Treatment of Muscle Fibrosis

Abstract

The present invention relates to compounds for use in treating a disease or disorder associated with muscle fibrosis in a subject. In particular, the present invention relates to inhibitors of the RhoA / ROCK pathway for use in treating a disease or disorder associated with muscle fibrosis in a subject, such as muscular dystrophy.

Description

[Technical field]

[0001] The present invention relates to compounds for use in treating a disease or disorder associated with muscle fibrosis in a subject. In particular, the present invention relates to inhibitors of the RhoA / ROCK pathway for use in treating a disease or disorder associated with muscle fibrosis in a subject, such as muscular dystrophy. [Background technology]

[0002] Fibrosis is a common and important process in the degeneration of skeletal muscle in patients with muscular dystrophy (MD), leading to muscle weakness, stiffness, contractures, and permanent disability.

[0003] MD is a group of muscle disorders in which muscle fibers are abnormally susceptible to damage. This results in the accumulation of defective muscle proteins, death of muscle cells and tissues, and progressive weakening of the musculoskeletal system of affected individuals. Nine major types of muscular dystrophies have been identified: Duchenne, Becker, limb-girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreifuss muscular dystrophies. Several conditions that resemble muscular dystrophies have also been identified.

[0004] In normal striated muscle, dystrophin associates with a large amount of proteins known as the dystrophin glycoprotein complex (DGC). The DGC serves to stabilize the sarcolemma by providing regular interval connections between the myofiber cytoskeleton and the extracellular matrix (part of the costamere cell adhesion complex). At the core of this cell adhesion complex is the adhesion receptor dystroglycan, which connects laminin in the extracellular matrix to dystrophin on the cytoplasmic surface. However, in many disorders, including muscular dystrophies, the production of functional dystrophin protein and / or functional DGC is impaired.

[0005] Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease that affects approximately 1 in every 3,500 male births and currently has no cure or effective treatment. Various molecular genetic approaches have been devised to combat DMD, but are unlikely to address the needs of all DMD patients. Gene replacement, compensatory gene upregulation, and cell-based therapies using numerous delivery methods have all had some success in the laboratory but have failed to translate to the clinic for a variety of reasons (Pichavant.C et al., Mol Ther, 2011). However, great success has been achieved recently by using an exon skipping approach to splice out the mutated portion of the DMD gene and restore some functional dystrophin gene (Kinali.M et al., Lancet Neurol., 2009. 8:918). This is a rapidly developing field, and phase II clinical trials of exon skipping are underway. These approaches offer real hope to the approximately 25% of DMD patients for whom there is no effective treatment. It is clear that there remains a need for therapeutic approaches that can be effective for all DMD patients. Ideally, small molecule therapies that are easy to administer, do not require customization for specific individuals, and have a good safety profile and are well tolerated. Such treatments do not currently exist.

[0006] No specific treatment has been approved to reduce fibrosis in muscular dystrophies so far. The only approved treatment for muscular dystrophies is corticosteroids used specifically for DMD. Treatment with corticosteroids can slow disease progression by reducing inflammation in muscle tissue and preserving muscle strength. Delazacort and prednisone are the main types of corticosteroids prescribed for DMD. Although the exact mechanism by which corticosteroids increase muscle strength in DMD is unclear, potential beneficial effects of corticosteroids include inhibition of muscle protein breakdown, stimulation of myoblast proliferation, stabilization of sarcolemmal membranes, increased myogenic repair, anti-inflammatory / immunosuppressive effects, reduction of cytoplasmic calcium concentration, upregulation of utrophin, and differential regulation of genes in muscle fibers.

[0007] Fibro / adipose precursor cells (FAPs) are mesenchymal muscle-resident stem cells characterized by expression of platelet-derived growth factor receptor α (PDGFRα). FAPs are the main cell type responsible for the expansion of fibrotic and adipose tissue in muscular dystrophies. Upon acute injury, FAPs become activated, proliferate, release components of the extracellular matrix (ECM), and serve as a scaffold for muscle regeneration. In healthy muscle, excess FAPs are eliminated by apoptosis, a process mediated by tumor necrosis factor α (TNF-α). However, in dystrophic muscle, the proliferation of FAPs is permanently activated by the continuous release of growth factors by M2 macrophages, which releases collagen I (among other components of the ECM), leading to the expansion of fibrotic tissue.

[0008] The molecular pathways driving FAP in muscular dystrophies are only just beginning to be elucidated. It is well known that TGF-β promotes the proliferation of FAP, inhibits TNF-α-mediated apoptosis of FAP, and induces the differentiation of FAP into fibroblasts. Furthermore, platelet-derived growth factor (PDGF) is involved in muscle regeneration and degeneration processes. PDGF-BB has been shown to activate satellite cell proliferation and chemotaxis, and its receptor, PDGFRβ, is highly expressed in dystrophic muscles compared to healthy muscles. PDGF-AA binds to the tyrosine kinase receptor PDGFRα, inducing receptor dimerization and autophosphorylation of the intracellular domain. Autophosphorylation of PDGFRα triggers signaling pathways, such as Ras-MAPK, PI3K, or PLC-γ, that are involved in different cellular responses, including proliferation, cell differentiation, apoptosis inhibition, intracellular calcium mobilization, or cell motility. PDGF-AA has been previously demonstrated to activate fibroblast proliferation and migration and increase the release of extracellular matrix components. Previous studies have shown that PDGF-AA expression is higher in dystrophic muscles compared with healthy muscles, and serum levels of PDGF-AA are elevated in DMD patients. However, the pathways activated by PDGF-AA in the muscular dystrophic FAP remain to be elucidated. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Pichavant. C et al., Mol Ther, 2011 [Non-Patent Document 2] Kinali.M et al., Lancet Neurol., 2009. 8:918 Summary of the Invention [Problem to be solved by the invention]

[0010] There is an urgent need for new means to treat diseases or disorders associated with muscle fibrosis (such as muscular dystrophies). [Means for solving the problem]

[0011] The inventors have used mass spectrometry to identify that the RhoA / ROCK pathway is activated by PDGF-AA in fibro / adipose precursor cells (FAPs) isolated from human skeletal muscle. Surprisingly, the inventors have shown that inhibiting this pathway can ameliorate muscle fibrosis in a mouse model of muscular dystrophy. Thus, RhoA / ROCK pathway inhibitors can be advantageously used to treat diseases or disorders associated with muscle fibrosis, such as muscular dystrophy.

[0012] The present invention provides a method of treating a disease or disorder associated with muscle fibrosis in a subject, the method comprising administering to the subject a therapeutically effective amount of a RhoA / ROCK pathway inhibitor.

[0013] Suitably, said disease or disorder associated with muscle fibrosis may be a muscular dystrophy.

[0014] Suitably, said disease or disorder associated with muscle fibrosis may be a dystrophinopathy.

[0015] Suitably, the disease or disorder associated with muscle fibrosis may be selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, recessive and dominant limb-girdle muscular dystrophy, myotonic dystrophy type I, myotonic dystrophy type II, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, inclusion body myositis, distal myopathy with dystrophic changes and myofibrillar myopathy.

[0016] Suitably, the inhibitor may inhibit the RhoA / ROCK pathway by directly inhibiting ROCK1 and / or ROCK2.

[0017] Preferably, the inhibitor is fasudil, Y27632, Y39983, Wf-536, AR-13324, AR-12286, AMA0076, PG324, azabenzimidazole-aminofurazan, DE-104, olefins, isoquinolines, indazoles, pyridine alkene derivatives, H-1152P, ROKα inhibitors, XD-4000, HMN-1152, 4-(1-aminoalkoxy)-4-(1-aminopropanediol), ... (n-phenyl)-N-(4-pyridyl)cyclohexane-carboxamide, quinazoline, ripasudil, VAS-012, Ki-23095, BA-2017, BA-215, BA-285, BA-1037, BA-210, Rhostatin, ROCK-IN-1-preclinical, ROCK inhibitor-2, ROCK2-IN-5, ROCK2-IN-2, ROCK-IN-2(azaindole 1, TC-S 7001), Chroman 1, Cotosudil, SAR407899 hydrochloride, SAR407899, Hydroxyfasudil, Ripasudil, Ripasudil free base, Belmosudil mesylate, Belmosudil, Sobesudil, RKI-1447, H-1152, GSK269962A, Rho-Kinase-IN-1, SB-772077B dihydrochloride, HSD1590, BDP5290, ZINC00881524, Belosudil, LX7101, GSK-25, GSK180736A, CRT0066854, Y-27632, CMPD101, Thiazovivin, SR-3677, and GSK429286A, and pharma- ceutically acceptable salts, solvates, or prodrugs thereof.

[0018] Suitably, the inhibitor may be a compound of formula (1) or a pharma- ceutically acceptable salt, solvate or prodrug thereof: [ka] During the ceremony, A is one or more R 1 is a monocyclic 5- to 7-membered heterocycloalkyl ring optionally substituted with Each R 1 are independently 1~6 Alkyl, Hydroxy-C 1~6 Alkyl, halo-C 1~6Alkyl and -C(O)OR 2 is selected from R 2 is C 1~6 is alkyl, Optionally, the compound of formula (1) is fasudil.

[0019] Suitably, the inhibitor may be selected from the group consisting of C3 exoenzyme, C3 transferase based, Rhosin, CCG-1423, CCG-203971, YS-49 monohydrate, YS-49, cerivastatin sodium, cerivastatin, Z62954982, Y16, MLS-573151, HA-100, HL07, DDO-5701, DDO-5713, DDO-5714, DDO-5715, DDO-5716, ML-7, MLCK18 and CT-04, and pharma- ceutically acceptable salts, solvates or prodrugs thereof.

[0020] Suitably, the inhibitor may inhibit the RhoA / ROCK pathway by ribosylating the RhoA protein.

[0021] Suitably, the inhibitor may be C3 exoenzyme.

[0022] The present invention further provides the use of a RhoA / ROCK pathway inhibitor in the treatment of a disease or disorder associated with muscle fibrosis.

[0023] Suitably, said disease or disorder associated with muscle fibrosis may be a muscular dystrophy.

[0024] Suitably, said disease or disorder associated with muscle fibrosis may be a dystrophinopathy.

[0025] Suitably, the disease or disorder associated with muscle fibrosis may be selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, recessive and dominant limb-girdle muscular dystrophy, myotonic dystrophy type I, myotonic dystrophy type II, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, inclusion body myositis, distal myopathy with dystrophic changes and myofibrillar myopathy.

[0026] Suitably, the inhibitor may inhibit the RhoA / ROCK pathway by directly inhibiting ROCK1 and / or ROCK2.

[0027] Preferably, the inhibitor is fasudil, Y27632, Y39983, Wf-536, AR-13324, AR-12286, AMA0076, PG324, azabenzimidazole-aminofurazan, DE-104, olefins, isoquinolines, indazoles, pyridine alkene derivatives, H-1152P, ROKα inhibitors, XD-4000, HMN-1152, 4-(1-aminoalkoxy)-4-(1-aminopropanediol), ... (n-phenyl)-N-(4-pyridyl)cyclohexane-carboxamide, quinazoline, ripasudil, VAS-012, Ki-23095, BA-2017, BA-215, BA-285, BA-1037, BA-210, Rhostatin, ROCK-IN-1-preclinical, ROCK inhibitor-2, ROCK2-IN-5, ROCK2-IN-2, ROCK-IN-2(azaindole 1, TC-S 7001), Chroman 1, Cotosudil, SAR407899 hydrochloride, SAR407899, Hydroxyfasudil, Ripasudil, Ripasudil free base, Belmosudil mesylate, Belmosudil, Sobesudil, RKI-1447, H-1152, GSK269962A, Rho-Kinase-IN-1, SB-772077B dihydrochloride, HSD1590, BDP5290, ZINC00881524, Belosudil, LX7101, GSK-25, GSK180736A, CRT0066854, Y-27632, CMPD101, Thiazovivin, SR-3677, and GSK429286A, and pharma- ceutically acceptable salts, solvates, or prodrugs thereof.

[0028] Suitably, the inhibitor may be a compound of formula (1) or a pharma- ceutically acceptable salt, solvate or prodrug thereof: [ka] During the ceremony, A is one or more R 1 is a monocyclic 5- to 7-membered heterocycloalkyl ring optionally substituted with Each R 1 are independently 1~6 Alkyl, Hydroxy-C 1~6 Alkyl, halo-C 1~6Alkyl and -C(O)OR 2 is selected from R 2 is C 1~6 is alkyl, Optionally, the compound of formula (1) is fasudil.

[0029] Suitably, the inhibitor may be selected from the group consisting of C3 exoenzyme, C3 transferase based, Rhosin, CCG-1423, CCG-203971, YS-49 monohydrate, YS-49, cerivastatin sodium, cerivastatin, Z62954982, Y16, MLS-573151, HA-100, HL07, DDO-5701, DDO-5713, DDO-5714, DDO-5715, DDO-5716, ML-7, MLCK18 and CT-04, and pharma- ceutically acceptable salts, solvates or prodrugs thereof.

[0030] Suitably, the inhibitor may inhibit the RhoA / ROCK pathway by ribosylating the RhoA protein.

[0031] Suitably, the inhibitor may be C3 exoenzyme.

[0032] Various aspects of the invention are described in further detail below. [Brief description of the drawings]

[0033] Embodiments of the present invention will be further described with reference to the accompanying drawings.

