ALK5 inhibitors as inducers of skeletal muscle hypertrophy

Abstract

The present invention relates to ALK5 inhibitors and their use as inducers of skeletal muscle hypertrophy, and to promote skeletal muscle regeneration, prevent skeletal muscle atrophy, or in the treatment or prevention of diseases or injuries that result in skeletal muscle tissue loss and / or muscle weakness. The present invention further relates to the non-therapeutic use of such compounds to increase muscle mass, strength, and / or performance in a subject. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates to therapeutic strategies for inducing skeletal muscle hypertrophy, preventing atrophy, or treating or preventing diseases or injuries that result in skeletal muscle tissue loss and / or muscle wasting. The present invention also relates to non-therapeutic uses of skeletal muscle hypertrophy inducers. [Background technology]

[0002] Muscle wasting and weakness can result from a large group of disease conditions and conditions, including metabolic, neurological, muscular diseases, acute or chronic illnesses (cachexia), aging, inactivity, starvation, and even intoxication. During the last 15 years, extensive research has led to a deeper understanding of the signaling pathways that are believed to be involved in muscle mass loss. However, currently, there is a paucity of therapeutic strategies that directly target muscle.

[0003] Muscle loss can occur especially with age and is a component of the frailty syndrome. This degenerative loss, termed "sarcopenia," leads to direct muscle atrophy and increases the risk of subsequent ill health, including falls, disability, hospitalization, and death. With an increasing elderly population, sarcopenia has become an increasingly global health concern, and there has been great interest in developing methods to counteract the effects of sarcopenia and reduce age-related decline and disability. Potential therapeutic interventions for sarcopenia may include physical activity and nutritional supplementation, but pharmacological interventions currently have limited efficacy.

[0004] Muscle weakness can also result directly from neuromuscular disorders such as myopathy, neuromuscular junction diseases, or motor neuron diseases.

[0005] Myopathies are neuromuscular disorders whose primary symptom is muscle weakness due to dysfunction of skeletal muscle fibers. Myopathies can be hereditary or acquired and include, for example, muscular dystrophies, metabolic myopathies such as mitochondrial myopathies or drug-induced myopathies, and autoimmune myopathies such as dermatomyositis, polymyositis or inclusion body myositis.

[0006] Among myopathies, muscular dystrophies represent a large group that cause progressive degeneration of muscle fibers resulting in muscle mass loss. Mutations in more than 30 genes that cause muscular dystrophies have been identified. Duchenne Muscular Dystrophy (DMD) is the most common form of muscular dystrophy, with an incidence of approximately 1 in 3500 males worldwide.

[0007] Treatment of neuromuscular disorders depends on the disease and the specific cause, but currently there are no specific treatments that halt or reverse any form of muscular dystrophy. Exercise and nutritional interventions have been shown to be beneficial in slowing the rate of muscle atrophy in some muscle-wasting conditions, but in many cases the wasting process cannot be halted.

[0008] Therefore, there is a strong need for novel therapeutic options that can effectively prevent muscle atrophy, promote muscle growth, increase muscle mass, and ultimately improve patients' quality of life. Summary of the Invention [Problem to be solved by the invention]

[0009] It is an object of the present invention to provide novel therapeutic strategies for inducing skeletal muscle hypertrophy or preventing muscle atrophy, promoting skeletal muscle regeneration, and treating or preventing skeletal muscle wasting. [Means for solving the problem]

[0010] Thus, the present invention relates to an ALK5 inhibitor, optionally in combination with a glucocorticoid, for use in the treatment or prevention of a disease or disorder that results in skeletal muscle tissue loss and / or muscle wasting.

[0011] The present invention also relates to pharmaceutical compositions comprising an ALK5 inhibitor, a glucocorticoid and a pharma- ceutically acceptable excipient, particularly for use in the treatment or prevention of diseases or disorders that result in skeletal muscle tissue loss and / or muscle wasting.

[0012] The present invention further relates to a kit comprising an ALK5 inhibitor and a glucocorticoid, preferably as a combined preparation to be used together, separately or sequentially, for use in the treatment of a disease or disorder that results in skeletal muscle tissue loss and / or muscle wasting.

[0013] The ALK5 inhibitor may be selected from the group consisting of RepSox, SB525334, SD208, SB505124, R268712, LY364947, GW788388, D4476, and A83-01, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof.Preferably, the ALK5 inhibitor is selected from the group consisting of RepSox, SB525334, SD208, SB505124, R268712, GW788388, D4476, and A83-01, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof. More preferably, the ALK5 inhibitor is selected from the group consisting of RepSox, SB505124, R268712, GW788388, D4476, and A83-01, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof. Even more preferably, the ALK5 inhibitor is selected from the group consisting of RepSox, and GW788388, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof. Even more preferably, the ALK5 inhibitor is RepSox, or any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof.

[0014] The glucocorticoid may be selected from the group consisting of prednisolone, prednisone, cortisone, corticosterone, hydrocortisone, fludrocortisone, fluorometholone, deoxycorticosterone, ketoconazole, budesonide, methylprednisolone, fluticasone, clobetasol, clobetasone, fluocinolone, flunisolide, mometasone, prednicarbate, amcinonide, diflucortolone, beclomethasone, betamethasone, dexamethasone, triamcinolone, and deflazacort, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof, preferably from the group consisting of prednisolone, prednisone, and deflazacort, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof. Preferably, the glucocorticoid is selected from the group consisting of prednisolone, prednisone, cortisone, corticosterone, fludrocortisone, fluorometholone, deoxycorticosterone, ketoconazole, budesonide, methylprednisolone, fluticasone, clobetasol, clobetasone, fluocinolone, flunisolide, mometasone, prednicarbate, amcinonide, diflucortolone, beclomethasone, betamethasone, triamcinolone, and deflazacort, and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof. More preferably, the glucocorticoid is prednisolone or prednisone, or any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof. Even more preferably, the glucocorticoid is prednisolone, or any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0015] Preferably, the ALK5 inhibitor, pharmaceutical composition, or kit of the present invention is not used in combination with an oxytocin receptor agonist.

[0016] The disease or disorder that leads to skeletal muscle tissue loss and / or muscle weakness can be selected from sarcopenia, cachexia, neuromuscular disease, disuse muscle atrophy, anorexic starvation-induced atrophy, and muscle damage including acute muscle damage or overuse muscle damage, preferably cachexia, neuromuscular disease, disuse muscle atrophy, anorexic starvation-induced atrophy, and muscle damage including acute muscle damage or overuse muscle damage. Preferably, the disease or disorder that leads to skeletal muscle tissue loss and / or muscle weakness is muscular dystrophy. More preferably, the disease or disorder that leads to skeletal muscle tissue loss and / or muscle weakness is muscular dystrophy selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, distal muscular dystrophy such as Miyoshi muscular dystrophy, and limb-girdle muscular dystrophy. Even more preferably, the disease or disorder resulting in skeletal muscle tissue loss and / or muscle weakness is Duchenne muscular dystrophy.

[0017] Furthermore, the disease or disorder resulting in skeletal muscle tissue loss and / or muscle weakness may be a motor neuron disease, preferably selected from the group consisting of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy, more preferably spinal muscular atrophy.

[0018] Alternatively, the disease or disorder resulting in skeletal muscle tissue loss and / or muscle weakness may be a neuromuscular junction disease, preferably selected from the group consisting of myasthenia gravis, autoimmune neuromyotonia (Isaacs syndrome), Lambert-Eaton myasthenic syndrome, and neuromuscular junction diseases resulting from a form of toxin that affects neuromuscular junction function, such as snake venom or neurotoxin.

[0019] The ALK5 inhibitor, pharmaceutical composition, or kit of the present invention may further comprise other active ingredients, preferably anti-inflammatory agents, anabolic agents, anti-neoplastic agents, antibiotics, local anesthetics, anabolic / androgenic steroids, glucocorticoids, appetite stimulants, cytokine modulators, angiotensin and β-adrenoreceptor inhibitors, NHE-1 inhibitors, anti-fibrotic agents, phosphodiesterase 5 (PDE5) inhibitors, dehydroepiandrosterone, vitamin D, ursolic acid, omega 3 acid (omega 3 ) The other active ingredient may be used in combination with an active ingredient selected from the group consisting of erythropoietin, angiotensin-converting enzyme (ACE) inhibitors, proteasome inhibitors, cyclophilin D inhibitors, PGC-1α (alpha) pathway modulators, myostatin and activin A antagonists, ghrelin agonists, β2-adrenoreceptor agonists, creatine supplements, antifibrotic agents, muscle ischemia therapeutics, mutation-specific therapeutics, therapeutic nonsense suppressors, utrophin upregulators, gene replacement therapeutics or cell therapeutics using muscle progenitor cells or stem cells. More preferably, the other active ingredient is a gene replacement therapeutic. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows RepSox activity in AchR cluster formation. Effects of IGF-1 and RepSox on AchR cluster formation in myotubes differentiated for 8 days. n=3-6 wells. *p≦0.05 vs. control myotubes by Mann-Whitney analysis. [Diagram 2] Figure 2 shows RepSox activity in calcium flux. A: Mean peak amplitude of Fluo-4 fluorescence in 20 μM ACh-stimulated myotubes differentiated for 8 days (7 of which in the presence of IGF-1 or RepSox). n=2-5 wells, ****p≦0.0001 vs. control myotubes by Mann-Whitney analysis. B: Representative pseudocolor images from calcium flux experiments showing levels of intracellular free Ca2+ in myotubes before (0 s) and after (3-19 s) stimulation with ACh. [Diagram 3]Figure 3 shows RepSox activity in contraction. Treatment of differentiating myotubes for 96 hours with 2 μM RepSox significantly increased the percentage of myotubes exhibiting robust contractions, detected as myotubes contracting from micropatterns when stimulated with 100 μM ACh. n=4 wells. *p≦0.05 vs. control myotubes by Mann-Whitney analysis. [Figure 4] FIG. 4 shows the fusion index obtained with prednisolone, RepSox, and the combination of prednisolone and RepSox. [Diagram 5] FIG. 5 shows the mean myotube area obtained with prednisolone, RepSox, and the combination of prednisolone and RepSox. [Figure 6] FIG. 6 shows the fusion index obtained with prednisolone, GW788388, and the combination of prednisolone and GW788388. [Figure 7] FIG. 7 shows the mean myotube area obtained with prednisolone, GW788388, and the combination of prednisolone and GW788388. [Figure 8] Figure 8 shows the response of myotubes from donor A (A) and donor 5 (B) to repeated twitch electrical stimulation. Fluorescence intensity (F) was normalized to the average intensity at the initial time point (F / F0). [Figure 9] Figure 9 shows the tetanic response of myotubes from donor 5 (A). Fluorescence intensity (F) was normalized to the average intensity at the initial time point (F / F0). The tetanic response of RepSox-treated myotubes increases with RepSox concentration, resulting in significant differences from control myotubes. *p≦0.05 and ***p≦0.001 vs. control myotubes by Mann-Whitney analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Activin receptor-like kinase (ALK-5), also known as transforming growth factor beta receptor 1 (Gene ID: 7046), is a serine / threonine protein kinase that, upon binding to TGF-β, forms a heterocomplex with the type II TGF-β receptor and transmits the TGF-β signal from the cell surface to the cytoplasm. This protein is involved in the regulation of many physiological and pathological processes, including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression, and carcinogenesis.

