafenib; HMSL10008-101-1); HG-6-64-01 (i.e., HMSL10017-101-1); HKI-272 (i.e., neratinib; HMSL10018-101-1); KIN001-055 (i.e., HY-11067; HMSL10033-101-1); SB239063 (i.e., HMSL10036-1 01-1); KIN001-242 (i.e., HMSL10044-104-1); SB590885 (i.e., GSK2118436; HMSL10046-101-1); AZ-628 (i.e., HMSL10050-101-1); MK2206 (i.e., HMSL10057-102-1); XMD11-50 (i.e., LRRK2-in-1; HMSL10086-101-1); XMD8-92 (i.e., HMSL10094-101-1); BIRB796; Dramapimod (i.e., HMSL10169-101 -1); sunitinib malate (i.e., SU11248; stent; HMSL10175-106-1); GDC-0879 (i.e., HMSL10181-101-1); XMD8-85 (i.e., HMSL10093-101-1); AMN-107 (i.e., nilotinib; HMSL10099-101-1); Y39983 (i.e., HMSL10149-102-1); SB203580 (i.e., RWJ64809; PB203580; HMSL10167-101-1); VX-745 (i.e., HMSL10 168-101-1); pseudoXL765 (i.e., HMSL10173-101-1); Y-27632 (i.e., HMSL10176-101-1); PH-797804 (i.e., HMSL10439-101); VX-702 (i.e., HMSL10440-101); NG25 (i.e., HMSL10419-101); SB202190 (i.e., HMSL10441-101); BI-D1870 (i.e., HMSL10423-101); BIX02565 (i.e., HMSL10434-101);URMC-099 (i.e., HMSL10453-101); staurosporine aglycone (i.e., K252C; HMSL10454-101); ralimetinib (i.e., LY2228820; HMSL10438-103); BMX-IN-1 (i.e., HMSL10427-101); PF3644022 (i.e., HMSL10476-101); NVP-BHG712 (i.e., KIN001-265; HMSL10200-101); bosutinib (i.e., SKI-606; HMSL10189-101) ; NVP-TAE226 (i.e., CHIR-265; HMSL10207-101); RAD001 (i.e., everolimus; HMSL10235-101); CC-401 (i.e., HMSL10185-101); CGP74514A (i.e., HMSL10355-101); KIN001-269 (i.e., HMSL10195-101); RAF265 (i.e., HMSL10206-101); OTSSP167 (i.e., HMSL10337-102); Dorsomorphin (i.e., Compound C; BML275; HMSL10399-102; losmapimod (i.e., GSK-AHAB; SB856553; GW856553X; HMSL10402-101); AZD5363 (i.e., HMSL10370-101); RO31-8220 (i.e., bisindolylmaleimide IX; HMSL10407-103); sotrastaurin (i.e., AEB071; HMSL10 408-101); TAK-632 (i.e., HMSL10409-101); FRAX597 (i.e., HMSL10400-101); GW2580 (i.e., HMSL10401-101); alisertib (i.e., MLN8237; HMSL10391-101), kinase inhibitors listed in Table 5, or derivatives, salts, metabolites, prodrugs, and stereoisomers thereof; 73. In some embodiments of any of paragraphs 1-41, 51-57, 59-60, 66, and 67-68, supra, the epigenetic modifier comprises an HDAC modifier (e.g., an HDAC1, HDAC3, and / or HDAC6 modifier), a BRD modifier (e.g., a BRD2 and / or BRD4 modifier), or an EGLN1 modifier. 73. In some embodiments of any of paragraphs 1-41, 51-57, 59-60, 66, and 67-68, supra, the epigenetic modifier comprises (+)-JQ1; S)-JQ1; belinostat (i.e., PXD101); MS-275 (i.e., entinostat; MS-27-275); vorinostat (i.e., suberoylanilide hydroxamic acid (SAHA); Zolinza); mocetinostat (i.e., MGCD0103); I-BET (i.e., GSK525762A); SB939 (i.e., pracinostat); PFI-1); rosilinostat (i.e., ACY-1215); I-BET151 (i.e., GSK1210151A); IOX2; or derivatives, salts, metabolites, prodrugs, and stereoisomers thereof.

[0362] Some selection definitions For convenience, certain terms used in the specification, description, examples, and appended claims are collected here. Unless otherwise stated or indicated by context, the following terms and phrases include the meanings set forth below. Unless otherwise stated or apparent from the context, the following terms and phrases are not intended to exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in the description of certain embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims. Moreover, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular.

[0363] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials can be used in the practice or testing of the present invention, methods, devices, and materials in connection therewith are described herein.

[0364] As used herein, the terms "comprising" or "comprising" are used in reference to compositions, methods, and their respective component(s) that are essential to the invention, yet are open to the inclusion of unspecified elements, whether essential or not.

[0365] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0366] Except in carrying out the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±5% of the recited value. For example, about 100 means 95 to 105.

[0367] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is a synonym of the term "for example."

[0368] The terms "decrease", "reduced", "reduction", "decrease" or "inhibit" are all used herein generally to mean a decrease in a substantially significant amount. However, for the avoidance of doubt, "reduced", "reduction" or "decrease" or "inhibit" means a decrease of at least 10% compared to a reference level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or 100%, including a decrease of 100% (e.g., non-existent levels compared to a reference sample), or any decrease between 10-100% compared to a reference level.

[0369] The terms "increased," "increase" or "enhance" or "activate" are all used herein generally to mean an increase in a statistically significant amount; for the avoidance of any doubt, the terms "increased," "increase" or "enhance" or "activate" mean an increase of at least 10% compared to a base level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or 100%, up to and including a 100% increase, or any increase between 10-100% compared to a base level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a base level.

