Use of SR-4370 and analogs thereof for the treatment of muscle-related disorders

Abstract

The current disclosure describes use of a histone deacetylase (HDAC) inhibitor, SR-4370 and analogs thereof, for the treatment of muscle-related disorders, such as Duchenne Muscular Dystrophy. Treatments disclosed herein can provide one or more of muscle regeneration, an increase in muscle strength, the healing of a muscle injury, an increase of the homogeneity of the diameter of a muscle fiber, and a decrease in muscle necrosis.

Description

USE OF SR-4370 AND ANALOGS THEREOFFOR THE TREATMENT OF MUSCLE-RELATED DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 672,994 filed July 18, 2024, which is incorporated herein by reference in its entirety as if fully set forth herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AR076978 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The current disclosure describes use of a histone deacetylase (HDAC) inhibitor, SR-4370, and analogs thereof for the treatment of muscle-related disorders, such as Duchenne Muscular Dystrophy.BACKGROUND OF THE DISCLOSURE

[0004] Duchenne muscular dystrophy (DMD) is a common, genetic neuromuscular disease associated with the progressive deterioration of muscle function and eventual death, first described by the French neurologist, Duchenne de Boulogne, after whom the disease is named. DMD has been characterized as an X-linked recessive disorder that affects 1 in 3,500 males caused by mutations in the dystrophin gene.SUMMARY OF THE DISCLOSURE

[0005] The current disclosure describes use of a histone deacetylase (HDAC) inhibitor, SR-4370, and analogs thereof for the treatment of muscle-related disorders, such as Duchenne Muscular Dystrophy.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0006] Some of the drawings may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present them in later proceedings.

[0007] FIG. 1. Compound list for MedChem Express Epigenetics Compound Library.

[0008] FIGs. 2A-2D. Drug-pool-based epigenetic library screening with dmd zebrafish. (2A) Schematic representation of the pooled screening approach. Compounds from each of the individual library plates (n=14) were pooled across rows as well as down columns, yielding 185unique total pools of 8-10 compounds each. Drug pools were named as [plate n_row x] or [plate n_column y]. Re-tested secondary pools were designated with the suffix “_sec”. Plate columns 1 and 12 were empty across the library (represented as crossed out wells). (2B) Timeline of small molecule treatments. Embryos from dmd+ / - crosses were collected, dechorionated, and transdermally exposed to compounds or 0.5% dimethylsulfoxide (DMSO) at a density of 25 animals / 3 mL media. Four replicates (ni-n4) were used per treatment pool. Media and compounds were replaced daily. At 96 hours post fertilization (hpf), animals were fixed and then scored using polarized light birefringence. Arrow points to muscle lesion. (2C) Brightfield, corresponding birefringence, and overlay images of 96 hpf larvae illustrating representative examples of unaffected and affected animals. Animal genotypes are shown. Affected animals are qualitatively scored as mild, moderate, and severe. Scale bar = 1 mm. * - swim bladder, an indicator of healthy larvae (Bradford et al., Genetics 220, iyac016, 2022). (2D) Distribution of screened library compounds. Over 86% (703 of 817 compounds) were successfully tested in intersecting row and column pools. Compounds at toxic row and column pool intersections were excluded from further analysis (n=71). Compounds in the 5 toxic pools following re-testing (n=43) were only tested in either row or column pools and thus were not assigned composite rescue scores (see FIGs. 1 and 6).

[0009] FIG. 3. Larval lethality and proportion of affected animals is consistent across DMSO control groups. Screening of pooled compounds occurred over 21 different batches, each with a DMSO control (Control batch, x axis). Each control batch included 4 wells of 25 animals each, with occasional loss of an animal (y axis). Stacked bars represent total qualitative ordinal scoring (unaffected, and mild, moderate, and severe affected) across 4 replicates. All DMSO control groups had >88% survival (average survival 98.9%±2.5 standard deviation [SD]) and followed an approximate Mendelian ratio of 25% (dashed line on y axis) affected individuals (average percent affected 23.5%±3.08 SD), as expected in the offspring from heterozygous dmd+Z- crosses. Chi- squared test of observed distribution with the 25% expected Mendelian ratio showed no significant difference (P>0.9999) across all 21 batches. Larvae with severe lesions represented the majority (80.3%±14.2 SD) of affected larvae and also showed no significant difference (P>0.9999) across all 21 batches.

[0010] FIGs. 4A-4D. Composite scoring identifies the histone deacetylase inhibitors (HDACi) SR- 4370 as a beneficial compound. (4A) Drug treatment pools with the lowest median odds of lesion development. Batch-corrected median odds ratios (OR) were calculated relative to DMSO control treatments. Median odds ratio is shown by a closed circle and whiskers represent the 95% confidence intervals. OR<1 indicates reduced chance of lesion development. Secondary pools(designated by the suffix “_sec”) refer to pools with suspected toxic compounds removed. Only one combination of the 8 pools with significantly reduced odds of lesion development intersected upon a compound (molibresib, [plate 10_row H], [plate 10_col 5]). *** P<0.0007. * P<0.05. (4B) Distribution of inferred rescue effects for individual compounds, as calculated using Mathematical Formula 1. The two highest ranked compounds are HDACi trichostatin A (TSA) and SR-4370. (4C) Lateral views of trunk muscle birefringence in 96 hpf wild type (WT; dmd+ / +) and dmd (dmd- / -) animals treated with DMSO, 1 pM TSA, or 1 pM SR-4370 from 24-96 hpf. Scale bar = 1mm. (4D) Normalized birefringence pixel intensities for DMSO, 1 pM TSA, and 1 pM SR-4370 treatments from 24-96 hpf. n=4 replicates for each treatment. Numbers of animals (per replicate; total across all 4 replicates) for each condition are as follows: WT +DMSO (4-5; 19), dmd +DMSO (4-9; 28), dmd +TSA (2-6; 17), dmd +SR-4370 (3-5; 19). ** P<0.01.

[0011] FIG. 5. Odds ratios of observing affected animals following 180 drug pool treatments.

[0012] FIG. 6. Compounds ranked by composite rescue scoring using Mathematical Formula 1.

[0013] FIG. 7. Validation tests of compounds based on composite scoring. Each control (DMSO) and treatment batch included 4 wells of 25 animals each, with occasional loss of an animal (represented on y axis). Control treatments shown (with batch number) represent the control treatments performed for the batches encompassing the individual compounds tested. Composite scoring ranks for each compound are shown in parentheses. Stacked bars represent total qualitative ordinal scoring (unaffected, and mild, moderate, and severe affected) across 4 replicates. All control and treatment groups had >99% survival (average survival 98.6%±0.55 SD for controls and 99.4%±1.24 SD for treatments) and followed an approximate Mendelian ratio of 25% (dashed line on y axis) affected individuals (average percent affected 24.3%±2.90 SD for controls and 24.1 %±5.90 SD for treatments). Larvae with severe lesions represented the majority (93.6%±4.85 SD) of affected larvae across all batches. TSA and SR-4370 showed lower percent of severe affected animals (0% and 25%, respectively) relative to controls and treatment group averages (93.6%±4.85 SD and 84.6%±21.99 SD).

[0014] FIG. 8. List of compounds.

[0015] FIGs. 9A, 9B. dmd rescue by SR-4370 is independent of chemical source and treatment site. (9A) Normalized birefringence pixel intensities for animals exposed to 1 pM SR-4370 from 24-96 hpf. Compounds were obtained from multiple sources (MCE, MedChemExpress; Selleck Chemicals, Houston, TX; Cayman Chemicals, Ann Arbor, Ml). n=4 replicates for each treatment. Numbers of animals (per replicate; total across all 4 replicates) for each condition are as follows: WT +DMSO (6-9; 27), dmd +DMSO (4-7; 24), dmd+MCE (4-9; 26), dmd+Selleck (6-10; 30), dmd +Cayman (6-7; 25). **** P<0.0001. (9B) Independent lab site comparison of normalizedbirefringence pixel intensities for animals exposed to 1 pM SR-4370 from 24-96 hpf. These independent tests, performed at distinct physical sites and separated by vertical dotted line on the graph, were separately normalized to their corresponding WT +DMSO pixel intensities. Numbers of animals for each condition are as follows: For Location 1 : WT +DMSO (n=19), dmd +DMSO (n=24), dmd+SR-4370 (n=18); For Location 2: WT +DMSO (n=18), dmd+DMSO (n=15), dmd +SR-4370 (n=20). Error bars represent standard deviation. Significance was determined using a one-way ANOVA comparing each treatment group to their dmd +DMSO control group. **** P0.0001. * P<0.05.

[0016] FIGs. 10A-10G. dmd rescue by SR-4370 follows a dose-response pattern and occurs during an early treatment window. (10A) Normalized birefringence pixel intensities for animals exposed to 100 nM-10 pM SR-4370 from 24-96 hpf. For 100 nM-1 pM treatments, n=4 replicates for each condition. Mortality in the 2.5 pM treatments reduced counts to n=3 replicates. Complete lethality was seen in the 5 pM and 10 pM groups (indicated by X). Numbers of animals (per replicate; total across all replicates) for each condition are as follows: WT +DMSO (5-6; 22), dmd +DMSO (5-8; 26), dmd +100nM (3-6; 20), dmd +250nM (6-10; 31), dmd +500nM (5-7; 24), dmd + 1pM (4-8; 21), dmd +2.5pM (1-4; 8). The 100 nM-1 pM treatments and the 2.5 pM-10 pM treatments were performed separately (represented by vertical dotted line) and thus their corresponding DMSO controls are shown. **** P<0.0001. ns = not significant. (10B) Timeline of alternate small molecule treatment conditions. For early pulse, animals were exposed from 24-48 hpf and then reared in plain embryo media (see Methods) until 96 hpf. Media and compounds were replaced daily for standard and delayed treatments. At end points, animals were fixed and then scored using birefringence. (10C) Normalized birefringence pixel intensities for animals exposed to 200 nM TSA or 1 pM SR-4370 from 24-48 hpf, 24-96 hpf, or 96-168 hpf. n=4 replicates for each treatment. Numbers of animals (per replicate; total across all 4 replicates) for each condition are as follows: early pulse WT +DMSO (4-7; 24), dmd +DMSO (3-6; 16), dmd +TSA (4- 10; 25), dmd +SR-4370 (5-11 ; 27); standard WT +DMSO (1-7; 16), dmd +DMSO (1-9; 22), dmd +TSA (4-12; 30), dmd +SR-4370 (3-10; 21); delayed WT +DMSO (5-9; 26), dmd +DMSO (3-7; 20), dmd +TSA (2-7; 19), dmd +SR-4370 (6-7; 26). **** P<0.0001. *** P<0.001. (10D) Lateral views of trunk muscle birefringence in 96 hpf WT (dmd+ / +) and dmd (dmd- / -) animals treated with DMSO, 200 nM TSA, or 1 pM SR-4370 from 24-48 hpf. Scale bar =1mm. (10E) Lateral views of trunk muscle of 4 dpf phalloidin-stained WT and dmd animals treated with DMSO or SR-4370 from 24-48 hpf and collected at 96 hpf. Arrows point to muscle lesions. Scale bar = 50 pm. (10F) Lateral views of trunk muscle of phalloidin-stained 10 dpf WT and dmd animals treated with DMSO or SR-4370 from 24-48 hpf. Arrows point to muscle lesions. Scale bar = 50 pm. (10G) Percentageof unaffected trunk myotomes in phalloidin-stained animals. Numbers of animals for each condition are as follows: WT +DMSO 96 hpf (n=4), dmd +DMSO 96 hpf (n=6), dmd +SR-4370 96 hpf (n=6); WT +DMSO 1O dpf (n=1O), dmd +DMSO 1O dpf (n=12), dmd +SR-4370 10 dpf (n=12). Error bars indicate standard deviation per treatment and time combination. **** P<0.0001. *** P<0.001.