[0022] With a solid knowledge in micropatterning technology and an own physiological human skeletal muscle model (MyoScreen, Saito) that allows sufficient maturation of human primary myoblasts and obtains myotubes with high levels of striations, high fusion index with aligned nuclei, and low morphological variability, the inventors here surprisingly showed that ALK5 inhibitors can induce skeletal muscle hypertrophy not only in healthy donor myotubes but also in DMD patient myotubes. The inventors showed that ALK5 inhibitors can increase myotube differentiation and size, provide myotubes with strong contractile ability, and prevent muscle atrophy. The inventors further showed that the combination of ALK5 inhibitors and glucocorticoids act together on myotube differentiation and size.

[0023] Thus, in a first aspect, the present invention relates to an ALK5 inhibitor for use as a skeletal muscle hypertrophy inducer for promoting skeletal muscle regeneration, preventing skeletal muscle atrophy, or in the treatment or prevention of diseases or injuries that result in skeletal muscle tissue loss and / or muscle wasting.

[0024] Thus, the present invention relates to ALK5 inhibitors for use as skeletal muscle hypertrophy inducers.

[0025] As used herein, the term "ALK5 inhibitor" refers to a molecule that inhibits or reduces the activity of ALK5, i.e., inhibits or reduces the transmission of TGF-β signals from the cell surface to the cytoplasm. In particular, ALK5 inhibitors may act in inhibiting or reducing TGF-β binding, in inhibiting or reducing ALK5 expression, or in inhibiting or reducing ALK5 kinase activity. ALK5 inhibitors that induce inhibition or reduction of TGF-β binding can bind to TGF-β and / or ALK5 to prevent ligand-receptor interaction. The ability of a compound to inhibit ALK5 can be assessed by any method known to one of skill in the art, such as an ALK5 binding assay, a TGF-β cell assay, or an ALK5 kinase assay as described in De Gouville et al. (Br J Pharmacol. 2005 May;145(2):166-77), Huynh et al. (Journal of biomolecular screening 16(7);2011) or Ma et al. (Expert Opin Drug Discov. 2008 June ;3(6):607-621).

[0026] ALK5 inhibitors can be covalently or non-covalently bound to ALK5 and alter its activity by steric hindrance or modification. The inhibitors can be, for example, small molecules, substrate-like peptide inhibitors (i.e., bait substrates), aptamers or antibodies against ALK5. The inhibition can also be by reducing or suppressing TGFBR1 gene expression, for example, by using specific RNAi, antisense, ribozymes, or knockout or gene editing tools such as CRISPR / Cas9, TALEN, or Zn finger nucleases.

[0027] Alternatively, the ALK5 inhibitor can bind covalently or non-covalently to TGF-β and inhibit its interaction with ALK5. The inhibitor can be, for example, a dominant-negative mutant of ALK5, i.e., a mutant form of ALK5 that can bind to TGF-β but cannot transmit a signal, or an aptamer or antibody against TGF-β. Examples of such dominant-negative mutants of ALK5 include, but are not limited to, those described by Schniewind et al. (Oncogene. 2007 Jul 19;26(33):4850-62), monoclonal antibodies LY2382770 against TGF-β, and fresolimumab.

[0028] Preferably, the ALK5 inhibitor is selected from the group consisting of a small molecule, a nucleic acid molecule that specifically interferes with ALK5 expression, a bait substrate, a dominant negative mutant of ALK5, and an aptamer or antibody against ALK5. More preferably, the ALK5 inhibitor is selected from the group consisting of a small molecule, preferably a small molecule that inhibits TGF-β binding or ALK5 kinase activity, a nucleic acid molecule that specifically interferes with ALK5 expression, and a bait substrate. Even more preferably, the ALK5 inhibitor is selected from the group consisting of a small molecule and a nucleic acid molecule that specifically interferes with ALK5 expression. In the most preferred embodiment, the ALK5 inhibitor is a small molecule, preferably a small molecule that inhibits TGF-β binding or ALK5 kinase activity, more preferably a small molecule that inhibits ALK5 kinase activity.

[0029] In an embodiment, the ALK5 inhibitor is a nucleic acid molecule that specifically interferes with ALK5 expression. As used herein, the term "nucleic acid molecule" includes, but is not limited to, RNAi, antisense, and ribozyme molecules. In the present invention, the "nucleic acid molecule that specifically interferes with ALK5 expression" is a nucleic acid molecule that can reduce or suppress TGFBR1 gene expression in a specific manner. As used herein, the term "RNAi" refers to any RNA that can downregulate the expression of a target protein. The term includes small interfering RNA (siRNA), double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), microRNA (miRNA), and short hairpin RNA (shRNA) molecules. Methods for designing, making, and using such interfering nucleic acid molecules are known to those skilled in the art. Examples of such interfering nucleic acid molecules include, but are not limited to, siRNAs targeting TGFBR1 described by Wang et al. (J Invest Dermatol. 2014 Jul;134(7):2016-2025), Cheng et al. (Mol Pharm. 2009 ;6(3):772-779), or Curado et al. (Mol Cell Biol. 2014 Dec;34(24):4389-403).

[0030] In other embodiments, ALK5 inhibitor is bait substrate.As used herein, the term "bait substrate" refers to substrate-like peptide inhibitor, i.e., peptide that can bind to ALK5 and inhibit the interaction of ALK5 with its substrate, particularly TGF-β.The example of such bait substrate includes, but is not limited to, TGF-β latent binding peptide (LAP) (Bottinger et al. Proc Natl Acad Sci US A. 1996 Jun 11;93(12):5877-82), which forms a complex with TGF-β and inhibits its interaction with ALK5.

[0031] In other embodiments, the ALK5 inhibitor is an antibody against ALK5 or against TGF-β, preferably an antibody against ALK5. The term "antibody" as used herein is used in the broadest sense, in particular monoclonal antibodies, polyclonal antibodies, antibody fragments, and derivatives thereof, so long as it specifically binds to a molecular target of interest. As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, generally a protein that includes an antigen-binding domain or a variable domain. Examples of antibody fragments include Fab, Fab', F(ab) and Fab'. 2 , F(ab') 2 , F(ab) 3 , Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (ScFv), dsFv, Fd (typically the VH and CH1 domains), and dAb (typically the VH domain) fragments, nanobodies, minibodies, diabodies, triabodies, tetrabodies, kappabodies, linear antibodies, and other antibody fragments that retain antigen-binding function (e.g., Holliger and Hudson, Nat Biotechnol. 2005 Sep;23(9):1126-36). Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant host cells (e.g., E. coli or phage). These techniques are known to those of skill in the art and are widely described in the literature. The term "antibody derivative" as used herein refers to an antibody provided herein, e.g., a full-length antibody or a fragment of an antibody, in which one or more amino acids have been chemically modified, e.g., by alkylation, pegylation, acylation, ester or amide formation, etc.

[0032] In another embodiment, the ALK5 inhibitor is an aptamer for ALK5. The aptamer may be a peptide aptamer or a nucleic acid aptamer. Peptide aptamers consist of a variable short peptide loop attached at both ends to a protein scaffold, such as the bacterial protein thioredoxin-A. Typically, the variable loop length consists of 10-20 amino acids. Peptide aptamers specific to a target of interest can be selected using any method known to those skilled in the art, such as the yeast two-hybrid system or phage display. Peptide aptamers can be produced by any method known to those skilled in the art, such as chemical synthesis or production in recombinant bacteria followed by purification. Nucleic acid aptamers are a group of small molecule nucleic acid ligands consisting of RNA or single-stranded DNA oligonucleotides, with high specificity and affinity for targets. Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology, which constructs nucleic acid aptamers specific to targets of interest, is known to those skilled in the art and can be used to obtain aptamers specific to specific molecular targets. Nucleic acid aptamers can be produced by any method known to those skilled in the art, such as chemical synthesis or in vitro transcription of RNA aptamers. Nucleic acid aptamers can be selected from the group consisting of DNA aptamers, RNA aptamers, XNA aptamers (nucleic acid aptamers containing xenonucleotides), and Spiegelmer® (composed entirely of non-natural L-ribonucleic acid backbones). Examples of anti-ALK5 aptamers include, but are not limited to, peptide aptamers that bind to the extracellular domain of ALK5, as described by Li et al. (Mol Biosyst. 2010 Dec;6(12):2392-402).

[0033] In a preferred embodiment, the ALK5 inhibitor is a small molecule that inhibits the activity of ALK5, preferably a small molecule that inhibits or reduces TGF-β binding to ALK5 or ALK5 kinase activity, more preferably a small molecule that inhibits or reduces ALK5 kinase activity.

[0034] As used herein, the term "small molecule" refers to small molecules that can be organic or inorganic compounds, typically less than 1000 Daltons. The small molecule can be derived from any known living organism, including but not limited to animals, plants, bacteria, fungi, and viruses, or can be a synthetic molecule.