[0034] [Figure 1 (1)]A) Representative images of DMD patient muscle-derived cells. CD56- were stained with desmin and TE-7 markers to validate the purification step. Scale bar 100 μm. B) Quantification of PDGFRα+ expression areas in muscle tissue obtained from healthy controls (n=2) and DMD patients (n=2). C) Representative images of healthy control and DMD patient muscle biopsies stained with PDGFRα antibody. Scale bar 100 μm. D) Flow cytometry isotype control staining and analysis of PDGFRα in CD56- isolated cells showing receptor positivity. D) Experimental design of screening. [Figure 1 (2)] F) Representative WB of total protein bands measured on Revert 700 and used as loading control for quantification of ArhGEF2 protein. G) Representative intracellular western images showing total cell number signal stained with TO-PRO-3. H) Viability measurements comparing the effects of PDGF-AA, C3 and Fasudil on FAP at 30 minutes (min), 24 hours (h), 48 hours and 72 hours using the PrestoBlue cell viability assay. Data are presented as the mean ± standard error of the mean of three replicates. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. [Diagram 2]Proteomic analysis of PDGF-AA treated DMS FAPs. (A) Volcano plot representing the total proteome. Data are presented as change in protein abundance in PDGF-AA treated FAPs relative to untreated FAPs. Proteins showing significantly different expression (Student's t-test, P ≤ 0.05) are highlighted in light grey. (B-D) Proteomap of upregulated proteins after PDGF-AA treatment. Proteomic maps visualize the organization of the proteome in terms of protein abundance and function. Each protein is represented by a polygon, and the area of ​​each polygon reflects the abundance of the protein (calculated in fold change). Functionally related proteins appear in adjacent regions. These three panels represent three hierarchical levels, the first panel shows the individual proteins detected (B), the second panel the proteins grouped into pathways (C), and the last panel shows the functional categories related to the groups of proteins (D). (E) The upregulated proteins shown in the proteomap were also represented in a heatmap showing the results from the Reactome database. [Diagram 3]We show that the RhoA pathway is upregulated in DMD and that PDGF-AA activates it. (A) Molecular pathway of RhoA / ROCK2 signaling downstream of ArhGEF2, which catalyzes the replacement of RhoA-GDP with RhoA-GTP and controls RhoA activation. GTP-bound RhoA activates rho-associated coiled-coil forming protein kinase (ROCK), a serine / threonine kinase that controls actin filament remodeling via phosphorylation of myosin light chain (MLC). (B) Quantification of WB bands of ArhGEF2 relative to total protein detection, and representative bands of ArhGEF2 expression in muscle tissue obtained from healthy controls (n=2) and DMD patients (n=2). Total protein detection was used as a loading control signal (Figure 1D). (C) Optical density at 490 nm showing RhoA activity relative to total RhoA protein detected by WB. (D) Representative images of p-MLC staining using a fluorescent microscope. Scale bar is 50 μm. (E) Quantification of p-MLC intensity of each cell after different treatments. (F) Representative images of FAP stained with phalloidin after different treatments. Scale bar is 50 μm. C- is untreated FAP. Data are expressed as the mean ± standard error of the mean of three replicates. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. Statistical significance was set at P<0.05. **P<0.01, ***P<0.001. [Figure 4] Figure 1 shows the effect of C3 exoenzyme, fasudil and PDGF-AA on FAP in DMD patients. (A) Analysis of FAP proliferation after 48 and 72 hours. (B) Analysis of FAP migration after 48 and 72 hours. (C) Representative images of migration assay at 48 and 72 hours. Scale bar is 200 μm. (D) Analysis of the amount of collagen delivered in the supernatant after 4 days of treatment. C- is untreated FAP. Data are presented as the mean ± standard error of the mean of three replicates. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. Statistical significance was set at P<0.05. *p<0.05, **P<0.01, ***P<0.001. [Figure 5(1)] A) Representative immunofluorescence images of PDGF-AA expression in quadriceps muscle samples from dba / 2J-WT and dba / 2J-mdx. Scale bar is 100 μm. B) Effect of fasudil treatment in dba / 2J-mdx mice. A) Body weight measurements after 6 weeks of fasudil treatment in dba-2J WT, untreated dba / 2J-mdx, and fasudil-treated dba / 2J-mdx mice. [Figure 5 (2)] C) Representative immunofluorescence images of collagen I expression in the heart. Scale bar is 100 μm. Quantification of collagen I expression area in the right and left ventricles of the heart. D) Percentage and mean intensity fluorescence (MFI) of CD209, CD80 and DR markers analyzed by flow cytometry. Data are expressed as mean ± standard error of the mean of the analyzed group. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. Statistical significance was set at P<0.05. *p<0.05, **P<0.01, ***P<0.001. [Figure 6(1)] A) Relative gene expression of ROCK2, LIMK2 and FHOD1 in quadriceps muscles of dba-2J WT mice, untreated dba / 2J-mdx and fasudil-treated dba / 2J-mdx. B) Quantification of ROCK activity in quadriceps muscles of dba-2J WT mice, untreated dba / 2J-mdx and fasudil-treated dba / 2J-mdx. C) Representative immunofluorescence images of LIMK2 expression in quadriceps muscles. Scale bar is 100 μm. E) Representative immunofluorescence images of MLC2 expression in quadriceps muscles. Scale bar is 100 μm. Data are expressed as the mean ± standard error of the mean of the analyzed group. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. Statistical significance was set at P<0.05. *p<0.05, **P<0.01, ***P<0.001. [Figure 6 (2)]D) Quantification of LIMK2 expression area in quadriceps muscle. D) Quantification of MLC2 expression area in quadriceps muscle. Data are expressed as the mean ± standard error of the mean of the analyzed groups. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. Statistical significance was set at P<0.05. *p<0.05, **P<0.01, ***P<0.001. [Figure 7(1)] Effect of fasudil treatment in dba / 2J-mdx mice. (A) Grip strength test in dba-2J WT mice, untreated dba / 2J-mdx, and fasudil-treated dba / 2J-mdx after 6 weeks of treatment with fasudil. Results are relative to animal weight. (B) Quantification of collagen I expression area in quadriceps muscle. Scale bar is 200 μm. (C) Quantification of mean muscle fiber size and frequency in dba-2J WT mice, untreated dba / 2J-mdx, and fasudil-treated dba / 2J-mdx. (D) Representative immunofluorescence images of collagen I expression in muscle samples. Scale bar is 200 μm. (F) Representative immunofluorescence images of PDGFRα+ (light gray arrow) and F4 / 80 (white arrow) expression in muscle samples. ECM is stained green with wheat germ agglutinin (WGA). Scale bar is 200 μm. Data are expressed as the mean ± standard error of the mean of the analyzed group. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. Statistical significance was set at P<0.05. *p<0.05, **P<0.01, ***P<0.001. [Figure 7(2)](E) Quantification of PDGFRα+ expression area in quadriceps. (G) Quantification of macrophage expression area in quadriceps. (H) Quantification of flow cytometric results to analyze F4 / 80 macrophages (Mo) accumulated in quadriceps and tibia anterior from mice. Data are expressed as number of F4 / 80 relative to weight of quadriceps and tibia anterior. (I) Analysis of percentage of total macrophages and mean fluorescence intensity (MFI) of CD163 and CD206 markers by flow cytometry. Data are expressed as mean ± standard error of the mean of the analyzed group. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. Statistical significance was set at P<0.05. *p<0.05, **P<0.01, ***P<0.001. [Figure 8(1)] A) Representative immunofluorescence images of T cells with CD3 (light grey arrows), neutrophils with Ly6G (white arrows) and B cells with CD19 cells. Scale bar is 100 μm. B) Quantification of T cells (CD3+ cells) in each section of quadriceps muscle. C) Quantification of neutrophils (Ly6G+ cells) in each section of quadriceps muscle. D) Quantification of B cells (CD19+ cells) in each section of quadriceps muscle. Results were statistically analyzed using one-way ANOVA followed by Tukey post-hoc test. Statistical significance was set at P<0.05. **P<0.01, ***P<0.001. [Figure 8 (2)] E) Heatmap representation of cytokine levels measured in three muscles from each of the dba / 2J-WT (n=3), dba / 2J-mdx (n=3) and fasudil-treated dba / 2J-mdx (n=3) groups. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention relates to compounds for use in treating a disease or disorder associated with muscle fibrosis in a subject. In particular, the present invention relates to inhibitors of the RhoA / ROCK pathway for use in treating a disease or disorder associated with muscle fibrosis.

[0036] The authors surprisingly found that the RhoA / ROCK pathway was activated in the myofibrosis process and that inhibition of this pathway ameliorated myofibrosis.

[0037] Treatment of the subject According to a first aspect, there is provided a method of treating a disease or disorder associated with muscle fibrosis in a subject, the method comprising administering to the subject a therapeutically effective amount of a RhoA / ROCK pathway inhibitor.

[0038] Inhibition of the RhoA / ROCK pathway blocks the activation of FAPs, which prevents them from proliferating, migrating and reorganizing actin, thus inhibiting / delaying the fibrotic process.

[0039] "Fibrosis" refers to the thickening and scarring of connective tissue and is well known to those skilled in the art. The term "muscle" refers to any type of muscle in the body, such as skeletal (striated), smooth, or cardiac muscle. Thus, as used herein, the term "muscle fibrosis" or "muscle fibrosis process" refers to any type of fibrosis affecting muscle. Muscle fibrosis can affect muscle function, impair muscle regeneration after injury, and increase muscle susceptibility to re-injury. "Disease or disorder associated with muscle fibrosis" refers to a disease or disorder in which muscle fibrosis occurs (e.g., muscle fibrosis is a pathological symptom or sign of the disease or disorder). Thus, the phrase "disorder associated with muscle fibrosis" encompasses any disease or disorder that results directly or indirectly from, and / or completely or partially from, muscle fibrosis. Muscle fibrosis may be the cause of the disease or disorder, but may also be a symptom that appears after the onset of the disease or disorder. Suitable diseases or disorders are well known to those skilled in the art. Suitable diseases or disorders associated with muscle fibrosis are described in detail elsewhere herein.

[0040] The RhoA / ROCK pathway inhibitors described herein may be used to treat any disease or disorder associated with muscle fibrosis in a subject. In one example, the disease or disorder associated with muscle fibrosis is muscular dystrophy.

[0041] Muscular dystrophies (MD) are a group of muscle disorders in which muscle fibers are abnormally susceptible to damage, resulting in the accumulation of defective muscle proteins, the death of muscle cells and tissue, and the progressive weakening of the musculoskeletal system of affected individuals.

[0042] Muscle tissue in muscular dystrophies is characterized by loss of muscle tissue and its replacement by adipose and fibrous tissue, leading to permanent weakness and disability. Thus, symptoms of MD include muscle weakness or degeneration, gastrocnemius muscle hypertrophy, loss of muscle fiber integrity, elevated serum creatine kinase levels, loss of dystrophin and dystrophin-associated proteins, and central nucleation of muscle fibers. Methods for assessing and / or identifying muscle weakness or degeneration, gastrocnemius muscle hypertrophy, loss of muscle fiber integrity, elevated serum creatine kinase levels, loss of dystrophin and dystrophin-associated proteins, and central nucleation of muscle fibers are well known. By way of example and not limitation, loss of dystrophin and dystrophin-associated proteins may be assessed histologically or at the molecular level (e.g., using PCR).

[0043] In one example, the disease or disorder associated with muscle fibrosis is a dystrophinopathy.

[0044] Dystrophinopathy refers to a group of diseases caused by mutations in the DMD gene, which codes for the dystrophin protein present in muscles. The severe end of the spectrum includes Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD-associated dilated cardiomyopathy. The mild end of the spectrum includes asymptomatic increases in serum creatine kinase and muscle spasms with myoglobinuria. Because dystrophin is located on the X chromosome, dystrophinopathy primarily affects males, but females range from carriers to those with late-onset mild disease to those with severe DMD.

[0045] As an example, dystrophinopathies are Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which are closely related and have similar treatment options.

[0046] In one example, the disease or disorder associated with muscle fibrosis is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, recessive and dominant limb-girdle muscular dystrophy, myotonic dystrophy type I, myotonic dystrophy type II, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, inclusion body myositis, distal myopathy with dystrophic changes, and myofibrillar myopathy.

[0047] As used herein, the terms "limb-girdle muscular dystrophy" and "LGMD" are intended to cover all muscular dystrophies included in the current classification published in 2018 (Straub et al., Neuromuscular Disorders DOI:10.1016 / j.nmd.2018.05.007), including autosomal dominant LGMD (LGMD-D) 1-4 and autosomal recessive LGMD (LGMD-R) 1-20. In one example, the limb-girdle muscular dystrophy may be selected from the group consisting of limb-girdle muscular dystrophy R3, limb-girdle muscular dystrophy R4, limb-girdle muscular dystrophy R6 and limb-girdle muscular dystrophy R7.

[0048] As used herein, the terms "congenital muscular dystrophy" and "CMD" are intended to include laminin alpha 2-deficient CMD (MDC1A), Ullrich congenital muscular dystrophy (UCMD 1, 2, and 3), Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB), Fukuyama CMD (FCMD), CMD plus secondary laminin deficiency 1 and 2 (MDC1B and MDC1C), CMD with midbrain retardation and pachygyria (MDC1D), stiff spine with muscular dystrophy type 1 (RSMD). In one example, the congenital muscular dystrophy may be selected from the group consisting of MDC1A, MDC1B, MDC1D, Fukuyama CMD (FCMD), muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS).

[0049] The term "RhoA" is an abbreviation for "Ras homolog gene family, member A." It refers to a small GTPase protein of the Rho family. In humans, RhoA is encoded by the gene RHOA, located on chromosome 3, and has an effector domain, four exons, a hypervariable region, and a CAAX box motif (C: Cys, A: aliphatic residue, X: any residue). The N-terminal region of RhoA contains two switch regions (switch I and switch II) with characteristic folds. The structure of these switches changes with the activation or inactivation of the RhoA protein. The C-terminus of RhoA is essential for the correct localization of the protein. RhoA protein is expressed in all tissues, including normal human tissues, embryonic tissues, and stem cells. RhoA is mainly localized in the cell membrane and cytoplasm, and also near cell-cell contacts and cell protrusions. RhoA plays an important role in multiple cellular processes, such as cell growth, transformation, and cytoskeleton control.

[0050] The term "ROCK" is an abbreviation for "Rho-associated protein kinase". ROCK is a downstream effector of RhoA and exists in two isoforms, ROCK1 and ROCK2. ROCK is a kinase that belongs to the AGC (PKA / PKG / PKC) family of serine-threonine-specific protein kinases. It is primarily involved in controlling cell shape and movement by acting on the cytoskeleton.

[0051] ROCKs (ROCK1 and ROCK2) are present in mammals (human, rat, mouse, cow), zebrafish, Xenopus, invertebrates (C. elegans, mosquito, Drosophila) and chicken. Human ROCK1 has a molecular mass of 158 kDa and is the major downstream effector of the small GTPase RhoA. Mammalian ROCKs contain a kinase domain, a coiled-coil region and a pleckstrin homology (PH) domain, which reduces the kinase activity of ROCK by an autoinhibitory intramolecular fold in the absence of RhoA-GTP.

[0052] The Rho / ROCK signaling pathway is an important signaling system that is critically involved in cell growth, differentiation, migration and development.

[0053] As used herein, the term "inhibitor" refers to a compound that directly or indirectly reduces, neutralizes, prevents, blocks, suppresses, delays or interferes with RhoA / ROCK pathway signaling. Several inhibitors of the RhoA / ROCK pathway are known. Suitable examples are described elsewhere herein. Inhibition may be reversible or irreversible.

[0054] As will be appreciated by those of skill in the art, inhibitors may function at the level of the target gene, transcript, or protein. In some instances, inhibitors may reduce RhoA / ROCK activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to control activity (e.g., activity in the absence of inhibitor).

[0055] An inhibitor may be an agent that inhibits RhoA / ROCK function (e.g., an inhibitory nucleic acid, binding molecule (e.g., a small molecule or antibody), or a peptide). One of skill in the art can readily identify suitable RhoA / ROCK pathway inhibitors using methods known in the art (e.g., based on assaying the effect of the potential inhibitor on known RhoA / ROCK activity).

[0056] In some examples, the inhibitor inhibits, reduces, slows, stops, blocks, suppresses, abolishes and / or prevents ROCK activity, hi other examples, the inhibitor inhibits, reduces, slows, stops, blocks, suppresses, abolishes and / or prevents RhoA activity.

[0057] In a particular example, the inhibitor is a direct ROCK inhibitor. In another example, the inhibitor is a direct RhoA inhibitor. As used herein, "direct inhibitor" refers to an inhibitor that directly targets a target gene, transcript or protein. In the context of ROCK, a direct inhibitor directly inhibits the ROCK protein, the ROCK transcript and / or the ROCK gene, thereby reducing the activity and / or expression of the ROCK protein. In the context of RhoA, a direct inhibitor directly inhibits the RhoA protein, the RhoA transcript and / or the RhoA gene, thereby reducing the activity and / or expression of the RhoA protein.

[0058] Thus, in some instances, the inhibitor directly targets the ROCK gene, RNA transcript or protein. For example, the inhibitor may directly bind to the ROCK gene, RNA transcript or protein. In some instances, the inhibitor directly targets the RhoA gene, RNA transcript or protein. For example, the inhibitor may directly bind to the RhoA gene, RNA transcript or protein.

[0059] Any suitable ROCK inhibitor and / or RhoA inhibitor may be used. In certain examples, the ROCK inhibitor and / or RhoA inhibitor may be selected from the group consisting of an inhibitory nucleic acid, a binding molecule (e.g., a small molecule or an antibody (including functional fragments thereof)) and a peptide. Suitable examples of inhibitory nucleic acids, binding molecules (e.g., a small molecule or an antibody (including functional fragments thereof)) or peptides will be readily identifiable to one of skill in the art.

[0060] In some examples, an inhibitor may be an "inhibitory nucleic acid" that, when present in a cell, degrades or inhibits the function, transcription or translation of its target gene in a sequence-specific manner. Exemplary inhibitory nucleic acids include aptamers, siRNAs, microRNA-adapted shRNAs, shRNAs, precursor microRNAs (pre-miRNAs), pri-miRNAs, miRNAs, amiRNAs, interfering RNAs or RNAi, dsRNAs, ribozymes, antisense oligonucleotides (ASOs), and DNA expression cassettes encoding the inhibitory nucleic acids.

[0061] In some examples, the inhibitor is a binding molecule. Preferably, the binding molecule binds to and inhibits the activity of ROCK or RhoA (e.g., inhibits one or more activities of ROCK or RhoA described elsewhere herein).

[0062] Examples of binding molecules are small molecules. Binding molecules also include antibodies and non-immunoglobulin binding agents such as phage display derived peptide binders and antibody mimetics (e.g., affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains, aptamers and affilins). The term "antibody" includes, for example, natural and non-natural antibodies, polyclonal and monoclonal antibodies, chimeric antibodies, and fully synthetic antibodies and fragments thereof (e.g., Fab', F(ab')2, Fv, or Fab fragments, or other antigen-recognizing immunoglobulin fragments, etc.). Antibodies that bind to a specific epitope can be generated by methods known in the art. For example, polyclonal antibodies can be generated by conventional methods of immunizing mammals (e.g., rabbits, mice, rats, sheep, goats). Polyclonal antibodies are then contained in the serum of the immunized animals and can be isolated using standard procedures (e.g., affinity chromatography, immunoprecipitation, size-exclusion chromatography, ion-exchange chromatography). Monoclonal antibodies can be generated by conventional methods of immunizing mammals, followed by isolation of plasma B cells producing the desired monoclonal antibody and fusing them with myeloma cells (see, e.g., Mishell et al., 1980). Screening for epitope recognition can be performed using standard immunoassays, including ELISA techniques, radioimmunoassays, immunofluorescence, immunohistochemistry, and Western blotting. In vitro methods of antibody selection, such as antibody phage display, may also be used to generate antibodies. Preferably, a nuclear localization signal is added to the antibody to enhance nuclear localization.