[0017] FIGs. 11A-11 D. Drug combinations containing the HDACi oxamflatin are beneficial for dmd zebrafish. (11 A) Normalized birefringence pixel intensities for 24-96 hpf treatments with DMSO, pool [plate 7_row F], and pool [plate 7_column C]. n=4 replicates for each treatment. Numbers of animals (per replicate; total across all 4 replicates) for each condition are as follows: WT +DMSO (5-10; 31), dmd +DMSO (4-10; 28), dmd +plate7_row F (5-9; 30); WT +DMSO (3-9; 24), dmd +DMSO (3-9; 25), dmd +plate7_col 10 (2-6; 16). * P<0.05. (11 B) Normalized birefringence pixel intensities for 24-96 hpf treatments with DMSO and the 10 individual compounds in pool [plate 7_row F], n=4 replicates for each treatment. Numbers of animals (per replicate; total across all 4 replicates) for each condition are as follows: WT +DMSO (3-8; 22), dmd +DMSO (2-6; 18), dmd +A-395 (4-6; 19), dmd +BRD-6929 (5-11 ; 35), dmd +EDO-S101 (3-9; 25), dmd +Olaparib (3-8; 24), dmd +Barasertib-HQPA (3-10; 28), dmd +HATi-ll (5-9; 26), dmd + Bl -9564 (3-8; 21), dmd +NSC663284 (4-10; 25), dmd +Oxamflatin (6-11 ; 35), dmd +FLLL32 (4-8; 23). (11C) Normalized birefringence pixel intensities for 24-96 hpf treatments with DMSO, pool [plate 7_row F], and [plate 7_row F] pools with each individual compound removed. n=4 replicates for each treatment. Numbers of animals (per replicate; total across all 4 replicates) for each condition are as follows: WT +DMSO (3-13; 28), dmd +DMSO (2-6; 19), dmd +plate 7_row F (2-6; 14), dmd -A-395 (5-9; 27), dmd -BRD-6929 (6-10; 30), dmd -EDO-S101 (6-11 ; 31), dmd -Olaparib (6-11 ; 30), dmd - Barasertib-HQPA (2-6; 16), dmd -HATi-ll (5-11 ; 31), dmd -BI-9564 (3-6; 16), dmd -NSC-663284 (6-9; 30), dmd -Oxamflatin (5-8; 27), dmd-FLLL32 (3-10; 26). * P<0.05. (11 D) Normalized birefringence pixel intensities for 24-96 hpf treatments with DMSO and pairwise combinations of HATi-ll, NSC 663284, oxamflatin, and FLLL32. n=4 replicates for each treatment. Numbers of animals (per replicate; total across all 4 replicates) for each condition are as follows: WT +DMSO (3-7; 21), dmd +DMSO (3-7; 21), dmd + plate 7_row F (3-5; 17), dmd +HATi-ll+NSC663284 (3- 10; 29), dmd +HATi-ll+Oxamflatin (3-9; 23), dmd +HATi-ll+FLLL32 (6-7; 25), dmd +NSC663284+Oxamflatin (4-8; 23), dmd +NSC663284+FLLL32 (2-10; 26), dmd +Oxamflatin+FLLL32 (2-6; 17).

[0018] FIGs. 12A-12F. HDACi rescue correlates with increased histone acetylation. (12A) Normalized birefringence pixel intensities for animals exposed to doses of TSA, givinostat, oxamflatin, or PCI-34051 from 24-96 hpf. n=4 replicates for each treatment. Numbers of animals(per replicate; total across all 4 replicates) for each condition are as follows: WT +DMSO (4-7; 24), dmd +DMSO (2-6; 17), dmd +TSA 20nM (5-9; 28), dmd +TSA 200nM (4-6; 20), dmd +Givinostat 10 M (3-8; 24), dmd +Givinostat 40pM (5-10; 29), dmd +Givinostat 200pM (4-8; 23), dmd +Oxamflatin 1 M (4-9; 26), dmd +Oxamflatin 10pM (5-9; 27), dmd +Oxamflatin 20pM (3-9; 20), dmd +PCI-34051 1 pM (4-7; 23), dmd + PC I -34051 15pM (3-9; 24), dmd +PCI-34051 50pM (3-7; 20). * P<0.05, *** P<0.001, **** P<0.0001. (12B) Lateral views of trunk muscle birefringence in 96 hpf WT (dmd+ / +) and dmd (dmd- / -') animals treated from 24-96 hpf with DMSO, 200 nM TSA, 1 M SR-4370, 200 M givinostat, 20 pM oxamflatin, or 50 pM PCI-34051. Scale bar =1 mm. (12C) Western blots of 48 hpf larval lysates from animals treated with different HDACi doses, as in FIG. 12A. (12D-12F) Quantification of western blots (n=3). (12D) Pan-H4ac levels in non- genotyped 48 hpf larvae from dmd+ / - clutches. (12E) H3K9ac levels in non-genotyped 48 hpf larvae from dmd+Z- clutches. (12F) Acetylated a-tubulin levels in non-genotyped 48 hpf larvae from dmd+Z- clutches. Pixel intensities were normalized to the anti-actin band in DMSO-treated animals. *** P<0.001 , **** P<0.0001.

[0019] FIGs. 13A, 13B. The HDACi SR-4370 increases longevity in dmd zebrafish. (13A) Survival curves of control (unaffected, presumed dmd+ / + and dmd+ / -, dashed lines) and dmd (affected, presumed dmd- / -, solid lines) larvae exposed to HDACi from 24-48 hpf. Control animals and dmd animals were clutch siblings and were treated together and reared in parallel. Arrow indicates early pulse treatment. For controls, n=100 animals per condition. For dmd treatments, n=100 animals (DMSO, TSA), 95 animals (SR-4370) and 94 animals (givinostat). Differences were calculated between the following comparisons: For controls: DMSO vs. TSA (P=0.2309), DMSO vs. SR-4370 (P=0.3530), and DMSO vs. givinostat (P=0.4138) . For dmd-. DMSO vs. TSA (P=0.1908), DMSO vs. SR-4370 (*** P<0.0001), and DMSO vs. givinostat (P=0.1036). (13B) Survival curves of control (unaffected, presumed dmd+ / + and dmd+ / -, dashed lines) and dmd (affected, presumed dmd- / -, solid lines) larvae exposed from 24-48 hpf and re-treated every 2-3 days for a total of 14 applications. Arrows indicate treatment times. For control and dmd treatments, n=50 animals per condition. Differences were calculated between the following comparisons: For controls: DMSO vs. SR-4370 (P=0.1948). For dmd: DMSO vs. SR-4370 (*** P<0.0001).

[0020] FIGs. 14A-14D. Uncropped images of original Western blots used to create FIGs. 12C- 12F. Each blot represents separate treatment replicates of the specified compounds and concentrations. The blot ing FIG. 14D was used for PCI-34051 quantitation of the 3 replicates shown. Representative bands shown in FIG. 12C are taken from the blot in FIG. 14A.DETAILED DESCRIPTION

[0021] Duchene muscular dystrophy (DMD) is an X-linked muscle wasting disease caused by a lack of dystrophin, a protein located in the sarcolemma and part of the dystrophin-associated protein complex (DAPC) (Monaco, et al., Nature. 1986, 323:646-50; Hoffman, et al., Cell. 1987, 51 :919-28). Mutations in the > 2,200-kb gene encoding for dystrophin, DMD, vary from point mutations to large deletions that result in dysfunctional dystrophin (Koenig, et al., Cell. 1987, 50(3):509-517; Flanigan, et al., Hum Mutat. 2009, 30(12):1657-66). Dystrophin’s role as a scaffolding protein connecting the extracellular matrix to the actin cytoskeleton is integral for functional myofiber contraction (Allen, et al., Physiol Rev. 2016;96(1):253-305). Disease progression is characterized by contraction-induced damage and inflammation, fibrosis, and necrosis (Grounds & Torrisi, FASEB J. 2004, 18(6):676-82; Reeve, et al., Muscle Nerve. 1997;20(3):357-60). Over time, patients become wheelchair-bound due to muscle weakness. Though life expectancy for DMD-afflicted individuals has increased from 15 years to the midtwenties to early forties, there is still no effective cure for the disease (Allen, supra; Kieny, et al., Ann Phys Rehabil Med. 2013, 56(6):443-54).

[0022] The current standard of care for DMD is to administer corticosteroids (Birnkrant et al., Lancet Neurology 2018; 17(3): 251-267). Corticosteroids such as prednisone, prednisolone, and deflazacort are administered to delay symptoms and ameliorate symptom severity, yet treatment may cause significant adverse side effects (Birnkrant et al., Lancet Neurology 2018, 17(3): 251- 267. The FDA approved use of several antisense oligonucleotides, which bind to complementary regions in exons that contain a mutation in the DMD gene, resulting in that exon region being skipped during translation (Nguyen & Yokota, Am J Transl Res. 2019;11(3):1202-18; Engelbeen, et al., Nucleic Acid Ther. 2023;33(6):348-60). Antisense oligonucleotides are designed to bind to complementary regions in the DMD sequence to restore the reading frame, which often leads to a truncated yet functional dystrophin protein (Nguyen & Yokota, supra; Shadid, et al., Expert Opin Drug Metab Toxicol. 2021, 17(11 ): 1281-92). However, an issue with this approach is pharmacological bioavailability, particularly in the heart muscles (Nguyen and Yokota, supra; Moulton & Moulton, Biochim Biophys Acta. 2010, 1798(12):2296-303). In 2016, the FDA approved eteplirsen (also Exondys 51) injection which restores the DMD gene reading frame by skipping exon 51 (Sarepta Therapeutics, Inc, 2020). The resulting gene product is a truncated yet functional dystrophin (Lim, et al., Drug Des Devel Ther. 2017, 11 :533-45). More recently in 2023, the FDA approved delandistrogene moxeparvovec (delandistrogene moxeparvovec-rokl; ELEVI DYS®), an adeno-associated virus (AAV) vector-based gene therapy that encodes a form of microdystrophin that restores core functionality of full-length dystrophin (Hoy, Drugs. 2023,83(14):1323-1329). However, since DMD can be caused by mutations in multiple exons of the DMD gene, gene therapy targeting one exon is only applicable to a subset of patients.

[0023] Treatment with some, but not all, histone deacetylase inhibitors (HDACi) have shown promising results in zebrafish (Johnson, et al., PLoS Curr. 2013 17, 5; Farr, 3rd, et al., Skelet Muscle. 2020,10(1):29; Spreafico, et al., Pharmacol Res. 2021 ,170), mice (Minetti , et al. Nat Med. 2006, 12(10):1147-50; Mozzetta, et al., EMBO Mol Med. 2013,5(4):626-39; Consalvi et al., Mol Med 2013, 19(1)), and DMD patients (Bettica, et al., Neuromuscul Disord. 2016;26(10):643- 9; www.Clinicaltrials.gov, clinical trial identifier: NCT01761292, accessed on 1 January 2024; Mercuri et al., Lancet Neurology, 2024,23(4): 393-403). Recently, the FDA approved Givinostat for DMD boys. In DMD patients, Givinostat treatment improves myofiber phenotype, including reduced fibrosis, reduced fatty tissue infiltration, and peripherally located nuclei (Bettica, supra; Mercuri 2024). Spreafico, et al. treated primary muscle cells derived from human DMD patients with PCI-34051 , an HDAC8 inhibitor, and observed increased myosin expression and myoblast differentiation efficacy (Spreafico, supra). In zebrafish embryos, the pan-HDACi Trichostatin A (TSA) and a combination of oxamflatin and salermide reduce the severity of muscle lesions (Johnson, supra; Farr, supra). In mice, Minetti et al (2006) showed improved muscle phenotype in mdx mice treated with TSA (Minetti, supra). While some HDACi show promise for DMD, there is still much to understand about how they work and how to identify improved compounds.

[0024] As disclosed herein, a commercial library of 817 epigenetic small molecules was tested on embryo dmd zebrafish, also known as sapje, which contain a nonsense mutation in exon 4 (Granato, et al., Development. 1996;123:399-413; Bassett, et al., Development. 2003;130(23):5851-60). The homozygous mutants exhibit DMD patient symptoms such as muscle lesions, inflammation, and fibrosis, making it a well-suited model for DMD (Berger, et al., Neuromuscul Disord. 2010;20(12):826-32). Drugs were pooled by library plate rows and columns and the pools were tested on embryos, thereby testing each compound twice. One HDACi, SR- 4370, consistently rescued the dmdta222a muscle lesion phenotype. Mutant embryos treated with SR-4370 showed significantly fewer and less severe muscle lesions compared to control group mutants. After testing low and high doses of SR-4370 on embryos, it was concluded that 1 M was the most effective dose. To determine a therapeutic time window when SR-4370 could rescue the DMD phenotype, separate groups of embryos were exposed to SR-4370 at time windows of 24 to 48, 24 to 96, and 96 to 168 hours post-fertilization. SR-4370 treatment significantly reduced muscle lesion severity from 24 to 48 hours and 24 to 96 hours.