[0370] The term "statistically significant" or "significantly" refers to statistical significance, which generally means at least two standard deviations (2SD) away from the reference level. The term refers to statistical evidence that a difference exists. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is in fact true.

[0371] The term "modulator" refers to a compound that alters or induces the activity of a molecule. For example, a modulator can result in an increase or decrease in the magnitude of a particular activity of a molecule compared to the magnitude of the activity in the absence of the modulator. In certain embodiments, a modulator is an inhibitor that reduces the magnitude of one or more activities of a molecule. In certain embodiments, an inhibitor completely prevents one or more activities of a molecule. In certain embodiments, a modulator is an activator that increases the magnitude of at least one activity of a molecule. In certain embodiments, the presence of a modulator results in an activity that does not occur in the absence of the modulator. Without limitation, a modulator can be selected from the group consisting of small or large organic or inorganic molecules; monosaccharides; disaccharides; trisaccharides; oligosaccharides; polysaccharides; biological macromolecules, such as proteins, peptides, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, enzymes, antibodies, antibody portions or fragments; extracts made from biological materials such as bacteria, plants, fungi, or animal cells or tissues; naturally occurring or synthetic compositions; and any combination thereof.

[0372] The term "selective modulator" refers to a compound that selectively modulates a target activity.

[0373] The term "selectively modulate" refers to the ability of a selective modulator to modulate a target activity to a greater extent than it modulates a non-target activity.

[0374] The term "target activity" refers to a biological activity that can be modulated by a modulator. Certain exemplary target activities include, but are not limited to, binding affinity, signal transduction, enzymatic activity, and the like.

[0375] The term "agonist" refers to a compound whose presence produces a biological activity of a receptor that is the same as the biological activity that results from the presence of a naturally occurring ligand for that receptor.

[0376] The term "partial agonist" refers to a compound whose presence results in a biological activity of a receptor that is of the same kind, but of a lesser magnitude, as that which results from the presence of a naturally occurring ligand for that receptor.

[0377] The term "antagonist" refers to a compound whose presence results in a decrease in the magnitude of a biological activity of a receptor. In certain embodiments, the presence of an antagonist results in complete inhibition of a biological activity of a receptor.

[0378] The term "inhibitor" refers to a molecule or substance or compound or composition or drug or any combination that can inhibit and / or reduce the activity of a target molecule. As used herein, the term "inhibitor" is interchangeable with the term "antagonist". The term "inhibitor" includes competitive, non-competitive, functional and chemical antagonists. The term "partial inhibitor" refers to a molecule or substance or compound or composition or drug or any combination that can incompletely block the action of an agonist, especially by a non-competitive mechanism.

[0379] As used herein, the term "ligand" refers to an agent that binds to a target molecule. Ligands are not limited to agents that bind to the recognition function regions of target molecules, such as the active site of an enzyme, the antigen binding site of an antibody, the hormone binding site of a receptor, the cofactor binding site, and the like. Ligands can also be agents that bind to any surface or conformational domain of a target compound. Thus, ligands include agents that may not have any obvious or known biological function themselves, other than their ability to bind to a target in the manner described above. The term ligand includes agents that react upon binding and agents that do not react other than by binding.

[0380] As used herein, the term "small molecule" can refer to compounds that are "natural product-like", however, the term "small molecule" is not limited to "natural product-like" compounds. Rather, small molecules are typically characterized in that they contain several carbon-carbon bonds and have a molecular weight of less than 5000 Daltons (5 kD), preferably less than 3 kD, even more preferably less than 2 kD, and most preferably less than 1 kD. In some cases, it is preferred that small molecules have a molecular weight equal to or less than 700 Daltons.

[0381] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The examples are merely illustrative and are not intended to limit any of the aspects described herein. The following examples are not intended to limit the present invention in any way. EXAMPLES

[0382] Example 1 Screening Assay Satellite cells were harvested from CAG-B-actin-GFP mice, 2-4 months of age, by FACS isolation methods outlined in Sherwood et al., 2004 and Cerletti et al., 2008. Briefly, all limb and abdominal muscles were harvested from the animals and digested first in 0.2% collagenase solution and then in 0.0125% collagenase / 0.05% dispase solution. The filtered solutions were stained for cell surface markers and subjected to FACS. Cells that were negative for Mac1, Sca1 and CD45 and positive for beta-integrin 1 and CXCR4 were used for screening assays. Cells were plated directly from the FACS machine at 50 cells / well into 96-well plates coated with 10ug / mL laminin (4-6 hours at 37°C, then partially removed). The medium for screening was Ham's F-10 supplemented with 10% heat-inactivated horse serum, 1x penicillin / streptomycin and 1x L-glutamine. Basic FGF (bFGF) was added only to the positive control wells; all other wells received medium only. The day of plating was designated day 0. Compounds were added the day after plating, day 1. All compounds were dissolved in DMSO and initially tested in duplicate at 10uM, 1uM or 0.1uM. The negative control for each plate was DMSO at the same concentration (no compound dissolved). Compounds were incubated with cells until day 4 without changing the medium. bFGF was added daily to the positive control wells. On day 4, plates were fixed in 4% paraformaldehyde and washed with phosphate-buffered saline. Plates were directly imaged on an Opera Confocal imager. Because the cells were easily visible from the CAG-EGFP-beta-actin transgene, the number of cells in each well was counted using Acapella script. Wells were scored for proliferation based on cell number (DMSO-treated wells typically had approximately 50 cells at the end of the assay, while bFGF-treated wells typically had 150-250 cells at the end).We selected compounds as hits if they had cell counts greater than or equal to one (or, in the second session of screening, three) standard deviation(s) from the DMSO control. Compounds so selected were then tested in an initial dose curve, usually at concentrations between 20 nM and 30 uM, corresponding to the concentrations that flagged them as hits. We called compounds active if they were able to grow to cell counts of at least two standard deviations relative to the DMSO negative control. Any compounds found to be active in the dose curve were resupplied from the original supplier. The resupplied compounds were tested again in the dose curve for growth potential. Compounds still found to be active were then queued for optimization and characterization. [Table 1] [Table 2]

