Treatment of Facioscapulohumeral Dystrophy (FSHD)

Abstract

Disclosed herein are methods and uses for treating, ameliorating, delaying progression, and / or preventing muscular dystrophy or cancer, including but not limited to facioscapulohumeral muscular dystrophy (FSHD) or sarcoma. More specifically, disclosed herein are methods of using small molecule protein arginine methylation (PRMT) inhibitors to inhibit methylation of amino acids, such as arginine, in dual homeobox 4 (DUX4) protein, and the use of these inhibitors. Even more specifically, the present disclosure provides methods of using such methylation inhibitors or arginine methylation inhibitors to inhibit methylation of DUX4 protein, resulting in reduced DUX4-activated cell death, including reduced DUX4-activated muscle cell death, and / or reduced DUX4 target gene activation. The present disclosure provides, in some embodiments, methods of using protein methylation inhibitors, including but not limited to, salvianolic acid A (SAA) or a derivative thereof, or adenosine dialdehyde (ADOX) or a derivative thereof, to inhibit methylation of arginine residues of DUX4 protein in a cell in vitro, ex vivo, or in vivo in a cell of a subject at risk for or suffering from muscular dystrophy or cancer associated with DUX4 overexpression.

Description

[Technical Field]

[0001] Incorporation by reference of sequence listing This application contains, as a separate part of the disclosure, a sequence listing in computer-readable form (filename: 56936_Seqlisting.txt, size: 6,005 bytes, created on June 22, 2022), which is incorporated by reference in its entirety into this specification.

[0002] The present disclosure relates to the field of muscular dystrophy or cancer treatment, including, but not limited to, facioscapulohumeral muscular dystrophy (FSHD) or sarcoma. More specifically, the present disclosure provides methods for treating, ameliorating, delaying progression, and / or preventing muscular dystrophy or cancer, including, but not limited to, FSHD or sarcoma. Specifically, the present disclosure provides methods for inhibiting methylation of arginine residues in dual homeobox 4 (DUX4) protein by administering a small molecule inhibitor of protein arginine methyltransferase (PRMT). More specifically, the present disclosure provides PRMT inhibitors for reducing DUX4-induced cell death and / or DUX4-induced gene activation, as well as methods of using the PRMT inhibitors to treat subjects suffering from or at risk of suffering from muscular dystrophy or cancer associated with DUX4 overexpression. [Background technology]

[0003] Muscular dystrophies (MD) are a group of genetic disorders characterized by progressive weakness and degeneration of the skeletal muscles that control movement. Some forms of MD have an onset in infancy or childhood, while others may not manifest until middle age or later. The disorders differ in the distribution and degree of muscle weakness (some forms of MD also affect the heart muscle), age at onset, rate of progression, and mode of inheritance.

[0004] Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common forms of muscular dystrophy, affecting an estimated 870,000 people worldwide. It is a significant cause of morbidity with many patients experiencing debilitating pain, fatigue, and progressive asymmetric muscle weakness. 1 Currently, there are no disease-modifying treatments, and the development of a therapy remains a critical unmet need. FSHD is caused by derepression of the transcription factor DUX4, which is normally expressed during early embryonic development and in the testis, but is epigenetically silenced in most somatic tissues. 2、3 Aberrant expression of DUX4 is toxic to muscle in vitro and in vivo 4 DUX4 activates multiple pathways that may contribute to myofiber toxicity, including oxidative stress, immune activation, and apoptosis. 5 Although significant progress has been made in linking DUX4 function to toxicity, the contribution of DUX4 regulation to FSHD pathology remains unclear. Despite progress in the FSHD field, there are still no approved treatments for FSHD, and therapeutic development remains a critical need in the field. There remains a need in the art for methods to treat muscular dystrophies, including, but not limited to, FSHD.

[0005] Post-translational modifications (PTMs) play an important role in protein regulation and function. Arginine methylation is a PTM involved in the regulation of proteins involved in transcription, pre-mRNA splicing, and myogenesis and skeletal muscle regeneration. 7 Small molecule inhibitors of protein arginine methyltransferases (PRMTs) are being explored in cancer. 6,8However, it is unclear whether arginine methylation inhibitors can be protective in FSHD disease models. The present disclosure provides methods for regulating DUX4 at the level of post-translational modification and for the therapeutic application of arginine methylation inhibition in FSHD disease. The present disclosure provides methods for inhibiting DUX4-induced cell death and DUX4-induced gene target activation in muscle cells and in subjects with MD and FSHD using inhibitors of PRMTs. Summary of the Invention

[0006] The present disclosure provides methods and uses for inhibiting DUX4 target gene expression (or target gene transactivation) and reducing DUX4-induced apoptotic cell death to treat, ameliorate, delay progression, and / or prevent muscular dystrophy (MD). In some embodiments, the muscular dystrophy is facioscapulohumeral dystrophy (FSHD). More specifically, the present disclosure provides small molecule inhibitors of protein arginine methyltransferase (PRMT or RMT) for inhibiting methylation of arginine residues of the double homeobox 4 (DUX4) protein in cells or in cells of subjects suffering from muscular dystrophy associated with DUX4 overexpression. The present disclosure provides such arginine methylation inhibitors as a means for modulating DUX4-mediated toxicity and for the treatment of muscular dystrophies, including, but not limited to, FSHD.

[0007] The present disclosure provides a method for inhibiting arginine methylation of dual homeobox 4 (DUX4) protein in a cell, comprising contacting the cell with an effective amount of at least one inhibitor of protein arginine methyltransferase (PRMT), wherein the inhibitor of PRMT is at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof;

[0008] [ka]

[0009] In the formula, R 1 is H or C1-C6 alkyl, and X 1 is CH or N. The present disclosure provides a method for reducing dual homeobox 4 (DUX4)-associated apoptotic cell death and / or reducing DUX4 target gene activation in a cell, comprising contacting the cell with an effective amount of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof;

[0010] [ka]

[0011] In the formula, R 1 is H or C1-C6 alkyl, and X 1 is CH or N. The present disclosure provides a method for treating an arginine methyltransferase (RMT) disorder in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof;

[0012] [ka]

[0013] In the formula, R 1 is H or C1-C6 alkyl, and X 1 is CH or N. In some embodiments, the cells are in a subject at risk for or suffering from muscular dystrophy. In some embodiments, the subject is a human subject.

[0014] In some embodiments, the PRMT disorder is muscular dystrophy. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, compound I is compound Ia,

[0015] [ka]

[0016] or a salt, hydrate, or stereoisomer thereof. In some embodiments, compound Ia is a hydrate of the formula:

[0017] [ka]

[0018] x is between 0.5 and 10. In some embodiments, compound II is compound IIa,

[0019] [ka]

[0020] or a salt, hydrate, or stereoisomer thereof. The present disclosure relates to the use of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof, in the treatment of a patient with atopic dermatitis, including the following:

[0021] [ka]

[0022] In the formula, R 1 is H or C1-C6 alkyl, and X 1 The present invention provides a use of a compound which is CH or N for the preparation of a pharmaceutical for inhibiting arginine methylation of dual homeobox 4 (DUX4) protein in a cell.

[0023] The present disclosure relates to the use of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof, in the treatment of a patient with atopic dermatitis, including the following:

[0024] [ka]

[0025] In the formula, R 1 is H or C1-C6 alkyl, and X 1 The present invention provides a use of the compound of the present invention, which is CH or N, for the preparation of a pharmaceutical agent for reducing dual homeobox 4 (DUX4)-associated apoptotic cell death in a cell and / or reducing DUX4 target gene activation.

[0026] The present disclosure relates to the use of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof, in the treatment of a patient with atopic dermatitis, including the following:

[0027] [ka]

[0028] In the formula, R 1 is H or C1-C6 alkyl, and X 1 The present invention provides a use of a compound selected from the group consisting of arginine methyltransferase (RMT) disorders, wherein the compound is CH or N, for the preparation of a medicament for treating an arginine methyltransferase disorder in a patient in need of such treatment.

[0029] In some embodiments, the cells are in a subject at risk for or suffering from muscular dystrophy. In some embodiments, the subject is a human subject.

[0030] In some embodiments, the PRMT disorder is muscular dystrophy. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, decreased DUX4 target gene activation is indicated by decreased expression of DUX4 target genes, including, but not limited to, PRAME family member 12 (PRAMEF12), zinc finger and SCAN domain-containing protein 4 (ZSCAN4), tripartite motif-containing 43 (TRIM43), and leucine twenty homeobox (LEUTX).

[0031] In some embodiments, compound I is compound Ia,

[0032] [ka]

[0033] or a salt, hydrate, or stereoisomer thereof. In some embodiments, compound Ia is a hydrate of the formula:

[0034] [ka]

[0035] x is between 0.5 and 10. In some embodiments, compound II is compound IIa,

[0036] [ka]

[0037] or a salt, hydrate, or stereoisomer thereof. The present disclosure provides the methods and uses described herein, wherein the small molecule inhibitor of PRMT is Compound A or a salt, hydrate, or stereoisomer thereof and / or Compound B or a salt, hydrate, or stereoisomer thereof.

[0038] The present disclosure also provides methods and uses, wherein the small molecule inhibitor of PRMT, or a composition comprising a small molecule inhibitor of PRMT, or a medicament comprising a small molecule inhibitor of PRMT, is formulated for intramuscular injection, transdermal delivery, or injection into the bloodstream.