[0035] Small molecules are [Table 1] JPEG0007653257000002.jpg138120 and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0036] Preferably, the small molecule is selected from the group consisting of RepSox, SB525334, SD208, SB505124, R268712, GW788388, D4476, and A83-01, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof.

[0037] More preferably, the small molecule is selected from the group consisting of RepSox, SB505124, R268712, GW788388, D4476, and A83-01, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof. Even more preferably, the small molecule is selected from the group consisting of RepSox, and GW788388, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof.

[0038] In a most preferred embodiment, the small molecule is RepSox, or any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0039] The inventors herein show that the combination of an ALK5 inhibitor and a glucocorticoid acts together on myotube differentiation and size.

[0040] Thus, in all aspects (uses, methods, compositions, etc.) of the present invention, ALK5 inhibitors can be used in combination with one or more glucocorticoids. In particular, ALK5 inhibitors can be used in combination with one or more glucocorticoids as skeletal muscle hypertrophy inducers to promote skeletal muscle regeneration, to prevent skeletal muscle atrophy, or in the treatment or prevention of diseases or injuries that result in skeletal muscle tissue loss and / or muscle wasting.

[0041] Thus, the present invention relates to ALK5 inhibitors for use as skeletal muscle hypertrophy inducers in combination with one or more glucocorticoids.

[0042] Glucocorticoids, or glucocorticosteroids, are corticosteroids that bind to the glucocorticoid receptor, which is where they exert their effects on cells and which distinguish them from mineralocorticoids and sex steroids. Glucocorticoids can be any glucocorticoid known to those skilled in the art, and preferably synthetic glucocorticoids.

[0043] Preferably, the glucocorticoid is selected from the group consisting of prednisolone, prednisone, cortisone, corticosterone, hydrocortisone, fludrocortisone, fluorometholone, deoxycorticosterone, ketoconazole, budesonide, methylprednisolone, fluticasone, clobetasol, clobetasone, fluocinolone, flunisolide, mometasone, prednicarbate, amcinonide, diflucortolone, beclomethasone, betamethasone, dexamethasone, triamcinolone, and deflazacort, and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0044] More preferably, the glucocorticoid is selected from the group consisting of prednisolone, prednisone, cortisone, corticosterone, fludrocortisone, fluorometholone, deoxycorticosterone, ketoconazole, budesonide, methylprednisolone, fluticasone, clobetasol, clobetasone, fluocinolone, flunisolide, mometasone, prednicarbate, amcinonide, diflucortolone, beclomethasone, betamethasone, triamcinolone, and deflazacort, and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0045] Even more preferably, the glucocorticoid is selected from the group consisting of prednisolone, prednisone, and deflazacort, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof.

[0046] In a most preferred embodiment, the glucocorticoid is prednisolone or prednisone, or any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0047] The ALK5 inhibitor and the glucocorticoid may be formulated and used in a single pharmaceutical composition or in separate pharmaceutical compositions.

[0048] They can be administered together, sequentially, or separately. As used herein, the term "together" refers to administration of a compound with less than 10 minutes between administration of a first compound and administration of another. As used herein, the term "sequentially" refers to administration of a compound with less than 1 hour between administration of a first compound and administration of another. As used herein, the term "separately" refers to administration of a compound with more than 1 hour between administration of a first compound and administration of another.

[0049] In some preferred embodiments, the ALK5 inhibitor is selected from the group consisting of RepSox, and GW788388, and any pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, and is used in combination with a glucocorticoid selected from the group consisting of prednisolone, prednisone, and deflazacort, and any pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. In some other preferred embodiments, the ALK5 inhibitor is selected from the group consisting of RepSox, and GW788388, and any pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof, and is used in combination with a glucocorticoid selected from the group consisting of prednisolone, and prednisone, and any pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof. In some other preferred embodiments, the ALK5 inhibitor is selected from the group consisting of RepSox, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof, and is used in combination with a glucocorticoid selected from the group consisting of prednisolone, and prednisone, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof.

[0050] The ALK5 inhibitor and the glucocorticoid can be used in the form of a pharmaceutically acceptable salt, hydrate, solvate, or prodrug, preferably in the form of a pharmaceutically acceptable salt, hydrate, or solvate, more preferably in the form of a pharmaceutically acceptable salt. In a preferred embodiment, all the ALK5 inhibitors and the glucocorticoids are used in the form of a pharmaceutically acceptable salt, hydrate, or solvate, more preferably in the form of a pharmaceutically acceptable salt.

[0051] Such pharma- ceutically acceptable salts, hydrates and solvates may, where appropriate, be formed in a manner known to those skilled in the art.

[0052] The term "pharmaceutically acceptable salt" refers to a salt that is non-toxic to a patient and suitable for maintaining the stability of a therapeutic agent and delivering the agent to a target cell or tissue. Pharmaceutically acceptable salts are well known in the art.

[0053] As used herein, the term "solvate" refers to a solvent addition form that contains a stoichiometric or non-stoichiometric amount of solvent. Some compounds form solvates because they tend to trap a certain molar ratio of solvent molecules in the crystalline solid state. When the solvent is water, the solvate formed is a hydrate. When the solvent is alcohol, the solvate formed is an alcoholate. A hydrate is a compound in which water is dissolved in H 2 It is formed by the combination of one of the substances that retains its molecular state as O with one or more water molecules, which combination can form one or more hydrates.

[0054] The ALK5 inhibitors and glucocorticoids can also be used in the form of prodrugs. Prodrugs are generally drug precursors that are converted to active or more active species by some process, such as conversion by metabolic pathways, after administration to an individual and subsequent absorption. Some prodrugs have chemical groups present on the prodrug that, for example, make them less active, increase their solubility, and / or improve safety characteristics over administration of the parent drug. In some embodiments, prodrugs are less likely to degrade in vivo and can exhibit a longer half-life than their parent drug. When the chemical group is cleaved and / or modified from the prodrug, an active drug is generated. Prodrugs can be effective in some situations because they are easier to administer than the parent drug. Prodrugs are bioavailable, for example, by oral administration, whereas the parent drug is not. In certain embodiments, prodrugs also have improved solubility in pharmaceutical compositions than the parent drug. A non-limiting example of a prodrug can be a compound as described in the present invention that, when administered, subsequently undergoes biotransformation in vivo to obtain a therapeutically effective concentration of the active agent. For further general examples, see "Design and Application of Prodrugs" in A Textbook of Drug Design and Development by Bundgaard (Krosgaard-Larsen and Bundgaard, Ed., 1991, Chapter 5, 113-191), which is incorporated herein by reference. Prodrugs can be prepared, for example, by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.

[0055] The present invention also relates to a pharmaceutical composition comprising an ALK5 inhibitor and a pharma- ceutically acceptable excipient, preferably for use as an agent for inducing skeletal muscle hypertrophy.

[0056] All of the embodiments described above for ALK5 inhibitors as skeletal muscle hypertrophy inducers are also encompassed in this aspect.

[0057] The pharmaceutical compositions of the present invention are formulated according to standard pharmaceutical standards known to those skilled in the art (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

[0058] Possible pharmaceutical compositions include those suitable for oral, mucosal (including nasal, rectal, or vaginal), topical (including transdermal, buccal, and sublingual), or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration. These formulations can use conventional excipients according to techniques known to those skilled in the art. Preferably, the pharmaceutical composition of the present invention is suitable for oral administration.

[0059] Compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions, which can be prepared immediately before use from solid or lyophilized forms. Adjuvants such as local anesthetics, preservatives, and buffers may be dissolved in the vehicle, and surfactants or wetting agents may be included in the composition to facilitate uniform dispersion of the active ingredient.

[0060] For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules, and liquid preparations such as syrups, elixirs, and concentrated drops. Non-toxic solid carriers or diluents can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonates, and the like. For compressed tablets, binders are also required, which are agents that give cohesive properties to the powdered material. For example, starch, gelatin, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also required in tablets to facilitate tablet disintegration. Disintegrants include starch, clay, cellulose, algins, gums, and cross-linked polymers. In addition, lubricants and glidants are also included in tablets to prevent tablet materials from adhering to the surface during the manufacturing process and to improve the flow properties of the powdered materials during manufacturing. Colloidal silicon dioxide is most commonly used as a lubricant, and compounds such as talc or stearic acid are most commonly used as lubricants.

[0061] For transdermal administration, the compositions can be formulated in the form of ointments, creams, or gels, and suitable penetrants or surfactants, such as dimethylsulfoxide, dimethylacetamide, and dimethylformamide, can be used to facilitate penetration.

[0062] For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any known suppository base by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycol (carbowax), polyethylene sorbitan monostearate, and mixtures thereof, as well as other suitable materials that modify the melting point or dissolution rate.

[0063] The pharmaceutical compositions of the present invention can be formulated to release the active agent substantially immediately upon administration, or at any desired time or period after administration.

[0064] The pharmaceutical compositions of the present invention can include one or more ALK5 inhibitors together with one or more pharma- ceutically acceptable excipients, which are selected according to the mode of administration as described above.

[0065] The pharmaceutical composition of the present invention may also contain one or more additional active compounds, such as, for example, anti-inflammatory agents, anabolic agents (e.g., growth hormone or insulin-like growth factor I), antineoplastic agents, antibiotics, local anesthetics, anabolic / androgenic steroids (e.g., testosterone), glucocorticoids, appetite stimulants (e.g., dronabinol), cytokine modulators (e.g., thalidomide), angiotensin and β-adrenoreceptor inhibitors, NHE-1 inhibitors (e.g., rimeporide), antifibrotic agents (e.g., losartan or lisinopril), phosphodiesterase 5 (PDE5) inhibitors (e.g., tadalafil or sildenafil), dehydroepiandrosterone, vitamin D, ursolic acid, omega 3 acid (omega 3 ), omega 3 hydroxylase (MEA) inhibitors (e.g., omega 3 hydroxylase ... acid), angiotensin converting enzyme (ACE) inhibitors, proteasome inhibitors, cyclophilin D inhibitors, PGC-1α (alpha) pathway modulators, myostatin and activin A antagonists, ghrelin agonists, β2-adrenoreceptor agonists, creatine supplements, anti-fibrotic agents such as losartan and lisinopril, muscle ischemia treatments such as tadalafil and sildenafil, mutation specific treatments such as exon skipping therapies (e.g. eteplirsen, a morpholino phosphorodiamidate antisense oligomer that targets mutations relevant to DMD cases), and therapeutic nonsense suppressors such as ataluren, and utrophin upregulators such as SMT-C1100.