[0383] As described above, we screened a set of 215 compounds, consisting mainly of kinase inhibitors and GPCR ligands. We assayed these compounds at 1 μM, and counted compounds that scored more than one standard deviation against the DMSO-treated negative control as potential hits. Using these criteria, we identified 20 compounds as hits from the set. We then tested each of these 20 compounds in a dose-response assay to verify their activity. Five compounds found to have activity using this method are shown in Table 3. Dose-response curves for the five compounds are shown in Figures 9A-13. [Table 3]

[0384] Three of the compounds (sunitinib (SU11248), JAK3 inhibitor VI, and lestaurtinib / CEP701) were retested and found to be active in the retest. Each compound promoted proliferation of input SMP cells similar to that seen with the bFGF positive control (4.7±1.4 for sunitinib and lestaurtinib, 3.7±1.1 for JAK3 inhibitor VI). DMSO-treated cells proliferated to a level of 1±0.3 in both tests.

[0385] In addition, we also tested whether CEP701 could synergize with bFGF to achieve higher levels of SMP proliferation. In these experiments, 300 cells were plated per well instead of the 50 cells / well used in the screening and subsequent dose-response assays. This change in protocol was introduced to reduce assay variability. Under these conditions, 0.05 μM CEP701 was added the day after the purified SMPs were plated, and 5 ng / mL bFGF was added daily to appropriate samples. Three days after plating, the medium was changed and fresh compound was added. This additional change in protocol was introduced to not only improve viability, but also reduce variability. Plates were fixed 4 days after plating.

[0386] Cultures treated with both CEP701 and bFGF showed an 8-fold increase in proliferation (Figure 14), indicating that CEP701 and bFGF can act additively to increase the expansion of SMPs in vitro. Since it was believed that the change of medium resulted in increased cell viability, we decided to try removing the compound a few days before fixation to allow the cells to recover. In the next experiment, the compound was added the day after plating (day 1). The compound was refreshed every day until it was completely removed from the culture on the days indicated below. The cells were then cultured in medium not supplemented with the compound until fixation on day 4. We found that a 2-day recovery time was favorable for the cultures. We confirmed the discovery that CEF-701 can act additively with bFGF (Figure 15). In addition, the Jak3 inhibitor VI also acts additively with bFGF (Figure 16), but sunitinib did not act additively with bFGF (Figure 17).

[0387] To evaluate the effect that these compounds have on the identity of SMPs, we studied the ability of treated SMPs to differentiate in culture. For these experiments, 300 cells were plated in each well on day 0 in our standard growth medium (10% horse serum in F-10). Compounds were added on day 1, and SMPs were allowed to grow for 3 days. On day 4, the medium was switched to differentiation medium (10% horse serum, 10% FBS, 0.5% chicken embryo extract in high glucose DMEM). Cultures were then incubated under differentiation conditions for 3 or 4 days and fixed on day 7 or 8. They were then stained with Hoechst and anti-myosin heavy chain antibodies.

[0388] As shown in Figures 18-20, treatment with CEP701, sunitinib, or the Jak3 inhibitor VI did not affect the ability of expanded SMPs to differentiate and fuse to form myotubes. In addition, satellite cells exposed to CEP701 for 3 days, allowed to recover for 2 days, and then placed in differentiation conditions were also able to fuse into myotubes (data not shown).

[0389] We focused on CEP701 for further study. Next, we investigated the additive effect of CEP701 and TGF-beta inhibitors. TGF-beta inhibitors are known to promote proliferation and prevent satellite cell differentiation. Addition of Alk5 inhibitor II to the cultures resulted in a slight increase in proliferation. However, little additive effect was observed with the addition of CEP701 and TGF-beta inhibitors (Figure 21).

[0390] Next, we tested the specificity of CEP701 for the proliferation of satellite cells. We isolated a Sca1 positive population of fibroblasts from muscle preparations (obtained via FACS). 500 or 3000 fibroblasts were plated in each well and cultured in DMEM supplemented with 10% FBS. Compounds were added the day after plating and refreshed every day. Plates were fixed 5 days after plating and stained for the proliferation marker Ki67. As can be seen in Figure 22, there was no difference in the percentage of proliferating cells between those exposed to CEP701 (right panel) and those exposed to DMSO (left panel). In addition, CEP701 had no effect on primary fibroblasts (Figure 23).

[0391] In another experiment, we tested the CEP701 compound in old tissues. Old mice were 15 months old at the time of the experiment. The conditions used in this assay were the same as those used in young animals. After FACS, 300 cells / well were plated on day 0 in our standard growth medium (10% horse serum in F-10). Compounds were added at the indicated concentrations on day 1 and refreshed every day. On day 4, compounds were stopped and only the medium was replaced. Plates were fixed and imaged on day 6.

[0392] As seen in Figures 24 and 25, 50 nM CEP701 appeared to be the optimal concentration for both old and young cells. In both cases, exposure to 50 nM CEP701 under the conditions described resulted in an approximately 6-fold increase in cell number. CEP701 and bFGF could act additively in cultures of old cells resulting in an approximately 10-fold increase in cell number.