[0039] Further aspects and advantages of the present disclosure will be apparent to those skilled in the art from a review of the following detailed description taken in conjunction with the drawings. However, the detailed description (including the drawings and specific examples), while indicating embodiments of the disclosed subject matter, is given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0040] [Figure 1A] DUX4 arginine methylation sites (Figure 1A) and mutagenesis strategy (Figure 1B) are shown. Figure 1A provides a schematic of the DUX4 arginine methylation PTM sites identified by mass spectrometry. Figure 1B provides an overview of the mutagenesis. [Figure 1B] Same as above. [Figure 2] Figure 1 shows that the DUX4 PTM R71A mutant protects against cell death. HEK293 cells were transfected with wild-type or DUX4 R71A mutant, and a caspase assay was performed 48 hours later to assess cell death. [Figure 3A] Figure 3A shows HEK293 cells transfected with wild-type DUX4, empty vector (pClneo), or DUX4 R71A. Quantitative RT-PCR was performed 24 hours later for the DUX4 target genes PRAMF12, ZSCAN4, TRIM43, and LEUTX, demonstrating reduced levels of DUX4 target gene expression in the R71A PTM mutant. [Figure 3B] Same as above. [Figure 4A]Figure 4A shows the localization of R71 in the crystal structure of DUX4 with DNA. Figure 4B shows GFP expression visualized 24 hours after cotransfection of HEK293 cells with the DUX4 activation fluorescent reporter (DRE) and wild-type DUX4, DUX4 R71A, or empty vector. The DUX4 R71A mutant showed reduced transactivation compared to the control. [Figure 4B] Same as above. [Figure 5A] We show that PRMT1 is a member of the DUX4 complex. Figure 5A provides a schematic diagram of the RIME assay. Figure 5B shows co-immunoprecipitation of DUX4 and PRMT1 in HEK293 cells. [Figure 5B] Same as above. [Figure 6A] We show that arginine methylation inhibitors protect against cell death in myoblasts. Myoblasts were transfected with no DNA or DUX4 in the presence or absence of increasing concentrations of SAA (Fig. 6A) or AdOX (Fig. 6B), and caspase assays were performed 48 hours later. [Figure 6B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present disclosure provides methods and uses for treating, ameliorating, delaying progression of, and / or preventing muscular dystrophy or cancer, including, but not limited to, facioscapulohumeral muscular dystrophy (FSHD) or sarcoma. More specifically, disclosed herein are methods and uses of protein arginine methylation inhibitors for inhibiting arginine methylation in dual homeobox 4 (DUX4) protein. Even more specifically, the present disclosure provides methods of using protein arginine methylation inhibitors for inhibiting methylation of DUX4 protein, resulting in reduced DUX4-activated cell death, including reduced DUX4-activated muscle cell death.

[0042] Thus, the present disclosure provides methods of using protein arginine methylation inhibitors, including but not limited to salvianolic acid A (SAA) or a derivative thereof, or adenosine dialdehyde (ADOX) or a derivative thereof, to inhibit methylation of arginine residues in DUX4 protein in cells, and in some examples, in cells of subjects at risk for or suffering from muscular dystrophy. In some embodiments, such muscular dystrophy is associated with overexpression of DUX4 protein. In some embodiments, such muscular dystrophy is FSHD.

[0043] The present disclosure provides methods for suppressing or inhibiting methylation of arginine amino acids in DUX4 protein, because such inhibition of DUX4 methylation in muscle cells is associated with a decrease in DUX4-induced cell death. Accordingly, in some embodiments, the products and methods described herein are used to treat, ameliorate, delay progression, and / or prevent muscular dystrophies associated with elevated levels of DUX4 protein, including, but not limited to, FSHD.

[0044] The DUX4 gene encodes a protein of approximately 45 kDa (see UniProtKB-Q9UBX2(DUX4_HUMAN)). Derepression of the DUX4 gene is involved in the pathogenesis of FSHD. Derepression can occur through two known mechanisms: D4Z4 repeat contraction or mutations in the chromatin-modifying genes SMCHD1 or DNMT3B. In the former, in unaffected subjects, the D4Z4 sequence consists of 11–100 repeats, whereas in FSHD1 patients, the sequence is reduced to 1–10 repeats (PubMed:19320656). Either condition can cause DNA hypomethylation at chromosome 4q35, thereby creating a permissive chromosomal environment for DUX4 expression.

[0045] DUX4 is located in the D4Z4 macrosatellite repeats, which are epigenetically repressed in somatic tissues. D4Z4 chromatin relaxation in FSHD1 leads to inefficient epigenetic repression of DUX4 and a divergent pattern of DUX4 protein expression in a subset of skeletal muscle nuclei. Ectopic expression of DUX4 in skeletal muscle activates the expression of stem cell and germ cell genes, and when overexpressed in somatic cells, DUX4 can ultimately lead to cell death.

[0046] Each D4Z4 repeat unit contains an open reading frame (designated DUX4) encoding two homeoboxes, and the repeat sequence and ORF are conserved in other mammals. The encoded protein has been reported to function as a transcriptional activator of many genes, including several thought to be FSHD disease biomarkers, including ZSCAN4, PRAMEF12, TRIM43, and MBD3L2 (PMID: 24861551). Contraction of macrosatellite repeats causes autosomal dominant FSHD. Alternative splicing results in multiple transcript variants.

[0047] In some embodiments of the present disclosure, DUX4 nucleic acids and proteins are provided. In some embodiments, a nucleic acid encoding human DUX4 is represented by the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of human DUX4 is represented by the amino acid sequence set forth in SEQ ID NO: 2. In various embodiments, the methods of the present disclosure also target isoforms and variants of DUX4 polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the methods of the present disclosure target isoforms and variants of human DUX4 represented by the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the variant comprises 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, and 70% identity to the amino acid sequence set forth in SEQ ID NO:2.

[0048] [Table 1]

[0049] Currently, there is no cure for FSHD, and despite its relative prevalence among muscular dystrophies, few FSHD-targeted translational trials have been published. Although several FSHD candidate genes have been identified, a number of recent studies support the pro-apoptotic DUX4 gene, which encodes a transcription factor, as a major contributor to FSHD pathogenesis. Thus, in brief, DUX4 overexpression is the primary pathogenic lesion underlying FSHD (Chen et al., (2016) Mol Ther 24, 1405-1411; Ansseau et al. (2017) Genes (Basel) 8; Lek et al. (2020) Sci Transl Med 12; Himeda et al. (2016) Mol Ther 24, 527-535; DeSimone et al. (2019) Sci Adv 5, 12; Lim et al. (2020) Proc Natl Acad Sci USA 117, 16509-16515; Wallace et al. (2018), supra; Rojas et al. (2020) J Pharmacol Exp Ther. Sep;374(3):489-498).

[0050] In some embodiments, the present disclosure provides small molecule inhibitors of arginine methylation for use in inhibiting or downregulating DUX4-induced cell death and / or treating muscular dystrophies associated with DUX4-induced cell death, such as FSHD. Arginine methylation is enzymatically catalyzed by a family of proteins, arginine methyltransferases (PRMTs), which can activate or repress gene expression depending on the cellular context. Given the strong correlation between PRMTs and pathophysiology, there is significant interest in understanding the molecular mechanisms of PRMTs in disease and in developing potent PRMT inhibitors.

[0051] The present disclosure provides small molecule inhibitors of arginine methylation (PRMT inhibitors or RMT inhibitors) for inhibiting arginine methylation of DUX4 to reduce DUX4-mediated cell death and DUX4-mediated gene activation associated with muscular dystrophy, including, but not limited to, FSHD. As used herein, the terms "protein arginine methyltransferase (PRMT) inhibitor" or "inhibitor of PRMT" and "arginine methyltransferase (RMT) inhibitor" or "inhibitor of RMT" are used interchangeably. In some embodiments, such small molecule PRMT inhibitor is salvianolic acid or a derivative thereof, or adenosine dialdehyde (ADOX) or a derivative thereof.

[0052] In some embodiments, the small molecule inhibitor of arginine methyltransferase (PRMT) is at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof;

[0053] [ka]

[0054] In the formula, R 1 is H or C1-C6 alkyl, and X 1 is CH or N. Suitable C1-C6 alkyl groups include straight-chain and branched C1-C6 alkyl groups, including, but not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, hexyl, isopropyl, isobutyl, isopentyl, isohexyl, sec-butyl, sec-pentyl, 3-pentyl, sec-isopentyl, sec-hexyl, neo-pentyl, tert-butyl, tert-pentyl, and tert-hexyl.

[0055] In some embodiments, compound I is compound Ia,

[0056] [ka]

[0057] or a salt, hydrate, or stereoisomer thereof. In some embodiments, compound Ia is a hydrate of the formula:

[0058] [ka]

[0059] x is between 0.5 and 10. In some embodiments, compound I or Ia is salvianolic acid or a derivative thereof.

[0060] In some embodiments, compound II is compound IIa,

[0061] [ka]

[0062] or a salt, hydrate, or stereoisomer thereof. In some embodiments, compound II or IIa is adenosine dialdehyde (ADOX) or a derivative thereof.