[0063] In some instances, introduction of a mutation that disrupts a target gene (e.g., introduction of a mutation that disrupts a ROCK or RhoA gene) may inhibit, reduce, slow, stop, block, suppress, abolish and / or prevent the activity and / or expression of a target protein (e.g., ROCK or RhoA) in a cell. As will be apparent to one of skill in the art, such a mutation may reduce expression of the protein encoded by the target gene (e.g., ROCK or RhoA), completely disable expression of the gene, or render the gene product non-functional. Thus, in some instances, the mutation may be a loss-of-function mutation. In some instances, the mutation may be a point mutation, insertion, substitution or deletion. In some instances, both alleles of the target gene are mutated. In some instances, the mutation may be located in the coding region (e.g., an exon for ROCK or RhoA) and / or in the non-coding region (e.g., the promoter region for ROCK or RhoA) of the target gene.

[0064] Mutation of ROCK gene and / or RhoA gene may be achieved by methods known in the art, including gene editing technology. For example, targeted gene editing technology (e.g., targeted gene editing construct) may be used to introduce mutation into ROCK gene and / or RhoA gene. For example, zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeat (CRISPR) nuclease (e.g., RNA-guided DNA endonuclease Cas9 (or its mutant)) or meganuclease may be used to introduce mutation into ROCK gene and / or RhoA gene.

[0065] In one example, the inhibitor inhibits the RhoA / ROCK pathway by directly inhibiting ROCK1 and / or ROCK2. In this context, "directly inhibiting ROCK" may refer to inhibiting the ability of ROCK to phosphorylate myosin light chain 2 (p-MLC2), which, when phosphorylated, leads to cytoskeletal reorganization (see FIG. 3A).

[0066] The ROCK inhibitor may be a "pan-ROCK inhibitor" that is a selective inhibitor of both ROCK1 and ROCK2 isoforms, or may be a specific inhibitor of ROCK1 or ROCK2. For example, a ROCK1 specific inhibitor can inhibit ROCK1 but not ROCK2, and a ROCK2 specific inhibitor can inhibit ROCK2 but not ROCK1. A pan-ROCK inhibitor can inhibit both isoforms with equal potency and have no selective effect, or can inhibit both but have higher potency against either ROCK1 or ROCK2. For example, a pan-ROCK inhibitor with higher potency against ROCK1 can inhibit both isoforms but inhibits ROCK1 more efficiently. ROCK2-IN-2 is an example of a selective ROCK2 inhibitor, and GSK429286A is an example of a selective ROCK1 inhibitor.

[0067] In one example, the ROCK inhibitor is a pan-ROCK inhibitor or a ROCK2 inhibitor. In one example, the ROCK inhibitor is a direct pan-ROCK inhibitor or a direct ROCK2 inhibitor.

[0068] In one example, the inhibitor is a RhoA / ROCK2 pathway inhibitor.

[0069] In one example, the inhibitor is fasudil, fasudil hydrochloride, Y27632, Y39983, Wf-536, AR-13324, AR-12286, AMA0076, PG324, azabenzimidazole-aminofurazan, DE-104, olefin, isoquinoline, indazole, pyridine alkene derivative, H-1152P, ROKα inhibitor, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-( 4-pyridyl)cyclohexanecarboxamide, Quinazoline, Ripasudil, Ripasudil hydrochloride, Ripasudil hydrochloride hydrate, VAS-012, Ki-23095, BA-2017, BA-215, BA-285, BA-1037, BA-210, Rhostatin, ROCK-IN-1-preclinical, ROCK inhibitor-2, ROCK2-IN-5, ROCK2-IN-2, ROCK-IN-2(azaindole-1, TC-S 7001), Chroman 1, Chroman 1 dihydrochloride, Cotosudil, SAR407899 hydrochloride, SAR407899, Hydroxyfasudil, Hydroxyfasudil hydrochloride, Ripasudil, Ripasudil free base, Belmosudil mesylate, Belmosudil, Sobesudil, Sobesudil hydrochloride, RKI-1447, RKI-1447 dihydrochloride, H-1152 dihydrochloride, H-1152, GSK269962A, GSK26996 2A hydrochloride, Rho-Kinase-IN-1, SB-772077B dihydrochloride, HSD1590, BDP5290, ZINC00881524, belosudil, LX7101, GSK-25, GSK180736A, CRT0066854, CRT0066854 hydrochloride, Y-27632 dihydrochloride, Y-27632, CMPD101, thiazovivin, SR-3677 and GSK429286A. These are examples of inhibitors that directly inhibit ROCK1 and / or ROCK2.

[0070] In one example, the inhibitor is a compound of formula (1), or a pharma- ceutically acceptable salt, solvate, or prodrug thereof: [ka] During the ceremony, A is one or more R 1is a monocyclic 5- to 7-membered heterocycloalkyl ring optionally substituted with Each R 1 are independently 1~6 Alkyl, Hydroxy-C 1~6 Alkyl, halo-C 1~6 Alkyl and -C(O)OR 2 is selected from R 2 is C 1~6 It is an alkyl. Compounds of formula (1) can be prepared using the method described in H. Li et al., Journal of Heterocyclic Chemistry, 56(1), 2019, pp. 260-267.

[0071] The term "compound of formula (1)" encompasses all stereoisomers of such compound, i.e., enantiomers (e.g., R and S enantiomers) and diastereomers, and all salts thereof, in substantially pure form and / or as mixtures of the foregoing in any ratio. Thus, reference to a compound of formula (1) refers to that enantiomer or diastereomer.

[0072] A monocyclic 5- to 7-membered heterocycloalkyl ring is typically fully saturated (eg, it has no double or triple bonds).

[0073] The monocyclic 5- to 7-membered heterocycloalkyl ring may contain a total of 1, 2, 3 or 4 heteroatoms. The monocyclic 5- to 7-membered heterocycloalkyl ring preferably has a total of 1 or 2 heteroatoms.

[0074] As shown in formula (1), one of the heteroatoms is a nitrogen atom. The other heteroatoms may each be selected from an oxygen atom and a nitrogen atom, and more preferably, the other heteroatoms are each nitrogen atoms.

[0075] The monocyclic 5- to 7-membered heterocycloalkyl ring is preferably a 5- or 7-membered heterocycloalkyl ring having a total of 1 or 2 heteroatoms.

[0076] Typically, A is [ka] and one or more R 1 The dashed bond indicates the point at which the nitrogen atom is attached to the sulfur atom in formula (1).

[0077] In general, halo-C 1~6 The alkyl is preferably chloroC 1~6 It is an alkyl group (for example, a halo group is a chloro group).

[0078] Each R 1 is independently preferably hydroxy-C 1~6 Alkyl and halo-C 1~6 alkyl, more preferably hydroxy-C 1~2 Alkyl and halo-C 1~2 It is an alkyl.

[0079] The compound of formula (1) [ka] or a pharma- ceutically acceptable salt, solvate or prodrug thereof. The group "Boc" refers to a tert-butyloxycarbonyl group.

[0080] The compound of formula (1) is preferably a compound selected from the above (1A), (1B), and (1D) to (1G), or a pharma- ceutically acceptable salt, solvate, or prodrug thereof.

[0081] Compound (1A) is known as fasudil (IUPAC name is 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline).

[0082] Preferably, the compound of formula (1) is fasudil (CAS number: 103745-39-7) or fasudil hydrochloride (CAS number: 105628-07-7).

[0083] Fasudil or fasudil hydrochloride is an over-the-counter vasodilator used to treat cerebral vasospasm. It is also useful in pulmonary hypertension. Fasudil inhibits the RhoA / ROCK pathway by blocking ROCK phosphorylating myosin light chain 2 (p-MLC2). Fasudil targets both ROCK1 and ROCK2 ATP-dependent kinase domains with equal potency and without selective effects.

[0084] The structural formula of fasudil hydrochloride is: [ka]

[0085] In one example, the inhibitor is fasudil, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0086] Y27632 (CAS:146986-50-7) is an orally active ATP-competitive inhibitor of ROCK-I and ROCK-II. Y-27632 attenuates doxorubicin-induced apoptosis of human cardiac stem cells. Y-27632 also inhibits dissociation-induced apoptosis of mouse prostate stem / progenitor cells. Y-27632 stimulates human induced pluripotent stem cells (hIPSCs) to selectively differentiate into mesendodermal lineages via modulation such as epithelial-mesenchymal transition. The structural formula of Y-27632 is: [ka]

[0087] In one example, the inhibitor is Y-27632, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0088] Y39983 (CAS: 199433-58-4), also known as Y-33075 and RKI-983, and its derivative Y-33075 dihydrochloride are selective ROCK inhibitors derived from Y-27632 and are more potent than Y-27632, with an IC50 of 3.6 nM. The formula of Y-39983 is: [ka]

[0089] In one example, the inhibitor is Y-39983, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0090] The structural formula of Y-33075 dihydrochloride (CAS number: 173897-44-4) is as follows. [ka]

[0091] In one example, the inhibitor is Y-33075, or a pharma- ceutically acceptable salt, solvate, or prodrug thereof. The inhibitor may be Y-33075 dihydrochloride.

[0092] The selective ROCK2 inhibitor (RXC007) is an orally available, highly selective, small molecule inhibitor targeting Rho Associated Coiled-Coil Containing Protein Kinase 2 (ROCK2). RXC007 is currently undergoing phase 1 clinical trials in healthy volunteers, with IPF as the first indication in clinical development.

[0093] Wf-536 (CAS: 539857-64-2) [(+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl)benzamide monohydrochloride] is a ROCK inhibitor that effectively reduces in vitro invasion and in vivo lung metastasis of B16 melanoma. The formula of Wf-536 is: [ka]

[0094] In one example, the inhibitor is Wf-536, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0095] Belmosudil (CAS:911417-87-3), also known as KD025 or SLx-2119, is a selective ROCK2 inhibitor with antifibrotic properties, with IC50 of 105 nM and 24 μM for ROCK2 and ROCK1, respectively. Belmosudil mesylate is a derivative of belmosudil (CAS:2109704-99-4). Belmosudil induces a significant downregulation of Tsp-1 and CTGF mRNA levels in PASMCs. Furthermore, belmosudil dose-dependently reduces infarct volume after transient middle cerebral artery occlusion. Belmosudil is as effective or more effective in aged, diabetic or female mice than in normal adult male mice. Several clinical trials using this molecule for diffuse cutaneous systemic sclerosis, chronic plaque psoriasis and chronic graft-versus-host disease are ongoing, all sponsored by Kadmon Corporation. The formula for belmosudil is: [ka]

[0096] In one example, the inhibitor is belmosudil, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0097] The structural formula of belmosudil mesylate (CAS number: 2109704-99-4) is as follows: [ka]

[0098] In one example, the inhibitor is belmosudil, or a pharma- ceutically acceptable salt, solvate, or prodrug thereof. The inhibitor may be belmosudil mesylate.

[0099] AR-13324 (CAS:2309668-15-1), also known as netarsudil, is a ROCK and norepinephrine transporter inhibitor that has been shown to reduce intraocular pressure in monkey eyes. Netarsudil primarily targets cells of the conventional outflow tract and effectively reduces IOP in both human and non-human primate eyes. In addition, netarsudil has been shown to increase the outflow capacity of non-human primate eyes and reduce episcleral venous pressure in rabbit eyes. It is currently undergoing clinical trials for the treatment of glaucoma and ocular hypertension. In 2019, Aerie Pharmaceuticals also marketed PG324 (Rocklatan®), an FDA-approved fixed-dose combination of netarsudil (0.02%) and latanoprost (0.005%). The structural formula of AR-13324 is: [ka]

[0100] In one example, the inhibitor is AR-13324, or a solvate, prodrug or alternative pharma- ceutically acceptable salt thereof.

[0101] Velosudil (also known as AR-11286) is a potent and selective Rho kinase (ROCK) inhibitor with Ki of 2 nM and 2 nM for ROCK1 and ROCK2, respectively. AR-12286 reduces intraocular pressure (IOP) primarily by increasing aqueous humor outflow through the trabecular meshwork. It is being tested in glaucoma in clinical trials sponsored by Aerie Pharmaceuticals. The formula for Velosudil is: [ka]

[0102] In one example, the inhibitor is belosudil, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0103] Sovesudil (CAS: 1333400-14-8), also known as PHP-201 or AMA0076, is a potent ATP-competitive locally acting Rho kinase (ROCK) inhibitor with IC50 of 3.7 nM and 2.3 nM against ROCK-I and ROCK-II, respectively. Sovesudil hydrochloride is a derivative of Sovedusil. Sovesudil reduces intraocular pressure (IOP) without inducing hyperemia. Sovesudil (PHP-201) (1 μM, 60 min) can induce changes in cell behavior in human trabecular meshwork (HTM) cells. In vivo experiments have shown that Sovesudil (0.1%, 0.3% and 0.5%) effectively reduces intraocular pressure (IOP) in normotensive and acutely hypertensive male New Zealand White rabbits without causing obvious hyperemia. [ka]

[0104] In one example, the inhibitor is sobesudil, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0105] Amino-furazan (oxadiazole)-azabenzimidazole is a potent ROCK1 inhibitor (IC50: 19 nM) that was described in 2007 (Stavenger et al., J Med Chem 2007) as having vasodilatory effects. Its formula is: [ka]

[0106] In one example, the inhibitor is an aminofurazan, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0107] Dihydropyrimidinylindazole amides have also been identified as potent ROCK inhibitors (Goodman et al., J Med Chem, 2007). Since this original description, many other pyridone derivatives have been developed with selective ROCK inhibitory capabilities.

[0108] H-1152 (CAS: 451462-58-1), also known as HMN-1152, is a membrane-permeable and selective ROCK inhibitor with a Ki value of 1.6 nM and an IC50 value of 12 nM for ROCK2. Several derivatives are available, such as H-1152 dihydrochloride or glycyl H-1152 hydrochloride. H-1152 potently inhibits Rho kinase with a Ki of 1.6 nM and slightly inhibits PKA, PKC, and MLCK with Kis of 0.63, 9.27, and 10.1 μM, respectively. H-1152 (0.5-10 μM) does not reduce neuronal viability. H-1152 (1, 5, or 10 μM) also does not alter the ratio of different neuronal morphologies. Furthermore, H-1152 (10 μM) increases the length of neurites in both BMP4 and LIF cultures. The structural formula of H-1152 is as follows: [ka]

[0109] In one example, the inhibitor is H-1152, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0110] XD-4000 is a selective ROCK2 inhibitor. In one example, the inhibitor is XD-4000, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0111] 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexanecarboxamides were described as Rho kinase inhibitors in 2004 (Gingrass et al., Biorganic & Medical Chemistry Letters 2004). Several different 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexanecarboxamides were examined for their ability to inhibit Rho kinase and to stimulate neurite outgrowth on treated surfaces for tissue culture in a semi-quantitative cell-based assay. Enantiomerically enriched (80% ee) compounds were tested directly without further resolution. All compounds inhibited Rho kinase activity at 10 μM by ≥ 65% compared to vehicle (DMSO) in the presence of 100 μM ATP. The two compounds showed similar results in the Rho kinase assay. The ability of these compounds to promote neurite outgrowth compared to vehicle was demonstrated. Their general formula is: [ka]

[0112] In one example, the inhibitor is a 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexanecarboxamide, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0113] Quinazoline is an organic compound with the formula CHN. It is a bicyclic heterocyclic aromatic ring consisting of two fused six-membered aromatic rings, a benzene ring, and a pyrimidine ring. Amino acid-derived quinazolines have Rock inhibitory capabilities. Studies have demonstrated that these amino acid-derived quinazolinones are primarily pan-Rock (I and II) inhibitors. Selectivity against other kinases has been achieved, but selectivity against PKA has not been achieved for most of these compounds. This differs from non-amino acid-derived quinazoline-based Rock inhibitors, which provide high selectivity against PKA. The formula for quinazoline is based on: [ka]

[0114] In one example, the inhibitor is a quinazoline, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0115] Ripasudil (CAS: 887375-67-9) and derivatives (Ripasudil hydrochloride, Ripasudil hydrochloride hydrate and Ripasudil free base) are derivatives of Fasudil, a Rho kinase inhibitor drug (previously known as K-115) used to treat glaucoma and ocular hypertension. The structural formula of Ripasudil is: [ka]

[0116] In one example, the inhibitor is Ripasudil, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0117] VAS-012 is a ROCK inhibitor being investigated in oncology, developed by VasGene Therapeutics.