[0393] In another experiment, we screened a new set of 200 compounds. This set is mainly focused on GPCR ligands, along with several kinase inhibitors and other annotated compounds. We screened the compounds in duplicate at 10 uM, except for the kinase inhibitors, which were screened in duplicate at 1 uM. Compounds were scored as promising hits if they increased the proliferation of input SMPs at a level higher than 3 standard deviations relative to the negative control in any of the replicates. Using these criteria, we identified two hits (N6-cyclopentyladenosine, an adenosinse receptor agonist, and budesonide, a glucocorticoid steroid) that were subsequently validated with dose-response curves (Figures 26 and 27).

[0394] After additional testing, we finally decided to resupply budesonide and N6-cyclopentyladenosine (CPA) from the original supplier to test different batches of compounds. We performed dose responses with all resupplied compounds, plating 50 cells per well, as we did during screening and the first dose response study.

[0395] In this experiment, the DMSO negative control had a normalized value of 1±0.4 and the bFGF positive control had a value of 5.6±1.7. Both CPA and budesonide were effective and produced proliferation levels significantly higher than those seen in the negative controls. Without wishing to be bound by theory, repeating these assays under optimized experimental conditions developed by the inventors (300 cells / well and medium change to refresh compound after 2 days of exposure) may improve both assay variability and proliferation responses.

[0396] As with the hits from the first set of screened compounds, we tested these compounds for their ability to synergize with bFGF. Using our standard culture conditions (10% horse serum in F-10), compounds were added to cells the day after plating and refreshed every day until removed from culture as indicated. Cells were then grown in medium not supplemented with compound until fixation on day 4.

[0397] CPA (30 μM) was found to synergize with bFGF in vitro. The combination of CPA and bFGF showed a 15-fold increase in cell number compared to the approximately 6-fold increase with bFGF alone (FIG. 28). However, the combination of budesonide and bFGF showed no further increase compared to bFGF alone (FIG. 29).

[0398] We also evaluated the effect that these compounds have on the identity of satellite cells and their ability to differentiate in culture. In these experiments, 300 cells were plated in each well on day 0 in our standard growth medium (10% horse serum in F-10). Compounds were added on day 1 and SMPs were allowed to grow for 3 days. On day 4, the medium was switched to differentiation medium (10% horse serum, 10% FBS, 0.5% chicken embryo extract in high glucose DMEM). Cultures were incubated under differentiation conditions for 3 or 4 days and fixed on day 7. They were then stained with Hoechst and anti-myosin heavy chain antibodies. As can be seen in Figures 29 and 30, satellite cells exposed to compounds were able to fuse into myosin heavy chain positive myotubes.

[0399] We then tested whether these compounds could act additively with TGF-beta inhibitors.When cells were treated with both CPA and Alk5 inhibitor II, a slight increase in proliferation was observed compared to CPA and Alk5 inhibitor II alone (Figure 31).

[0400] In this study, the inventors performed a screen designed to identify compounds that can expand satellite cells in vitro. The inventors discovered several compounds that could result in expansion both in the presence and absence of bFGF. The compounds resulted in a satellite cell population that differentiated normally and had normal markers for its differentiation state. These results indicate that treatment with these compounds allows the satellite cell population to expand normally and contribute to muscle repair in pathological conditions.

[0401] Example 2 Satellite cells were harvested from CAG-B-actin-GFP mice, 2-4 months old, by FACS isolation as outlined in Example 1. Briefly, all limb and abdominal muscles were harvested from the animals and digested first in 0.2% collagenase solution, then in 0.0125% collagenase / 0.05% dispase solution. The filtered solution was stained for cell surface markers and subjected to FACS. Cells that were negative for Mac1, Sea1 and CD45, and positive for beta-integrin 1 and CXCR4 were used for screening assays. Cells were plated directly from the FACS machine at 50 cells / well into 96-well plates coated with 10ug / mL laminin (4-6 hours at 37°C, then partially removed). The medium for screening was Ham's F-10 supplemented with 10% heat-inactivated horse serum, 1x penicillin / streptomycin and 1x L-glutamine. Basic FGF (bFGF) was added only to positive control wells; all other wells received medium only. The day of plating was designated day 0.

[0402] The day after plating (day 1), compounds were added as diagrammed in Figure 33. All compounds were dissolved in DMSO and initially tested in duplicate at 10 uM, 1 uM or 0.1 uM. The negative control for each plate was the same concentration of DMSO (no compound dissolved). Compounds were incubated with cells until day 4 without changing the medium. bFGF was added daily to the positive control wells. On day 4, plates were fixed in 4% paraformaldehyde and washed with phosphate buffered saline. Plates were imaged directly on an Opera Confocal imager because cells were easily visible from the CAG-EGFP-beta-actin transgene. The number of cells in each well was counted using Acapella script. Wells were scored for proliferation based on cell number (DMSO-treated wells usually had about 50 cells at the end of the assay, bFGF-treated wells usually had 150-250 cells at the end). Compounds were selected as hits if they had cell numbers greater than or equal to one (or, in the second session of screening, three) standard deviation(s) from the DMSO control. Compounds so selected were then tested in an initial dose curve, usually at concentrations between 20 nM and 30 uM, corresponding to the concentrations that had flagged them as hits. Compounds were considered active if they were able to grow to cell numbers at least two standard deviations relative to the DMSO negative control. Any compounds found to be active in the dose curve were resupplied from the original supplier. Resupplied compounds were again tested in the dose curve for growth potential. Compounds still found to be active were then queued for optimization and characterization.