[0063] Salvia miltiorrhiza (SM) has long been used in traditional Chinese medicine for the treatment of cardiovascular and liver diseases. Extracts from this plant confer potent hepatoprotective activity both in vitro and in vivo. Hase et al., Planta Med. 63:22-6 (1997). Magnesium lithospermate B may be one of the main active components of SM that protects the liver (Liu et al., Chung Kuo Chung Hsi I Chih Ho Tsa Chih 13:352-3, 326 (1993)). SM also contains antioxidants that apparently aid in membrane damage repair when treating viral myocarditis (Meng et al., Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 12:345-7, 324-5 (1992)). Patients suffering from chronic hepatitis B have responded to treatment with SM and / or Polyporus Umbellatus polysaccharidic (PUP) (Xiong, Chung Kuo Chung Hsi I Chieh Ho Tsa Chih 13:33-5, 516-7 (1993)). Herbal extracts from SM have also demonstrated anti-HIV activity (U.S. Pat. No. 5,178,865) and anti-hepatitis activity (International PCT Application No. 98 / 24460, Chinese Patent Application No. 1,192,922, and Chinese Patent Application No. 1,192,918). Antiviral agents active against herpes, polio, measles, varicella-zoster, cytomegalovirus, DNA viruses, and RNA viruses have been described, containing at least one herbal medicine from the roots of Salvia miltiorrhiza Bunge (European Patent No. 568,001). Salvia extracts have also been prepared as anti-herpes virus agents (US Pat. No. 5,411,733).

[0064] Some forms of salvianolic acid (e.g., salvianolic acid A and acetylsalvianolic acid) have been described to have antioxidant properties (Lin et al., J. Biochem. Pharmacol. 51:1237-1241 (1996)). Salvianolic acid has also been indicated for the prevention of liver damage and fibrosis associated with its anti-lipid peroxidation effects (Hu et al., Acta Pharmacol. Sin. 18:478-480 (1997)) and for use in the treatment of coronary artery disease (Japanese Patent No. 2,131,423). Additional forms of salvianolic acid described in the literature include those isolated from aqueous extracts of Salvia cavaleriei (e.g., salvianolic acids A, B, CH, and I) (Zhang et al., Planta Med. 60:70-72 (1994)) or from S. miltiorrhiza (e.g., salvianolic acid K, caffeic acid trimer) (Kasimu et al., J. Biochem. Pharmacol. 51:1237-1241 (1996)). al., Chem. Pharm. Bull. 46:500-504 (1998) and Tezuka et al., Chem Pharm. Bull. 46:107-112 (1998). Salvianolic acids F2 and F3 can be synthetically prepared as described in Dalla et al., Tetrahedron 55:6923-6930 (1999) and Dalla et al., Tetrahedron Lett. 39:8285-8286 (1998). Additional members of the Salvia family include S. bowleyana, S. deserta, S. miltiorhiza var. miltiorhiza f. alba, S. paramiltiorhiza, S. paramiltiorhiza f. purpureo-rubra, S. przewalskii, S. przewalskii var. mandarinorum, and S. sinica. The various species of Salvia officinalis, including Salvia officinalis (S. purpurea), and Salvia trijuga (S. trijuga) (Kasiumu et al., 1998), can be used as sources for obtaining Salvia miltiorrhiza. Methods for producing plants with elevated levels of secondary metabolites, such as salvianolic acid, have also been described. See U.S. Patent No. 5,869,340 (1999).In various embodiments, salvianolic acid A and its derivatives are used in the methods of the present disclosure as inhibitors of PRMTs.

[0065] Adenosine dialdehyde (ADOX or AdOx) is an indirect methyltransferase inhibitor widely used to accumulate methyl-accepting proteins in a hypomethylated state for protein methylation analysis. ADOX is an adenosine analog and an S-adenosylmethionine-dependent methyltransferase inhibitor. ADOX inhibits S-adenosyl-L-homocysteine ​​hydrolase, resulting in the accumulation of S-adenosyl-L-homocysteine ​​(AdOx), a product inhibitor of methyltransferases that utilize S-adenosyl-L-methionine (AdoMet) as the methyl group donor. ADOX inhibited the Tax-activated NF-κB pathway, resulting in the reactivation of p53 and the induction of p53 target genes. Analysis of the NF-κB pathway showed that ADOX treatment led to the disassembly of the IκB kinase complex and the inhibition of NF-κB through the stabilization of the NF-κB inhibitor IκBα. ADOX induced G2 / M cell cycle arrest and cell death in HTLV-1 transformed lymphocytes, but not in control lymphocytes (Dasgupta, et al. J Virol. 2008, 82(1):49-59). In various embodiments, ADOX and its derivatives are used in the methods of the present disclosure as inhibitors of PRMTs.

[0066] In some embodiments, the present disclosure includes compositions comprising a small molecule inhibitor of PRMT described herein in combination with a pharmaceutically acceptable carrier. In various aspects, such compositions comprise or further comprise other ingredients, such as diluents, excipients, and / or adjuvants. Acceptable carriers, diluents, excipients, and adjuvants are nontoxic to recipients and preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparaginine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronic®, or polyethyl glycol (PEG).

[0067] The present disclosure also includes compositions described herein comprising any small molecule inhibitor of a PRMT described herein, alone or in combination with another small molecule inhibitor of a PRMT, or in combination with another known therapy for treating MD, including but not limited to FSHD.

[0068] Sterile injectable solutions are prepared by incorporating the small molecule inhibitor of PRMT in the required amount into a suitable solvent, along with various other ingredients listed above, as needed, followed by filtration sterilization.Generally, dispersions are prepared by incorporating sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other ingredients listed above that are required.For the preparation of sterile powders for sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of the active ingredient plus any additional desired ingredients from their previously sterile-filtered solutions.

[0069] The present disclosure provides a method for inhibiting arginine methylation of dual homeobox 4 (DUX4) protein in a cell, comprising contacting the cell with an effective amount of at least one inhibitor of protein arginine methyltransferase (PRMT), wherein the inhibitor of PRMT is at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof;

[0070] [ka]

[0071] In the formula, R 1 is H or C1-C6 alkyl, and X 1 is CH or N. The present disclosure provides a method for reducing dual homeobox 4 (DUX4)-associated apoptotic cell death and / or reducing DUX4 target gene activation in a cell, comprising contacting the cell with an effective amount of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof;

[0072] [ka]

[0073] In the formula, R 1 is H or C1-C6 alkyl, and X 1 is CH or N. The present disclosure provides a method for treating an arginine methyltransferase (RMT) disorder in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof;

[0074] [ka]

[0075] In the formula, R 1 is H or C1-C6 alkyl, and X 1 is CH or N. In some embodiments, the cells are in a subject at risk for or suffering from muscular dystrophy. In some embodiments, the subject is a human subject.

[0076] In some embodiments, the PRMT disorder is muscular dystrophy. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, compound I is compound Ia,

[0077] [ka]

[0078] or a salt, hydrate, or stereoisomer thereof. In some embodiments, compound Ia is a hydrate of the formula:

[0079] [ka]

[0080] x is between 0.5 and 10. In some embodiments, compound II is compound IIa,

[0081] [ka]

[0082] or a salt, hydrate, or stereoisomer thereof. In some embodiments, the present disclosure provides methods for inhibiting protein arginine methylation of Dual Homeobox 4 (DUX4) protein in a cell, methods for reducing Dual Homeobox 4 (DUX4)-associated apoptotic cell death and / or reducing DUX4 target gene activation in a cell, methods for treating an arginine methyltransferase disorder, or methods for treating muscular dystrophy or cancer, comprising using a compound obtained from an extract of a Salvia plant (e.g., Salvia miltiorrhiza).

[0083] In some embodiments, the compound comprises Compound A or a salt, hydrate, or stereoisomer thereof, and / or Compound B or a salt, hydrate, or stereoisomer thereof;

[0084] [ka]

[0085] In the formula, R 1 is H or C1-C6 alkyl. In some embodiments, the compound is salvianolic acid or a derivative thereof. For example, in some embodiments, the compound is Compound I or a salt, hydrate, or stereoisomer thereof:

[0086] [ka]

[0087] In the formula, R 1 is H or C1-C6 alkyl. In some embodiments, R 1 is H. The compounds described herein can exist in free form or as salts as needed.These pharmaceutically acceptable salts are particularly interesting because they are useful for administering the compounds described below for medical purposes.Pharmaceutically unacceptable salts are useful in the manufacturing process, for the purpose of isolation and purification, and in some cases, for the separation of stereoisomeric forms of the compounds described herein or their intermediates.

[0088] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and lower animals without undue adverse side effects, such as toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio.

[0089] Pharmaceutically acceptable salts are well known in the art.For example, S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference.Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases.These salts can be prepared in situ during the final isolation and purification of compounds.

[0090] When the compounds described herein contain a basic group, or a sufficiently basic bioequivalent, an acid addition salt can be prepared by 1) reacting the purified compound in its free base form with a suitable organic or inorganic acid, and 2) isolating the salt so formed. In practice, the acid addition salt may be in any convenient form for use, and use of the salt amounts to use of the free base form.

[0091] Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxybenzoate, benzoic acid, benzoic acid, bisulfate, borate, butyrate, camphor ...sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxybenzoate, -ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.