[0025] Aspects of the disclosure are now described with additional detail and options as follows: (i) SR-4370 and Analogs Thereof; (ii) Compositions; (iii) Methods of Use; (iv) ExemplaryEmbodiments; (v) Experimental Example; and (vi) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0026] (i) SR-4370 and Analogs Thereof. SR-4370 is a small molecule histone deacetylase (HDAC) inhibitor (i) having the structure:O SR-4370

[0027] SR-4370 can be obtained commercially from a variety of vendors (e.g., Medchem Express, Selleckchens, MedKoo Biosciences, Adooq Bioscience, GIpBio). SR-4370 can be synthesized using a scintillation vial containing a Teflon-coated magnetic stir bar charged with 2', 3'- difluorobiphenyl-4-carbohydrazide (50 mg, 0.25 mmol), 1 ml. of methanol, 22 pL of butyraldehyde (1 eq, 0.25 mmol) and catalytic p-toluenesulfonic acid can subsequently be added to the vial. The resultant solution can be allowed to stir for 12 h at room temperature. The reaction mixture can then be acidified to pH 5 through the addition of 4M HCI in dioxane. Sodium cyanoborohydride (19 mg, 1.2 eq, 0.31 mmol) can be added and the reaction mixture can be allowed to stir for 3 h at room temperature. The reaction mixture can then be concentrated in vacuo. Water can be added to the resultant residue followed by diethyl ether. The aqueous layer can be extracted three times with diethyl ether. The organic layers can be combined, dried and concentrated. The resultant white solid can be further purified by flash chromatography utilizing a solvent gradient from 0-65% EtOAc in hexanes.

[0028] In particular embodiments, analogs of SR-4370 include formula I: Formula Iwherein X and Y are independently N or CR2, wherein R1 and R2 are independently hydrogen, an alkyl group, a halo group, or an ester group, R5 is a Ci to Cio alkyl group, andn is an integer from 0 to 3.

[0029] In particular embodiments, X and Y in formula I are each N. In particular embodiments, X and Y in formula I are each N, and n is 0.

[0030] In particular embodiments, X and Y in formula I are each CR2. In particular embodiments, X in formula I is CF, CH, or C-C(0)OR3, wherein R3 is an alkyl group. In particular embodiments, X in formula I is CF, CH, or C-C(0)OR3, wherein R3 is an alkyl group, and n is 0.

[0031] In particular embodiments, Y in formula I is CH or CR4, wherein R4 is an alkyl group. In particular embodiments, Y in formula I is CH or CR4, wherein R4 is an alkyl group, and n is 0. In particular embodiments, X is CF and Y is CH or CR4, wherein R4 is an alkyl group. In particular embodiments, X is CF, Y is CH, R1 is fluoro, and n is 1.

[0032] In particular embodiments, analogs of SR-4370 include formula II: Formula IIwherein X is C-C(0)0R3, wherein R3is an alkyl group.

[0033] In particular embodiments, the analog of SR-4370 includes SR-4373, SR-3558, UF010, SR-4369, SR-4360, SR-4372, or SR-3299:

[0034] Analogs of SR-4370 can be synthesized using the methods described in U.S. Patent No. 10,897,944.

[0035] SR-4370 and analogs thereof can be used in the form of salts derived from inorganic or organic acids. Pharmaceutically acceptable salts include salts of acidic or basic groups present in the disclosed compounds. Suitable pharmaceutically acceptable salts include base addition salts, including alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts, which may be similarly prepared by reacting the drug compound with a suitable pharmaceutically acceptable base. The salts can be prepared in situ during the final isolation and purification of the compounds of the present disclosure; or following final isolation by reacting a free base function, such as a secondary or tertiary amine, of a disclosed compound with a suitable inorganic or organic acid; or reacting a free acid function, such as a carboxylic acid, of a disclosed compound with a suitable inorganic or organic base.

[0036] Acidic addition salts can be prepared in situ during the final isolation and purification of SR-4370 or analogs thereof, or separately by reacting moieties including one or more nitrogen groups with a suitable acid. In various aspects, acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulfuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid. In a further aspect, salts further include the following: hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate, butyrate, camphorate, camphorsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, 2- hydroxyethanesulfonate (isethionate), nicotinate, 2-naphthalenesulfonate, oxalate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, undecanoate, and pamoate (i.e., 1,T-methylene-bis-(2- hydroxy-3-naphthoate)) salts. Also, basic nitrogen-containing groups can be quatemized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others.

[0037] Basic addition salts can be prepared in situ during the final isolation and purification of SR- 4370 or analogs thereof, or separately by reacting carboxylic acid moieties with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutical acceptable metal cation or with ammonia, or an organic primary, secondary or tertiary amine. Pharmaceutical acceptable salts include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminum salts and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines useful for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. In further aspects, bases which may be used in the preparation of pharmaceutically acceptable salts include the following: ammonia, L-arginine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylenediamine, N-methyl-glucamine, hydrabamine, 1 H-imidazole, L-lysine, magnesium hydroxide, 4-(2- hydroxyethyl)-morpholine, piperazine, potassium hydroxide, 1-(2-hydroxyethyl)-pyrrolidine, secondary amine, sodium hydroxide, triethanolamine, tromethamine and zinc hydroxide.

[0038] The term “substituted” includes all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit provisothat such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0039] The term “alkyl” refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n- pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also be a C1 alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C1-C24 alkyl.

[0040] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. Alternatively, the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g. fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted with two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “aminoalkyl” specifically refers to an alkyl group that is substituted with one or more amino groups. The term “hydroxyalkyl” specifically refers to an alkyl group that is substituted with one or more hydroxy groups. When “alkyl” is used in one instance and a specific term such as “hydroxyal kyl” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like.

[0041] The term “alkenyl” refers to a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structuressuch as (A1A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0042] The term “cycloalkenyl” refers to a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0043] The term “alkynyl” refers to a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0044] The term “cycloalkynyl” refers to a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0045] The term “aromatic group” refers to a ring structure having cyclic clouds of delocalized p electrons above and below the plane of the molecule, where the p clouds contain (4n+2) pelectrons. A further discussion of aromaticity is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497. The term “aromatic group” is inclusive of both aryl and heteroaryl groups.

[0046] The term “aryl” refers to a group that contains any carbon-based aromatic group including benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, — NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” In addition, the aryl group can be a single ring structure or include multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carboncarbon bond.

[0047] The terms “halo,” “halogen” or “halide,” can be used interchangeably and refer to F, Cl, Br, or I.

[0048] The term “ester” refers to the formula — 0C(0)A1 or — C(0)0A1 , where A1 can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0049] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present disclosure includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0050] (ii) Compositions. SR-4370 and / or analogs thereof can be formulated alone or in combination into compositions for administration to subjects. As indicated previously, salts of the SR-4370 and / or analogs thereof can also be used. SR-4370 and / or analogs and / or salts thereof are referred to herein as “active ingredients”.

[0051] A pharmaceutically acceptable salt includes any salt that retains the activity of the reference compound and is acceptable for pharmaceutical use. A pharmaceutically acceptablesalt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt. A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage or by hydrolysis of a biologically labile group.

[0052] In particular embodiments, the compositions include active ingredients of at least 0.1% w / v or w / w of the composition; at least 1% w / v or w / w of composition; at least 10% w / v or w / w of composition; at least 20% w / v or w / w of composition; at least 30% w / v or w / w of composition; at least 40% w / v or w / w of composition; at least 50% w / v or w / w of composition; at least 60% w / v or w / w of composition; at least 70% w / v or w / w of composition; at least 80% w / v or w / w of composition; at least 90% w / v or w / w of composition; at least 95% w / v or w / w of composition; or at least 99% w / v or w / w of composition.

[0053] Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants, binders, buffering agents, bulking agents or fillers, chelating agents, coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles.

[0054] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0055] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0056] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0057] Exemplary isotonic agents include polyhydric sugar alcohols, including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0058] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, and benzalkonium halides.

[0059] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the compounds or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols, amino acids, organic sugars or sugar alcohols, polyethylene glycol (PEG), amino acid polymers, sulfur- containing reducing agents, low molecular weight polypeptides (i.e. , <10 residues), proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins, hydrophilic polymers, monosaccharides, disaccharides, trisaccharides, and polysaccharides.

[0060] Compositions can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further beformulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, sublingual, and / or subcutaneous administration.

[0061] For injection, compositions can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline. The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the formulation can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0062] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like.

[0063] Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid, or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.

[0064] Any composition disclosed herein can advantageously include any other pharmaceutically acceptable carriers, which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by the U.S. FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0065] (iii) Methods of Use. Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds), livestock (e.g., horses, cattle, goats, pigs, chickens), and research animals (e.g., monkeys, rats, mice, fish) with compositions disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments, and / or therapeutic treatments.

[0066] An "effective amount" is the amount of a composition necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an animal model or in vitro assay relevant to the assessment of the development, progression, and / or resolution of a muscle disorder.

[0067] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a muscle disorder or displays only early signs or symptoms of amuscle disorder such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the muscle disorder further. Thus, a prophylactic treatment functions as a preventative treatment against a muscle disorder. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of a muscle disorder.

[0068] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of a muscle disorder and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the muscle disorder. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the muscle disorder and / or reduce control or eliminate side effects of the muscle disorder.

[0069] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0070] Particular embodiments of muscle disorders that can be treated with compositions disclosed herein include muscular dystrophies such as Duchenne Muscular Dystrophy (DMD), Becker muscular dystrophy (BMD), Congenital Muscular Dystrophy (CMD), dystroglycanopathy, Limb-girdle muscular dystrophy (LGMD) (e.g., LGMD2A due to calpain-3 deficiency, ACTA1 LGMD), Myotonic muscular dystrophy (MMD) and Facioscapulohumeral muscular dystrophy (FSHD). In particular embodiments, CMD includes merosin-deficient CMD (MDC1A), Fukuyama CMD, L-CMD (LMNA-related CMD), or a dystroglycanopathy (defects in proteins that attach to dystroglycan).

[0071] In particular embodiments, therapeutically effective amounts alleviate one or more symptom(s) of a muscle-related disorder, such as DMD. Success in alleviating one or more symptom(s) of a muscle-related disorder can be assessed by any of the following indications: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, prevention of muscle lesion formation, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of pulmonary function, improvement of cardiac function, and improvement of the quality of life. Each of these indications can be determined by assessments known to the skilled person. For each of these indications, as soon as a detectable improvement or prolongation of a parameter measured in an assessment has been found, it will generally mean that one or more symptoms of a muscle-related disorder has been alleviated.

[0072] Additionally or alternatively, the alleviation of one or more symptom(s) of a muscle-related disorder may be assessed by measuring an improvement of a muscle fiber function, integrityand / or survival. Such symptoms or characteristics may be assessed at the cellular, tissue, or selflevel.

[0073] An alleviation of one or more characteristics of a muscle cell from a subject may be assessed by any of the following assays on a myogenic cell or muscle cell from a patient: reduced calcium uptake by muscle cells, decreased collagen synthesis, altered morphology, altered lipid biosynthesis, and / or decreased oxidative stress. These parameters are usually assessed using immunofluorescence and / or histochemical analyses of cross sections of muscle biopsies.

[0074] The improvement of muscle fiber function, integrity and / or survival may be assessed using at least one of the following assays: a detectable decrease of creatine kinase in blood, a detectable decrease of necrosis of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic, and / or a detectable increase of the homogeneity of the diameter of muscle fibers in a biopsy cross-section of a muscle suspected to be dystrophic. Each of these assays is known to the skilled person.

[0075] A detectable decrease in creatine kinase may include a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the concentration of creatine kinase in a same subject before treatment.

[0076] A detectable decrease of necrosis may include a decrease of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the area wherein necrosis has been identified using biopsy cross-sections. The decrease is measured by comparison to the necrosis as assessed in a same subject before treatment.

[0077] A detectable increase of the homogeneity of the diameter of a muscle fiber is generally assessed by comparison to the homogeneity of the diameter of a muscle fiber in a same subject before treatment.

[0078] The term “muscle” means a structure, which is composed of myoblasts, myotubes, myofibers, stem cells that could produce myoblasts, and proteins that support those structures. The muscle includes skeletal, cardiac, and smooth muscles.