[0403] We screened a set of about 400 compounds from a custom screening library, which is illustrated in FIG. 34. We assayed these compounds at 1 μM, and counted compounds that scored more than one standard deviation against the DMSO-treated negative control as potential hits. Using these criteria, we identified about 10 compounds from the set as hits that could increase satellite cell proliferation in vitro. We then tested each of these compounds in a dose-response assay to verify their activity. As illustrated in FIG. 35, the four compounds that were found to increase satellite cell proliferation in vitro were lestaurtinib (CEP701), sunitinib (SU11248), JAK3 inhibitor VI, and N6-cyclopentyladenosine (CPA). Lestaurtinib (CEP701) was identified as the best hit, was effective at nanomolar doses, and had target overlap with several other hit compounds. In addition, lestaurtinib (CEP701) increased the proliferation of senescent satellite cells in vitro (Figure 36), and increased the proliferation of human satellite cells, but not fibroblasts.

[0404] We then tested each of these compounds in a dose-response assay to verify their activity, as illustrated in Figure 37A. Dose-response curves with lestaurtinib (CEP701), sunitinib (SU11248), JAK3 inhibitor VI, and N6-cyclopentyladenosine (CPA) are shown in Figure 37B.

[0405] In addition to lestaurtinib (CEP701), quizartinib (AC220), a small molecule receptor tyrosine kinase inhibitor, also demonstrated the ability to expand human satellite cells at low doses. As shown in Figure 38, both CEP-701 and AC220 increased human satellite cells by more than 2-fold at a concentration of 1 nM relative to the DMSO control.

[0406] As illustrated in Figure 39, differentiation medium containing 5% horse serum and compounds identified as hits (e.g., CEP701, SU11248, JAK3 inhibitor VI, CPA and Tyr AG490) drives myoblast differentiation. Similarly, Figure 40 demonstrates that CEP701 enhances myoblast differentiation in differentiation medium relative to DMSO control, as evidenced by the observed increases in both myoblast area and length.

[0407] Next, we sought to determine whether CEP701-treated cells maintained engraftability by performing the experimental protocol illustrated in Figure 41 A by transplanting Tubulin>GFP satellite cells into injured mdx muscle and administering CEP701 or DMSO control. As illustrated in Figure 41 B, CEP701-treated cells resulted in an increased number of GFP+ fibers per section relative to the DMSO control.

[0408] Next, we sought to determine whether CEP701 treatment would increase the size of regenerating fibers and the number of satellite cells in vivo. As shown in Figure 42A, CEP701 was administered subcutaneously to mice after CTX injury, followed by tissue harvest. Regenerating muscle fibers are eMHC+. As shown in Figures 42B-42D, treatment with CEP701 increased both the size of regenerating fibers and the number of satellite cells in vivo in both adult and aged mice.

[0409] We also performed in vitro binding assays (KINOME scan) on active site fragments in an attempt to identify multiple hit compounds that inhibit receptor tyrosine kinases (RTKs). As shown in Table 4 below, CEP701, AC220 and sunitinib each inhibit multiple RTKs (numbers are Kd in nM; expression in primary myoblasts).

[0410] [Table 4]

[0411] The RET proto-oncogene is a receptor for the GDNF ligand family, which includes glial cell line-derived neurotrophic factor, neurturin, artemin, and persephin, and is important for the development and function of several tissue types, including the nervous system. The RET proto-oncogene activates several downstream pathways, including JAK / STAT. As shown in Figure 43A, the multiple hit compounds CEP701, AC220, and sunitinib identified in Table 4 inhibit the PDGFR family of RTKs. As shown in Figure 43B, phospho-RET levels are elevated in injured muscles, which compares the fold change of phospho-RET in both intact contralateral tibial anterior (TA) muscles to that observed 2 days after cardiotoxin injury. As shown in Figure 43B, an approximately 8-fold increase in phospho-RET was observed by ELISA 2 days after cardiotoxin injury versus intact contralateral TA muscles.

[0412] As shown in Figures 44A-44B, satellite cells express RET in vitro, and as shown in Figures 45A-45D, CEP701 treatment inhibits RET phosphorylation in vitro.

[0413] Figure 46 shows the results of a study evaluating the effect of in vitro deletion of RET. Using a conditional RET mutant and reporter, we were able to determine that the RET promoter is active in at least 25% of satellite cells (Figure 47) and that RET knockout cells grow better in vitro than wild-type cells (Figure 48). Figure 49 demonstrates the fold change relative to controls for untreated FLT3 and RET knockout cells.

[0414] Example 3 This example includes a list of primary hits from a screen designed to identify compounds that promote satellite cell proliferation in vitro. Satellite cells are skeletal muscle stem cells responsible for postnatal muscle growth and regeneration. Compounds that proliferate satellite cells in vitro have important relevance for muscle regeneration because they have the potential to proliferate cells that are equally effective in vivo and can be transplanted for cell replacement therapy. The four compound libraries tested, LINCS1, 2, 3, and 4, were provided to us by the Sorger lab at Harvard Medical School. The Sorger lab is a member of the Library of Integrated Network-Based Cellular Signatures (LINCS) Consortium, an NIH initiative that aims to generate public data for further investigation of therapeutically relevant cellular pathways. LINCS1, 2, and 4 contain kinase inhibitors, while LINCS3 contains epigenetic modifiers. The results of the screen are shown in Table 5.