[0092] When the compounds described herein contain a carboxy group or a sufficiently acidic bioequivalent, a base addition salt can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base, and 2) isolating the salt thus formed. In practice, the use of a base addition salt may be more convenient, and the use of the salt form corresponds to the use of the free acid form. Salts derived from appropriate bases include alkali metals (e.g., sodium, lithium, and potassium), alkaline earth metals (e.g., magnesium and calcium), ammonium, and N +(Ci-4 alkyl) salts are included. The present disclosure also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

[0093] Base addition salts include pharmaceutically acceptable metal salts and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum. Sodium and potassium salts are generally preferred. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates. Suitable inorganic base addition salts are prepared from metal bases including sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, and the like. Suitable amine base addition salts are prepared from amines, which are frequently used in pharmaceutical chemistry because of their low toxicity and acceptability for medical use. Ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, dicyclohexylamine, etc.

[0094] Other acids and bases may be used in the preparation of salts which, while not themselves pharmaceutically acceptable, are useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts.

[0095] It is to be understood that the present disclosure includes mixtures / combinations of different pharmaceutically acceptable salts, as well as mixtures / combinations of compounds in free form and pharmaceutically acceptable salts. In some embodiments, the compound is compound Ia (ie, salvianolic acid A (SAA)).

[0096] [ka]

[0097] In some embodiments, the compound is a hydrate of compound Ia of the following formula:

[0098] [ka]

[0099] In the formula, x is 0.5 to 10. As used herein, the term "hydrate" refers to a chemical compound formed by the interaction of water with a compound, including, for example, a hemihydrate, a monohydrate, a dihydrate, a trihydrate, etc.

[0100] In some embodiments, the compound is Compound II or a salt, hydrate, or stereoisomer thereof;

[0101] [ka]

[0102] In the formula, X 1 is CH or N. In some embodiments, X is CH or N, such that compound II is compound IIa or a salt, hydrate, or stereoisomer thereof. 1 is N.

[0103] [ka]

[0104] In some embodiments, the present disclosure provides a method of inhibiting protein arginine methyltransferase (PRMT or RMT), comprising contacting PRMT with an effective amount of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof.

[0105] In some embodiments, the present disclosure provides a method for reducing DUX4-induced apoptosis or cell death and / or DUX4-induced transactivation or gene expression, comprising contacting a DUX4 protein with an effective amount of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof. In some embodiments, the DUX4 protein is in a cell. In some embodiments, the DUX4 protein is in a subject's cell. In some embodiments, the cell is in a human subject. In some embodiments, the human subject has or is at risk of having muscular dystrophy. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

[0106] In some embodiments, the present disclosure provides a method of treating a protein arginine methyltransferase (PRMT or RMT) disorder in a patient, comprising administering to the patient a therapeutically effective amount of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof. In some embodiments, the PRMT disorder is a muscular dystrophy, including, but not limited to, FSHD.

[0107] In some embodiments, in conjunction with any of the above or below embodiments, the disclosed methods include using compound Ia, or a salt, hydrate, or stereoisomer thereof. In some embodiments, the disclosed methods include using a hydrate of compound Ia.

[0108] In some embodiments, in conjunction with any other embodiment above or below, the disclosed methods include using compound II or a salt, hydrate, or stereoisomer thereof. In some embodiments, the disclosed methods include using compound IIa or a salt, hydrate, or stereoisomer thereof.

[0109] In some embodiments, DUX4-induced apoptosis or cell death and / or DUX4-induced transactivation is reduced by at least or about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 99, or 100 percent in a cell or subject by the methods provided herein.

[0110] The method includes administering an effective dose or effective doses of a composition comprising a small molecule inhibitor of PRMT of the present disclosure to a subject, including an animal (such as a human) in need thereof. If the dose is administered before the onset of muscular dystrophy, the administration is prophylactic. If the dose is administered after the onset of muscular dystrophy, the administration is therapeutic. In embodiments of the present disclosure, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the muscular dystrophy being treated, slows or prevents the progression of muscular dystrophy, slows or prevents the progression of muscular dystrophy, reduces the extent of disease, results in remission (partial or complete) of muscular dystrophy, and / or prolongs survival. In some embodiments, the muscular dystrophy is FSHD.

[0111] Combination therapies are also contemplated by the present disclosure. As used herein, combination includes simultaneous or sequential treatment. Combinations of the methods of the present disclosure with standard medical treatments (e.g., corticosteroids and / or immunosuppressants) or other inhibitory RNA constructs are specifically contemplated, as are combinations with other therapies, such as those disclosed in WO 2013 / 016352, the entire contents of which are incorporated herein by reference.

[0112] Administration of an effective dose of a small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT can be by routes standard in the art, including, but not limited to, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraventricular, intrathecal, intraosseous, intraocular, rectal, or vaginal. The route of administration can be selected and / or adapted by one skilled in the art taking into consideration the disease state being treated and the target cells / tissues, such as cells expressing DUX4. In some embodiments, the route of administration is intramuscular. In some embodiments, the route of administration is intravenous. In some aspects, an effective dose is delivered by a systemic route of administration, i.e., systemic administration. Systemic administration is a route of administration into the circulatory system such that the entire body is affected. In various aspects, such systemic administration is via enteral administration (drug absorption through the gastrointestinal tract) or parenteral administration (generally via injection, infusion, or implantation). In various aspects, an effective dose is delivered by a combination of routes. For example, in various embodiments, the effective dose is delivered intravenously and / or intramuscularly, or intravenously and intracerebroventricularly, etc. In some embodiments, the effective doses are delivered sequentially or sequentially. In some embodiments, the effective doses are delivered simultaneously. In various embodiments, the route of administration is selected and / or adapted by one skilled in the art taking into consideration the status or condition of the disease or disorder being treated, the status, condition, or age of the subject, and the target cells / tissues targeted by the small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT.

[0113] In some embodiments, the actual administration of the small molecule inhibitor of a PRMT disclosed herein or a composition comprising a small molecule inhibitor of a PRMT can be achieved by using any physical method that will deliver the small molecule inhibitor of a PRMT to the target tissue of a subject, i.e., a human or animal subject. Administration according to the present disclosure includes, but is not limited to, injection into muscle, the bloodstream, the central nervous system, and / or direct injection into the brain, liver, or other organs. Simply resuspending a small molecule inhibitor of a PRMT in phosphate-buffered saline has been shown to be sufficient to provide a useful vehicle for muscle tissue expression, and there are no known limitations on the carriers or other components that can be co-administered with the small molecule inhibitor of a PRMT. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have already been developed and can be used in the practice of the present disclosure. The small molecule inhibitor of a PRMT can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0114] For intramuscular injection, adjuvant solutions such as sesame oil or peanut oil, or aqueous propylene glycol solutions, as well as sterile aqueous solutions, can be used. Such aqueous solutions can be buffered, if necessary, and the liquid diluent can first be made isotonic with saline or glucose. Solutions of small molecule inhibitors of PRMTs or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions of small molecule inhibitors of PRMTs can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary storage and use conditions, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed can be readily obtained by standard techniques well known to those skilled in the art.

[0115] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. In some embodiments, the proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0116] In some embodiments, the formulation contains a stabilizer. The term "stabilizer" refers to a substance or excipient that protects the formulation from harmful conditions, such as those encountered during heating or freezing, and / or extends the stability or shelf life of the formulation in a stable state. Examples of stabilizers include, but are not limited to, sugars such as sucrose, lactose, and mannose, sugar alcohols such as mannitol, amino acids such as glycine or glutamic acid, and proteins such as human serum albumin or gelatin.

[0117] In some embodiments, the formulation includes an antimicrobial preservative. The term "antimicrobial preservative" refers to any substance added to a composition that inhibits the growth of microorganisms that may be introduced upon repeated puncture of the vial or container used. Examples of antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.

[0118] Sterile injectable solutions are prepared by incorporating the small molecule inhibitor of PRMT in the required amount into a suitable solvent, along with various other ingredients listed above, as needed, followed by filtration sterilization.Generally, dispersions are prepared by incorporating sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other ingredients listed above that are required.For the preparation of sterile powders for sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces a powder of the active ingredient plus any additional desired ingredients from their previously sterile-filtered solutions.

[0119] The present disclosure provides methods for administering to a cell or a subject in need thereof an effective dose (or doses administered essentially simultaneously or at intervals) of a small molecule inhibitor of PRMT designed to reduce DUX4-induced apoptosis or cell death and / or DUX4-activated gene expression (or DUX4 target gene activation or transactivation). In some embodiments, an effective dose is therefore a therapeutically effective dose. A therapeutically effective dose is determined by a clinician or team of clinicians depending on the subject's age, sex, height, weight, and condition.

[0120] In some embodiments, the initial dose is followed by a second, higher dose. In some embodiments, the initial dose is followed by a second, same dose. In some embodiments, the initial dose is followed by one or more smaller doses. In some embodiments, the initial dose is followed by multiple doses, which may be the same dose or larger doses.

[0121] The in vivo method comprises administering an effective dose or effective doses of the small molecule inhibitor of PRMT disclosed herein or a composition comprising a small molecule inhibitor of PRMT to a subject (including a human subject) in need thereof. Thus, provided are methods of administering an effective dose (or doses administered essentially simultaneously or at intervals) of a small molecule inhibitor of PRMT described herein or a composition comprising a small molecule inhibitor of PRMT to a subject in need thereof. Administration is prophylactic when the dose or doses are administered before the onset of a disorder / disease. Administration is therapeutic when the dose or doses are administered after the onset of a disorder / disease. An effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, delays or prevents progression to the disorder / disease state, delays or prevents progression of the disorder / disease state, reduces the extent of the disease, results in remission (partial or complete) of the disorder / disease state, and / or prolongs survival.