[0079] The term “muscle injury” refers to the condition that muscle does not function normally. The injury could be caused by excessive impact to a muscle where muscle fibers compressed in this manner can become irritated and even torn, caused by a muscle being stretched beyond its capacity and caused by intense and rapid contraction of a muscle. In particular embodiments, therapeutically effective amounts, heal a muscle injury, partially or fully.

[0080] The term “muscle lesion” refers to the damage or injury to the muscle tissue that occurs as a result of the progressive degeneration and inflammation associated with the muscle disorder (e.g., the absence or malfunction of dystrophin in DMD). Example muscle lesions includemicroscopic muscle fiber tears, infiltration of inflammatory cells, fibrosis and fatty infiltration.

[0081] The terms “muscle atrophy’’ and “muscle loss” refer to the condition which is caused by disuse of muscles, e.g. a lack of physical activity. For example, a subject under the medical conditions that limit their movement can lose muscle tone and develop atrophy. In particular embodiments, therapeutically effective amounts reverse muscle atrophy, partially or fully.

[0082] The term “muscle strength” means the amount of force that a muscle can produce with maximal efforts. In particular embodiments, therapeutically effective amounts increase muscle strength.

[0083] The term “regeneration” means the repair of cells, tissues, or organs. In the present disclosure, the term regeneration refers to the repair of myoblasts, myofibers, and muscular environment, which can provide an optimal environment to generate myofibers. In particular embodiments, therapeutically effective amounts lead to regeneration.

[0084] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher, taking into account parameters such as physical and physiological factors including body weight, severity of condition, type of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0085] Useful doses can range from 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other non-limiting examples, a dose can include 1 pg / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other non-limiting examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0086] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).

[0087] In particular embodiments, the compositions disclosed herein are administered prior to muscle lesion formation. In particular embodiments, the compositions disclosed herein are administered early after diagnosis of the muscle disorder. In particular embodiments, the compositions disclosed herein are administered before diagnosis of the muscle disorder. In particular embodiments, a single early pulse of a composition including active ingredientsimproves muscle phenotype and increases lifespan. In particular embodiments, administering a composition including active ingredients increases histone acetylation.

[0088] The pharmaceutical compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intraparenteral, intranasal, intralesional, intramuscular, oral, subcutaneous, and / or sublingual administration.

[0089] In particular embodiments, the methods described herein can include combination with a secondary treatment. In particular embodiments, a method of treating a subject with a muscle disorder includes administering a therapeutically effective amount of SR-4370 or an SR-4370 analog to the subject, and administering a therapeutically effective amount of secondary treatment. In particular embodiments, the secondary treatment is a corticosteroid. In particular embodiments, the corticosteroid includes prednisone, prednisolone, or deflazacort. In particular embodiments, the secondary treatment includes an antisense oligonucleotide. In particular embodiments, the antisense oligonucleotide binds to complementary regions in exons that contain a gene mutation. In particular embodiments, the antisense oligonucleotide includes eteplirsen. In particular embodiments, the secondary treatment includes an adeno-associated virus (AAV) vector-based gene therapy. In particular embodiments, the AAV vector-based gene therapy encodes a form of microdystrophin. In particular embodiments, the AAV vector-based gene therapy includes delandistrogene moxeparvovec. In particular embodiments, the secondary treatment includes other HDAC inhibitors. In particular embodiments, other HDAC inhibitors include givinostat, trichostatin A (TSA), PCI-34051 , oxamflatin, and / or salemide.

[0090] (iv) Exemplary Embodiments.1. A method of treating a muscle disorder in a subject in need thereof including administering a therapeutically effective amount of SR-4370 or an SR-4370 analog to the subject, thereby treating the muscle disorder in the subject.2. The method of embodiment 1 , wherein the method includes administering the therapeutically effective amount of SR-4370.3. The method of embodiments 1 or 2, wherein the method includes administering the therapeutically effective amount of the SR-4370 analog.4. The method of embodiment 3, wherein the SR-4370 analog includes formula IwhereinX and Y are independently N or CR2,R1 and R2 are independently hydrogen, an alkyl group, a halo group, or an ester group,R5 is a Ci to Cio alkyl group, and n is an integer from 0 to 3. The method of embodiment 4, wherein X and Y are each N. The method of embodiments 4 or 5, wherein X and Y are each N and n is 0. The method of embodiment 4, wherein X and Y are each CR2. The method of embodiment 4, wherein X is CF, CH, or C-C(0)OR3 and R3 is an alkyl group. The method of embodiments 4 or 8, wherein X is CF, CH, or C-C(0)OR3; R3 is an alkyl group; and n is 0. The method of embodiment 4, wherein Y is CH or CR4 and R4 is an alkyl group. The method of embodiments 4 or 10, wherein Y is CH or CR4, R4 is an alkyl group, and n is 0. The method of any of embodiments 4-11 , wherein X is CF, Y is CH or CR4, and R4 is an alkyl group. The method of any of embodiments 4-13, wherein X is CF, Y is CH, R1 is fluoro, and n is 1. The method of embodiment 3, wherein the SR-4370 analog includes formula IIwherein X is C-C(0)0R3and R3is an alkyl group. The method of embodiment 3, wherein the SR-4370 analog includes SR-4373, SR-3558, UF010, SR-4369, SR-4360, SR-4372, or SR-3299.The method of any of embodiments 1-15, wherein the muscle disorder includes a muscular dystrophy. The method of embodiment 16, wherein the muscular dystrophy includes Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, or dystroglycanopathy. The method of embodiments 16 or 17, wherein the muscular dystrophy includes Duchenne muscular dystrophy. The method of any of embodiments 1-18, wherein the therapeutically effective amount is administered as a single dose. The method of any of embodiments 1-18, wherein the therapeutically effective amount is administered as multiple doses. The method of embodiment 20, wherein the multiple doses are administered daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly. The method of any of embodiments 1-21 , wherein the therapeutically effective amount is administered via injection, inhalation, infusion, perfusion, lavage, or ingestion. The method of any of embodiments 1-22, wherein the administering includes intravenous, intradermal, intraarterial, intraparenteral, intranasal, intramuscular, oral, or subcutaneous administering. The method of any of embodiments 1-23, wherein the administering occurs prior to muscle lesion formation. The method of any of embodiments 1-24, wherein the method increases histone acetylation. The method of any of embodiments 1-25, further including administering a therapeutically effective amount of a secondary treatment. The method of embodiment 26, wherein the secondary treatment includes a corticosteroid, a second histone deacetylase inhibitor, an antisense oligonucleotide, or an adeno-associated virus (AAV) vector. The method of embodiment 27, wherein the corticosteroid includes prednisone, prednisolone, or deflazacort. The method of embodiments 27 or 28, wherein the second histone deacetylase inhibitor includes givinostat, trichostatin A (TSA), PCI-34051 , oxamflatin, or salemide. The method of any of embodiments 27-29, wherein the antisense oligonucleotide includeseteplirsen. The method of any of embodiments 27-30, wherein the adeno-associated (AAV) vector includes delandistrogene moxeparvovec. A composition including a therapeutically effective amount of SR-4370 or an SR-4370 analog and a pharmaceutically acceptable carrier. The composition of embodiment 32, wherein the composition includes the therapeutically effective amount of SR-4370. The composition of embodiments 32 or 33, wherein composition includes the therapeutically effective amount of the SR-4370 analog. The composition of embodiment 34, wherein the SR-4370 analog has formula IwhereinX and Y are independently N or CR2,R1 and R2 are independently hydrogen, an alkyl group, a halo group, or an ester group, R5 is a Ci to Cio alkyl group, and n is an integer from 0 to 3. The composition of embodiment 35, wherein X and Y are each N. The composition of embodiments 35 or 36, wherein X and Y are each N and n is 0. The composition of embodiment 35, wherein X and Y are each CR2. The composition of embodiment 35, wherein X is CF, CH, or C-C(0)OR3 and R3 is an alkyl group. The composition of embodiments 35 or 39, wherein X is CF, CH, or C-C(0)OR3; R3 is an alkyl group; and n is 0. The composition of embodiment 35, wherein Y is CH or CR4 and R4 is an alkyl group. The composition of embodiments 35 or 41 , wherein Y is CH or CR4, R4 is an alkyl group, and n is 0. The composition of any of embodiments 35-42, wherein X is CF, Y is CH or CR4, and R4 isan alkyl group. The composition of any of embodiments 35-43, wherein X is CF, Y is CH, R1 is fluoro, and n is 1. The composition of embodiment 34, wherein the SR-4370 analog includes formula IIwherein X is C-C(0)0R3and R3is an alkyl group, The composition of embodiment 34, wherein the SR-4370 analog includes SR-4373, SR- 3558, UF010, SR-4369, SR-4360, SR-4372, or SR-3299. The composition of any of embodiments 32-46, wherein the therapeutically effective amount treats a muscle disorder. The composition of embodiment 47, wherein the muscle disorder includes a muscular dystrophy. The composition of embodiment 48, wherein the muscular dystrophy includes Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, or dystroglycanopathy. The composition of embodiments 48 or 49, wherein the muscular dystrophy includes Duchenne muscular dystrophy. The composition of any of embodiments 32-50, wherein the therapeutically effective amount includes from 0.1 pg / kg to 1000 mg / kg. The composition of any of embodiments 32-51 , wherein the therapeutically effective amount includes 1 pg / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, or 1000 pg / kg. The composition of any of embodiments 32-52, wherein the therapeutically effective amount includes 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, or 1000 mg / kg. The composition of any of embodiments 32-53, further including a secondary treatment. The composition of embodiment 54, wherein the secondary treatment includes a corticosteroid, a second histone deacetylase inhibitor, an antisense oligonucleotide, or an adeno-associated (AAV) vector.56. The composition of embodiment 55, wherein the corticosteroid includes prednisone, prednisolone, or deflazacort.57. The composition of embodiments 55 or 56, wherein the second histone deacetylase inhibitor includes givinostat, trichostatin A (TSA), PCI-34051 , oxamflatin, or salemide.58. The composition of any of embodiments 55-57, wherein the antisense oligonucleotide includes eteplirsen.59. The composition of any of embodiments 55-58, wherein the adeno-associated (AAV) vector includes delandistrogene moxeparvovec.

[0091] (v) Experimental Example. Epigenetic small molecule screening identifies a new HDACi compound for ameliorating Duchenne muscular dystrophy.

[0092] Abstract. Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease. There are currently few effective therapies to treat the disease, although many approaches are being pursued. Certain histone deacetylase inhibitors (HDACi) to ameliorate DMD phenotypes in mouse and zebrafish models, and the HDACi givinostat recently gained FDA approval for DMD. The goal was to identify additional HDACi, or other classes of epigenetic small molecules, that are beneficial for the treatment of DMD. Using an animal model for DMD, the zebrafish dmd mutant strain sapje, a library of over 800 epigenetic small molecules was screened. The screening identified a new HDACi, SR-4370, that ameliorated dmd mutant zebrafish skeletal muscle degeneration, as well as additional HDACi that have previously been shown to improve dmd zebrafish. A single early treatment of HDACi can ameliorate the muscle phenotype and increase lifespan in dmd zebrafish. Furthermore, HDACi treatments that improve dmd muscle also cause increased histone acetylation in zebrafish larvae. The results also demonstrate the effectiveness of small-molecule screening in dmd zebrafish.

[0093] Introduction. Duchenne muscular dystrophy (DMD) is an X-linked muscle degeneration disease caused by mutations in the DMD gene, which encodes dystrophin (Hoffman et al., Cell 51 , 919-928, 1987; Burghes et al., Nature 328, 434-437, 1987; Duan et al., Nat Rev Dis Primers 7, 13, 2021 (“Duan 2021”)). Dystrophin is associated with the sarcolemma and is part of the dystrophin-associated protein complex (DAPC), which connects the extracellular matrix to the actin cytoskeleton. Mutations in the 2.4 Mb DMD gene that result in loss or dysfunction of dystrophin can vary from point mutations to large deletions (Duan 2021 ; Elasbali et al., International Journal of Biological Macromolecules 264, 130544, 2024). Loss of dystrophin leads to contraction-induced structural myofiber damage and the dysregulation of intracellular signaling pathways (Allen et al., Physiological Reviews 96, 253-305, 2016; Dowling et al., Eur J Transl Myol, 33(4):11856, 2023). DMD disease is characterized by progressive muscle damage andinflammation, fibrosis, and necrosis (Duan 2021 ; McDonald et al., JND 11 , 499-523, 2024 (“McDonald 2024”); Birnkrant et al., Lancet Neurol 17, 251-267, 2018). Over time, patients become wheelchair-bound due to muscle weakness and eventually experience cardiorespiratory failure. While advances in palliative care have increased the life expectancy of DMD boys from 15 years to the mid-twenties to early forties, there is still no effective cure for the disease.