[0118] Ki-23095 is a ROCK inhibitor being investigated in cardiovascular and renal diseases.

[0119] BA-2017, BA-215, BA-285, BA-1037, and BA-210 are all investigational ROCK inhibitors whose in vivo or in vitro results have not yet been published.

[0120] Rhosin hydrochloride (CAS:1173671-63-0), also known as Rhostatin, is a potent specific inhibitor of RhoA subfamily Rho GTPases with a Kd of approximately 0.4 μM. In vitro experiments have shown that this drug inhibits RhoA activity and RhoA-mediated cellular functions without affecting Cdc42 or Rac1 signaling activity. By suppressing RhoA or RhoC activity, Rhosin can inhibit mammary sphere formation by breast cancer cells, inhibit invasion of mammary epithelial cells, and synergize with NGF to induce neurite outgrowth in PC12 cells. Treatment with Rhosin inhibits lung metastasis of B16BL6 and 4T1 cells in vivo in mouse animal models. The structural formula of Rhosin hydrochloride is: [ka]

[0121] In one example, the inhibitor is Rhosin, or a pharma- ceutically acceptable salt, solvate, or prodrug thereof. The inhibitor may be Rhosin hydrochloride.

[0122] ROCK inhibitor-2 (CAS:1127308-52-4) is a selective dual ROCK1 and ROCK2 inhibitor with IC50 of 17 nM and 2 nM, respectively. Its structural formula is as follows: [ka]

[0123] In one example, the inhibitor is ROCK inhibitor-2, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0124] ROCK-IN-1 (CAS:934387-35-6) is a potent ROCK inhibitor with an IC50 of 1.2 nM against ROCK2. Its structural formula is as follows: [ka]

[0125] In one example, the inhibitor is ROCK-IN-1, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0126] ROCK2-IN-5 (compound 1d) is a hybrid compound that contains a structural fragment of the Rho kinase inhibitor fasudil and the NRF2 inducers caffeic acid and ferulic acid. ROCK2-IN-5 has an excellent multi-target profile and good tolerability. ROCK2-IN-5 has potential for the study of amyotrophic lateral sclerosis (ALS) with SOD1 mutations.

[0127] ROCK2-IN-2 (CAS:867017-68-3) is a selective ROCK2 inhibitor, which is compound A-30 with an IC50 of less than 1 μM, extracted from US Patent Application Publication No. 20180093978A1. Its structural formula is as follows: [ka]

[0128] In one example, the inhibitor is ROCK2-IN-2, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0129] Hydroxyfasudil (CAS number: 105628-72-6) is a ROCK inhibitor with IC50 values ​​of 0.73 μM and 0.72 μM for ROCK1 and ROCK2, respectively, and its structural formula is as follows: [ka]

[0130] In one example, the inhibitor is hydroxyfasudil, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0131] The structural formula of hydroxyfasudil hydrochloride (CAS number: 155558-32-0) is as follows: [ka]

[0132] In one example, the inhibitor is hydroxyfasudil, or a pharma- ceutically acceptable salt, solvate, or prodrug thereof. The inhibitor may be hydroxyfasudil dichloride.

[0133] Ripasudil (K-115) (CAS number: 887375-67-9) is a ROCK-specific inhibitor with IC50 values ​​of 19 nM and 51 nM against ROCK2 and ROCK1, respectively, and the structural formula of its hydrochloride salt is as follows. [ka]

[0134] In one example, the inhibitor is ripasudil hydrochloride, or a solvate, prodrug or alternative pharma- ceutically acceptable salt thereof.

[0135] The structural formula of Ripasudil free base (CAS number: 223645-67-8) is as follows: [ka]

[0136] In one example, the inhibitor is ripasudil free base, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0137] The inhibitor Chroman 1 (CAS number: 1273579-40-0), as well as its salts, solvates and solvate salts, are highly potent and selective ROCK inhibitors. Chroman 1 is more potent against ROCK2 (IC50=1 pM) than ROCK1 (IC50=52 pM). Chroman 1 also has inhibitory activity against MRCK with an IC50 of 150 nM. The structural formula of Chroman 1 is: [ka]

[0138] In one example, the inhibitor is chroman 1, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0139] The structural formula of chroman 1 dihydrochloride is: [ka]

[0140] In one example, the inhibitor is chroman 1 dihydrochloride, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0141] The inhibitor Cotosudil (CAS number: 1258833-31-6) is a ROCK kinase inhibitor and can be used in the study of glaucoma or ocular hypertension. The structural formula of Cotosudil chroman 1 is as follows: [ka]

[0142] In one example, the inhibitor is Cotosudil 1, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0143] The inhibitor SAR407899 (CAS number: 923359-38-0), as well as its salts, solvates and solvate salts, is a selective and potent ATP-competitive ROCK inhibitor with an IC50 of 135 nM for ROCK-2 and Ki of 36 nM and 41 nM for human and rat ROCK-2, respectively. In vivo, SAR407899 inhibits ROCK-mediated phosphorylation of MYPTT696 in the thoracic aorta of spontaneously hypertensive rats and effectively attenuates the pressor response to vasoconstrictors in rats. The structural formula of SAR407899 is: [ka]

[0144] In one example, the inhibitor is SAR407899, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0145] The structural formula of SAR407899 hydrochloride is: [ka]

[0146] In one example, the inhibitor is SAR407899, or a pharma- ceutically acceptable salt, solvate or prodrug thereof. The inhibitor may be SAR407899 hydrochloride.

[0147] Sobesudil (PHP-201) (CAS number: 1333400-14-8), its salts, solvates and solvate salts, is a potent ATP-competitive locally acting Rho kinase (ROCK) inhibitor with IC50 of 3.7 nM and 2.3 nM against ROCK-I and ROCK-II, respectively. Sobesudil reduces intraocular pressure (IOP) without inducing hyperemia and has the following structural formula: [ka]

[0148] In one example, the inhibitor is sobesudil, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0149] The structural formula of sobesudil hydrochloride is: [ka]

[0150] In one example, the inhibitor is sobesudil hydrochloride, or a solvate, prodrug or a pharma- ceutically acceptable salt thereof.

[0151] RKI 1447 (CAS number: 1342278-01-6), as well as its salts, solvates and solvate salts, are potent Rho-associated ROCK kinase inhibitors with anti-invasive and anti-tumor activity against breast cancer (IC50 values ​​of 14.5 nM and 6.2 nM for ROCK1 and 2, respectively). RKI 1447 is a type 1 inhibitor that binds to both the hinge region and the DFG motif of the ROCK ATP binding site. The structural formula of RKI 1447 is: [ka]

[0152] In one example, the inhibitor is RKI 1447, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0153] The structural formula of RKI 1447 dihydrochloride (CAS number: 1782109-09-4) is as follows: [ka]

[0154] In one example, the inhibitor is RKI 1447 or a pharma- ceutically acceptable salt, solvate or prodrug thereof. The inhibitor may be RKI 1447 hydrochloride.

[0155] H-1152 (CAS number: 451462-58-1), its salts, solvates and solvate salts, is a membrane-permeable and selective ROCK inhibitor with a Ki value of 1.6 nM and an IC50 value of 12 nM for ROCK2. H-1152 has also been shown to have low inhibitory activity against CaMKII, PKG, AuroraA, PKA, Src, PKC, MLCK, Abl, EGFR, MKK4, GSK3α, AMPK and P38α. The structural formula of H-1152 is as follows: [ka]

[0156] In one example, the inhibitor is H-1152, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0157] The structural formula of H-1152 dihydrochloride (CAS number: 871543-07-6) is as follows. [ka]

[0158] In one example, the inhibitor is H-1152 dichloride, or a solvate, prodrug or alternative pharma- ceutically acceptable salt thereof.

[0159] GSK269962A (GSK 269962) (CAS number: 850664-21-0), as well as its salts, solvates and solvate salts, are potent ROCK inhibitors with IC50 of 1.6 nM and 4 nM against recombinant human ROCK1 and ROCK2, respectively. GSK269962A has anti-inflammatory and vasodilatory activity. GSK269962A is a potent antihypertensive agent that induces a dose-dependent reduction in blood pressure in spontaneously hypertensive rats. Its structural formula is: [ka]

[0160] In one example, the inhibitor is GSK269962A, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0161] The structural formula of GSK269962A hydrochloride (GSK 269962 hydrochloride) (CAS number: 2095432-71-4) is as follows. [ka]

[0162] In one example, the inhibitor is GSK269962A or a pharma- ceutically acceptable salt, solvate or prodrug thereof. The inhibitor may be GSK269962A hydrochloride.

[0163] Rho-Kinase-IN-1 (CAS number: 1035094-83-7) is a Rho kinase (ROCK) inhibitor, which is compound 1.008 extracted from US Patent Publication No. 20090325960A1 (Ki values ​​for ROCK1 and ROCK2 are 30.5nM and 3.9nM, respectively). Rho-Kinase-IN-1 can be useful in the treatment of diseases or conditions associated with excessive cell proliferation, remodeling, edema, and inflammation, and its structural formula is as follows: [ka]

[0164] In one example, the inhibitor is Rho-Kinase-IN-1, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0165] SB-772077B dihydrochloride (CAS number: 607373-46-6) is an aminofurazan Rho kinase (ROCK) inhibitor with IC50 values ​​of 5.6 nM and 6 nM against ROCK1 and ROCK2, respectively, and its structural formula is as follows: [ka]

[0166] In one example, the inhibitor is SB-772077B or a pharma- ceutically acceptable solvate or prodrug thereof. The inhibitor may be SB-772077B dihydrochloride.

[0167] HSD1590 (CAS number: 2379279-96-4) is a potent ROCK inhibitor with IC50 of 1.22 nM and 0.51 nM for ROCK1 and ROCK2, respectively. HSD1590 exhibits single-digit nanomolar binding to ROCK (Kds<2 nM). In vitro studies have shown that HSD1590 exhibits significant attenuation of migration. The structural formula of HSD1590 is: [ka]

[0168] In one example, the inhibitor is HSD1590, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0169] BDP5290 (CAS number: 1817698-21-7) is a potent inhibitor of both ROCK and MRCK with IC50 values ​​of 5nM, 50nM, 10nM, and 100nM against ROCK1, ROCK2, MRCKα, and MRCKβ, respectively. In vitro studies have shown that BDP5290 completely inhibits MRCKβ-induced phosphorylation of myosin II light chain (MLC). The structural formula of BDP5290 is: [ka]

[0170] In one example, the inhibitor is BDP5290, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0171] ZINC00881524 (CAS number: 557782-81-7) is a Rho-associated kinase (ROCK) inhibitor. In vitro, it reduces the levels of ROCK1 in T47D and CAMA-1 breast cancer cells. When used in combination with CNN1 knockdown, ZINC00881524 reduces the proliferation of T47D and CAMA-1 cells. The structural formula of ZINC00881524 is: [ka]

[0172] In one example, the inhibitor is ZINC00881524, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0173] Velosudil (AR-12286) (CAS number: 1414854-42-4) is a potent and selective Rho kinase (ROCK) inhibitor with Ki of 2 nM and 2 nM for ROCK1 and ROCK2, respectively. AR-12286 reduces intraocular pressure (IOP) primarily by increasing aqueous humor outflow through the trabecular meshwork. The structural formula of Velosudil is: [ka]

[0174] In one example, the inhibitor is belosudil, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0175] LX7101 (CAS number: 1192189-69-7) is a potent LIMK and ROCK2 inhibitor with IC50 values ​​of 24 nM, 1.6 nM, and 10 nM for LIMK1, LIMK2, and ROCK2, respectively, and also inhibits PKA with an IC50 of less than 1 nM. The structural formula of LX7101 is as follows: [ka]

[0176] In one example, the inhibitor is LX7101, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0177] GSK-25 (CAS number: 874119-56-9) is a potent, selective, and orally bioavailable inhibitor of ROCK1 (IC50=7nM). GSK-25 maintains good selectivity for RSK1 and p70S6K (RSK1:IC50=398nM, p70S6K:IC50=1μM). GSK-25 inhibits the P450 profile (IC50 for CYP2C9, CYP2D6, and CYP3A4 is 2.5μM, 5.2μM, and 2.5μM, respectively). GSK-25 has been profiled in the spontaneously hypertensive rat (SHR) model of hypertension and induces blood pressure reduction. The structural formula of GSK-25 is: [ka]

[0178] In one example, the inhibitor is GSK-25, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0179] GSK180736A (CAS number: 817194-38-0) is a potent Rho-associated coiled-coil kinase 1 (ROCK1) inhibitor with an IC50 of 100 nM. GSK180736A is also a selective ATP-competitive G protein-coupled receptor kinase 2 (GRK2) inhibitor with an IC50 of 0.77 μM. GSK180736A is a structurally similar compound to paroxetine, which was developed as a ROCK inhibitor, and has been shown to be a more potent and selective GRK2 inhibitor with an IC50 of 0.77 μM and more than 100-fold more selective than other GRKs. ROCK1 is a potential therapeutic target in the treatment of cardiovascular diseases such as hypertension, and its structural formula is: [ka]

[0180] In one example, the inhibitor is GSK180736A, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0181] CRT0066854 (CAS number: 1438881-19-6), as well as its salts, solvates and solvate salts, are potent and selective atypical PKC isozyme inhibitors. CRT0066854 has IC50 values ​​of 132 nM, 639 nM and 620 nM against full-length (FL) PKCι, PKCζ and ROCK-II kinases, respectively. The structural formula of CRT0066854 is: [ka]

[0182] In one example, the inhibitor is CRT0066854, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0183] The structural formula of CRT0066854 hydrochloride (CAS number: 2250019-91-9) is as follows. [ka]

[0184] In one example, the inhibitor is CRT0066854, or a pharma- ceutically acceptable salt, solvate or prodrug thereof. The inhibitor may be CRT0066854 hydrochloride.

[0185] In one example, the inhibitor is selected from the group consisting of C3 exoenzyme, C3 transferase-based, Rhosin, Rhosin hydrochloride, CCG-1423, CCG-203971, YS-49 monohydrate, YS-49, cerivastatin sodium, cerivastatin, Z62954982, Y16, MLS-573151, HA-100 hydrochloride, HA-100, HL07, DDO-5701, DDO-5713, DDO-5714, DDO-5715, DDO-5716, ML-7, MLCK18, and CT-04. These inhibitors inhibit various steps in the RhoA / ROCK pathway but do not directly inhibit ROCK.

[0186] The RhoA / ROCK2 signaling pathway is well known for controlling the organization of the actin cytoskeleton and cell dynamics in several cell types. RhoA acts through two molecular structures, becoming inactive when bound to guanosine diphosphate (GDP) and active when bound to GTP. ROCK, a Rho downstream effector molecule, is a serine / threonine kinase protein that controls actin filament remodeling by phosphorylating a number of downstream target proteins, including the myosin-binding subunit of myosin light chain 2 (MLC2) phosphatase. Thus, inactivation of the RhoA / ROCK pathway can be promoted by targeting different proteins in the signaling pathway.

[0187] In one example, the inhibitor inhibits the RhoA / ROCK pathway by ribosylating the RhoA protein, in other words, the inhibitor may be an ADP-ribosyltransferase.

[0188] Ribosylation of the RhoA protein, which is involved in the RhoA / ROCK pathway, inactivates RhoA, blocking the entire pathway (Fig. 3A), and inhibits cytoskeletal reorganization.

[0189] In one example, the inhibitor is C3 exoenzyme, which inhibits the RhoA / ROCK pathway by ribosylating RhoA. C3 exoenzyme is an ADP-ribosyltransferase.

[0190] "C3 exoenzyme" or "C3 transferase" (UniProtKB-Q7M0L1(Q7M0L1_CLOBO) is an enzyme from Clostridium botulinum that blocks RhoA by locking it in an inactive state and leaving it bound to a RhoA dissociation inhibitor. C3 exoenzyme is an enzyme from Clostridium botulinum, C3 transferase, that selectively blocks the function of RhoA, B, and C by ADP-ribosylation of asparagine 41, without affecting Rac or Cdc42 (Figure 3A).