[0122] In some embodiments, the compositions and methods of the present disclosure are used in treating, ameliorating, or preventing diseases such as muscular dystrophy (MD). In various aspects, the MD is FSHD. FSHD is the most commonly inherited muscular dystrophy, affecting an estimated 870,000 people. Classic descriptions of FSHD symptoms include progressive muscle weakness in the face, shoulder girdle, and arms, but the disease can manifest more broadly, including muscles of the trunk and lower limbs. Variability is also common within individuals, and asymmetric weakness is common. Age of onset can range from early childhood to adulthood and is usually related to disease severity, with earlier onset often associated with more severe muscle weakness. Most patients with FSHD have a normal lifespan, but respiratory failure can occur, and the disease can be debilitating, with approximately 25% of affected individuals becoming wheelchair dependent by their 50s, and even earlier in more severe forms of the disease, while others remain ambulatory throughout their lives.

[0123] FSHD is caused by abnormal expression of the double homeobox 4 gene (DUX4), which produces a transcription factor toxic to skeletal muscle. DUX4 normally functions during the two-cell stage of human development but is subsequently repressed in essentially all other tissues, except perhaps the testis. In the skeletal muscle of people with FSHD, specific genetic and epigenetic factors simultaneously enable DUX4 derepression, which then initiates several aberrant gene expression cascades, including those involved in abnormal differentiation, oxidative stress, inflammatory infiltration, cell death, and muscle atrophy.

[0124] In families known to have pathological FSHD, the disclosed methods, in various embodiments, are methods of preventing the disease, being performed before the onset of the disease. In various other embodiments, the disclosed methods are performed after diagnosis, and thus are methods of treating or ameliorating the disease. Thus, the small molecule inhibitors of PRMTs described herein and compositions comprising the small molecule inhibitors of PRMTs described herein are used to inhibit DUX4-induced cell death and DUX4-induced target gene expression and DUX4 signaling activated by arginine methylation in the treatment, amelioration, or prevention of muscular dystrophies associated with DUX4 overexpression, such as FSHD.

[0125] In some embodiments, the compositions and methods of the present disclosure are used to treat, ameliorate, or prevent diseases such as cancer. DUX4 has been shown to be activated in several cancer types, where it functions to mask tumor cells from the immune system (Chew et al., Dev. Cell 2019 Sep 9;50(5):658-71). For example, DUX4 protein fusions are known to cause cancers such as rhabdomyosarcoma and Ewing's sarcoma. CIC-DUX4 gene fusions induce sarcomas and promote sarcoma metastasis (Yoshimoto et al., Cancer Res. 2017 Jun 1;77(11):2927-2937, Okimoto et al., J Clin Invest. 2019;129(8):3401-3406). Thus, the small molecule inhibitors of PRMTs described herein, and compositions comprising the small molecule inhibitors of PRMTs described herein, are used to inhibit DUX4-induced cell death and DUX4-induced gene expression and DUX4 signaling activated by arginine methylation in the treatment, amelioration, or prevention of cancer.

[0126] Molecular, biochemical, histological, and functional outcome measures indicate the therapeutic efficacy of the products and methods disclosed herein for (1) reducing apoptosis or cell death, (2) reducing DUX4-induced gene expression or transactivation following arginine methylation of the DUX4 protein, and (3) treating muscular dystrophies, such as FSHD. Outcome measures are described, for example, in Dyck and Thomas, Peripheral Neuropathy, Elsevier Saunders, Philadelphia, PA, 4 thEdition, Volume 1 (2005), Chapters 32, 35, and 43, and Burgess et al., Methods Mol. Biol., 602:347-393 (2010). Outcome measures include, but are not limited to, reduction or elimination of DUX4-induced cell death or gene activation in affected cells and tissues. Inhibition of arginine methylation of DUX4 in cells is detected by methods known in the art, including but not limited to, those described herein, including in the Examples, before and after administration of a small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT to determine improvement.

[0127] In some embodiments, methylation of DUX4 protein in cells of a subject is reduced after administration of a small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT compared to methylation of DUX4 protein before administration of the small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT. In some embodiments, methylation of DUX4 protein is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, or more than at least about 100%. In various embodiments, improved muscle strength, improved muscle function, and / or improved mobility and stamina represents an improvement of at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, or at least more than about 100%.

[0128] In some embodiments, apoptosis or cell death of cells in a subject is reduced after administration of a small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT compared to apoptosis or cell death of cells in the subject before administration of the small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT, hi some aspects, apoptosis or cell death of cells in a subject is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, or more than at least about 100%. In various embodiments, improved muscle strength, improved muscle function, and / or improved mobility and stamina represents an improvement of at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100%, or at least more than about 100%.

[0129] Other outcome measures include measuring serum creatine kinase (CK) levels in subjects before and after treatment. Increased CK levels are a hallmark of muscle damage. In patients with muscular dystrophy, CK levels are significantly elevated above the normal range (10-100 times normal levels since birth). When elevated CK levels are found in a blood sample, it usually indicates that muscles are being destroyed by some abnormal process, such as muscular dystrophy or inflammation. Therefore, a positive therapeutic outcome of treatment using the disclosed method is a decrease in serum creatine kinase levels after administration of a small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT, compared to the serum creatine kinase level before administration of a small molecule inhibitor of PRMT or a composition comprising a small molecule inhibitor of PRMT.

[0130] Other outcome measures include measuring muscle strength, muscle function, mobility, stamina, or a combination of two or more thereof in a subject after treatment. Such outcome measures are important in determining the progression of muscular dystrophy in a subject and are measured by various tests known in the art. Some of these tests include, but are not limited to, the 6-minute walk test, the stand-up test, the 4-step ascent test, the 4-step ascent and descent test, the North Star Gait Assessment (NSAA) test, the 10-meter timed test, the 100-meter timed test, the handheld dynamometer (HHD) test, the timed up-and-go test, the Bayley-III gross motor subscale score, the maximal voluntary isometric contraction test (MVICT), or a combination of two or more thereof.

[0131] Combination therapies are also encompassed by the present disclosure. As used herein, combination includes both simultaneous and sequential treatment. Combination of the methods described herein with standard medical and supportive care is specifically contemplated, as is combination with therapies such as glucocorticoids. All types of glucocorticoids are encompassed for use in the combination therapies disclosed herein. Such glucocorticoids include, but are not limited to, prednisone, prednisolone, dexamethasone, deflazacort, beclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, and triamcinolone.

[0132] Other combination therapies included in the present disclosure are combinations of small molecule inhibitors of PRMTs or compositions comprising small molecule inhibitors of PRMTs described herein with U7-snRNA, miRNA-based gene therapy, small molecule inhibitors of DUX4 expression, oligonucleotides that inhibit DUX4 via RNAi or RNAse H or exon skipping mechanisms, or U7-snRNA plus a theoretical CRISPR-based gene therapy approach.

[0133] "Treating" includes improving or inhibiting one or more symptoms of muscular dystrophy, including, but not limited to, muscle wasting, muscle weakness, muscle tonotonia, skeletal muscle problems, retinal abnormalities, hip weakness, facial weakness, abdominal muscle weakness, joint and spinal abnormalities, leg weakness, shoulder weakness, hearing loss, muscle inflammation, and asymmetric weakness.

[0134] The present disclosure also provides a kit comprising an inhibitor of PRMT or a composition comprising the inhibitor of PRMT of the present disclosure. In the context of the present disclosure, the term "kit" refers to two or more components, one of which corresponds to the inhibitor of PRMT of the present disclosure or a composition comprising the inhibitor of PRMT, and the other of which corresponds to a container, recipient, instructions, or other item. Thus, in various embodiments, a kit is a set of products sufficient to achieve a particular goal and can be sold as a single unit.

[0135] The kit may comprise one or more recipients (vials, ampoules, containers, syringes, bottles, bags, etc.) of any suitable shape, size, and material containing the PRMT inhibitor or a composition comprising a PRMT inhibitor of the present disclosure in a suitable dosage amount for administration (see above). The kit may additionally comprise instructions or instructions for use (e.g., in the form of a leaflet or instruction manual), a means for administering the PRMT inhibitor or a composition comprising a PRMT inhibitor, such as a syringe, pump, infuser, etc., a means for reconstituting the PRMT inhibitor or a composition comprising a PRMT inhibitor, and / or a means for diluting the PRMT inhibitor or a composition comprising a PRMT inhibitor.

[0136] In some embodiments, the kits include labels and / or instructions that describe the use of the reagents provided in the kit. The kits also optionally include a catheter, syringe, or other delivery device for delivering one or more of the compositions used in the methods described herein.

[0137] The present invention also provides kits for single-dose administration units or for multiple doses. In some embodiments, the present disclosure provides kits including single-chamber and multi-chamber pre-filled syringes.