[0094] The current standard of care for DMD is corticosteroid treatment (McDonald 2024; Birnkrant et al., Lancet Neurol 17, 251-267, 2018; D’Ambrosio et al., Neurotherapeutics 20, 1669-1681, 2023 (“D’Ambrosio 2023”)). Corticosteroids such as prednisone, prednisolone, and deflazacort are administered to delay symptoms and ameliorate symptom severity, yet these treatments cause significant adverse side effects (D’Ambrosio 2023). Additional treatment approaches currently underdevelopment include using antisense oligonucleotides to induce exon skipping of DMD mutations, CRISPR editing, and DMD gene therapy (D’Ambrosio 2023; Happi Mbakam et al., Expert Rev Neurother 23, 905-920, 2023 (“Happi Mbakam 2023”); Chemello et al., Hum Gene Ther 34, 379-387, 2023 (“Chemello 2023”); Aartsma-Rus et al., Hum Gene Ther 34, 372-378, 2023 (“Aartsma-Rus 2023”)). While these approaches are gaining FDA approvals, many issues remain, including efficacy, safety, and delivery (D’Ambrosio 2023; Happi Mbakam; Chemello 2023; Aartsma-Rus 2023). Furthermore, because DMD can be caused by a spectrum of mutations, DMD gene-directed therapies that target specific exons or mutations are only applicable to a subset of patients.

[0095] There have been significant efforts to develop small molecule therapies that target various disease mechanisms downstream of the loss of dystrophin (Spinazzola et al., Expert Opinion on Orphan Drugs 4, 1179-1194, 2016; Deng et al., Front. Pharmacol. 13, 950651, 2022). Treatment with some, but not all, histone deacetylase inhibitors (HDACi) have shown promising results for DMD in mice and zebrafish animal models as well as in DMD patients (Bajanca and Vandel, PLoS Curr 9, 2017; Johnson et al., PLoS Curr 5, 2013 (“Johnson 2013”); Farr et al., Skeletal Muscle 10, 29, 2020 (“Farr 2020”); Spreafico et al., Pharmacological Research 170, 105750, 2021 (“Spreafico 2021”); Minetti et al., Nat Med 12, 1147-1150, 2006 (“Minetti 2006”); Consalvi et al., Mol Med 19, 79-87, 2013 (“Consalvi 2013”); Bettica et al., Neuromuscular Disorders 26, 643- 649, 2016 (“Bettica 2016”); Mercuri et al., The Lancet Neurology 23, 393-403, 2024 (“Mercuri 2024”)). For example, Minetti et al. showed improved muscle phenotype in mdx mice treated with the pan-HDACi trichostatin A (TSA) (Minetti 2006). In zebrafish, both TSA and a combination of oxamflatin and salermide have been shown to reduce the severity of muscle lesions in dmd mutant embryos (Johnson 2013; Farr 2020). In a phase 2 and phase 3 clinical trial, treatment with givinostat, a pan-HDACi, improved muscle histology and also improved a stair-climb functionalassessment in DMD boys (Bettica 2016; Mercuri 2024). Recently, the FDA approved the use of givinostat for DMD boys.

[0096] In this example, a commercial library of over 800 epigenetic small molecules was tested, including HDACi, for their ability to improve the muscle degeneration phenotype of dmd zebrafish, a valuable DMD animal model for drug screening (Granato et al., Development 123, 399-413, 1996 (“Granato 1996”); Bassett et al., Development 130, 5851-5860, 2003 (“Bassett 2003”); Kawahara and Kunkel, Drug Discovery Today: Technologies 10, e91-e96, 2013 (“Kawahara and Kunkel 2013”); Wasala et al., Expert Opinion on Drug Discovery 15, 443-456, 2020). Zebrafish dmd mutants exhibit DMD phenotypes such as skeletal muscle lesions, inflammation, fibrosis, and lethality during the early juvenile phase, making dmd zebrafish a well-suited model for DMD (Bassett 2003; Berger et a / ., Neuromuscular Disorders 20, 826-832, 2010; Kawahara et al., Proc Natl Acad Sci 108, 5331-5336, 2011 (“Kawahara 2011”)). The drug library screen was performed by treating dmd zebrafish with compounds pooled from each row and column of each library plate, thereby testing each compound twice. An HDACi for DMD was identified, SR-4370, that rescues the dmd zebrafish muscle lesion phenotype and improves survival. Optimal doses and timing of HDACi treatments were also identified for dmd zebrafish. Furthermore, a correlation between HDACi rescue of dmd muscle lesions and histone acetylation levels in zebrafish larvae were identified. The results demonstrate that SR-4370 is a previously unidentified beneficial small molecule for DMD and underscores the value of using dmd zebrafish for drug screening.

[0097] Results. Drug-pool-based epigenetic library screening identifies beneficial drug pools for dmd zebrafish. In previous work, a pilot screen of a commercial 94-chemical epigenetic small molecule library was used to identify a combination of two HDACi compounds, oxamflatin and salermide, that ameliorated dmd mutant zebrafish skeletal muscle degeneration (Farr 2020). Here, it was tested whether additional epigenetic small molecules could be identified that improve zebrafish dmd muscle lesions. The commercially-available MedChemExpress Epigenetics Compound Library was selected, which contains a variety of compounds, including HDACi. The version of the library that was obtained contained 817 chemicals distributed over fourteen 96-well plates (FIG. 1). To efficiently screen this large library, an orthogonal drug pooling approach was used (FIG. 2A). Each library plate was divided into plate-specific row- and column-based drug pool combinations, resulting in 185 unique pools across the library. Each compound was thus included in two separate pools: a row pool and a column pool (FIG. 2A).

[0098] Based on previous work that identified optimal dosing for epigenetic small molecules (Farr 2020), a dose of 1 M of each compound was used in a row or column pool for dmd zebrafish screening treatments. Embryos from dmd+ / - crosses were treated from 24-96 hours postfertilization (hpf), based on the timing used for previous zebrafish dmd drug screens (FIG. 2B) (Farr 2020; Kawahara and Kunkel 2013; Kawahara 2011 ; Waugh et al., Human Molecular Genetics 23, 4651-4662, 2014 (“Waugh 2014’’)). Each row- and column-based drug pool was tested in quadruplicate, with 25 animals per well for each replicate, based on power calculations (FIG. 2B; see Materials and methods). After treatments, animals were fixed to preserve them for scoring muscle birefringence, whereby polarized light transmission appears reduced in the damaged muscle of diseased animals (FIGs. 2B, 20). The approach for assessing the dystrophic phenotype in this drug screen follows the approach used in previous zebrafish dmd chemical screens (Farr 2020; Kawahara 2011 ; Waugh 2014; Kawahara et al., Human Molecular Genetics 23, 1869-1878, 2014). In this approach, animals were scored as “affected”, showing a dmd mutant (dmd- / ~) muscle lesion phenotype, or “unaffected”, showing normal birefringence (FIG. 2C).

[0099] During the screening, general embryo lethality (unrelated to dmd) was observed for 51 of the 185 unique treatment pools, thereby precluding birefringence-based phenotypic scoring of larvae at 96 hpf. Within the corresponding dimethylsulfoxide (DMSO)-vehicle control groups, only low levels of mortality occurred (FIG. 3). The intersections of the 51 “lethal” row and column pools identified 71 compounds (“excluded compounds” in FIG. 1). To circumvent the embryonic lethality 71 compounds were excluded from their respective drug pools. Following the exclusion of the 71 suspected toxic compounds, re-screening of the 51 pools showed that only 5 pools remained toxic, thus allowing birefringence scoring of 46 of the 51 lethal pools. Overall, 180 unique pools were evaluated, and 86% (703 / 817) of compounds in the library, in >2 (initial or secondary row and column) treatment pools (FIGs. 1 , 2D).

[0100] For the 180 scoreable treatment pools, the average percentage of affected animals from each pool treatment and from each corresponding DMSO control treatment was determined. By fitting the normalized binary (affected vs. unaffected) phenotypic scoring results to a logistic regression model, the odds of observing affected animals were significantly reduced in 8 of 180 pools (FIGs. 4A, 5). The pool with the second lowest odds of affected animals, [plate 11_row H], which had 39% decreased odds of lesions compared to controls, is noteworthy because it contains trichostatin A (TSA), a pan-HDAC inhibitor previously shown to improve mouse and zebrafish DMD models (Johnson 2013; Farr 2020; Minetti 2006). However, only one compound, molibresib, emerged through the intersection of these top-ranked pools ([plate 10_row H], [plate 10_col 5]; FIG. 4A). The inability to identify multiple positive row- and column-intersecting compounds from this logistic regression model suggests that requiring both row and column drug pool treatments to exhibit rescue is too stringent, as some drug pools may have inhibitoryinteractions. In addition, binary scoring of larvae as affected / unaffected may be insensitive to drug pool treatments that cause modest or incomplete rescue in dmd mutants.

[0101] Composite scoring identifies the HDACi SR-4370 as a beneficial compound. In order to take into account both row and column treatment effects, as well as treatments that cause a reduction in lesion severity without achieving complete rescue, a composite scoring approach was developed that allowed for the incorporation of both ordinal scoring (unaffected, mild, moderate, or severe; FIG. 2C) and binary scoring (percentage of affected animals) for both row and column treatments (see Mathematical Formula 1 in Materials and methods). In control DMSO treatments, 25% of larvae from dmd+ / - crosses appear affected and exhibit muscle lesions that are predominantly scored as severe at 96 hpf (FIG. 3). Compounds in row and / or column pool treatments with less than 25% affected larvae and / or a greater proportion of mild vs. severe lesions were calculated as having higher composite scores (FIG. 6). These composite scores were used to generate a ranked plot of the inferred rescue effects of individual library compounds (FIG. 4B). TSA emerged as the compound with the greatest inferred rescue effect (93% average percent increase in larvae with mild lesions in [plate 11_row H] and [plate 11_col 6] treatments compared to DMSO controls) (FIGs. 4B, 6), supporting the utility of using the formula to rank the rescue effects of individual compounds.

[0102] The composite scoring identifies the HDACi SR-4370 as the compound with the second highest inferred rescue effect (83% average percent increase in larvae with mild lesions compared to DMSO control) (FIGs. 4B, 6). This score primarily reflected SR-4370’s inclusion in the pool [plate 8_col 7], which had the overall lowest odds of dystrophic lesion development (median odds ratio (OR)=0.319; FIGs. 4A, 5). SR-4370 was tested for its ability to rescue dmd lesions as an individual compound. 1 M treatments of SR-4370, from 24-96 hpf, led to significantly improved dmd birefringence (FIGs. 4C, 4D). Additional individual compounds from the composite scoring were then tested, including the next top 10 compounds with the highest composite scores, as well as molibresib (FIG. 4A) and other compounds with a range of composite score ranks (FIG. 7). Aside from TSA and SR-4370, no other individual compounds tested showed significant effects on dmd rescue (FIG. 7). The lack of effect of these additional compounds correlates with the steep reduction in inferred rescue effect between SR-4370 (rank 2) and the next highest ranked compound (CC-90005, rank 3) (FIGs. 4B, 6). Thus, through pool-based screening and the composite scoring approach, SR-4370 was identified as an HDACi compound that improves zebrafish dmd muscle lesions.

[0103] Additional validations corroborate the beneficial effects of SR-4370. Although the effects of SR-4370 and SR-4370-containing treatment pools were consistent across multipleexperimental batches, all compounds were sourced from a single vendor, thereby representing a potential source of confirmation bias. The source-independent effects of SR-4370 was validated by ordering compounds from 3 different vendors (FIG. 8) and testing them using standard 1 M treatments from 24-96 hpf. dmd muscle birefringence was significantly improved across all treatments of SR-4370 from multiple sources (FIG. 9A). Normalized birefringence ranged between 82%-88%, indicative of consistent phenotypic improvement across groups (FIG. 9A).