[0191] Rhosin (CAS number: 1173671-63-0) is a potent and specific RhoA subfamily Rho GTPase inhibitor that specifically binds to RhoA and inhibits RhoA-GEF interactions with a Kd of approximately 0.4 μM, and does not interact with Cdc42 or Rac1, GEFs, or LARGs. Rhosin induces cell apoptosis. Rhosin promotes stress resilience by enhancing D1-MSN plasticity and reducing hyperexcitability. Rhosin compounds are: [ka]

[0192] In one example, the inhibitor is Rhosin, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0193] CCG-1423 (CAS number: 285986-88-1) is a novel inhibitor of RhoA / C-mediated gene transcription and can inhibit invasion of PC-3 prostate cancer cells in the Matrigel model of metastasis. With an IC50 value of 1.5 μM, the target is a Rho signaling inhibitor, CCG-1423 selectively inhibits spontaneous PC-3 prostate cancer cell invasion through Matrigel matrix in vitro, but does not inhibit Gαi-dependent LPA-stimulated SKOV-3 ovarian cancer cell invasion in vitro. At 100 μM, a near complete inhibition of invasion is achieved, with a degree of toxicity lower than that induced by CCG-1423 at 10 μM. SRF binds to this site in vivo, and CCG-1423, an SRF inhibitor, completely blocks STARS adjacent reporter activity in H9c2 cells. Pharmacological MKL inhibition with CCG-1423 significantly inhibits CCN1 promoter activity, as well as mRNA and protein expression. In vivo, pharmacological SRF inhibition with CCG-1423 reduces nuclear MKL1 and improves glucose uptake and tolerance in insulin-resistant mice in vivo. The compounds of CCG-1423 are: [ka]

[0194] In one example, the inhibitor is CCG-1423, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0195] CCG-203971 (CAS number: 1443437-74-8) is a second generation Rho / MRTF / SRF pathway inhibitor. CCG-203971 potently targets SRE luciferase activated by RhoA / C (IC50=6.4μM). CCG-203971 inhibits migration of PC-3 cells with IC50 of 4.2μM. It is a potential anti-metastatic agent. In vitro, CCG-203971 is a second generation Ras homolog gene family, member A (RhoA) / myocardin-related transcription factor A (MRTF-A) / serum response factor (SRF) pathway inhibitor that dose-dependently suppresses both matrix stiffness and transforming growth factor-β-mediated fibrogenesis as determined by protein and gene expression. CCG-203971 significantly suppresses TGF-β-induced MKL1 expression at a concentration of 25μM. The compound of CCG-203971 is as follows: [ka]

[0196] In one example, the inhibitor is CCG-203971, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0197] YS-49 (CAS number: 132836-42-1) is a PI3K / Akt (downstream target of RhoA) activator and reduces RhoA / PTEN activation in 3-methylcholanthrene-treated cells. YS-49 inhibits angiotensin II (Ang II)-stimulated VSMC proliferation through induction of heme oxygenase (HO)-1. YS-49 is also an isoquinoline compound alkaloid, which has a potent positive inotropic effect via activation of cardiac β-adrenergic receptors.

[0198] In vitro, YS-49 (1–100 μM, 18 h, RAVSMC and RAW 264.7 cells) concentration-dependently inhibited nitrite accumulation in both RAVSMC and RAW 264.7 exposed to lipopolysaccharide (LPS) and INF-γ, with IC50 values ​​of 22 μM and 30 μM, respectively.

[0199] YS-49 (10-100 μM, 18 h, RAVSMC and RAW264.7 cells) suppresses LPS- and / or cytokine-induced iNOS gene expression at the transcriptional level in RAVSMC and RAW264.7 cells. The compounds of YS-49 are as follows: [ka]

[0200] In one example, the inhibitor is YS-49, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0201] Cerivastatin (CAS number: 145599-86-6) is a synthetic lipid-lowering agent, a highly potent, well-tolerated, orally active HMG-CoA reductase inhibitor with a Ki of 1.3 nM / L. Cerivastatin reduces low-density lipoprotein cholesterol levels. Cerivastatin also inhibits the proliferation and invasion of MDA-MB-231 cells, mainly through RhoA inhibition, and has anticancer effects. In vitro, cerivastatin induces a decrease in cell proliferation, an increase in p21 levels, an inhibition of cell invasion, and an inactivation of NFkB. The compound of cerivastatin is as follows: [ka]

[0202] In one example, the inhibitor is cerivastatin, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0203] Z62954982 (CAS number: 1090893-12-1) is a potent, selective, cell-permeable Rac1 (IC50=12 μM) inhibitor that is 4-fold more effective than NSC23766 (HY-15723A) (IC50=50 μM). Z62954982 disrupts the Rac1 / Tiam1 complex, reducing the cytoplasmic levels of active Rac1 (GTP-bound Rac1) without affecting the activity of other Rho GTPases (such as Cdc42 or RhoA). The compound Z62954982 is: [ka]

[0204] In one example, the inhibitor is Z62954982, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0205] Y16 (CAS number: 429653-73-6) is a specific inhibitor of leukemia-associated Rho guanine nucleotide exchange factor (LARG) with a Kd value of 76 nM. Y16 has activity in blocking the interaction of LARG and related G protein-coupled Rho GEFs with RhoA. Y16 shows no detectable effect on other diffuse B cell lymphoma (Dbl) family Rho GEFs, Rho effectors, or RhoGAPs. In vitro, Y16 (10-30 μM, 24 h, NIH 3T3 cells) could dose-dependently inhibit serum-induced RhoA-GTP formation and is specific for RhoA. Y16 (10-30 μM, 24 h, NIH 3T3 cells) efficiently inhibits serum- or SDF-1α-induced phosphorylated MLC and phosphorylated FAK formation downstream of RhoA. The compounds of Y16 are as follows: [ka]

[0206] In one example, the inhibitor is Y16, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0207] MLS-573151 (CAS number: 10179-57-4) is a selective GTPase Cdc42 inhibitor with an EC50 of 2 μM. MLS-573151 is inactive against other GTPase family members such as Rab2, Rab7, H-Ras, Rac1, Rac2 and RhoA wild type. MLS-573151 acts by blocking the binding of GTP to Cdc42. In vitro, the fluorescence intensity of phagocytosed beads or bacteria in hemocytes, measured as a measure of phagocytic efficiency, was significantly reduced in MLS-573151 (50 μM, 15 min) treated granulocytes compared to the control group. MLS-573151 may effectively inhibit the phagocytic ability of granulocytes. The compound of MLS-573151 is as follows: [ka]

[0208] In one example, the inhibitor is MLS-573151, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0209] HA-100 (CAS number: 84468-24-6) is a potent protein kinase inhibitor with IC50 values ​​of 4 μM, 8 μM, 12 μM, and 240 μM against cGMP-dependent protein kinase (PKG), cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and MLC kinase, respectively. HA-100 is also used as a ROCK inhibitor. In vitro, HA-100 competitively inhibits MLC kinase and PKC against ATP with Ki values ​​of 61 μM and 6.5 μM, respectively. The compounds of HA-100 are as follows: [ka]

[0210] In one example, the inhibitor is HA-100, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0211] HL-07 inhibits RhoA activation preferentially over GNPs. HL07 inhibited phenylephrine (PE)-induced contraction in rat aorta and blocked PE-stimulated RhoA activation in human cerebrovascular smooth muscle cells. The compounds of HL-07 are as follows: [ka]

[0212] In one example, the inhibitor is HL-07, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0213] DDO-5701 or Proglumide (CAS Number: 6620-60-6) (approved drug) is a non-peptide, orally active cholecystokinin (CCK)-A / B receptor antagonist. Proglumide selectively blocks the effects of CCK in the central nervous system. Proglumide has the ability to inhibit gastric secretion and protect the gastroduodenal mucosa. Proglumide also has antiepileptic and antioxidant activities. The chemical compound of DDO-5701 is: [ka]

[0214] In one example, the inhibitor is DDO-5701, or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0215] DDO-5713, DDO-5714, DDO-5715, and DDO-5716 are DDO-5701 derivatives with structural modifications for binding activity. They are small molecule inhibitors of RhoA and exhibit higher RhoA inhibitory activity than DDO-5701. Of these four inhibitors, DDO-5716 has been shown to have potential applications in the treatment of breast cancer. More specifically, DDO-5716 can effectively reverse the functions of breast cancer cells controlled by RhoA.

[0216] ML-07 hydrochloride (CAS number: 110448-33-4) is a naphthalenesulfonamide derivative that potently inhibits MLCK (IC50=300nM). ML-7 hydrochloride also inhibits YAP / TAZ. It shows more potent inhibition than ML9 hydrochloride. Ca 2+ -Exhibits reversible, ATP-competitive inhibition of calmodulin-dependent and -independent smooth muscle MLCK. Inhibits formation and stabilization of megakaryocyte plasmoblast processes. The compounds of ML-07 are: [ka]

[0217] In one example, the inhibitor is ML-07, or a pharma- ceutically acceptable salt, solvate, or prodrug thereof. The inhibitor may be ML-07 hydrochloride.

[0218] MLCK18 (CAS number: 224579-74-2): MLCK inhibitor peptide 18 is a myosin light chain kinase (MLCK) inhibitor with an IC50 of 50 nM and inhibits CaM kinase II with 4000-fold selectivity without inhibiting PKA.

[0219] The amino acid sequence of MLCK18 is RKKYKYRRK (SEQ ID NO:1).

[0220] use The RhoA / ROCK pathway inhibitors described herein can be used to treat diseases or disorders associated with muscle fibrosis.

[0221] The RhoA / ROCK pathway inhibitors described herein can be used in the manufacture of a medicament for the treatment of a disease or disorder associated with muscle fibrosis.

[0222] Details regarding inhibitors, treatments, and diseases or disorders are provided elsewhere herein and apply to this use as well.

[0223] Also provided herein are pharmaceutical compositions comprising at least one RhoA / ROCK pathway inhibitor as described herein and a pharma- ceutically acceptable excipient, adjuvant, diluent, and / or carrier. Compositions may typically include pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and supplemental immune enhancing agents such as cytokines, and optionally other therapeutic agents or compounds.

[0224] In one embodiment, the pharmaceutical composition comprises only one RhoA / ROCK pathway inhibitor as an active agent, hi a preferred embodiment, the only RhoA / ROCK pathway inhibitor in the composition is fasudil or C3 exoenzyme.

[0225] In one embodiment, a pharmaceutical composition is provided comprising a RhoA / ROCK pathway inhibitor as an active agent for the treatment of a disease or disorder associated with muscle fibrosis, in particular DMD or Becker muscular dystrophy.

[0226] Details regarding inhibitors, treatments, and diseases or disorders are provided elsewhere herein and apply to the pharmaceutical compositions as well.

[0227] The inhibitor of the present invention can be used or administered alone or in combination with at least one or more other compounds. Administration "in combination" with at least one or more other compounds includes simultaneous (concurrent) and sequential administration in any order. "Concomitant" and "combination" in the context of the present invention also include pharmaceutical preparations that contain both the inhibitor and at least one or more other compounds as separate individual dosage forms, in separate containers, or, for example, in blisters that contain both types of drugs in separate solid dosage units (e.g., dosage units that must be taken together or within one day are conveniently combined for the patient). The pharmaceutical preparation itself, or as part of a kit, may include instructions for administering the separate dosage units simultaneously, sequentially, or separately to a subject in need thereof.

[0228] Outcome measures and dosage Outcome measures for patients with MD vary depending on the progression of the disease and the age of the patient.

[0229] In certain embodiments, administering the inhibitors of the invention affects the clinical outcome of the disease. Some commonly used outcome measures are described in the next section. Generally, these assessments are repeated over time, and performance declines during the natural history of MD. Generally, patients treated according to the invention deteriorate at a slower rate than patients treated according to standard treatment. Some patients may improve. In some embodiments, patients treated according to the invention have stable scores on one or more of these measures for 3, 6, 9, or 12 months or more. In certain embodiments, patients treated according to the invention show at least a 5%, 10%, 20%, 30%, 40% or more percentage worsening rate on one or more of these measures compared to patients treated with standard treatment over a period of 6, 12, 18, 24 months or even longer.

[0230] The 6-minute walk test measures the distance a subject can walk in 6 minutes. A shorter distance indicates decreased function and a longer distance indicates improved function. This test is one of the most commonly used tests and has been used as the basis for approval of controlled substances.

[0231] The North Star ambulatory assessment scale (NSAA) is a 17-item rating scale used to measure the functional motor skills of ambulatory children with MD. Each item is scored 0, 1, or 2, with a score of 2 assigned to "normal" performance where the subject performs the activity as instructed, a score of 1 assigned if the subject does not perform as instructed but achieves the goal without the physical assistance of another person, and a score of 0 assigned if the subject is unable to achieve the goal independently. The NSAA is also very commonly used with ambulatory patients.

[0232] The Performance of upper limb (PUL) assesses upper limb performance and can therefore be applied to non-ambulatory patients. There are two commonly used versions of the PUL, 1.2 and 2.0, each assessing three "dimensions" of upper limb performance: high / shoulder level, mid / elbow level, and distal / wrist and finger level. PUL 1.2 assesses 22 items with a maximum score of 72 (high=16 points for 4 items, mid=34 points for 9 items, distal=24 points for 8 items). PUL 2.0 largely overlaps with PUL 1.2, but some assessments have been removed as redundant and the score range of many assessments has been reduced to 0, 1, or 2, similar to NSAA scoring. Thus, the total possible score of the PUL 2.0 is 42 (12 for high / shoulder level, 17 for mid / elbow level, and 13 for distal / wrist / hand level). In both versions, higher scores indicate better function.

[0233] Other simple motor assessments may also be used, e.g., time taken to climb 4 floors, time taken to rise from the floor, etc., with shorter times indicating better function. Measures of respiratory performance, e.g., predicted forced vital capacity, predicted peak expiratory flow, or slow vital capacity, may be assessed with spirometry. Changes in hand grip strength may be assessed with handheld dynamometry / dynamometry.

[0234] MD patients often suffer from cardiomyopathy, and the method of the present invention is intended to improve cardiac function. Cardiomyopathy is a marked deterioration of the contractile ability of the myocardium, leading to heart failure. The disease progresses over time with variable onset of arrhythmias and ventricular dysfunction. Electrocardiographic abnormalities are detected early in the disease and progress with age. The development of cardiomyopathy is characterized by early diastolic dysfunction followed by eccentric hypertrophy. Symptoms of cardiomyopathy may be measured using, for example, echocardiographic evaluation, cardiac magnetic resonance imaging (MRI), and cardiac MRI with delayed gadolinium enhancement. In particular, left ventricular size, thickness, volume, ejection fraction, myocardial scar / inflammatory burden, and strain may be evaluated. Strain, changes in left ventricular size and volume, and scar burden are key measures.

[0235] As MD is common in younger patients, some specific assessments may be applied in the evaluation of pediatric patients. The Bayley Scales of Infant and Toddler Development-III (Bayley-III) gross motor scale is a functional assessment that can be used to evaluate muscle strength in patients with MD aged 2 to 4 years. The minimum score value is 0 and the maximum score value is 72. Higher scores indicate better outcomes. The Personal Adjustment and Role Skills Scale, ed. 3 (PARS III) questionnaire is a scale designed to evaluate the behavior and measure psychosocial adjustment of children with chronic physical illness. PARS III is completed by the parent / guardian. The minimum score value is 28 and the maximum score value is 112. Higher scores indicate better personal adjustment. The Pediatric Outcome Data Collection Instrument (PODCI) measures the physical functioning of children. The minimum score value is 0 and the maximum score value is 100. A higher score means a better outcome.

[0236] In accordance with the therapeutic methods of the invention, a therapeutically effective amount / dose of a ROCK inhibitor, or a pharma- ceutically acceptable salt, solvent, or prodrug thereof, is administered to a patient one or more times per day. The inhibitor may be administered once per day, twice per day, three times per day, four times per day, or five or more times per day.

[0237] The following dosage description is for adult subjects, and dosage in pediatric populations can be reduced using standard conversion based on differences in body weight, body volume or surface area.Thus, for example, if the daily dosage is 90mg for a 70kg adult, it is 1.29mg / kg, and the equivalent pediatric dosage for a 25kg child is 32.25mg.The following description is for Fasudil, but dosage can be easily extrapolated to other ROCK inhibitors described herein based on their relative activity in Rho kinase inhibitors.