[0138] This entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of this document. The disclosure also includes all embodiments of the disclosure that are somewhat narrower in scope than, for example, the variations specifically mentioned above. With respect to aspects of the disclosure described as genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure described or claimed as "a" or "an," these terms should be understood to mean "one or more" unless the context clearly dictates a more limited meaning.

[0139] Unless otherwise specified, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the present disclosure.

[0140] Whenever the term "and / or" is used herein, it includes the meaning of "and", "or" and "all or any other combination of the elements connected by that term". As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, it also includes the specific number, for example, about 10 includes 10.

[0141] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," should be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," or, as sometimes used herein, with the term "having."

[0142] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0143] In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0144] It is understood that this disclosure is not limited to the particular methodology, protocols, materials, reagents, and substances, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the presently disclosed subject matter, which is defined solely by the claims.

[0145] All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) cited throughout the body of this specification, whether supra or infra, are hereby incorporated by reference in their entirety. To the extent that material incorporated by reference is inconsistent or contradictory with this specification, this specification takes precedence over any such material.

[0146] A better understanding of the present disclosure and its advantages will be obtained from the following examples, which are provided for illustrative purposes only. The examples are not intended to limit the scope of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes may be suggested to those skilled in the art in light of these, but are within the spirit and scope of the present application and the scope of the appended claims. [Example]

[0147] Additional aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. Example 1 Materials and Methods Mutagenesis DUX4-modified mutant plasmids (single, double, triple, HOX1_methyl-null, basic, and HOX1_methyl-mimetic mutant constructs) were constructed using recombinant PCR. Mutations were constructed by amplifying the DUX4 ORF using PCR with primers containing the mutation site, using the CMV-driven wild-type DUX4 plasmid (AAV.DUX4.V5) as a template. The entire mutant DUX4 ORF was amplified, gel-purified, and cloned into PCR-blunt II-topo vector prior to sequence verification. The DUX4 mutant ORF was then cloned into AAV.CMV.DUX4 or AAV.CMV.eGFP to replace either wild-type DUX4 or eGFP, respectively. Using AAV.CMV.eGFP as the base vector, phospho-null, phospho-mimetic, methyl-null, basic, and methyl-mimetic mutants were synthesized by Genscript with N-terminal NheI and C-terminal Acc65I restriction enzyme sites flanking the DUX4 ORF.

[0148] cell culture Human embryonic kidney cells (HEK293) and human immortalized myoblasts (WS236, 15V, biceps, and unaffected control cells) were maintained as previously described. 9 Briefly, HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum, L-glutamine, and penicillin / streptomycin at 37°C in 5% CO. Human immortalized myoblasts were cultured in LHCN medium containing DMEM supplemented with 16% Medium 199, 15% fetal bovine serum, 30 ng / ml zinc sulfate, 1.4 μg / ml vitamin B12, 55 ng / ml dexamethasone, 2.5 ng / ml human growth factor, 10 ng / ml fibroblast growth factor, 20 mM HEPES, and penicillin / streptomycin.

[0149] Protein immunoprecipitation HEK293 cells were transfected with a total of 4 μg of AAV.CMV.DUX4.V5, PRMT1-GFP, or PRKACA-Flag (1 × 10) using Lipofectamine 2000. 6Cells were transfected in suspension at 1000 x g (1000 x g) at 1000 x g (1000 x g) and harvested 16 hours later using cold 1x PBS. Cells were pelleted and lysed in Buffer A containing 137 mM NaCl, 50 mM Tris, pH 7.5, 1% NP-40, a protease inhibitor cocktail (Sigma), and phosphatase inhibitors including sodium pyrophosphate, β-glycerol phosphate, sodium fluoride, and sodium orthovanadate. All steps were performed at 4°C or on ice. The lysate was incubated with protein agarose G for 1 hour with rotation. The supernatant was then incubated overnight with anti-V5 antibody conjugated to agarose resin with rotation. The resin was washed five times with Buffer A and then resuspended in Buffer A supplemented with 1 mM DTT and 1x LDS-PAGE (Invitrogen) sample buffer. DUX4 complexes were eluted by boiling at 95°C for 10 minutes.

[0150] High-resolution mass spectrometry sample preparation and peptide digestion Immunoprecipitated protein samples were loaded onto TGX 4-15% precast gels (Bio-Rad), separated, and stained with Bio-Safe Coomassie (Bio-Rad). The band corresponding to DUX4 was excised. Potential disulfide bonds were reduced and alkylated. Gel pieces were subjected to overnight digestion with 800 ng of trypsin (Promega) and / or chymotrypsin (Promega) in 100 mM ammonium bicarbonate (Sigma) at 37°C. Peptides were extracted from the gel matrix, dried by vacuum centrifugation, and resuspended in loading buffer (2% acetonitrile, 0.1% formic acid).

[0151] Liquid chromatography and mass spectrometry (LC-MS / MS) Peptides were separated on a Thermo Dionex UltiMate 3000 RSLC HPLC system connected to a Thermo Orbitrap Fusion Tribrid mass spectrometer. Peptides were loaded onto a PepMap100 C18 microcolumn (5 μm, 100 Å, 0.3 x 50 mm) and desalted with 0.5% TFA in 2% acetonitrile for 4 min. The mobile phase solvents were Buffer A: 0.1% formic acid in water and Buffer B: 0.1% formic acid in acetonitrile. Peptides were eluted with a linear gradient of Buffer B (5–30%) at a flow rate of 300 nL / min over 140 min, followed by column wash and equilibration. Peptide separation was performed on a Thermo EASY-Spray PepMap C18 column (3 μm, 100 Å, 0.75 x 150 mm) operated at 275 °C and a spray voltage of 1.7 kV.

[0152] MS / MS data were collected on an Orbitrap Fusion operated in top speed mode with a 3-second cycle time. MS1 scans were collected at 60K resolution in the Orbitrap before HCD fragmentation at a normalized collision energy of 27%. Fragment ions were isolated in the quadrupole with an isolation window of 1.6 m / z and detected at 15K resolution in the Orbitrap. The AGC target was set to a maximum injection time of 4E5 or 50 ms for MS1 scans and 5E4 or 500 ms for MS2 scans to maximize ion series coverage. Dynamic exclusion was set to ±10 ppm for 30 seconds.

[0153] Mass spectrometry data analysis The raw data were converted to mxXML format using the MSConvert tool in ProteoWizard (v3.0.4624) and searched using MassMatrix search engine v2.4.2 against a database containing DUX4 sequences downloaded from UniProt (accession Q9UBX2) along with the C-terminal V5 epitope tag (GKPIPNPLLGLDST (SEQ ID NO: 3)) and common contaminating proteins (downloaded on June 22, 2015, total 234 entries). 10-14The peptide mass tolerance was set to 20 ppm with a fragment mass tolerance of 0.02 Da. Data were also searched using MASCOT (version 2.6.0, Matrix Science) against the UniProt human database containing the DUX4 sequence downloaded from UniProt (accession Q9UBX2) along with a C-terminal V5 epitope tag (GKPIPNPLLGLDST (SEQ ID NO: 3)). The peptide mass tolerance was set to 10 ppm with a fragment mass tolerance of 0.05 Da. For both search engines, variable modifications included acetylation of K, mono-, di-, or tri-methylation of K, mono- or di-methylation of R, oxidation of M, and phosphorylation of S, T, and Y. Carbamidomethylation of C was included as a fixed modification. Enzyme specificity was set for trypsin and chymotrypsin with a maximum of four missed cleavages. Search-generated peptides that also passed manual validation were included in the sequence coverage calculation.

[0154] The raw data were converted to mxXML format using the MSConvert tool in ProteoWizard (v3.0.4624) and searched using the MassMatrix search engine v2.4.2 against a database containing the DUX4 sequence downloaded from UniProt (accession Q9UBX2) along with the C-terminal V5 epitope tag (GKPIPNPLLGLDST (SEQ ID NO: 3)) and common contaminating proteins (downloaded June 22, 2015, a total of 234 entries). The peptide mass tolerance was set to 20 ppm with a fragment mass tolerance of 0.02 Da. Variable modifications included acetylation of K; mono-, di-, or tri-methylation of K; mono- or di-methylation of R; oxidation of M; and phosphorylation of S, T, and Y. Carbamidomethylation of C was included as a fixed modification. Enzyme specificity was set for trypsin and chymotrypsin with a maximum of four missed cleavages. Search-generated peptides that also passed manual validation were included in the calculation of sequence coverage.

[0155] quantitative PCR HEK293 cells and human myoblasts (5x10 cells / well) were transfected with 2µg of AAV.DUX4.V5 (wild-type or mutant construct) using Lipofectamine 2000 (Thermo Scientific). Cells were harvested 24 hours post-transfection in TRIzol RNA Isolation Reagent (Life Technologies). RNA was isolated, DNase-treated, and reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Human RPL13A (Hs01494366_g1), human ZSCAN4 (Hs00537549_m1), and human PRAMEF12 (Hs04193637_mH) were quantified using TaqMan gene expression assays (Applied Biosystems). Efficiency was comparable across all probes. RPL13A was used as a reference gene for normalization. Normalized expression (ΔΔCq) was calculated relative to control pClNeo-transfected cells.