[0104] To independently validate the effects of SR-4370, treatments were performed on dmd zebrafish raised at a different geographical location (Location 2), using standard 1 M SR-4370 treatments from 24-96 hpf. Although normalized dmd birefringence was lower in animals at Location 2 compared to the original location of the work (Location 1), dmd muscle was significantly improved at both locations (FIG. 9B). By demonstrating reproducibility, these independent tests are a strong validation of the benefits of SR-4370 in dmd mutant zebrafish.

[0105] HDACi treatments on dmd embryos are effective prior to muscle lesion formation. The optimal SR-4370 treatment dose and timing necessary for dmd muscle lesion improvement was determined. Doses of SR-4370 from 0.1-10 pM were tested using the standard 24-96 hpf treatment conditions. A dose response between 0.1-1 pM was determined, with 500 nM being the minimum threshold for effective rescue of birefringence (FIG. 10A). 2.5 pM treatment did not significantly improve dmd muscle birefringence and caused substantial lethality, and embryonic lethality (of both WT and dmd embryos) occurred with 5 pM and 10 pM treatments (FIG. 10A). These results identify 1 pM as the optimal dose for 24-96 hpf treatments.

[0106] Muscle lesions in dmd zebrafish are initially visible at 48 hpf but become progressively more conspicuous through 96 hpf (Bassett 2003). Next, it was evaluated whether shortened administration of beneficial compounds, prior to lesion formation, could affect muscle morphology at later stages (FIG. 10B). Using birefringence as a readout, it was observed that an abbreviated 24-48 hpf “early pulse” administration of either SR-4370 or TSA significantly improved the dystrophic phenotype at 96 hpf (FIGs. 10C, 10D). Using phalloidin staining to image trunk muscle, it was observed that an early pulse treatment of SR-4370 led to improved muscle morphology at 96 hpf as well as at 10 days post-fertilization (dpf) (FIGs. 10E-10G). Thus, a single HDACi administration from 24-48 hpf, prior to lesion formation, improves dmd muscle structure for at least several days past the treatment.

[0107] Finally, it was tested whether the standard 3-day treatment length could be applied after lesion formation to improve dmd birefringence. Treatments from 96-168 hpf did not provide any detectable improvement of dmd birefringence (FIG. 10C). These results suggest that HDACi compounds, and specifically SR-4370, are effective during early development.

[0108] Drug pool screening identifies complex drug combinations beneficial for dmd zebrafish. Because the previous studies identified the HDACi oxamflatin as beneficial for dmd zebrafish, oxamflatin-containing drug pools from the current library screen were re-examined to determine whether there was evidence of phenotypic improvement. Oxamflatin is located at library position [plate 7_F10] (FIG. 1) and only achieved a composite rescue effect of 13% (FIG. 6). Pool [plate 7_col 10] had slightly decreased odds of lesion development (median OR=0.725), while the corresponding row pool containing oxamflatin, [plate 7_row F], demonstrated increased odds of lesion development (median OR=1.290) compared to controls (FIG. 5). Quantitative birefringence from these two pool treatments were assessed and it was found that [plate 7_row F] significantly improved dmd birefringence, while [plate 7_col 10] did not (FIG. 11A). Even though [plate 7_row F] had increased odds of lesions from binary scoring, the quantitative birefringence results suggest that the lesions in affected animals are less severe. Individual compounds from pool [plate 7_row F], at 1 pM each, were then tested and it was found that none of the 10 compounds in pool [plate 7_row F], including oxamflatin, significantly improved dmd muscle birefringence (FIG. 11 B). These results therefore suggest that a combination of drugs within pool [plate 7_row F] is contributing to the rescue effects of this drug pool.

[0109] Next, the goal was to narrow down which compounds from pool [plate 7_row F] were necessary for improving dmd birefringence by systematically removing individual compounds from the drug pool. A series of drug pools were tested, containing 9 compounds each, and their effects were compared to the full 10-compound pool from [plate 7_row F], Drug pools lacking HATi-ll, NSC 663284, or FLLL32 did not significantly improve dmd muscle birefringence, identifying these three compounds as necessary for the rescue effects of [plate 7_row F] (FIG. 11C). However, pairwise combinations of these compounds, including combinations with oxamflatin, exhibited no rescue effects (FIG. 11 D), indicating that a combination of greater than two compounds from [plate 7_row F] was likely necessary to improve the muscle phenotype in dmd mutants. These results underscore some of the complexities of screening drug pools. Even though in previous studies a beneficial two-drug combination was identified that includes oxamflatin, these results suggest that multi-epigenetic compound combinations also have the potential to provide modest rescue of dmd birefringence.

[0110] HDACi-induced muscle improvement correlates with increased histone acetylation. Next, the effects of different HDACi treatments in dmd zebrafish were compared and mechanistically validated. Effective doses of the HDACi TSA and oxamflatin, as well as givinostat and PCI-34051 , which have both been shown to improve a zebrafish dmd-knock-down model (Spreafico 2021). Using 24-48 hpf treatments, 200 nM TSA and 10 pM and 20 pM oxamflatin treatments increaseddmd birefringence, whereas 20 nM TSA and 1 pM oxamflatin did not induce any rescue (FIGs. 12A, 12B), consistent with previous studies (Johnson 2013; Farr 2020). However, no birefringence improvement was seen with any tested doses of givinostat or PCI-34051 (FIGs. 12A, 12B). 200 pM givinostat and 50 pM PCI-34051 treatments both resulted in precipitation and the formation of a crystalline film in each well, suggesting that the highest possible treatment dose for these compounds was exceeded. These results reveal variation among different HDACi in their rescue ability and effective doses for dmd zebrafish.

[0111] Next, it was examined whether the effective and ineffective doses of these different HDACi led to altered histone and tubulin acetylation levels. As pan-HDACi, TSA, SR-4370, givinostat, and oxamflatin have been shown to induce increased histone H4 and histone H3 acetylation (Farr 2020; Minetti 2006; Mozzetta 2024; Kim et al., Oncogene 18, 2461-2470, 1999; lezzi et al., Proc Natl Acad Sci U S A 99, 7757-7762, 2002 (“lezzi 2002”); lezzi et al., Dev Cell 6, 673-684, 2004; Consalvi et al., EMBO Rep 23, e54721 , 2022 (“Consalvi 2022”)). PCI-34051 is a selective HDAC8 inhibitor that has been shown to increase tubulin acetylation (Spreafico 2021). Western blot analysis revealed that both pan-H4 acetylation and H3K9 acetylation were significantly elevated in larvae exposed to 200 nM TSA, 1 pM SR-4370, and 20 pM oxamflatin, doses that rescue dmd birefringence (FIGs. 10A, 12A, and 12C-12E). Increased histone acetylation was not observed following givinostat treatments (40 and 200 pM), PCI-34051 treatments (1 and 50 pM), or non- dmd-rescuing doses of TSA, SR-4370, and oxamflatin (FIGs. 12A, 12C). Increased tubulin acetylation was observed in larvae exposed to 200 pM givinostat and 50 pM PCI-34051 , suggesting that these compounds were active within the animals (FIG. 12F). HDACi treatments and doses that improve dmd muscle birefringence thus correlate with increased histone acetylation but do not correlate with increased tubulin acetylation. Therefore, HDACi treatments that improve the muscle phenotype of dmd zebrafish are strongly associated with increased histone acetylation.

[0112] SR-4370 increases longevity in dmd zebrafish. Due to the mechanistic and phenotypic differences seen after HDACi treatments during embryonic and larval development, dmd survival was evaluated as an indirect indicator of cumulative, long-term motor function. It was first tested whether a single early pulse treatment of HDACi from 24-48 hpf could improve dmd survival. HDACi doses were selected based on their ability to improve birefringence (FIG. 12A) or their maximum solubility in 0.5% DMSO. After receiving a single exposure from 24-48 hpf, animals from dmd+ / - crosses were sorted via birefringence at 4 dpf into control (dmd+ / + and dmd+ / -) and dmd groups. Animals were then monitored for 50 days. Survival in control siblings (dmd+ / + and dmd+ / -) was high (>92% through 50 dpf) and did not significantly differ across treatments (FIG.13A). The DMSO-treated dmd group had a median survival time of 27 days, and complete mortality, or 0% survival, was observed by 39 dpf (FIG. 13A). Relative to the DMSO dmd group, no significant differences were seen in dmd animals treated with either TSA or givinostat (median survival of 23 and 25 days, respectively). However, median survival for the SR-4370 dmd group was significantly increased to 31 days, or +14.8%, compared to the DMSO dmd group (FIG. 13A). Additionally, the TSA- and SR-4370-treated dmd groups each had animals surviving through 50 dpf (3 and 7 animals, respectively). These results show that a single early application of HDACi can improve the muscle phenotype and increase lifespan in dmd zebrafish.

[0113] It was next investigated whether reapplication of SR-4370 beyond the initial 24-48 hpf early pulse treatment could further increase lifespan. Animals in these experiments were exposed to multiple treatments beginning at 24-48 hpf and repeated every 3-4 days (FIG. 13B). The DMSO-treated dmd group had a median survival time of 32 days, and complete mortality was observed by 41 dpf (FIG. 13B). Median survival in the SR-4370-treated dmd group was significantly increased to 36 days, or +12.5%, relative to the DMSO dmd group (FIG. 13B). Additionally, the SR-4370-treated dmd group had 1 animal surviving through 50 dpf (FIG. 13B). While both SR-4370 treatment regimens significantly improved survival, the repeated reapplication of SR-4370 did not appear to have a greater beneficial effect than the single early pulse, consistent with the finding that delayed treatment, post lesion appearance, did not improve dmd birefringence at 168 hpf (FIG. 10C). These collective results show that SR-4370 can improve early muscle lesions, increase histone acetylation, and increase dmd zebrafish lifespan in an application-dependent manner.

[0114] Discussion. In this investigation, a drug-pooling approach was used to screen over 800 epigenetic small molecules for their ability to alleviate muscle lesions in the zebrafish dmd model. An HDACi, SR-4370, that significantly improved muscle structure in dmd zebrafish larvae was identified. After conducting dose-response experiments, it was found that 500 nM and 1 M doses of SR-4370 significantly ameliorated muscle lesion severity. Treatments with the HDACi SR-4370 and TSA significantly rescued the dmd phenotype between 4 and 10 dpf and when embryos were exposed from 24 to 48 hpf, prior to dmd muscle lesion appearance. Furthermore, the ability of the HDACi TSA, SR-4370, and oxamflatin to improve dmd zebrafish muscle correlated with the ability of these HDACi to cause increased levels of histone acetylation in zebrafish larvae. SR-4370 can also increase the lifespan of dmd zebrafish larvae following single and multiple applications. The findings support the use of zebrafish as an efficient screening tool for identifying small molecules that have in vivo alleviatory effects in damaged muscle.

[0115] Zebrafish are a well-established model for drug screening and drug discovery for humandisease and, in particular, muscular dystrophy (Karuppasamy et al., Dis Model Meeh 17, dmm050339, 2024 (“Karuppasamy 2024”); Widrick et al., J Neuromuscul Dis 6, 271-287, 2019; Patton et al., Nat Rev Drug Discov 20, 611-628, 2021 ; Lee et al., Pharmaceuticals (Basel) 14, 500, 2021). The orthogonal drug pooling method that was employed here is similar to the approach used in a previous screen for antiangiogenic compounds in zebrafish larvae (Ohnesorge et al., Front Pharmacol 10, 508, 2019). Pooled chemical screens can save time and resources as well as significantly reduce the number of animals used relative to single compound efforts. One limitation of the pooled-compound approach is that overly toxic individual chemicals can cause drug pools to be unfeasible to screen. This issue was addressed by orthogonally identifying likely toxic candidate compounds, removing these compounds, and rescreening the drug pools. Another limitation of the pooled-compound approach is that pooled compounds may inhibit or mask each other’s activity without apparent toxicity. This issue was addressed by generating a composite scoring approach, which allowed us to identify SR-4370, based largely on its activity in its column pool. Oxamflatin was found to exhibit dmd rescue effects at >10 pM, but not at 1 M (FIGs. 12A-12F). However, aside from the positive effects shown here for the HDACi TSA, SR-4370, and oxamflatin, 37 additional compounds were tested individually (FIGs. 7, 11A-11 D) and no clear dmd rescue effects were found from these compounds. Of the 37 compounds tested where no clear dmd rescue effects were found, tucidinostat, SW-100, mocetinostat, and entinostat are HDACi.