[0238] For example, the minimum therapeutically effective dose of fasudil is 90 mg per day, and is generally administered in 2-3 doses as an immediate release formulation to meet the daily dose. The maximum therapeutically effective dose may be empirically determined as the maximum dose that is effective in alleviating one or more MD symptoms while not inducing unacceptable levels or adverse events. For example, generally, the daily dose of fasudil does not exceed 240 mg.

[0239] Suitable daily doses of Fasudil are 90 mg, 100 mg, 110 mg, 1120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg or 240 mg, which may be administered in equal amounts once, twice, three times, four times or more per day.

[0240] As described elsewhere herein, the route of administration can be selected to best suit the requirements. The dosage form can be a non-modified release dosage form or a modified release dosage form. For example, the dosage form can be an immediate release formulation or a sustained release formulation.

[0241] One preferred dosing regimen involves administering 60 mg of fasudil hydrochloride hemihydrate three times a day using an oral immediate release formulation for a total daily dose of 180 mg. Other daily doses range from 90 mg to 180 mg twice a day.

[0242] A further dosing regimen is treatment with 90 mg of fasudil hydrochloride hemihydrate administered twice daily using an immediate release formulation for a total daily dose of 180 mg.

[0243] In other embodiments, 180 mg or 240 mg of Fasudil hydrochloride may be administered once a day using an immediate release formulation. Above 240 mg per day, the renal effects of the drug are generally unacceptable.

[0244] Based on ROCK inhibitory activity, one of skill in the art can readily extrapolate the dosage ranges provided for fasudil to other ROCK inhibitors.

[0245] Alternative embodiments include treatment with 90-240 mg, 120-240 mg, 140-240 mg, 160-240 mg, 180-240 mg, 200-240 mg, or 220-240 mg of fasudil hydrochloride hemihydrate once daily in a sustained release dosage form. Typically, the sustained release dosage form contains 180-240 mg of fasudil hydrochloride hemihydrate. Treatment with sustained release administration of fasudil hydrochloride hemihydrate at a total daily dose of 180 mg is preferred.

[0246] The method of administering the composition according to the present invention generally continues for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 2 years, at least 3 years, or at least 4 years. In some preferred methods, treatment is administered for up to 30 days, up to 60 days, or even up to 90 days or more. Treatment for more than 60 days is preferred, with treatment for at least 6 months being particularly preferred. The exact duration of treatment will depend on the patient's condition and response to treatment. In the most preferred methods, it is envisaged that treatment will commence after the onset or appearance of symptoms.

[0247] It is understood that the dosage ranges described herein are guidelines for administering the provided pharmaceutical compositions to adults. For example, the amount administered to a child or adolescent may be less than or the same as that administered to an adult, as determined by a physician or person skilled in the art.

[0248] general definition Throughout the description and claims of this specification, the terms "comprise" and "contain" and variations thereof mean "including, but not limited to," and are not intended to (and do not) exclude other parts, appendants, components, integers or steps.

[0249] Throughout the description and claims of this specification, the singular includes the plural unless the context requires not to include the plural. In particular, when using the indefinite article, the specification should be understood to contemplate the plural as well as the singular, unless the context requires that only the singular is intended.

[0250] As used herein, the singular forms "a," "an," "the" without a specified quantity include plural referents unless the context clearly dictates not to include plural referents. Unless otherwise specified, nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation. It is to be understood that the present invention is not limited to the particular methodology, protocols, and reagents described, which may vary depending on the context in which they are used by those of skill in the art.

[0251] It is to be understood that any feature, integer, property, compound, chemical moiety or group described in connection with a particular aspect, embodiment, or example of the invention is also applicable to any other aspect, embodiment, or example described herein, unless incompatible.

[0252] As understood by those skilled in the medical arts, the terms "treat" and "treatment" refer to the medical management of a disease, disorder, or condition in a subject (i.e., a patient) (see, e.g., Stedman's Medical Dictionary). In general, an appropriate dosage and treatment regimen provides an amount of inhibitor sufficient to provide a therapeutic and / or prophylactic effect. The therapeutic effect of a subject to whom an inhibitor described herein is administered includes, for example, improved clinical outcome, with the purpose of preventing or slowing or arresting (reducing) undesirable physiological changes associated with a disease, or preventing or slowing or arresting (reducing) the spread or severity of such a disease. As discussed herein, efficacy of an inhibitor may include beneficial or desirable clinical results, including, but not limited to, reduction, alleviation or amelioration of symptoms resulting from or associated with the disease being treated, reduction in the occurrence of symptoms, improved quality of life, prolonged disease-free state (i.e., reduction in the likelihood or tendency of a subject to exhibit symptoms that provide the basis for a diagnosis of a disease), reduction in the extent of disease, stabilization of the disease state (i.e., not getting worse), slowing or delaying the progression of disease, improvement or palliative treatment of the disease state, and remission (whether partial or complete), whether detectable or undetectable, and / or overall survival.

[0253] As used herein, the phrase "disorder associated with muscle fibrosis" refers to, but is not limited to, any disease or disorder that results directly or indirectly, and / or completely or partially, from muscle fibrosis. Muscle fibrosis may be the cause of the disease or disorder, but may also be a symptom that appears after the onset of the disease or disorder.

[0254] As used herein, the terms "disease," "disorder," and "condition" are used interchangeably to refer to a disturbance in the structure or function of a human or animal, particularly a disturbance that produces specific symptoms or affects a specific area and is not merely the direct result of a physical injury.

[0255] Diseases that can be treated and / or prevented with the inhibitors described herein are described in detail elsewhere herein.

[0256] As used herein, the term "subject" refers to an individual having or at risk of having a particular condition, disorder or symptom, such as a human, dog, cat, pig, horse, mouse, cow, rat, etc. The subject may be a patient, i.e., a subject in need of treatment according to the present invention. The subject may be undergoing treatment for a condition, disorder or symptom. Alternatively, the subject has not undergone treatment prior to treatment according to the present invention. The subject is preferably in need of administration of an inhibitor of the present invention.

[0257] As used herein, "administration" or "administering" of the (pharmaceutical) compositions described herein to a subject includes any route of introduction or delivery to a subject that allows the composition to perform its intended function. Administration can be performed by any suitable route, including oral, intranasal, intraocular, ophthalmic, parenteral (intravenous, intramuscular, intraperitoneal or subcutaneous), or topical. Administration includes self-administration and administration by another. The composition can be administered in a therapeutically effective amount. As used herein, the phrase "therapeutically effective amount" refers to a dose or plasma concentration in a subject that results in the particular pharmacological effect that the described composition is intended to achieve by administration, for example to treat a disease of interest in the subject. The therapeutically effective amount can vary based on the route and form of administration, the age and weight of the subject, and / or the disease or condition being treated.

[0258] The composition may be a pharmaceutical composition or formulation comprising the inhibitor and pharma- ceutical acceptable excipients, adjuvants, diluents and / or carriers. Pharmaceutical compositions or formulations may typically contain pharma- ceutical acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and supplementary immune enhancing agents such as cytokines, and optionally other therapeutic agents or compounds.

[0259] As used herein, "pharmaceutical acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., that may be administered to an individual, together with a selected inhibitor, without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0260] Excipients are natural or synthetic substances that are formulated with an active ingredient (such as a neurotoxin provided herein) to bulk the formulation or to impart a therapeutic effect to the active ingredient in the final dosage form (such as facilitating drug absorption or solubility). Excipients are also useful in the manufacturing process, aiding in the handling of the active agent involved, such as by promoting powder flowability or non-stickiness, as well as aiding in in vitro stability, such as preventing degradation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. Thus, suitable excipients are readily identifiable by one of ordinary skill in the art. By way of example, suitable pharma-ceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0261] Adjuvants are pharmacological and / or immunological agents that modify the effects of other agents in the formulation. Pharmaceutically acceptable adjuvants are well known in the art and include cell-penetrating peptides. Thus, suitable adjuvants can be easily identified by those skilled in the art.

[0262] A diluent is a diluting agent. Pharmaceutically acceptable diluents are well known in the art and include water or saline. Thus, suitable diluents are readily identifiable by those skilled in the art.

[0263] Carrier is non-toxic to recipient at the dosage and concentration used, and is compatible with other ingredients of the formulation. The term "carrier" refers to an organic or inorganic ingredient, natural or synthetic, that is combined with active ingredient to facilitate application. Pharmaceutically acceptable carriers are well known in the art and include serum albumin. Therefore, suitable carriers can be easily identified by those skilled in the art.

[0264] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994), and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in the present invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. Thus, the terms defined below are described in more detail by reference to the specification as a whole. It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described, which may vary depending on the context in which they are used by those skilled in the art.

[0265] As used herein, the term "alkyl," alone or in combination with other terms, refers to a branched or unbranched saturated hydrocarbon chain. Alkyl groups are described using prefixes that specify the minimum and maximum number of carbon atoms in the moiety (e.g., "C a~b "). For example, C a~bAlkyl refers to an alkyl moiety of integer "a" to integer "b" carbon atoms, inclusive. Unless otherwise specified, alkyl groups are unsubstituted. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

[0266] As used herein, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced with a halogen atom. The alkyl group of a "haloalkyl" is a saturated alkyl group. The alkyl group is described using a prefix that specifies the minimum and maximum number of carbon atoms in the alkyl portion. Representative examples of "haloalkyl" include, but are not limited to, -CF3, -CHF2, -CH2F, -CH2Cl, -CH2Br, -CF2CF3, -CHFCF3, -CH2CF3, -CF2CH3, -CHFCH3, -CF2CF2CF3, -CF2CH2CH3, CHFCH2CH3, and -CHFCH2CF3. A haloalkyl group is unsubstituted.

[0267] As used herein, the term "heterocycloalkyl", alone or in combination with other terms, refers to a monocyclic non-aromatic ring system containing at least one nitrogen atom (as shown in formula (1)) and carbon atoms. In addition to the nitrogen atom, the heterocycloalkyl group may contain one or more additional heteroatoms, such as nitrogen, oxygen, sulfur or phosphorus, preferably nitrogen, oxygen or sulfur, especially nitrogen or oxygen. The heterocycloalkyl group may be fully saturated or may contain unsaturated portions. In some cases, the heterocycloalkyl group may contain a single nitrogen heteroatom (as shown in formula (1)) or at least two or more heteroatoms (which may be the same or different). The heterocycloalkyl group may be substituted or unsubstituted. In some cases, the heterocycloalkyl group may contain five ring atoms, six ring atoms, or seven ring atoms.

[0268] As used herein, the term "hydroxyalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced with a hydroxy group. The alkyl group of a "hydroxyalkyl" is a saturated alkyl group. The alkyl group is described using a prefix that specifies the minimum and maximum number of carbon atoms in the alkyl portion. Representative examples of "hydroxyalkyl" include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH(OH)CH3, -CH2CH2CH3, OH. Hydroxyalkyl groups are unsubstituted. In general, it is preferred that a hydroxyalkyl group has one hydroxy group.

[0269] As used herein, the term "pharmaceutically acceptable salt" generally refers to a salt of a compound, particularly a compound of formula (1), that is chemically and / or physically compatible with other ingredients that make up the formulation and / or generally physiologically compatible with the recipient. Suitable pharmaceutically acceptable salts of compounds of formula (1) are acid addition salts of the compounds, for example, acid addition salts of inorganic or organic acids. Examples of such acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, trifluoroacetic acid, formic acid, citric acid or maleic acid. In some cases, compounds of formula (1) may be acidic and may form a salt with a counter cation. The salt may be an alkali metal salt (e.g., sodium salt or potassium salt), an alkaline earth metal salt (e.g., calcium salt or magnesium salt), an ammonium salt, or a salt with an organic base that provides a physiologically acceptable cation (e.g., a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris(2-hydroxyethyl)amine). Generally, pharmaceutically acceptable salts of compounds of formula (1) are acid addition salts.

[0270] Representative salts include, but are not limited to, acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, hydrogensulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, trifluoroacetate, and the like. Other examples of representative salts include alkali metal or alkaline earth metal cations, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, lysine, arginine, benzathine, choline, tromethamine, diolamine, glycine, meglumine, olamine, and the like.

[0271] The compounds of formula (1) may exist in solvated (e.g., hydrated) or unsolvated forms. It is to be understood that the present invention encompasses all such solvated forms that possess RhoA / ROCK pathway inhibitory activity.

[0272] The compounds of formula (1) may be administered in the form of prodrugs that are broken down in the human or animal body to release the compound. Prodrugs may be used to modify the physical properties and / or pharmacokinetic properties of the compound. Prodrugs may be formed when the compounds of formula (1) contain a suitable group or substituent to which a property-modifying group can be attached. Examples of prodrugs include in vivo cleavable ester derivatives that may be formed at a carboxy or hydroxy group in the compounds of formula (1), in vivo cleavable amide derivatives that may be formed at a carboxy or amino group in the compounds of formula (1), or N-oxides formed at an amine moiety in the compounds of formula (1).

[0273] References to prodrugs of compounds of formula (1) may be esters, amides, or N-oxides thereof.

[0274] Aspects of the present invention are illustrated by the following non-limiting examples. EXAMPLES

[0275] The invention will now be further described with reference to the following examples and the accompanying drawings.

[0276] result PDGF-AA induces activation of the Rho-A pathway in human muscle FAP First, we confirmed that the number of FAPs was increased in DMD muscle biopsies (n=2) compared to healthy controls (n=2) (Figure 1A-B). Next, we isolated FAPs from muscle biopsies of three DMD patients and treated them with 50 ng / ml PDGF-AA for 4 days. To analyze the changes in protein profiles, we compared PDGF-AA-treated FAPs to untreated FAPs using quantitative proteomic analysis by mass spectrometry. We observed that 1890 proteins were differentially expressed (Volcano plot in Figure 2A). We created a proteomap of upregulated proteins according to functional gene classification (Kyoto Encyclopedia Genes and Genomes, KEGG) (Figure 2B). We observed an increase in the expression of proteins involved in various signaling pathways (MAPK, PI3K-Akt, FoxO or Hedgehog), proteins involved in cellular metabolism, and proteins involved in genetic information processing (Figure 2C-D). Among the upregulated distinct protein groups, the inventors were interested in proteins involved in cellular functions related to fibrosis, such as cytoskeleton reorganization, cell adhesion, and actin filament reorganization. Proteins related to these cellular functions, such as proteins of the Ras homolog gene pathway (Rho), were increased in PDGF-AA-treated FAP, as displayed in the heatmap showing the difference between untreated and PDGF-AA-treated DMD-FAP (Figure 2E). Among the distinct proteins of this family, the Ras homolog gene family member A (RhoA) pathway was focused on, as it is involved in fibroblast activation and fibrosis in several other pathologies.

[0277] C3 exoenzyme and fasudil treatment block PDGF-AA-mediated activation of the Rho-kinase pathway The mass spectrometry results were validated in two different experiments. First, the inventors studied the expression of the guanine nucleotide exchange factor Rho 2 (ArhGEF2), an effector protein of the Rho kinase pathway (Figure 3A), in DMD muscle samples and age- and sex-matched controls (loading control in Figure 1E). A 7.7-fold increase in ArhGEF2 expression was observed in DMD muscles compared to control muscles (n=2) (Figure 3B). In a second step, they analyzed whether the addition of PDGF-AA to DMD-FAP in culture leads to a significant increase in RhoA bound to guanosine triphosphate (GTP) (RhoA-GTP) compared to untreated FAP (c-). To further confirm whether the increase in RhoA-GTP is mediated by activation of the RhoA pathway, the effect of pretreatment with exoenzyme C3 (C3) was also analyzed. The exoenzyme C3 is an ADP-ribosyltransferase that selectively ribosylates RhoA protein at asparagine residue 41, rendering it inactive. As expected, PDGF-AA treatment significantly increased RhoA-GTP levels by 1.88-fold (n=3, SEM=0.206, p<0.01), whereas pretreatment with C3 blocked the above effects induced by PDGF-AA (n=3, SEM=0.07, p<0.05) (Figure 3C). Treatment with PDGF-AA, C3, or fasudil did not affect cell viability at any of the time points tested (Figure 1H).

[0278] Fasudil is an over-the-counter vasodilator used to treat cerebral vasospasm and is also useful in pulmonary hypertension. Fasudil inhibits the RhoA pathway by blocking Rho-associated protein kinase (ROCK), which phosphorylates myosin light chain 2 (p-MLC2). Treatment of DMD-FAP with PDGF-AA resulted in a statistically significant 2.3-fold increase in MLC2 phosphorylation (n=3, SEM=0.33, p<0.001), which was reversed by pretreatment with C3 exoenzyme (n=3, SEM=0.11, p<0.001) and fasudil (n=3, SEM=0.09, p<0.001), as shown in Figure 3D and Figure 3E. The final step in the Rho kinase pathway is the polymerization of F-actin filaments, mediated by MLC2 phosphorylation. Treatment of FAPs with PDGF-AA induced polymerization of intracellular actin filaments, which was affected by pretreatment with C3 or fasudil ( Fig. 3F ).