[0156] Caspase-3 / 7 activation assay HEK293 cells were transfected with 100 ng of plasmid DNA (65,000 cells / well) using Lipofectamine 2000. 48 hours later, the assay was performed using the Apo-ONE Homogeneous Caspase-3 / 7 Assay (Promega). The protocol was followed according to the manufacturer's instructions. Briefly, 100 μl of reagent was added to each well, and the 96-well plate was gently rotated for 20 minutes. Relative fluorescence was monitored every hour for 4–6 hours.

[0157] Inhibitor studies were performed in human myoblasts transfected with 100 ng of DUX4 DNA (65,000 cells / well) using Lipofectamine 2000. The inhibitors adenosine dialdehyde or salvianolic acid A were added after 1.5 hours, and caspase assays were performed 48 hours later as described above.

[0158] Intracellular DUX4 activation reporter HEK293 cells (65,000 cells / well) were transfected in suspension with 100 ng of plasmid DNA (AAV.CMV.DUX4.V5 or mutants) and 100 ng of pLenti.DUX4-activated GFP using Lipofectamine 2000 (Thermo Scientific) and plated simultaneously in 96-well plates, as previously described (reference). GFP expression was quantified 24 and 48 hours after transfection using a SPECTRAmax M2 instrument (Molecular Devices). GFP expression was monitored visually with a fluorescence stereomicroscope (Leica M165 FC microscope, Leica Microsystems).

[0159] Rapid immunoprecipitation mass spectrometry (RIME) of endogenous proteins Cells were fixed with 1% formaldehyde for 8 minutes and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer followed by disruption using a Dounce homogenizer. The lysate was sonicated to shear the DNA to an average length of 300–500 base pairs. Genomic DNA (input) was prepared by treating an aliquot of chromatin with RNase, proteinase K, and heat to reverse crosslinks, followed by ethanol precipitation. The pellet was resuspended, and the resulting DNA was quantified using a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed for quantification of the total chromatin yield.

[0160] An aliquot of chromatin (100 μg) was precleared with protein G agarose beads (Invitrogen). The protein of interest was immunoprecipitated using 10 μg of antibody against V5 (Abcam, ab15828) and protein G magnetic beads. The protein complex was washed, and then trypsin was used to remove the immunoprecipitate from the beads and digested protein sample. The protein digest was separated from the beads and purified using a C18 spin column (Harvard Apparatus). Peptides were vacuum dried using a speedvac.

[0161] The digested peptides were analyzed by LC-MS / MS on a Thermo Scientific Q Exactive Orbitrap mass spectrometer coupled to a Proxeon Easy-nLC II HPLC (Thermo Scientific) and a Proxeon nanospray source. The digested peptides were loaded onto a 100 micron x 25 mm Magic C18 100 Å 5U reversed-phase trap, where they were desalted online, and then separated using a 75 micron x 150 mm Magic C18 200 Å 3U reversed-phase column. Peptides were eluted using a 90-minute gradient at a flow rate of 300 nL / min. MS survey scans were acquired over the m / z range of 300-1600, and MS / MS spectra were acquired using the top 15 method, in which the top 15 ions in the MS spectrum were subjected to HCD (higher-energy collisional dissociation). A 1.6 m / z isolation mass window was used for precursor ion selection, and a normalized collision energy of 27% was used for fragmentation. A 5-second duration was used for dynamic exclusion. Tandem mass spectra were extracted. Charge state deconvolution and disotoping were not performed. All MS / MS samples were analyzed using X!Tandem (The GPM, thegpm.org; version CYCLONE (2013.02.01.1)). X!Tandem was configured to search the uniprot-_20160718_rlT5G3 database (unknown version, 141,320 entries) assuming the digestive enzyme trypsin. X!Tandem searches were performed with a fragment ion mass tolerance of 20 ppm and a parent ion tolerance of 20 ppm. Carbamidomethyl cysteine ​​was specified as a fixed modification in X!Tandem. n-terminal Glu->pyro-Glu, n-terminal ammonia loss, n-terminal gln->pyro-Glu, asparagine and glutamine deamidation, methionine and tryptophan oxidation, methionine and tryptophan dioxide, and n-terminal acetyl were designated with X!Tandem as variable modifications.

[0162] MS / MS-based peptide and protein identifications were validated using Scaffold (version Scaffold_4.6.1, Proteome Software Inc., Portland, OR). Peptide identifications were accepted if they exceeded certain database search engine thresholds. X!Tandem identifications required a -Log(Expected Scores) score of at least 1.5. Protein identifications were accepted if they contained at least one identified peptide. Proteins that contained similar peptides and could not be distinguished based on MS / MS analysis alone were grouped to satisfy the parsimony principle. Proteins sharing significant peptide evidence were grouped into clusters.

[0163] Final list generation was done by taking all proteins with a spectral count of 5 or greater from each replicate reaction and comparing them in a Venn diagram against the IgG control replicates. Example 2 Inhibition of arginine methylation is associated with reduced apoptotic cell death and decreased activation of DUX4 target genes DUX4 post-translational modifications (PTMs) were detected by overexpressing DUX4 in HEK293 cells, immunoprecipitating DUX4, and performing mass spectrometry analysis. Using this method, we identified several DUX4 arginine methylation PTMs (Figure 1A) and generated methylation-mimetic and null mutants for further characterization (Figure 1B). We identified an arginine methylation null mutant, DUX4 R71A. The DUX4 R71A mutant protects against DUX4-mediated apoptotic cell death, as indicated by reduced caspase cleavage (Figure 2).

[0164] Next, we performed experiments to determine whether the arginine methylation null mutant affected DUX4's ability to activate target genes. Wild-type or DUX4 R71A was expressed in HEK293 cells and human myoblasts. DUX4 target gene expression was examined by quantitative RT-PCR. DUX4 R71A resulted in decreased expression of DUX4 target genes, namely, PRAME family member 12 (PRAMEF12), zinc finger and SCAN domain-containing protein 4 (ZSCAN4), tripartite motif-containing 43 (TRIM43), and leucine twenty homeobox (LEUTX) (Figure 3A-B). Interestingly, R71 is located close to where DUX4 binds to DNA (Figure 3A). We hypothesized that DUX4 R71A might interfere with DUX4's ability to bind to DNA, and therefore, we performed a DUX4 activation fluorescent reporter (DRE) assay. 4 This was further investigated using a cytochrome P454 inhibitor. Consistent with the hypothesized hypothesis, DUX4 R71A was associated with decreased reporter gene expression (Figure 4B). These results indicate that inhibition of arginine methylation is associated with decreased apoptotic cell death and decreased DUX4 target gene activation.

[0165] Example 3 Inhibition of arginine methylation protects against DUX4-mediated cell death in myoblasts To isolate arginine methyltransferases associated with the DUX4 complex, we performed a proteomics approach. Rapid immunoprecipitation mass spectrometry (RIME) of endogenous proteins combines cross-linking, immunoprecipitation, and mass spectrometry to identify transient or distant interactions. 16 RIME was performed in human myoblasts expressing wild-type DUX4 and the protein arginine methyltransferase 1 (PRMT1), which was identified as a component of the DUX4 complex (Fig. 5A). Furthermore, DUX4 and PRMT1 interact when overexpressed in HEK293 cells (Fig. 5B).

[0166] The observation that DUX4 arginine methylation null mutants protect against cell death and that DUX4 interacts with arginine methyltransferases indicates a role for arginine methylation inhibitors as protective in FSHD disease models. Therefore, adenosine dialdehyde (AdOx), a global methylation inhibitor, 17 and salvianolic acid A (SAA), a PRMT1 inhibitor. 18 We tested these compounds for their ability to protect against DUX4-mediated cell death. Caspase assays were performed in human myoblasts expressing DUX4 in the presence or absence of AdOx or SAA. SAA and AdOx resulted in a dose-dependent decrease in caspase cleavage (Figures 6A-B), indicating that arginine methylation inhibitors protect against DUX4-mediated cell death in myoblasts.

[0167] This study identifies arginine methylation as a key regulator of DUX4-mediated toxicity. The arginine methylation null mutant DUX4 R71A results in reduced apoptotic cell death and decreased expression of DUX4 target genes. DUX4 forms a complex with the arginine methyltransferase PRMT1, and arginine methylation inhibitors protect against DUX4-mediated toxicity in human myoblasts. Together, these results indicate that inhibition of arginine methylation is a target for FSHD therapy.

[0168] Example 4 SAA and / or AdOx decrease DUX4 activation biomarker expression in a mouse model of FSHD SAA and AdOx or their derivatives described herein are injected intramuscularly (IM) or intravenously (IV), respectively, into the FSHD mouse model (TIC-DUX4) or any other mouse model of FSHD. After 4, 8, 12, 16, 20, and 24 weeks, DUX4 biomarkers such as Wfdc3 or Trim36, DUX4 expression levels, and DUX4 target gene expression are measured by qRT-PCR, RNAscope, or ddPCR.

[0169] Decreased levels of DUX4 biomarker expression are observed in the muscles of mice treated with SAA or AdOx compared to levels in the muscles of untreated mice. Treatment with SAA or its derivatives or AdOx or its derivatives also causes decreased expression of DUX4 target genes, namely PRAMEF12, ZSCAN4, TRIM43, and LEUTX, in treated mice compared to untreated mice. Decreased levels or decreased expression of these genes are associated with improvements in muscle strength, morphology, and overall gait and motor function.