[0415] Figure 50 identifies small molecules that promote satellite cell proliferation screening in this example. (A) Diagram of chemical screening experiment outlining FACS isolation of satellite cells and compound library treatment. (B) Representative dose-response curves from 4 of the top 10 compounds. The top 10 compounds were selected based on maximum fold change in cell proliferation relative to vehicle control. Proliferation was assessed by high content imaging using Hoechst33342 as a cell marker. (C) Representative fluorescent images of FACS-sorted satellite cells from Tg:Pax7-nGFP mice, cultured on 96w plates for 4 days and treated with vehicle, compound or positive control (Jak3 ​​inhibitor 6). The optimal treatment concentration for each compound was determined by dose response; 3uM for XMD8-92, 5uM for SB23906, 800nM for XMD11-50, and 400nM for vorinostat. Hoechst33342 was used as a cell marker. Scale bar 100 um. (D) Fold change over vehicle control for several compounds that promote satellite cell expansion. [Table 5]

[0416] material and method Satellite cell isolation and chemical screening. Satellite cells were isolated from intact limb muscles and prepared for fluorescence-activated cell sorting (FACS) as previously described (Rocheteau et al., 2012). FACS was performed on a Beckman Coulter MoFlo Legacy (Beckman Coulter) at the Bauer Flow Cytometry Core Facility at Harvard University. Satellite cells were sorted into Eppendorf tubes and manually plated into 96-well plates (Greiner). Plates were pre-coated with 10ug / uL laminin diluted in PBS for 4 hours at 37°C. Sorted cells were cultured on StemSpan SFEM supplemented with 0.05ng / mL basic fibroblast growth factor (bFGF, Life Technologies). II basal medium (Stem Cell Technologies). Wells for positive controls were treated with 600 nM Jak3 inhibitor IV (EMD Millipore). For screening and dose-response follow-up, cells were plated at 150 cells / well.

[0417] Compound addition The day the cells were plated was considered day 0. Compounds were added on day 1. For screening and dose-response follow-up, cells were incubated without changing medium until day 4, when plates were prepared for imaging. All compounds were suspended in dimethyl sulfoxide (DMSO, Sigma Aldrich); 0.1% DMSO was added as a negative control in all assays. [ka]

[0418] All patents and other publications identified in the specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. Any statement as to the date or representation as to the content of these documents is based on the information available to the applicant and does not constitute any admission as to the accuracy of the dates or contents of these documents.