[0279] Fasudil blocks PDGF-AA-mediated increases in proliferation, migration, and collagen I expression in human FAP in vitro The effects of PDGF-AA, C3 exoenzyme and fasudil on proliferation, migration and collagen I production were studied in vitro. Addition of PDGF-AA to the culture medium significantly increased proliferation of FAPs at both 48 hours (n=3, SEM=0.02, p<0.001) and 72 hours (n=3, SEM=0.05, p<0.001) compared to untreated FAPs (3.6-fold and 6-fold increase, respectively). In contrast, treatment of FAPs with C3 exoenzyme (n=3, SEM=0.01, p<0.001) and fasudil (n=3, SEM=0.02, p<0.001) prior to stimulation with PDGF-AA significantly decreased the proliferation rate at 72 hours compared to PDGF-AA-treated FAPs (3.27-fold and 2.6-fold decrease, respectively) (Figure 4A). Cell viability was tested for all treatment doses and different cell exposure times. PrestoBlue data showed that neither C3 exoenzyme nor fasudil affected cell viability (Figure 1H).

[0280] Treatment with PDGF-AA significantly enhanced in vitro migration of FAPs (2.48- and 3.37-fold increases, respectively) at both 48 h (n=3, SEM=0.51, p<0.001) and 72 h (n=3, SEM=0.66, p<0.001) compared to untreated FAPs. This effect was significantly blocked by the addition of C3 exoenzyme and fasudil to the medium at either the 48 or 72 h time points (Figure 4B-C). C3 exoenzyme reduced migration rates by 2.1-fold (n=3, SEM=0.47, p<0.001) at 48 h and 3-fold (n=3, SEM=0.25, p<0.001) at 72 h. Fasudil reduced migration rate 2.43-fold (n=3, SEM=0.41, p<0.001) at 48 hours and 4-fold (n=3, SEM=0.22, p<0.001) at 72 hours.

[0281] PDGF-AA treatment also non-significantly increased collagen I release into the culture medium, but this effect was reversed by the addition of C3 exoenzyme and fasudil (Figure 4D). C3 exoenzyme significantly reduced collagen I release by 1.7-fold (n=3, SEM=0.33, p<0.05).

[0282] Fasudil treatment improves muscle function and reduces muscle fibrosis in the dba / 2J-mdx mouse model of DMD Based on the in vitro results, it was decided to test whether fasudil has an anti-fibrotic effect in vivo in the dba / 2J-mdx mouse model of DMD. First, it was confirmed that PDGF-AA levels were increased in muscle samples from dba / 2J-mdx mice compared to healthy WT controls, similar to the case in DMD patients (Figure 5A). Based on these results, six 7-week-old dba / 2J-mdx mice were orally administered fasudil at a dose of 100 mg / kg / day for 6 weeks. The mice did not show any changes in behavior during the treatment period, suggesting that there were no significant side effects. However, when comparing treated (n=6, SEM=0.94) to untreated (n=5, n=0.60) mice, a decrease in body weight was observed (Figure 5B), although these differences were not statistically significant. Inhibition of the RhoA pathway by fasudil was confirmed by analyzing different downstream effector targets of RhoA, such as ROCK2, LIM domain kinase 2 (LIMK2), and Formin Homology 2 Domain Containing 1 (FHOD1), the latter two proteins involved in the control of actin filament dynamics (Figure 6A). We further quantified the enzymatic activity of ROCK, which was reduced in the quadriceps of treated mice (n=6, SEM=0.12) compared to untreated mice (n=5, SEM=0.14), although these changes were not significant (Figure 6B). However, the protein expression of LIMK2 and MLC2 was significantly decreased in treated mice (n=6, SEM=0.45 and n=6, SEM=0.05, respectively) compared to untreated mice (n=5, SEM=0.97 and n=5, SEM=0.64, respectively) (Figure 6C-F). Muscle strength was analyzed using the forelimb grip strength 6 weeks after the start of treatment and the results obtained in untreated dba / 2J-mdx mice and healthy control mice were compared. It was observed that the forelimb muscle strength of treated mice (n=6, SEM=1.63) was significantly increased by 1.76-fold compared to untreated mice (n=5, SEM=2.29, p<0.01) (Figure 7A). Interestingly, the difference between treated and WT mice did not reach significance. The inventors further analyzed the effect of fasudil on the histology of the quadriceps muscle.Fasudil induced a significant 23% decrease in collagen I area in the quadriceps of treated mice (n=6, SEM=1.63, p<0.05) (Figure 7B and D). A significant increase in muscle fiber size was detected in treated mice (n=6, SEM=0.60) compared to untreated mice (n=5, SEM=0.64, p<0.001) (Figure 7C). Furthermore, PDGFRα area, a measure of the number of FAPs present in the quadriceps of treated mice (n=6, SEM=0.38), was observed to be decreased by 42% compared to untreated mice (n=5, SEM=0.95), although these differences did not reach significance (Figure 7E and F).

[0283] Effect of Fasudil Treatment on Inflammation Quantification of macrophages was performed by analyzing areas of the quadriceps muscle stained with the F4 / 80 marker. Fasudil induced a 74% reduction in treated mice (n=6, SEM=0.15) compared to untreated mice (n=5, SEM=0.73) (Figure 7F and G). These results were consistent with flow cytometry analysis showing a 18.3% reduction in the number of F4 / 80 positive cells in treated mice (n=6, SEM=33.49) compared to untreated mice (n=5, SEM=39.82) (Figure 7H). Indeed, a trend towards reduced expression of profibrotic M2 markers such as CD163 and CD206 was observed in macrophages obtained from fasudil-treated mice. In detail, in macrophages obtained from skeletal muscle of treated mice (n=6, SEM=1.10), a 31% decrease in the expression level of the CD163 marker was observed, whereas the number of CD163+ cells was only 3% decreased compared to untreated mice (n=5, SEM=2.58). Similarly, in macrophages obtained from treated mice (n=6, SEM=1.43), a 18% decrease in the expression level of the CD206 marker was observed, whereas the number of CD206+ cells was only 10% decreased compared to untreated mice (n=5, SEM=5.91) (Figure 7I). The inventors did not observe differences in the expression level or number of CD209 cells, which also distinguish M2 macrophages. The inventors did not observe any changes in the percentage of positive cells or in the expression of two well-known M1 markers, CD80 and DR, in macrophages obtained from skeletal muscle of treated mice compared to untreated mice (Figure 5C).

[0284] The populations of T cells, B cells and neutrophils were also studied, and a trend towards a reduced population of neutrophils was observed in treated mice (n = 6, SEM = 0.72) compared to untreated mice (n = 5, SEM = 0.74), whereas the numbers of T and B cells remained unchanged (Figure 8A-D). Finally, we studied whether fasudil treatment modifies the expression of several cytokines involved in the inflammatory process in muscular dystrophy. As shown in the heatmap in Figure 8E, we observed a reduction in TNFα, IL-4, IL-17, CXCL1, CXCL2, CXCL10, CCL2, CCL3 or CCL17, among others, in fasudil-treated mice compared to untreated mice.

[0285] Consideration In this study, the inventors investigated the molecular pathways activated by PDGF-AA in skeletal muscle of DMD patients, leading to myofibrosis. The inventors observed that PDGF-AA activates the RhoA / ROCK2 pathway, which is effectively blocked by fasudil, a well-known Rho kinase inhibitor that reduces FAP activation in vitro. To validate these findings, they performed a proof-of-concept preclinical study and observed a reduction in myofibrosis in a mouse model of DMD. These results expand the molecular pathways involved in the process of myofibrosis in DMD and support the efficacy of Rho kinase inhibitors as therapeutic agents for DMD in particular and muscular dystrophies in general.

[0286] The absence of dystrophin in DMD causes sarcolemma instability, leading to the expansion of fibrotic and adipose tissue. Although several growth factors have been associated with fibrotic expansion, the role of PDGF-AA has not been thoroughly studied. Previous studies have revealed that persistent signaling through PDGFRα in DMD leads to fibrosis and impedes repair, but the molecular consequences of persistent exposure of FAP to PDGF-AA have not been examined. To study the molecular changes that occur in DMD, we treated FAP from DMD patients with PDGF-AA and observed upregulation of several proteins belonging to the RhoA / ROCK2 pathway. The RhoA / ROCK2 signaling pathway is well known for controlling the organization of the actin cytoskeleton and cell dynamics in several cell types. RhoA acts through two molecular structures, becoming inactive when bound to guanosine diphosphate (GDP) and active when bound to GTP. ROCK2, a Rho downstream effector molecule, is a serine / threonine kinase protein that controls actin filament remodeling by phosphorylating numerous downstream target proteins, including the myosin-binding subunit of myosin light chain 2 (MLC2) phosphatase. Actin rearrangements triggered by the RhoA / ROCK2 signaling pathway lead to fibroblast activation in different pathological conditions, such as cardiac fibrogenesis, tumor cell growth, and pulmonary arterial hypertension. Actin polymerization controls cell polarization, organization of adhesion structures, and force generation that is essential for cell migration. These events allow cells to migrate to injury sites, where they proliferate and release components of the ECM to regenerate damaged tissues. However, excessive activation of fibroblasts increases ECM deposition, leading to the accumulation of fibrogenesis. Although actin rearrangements generated by fibroblast activation have not been characterized in FAP, previous studies suggest that fibroblasts and FAP are phenotypically and biochemically equivalent, supporting the possibility that Rho kinase activation may have similar effects on both cells. The inventors demonstrated that the RhoA / ROCK2 pathway is increased in muscle of DMD patients compared to age- and sex-matched healthy control subjects.Therefore, it was decided to analyze the effect of PDGF-AA on the RhoA / ROCK2 signaling pathway in FAPs isolated from DMD patients. First, it was confirmed that the RhoA / ROCK2 pathway could be regulated by PDGF-AA, since C3 exoenzyme and fasudil significantly reduced the activation of the signaling pathway. The results show that PDGF-AA activated the RhoA / ROCK2 pathway, leading to increased actin filament polymerization, proliferation, and migration, and both C3 and fasudil attenuated these effects. PDGF-AA induced a higher proliferation rate at the 48 and 72 h time points, whereas the blocking effect of the RhoA / ROCK2 inhibitor was only observed at the 72 h time point. The lack of proliferation inhibition at 48 h after C3 exoenzyme or fasudil treatment may be due to other signaling pathways activated after PDGF-AA treatment. PDGF-AA is a mitogenic growth factor that not only activates the RhoA pathway, but also triggers other signaling pathways, such as MAPK, PLC, and PI3K. These molecular pathways are primarily involved in cell proliferation, and RhoA / ROCK2 is involved in actin remodeling.

[0287] Because the Rho / ROCK-mediated pathway interacts with other signaling pathways known to contribute to fibrosis, it was tested whether fasudil reduces fibrosis in dba / 2J-mdx mice. Fasudil is a selective inhibitor of ROCK1 and ROCK2 isoforms and is used in clinical trials as a first-generation selective Rho / ROCK inhibitor. Fasudil has been tested in different diseases, such as pulmonary hypertension, amyotrophic lateral sclerosis, and cardiovascular disease. The inventors observed that dba / 2J-mdx mice treated with fasudil had better performance in grip strength tests than untreated mice. These results were associated with a decreased amount of collagen I and a trend toward a decreased number of FAP cells in the skeletal muscles of treated animals compared to untreated animals.

[0288] Since the inflammatory response is another hallmark of DMD muscles, the inventors also analyzed the inflammatory cell profile after treatment. Fasudil induced a trend towards a decrease in the number of neutrophils and macrophages, especially the number of CD163- and CD206-positive macrophages, without modifying the M1 population. Furthermore, fasudil reduced the expression of several inflammatory cytokines, some of which are directly involved in the fibrotic process, e.g., IL4, IL17, and CCL17. Previous studies have shown that RhoA can be expressed in macrophages, but the inventors only observed that blocking this pathway with fasudil did not significantly reduce the number of macrophages. The observed changes in the number of infiltrating inflammatory cells and the reduction in cytokine levels could be explained either by a direct effect of fasudil on inflammatory cells, or by an indirect effect through a reduction in the fibrotic content of the muscle. It is well known that in muscular dystrophies, ongoing muscle damage sustains the inflammatory response, so a treatment that improves the overall structure of the muscle to reduce muscle degeneration may have the effect of indirectly reducing inflammation. For example, attenuation of muscle fibrotic processes may improve muscle regeneration in treated animals, reduce the number of degenerating fibers, and prevent macrophages and neutrophils from infiltrating damaged muscles. Indeed, the increase in fiber size observed in treated mice could be due to improved muscle regeneration and reduced effects on muscle structure. In summary, treatment of dba-2J mdx mice with fasudil improved grip strength tests, reduced collagen I expression, and showed a trend toward a decrease in the number of PDGFRα+ cells in skeletal muscles, confirming the in vitro results. The results suggest that these effects are mainly explained by the effect of RhoA / ROCK2 inhibition on fibrotic deposition.

[0289] Although ROCKs are expressed in different cell types, the side effects caused by fasudil do not pose major safety concerns. Fasudil targets the ATP-dependent kinase domains of either ROCK1 or ROCK2 with equal potency and without selective effects. It is noteworthy that ROCK2 is the isoform detected in our proteomic analysis, and previous studies have shown that ROCK2 is the predominant isoform in skeletal muscle.

[0290] Muscle fibrosis is an irreversible process that occurs in muscular dystrophies and needs to be prevented before ECM accumulation disrupts muscle structure and function. Because FAP activation and ECM remodeling begin early in skeletal muscle degeneration, inhibiting the early cellular changes that occur in FAP could prevent increased ECM deposition. Therapies focused on targeting actin reorganization to prevent migration of FAP to injury sites may help delay fibrosis progression. Combining antifibrotic therapy with cell or gene therapy may also result in better outcomes from these experimental treatments.

[0291] In summary, this study demonstrates that PDGF-AA induces RhoA / ROCK2 pathway signaling in DMD-FAP, leading to their proliferation, migration and actin reorganization. Treatment with fasudil, a well-known ROCK inhibitor, blocks the cellular effects of PDGF-AA in vitro, attenuates muscle fibrosis and enhances muscle strength in DMD mouse models.

[0292] Materials and Methods cell culture Muscle biopsies (Table 1) from three DMD patients seen at the Hospital de la Santa Creu i Sant Pau (HSCSP), Barcelona, ​​were used. The Ethics Committee of the HSCSP approved the study and all participants signed an informed consent form. All study procedures were performed in accordance with Spanish regulations.

[0293] Muscle explants were cultured and FAPs were isolated as described herein. FAPs were treated with 50 ng / ml PDGF-AA daily for 4 days (Figure 1E). To test activation of the RhoA pathway by PDGF-AA, the pathway was first inhibited with 50 μM fasudil (Bio-Techne, MN, USA) or 2 μg / ml C3 exoenzyme (Cytoskeleton, Denver, USA) for 15 h, then activated with 50 ng / ml PDGF-AA for 20 min, proceeding to G-LISA or IF assays for phosphorylated myosin light chain (p-MLC). To test the functional effect of the PDGF-AA pathway and its inhibition, 2 μg / ml C3 exoenzyme or 50 μM fasudil were first added to the cultures, followed by 50 ng / ml PDGF-AA after 4 h. This inhibition / induction cycle was repeated for as many days as the test lasted.

[0294] Quantitative Proteomics Protein samples were extracted and analyzed using a Lumos Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) connected to an EASY-nLC 1000 (Thermo Fisher Scientific (Proxeon), Odense, Denmark). Samples were analyzed using MaxQuant software (version 1.6.1.0) via the human Swissprot database.

[0295] Cell viability The effect of each treatment on FAP viability was measured with PrestoBlue reagent (Invitrogen) according to the manufacturer's instructions. Fluorescence was measured at an excitation wavelength of 570 nm and an emission wavelength of 600 nm using a microplate reader (INFINITE M1000 PRO, Tecan Trading AG, Switzerland).