[0066] As mentioned above, the pharmaceutical compositions of the present invention may contain one or more glucocorticoids or may be used in combination with one or more glucocorticoids.

[0067] In certain embodiments, pharmaceutical compositions herein comprise one or more ALK inhibitors and one or more glucocorticoids, preferably one ALK inhibitor and one glucocorticoid.

[0068] The present invention also relates to a kit comprising an ALK5 inhibitor and a glucocorticoid, preferably as a combined preparation, used together, separately or sequentially, for use as a skeletal muscle hypertrophy inducer. In particular, the kit can comprise a pharmaceutical composition comprising an ALK5 inhibitor, a pharma- ceutical acceptable excipient, and another pharmaceutical composition comprising a glucocorticoid and a pharma- ceutical acceptable excipient.

[0069] Preferably, the pharmaceutical composition or kit comprises an ALK5 inhibitor selected from the group consisting of RepSox, SB525334, SD208, SB505124, R268712, LY364947, GW788388, D4476, and A83-01, and any pharma- ceutically acceptable salts, hydrates, solvates, or prodrugs thereof, and prednisolone, prednisone, cortisone, corticosterone, hydrocortisone, fludrocortisone, fluorometholone, deoxycortisone, benzodiazepine, benzoic acid ... and a glucocorticoid selected from the group consisting of cicorticosterone, ketoconazole, budesonide, methylprednisolone, fluticasone, clobetasol, clobetasone, fluocinolone, flunisolide, mometasone, prednicarbate, amcinonide, diflucortolone, beclomethasone, betamethasone, dexamethasone, triamcinolone, and deflazacort, and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0070] More preferably, the pharmaceutical composition or kit comprises an ALK5 inhibitor selected from the group consisting of RepSox, and GW788388, and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof, and a glucocorticoid selected from the group consisting of prednisolone, prednisone, and deflazacort, and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof, preferably selected from the group consisting of prednisolone, and prednisone, and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0071] Even more preferably, the pharmaceutical composition or kit comprises an ALK5 inhibitor selected from the group consisting of RepSox and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof, and a glucocorticoid selected from the group consisting of prednisolone, and prednisone, and any pharma- ceutically acceptable salt, hydrate, solvate, or prodrug thereof.

[0072] In the experimental section, the inventors showed that ALK5 inhibitors can promote the differentiation of myoblasts into myotubes, increase the number and size of myotubes, and / or increase the fusion index, which reflects the ability of cells to regenerate.

[0073] Thus, the present invention relates to the ALK5 inhibitor, pharmaceutical composition, or kit of the present invention for use as a skeletal muscle hypertrophy inducer.

[0074] Skeletal muscle fibers are syncytia that develop from the successive fusion of myoblasts. The processes involve i) differentiation of myoblasts into myocytes, ii) fusion of myocytes to form nascent myotubes, and iii) further fusion of myocytes with nascent myotubes to form more mature myotubes. Thus, as used herein, the expression <skeletal muscle hypertrophy> refers to an increase in skeletal muscle mass characterized by an increase in the size of existing myofibers, and / or an increase in the number of myofibers, and / or an increase in the average number of nuclei per myotube, and / or an increase in the fusion index (number of nuclei in myotubes divided by the total number of nuclei in myoblasts and myotubes). Preferably, the expression <skeletal muscle hypertrophy> is indicated by an increase in the size of existing myofibers, and / or an increase in the number of myofibers, and / or an increase in the fusion index. As used herein, the terms "myotube" and "myofiber" are used interchangeably.

[0075] The present invention also relates to a method for inducing skeletal muscle hypertrophy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an ALK5 inhibitor or pharmaceutical composition of the present invention.

[0076] The therapeutically effective amount administered can be readily selected by one of skill in the art, and should be sufficient to increase skeletal muscle mass or strength in the subject.

[0077] As used herein, a subject is an animal, preferably a mammal, more preferably a human. Preferably, the subject is a patient suffering from muscle wasting or weakness due to a disease or disorder that results in skeletal muscle tissue loss and / or muscle weakness, such as the diseases or disorders described below.

[0078] The present invention further relates to the use of an ALK5 inhibitor or pharmaceutical composition of the present invention for the preparation of a medicament for inducing skeletal muscle hypertrophy, optionally in combination with one or more glucocorticoids.

[0079] As mentioned above, the ALK5 inhibitors or pharmaceutical compositions of the invention can be used in combination with one or more glucocorticoids.

[0080] The present invention also relates to an ALK5 inhibitor, or a pharmaceutical composition, or a kit of the present invention for use in preventing involuntary loss of skeletal muscle mass, preferably due to degeneration of muscle fibers, for use in promoting or stimulating an increase in skeletal muscle mass, for use in having sufficient skeletal muscle mass, and / or for use in increasing skeletal muscle mass and / or skeletal muscle strength.

[0081] The present invention also relates to a method for preventing involuntary loss of skeletal muscle mass, preferably due to degeneration of muscle fibers, promoting or stimulating skeletal muscle mass gain, having sufficient skeletal muscle mass, and / or increasing skeletal muscle mass and / or strength in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an ALK5 inhibitor or pharmaceutical composition of the present invention.

[0082] The therapeutically effective amount administered can be readily selected by one of skill in the art and should be sufficient to prevent involuntary loss of skeletal muscle mass, promote or stimulate skeletal muscle mass gain, provide sufficient skeletal muscle mass, and / or increase skeletal muscle mass and / or strength.

[0083] The subject may be as described above.

[0084] The present invention further relates to the use of an ALK5 inhibitor or pharmaceutical composition of the present invention for preparing a medicament for preventing involuntary loss of skeletal muscle mass, preferably due to degeneration of muscle fibers, promoting or stimulating skeletal muscle mass increase, ensuring sufficient skeletal muscle mass, and / or increasing skeletal muscle mass and / or strength, optionally in combination with one or more glucocorticoids.

[0085] As mentioned above, the ALK5 inhibitors or pharmaceutical compositions of the invention can be used in combination with one or more glucocorticoids.

[0086] In the experimental section, we showed that ALK5 inhibitors can not only promote the differentiation of myoblasts into myotubes and increase the fusion index, which reflects the ability of cells to regenerate, but also prevent skeletal muscle atrophy, especially atrophy induced by IL-1β, TNF-α, myostatin, TGF-β, and dexamethasone.

[0087] Therefore, the present invention also relates to an ALK5 inhibitor according to the present invention, or a pharmaceutical composition, or a kit for use in promoting skeletal muscle regeneration and / or preventing skeletal muscle atrophy.

[0088] All of the embodiments described above for ALK5 inhibitors as skeletal muscle hypertrophy inducers are also encompassed in this aspect.

[0089] As used herein, the term "skeletal muscle regeneration" refers to the ability of muscle cells or muscle tissue to regenerate, i.e., generate new myotubes from myoblasts. Thus, the term "promoting skeletal muscle regeneration" refers to the ability of an ALK5 inhibitor to promote the differentiation of myoblasts into myotubes and / or increase the number of myotubes and / or improve the regenerative capacity of muscle tissue, and in particular myotubes.

[0090] As used herein, the term "prevent skeletal muscle atrophy" refers to the ability of an ALK5 inhibitor to prevent, stop or slow muscle wasting.Muscle atrophy can be caused by, for example, disease states, or certain physiological conditions such as aging, starvation or inactivity, or by atrophic agents such as drugs (statins) or poisons (botulinum toxin).Prevention of muscle atrophy is preferably achieved by increasing muscle mass production to compensate for muscle loss.

[0091] As mentioned above, the ALK5 inhibitors or pharmaceutical compositions of the invention can be used in combination with one or more glucocorticoids.

[0092] The present invention also relates to a method for promoting skeletal muscle regeneration and / or preventing skeletal muscle atrophy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an ALK5 inhibitor or pharmaceutical composition of the present invention.

[0093] The therapeutically effective amount administered can be readily selected by one of skill in the art and should be sufficient to stimulate skeletal muscle regeneration and / or prevent, arrest or slow muscle wasting, preferably by increasing muscle mass production to compensate for muscle loss.

[0094] The subject may be as described above.

[0095] The present invention further relates to the use of an ALK5 inhibitor or pharmaceutical composition of the present invention for preparing a medicament for promoting skeletal muscle regeneration and / or preventing skeletal muscle atrophy, optionally in combination with one or more glucocorticoids.

[0096] As mentioned above, the ALK5 inhibitors or pharmaceutical compositions of the invention can be used in combination with one or more glucocorticoids.

[0097] The invention further relates to an ALK5 inhibitor, or a pharmaceutical composition, or a kit according to the invention for use in the treatment or prevention of muscle wasting, in particular in the treatment or prevention of diseases or disorders that result in skeletal muscle tissue loss and / or skeletal muscle weakness.

[0098] The present invention also relates to the use of an ALK5 inhibitor or a pharmaceutical composition according to the present invention for the preparation of a medicament for treating a disease or disorder resulting in muscle wasting, in particular skeletal muscle tissue loss and / or skeletal muscle weakness, optionally in combination with one or more glucocorticoids.

[0099] The present invention also relates to a method for treating a disease or disorder resulting in muscle wasting, in particular skeletal muscle tissue loss and / or skeletal muscle weakness, in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an ALK5 inhibitor or pharmaceutical composition of the present invention.

[0100] All of the embodiments described above for ALK5 inhibitors as skeletal muscle hypertrophy inducers are also encompassed in this aspect.

[0101] As mentioned above, the ALK5 inhibitors or pharmaceutical compositions of the invention can be used in combination with one or more glucocorticoids.