[0419] While preferred embodiments have been shown and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention, and are therefore deemed to be within the scope of the invention as defined in the following claims. Moreover, to the extent not already indicated, it will be understood by those skilled in the art that any one of the various embodiments described and illustrated herein can be further modified to incorporate features shown in any of the other embodiments disclosed herein. Examples of embodiments of the present invention include the following: (Item 1) A method for increasing satellite cell proliferation, comprising contacting satellite cells with a compound selected from the group consisting of a kinase inhibitor, a G protein-coupled receptor (GPCR) modulator, a histone deacetylase (HDAC) modulator, an epigenetic modifier, a hedgehog signaling pathway modulator, a neuropeptide, a dopamine receptor modulator, a serotonin receptor modulator, a histamine receptor modulator, an adenosine receptor agonist, an ionophore, an ion channel modulator, a gamma-secretase modulator, a corticosteroid, and any combination thereof. (Item 2) The method of claim 1, wherein the compound is selected from the group consisting of small organic or inorganic molecules; saccharin; oligosaccharides; polysaccharides; peptides, proteins, peptide analogs and derivatives; antibodies, antibody fragments, peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; extracts made from biological materials; naturally occurring or synthetic compositions; and any combination thereof. (Item 3) 3. The method of any of items 1 to 2, wherein the compound is one or more of a B-Raf inhibitor, a JAK3 inhibitor, a p38MAPK inhibitor, a C-Raf1 inhibitor, an Akt inhibitor, an ERK inhibitor, a BMK1 / ERK5 inhibitor, a p38MAPK inhibitor, a RTK inhibitor, an ERK5 inhibitor, a Bcr-Abl inhibitor, a RhoK inhibitor, a p38 inhibitor, a p110 inhibitor, a FAK inhibitor, an ATP-competitive JNK inhibitor, a MELK inhibitor, an inhibitor of a pathway identified in Table 5, an Flt3 kinase inhibitor, a PDGFR / EGFR inhibitor, a Bcr-abl inhibitor, a Jak3 inhibitor, an SRC kinase inhibitor, an HDAC1 modulator, an HDA31 modulator, an HDAC6 modulator, a BRD2 modulator, an EGLN1 modulator, or a derivative, salt, metabolite, prodrug, or stereoisomer thereof. (Item 4) 4. The method according to any of items 1 to 3, wherein the compound is selected from the group consisting of protein kinase inhibitors and receptor kinase inhibitors. (Item 5) The compound is [ka] [ka] BAY-439006 (i.e., sorafenib; HMSL10008-101-1); HG-6-64-01 (i.e., HMSL10017-101-1); HKI-272 (i.e., neratinib; HMSL10018-101-1); KIN001-055 (i.e., HY-11067; HMSL10033-101-1); SB239063 (i.e., HMSL10036-101-1); KIN001-242 (i.e., HMSL10044-104-1); SB590885 (i.e., GSK2118436; HMSL10046 -101-1); AZ-628 (i.e., HMSL10050-101-1); MK2206 (i.e., HMSL10057-102-1); XMD11-50 (i.e., LRRK2-in-1; HMSL10086-101-1); XMD8-92 (i.e., HMSL10094-101-1); BIRB796; Dramapimod (i.e., HMSL10169-101-1); Sunitinib Malate (i.e., SU11248; Stent; HMSL10175-106-1); GDC-0879 (i.e., HMSL10181-101-1 );XMD8-85 (i.e., HMSL10093-101-1);AMN-107 (i.e., nilotinib; HMSL10099-101-1);Y39983 (i.e., HMSL10149-102-1);SB203580 (i.e., RWJ64809;PB203580;HMSL10167-101-1);VX-745 (i.e., HMSL10168-101-1);pseudoXL765 (i.e., HMSL10173-101-1);Y-27632 (i.e., HMSL10176-101-1);PH-797804 ( i.e., HMSL10439-101); VX-702 (i.e., HMSL10440-101); NG25 (i.e., HMSL10419-101); SB202190 (i.e., HMSL10441-101); BI-D1870 (i.e., HMSL10423-101); BIX02565 (i.e., HMSL10434-101); URMC-099 (i.e., HMSL10453-101); staurosporine aglycone (i.e., K252C; HMSL10454-101); ralimetinib (i.e., LY2228820;HMSL10438-103); BMX-IN-1 (i.e., HMSL10427-101); PF3644022 (i.e., HMSL10476-101); NVP-BHG712 (i.e., KIN001-265; HMSL10200-101); bosutinib (i.e., SKI-606; HMSL10189-101); NVP-TAE226 (i.e., CHIR-265; HMSL10207-101); RAD00 1 (i.e., everolimus; HMSL10235-101); CC-401 (i.e., HMSL10185-101); CGP74514A (i.e., HMSL10355-101); KIN001-269 (i.e., HMSL10195-101); RAF265 (i.e., HMSL10206-101); OTSSP167 (i.e., HMSL10337-102); dorsomorphin (i.e., Compound C; BML275; HMSL10399-102; losmapimod (i.e., GSK-AHAB; SB856553; GW856553X; HMSL10402-101); AZD5363 (i.e., HMSL10370-101); RO31-8220 (i.e., bisindolylmaleimide IX; HMSL10407-103); sotrastaurin (i.e., AEB071; HMSL10408-101); TAK-632 (i.e., HMSL10409-101); FRAX597 (i.e., HMSL10400-101); GW2580 (i.e., HMSL10401-101); alisertib (i.e., MLN8237; HMSL 10391-101), (+)-JQ1; S)-JQ1; belinostat (i.e., PXD101); MS-275 (i.e., entinostat; MS-27-275); vorinostat (i.e., suberoylanilide hydroxamic acid (SAHA); Zolinza); mocetinostat (i.e., MGCD0103); I-BET (i.e., GSK525762A); SB939 (i.e., prasinostat); PFI-1); rosilinostat (i.e., ACY-1215); I-BET151 (i.e., GSK1210151A); IOX2; and any combination thereof. (Item 6) 6. The method of any of items 1 to 5, wherein the compound is contacted with the satellite cells at a concentration of about 0.01 nM to about 100 μM. (Item 7) 7. The method of any of items 1 to 6, wherein the contacting step is for at least 1 hour. (Item 8) 8. The method according to any of items 1 to 7, wherein the contacting step lasts for 1 to 7 days. (Item 9) 9. The method of any of items 1 to 8, wherein the contacting is in vitro. (Item 10) 10. The method of any of items 1 to 9, wherein said contacting is ex vivo. (Item 11) 11. The method of any of items 1 to 10, wherein the contacting is in vivo. (Item 12) 12. The method of claim 11, wherein the in vivo contacting is in a mammal. (Item 13) 13. The method according to item 11 or 12, wherein the in vivo contacting is in a human. (Item 14) 14. The method of any of items 11 to 13, wherein the in vivo contacting is in a subject, and the subject is in need of treatment for damaged muscle tissue. (Item 15) 15. The method of claim 14, wherein the damaged muscle tissue is the result of physical injury or accident, disease, infection, abuse, loss of blood circulation, or muscle atrophy or wasting. (Item 16) 16. The method according to item 14 or 15, wherein the damaged muscle tissue is dystrophic muscle or aging muscle. (Item 17) 17. The method according to any of items 14 to 16, wherein the damaged muscle tissue is a result of muscle atrophy / wasting. (Item 18) 1. A method for repairing or regenerating muscle in a subject, comprising administering to the subject a therapeutically effective amount of a compound, wherein the subject has damaged muscle tissue, and the compound