[0170] Example 5 SAA and / or AdOx reduce DUX4-activated cell death in muscle SAA and AdOx or their derivatives as described herein are injected intramuscularly (IM) or intravenously (IV) into patients with FSHD. DUX4 activation biomarkers and / or DUX4 target genes in the patient's muscles are measured in biopsied muscles as described herein before and 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, and 52 weeks after treatment.

[0171] Decreased expression levels of DUX4 activation biomarkers and / or DUX4 target genes, i.e., PRAMEF12, ZSCAN4, TRIM43, and LEUTX, are observed in the muscles or muscle cells of patients treated with SAA and AdOx or their derivatives, compared to the levels of DUX4 activation biomarkers and / or DUX4 target genes in the same patients before treatment. Improvement in FSHD disease symptoms is also observed. Measures of such improvement include, but are not limited to, reduced fibrosis and improved function as measured by accessible workspace and patient-reported outcomes.

[0172] The foregoing description is given for clarity of understanding only, and no unnecessary limitations should be understood therefrom, since modifications within the scope of the invention may be apparent to those skilled in the art. Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" should be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.

[0173] Throughout this specification, when a composition is described as comprising components or materials, it is contemplated that the composition can consist essentially of, or consist of, any combination of the listed components or materials, unless otherwise stated. Similarly, when a method is described as comprising particular steps, it is contemplated that the method can also consist essentially of, or consist of, any combination of the listed steps, unless otherwise stated. The inventions illustratively disclosed herein may suitably be practiced in the absence of any element or step not specifically disclosed herein.

[0174] The implementation of the methods disclosed herein, and their individual steps, can be performed manually and / or with the assistance of or automation provided by electronic devices. While the processes are described with reference to specific embodiments, those skilled in the art will readily understand that other ways of performing the acts associated with the methods may be used. For example, the order of various steps may be changed without departing from the scope or spirit of the methods, unless otherwise noted. In addition, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

[0175] All patents, publications, and references cited herein are incorporated by reference in their entirety. In the event of a conflict between this disclosure and the incorporated patents, publications, and references, this disclosure shall control. References mentioned herein with numbering are provided with the full citation as set forth herein below.

[0176] References 1. Hamel, J. & Tawil, R. Facioscapulohumeral Muscular Dystrophy: Update on Pathogenesis and Future Treatments. Neurotherapeutics: the journal of the American Society for Experimental NeuroTherapeutics 15, 863 - 871 (2018). 2. Lemmers, R. J. L. F. et al. A unifying genetic model for facioscapulohumeral muscular dystrophy. Science 329, 1650 - 1653 (2010). 3. Snider, L. et al. Facioscapulohumeral dystrophy: incomplete suppression of a retrotransposed gene. PLoS genetics 6, e1001181 (2010). 4. Rickard, A. M., Petek, L. M. & Miller, D. G. Endogenous DUX4 expression in FSHD myotubes is sufficient to cause cell death and disrupts RNA splicing and cell migration pathways. Human molecular genetics 24, 5901 - 5914 (2015). 5. Geng, L. N. et al. DUX4 activates germline genes, retroelements, and immune mediators: implications for facioscapulohumeral dystrophy. Developmental cell 22, 38 - 51 (2012). 6.Guccione,E.& Richard,S.The regulation,functions and clinical relevance of arginine methylation.Nat Rev Mol Cell Bio 20,642-657(2019). 7.Blanc,R.S.& Richard,S.Arginine Methylation:The Coming of Age.Mol Cell 65,8-24(2017). 8.Smith,E.et al.Recent advances in targeting protein arginine methyltransferase enzymes in cancer therapy.Expert Opin Ther Tar 22,1-19(2018). 9.Eidahl,J.O.et al.Mouse Dux is myotoxic and shares partial functional homology with its human paralog DUX4.Hum Mol Genet 25,4577-4589(2016). 10.Kessner,D.,Chambers,M.,Burke,R.,Agus,D.& Mallick,P.ProteoWizard:open source software for rapid proteomics tools development.Bioinformatics 24,2534-2536(2008). 11.Craig,R.,Cortens,J.P.& Beavis,R.C.Open Source System for Analyzing,Validating,and Storing Protein Identification Data.J Proteome Res 3,1234-1242(2004). 12.Xu,H.& Freitas,M.A.A mass accuracy sensitive probability based scoring algorithm for database searching of tandem mass spectrometry data.Bmc Bioinformatics 8,133(2007). 13.Xu,H.,Yang,L.& Freitas,M.A.A robust linear regression based algorithm for automated evaluation of peptide identifications from shotgun proteomics by use of reversed-phase liquid chromatography retention time.Bmc Bioinformatics 9,347(2008). 14.Xu,H.& Freitas,M.A.Monte Carlo Simulation-Based Algorithms for Analysis of Shotgun Proteomic Data.J Proteome Res 7,2605-2615(2008). 15.Wallace,L.M.et al.DUX4,a candidate gene for facioscapulohumeral muscular dystrophy,causes p53-dependent myopathy in vivo.Ann Neurol 69,540-552(2011). 16.Mohammed,H.et al.Endogenous Purification Reveals GREB1 as a Key Estrogen Receptor Regulatory Factor.Cell Reports 3,342-349(2013). 17. Bartel, RL & Borchardt, RTE effects of adenosine dialdehyde on S-adenosylhomocysteine ​​hydrolase and S-adenosylmethionine-dependent transmethylations in mouse L929 cells.Mol Pharmacol 25,418-24(1984). 18.Li,T.et al.Protein arginine methyltransferase 1 may be involved in pregnane x receptor-activated overexpression of multidrug resistance 1 gene during acquired multidrug resistance.Oncotarget 7,20236-20248(2015). The present disclosure has been described in terms of particular embodiments found or suggested to include particular modes for carrying out the disclosure. Various modifications and variations of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the present disclosure has been described in connection with particular embodiments, it should be understood that the claimed methods of the present disclosure should not be unduly limited to such particular embodiments. Indeed, various modifications of the described modes for carrying out the methods that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

1. A composition for inhibiting arginine methylation of double homeobox 4 (DUX4) protein in cells, comprising at least one inhibitor of protein arginine methyltransferase (PRMT), wherein the inhibitor of PRMT is at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof, 【Chemical 1】 In the formula, R １ is H or C １ to C ６ alkyl, and X １ is CH or N, a composition.

2. A composition for reducing double homeobox 4 (DUX4)-related apoptotic cell death in cells and / or reducing DUX4 target gene activation, comprising at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof, [Chemical 2] In the formula, R １ is H or C １ to C ６ alkyl, and X １ is CH or N, a composition.

3. A composition for treating protein arginine methyltransferase disorder in a patient in need of treatment for protein arginine methyltransferase disorder, comprising at least one of Compound I and Compound II, or a salt, hydrate or stereoisomer thereof, 【Chemical Formula 3】 In the formula, R １ is H or C １ to C ６ alkyl, and X １ is CH or N, a composition.

4. The composition according to claim 1, wherein the cells are within a subject at risk of or suffering from muscular dystrophy.

5. The composition according to claim 2, wherein the cells are within a subject at risk of or suffering from muscular dystrophy.

6. The composition according to claim 3, wherein the PRMT disorder is muscular dystrophy.

7. The composition according to any one of claims 4 to 6, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

8. Compound I is Compound Ia, [Chemical Formula 4] or a salt, hydrate, or stereoisomer thereof, and the composition according to any one of claims 1 to 3.

9. Compound Ia is a hydrate of the following formula, [Chemical Formula 5] x is from 0.5 to 10, and the composition according to claim 8.

10. Compound II is Compound IIa, 【Chemical Formula 6】 or a salt, hydrate, or stereoisomer thereof, and the composition according to any one of claims 1 to 3.

11. Use of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof, for the preparation of a medicament for inhibiting arginine methylation of double homeobox 4 (DUX4) protein in cells, 【Chemical Formula 7】 In the formula, R １ is H or C １ to C ６ alkyl, and X １ is CH or N, for use. Use of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof, for the preparation of a medicament for reducing double homeobox 4 (DUX4)-related apoptotic cell death in cells and / or reducing DUX4 target gene activation, [Chemical Formula 8] In the formula, R １ is H or C １ to C ６ alkyl, and X １ is CH or N, use. Use of at least one of Compound I and Compound II, or a salt, hydrate, or stereoisomer thereof, for the preparation of a medicament for treating protein arginine methyltransferase (PRMT) disorders in a patient in need thereof, 【Chemical Formula 9】 In the formula, R １ is H or C １ to C ６ alkyl, and X １ is CH or N, for use.

14. The use according to claim 11, wherein the cell is within a subject at risk of or suffering from muscular dystrophy. The use according to claim 12, wherein the cell is within a subject at risk of or suffering from muscular dystrophy.

16. The use according to claim 13, wherein the PRMT disorder is muscular dystrophy.

17. The use according to any one of claims 14 to 16, wherein the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD).

18. Compound I is Compound Ia, 【Chemical Formula 10】 or a salt, hydrate, or stereoisomer thereof, the use according to any one of claims 11 to 13.

19. Compound Ia is a hydrate of the following formula, 【Chemical Formula 11】 The use according to claim 18, wherein x is from 0.5 to 10.

20. Compound II is Compound IIa, 【Chemical 12】 or a salt, hydrate, or stereoisomer thereof, the use according to any one of claims 11 to 13.

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