[0102] As used herein, the terms "treatment," "treat," or "treating" refer to any action intended to improve the well-being of a patient, such as the cure, prevention, prophylaxis, and delay of disease. In certain embodiments, such terms refer to the amelioration or eradication of symptoms of a disease or associated with a disease. In other embodiments, the terms refer to the minimization of the spread or worsening of a disease brought about by the administration of one or more therapeutic agents to a subject with such a disease.

[0103] As used herein, the term "treatment of muscle wasting" may refer to the treatment, prevention, or delay of involuntary loss of skeletal muscle mass, preferably due to degeneration of muscle fibers.

[0104] In particular, the term "treatment of a disease or disorder that results in skeletal muscle tissue loss and / or skeletal muscle weakness" can refer to maintaining or increasing skeletal muscle mass and / or strength in a patient, or slowing the loss of skeletal muscle mass and / or strength in a patient.

[0105] An effective amount can be a therapeutically or prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective at the dosage and for the period of time necessary to obtain a desired therapeutic or prophylactic result. A therapeutically effective amount can vary depending on factors such as the individual's disease or disorder, stage of the disease, age, sex, and weight. A therapeutically effective amount includes an amount in which any toxic or detrimental effects are outweighed by therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective at the dosage and for the period of time necessary to obtain a desired prophylactic result. Generally, but not necessarily, a prophylactic dose is used in subjects prior to or at an early stage of disease, so a prophylactically effective amount can be less than a therapeutically effective amount.

[0106] The disease or disorder to be treated can be any disease or disorder that results in a loss of skeletal muscle tissue or mass and / or skeletal muscle wasting.

[0107] Muscle wasting (i.e., loss of skeletal muscle tissue) and muscle weakness can result from a large group of diseases or disorders, such as metabolic diseases (e.g., glycogen storage diseases, adiposity diseases, or disorders of purine nucleotide metabolism), neurological diseases (e.g., hereditary sensorimotor neuropathy type III) and neuromuscular diseases, cachexia (i.e., muscle wasting due to diseases such as cancer, AIDS, congestive heart failure, chronic obstructive pulmonary disease, severe burns, renal failure, or liver failure), sarcopenia, disuse muscle atrophy (i.e., atrophy due to prolonged inactivity), atrophy induced by excessive starvation, such as starvation due to anorexia nervosa, or muscle injury, including acute muscle injury, overuse muscle injury, or battle injury.

[0108] Preferably, the disease or disorder to be treated is selected from neuromuscular diseases, cachexia, sarcopenia, disuse muscle atrophy, anorexic starvation-induced atrophy, and muscle damage, including acute muscle damage or overuse muscle damage. More preferably, the disease or disorder to be treated is selected from neuromuscular diseases, cachexia, and sarcopenia.

[0109] In certain embodiments, the disease or disorder is a neuromuscular disease, preferably selected from a muscle disease (ie, a myopathy), a neuromuscular junction disease, or a motor neuron disease.

[0110] Myopathies are neuromuscular disorders whose primary symptom is muscle weakness due to dysfunction of skeletal muscle fibers. Myopathies can be hereditary or acquired and include, for example, muscular dystrophies, metabolic myopathies such as mitochondrial myopathies or drug-induced myopathies, and autoimmune myopathies such as dermatomyositis, polymyositis or inclusion body myositis.

[0111] Muscular dystrophies represent a large group of myopathies that cause progressive degeneration of muscle fibers resulting in muscle loss. Mutations in over 30 genes that cause muscular dystrophies have been identified. Examples of muscular dystrophies include, but are not limited to, Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy, distal muscular dystrophies such as Miyoshi muscular dystrophy, Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0112] Motor neuron diseases are disorders characterized by the gradual degeneration and death of motor neurons that control voluntary muscles. That is, the motor neurons stop sending messages to the muscles, which slowly weaken and atrophy. Examples of motor neuron diseases include amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy.

[0113] Neuromuscular junction diseases are disorders that share a common disruption of neurotransmission through the neuromuscular junction and result in progressive debilitation due to muscle weakness. Neuromuscular junction diseases include, for example, myasthenia gravis, autoimmune neuromyotonia (Isaacs syndrome), Lambert-Eaton myasthenic syndrome, or can be due to forms of toxins that affect neuromuscular junction function, such as snake venoms or neurotoxins (e.g., Clostridium botulinum toxins).

[0114] Preferably, the disease or disorder is selected from muscular dystrophies, in particular from Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, distal muscular dystrophies such as Miyoshi muscular dystrophy, and limb-girdle muscular dystrophies.

[0115] More preferably, the disease or disorder is Duchenne muscular dystrophy.

[0116] In other particular embodiments, the disease or disorder is selected from cachexia and sarcopenia, preferably sarcopenia.

[0117] In all methods and aspects of the present invention, the ALK5 inhibitor or pharmaceutical composition of the present invention may be used in combination with other active ingredients, which may be selected depending on the desired effect or the disease to be prevented or treated. Examples of other active ingredients include, for example, anti-inflammatory agents, anabolic agents (e.g., growth hormone or insulin-like growth factor I), antineoplastic agents, antibiotics, local anesthetics, anabolic / androgenic steroids (e.g., testosterone), glucocorticoids, appetite stimulants (e.g., dronabinol), cytokine modulators (e.g., thalidomide), angiotensin and β-adrenoreceptor inhibitors, NHE-1 inhibitors (e.g., rimeporide), antifibrotic agents (e.g., losartan or lisinopril), phosphodiesterase 5 (PDE5) inhibitors (e.g., tadalafil or sildenafil), dehydroepiandrosterone, vitamin D, ursolic acid, omega 3 acid (omega 3 These include anti-fibrotic agents such as tadalafil and sildenafil, angiotensin-converting enzyme (ACE) inhibitors, proteasome inhibitors, cyclophilin D inhibitors, PGC-1α (alpha) pathway modulators, myostatin and activin A antagonists, ghrelin agonists, β2-adrenoreceptor agonists, creatine supplements, anti-fibrotic agents such as losartan and lisinopril, muscle ischemia treatments such as tadalafil and sildenafil, mutation-specific therapies such as exon skipping therapies (e.g., targeting mutations relevant to DMD cases), and Antisense oligomers that target specific nucleotides, such as eteplirsen, a morpholino phosphorodiamidate antisense oligomer that targets specific nucleotides, therapeutic nonsense suppressors such as ataluren, utrophin upregulators such as SMT-C1100, gene replacement therapies using muscle progenitor or stem cells (such as using rAAV2.5-CMV-Mini-dystrophy, rAAVrh74.MCK.Mini-dystrophy, or rAAV1.CMV.huFollistatin344), or cell therapies.

[0118] Such combination therapy as described above includes combined administration (comprising two or more therapeutic agents in the same or separate formulations) and separate administration (wherein administration of an ALK5 inhibitor can occur prior to, together with, and / or after administration of the additional therapeutic agent).

[0119] The ALK5 inhibitor (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, oral, transmucosal, or topical administration, preferably oral administration.

[0120] The doses used for administration can be adapted depending on various parameters, in particular depending on the mode of administration used, the pathology involved, or the duration of treatment desired. The ALK5 inhibitor (and any further therapeutic agents) can be administered as a single dose or in multiple doses.

[0121] The amount of ALK5 inhibitor that will be effective in the treatment of a particular disorder or condition will depend on the characteristics of the disorder or condition, and can be determined by standard clinical techniques.

[0122] In a preferred embodiment, each dose may range from about 0.0001 mg to about 300 mg of ALK5 inhibitor per kilogram of body weight, more preferably from about 0.0005 mg to about 50 mg of ALK5 inhibitor per kilogram of body weight, and even more preferably from about 0.001 mg to about 5 mg of ALK5 inhibitor per kilogram of body weight. Preferably, this dose range is a daily dose range.

[0123] In some embodiments where an ALK5 inhibitor is used in combination with a glucocorticoid, each dose may range from about 0.05 mg to about 100 mg of glucocorticoid per kilogram of body weight, preferably from about 0.05 mg to about 10 mg of glucocorticoid per kilogram of body weight, more preferably from about 0.1 mg to about 5 mg of glucocorticoid per kilogram of body weight, and even more preferably from about 0.1 mg to about 2 mg of glucocorticoid per kilogram of body weight. Preferably, this dose range is a daily dose range.

[0124] In certain embodiments, the ALK5 inhibitor, preferably RepSox, is used in combination with a glucocorticoid, preferably prednisolone or prednisone, and the dose of the ALK5 inhibitor is from about 0.001 mg to about 5 mg of ALK5 inhibitor per kilogram of body weight and the dose of the glucocorticoid is from about 0.1 mg to about 2 mg of glucocorticoid per kilogram of body weight.

[0125] The dosing schedule of administration can vary from once a month to daily, depending on many clinical factors, including the type of disease, the severity of the disease, and the subject's sensitivity to the therapeutic agent.

[0126] In certain embodiments of all aspects of the invention in which an ALK5 inhibitor is used in combination with one or more glucocorticoids (in the same or separate pharmaceutical compositions), the amounts or relative amounts of the ALK5 inhibitor and glucocorticoid are such that they act together when administered to a subject.

[0127] As used herein, the term "act together" means that the effect, for example therapeutic effect, of the combination of ALK5 inhibitor and glucocorticoid is greater than the sum of the effects of each compound used alone.In other words, the effect of the combination as a skeletal muscle hypertrophy inducer for promoting skeletal muscle regeneration, preventing skeletal muscle atrophy, or treating or preventing diseases or injuries that cause skeletal muscle tissue loss and / or muscle wasting is greater than the sum of the effects of each compound alone.

[0128] The potentiation effect allows for the use of lower amounts of ALK5 inhibitors and / or glucocorticoids. Thus, by combining an ALK5 inhibitor with a glucocorticoid, the efficacy of treatment can be maintained or even improved while reducing adverse or toxic effects.

[0129] Thus, in a preferred embodiment, the ALK5 inhibitor and glucocorticoid are present in a co-acting composition or combination or are administered in sub-therapeutic amounts to a subject in need of treatment.