[0116] HDACi treatments are effective at improving zebrafish dmd birefringence when they are initiated prior to overt dmd muscle lesion formation. These results are consistent with previous studies showing that young (1.5 or 3 month old) mdx mice treated with an effective HDACi (TSA) show improved muscle morphology and function, including increased myofiber size and integrity (while older (12 month old) mdx mice are resistant to the beneficial effects of otherwise effective HDACi (Minetti 2006; Consalvi 2013; Mozzetta et al., EMBO Molecular Medicine 5, 626-639, 2013 (“Mozzetta 2013”); Saccone et al., Genes Dev. 28, 841-857, 2014 (“Saccone 2014”)). The age-dependent effects of otherwise effective HDACi in mdx mice may be mediated by the age- and disease-dependent plasticity of fibro-adipogenic progenitors (FAPs), which are multipotent muscle mesenchymal cells (Mozzetta 2024; Consalvi 2022; Mozzetta 2013; Saccone 2014). In early-stage mdx mice, otherwise effective HDACi can induce FAPs toward a pro-myogenic and proliferative phenotype, whereas late-stage FAPs are resistant to these HDACi effects (Mozzetta 2024; Consalvi 2022; Mozzetta 2013; Saccone 2014; Sandona et al., UMS 24, 4306, 2023). The effectiveness of HDACi during zebrafish embryogenesis may also be related to the stagedependent ability of HDACi to promote myogenesis and muscle differentiation (lezzi 2002).

[0117] The ability of HDACi treatments to improve dmd zebrafish muscle structure correlates with the ability of HDACi to cause increased histone acetylation in zebrafish larvae. Zebrafish treated with the HDACi TSA, SR-4370, and oxamflatin, showed levels of H3K9 and pan-H4 acetylation increased relative to control animals. A combination treatment of the HDACi salermide and oxamflatin caused an increase in pan-H4 acetylation and H4K16 acetylation (Farr 2020). HDACi administration similarly causes histone hyperacetylation in mdx mice (Minetti 2006; Consalvi 2022; Colussi et al., PNAS 105, 19183-19187, 2008 (“Colussi 2008”)).

[0118] A third finding is the long-term effectiveness of early HDACi treatment at increasing the lifespan of dmd zebrafish. Both single and repeated applications of SR-4370, initiated prior to lesion development, increased longevity relative to dmd animals treated with DMSO or with other HDACi.

[0119] Changes in tubulin acetylation was observed with a high dose of givinostat or PCI-34051 , reflecting activity of these compounds in the animals.

[0120] SR-4370 is reported to inhibit HDAC1 , 2, 3, 6, and 8 (Zutz et al., Antimicrob Agents Chemother 65, e01815-20, 2020 (“Zutz 2020”)). HDAC2 down-regulation leads to improved muscle structure and function in mdx mice (Colussi 2008). Inhibiting HDAC6 in mdx mice results in improved muscle phenotype through Smad3 acetylation and downregulation of transforming growth factor beta signaling (Osseni et al., Nat Commun 13, 7108, 2022). HDAC8 inhibition with PCI-34051 in dystrophic zebrafish results in a-tubulin acetylation and improved cytoskeleton organization (Spreafico 2021). SR-4370 is fluorinated. A comparison of inhibitory activity of the HDACi vorinostat to that of several synthesized 1 ,2-difluoro versions showed that the fluorinated compounds had increased potency against HDAC1 and HDAC6 compared to the parent compound (Erdeljac et al., ACS Med Chem Lett 10, 1336-1340, 2019). Fluorinated analogues of the class I HDAC inhibitor largazole had stronger inhibition towards class I HDACs than unmodified largazole (Zhang et al., Eur J Med Chem 182, 111672, 2019).

[0121] A biochemical readout of the compounds being tested, in this case histone or tubulin acetylation levels in zebrafish larvae treated with HDACi was assayed. In cases where positive benefits of HDACi on dmd muscle structure were observed, increased histone acetylation levels were also observed.

[0122] This example identifies SR-4370 as a therapeutic HDACi for the treatment of muscle- related disorders.

[0123] Materials and methods. Zebrafish husbandry. All animal experiments were carried out in accordance with controlling Institutional Animal Care and Use Committees (IACUC). In general, the approaches followed the recommended standards for zebrafish drug screening(Karuppasamy 2024). Zebrafish were raised and staged as described in Westerfield, M. (The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio Rerio). (University of Oregon Press, Eugene, 2007). Time (hpf or dpf) refers to hours or days post-fertilization at 28.5°C. Eggs were collected from 20-30 min spawning intervals and raised in Petri dishes in ICS water (Farr 2020) in a dark 28.5°C incubator, up to 5 dpf. After 5 dpf, the fish were maintained on a recirculating water system (Aquaneering, San Diego, CA) under a 14 h on, 10 h off light cycle. From 6-30 dpf, the fish were raised in 2.8 L tanks with a density of no more than 50 fish per tank and were fed a standard diet of paramecia (Carolina) one time per day and Zeigler AP100 dry larval diet two times per day. From 30 dpf onwards, the fish were raised in 6 L tanks with a density of no more than 50 fish per tank and were fed a standard diet of Artemia nauplii (Brine Shrimp Direct) and Zeigler adult zebrafish feed, each two times per day. The zebrafish dmd132223mutant strain (also known as sapje hereafter referred to as dmd) has been previously described and is a recessive, non-sex-linked null allele (Granato 1996; Bassett 2003). The dmd132223strain was originally derived on a TU genetic background (Granato 1996) and has been maintained at SCRI for several generations through outcrosses with an AB wild-type strain, thus the dmd132223genetic background for this example is considered mixed but largely AB. dmdta222agenotyping was performed as previously described (Berger et al., Journal of Cellular and Molecular Medicine 15, 2643-2651, 2011). Experimental zebrafish embryos and larvae were used at stages prior to sexual maturity, and zebrafish lack a definite sex-determining chromosome (Aharon et al., Cell. Mol. Life Sci. 79, 8, 2021).

[0124] Small molecules. Chemical screening was performed using an epigenetic compound library (MedChemExpress, Monmouth Junction, NJ; Catalog #HY-L005) including 817 compounds distributed over fourteen 96-well plates. Most compounds were received as 10 mM stocks pre-dissolved in dimethyl sulfoxide (DMSO). The identity, plate, and well location of each compound is provided in FIG. 1.

[0125] For each plate, compounds across a single row ( / .e., rows A-H) or down a single column (i.e., columns 2-11) were combined in equal parts (FIG. 2A), resulting in 185 unique pools. 1000X stocks of chemicals were made in DMSO just prior to each treatment experiment.

[0126] Select compounds were separately ordered from MedChemExpress or other sources and resuspended in DMSO (Sigma-Aldrich, St. Louis, MO) to make 10 mM stocks. The individually- ordered compounds, sources, and corresponding PubChem Cl Ds are listed in FIG. 8.

[0127] Small molecule treatments. For epigenetic library screening for dmd birefringence rescue tests, compounds were administered from 24-96 hpf, the same timing used for previous zebrafish dmd drug screens (FIG. 2B) (Farr 2020; Kawahara and Kunkel 2013; Kawahara 2011 , Waugh2014). Embryos from dmdta222ai+crosses were produced via group spawnings, collected at 20-30 min intervals, sorted to a density of <100 embryos / IOOmm Petri dish, and raised at 28.5°C. Clutches were selected for both appropriate and synchronous development at 24 hpf as a requirement for experimental inclusion. At 24 hpf, embryos were enzymatically dechorionated with 0.5 mg / mL Pronase (Sigma-Aldrich, St. Louis, MO) for 14 min and sorted into 12-well plates at a density of 25 embryos / well, as previously described (Hasegawa et al., JDB 11 , 16, 2023). Quadruplicate wells each received 3 mL of embryo medium (EM; 14.97 mM NaCI, 0.50 mM KCI, 0.98 mM CaCI2.2H2O, 0.15 mM KH2PO4, 0.99 mM MgSO4.7H2O, 0.05 mM Na2HPO4, 0.83 mM NaHCOs) with either vehicle control or chemical treatment, and treatment media were refreshed every 24 hr. Unless otherwise noted, vehicle control was 0.5% DMSO and the working concentration of compound(s) in a treatment group was 1 pM / compound, based on Farr et al., which identified 1 pM as an optimal dose for zebrafish embryo survival with epigenetic small molecule treatments (Farr 2020). Larvae at 96 hpf were fixed in 10% formalin in phosphate buffered saline (PBS) and stored at 4°C until phenotypic scoring and imaging.

[0128] For treatments of individual drugs, the steps described above were followed, except that drug concentrations and / or timing of treatments were altered, as described in the Results. “Early pulse” treatment was from 24-48 hpf while “delayed” treatment was from 96-168 hpf (FIG. 10B). To obtain tissue for genotyping following treatments, larval heads were removed with a scalpel at the level of the pectoral fins. Larval tails with trunk skeletal muscle remained intact and were maintained in 10% formalin in PBS until imaging was performed.

[0129] Validation treatments at the University of Maine were carried out as described above except for the following modifications: Embryos were raised in a 28°C incubator with a 14 hr on, 10 hr off light cycle. At 24 hpf, embryos were manually dechorionated with forceps and sorted into 12-well plates at a density of 4-6 embryos / well. Vehicle control was 0.1 % DMSO, while working concentration of SR-4370 remained at 1 pM. At 96 hpf, larvae were fixed in 4% paraformaldehyde (PFA) and stored at 4°C for 48 hours. Larvae were then rinsed in PBS- 0.1 % Tween 20 prior to imaging.

[0130] Muscle birefringence imaging and quantitation. Qualitative and quantitative scoring of dmd zebrafish larval muscle lesions using polarized light birefringence was performed as previously described (Farr 2020). Birefringence was used to qualitatively sort and score larvae based on the binary presence (muscle lesions vs. no muscle lesions) and ordinal severity (unaffected / no lesions or mild, moderate, or severe lesions) of dystrophic muscle disease (FIG. 2C) (Farr 2020; Smith et al., JoVE 50925, 2013). Quantitative measurements of birefringence were performed by mounting larvae in 2.5% methyl cellulose and adjusting camera and software settings (SZX16stereomicroscope, DP72 camera, cellSens Dimension v4.1 ; Olympus Life Sciences, Bethlehem, PA) so that grayscale images had few to no saturated pixels across the entirety of the trunk, as previously described (Farr 2020). Imaged was used to outline the trunk musculature, using the wand tool, and to calculate the average pixel intensity within the resulting outlined selection. Average pixel intensity values (mean gray values) across the trunk muscle birefringence of individual larvae were calculated, and values were then normalized to the WT +DMSO control average values.

[0131] Pool-specific risk ratio and composite scoring to obtain inferred rescue effect of individual compounds. Because of the number of treatments and animals needed, screening of the 185 drug pools of the epigenetic library was performed over multiple batches and zebrafish breedings. Therefore, frequencies of affected animals with lesions (using binary scoring) across chemical treatment pools were normalized relative to the batch-specific vehicle control average. These normalized results were then fitted to a logistic regression model (R package: GLM, v14) that assigned each drug pool a risk ratio reflective of the likelihood of affected animals compared to DMSO controls (FIG. 5).

[0132] To assign individual compounds an inferred rescue score based on the effects of their corresponding row and column drug pools, a composite score that incorporated both binary scoring (affected vs. unaffected) and ordinal scoring (lesion severity), using the following formula (Mathematical Formula 1):

[0133] Mathematical Formula 1: Average percent mild for an exposed group or control treatment with n replicates: number of mild in a replicate bi -> number of affected in a replicateL = 1 / n £"=1Oi)

[0134] For a given exposed row or column group, difference from the corresponding batch DMSO control:Drow ~ / -row / -DMSODcol = / -col — -DMSO

[0135] Inferred rescue from intersecting pools:R ~ (DrOw + Drow) /

[0136] The composite score thus accounts for results from intersecting row and column drug pool treatments. Mathematical Formula 1 values for individual compounds (composite scores) are provided in FIG. 6 and are plotted in FIG.4B.