is selected from the group consisting of a kinase inhibitor, a G protein-coupled receptor (GPCR) modulator, a histone deacetylase (HDAC) modulator, an epigenetic modifier, a hedgehog signaling pathway modulator, a neuropeptide, a dopamine receptor modulator, a serotonin receptor modulator, a histamine receptor modulator, an ionophore, an ion channel modulator, a gamma-secretase modulator, and any combination thereof. (Item 19) 20. The method of claim 18, wherein the damaged muscle tissue is the result of physical injury or accident, disease, infection, abuse, loss of blood circulation, or muscle atrophy or wasting. (Item 20) 20. The method according to any of items 18 to 19, wherein the damaged muscle tissue is dystrophic muscle or aging muscle. (Item 21) 21. The method according to any of items 18 to 20, wherein the damaged muscle tissue is a result of muscle atrophy / wasting. (Item 22) 22. The method according to any of items 18 to 21, wherein the subject is a mammal. (Item 23) 23. The method of any of items 18 to 22, wherein the subject is a human. (Item 24) 24. The method of any of items 18 to 23, wherein the compound is co-administered with a therapeutic agent. (Item 25) 25. The method of claim 24, wherein the compound and the therapeutic agent are administered in the same formulation. (Item 26) 26. The method according to any of items 18 to 25, wherein the compound is administered at a dosage of 1 μg / kg to 150 mg / kg. (Item 27) 27. The method of any of items 18 to 26, wherein the administering step is by injection, infusion, instillation, inhalation, or ingestion. (Item 28) 29. The method according to any one of claims 18 to 27, wherein the administering step is once a day. 29. The method of any of items 18 to 28, further comprising the step of diagnosing the subject for muscle damage or muscle atrophy / wasting prior to treating the subject for muscle repair or regeneration. (Item 30) The compound is [ka] [ka] BAY-439006 (i.e., sorafenib; HMSL10008-101-1); HG-6-64-01 (i.e., HMSL10017-101-1); HKI-272 (i.e., neratinib; HMSL10018-101-1); KIN001-055 (i.e., HY-11067; HMSL10033-101-1); SB239063 (i.e., HMSL10036-101-1); KIN001-242 (i.e., HMSL10044-104-1); SB590885 (i.e., GSK2118436; HMSL10046 -101-1); AZ-628 (i.e., HMSL10050-101-1); MK2206 (i.e., HMSL10057-102-1); XMD11-50 (i.e., LRRK2-in-1; HMSL10086-101-1); XMD8-92 (i.e., HMSL10094-101-1); BIRB796; Dramapimod (i.e., HMSL10169-101-1); Sunitinib Malate (i.e., SU11248; Stent; HMSL10175-106-1); GDC-0879 (i.e., HMSL10181-101-1 );XMD8-85 (i.e., HMSL10093-101-1);AMN-107 (i.e., nilotinib; HMSL10099-101-1);Y39983 (i.e., HMSL10149-102-1);SB203580 (i.e., RWJ64809;PB203580;HMSL10167-101-1);VX-745 (i.e., HMSL10168-101-1);pseudoXL765 (i.e., HMSL10173-101-1);Y-27632 (i.e., HMSL10176-101-1);PH-797804 ( i.e., HMSL10439-101); VX-702 (i.e., HMSL10440-101); NG25 (i.e., HMSL10419-101); SB202190 (i.e., HMSL10441-101); BI-D1870 (i.e., HMSL10423-101); BIX02565 (i.e., HMSL10434-101); URMC-099 (i.e., HMSL10453-101); staurosporine aglycone (i.e., K252C; HMSL10454-101); ralimetinib (i.e., LY2228820;HMSL10438-103); BMX-IN-1 (i.e., HMSL10427-101); PF3644022 (i.e., HMSL10476-101); NVP-BHG712 (i.e., KIN001-265; HMSL10200-101); bosutinib (i.e., SKI-606; HMSL10189-101); NVP-TAE226 (i.e., CHIR-265; HMSL10207-101); RAD00 1 (i.e., everolimus; HMSL10235-101); CC-401 (i.e., HMSL10185-101); CGP74514A (i.e., HMSL10355-101); KIN001-269 (i.e., HMSL10195-101); RAF265 (i.e., HMSL10206-101); OTSSP167 (i.e., HMSL10337-102); dorsomorphin (i.e., Compound C; BML275; HMSL10399-102; losmapimod (i.e., GSK-AHAB; SB856553; GW856553X; HMSL10402-101); AZD5363 (i.e., HMSL10370-101); RO31-8220 (i.e., bisindolylmaleimide IX; HMSL10407-103); sotrastaurin (i.e., AEB071; HMSL10408-101); TAK-632 (i.e., HMSL10409-101); FRAX597 (i.e., HMSL10400-101); GW2580 (i.e., HMSL10401-101); alisertib (i.e., MLN8237; HMSL10402-101); 0391-101), (+)-JQ1; S)-JQ1; belinostat (i.e., PXD101); MS-275 (i.e., entinostat; MS-27-275); vorinostat (i.e., suberoylanilide hydroxamic acid (SAHA); Zolinza); mocetinostat (i.e., MGCD0103); I-BET (i.e., GSK525762A); SB939 (i.e., prasinostat); PFI-1); rosilinostat (i.e., ACY-1215); I-BET151 (i.e., GSK1210151A); IOX2; and any combination thereof. (Item 31) A high throughput assay for screening compounds that induce, stimulate, enhance or increase satellite cell proliferation, comprising: (a) contacting satellite cells with a test compound; (b) assessing satellite cell proliferation; (c) selecting a compound that induces, stimulates, enhances, or increases satellite cell replication or growth; A high throughput assay comprising: (Item 32) 32. The assay of claim 31, wherein the step of assessing satellite cell proliferation comprises detecting a cell marker. (Item 33) 33. The assay of item 32, wherein the cell marker is selected from the group consisting of CXCR4, β1-integrin, Sca-1, Mac-1, CD45, PAX7, PAX3, Myf5, MyoD, desmin, and any combination thereof. (Item 34) 34. The assay of any of items 31 to 33, wherein the test compound has a concentration in the range of 0.1 nM to 1000 mM. (Item 35) 35. The assay according to any of items 31 to 34, wherein the assay is carried out at a temperature in the range of about 15° C. to about 55° C. (Item 36) 36. The assay of any of items 31 to 35, wherein the test compound is contacted with the pancreatic cells for a period of from 1 hour to 7 days. (Item 37) 37. The assay of any of paragraphs 31 to 36, wherein the test compound increases satellite cell proliferation by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more relative to an untreated control. (Item 38) 38. The assay according to any of items 31 to 37, wherein the satellite cells are isolated from a mammal. (Item 39) 39. The assay of any of items 31 to 38, wherein the satellite cells are isolated from a subject, the subject being in need of treatment for damaged muscle tissue. (Item 40) 40. The method of claim 39, wherein the damaged muscle tissue is the result of physical injury or accident, disease, infection, abuse, loss of blood circulation, or muscle atrophy or wasting. (Item 41) 41. The method according to item 39 or 40, wherein the damaged muscle tissue is dystrophic muscle or aging muscle. (Item 42) 42. The method according to any of items 39 to 41, wherein the damaged muscle tissue is a result of muscle atrophy / wasting.

Claims

[Claim 1] The invention described in the specification.

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