[0130] As used herein, the term "sub-therapeutic" refers to an amount or dose of a therapeutic agent that is less than the conventional dose administered to a subject for the same indication and same route of administration when used alone. In particular, the term "sub-therapeutic" refers to an amount or dose of a therapeutic agent that has no or little effect on skeletal muscle cells when used alone.

[0131] One or more compounds may be administered in therapeutic amounts, while the others may be administered subtherapeutic amounts. In particular, the ALK5 inhibitor is administered in therapeutic amounts, while the glucocorticoid is administered subtherapeutic amounts, or vice versa. Preferably, to prevent side effects induced by administration of glucocorticoid, the ALK5 inhibitor is administered in therapeutic amounts, while the glucocorticoid is administered subtherapeutic amounts of about 0.01 mg to about 0.5 mg of glucocorticoid per kilogram of body weight, preferably about 0.1 mg to about 0.5 mg of glucocorticoid per kilogram of body weight. In certain embodiments, the ALK5 inhibitor, preferably RepSox, is used in combination with a glucocorticoid, preferably prednisolone or prednisone, and the dose of the ALK5 inhibitor is from about 0.001 mg to about 5 mg of ALK5 inhibitor per kilogram of body weight and the dose of the glucocorticoid is from about 0.1 mg to about 0.5 mg of glucocorticoid per kilogram of body weight.

[0132] Alternatively, the amount or dosage of the combination compound may be reduced, but instead the frequency of administration or duration of treatment may be reduced.

[0133] As shown in the experimental section, ALK5 inhibitors can prevent muscle atrophy.

[0134] Thus, the present invention also relates to a product comprising an ALK5 inhibitor as described above and a compound that induces skeletal muscle atrophy as a combined preparation for joint, separate or sequential use.

[0135] All of the embodiments described above for ALK5 inhibitors as skeletal muscle hypertrophy inducers are also encompassed in this aspect.

[0136] In certain embodiments, the compound that induces skeletal muscle atrophy is a therapeutic agent, in this embodiment, an ALK5 inhibitor is used to prevent or limit drug-induced myopathy.

[0137] The present invention further relates to a method for preventing or limiting skeletal muscle atrophy induced by a therapeutic agent in a subject, comprising administering to the subject a therapeutically effective amount of an ALK5 inhibitor as described above, either simultaneously, separately or sequentially to administration of the therapeutic agent that induces skeletal muscle atrophy.

[0138] Examples of therapeutic agents that induce skeletal muscle atrophy include, but are not limited to, therapeutic agents administered by intramuscular route, such as corticosteroids, colchicine, chloroquine, hydroxychloroquine, D-penicillamine, antibiotics, beta-blockers, amiodarone, cimetidine, zidovudine, vincristine, clofibrate, statins, fibrates, cyclosporine, L-tryptophan, hypokalemia-inducing agents, and lipid-lowering agents, or combinations of agents such as fibrates and statins, or cyclosporine and colchicine, and vaccines.

[0139] In a preferred embodiment, the therapeutic agent that induces skeletal muscle atrophy is a lipid-lowering agent, preferably selected from statins and fibrates.

[0140] The ALK5 inhibitor and the therapeutic agent that induces skeletal muscle atrophy can be administered together. Alternatively, the ALK5 inhibitor can be administered to the subject before or after the administration of the therapeutic agent that induces skeletal muscle atrophy. Preferably, when the therapeutic agent and the ALK5 inhibitor are administered separately, they are administered within 24 hours.

[0141] Optionally, as mentioned above, the ALK5 inhibitors can be used in combination with other active ingredients, preferably in combination with one or more glucocorticoids.

[0142] ALK5 inhibitors, ie skeletal muscle hypertrophy inducers, as described above may also find application in the feed and food industry, particularly as dietary supplements.

[0143] Thus, in a further aspect, the present invention also relates to a dietary supplement composition comprising an ALK5 inhibitor and, optionally, one or more glucocorticoids.

[0144] The present invention also relates to non-therapeutic uses of an ALK5 inhibitor or a kit as described above, or a dietary supplement composition of the invention, for increasing muscle mass, strength, and / or muscle performance in a subject.

[0145] The present invention further relates to non-therapeutic uses of an ALK5 inhibitor or a kit as described above, or a dietary supplement composition of the present invention, for use in preventing skeletal muscle mass loss, preferably involuntary and / or undesirable skeletal muscle mass loss.

[0146] Optionally, as mentioned above, the ALK5 inhibitors can be used in combination with other active ingredients, preferably in combination with one or more glucocorticoids.

[0147] The subject is preferably a mammal, more preferably a human.

[0148] In an embodiment, the subject is a non-human animal, preferably a mammal, even more preferably a livestock animal or a sport or leisure animal such as a race horse. The livestock animal is a non-human mammal, preferably a meat-producing mammal. In particular, the livestock animal can be selected from pigs, cows, goats, sheep, horses, bison, deer, elk, or moose.

[0149] In other embodiments, the subject is a human, preferably an adult human.

[0150] In certain embodiments, the subject is an older adult human, for example over 60 years of age, and the dietary supplement composition is used or suitable for arresting, slowing, or preventing loss of muscle function and / or muscle mass.

[0151] The subject is preferably a healthy subject, ie, a subject not suffering from a disease or disorder that results in skeletal muscle tissue loss and / or muscle wasting.

[0152] The dietary supplement composition may be in powder, liquid, or solid form.

[0153] Preferably, the dietary supplement composition is formulated for oral administration. In particular, the dietary supplement composition can be formulated into conventional oral administration forms, such as tablets, capsules, powders, granules, and liquid preparations, such as syrups, elixirs, and concentrated drops. Non-toxic solid carriers or diluents can be used, including, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium, carbonates, and the like. In compressed tablets, binders are also required, which are agents that give cohesive properties to powder materials. For example, starch, gelatin, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also required in tablets to facilitate tablet disintegration. Disintegrants include starch, clay, cellulose, algins, gums, and cross-linked polymers. In addition, lubricants and glidants are also included in tablets to prevent tablet materials from adhering to the surface during the manufacturing process, and to improve the flow properties of powder materials during manufacturing. Colloidal silicon dioxide is most commonly used as a lubricant, and compounds such as talc or stearic acid are most commonly used as lubricants.

[0154] The dietary supplement composition can include additional ingredients that provide a beneficial effect to a subject, such as vitamins (e.g., vitamin D), amino acids, proteins, lipids (omega-3 fatty acids), ursolic acid, tomaditine, antioxidants, polyphenols, isoflavones and derivatives present in soybeans, tea leaf components, and garlic compounds.

[0155] All embodiments described above regarding ALK5 inhibitors and in particular their use as skeletal muscle hypertrophy inducers are also encompassed in this aspect.

[0156] The present invention also relates to the use of the ALK5 inhibitor as described above as a material for animal feed compositions or an additive for animal feed compositions.The present invention also relates to the use of the ALK5 inhibitor for preparing a material or an additive for animal feed compositions.Optionally, as described above, the ALK5 inhibitor can be used in combination with other active ingredients, preferably in combination with one or more glucocorticoids.

[0157] The present invention further relates to a material or additive for an animal feed composition comprising an ALK5 inhibitor as described above, and optionally one or more glucocorticoids.

[0158] The present invention further relates to livestock feed compositions comprising as ingredients or additives an ALK5 inhibitor as described above, and optionally one or more glucocorticoids.

[0159] The feed composition, material, additive, or dietary supplement of the present invention may be any edible GRAS (generally recognized as safe) feed, such as, for example, corn gluten feed, sunflower hulls, distillers grains, guar hulls, wheat middlings, rice hulls, rice bran, oilseed meal, dried blood meal, animal by-product meal, fish by-products, fish meal, soluble dried fish, feather meal, poultry by-products, meat meal, bone meal, dried whey, soy protein concentrate, soy flour, yeast, wheat, oats, grain sorghum, corn feed meal, rye, corn, barley, aspirated grain fraction, dried brewers grains, corn flour, corn gluten meal, feed oat meal, sorghum grain meal, wheat mill run, wheat red dog, hominy feed, wheat flour, wheat bran, wheat germ meal, oat groats, rye middlings, cotyledon fiber, ground grains, or mixtures thereof. The material may further comprise a safe material.

[0160] Preferably, the feed composition, material, additive, or dietary supplement of the present invention is used as a non-therapeutic skeletal muscle hypertrophy inducer, particularly for improving the performance of livestock, i.e., increasing weight gain.Thus, preferably, the feed composition, material, additive, or dietary supplement is used for administration to healthy subjects, i.e., subjects not suffering from diseases or disorders that lead to skeletal muscle tissue loss and / or muscle wasting.Subjects may be as described above for the dietary supplement composition of the present invention.

[0161] The present invention also relates to a method for improving the performance and / or health of livestock, comprising feeding said livestock with an ALK5 inhibitor as described above, and optionally one or more glucocorticoids, in particular the feed composition, material, additive, or dietary supplement of the present invention.Preferably, as used herein, the term "improving livestock performance" refers to increasing weight gain.This use is intended to be a non-therapeutic use as described above, and preferably the compound, feed composition, material, additive, or dietary supplement is used for administration to healthy livestock, i.e. livestock not suffering from a disease or disorder that leads to skeletal muscle tissue loss and / or muscle wasting.

[0162] The feed composition, ingredient, additive, or dietary supplement may be in powder, liquid, or solid form.

[0163] The ingredients of the feed composition of the present invention other than the ALK5 inhibitor depend on the characteristics of the livestock and can be easily selected by those skilled in the art.

[0164] Preferably, the feed composition of the present invention is in a form and / or composition approved by a government agency, such as a national food authority (eg ANSES in France, ACIA in Canada, or FAD in the United States).

[0165] All embodiments described above regarding ALK5 inhibitors and in particular their use as skeletal muscle hypertrophy inducers are also encompassed in this aspect.

[0166] Preferably, in all aspects of the present invention, the ALK5 inhibitor, optionally in combination with a glucocorticoid, is not used in combination with an oxytocin receptor agonist. In particular, in a preferred embodiment, the pharmaceutical composition of the present invention does not comprise an oxytocin receptor agonist, and the ALK5 inhibitor, optionally in combination with a glucocorticoid, is not used in combination with an oxytocin receptor agonist.