[0137] Phalloidin staining and imaging. Larvae were fixed for 4 hours at room temperature in 4% paraformaldehyde in PBS, after which they were washed in PBS + 0.1% Tween 20 (PBTw) and stored in PBS + 0.02% sodium azide at 4°C until the staining procedure was begun. The heads were removed for genotyping, after which the trunks / tails were pooled by genotype for imaging. The trunk / tail samples were permeabilized in 2% Triton X-100 at room temperature (2 hours for 96 hpf animals; 6 hours for 10 dpf animals) and then washed in PBTw. Muscle was labeled with phalloidin (Biotium Phalloidin CF 488A; Cat # 00042) diluted 1 :30 in antibody block (5% bovine serum albumin; 2% heat-inactivated normal goat serum; 1 % DMSO; 1% Triton X-100; 0.2% saponin in PBS) at 4°C (16 hrs for 96hpf animals; 64 hrs for 10dpf animals). The samples were then washed in PBTw, partially cleared in 80% glycerol, and mounted laterally in 80% glycerol + 4% propyl gallate (Sigma; Cat # P3130). Images were obtained on a Leica TCS SP5 confocal with a 40X, 1.3 NA oil immersion objective (96 hpf images) or a 20X, 0.7 NA non-immersion objective (10 dpf images). Image stacks were processed in Imaged.

[0138] Immunoblotting. Following HDACi exposure from 24-48 hpf, 25 larvae / replicate were de- yolked by rinsing in cold Ringer’s solution then lysed in 12.5 pL / larva of 1.5x LDS sample buffer (Cat. No. NP0007; Invitrogen, Waltham, MA). Lysates of 1 larva equivalent were then separated on reducing 12% NuPage Bis-Tris gels (NP0342BOX; Invitrogen), run in NuPage MES SDS buffer (Cat. No. NP0002; Invitrogen), and blocked in LI-COR TBS Intercept block (Cat. No. 927-660003; LI-COR Biosciences, Lincoln, NE). Blots were probed using the following antibodies: anti-acetyl- histone H3 (K9) (1 :4000; Cat. No. 07-352; MilliporeSigma, Burlington, MA), anti-acetyl-histone H4 (recognizes acetylated K5, K8, K12, and K16; 1 :2000; Cat. No. 06-866; MilliporeSigma), and antiacetyl a-tubulin (1 :1000; Cat. No. 5335; Cell Signaling Technology, Danvers, MA). Anti-a-actin (1 :4000; Cat. No. 0869100; MP Biomedical, Irvine, CA) was included as a loading control. Infrared-labeled secondary antibodies (Anti-rabbit IgG DyLight 800, Cat. No. 611-145-002, used at 1 :10,000, and Anti-mouse IgG DyLight 680, Cat. No. 610-144-002, used at 1 :20,000; Rockland Immunochemicals, Philadelphia, PA) were visualized using an Odyssey infrared imager (LI-COR Biosciences) and quantified using Image Studio Lite (version 5.2). See FIGs. 14A-14Dfor uncropped images of original blots. Replicates (n=3) for all DMSO, SR-4370, givinostat, and oxamflatin treatments were run on different gels, with separate a-actin normalization per gel. As part of an independent experimental batch, DMSO and PCI-34051 treatment replicates (n=3) were run on a single gel, with separate a-actin normalization per treatment.

[0139] Survival analyses. Early pulse HDACi treatments for survival analysis followed the steps described above for “Early pulse” treatment, from 24-48 hpf. Vehicle control was 0.5% DMSO, TSA was used at 200 nM, SR-4370 was used at 1 pM, and givinostat was used at 200 pM. At 4dpf, phenotypic sorting (affected vs. unaffected) was performed under brief anesthesia using a 1 :50 dilution of 0.4% MS-222 (Finquel, Merck & Co, Rahway, NJ) for a final concentration of 0.008% in 5 mL of ICS water. From 5 dpf through the end of the experiment, animals were reared and fed as described above under Zebrafish husbandry, except that a reduced housing density of 25 animals per 2.8 L tank was used. Animals were counted every 2-3 days through 50 dpf.

[0140] Repeated SR-4370 treatments for survival analysis were initiated with an early pulse treatment from 24-48 hpf as above. At 4 dpf, phenotypic sorting was performed as above. From 5 dpf, animals were reared as described above. For treatments beyond 4 dpf, animals were decanted from 2.8 L housing tanks into 125 mL of media in static containers at a maximum density of 25 animals / container. These treatments were conducted overnight (16 hrs duration), every 3- 4 days, for a total of 14 applications.

[0141] Statistical analyses. A priori power calculation was performed for library screening using the online software G*Power (version 3.1.9.7; Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Germany; www.gpower.hhu.de / ) (Kang et al., Journal of Educational Evaluation for Health Professions 18, 17, 2021). Using a 2-tailed t test, with an alpha of 0.05, and an effect size of 2.6 based on Farr et al., (Farr 2020), we calculated that with n=4 replicates, of 25 animals each, per treatment, a power of 0.86 is achieved.

[0142] Unless otherwise noted, data was analyzed using a one-way ANOVA comparing each treatment group / condition to the dmd DMSO control group, with Dunnett’s correction for multiple comparisons, and a cutoff of p < 0.05. Unless otherwise noted, error bars represent standard error of the mean between replicates. Survival curves were generated using a Kaplan-Meier estimate. A Log-rank (Mantel-Cox) test with a Bonferroni corrected threshold of aBonferroni = 0.0167 or (0.05 / 3) was used to calculate significant differences between comparison groups. Graphs and statistics were generated in GraphPad Prism (version 10.0.3).

[0143] (vi) Closing Paragraphs. As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. A material effect would cause a statistically significant reduction in the ability toobtain a claimed effect according to a relevant experimental method described in the current disclosure.

[0144] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11 % of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0145] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0146] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. Nolanguage in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0147] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0148] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0149] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0150] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0151] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention maybe embodied in practice.

[0152] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

CLAIMSWhat is claimed is:

1. A method of treating a muscle disorder in a subject in need thereof comprising administering a therapeutically effective amount of SR-4370 or an SR-4370 analog to the subject, thereby treating the muscle disorder in the subject.

2. The method of claim 1, wherein the method comprises administering the therapeutically effective amount of SR-4370.

3. The method of claim 1 , wherein the method comprises administering the therapeutically effective amount of the SR-4370 analog.

4. The method of claim 3, wherein the SR-4370 analog comprises formula IwhereinX and Y are independently N or CR2,R1 and R2 are independently hydrogen, an alkyl group, a halo group, or an ester group, R5 is a Ci to Cio alkyl group, and n is an integer from 0 to 3.

5. The method of claim 4, wherein X and Y are each N.

6. The method of claim 4, wherein X and Y are each N and n is 0.

7. The method of claim 4, wherein X and Y are each CR2.

8. The method of claim 4, wherein X is CF, CH, or C-C(0)OR3 and R3 is an alkyl group.

9. The method of claim 4, wherein X is CF, CH, or C-C(0)OR3; R3 is an alkyl group; and n is 0.

10. The method of claim 4, wherein Y is CH or CR4 and R4 is an alkyl group.

11. The method of claim 4, wherein Y is CH or CR4, R4 is an alkyl group, and n is 0.

12. The method of claim 4, wherein X is CF, Y is CH or CR4, and R4 is an alkyl group.

13. The method of claim 4, wherein X is CF, Y is CH, R1 is fluoro, and n is 1.

14. The method of claim 3, wherein the SR-4370 analog comprises formula IIwherein X is C-C(0)0R3and R3is an alkyl group.

15. The method of claim 3, wherein the SR-4370 analog is selected from the group consisting of SR-4373, SR-3558, UF010, SR-4369, SR-4360, SR-4372, and SR-3299.

16. The method of claim 1 , wherein the muscle disorder comprises a muscular dystrophy.

17. The method of claim 16, wherein the muscular dystrophy comprises Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, or dystroglycanopathy.

18. The method of claim 16, wherein the muscular dystrophy comprises Duchenne muscular dystrophy.

19. The method of claim 1 , wherein the therapeutically effective amount is administered as a single dose.

20. The method of claim 1 , wherein the therapeutically effective amount is administered as multiple doses.

21. The method of claim 20, wherein the multiple doses are administered daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly.

22. The method of claim 1 , wherein the therapeutically effective amount is administered via injection, inhalation, infusion, perfusion, lavage, or ingestion.

23. The method of claim 1 , wherein the administering comprises intravenous, intradermal, intraarterial, intraparenteral, intranasal, intramuscular, oral, or subcutaneous administering.

24. The method of claim 1 , wherein the administering occurs prior to muscle lesion formation.

25. The method of claim 1 , wherein the method increases histone acetylation.

26. The method of claim 1 , further comprising administering a therapeutically effective amount of a secondary treatment.

27. The method of claim 26, wherein the secondary treatment comprises a corticosteroid, a second histone deacetylase inhibitor, an antisense oligonucleotide, or an adeno-associated virus (AAV) vector.

28. The method of claim 27, wherein the corticosteroid comprises prednisone, prednisolone, ordeflazacort.

29. The method of claim 27, wherein the second histone deacetylase inhibitor comprises givinostat, trichostatin A (TSA), PCI-34051 , oxamflatin, or salemide.

30. The method of claim 27, wherein the antisense oligonucleotide comprises eteplirsen.

31. The method of claim 27, wherein the adeno-associated (AAV) vector comprises delandistrogene moxeparvovec.

32. A composition comprising a therapeutically effective amount of SR-4370 or an SR-4370 analog and a pharmaceutically acceptable carrier.

33. The composition of claim 32, wherein the composition comprises the therapeutically effective amount of SR-4370.

34. The composition of claim 32, wherein composition comprises the therapeutically effective amount of the SR-4370 analog.

35. The composition of claim 34, wherein the SR-4370 analog has formula IwhereinX and Y are independently N or CR2,R1 and R2 are independently hydrogen, an alkyl group, a halo group, or an ester group, R5 is a Ci to Cio alkyl group, and n is an integer from 0 to 3.

36. The composition of claim 35, wherein X and Y are each N.

37. The composition of claim 35, wherein X and Y are each N and n is 0.

38. The composition of claim 35, wherein X and Y are each CR2.

39. The composition of claim 35, wherein X is CF, CH, or C-C(0)OR3 and R3 is an alkyl group.

40. The composition of claim 35, wherein X is CF, CH, or C-C(0)OR3; R3 is an alkyl group; and n is 0.

41. The composition of claim 35, wherein Y is CH or CR4 and R4 is an alkyl group.

42. The composition of claim 35, wherein Y is CH or CR4, R4 is an alkyl group, and n is 0.

43. The composition of claim 35, wherein X is CF, Y is CH or CR4, and R4 is an alkyl group.

44. The composition of claim 35, wherein X is CF, Y is CH, R1 is fluoro, and n is 1.

45. The composition of claim 34, wherein the SR-4370 analog comprises formula IIwherein X is C-C(0)0R3and R3is an alkyl group.

46. The composition of claim 34, wherein the SR-4370 analog is selected from the group consisting of SR-4373, SR-3558, UF010, SR-4369, SR-4360, SR-4372, and SR-3299.

47. The composition of claim 32, wherein the therapeutically effective amount treats a muscle disorder.

48. The composition of claim 47, wherein the muscle disorder comprises a muscular dystrophy.

49. The composition of claim 48, wherein the muscular dystrophy comprises Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophy, or dystroglycanopathy.

50. The composition of claim 48, wherein the muscular dystrophy comprises Duchenne muscular dystrophy.

51. The composition of claim 32, wherein the therapeutically effective amount comprises from 0.1 pg / kg to 1000 mg / kg.

52. The composition of claim 32, wherein the therapeutically effective amount comprises 1 pg / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, or 1000 pg / kg.

53. The composition of claim 32, wherein the therapeutically effective amount comprises 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, or 1000 mg / kg.

54. The composition of claim 32, further comprising a secondary treatment.

55. The composition of claim 54, wherein the secondary treatment comprises a corticosteroid, a second histone deacetylase inhibitor, an antisense oligonucleotide, or an adeno-associated (AAV) vector.

56. The composition of claim 55, wherein the corticosteroid comprises prednisone, prednisolone, or deflazacort.

57. The composition of claim 55, wherein the second histone deacetylase inhibitor comprisesgivinostat, trichostatin A (TSA), PCI-34051 , oxamflatin, or salemide.

58. The composition of claim 55, wherein the antisense oligonucleotide comprises eteplirsen.

59. The composition of claim 55, wherein the adeno-associated (AAV) vector comprises delandistrogene moxeparvovec.

Images