[0167] The oxytocin receptor (OXTR) is a G-protein-coupled receptor for the peptide oxytocin, which acts as both a hormone and a neurotransmitter.

[0168] An OXTR agonist is any agent that specifically enhances OXTR expression, OXTR signaling, or downstream signaling of OXTR.

[0169] In particular, OXTR agonists can be small molecules, nucleic acid molecules that specifically interfere with OXTR expression, bait substrates, dominant-negative mutants of OXTR, and aptamers or antibodies against OXTR.

[0170] In particular, OXTR agonists may include oxytocin and its analogs. Examples of OXTR agonists, particularly oxytocin analogs, are published in WO 2016 / 025629, U.S. Pat. No. 8,748,564, U.S. Pat. App. Pub. No. 2007 / 117794, and U.S. Pat. App. Pub. No. 2013 / 085106, and include, for example, demoxitocin, carbetocin, TC OT 39, and WAY-267464.

[0171] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0172] Example 1 Materials and Methods <Cell Source and Cell Culture> Primary skeletal cells from healthy donors (Donor 1, Donor 3, and Donor 5) were Clonetics Human Skeletal Muscle Myoblasts (HSMM). In addition, cells from three DMD (Duchenne muscular dystrophy) donors (Donors Z, W, and D1) and a healthy donor (Donor A) were obtained.

[0173] Donor characteristics are detailed in Tables 2 and 3 below. [Table 2] [Table 3]

[0174] Myocytes were maintained in culture with supplements and fetal bovine serum (FBS) from Lonza according to the supplier's instructions. An expansion step was performed to obtain enough cells to seed the screening plates.

[0175] <Hypertrophy assay and atrophy recovery assay> Hypertrophy and atrophy recovery assays were performed using a fully automated in vitro human myotube model called MyoScreen (Saito, France), which is based on the strict control of the microenvironment to guide the differentiation of human primary myoblasts. Myotubes formed on MyoScreen micropatterns show a high level of maturity with a highly standardized morphology.

[0176] Donor human primary myoblasts were seeded in MyoScreen micropatterned 96-well plates (Cyto, France), allowed to attach for 24 h in growth medium, and differentiated for at least 5 days in horse reduced serum medium.

[0177] On day 0, MyoScreen plates (Saito, France) containing micropatterns were pre-filled with 200 μl / well of growth medium and kept in an incubator at 37° C. Human primary myoblasts were detached from flasks, counted and plated at 15000 cells per well in 100 μl of growth medium.

[0178] On day 1, the growth medium was changed to 300 μl / well of differentiation medium (DMEM containing 0.1% horse serum) in which myoblasts initiated differentiation and myotube formation.

[0179] On day 2, the differentiation medium was changed. Then, candidate compounds were diluted in differentiation medium and transferred to the plate. The final concentration of DMSO should be 0.1% or less. At least 6 wells were treated with vehicle as basal control and 6 wells were treated with 100ng / ml IGF-1 as hypertrophy positive control.

[0180] In the atrophy recovery assay, one hour after addition of the candidate compounds, atrophy inducers were added at final concentrations of 150 ng / mL myostatin, 25 ng / mL IL-1β, 2 ng / mL TNF-α, 0.5 ng / mL TGF-β, and 100 μM dexamethasone.

[0181] On day 6, cells were fixed with formalin 5% for 30 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with PBS+BSA 1% for 20 min. Myotubes were incubated with primary antibody against troponin T in blocking buffer for 1.5 h, washed 3 times with PBS, incubated with secondary antibody and Hoechst (1 / 10000) for 1.5 h, and washed 3 times with PBS.

[0182] <Image analysis> Images were acquired at 10x magnification on an Operetta High Content Imaging System. Image processing and analysis were performed using proprietary algorithms developed in Acapella High Content Imaging Software by Saito (Perkin Elmer). Eleven fields of view were acquired per well.

[0183] First, myotubes and nuclei were segmented using troponin T staining intensity and Hoechst staining, respectively. Typically, 1-2 myotubes were identified per micropattern (myotubes are troponin T stained areas containing at least two nuclei). A segmentation threshold was set to avoid detection of background noise and to exclude aberrant small myotube structures. At the end of this first step, specific readouts were calculated for all wells, such as nuclei count and fusion index (percentage of nuclei contained in troponin T staining). Typically, about 50-60 myotubes were detected per well in the control condition.

[0184] An image cleanup step was then performed on the Troponin T images to remove myotubes bordering the image border. The final valid myotubes were used to extract myotube morphology parameters including myotube width and area, and the number of nuclei per myotube.

[0185] Nuclei counts, fusion index, mean myotube area, and number of nuclei per myotube were examined as readouts related to and affected by myotube differentiation and atrophy and hypertrophy induction.

[0186] <First screening> A primary screen was performed to identify hypertrophic compounds that increase myotube differentiation and myotube size. Candidate compounds were tested at 10 μM in monoduplicates in donor 3 cells.

[0187] <Retest> Retesting was performed by cherry-picking (same group of compounds as in the primary screen). Each hit was tested in replicates of 6 wells under the same conditions as in the primary screen (donor 3, 10 μM).

[0188] Dose response in three healthy donors Dose-response assays were performed in three healthy donors (donors 3, 5, and 1) with eight doses of candidate compounds ranging from 33 μM to 0.015 μM, with two wells per dose in replicates.

[0189] <EC 50 Calculation> Compounds of interest were tested in dose-response at several doses in triplicate wells. Results were normalized to control conditions (basal levels) and plotted using GraphPad Prism. The "Nuclei Count" reading allowed detection of any toxic effects. The "Fusion Index" and "Myosin Area" readings were fitted to the EC 50 was used to determine the value.

[0190] <Atrophy recovery evaluation> Atrophy reversal assays were performed in triplicate in the presence of atrophy inducers, namely TNF-α, IL-1β, myostatin, TGF-β, and dexamethasone.

[0191] [result] As shown below in Table 4, RepSox was identified as an inducer of skeletal muscle hypertrophy upon primary screening and retesting, resulting in increased fusion index and / or myotube area readouts of greater than +170%. Table 4 shows the hypertrophic activity in healthy donor 3. [Table 4] (% activity = myotube fusion index (compound) x 100 / myotube fusion index (basal control))

[0192] A dose response was performed in three healthy donors, and the results are shown in Table 5. RepSox was active in all donors. Table 5 shows the results of the first dose-response assay in various donors. [Table 5]

[0193] Eight additional ALK5 inhibitors were also tested, namely, SB525334, SD208, SB505124, R268712, LY364947, GW788388, D4476, and A83-01. The results are shown in Table 6. EC 50 Value confirmed. Table 6 shows the results of a second dose-response assay in healthy donor 3. [Table 6]

[0194] The activity of RepSox and GW788388 was also assayed in cells from DMD donors, namely donors Z, W, and D1, and the results are shown in Table 7. Table 7 shows the hypertrophic activity in DMD donors. [Table 7] (% activity = myotube fusion index (compound) x 100 / myotube fusion index (basal control))

[0195] Atrophy reversal assays were performed on RepSox. Healthy donor 3 myotubes were subjected to atrophy using various inducers, TNF-α, IL-1β, myostatin, TGF-β, and dexamethasone. The ability of RepSox to suppress atrophy induced by these various atrophy inducers was measured.

[0196] The results shown in Table 8 indicate that RepSox is able to inhibit atrophy induced by IL-1β, TNF-α, myostatin, TGF-β, and dexamethasone. Table 8 shows the results of the atrophy recovery assay. [Table 8] (+++: Full recovery, ++: Partial recovery, +: Low recovery)

[0197] Example 2: Acetylcholine receptor clustering assay Materials and Methods The MyoScreen protocol was performed as described in the hypertrophy and atrophy recovery assay in Example 1, but was stopped on day 9 instead of day 6. At the end of the assay, AchR was immunostained using a specific antibody in addition to troponin T and nuclei. Images were acquired at 20x magnification on a PerkinElmer Operetta high content imaging system. Image processing and analysis were performed using a proprietary algorithm developed in Acapella high content imaging software (Perkin Elmer). Specific readings were calculated for each well: nuclei count and myotube fusion index, number of AchR, average AchR area, total AchR area normalized by total myotube area.

[0198] [result] Remarkably, when labeled with a specific anti-AchR antibody, MyoScreen myotubes at 6 days post-differentiation show AchR clusters spaced along the sarcolemma membrane in specific regions in the middle of the myotube fiber and at the myotube ends. To develop an automated AchR cluster assay, we developed an AchR detection routine that was sensitive and robust enough to analyze the number and size of cluster aggregates in images using the Operetta / Acapella system. The effect of RepSox on AchR cluster formation was evaluated to show a positive effect of the compound at the neuromuscular junction. After 8 days of differentiation, including 7 days of drug treatment, RepSox-treated myotubes showed substantially more AChR clusters and a 6-fold increase in total AChR area, while IGF-1 had no effect (Figure 1).

[0199] Example 3: Calcium flux assay Materials and Methods The cells were washed with calcium buffer containing (mM) 130 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgCl2, 5.6 D-glucose, 10 Hepes, pH 7.4. The cells were then incubated with 2 μM Fluo-4AM (F14201-Invitrogen) for 1 h in an incubator (37 °C, 5% CO2). After washing to remove unloaded dye, video images were obtained of the response of myotubes to 20 μM ACh (A6625-Sigma-Aldrich). Stream acquisition was set at 1 s intervals, and one field of view was acquired per well. Images were automatically processed and analyzed using dedicated software developed in-house integrating the open source Image J framework. Image analysis tools segment myotubes in images acquired from the same well. Fluo-4 total fluorescence intensity was measured within each myotube and corrected for background. The total intensity was normalized to the myotube area to obtain the mean fluorescence intensity reading. For data presentation, the fluorescence intensity (F) was normalized to the mean intensity of the three initial time points (F / F0).

[0200] [result] Interestingly, when measuring Ca2+ flux after ACh stimulation, only myotubes differentiated in the presence of RepSox showed a statistically significant increase of 1.56-fold in [Ca2+]i peak amplitude compared to control myotubes. Again, IGF-1 had no effect. Time-lapse video of calcium flux also suggested that myotubes differentiated in the presence of RepSox showed a significant increase in the number of contracting myotubes after ACh stimulation (Figure 2).

[0201] Example 4: Contractile Activity Assay Materials and Methods Cells were washed with calcium buffer as described above. Myotube contraction was induced by adding 100 μM ACh for 3 min, after which myotubes were fixed and immunostained with troponin T antibody. Automated acquisition was performed on an Operetta platform with a ×10 / 0.3NA objective. Acapella was used to automatically detect and quantitate detached myotubes based on detection of high intensity areas within the edges of segmented myotubes, which mark the sites where myotubes have detached.

[0202] [result] The effect on contractile force was quantified after 5 days of differentiation, including 4 days of RepSox or IGF-1 treatment. When stimulated by the addition of 100 μM ACh, effective contractile force was increased in RepSox (77 ± 5%) and IGF-1 (67 ± 5%) treated myotubes. RepSox enhances ACh-stimulated [Ca2+]i influx and contractile force (Figure 3).

[0203] Example 5: Combination of ALK5 inhibitors with glucocorticoids Materials and Methods Hypertrophy assays were performed in DMD donor Donor Z as described in Example 1. Treatments were performed on day 2 to add 10 nM prednisolone, 300 nM RepSox or GW788388, or a combination of prednisolone and one of these two ALK5 inhibitors. After 4 days of treatment, myotube fusion index and average area were measured by image analysis as described in Materials and Methods in Example 1.

[0204] [result] As shown in Figures 4-7, the positive control IGF-1 increased both the fusion index (+160%) and myotube average area (+54%), confirming the hypertrophy assay in DMD donor Z.

[0205] Both the ALK5 inhibitors RepSox and GW788388 induce significant hypertrophy when added at 300 nM and alone. It is noted that RepSox is the most potent ALK5 inhibitor, increasing the fusion index by +100% (vs. +41% for GW788388) and myotube area by +68% (vs. +21% for GW788388).

[0206] 10 nM prednisolone alone results in a small increase in the fusion index of donor Z myotubes (+25%) but does not significantly increase the average myotube area.

[0207] The combination of one ALK5 inhibitor with prednisolone potentiates the effect of each compound alone, with both the fusion index and myotube area being significantly greater in the combination condition than with treatment with either compound. For example, the combination of prednisolone with RepSox increases the fusion index by +148% (Figure 4) and the myotube area by +102% (Figure 5).

[0208] Example 6: Electrical Pulse Stimulation Assay Materials and Methods Donor 5 and A myotubes (see Table 2) were differentiated for 6–7 days, including treatment with 3–500 nM RepSox on days 5–6. Cells were washed with calcium buffer containing (in mM): 130 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgCl2, 5.6 D-glucose, 10 Hepes, pH 7.4. Cells were then placed in an incubator (37 °C, 5% CO2) for 24 h. 2 ) for 1 h with 2 μM Fluo-4AM (F14201-Invitrogen). After washing to remove unloaded dye, the response of myotubes to electrical pulse stimulation was monitored using a Hamamatsu Photonics FDSS / μCELL / EFS. Twitch stimulation was applied at 5 V with a pulse width of 50 ms, at 0.2 Hz, for 60 s. Tetanic stimulation was applied at 3 V with a pulse width of 15 ms, at 30 Hz, for 20 s. Fluo-4 total fluorescence intensity was measured in each well. For data presentation, fluorescence intensity (F) was normalized to the mean intensity at the initial time point (F / F0).

[0209] [result] Donor A and donor 5 myotubes responded to repeated twitch electrical stimulation as shown in Figures 8A and 8B. When myotubes were treated with 300 nM RepSox for 5 days, the calcium peak amplitude was 2-fold greater.

[0210] Increasing the stimulation frequency resulted in wave summation and full tetanus, indicative of the in vivo phenomenon. The tetanic response of RepSox-treated myotubes increased with RepSox concentration, resulting in a significant difference from control myotubes (FIG. 9B).

[0211] These results confirm that RepSox enhances calcium influx in myotubes after chemical (ACh) or electrical (FDSS / μCELL / EFS) stimulation.

Claims

1. 1. A pharmaceutical composition for use in the treatment or prevention of a disease or disorder of skeletal muscle selected from the group consisting of sarcopenia, cachexia, neuromuscular diseases, disuse muscle atrophy, anorexic starvation-induced muscle atrophy, and muscle damage, comprising an ALK5 inhibitor, The pharmaceutical composition, wherein the ALK5 inhibitor is selected from the group consisting of RepSox, and GW788388, and pharma- ceutically acceptable salts, hydrates, or solvates thereof.

2. The pharmaceutical composition of claim 1 , wherein the ALK5 inhibitor is used in combination with a glucocorticoid.

3. 1. A pharmaceutical composition for use in the treatment or prevention of a disease or disorder of skeletal muscle selected from the group consisting of sarcopenia, cachexia, neuromuscular diseases, disuse muscle atrophy, anorexic starvation-induced muscle atrophy, and muscle damage, comprising an ALK5 inhibitor, a glucocorticoid, and a pharma- ceutically acceptable excipient, the ALK5 inhibitor is selected from the group consisting of RepSox, and GW788388, and pharma- ceutically acceptable salts, hydrates, or solvates thereof; The pharmaceutical composition, wherein the glucocorticoid is selected from the group consisting of prednisolone, prednisone, and pharma- ceutically acceptable salts, hydrates, or solvates thereof.

4. A kit for use in the treatment or prevention of a disease or disorder of skeletal muscle selected from the group consisting of sarcopenia, cachexia, neuromuscular diseases, disuse muscle atrophy, anorexic starvation-induced muscle atrophy, and muscle damage, comprising an ALK5 inhibitor and a glucocorticoid as a combined preparation used together, either individually or sequentially, the ALK5 inhibitor is selected from the group consisting of RepSox, and GW788388, and pharma- ceutically acceptable salts, hydrates, or solvates thereof; The glucocorticoid is selected from the group consisting of prednisolone, prednisone, and pharma- ceutically acceptable salts, hydrates, or solvates thereof.

5. The pharmaceutical composition according to any one of claims 1 to 3, or the kit according to claim 4, wherein the ALK5 inhibitor is RepSox, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof.

6. The pharmaceutical composition according to any one of claims 1 to 3, or the kit according to claim 4, wherein the ALK5 inhibitor is GW788388, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof.

7. The pharmaceutical composition according to any one of claims 2, 3, 5 and 6, or the kit according to any one of claims 4 to 6, wherein the glucocorticoid is prednisolone, or a pharma- ceutically acceptable salt, hydrate, or solvate thereof.

8. The pharmaceutical composition according to any one of claims 2, 3, 5 and 6, or the kit according to any one of claims 4 to 6, wherein the glucocorticoid is prednisone or a pharma- ceutically acceptable salt, hydrate or solvate thereof.

9. The pharmaceutical composition of any one of claims 1 to 3 and 5 to 8, or the kit of any one of claims 4 to 8, wherein the ALK5 inhibitor, the pharmaceutical composition, or the kit is not used in combination with an oxytocin receptor agonist.

10. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 9, or the kit according to any one of claims 4 to 9, wherein the disease or disorder of skeletal muscle is selected from cachexia, neuromuscular disease, disuse muscle atrophy, anorexic starvation-induced muscle atrophy, and muscle damage including acute muscle injury or overuse muscle injury.

11. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 10, or the kit according to any one of claims 4 to 10, wherein the disease or disorder of skeletal muscle is muscular dystrophy.

12. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 11, or the kit according to any one of claims 4 to 11, wherein the disease or disorder of skeletal muscle is a muscular dystrophy selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, distal muscular dystrophy such as Miyoshi muscular dystrophy, and limb-girdle muscular dystrophy.

13. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 12, or the kit according to any one of claims 4 to 12, wherein the skeletal muscle disease or disorder is Duchenne muscular dystrophy.

14. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 10, or the kit according to any one of claims 4 to 10, wherein the skeletal muscle disease or disorder is sarcopenia.

15. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 10, or the kit according to any one of claims 4 to 10, wherein the disease or disorder of skeletal muscle is cachexia.

16. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 10, or the kit according to any one of claims 4 to 10, wherein the disease or disorder of skeletal muscle is a motor neuron disease selected from the group consisting of amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy.

17. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 10, or the kit according to any one of claims 4 to 10, wherein the skeletal muscle disease or disorder is spinal muscular atrophy.

18. The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 10, or the kit according to any one of claims 4 to 10, wherein the disease or disorder of skeletal muscle is a neuromuscular junction disease, preferably selected from the group consisting of myasthenia gravis, autoimmune neuromyotonia (Isaacs syndrome), Lambert-Eaton myasthenic syndrome, and neuromuscular junction diseases caused by a form of toxin that affects neuromuscular junction function, such as snake venom or neurotoxin.

19. The ALK5 inhibitor, the pharmaceutical composition, or the kit may further comprise other active ingredients, preferably anti-inflammatory agents, anabolic agents, antineoplastic agents, antibiotics, local anesthetics, anabolic / androgenic steroids, glucocorticoids, appetite stimulants, cytokine modulators, angiotensin and β-adrenoreceptor inhibitors, NHE-1 inhibitors, antifibrotic agents, phosphodiesterase 5 (PDE5) inhibitors, dehydroepiandrosterone, vitamin D, ursolic acid, omega 3 acid (omega 3 The pharmaceutical composition according to any one of claims 1 to 3 and 5 to 18, or the kit according to any one of claims 4 to 18, used in combination with an active ingredient selected from the group consisting of an anti-inflammatory drug, an anti-inflammatory drug, an anti-inflammatory drug, an anti-inflammatory drug, a steroid therapy, a steroid agonist ...

20. The pharmaceutical composition of any one of claims 1 to 3 and 5 to 18, or the kit of any one of claims 4 to 18, wherein the ALK5 inhibitor, the pharmaceutical composition, or the kit is used in combination with a gene replacement therapy agent.

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