[0296] G-LISA, ROCK activity, Sircol assay and cytokine array RhoA-GTP protein was analyzed by colorimetric assay using the G-LISA assay (Cytoskeleton). ROCK enzymatic activity was assessed by colorimetric method using the ROCK activity assay (Abcam) in protein extracts from quadriceps. The total amount of soluble collagen released by FAP was measured using the Sircol kit (Biocolor, UK). Mouse cytokines were measured using the Proteome Profiler Cytokine Array Panel A (R&D Systems, USA) (see Supplementary Information).

[0297] Proliferation and migration assays Proliferation assays were performed at 24 and 72 h according to the manufacturer's instructions (Roche, Indianapolis, IN). Migration assays were performed at 48 and 72 h using 24-well plates with inserts (Ibdi, Munich, Germany).

[0298] Western blot Cells or muscle tissues corresponding to each condition were lysed using RIPA buffer (Sigma-Aldrich) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland), and specific bands corresponding to proteins of interest were visualized using an Odyssey infrared detection system (Li-Cor) and Image Studio software (Li-Cor). Total protein bands were measured using a Revert 700 kit (Li-Cor) (Figure 1F). Primary and secondary antibodies used in this study are listed in Table 1.

[0299] Histology and immunofluorescence Cultured FAPs were fixed with ethanol and incubated in blocking solution (Santa Cruz Biotech, Dallas, TX, USA). Frozen muscle sections were obtained by cryostat (Leica Microsystems, Wetzlar, Germany), fixed with acetone, and incubated in blocking solution (Santa Cruz Biotech). Primary and secondary antibodies used in this study are listed in Table 2.

[0300] Images were acquired with an Olympus BX51 microscope connected to an Olympus DP72 camera. Image J software was used to quantitate the intensity of individual cells and the area of ​​positive staining in muscle tissue. At least five independent fields were quantified per staining.

[0301] Mouse model Animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Ethics Committee of the Universitat Autonoma de Barcelona. All animals used in this study were 7-week-old males. In accordance with previous studies, six dba / 2J-mdx male mice were orally administered fasudil (LC Laboratories) diluted in water by gavage at 100 mg / kg / day for 6 weeks. Five untreated dba / 2J-mdx male mice were used as controls and six dba / 2J male mice were used as healthy controls.

[0302] Grip strength test Grip strength was measured using a grip strength meter. Mice were first held by their tails over a grid. Once the mouse had grasped the grid, it was pulled out until it released its claws. This procedure was repeated five times and the average of the highest three values ​​was used. The average results were normalized to the animal's weight and expressed as grip strength / body weight (N / g).

[0303] Flow cytometry For flow cytometry, single cell suspensions from cell cultures, quadriceps and anterior tibiae were acquired. Anti-PDGFRα, followed by streptavidin-PECy5 and anti-CD56 were used to label cultured human muscle-derived cells. Anti-sca1, anti-CD45, anti-F4 / 80, anti-CD163 and viability markers were used to label cells acquired from mice (Table 1). Samples were acquired using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). Forward scatter area and height were used to exclude doublet cells. Compensation was adjusted according to single stained controls. Total numbers of viable FAPs (viable, Ter119- / CD45 / CD31- / integrin-α7- / sca1+) and macrophages (viable, CD45+F4 / 80+) were quantified using MACSQuantify software and normalized according to grams of muscle tissue. FlowJo v10 software was used for data analysis and data preparation.

[0304] statistical analysis Results are expressed as mean ± standard error of the mean (SEM). One-way ANOVA test was used to analyze differences between groups. Tukey post-hoc test was performed when ANOVA revealed significant differences. The significance level was set at p < 0.05. Statistical analysis was calculated using GraphPad Prism version 7 for Windows (San Diego, CA: GraphPad Software, Inc.).

[0305] Additional information regarding human tissue processing To obtain FAP cells, an open muscle biopsy of the left biceps brachii was performed in all patients. Muscle biopsies were obtained by linearly incising the skin along the longitudinal axis of the muscle fibers. Once the fascia was reached, the muscle fragment of interest was excised with a scalpel. The biopsy tissue was cleared from the adipose tissue and cut into pieces 1 mm thick. 2 The tissue was cut into pieces to prepare explants.

[0306] Muscle explants were cultured and subjected to immunomagnetic separation using a specific anti-CD56 antibody (Miltenyi Biotec, Bergisch-Gladbach, Germany). The negative fraction (CD56-) was cultured and its purity was verified by immunofluorescence (IF) and flow cytometry. IF was used to analyze the positive expression of the fibroblast marker TE-7 and the negative expression of desmin. Flow cytometry was used to analyze the expression of PDGFRα. Representative images of IF and the quantification of PDGFRα expression are shown in Figures 1A and 1B. Histograms obtained from the flow cytometry experiments are shown in Figure 1C. Stained cells were analyzed with a MACSQuant 10 (Miltenyi Biotec). Compensation was adjusted according to single stained controls. FlowJo v10 software was used for data analysis and data preparation.

[0307] More information on quantitative proteomics Protein extraction, quantification and digestion Protein samples were extracted with 6 M urea / 200 mM ammonium bicarbonate (ABC) and precipitated with cold acetone. The washed samples were dissolved in the aforementioned buffer and then quantified with the RCDC Protein Assay kit (Biorad, Hercules, CA). 10 μg of protein from each sample was digested in solution using both Lys-C and trypsin. Briefly, samples were reduced with 10 mM dithiothreitol (DTT, in 200 mM ABC) in a thermomixer at 30 °C and 650 rpm for 1 h and alkylated with 20 mM chloroacetamide (CAA, in 200 mM ABC) in a thermomixer at room temperature in the dark at 650 rpm for 30 min. Samples were then diluted to 2 M urea (final concentration) and the required amount of 1 μg / μl LysC (WAKO, Osaka, Japan) was added to achieve an enzyme:protein (w:w) ratio of 1:10. Digestion was performed overnight at 37 °C and 650 rpm in a thermomixer. Afterwards, samples were diluted to 1 M urea (final concentration). Finally, the required amount of 1 μg / μl trypsin (Promega, Madison, WI) was added to achieve an enzyme:protein (w:w) ratio of 1:10 and incubated for 8 h at 37 °C and 650 rpm in a thermomixer. The peptide mixture was desalted using a commercial Ultra Microspin C18, 300A silica column (The Nest Group, MA, USA) according to the manufacturer's instructions. Finally, samples were dried in a SpeedVac and stored at -20 °C until LC-MS / MS analysis.

[0308] LC-MS / MS analysis Peptide samples were analyzed using a Lumos Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) connected to an EASY-nLC 1000 (Thermo Fisher Scientific (Proxeon), Odense, Denmark). Peptides were loaded directly onto the analytical column and separated for 110 min by reversed-phase chromatography using a 50 cm column with an internal diameter of 75 μm packed with 2 μm C18 particle spectrometer (Thermo Fisher Scientific) with successive steps of a linear gradient from 5% to 22% B in 80 min, 22% to 32% B in 10 min, and 32% to 95% B in 20 min at a flow rate of 250 nL / min, and a binary solvent system of 0.1% formic acid in HO (solvent A) and 0.1% formic acid in acetonitrile (solvent B). After each analysis, the column was washed with 5% buffer A and 95% buffer B for 10 min.

[0309] The mass spectrometer was operated in data-dependent acquisition (DDA) mode, with detection in the Orbitrap using a resolution of 120000, 1 microscan full MS scan over a mass range of m / z 350-1500. Automatic gain control (AGC) was set to 2E5, and dynamic exclusion was set to 60 s. In each cycle of DDA analysis, after each survey scan, top speed ions with charges between 2 and 7 above a threshold ion number of 1e4 were selected for fragmentation at a normalized collision energy of 28%. Fragment ion spectra generated by higher energy collisional dissociation (HCD) were acquired in the ion trap, with AGC set to 3e4, an isolation window of 1.6 m / z, and a maximum injection time of 40 ms. All data were acquired using Xcalibur software v3.0.63.

[0310] Raw data processing and database searching Samples were analyzed using MaxQuant software (version 1.6.1.0) via the human Swissprot database. Trypsin was selected as the enzyme, allowing up to two missed cleavages. Carbamidomethylation (C) was set as a fixed modification, while oxidation (M) and acetylation (N-terminus) were used as variable modifications. Searches were performed using a peptide tolerance of 10 ppm and a product ion tolerance of 0.5 Da. The resulting data files were filtered to have an FDR < 1% at both the peptide and protein levels. Non-unique peptides were assigned to the corresponding protein groups according to the Razor peptide rules implemented in the software. The "match between runs" option was also enabled. Intensity values ​​were normalized using the LFQ algorithm.

[0311] Statistical and bioinformatic analyses The final protein list was analyzed using R. First, the protein list was filtered to remove peptides / proteins tagged as "Reverse" (significantly identified in the reverse database), "potential contaminant" (items identified as contaminants in the "contaminants.fasta" file), and "Only identified by site" (proteins identified only by modified peptides). Then, proteins with at least 75% validity values ​​in each experimental condition were selected. Missing values ​​were imputed using the knn algorithm. Comparisons between groups were performed using t-tests. p-values ​​were adjusted using the Benjamini-Hochberg procedure.

[0312] Total proteins analyzed were represented in a Volcano plot generated by Graphpad Prism software. A proteomap was performed using the upregulated proteins in PDGF-AA treated cells and generated to visualize the differential contribution of biological pathways (bionic-vis.biologie.uni-greifswald.de / , v2.0). The map was created from the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classification and shows the quantitative organization of the proteome. Each protein is represented by a polygon. The area reflects the abundance of the protein, and functionally related proteins are placed in areas of common similar color (34). The area of ​​each protein reflects the magnitude of the fold change in PDGF-AA treated cells compared to untreated cells. The upregulated proteins represented in the proteomap were subjected to pathway enrichment analysis of the Reactome database and represented in a heatmap generated by the Clustvis online tool (biit.cs.ut.ee / clustvis / ).

[0313] For more information on G-LISA, ROCK activity assays, Sircol and cytokine arrays: G-LISA RhoA-GTP protein was analyzed by colorimetric assay using the G-LISA assay (Cytoskeleton). The pathway was inhibited overnight with 2 μg / ml C3 exoenzyme (Cytoskeleton) inhibitor. The pathway was then activated with 50 ng / ml PDGF-AA for 20 min at 37°C and the G-LISA assay (Cytoskeleton) was performed according to the manufacturer's instructions. RhoA-GTP measurements were normalized to total RhoA amounts measured by Western blot.

[0314] ROCK activity assay The enzymatic activity of ROCK was assessed colorimetrically using the ROCK activity assay (Abcam) in protein extracts from quadriceps muscle. Experiments were performed according to the protocol provided by the manufacturer, and ROCK measurements were normalized to the total amount of protein used as determined by Western blotting.

[0315] Sircol A colorimetric assay was used to measure the total amount of soluble collagen released by the cultured FAPs. This test was performed using a commercially available Sircol kit, following the manufacturer's instructions (Biocolor, UK). Results were measured using a Coulter AD 340 plate reader (Beckam-Coulter, Brea, CA, USA) with the AD-LD program. At the end of the assay, nuclei were stained with TO-PRO-3 (Thermo Fisher Scientific) and the emitted signal was measured with an Odyssey reader (LI-COR). To subtract the effect of increased proliferation after PDGF-AA treatment, the signal emitted by TO-PRO-3 was used to normalize the results obtained with the Sircol assay. A representative image of TO-PRO-3 staining is shown in Supplementary Figure 1G.

[0316] Cytokine Array Relative expression of proinflammatory cytokines was measured by Proteome Profiler Array, Panel A (R&D Systems, USA) using 100 μg protein extracted from mouse quadriceps muscles according to the manufacturer's protocol. Three random samples from each mouse group were used for analysis. Quantitative analysis of blot spots was performed using Image Studio 5.2 (LI-COR, Lincoln, NE). Relative results were normalized to the DBA / 2J-WT group.

[0317] Additional information about proliferation and migration assays Proliferation assay Proliferation assays were performed at 24 and 72 h according to the manufacturer's instructions (Roche, Indianapolis, IN). Results were measured using a Coulter AD 340 plate reader with the AD-LD program (Beckam-Coulter, Brea, CA, USA).

[0318] Migration assay Cells were seeded in 24-well plates (Ibidi, Munich, Germany) with an insert separating the two chambers and treated with 25 μg / ml mitomycin C (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. After the corresponding treatment at 48 and 72 h, cells were fixed with 4% paraformaldehyde (PFA) and stained with Hoechst 33342. A total of three images were taken at 10x magnification for each well, and quantification of cells that migrated into the 500 μm space was performed with Fiji software.

[0319] Additional information about Western Blot Thirty micrograms of total protein was loaded onto a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Nonspecific binding sites were blocked with casein diluted 1:1 in Tris-buffered saline (TBS) for 1 h. Membranes were incubated overnight with primary antibodies corresponding to each experimental condition. Corresponding secondary antibodies conjugated to Dye680 or Dye800 (Li-Cor) were incubated at a 1:7500 dilution for 1 h (Table 1).

[0320] RNA extraction, reverse transcription and real-time quantitative PCR Total RNA was extracted from mouse quadriceps samples using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA was quantified using a nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA). 500 ng of total RNA was reverse transcribed into complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR (qPCR) was performed using TaqMan R Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All mRNA-specific FAM-labeled primers / probes were purchased from Applied Biosystems. The mRNA-specific probes used were Gapdh (Mm99999915_g1), Fhod1 (Mm00624707_m1), Rock2 (Mm01270843_m1), and Limk2 (Mm01187665_m1). Relative quantification was performed using the comparative Ct method, and all results were compared to control samples. GAPDH was used as an endogenous control.

[0321] [Table 1]

[0322] [Table 2(1)] [Table 2(2)]

[0323] array MLCK inhibitory peptide 18 (SEQ ID NO:1): RKKYKYRRK

Claims

1. A method for treating a disease or disorder associated with muscle fibrosis in a subject, comprising administering to the subject a therapeutically effective amount of a RhoA / ROCK pathway inhibitor.

2. 2. The method of claim 1, wherein the disease or disorder associated with muscle fibrosis is a muscular dystrophy.

3. The method of claim 1 or 2, wherein the disease or disorder associated with muscle fibrosis is a dystrophinopathy.

4. 4. The method of any one of claims 1 to 3, wherein the disease or disorder associated with muscle fibrosis is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, recessive and dominant limb-girdle muscular dystrophy, myotonic dystrophy type I, myotonic dystrophy type II, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, inclusion body myositis, distal myopathy with dystrophic changes and myofibrillar myopathy.

5. The method according to any one of claims 1 to 4, wherein the inhibitor inhibits the RhoA / ROCK pathway by directly inhibiting ROCK1 and / or ROCK2.

6. The inhibitors include fasudil, Y27632, Y39983, Wf-536, AR-13324, AR-12286, AMA0076, PG324, azabenzimidazole-aminofurazan, DE-104, olefins, isoquinolines, indazoles, pyridine alkene derivatives, H-1152P, ROKα inhibitors, XD-4000, HMN-1152, 4-(1-aminoalkyl) -N-(4-pyridyl)cyclohexane-carboxamide, quinazoline, ripasudil, VAS-012, Ki-23095, BA-2017, BA-215, BA-285, BA-1037, BA-210, Rhostatin, ROCK-IN-1-preclinical, ROCK inhibitor-2, ROCK2-IN-5, ROCK2-IN-2, ROCK-IN-2 (azaindole 1, TC-S 7001), Chroman 1, Cotosudil, SAR407899 hydrochloride, SAR407899, hydroxyfasudil, ripasudil, ripasudil free base, belmosudil mesylate, belmosudil, sobesudil, RKI-1447, H-1152, GSK269962A, Rho-Kinase-IN-1, SB-772077B dihydrochloride, HSD1590, BDP 5290, ZINC00881524, berosudil, LX7101, GSK-25, GSK180736A, CRT0066854, Y-27632, CMPD101, thiazovivin, SR-3677, and GSK429286A, and pharma- ceutically acceptable salts, solvates, or prodrugs thereof.

7. The inhibitor is a compound of formula (1) or a pharma- ceutically acceptable salt, solvate or prodrug thereof, 【Chemistry 1】 During the ceremony, A is one or more R １ is a monocyclic 5- to 7-membered heterocycloalkyl ring optionally substituted with Each R １ are independently １～６ Alkyl, Hydroxy-C １～６ Alkyl, halo-C １～６ Alkyl and -C(O)OR ２ is selected from R ２ is C １～６ is alkyl, 6. The method of any one of claims 1 to 5, optionally wherein the compound of formula (1) is fasudil.

8. 5. The method of any one of claims 1 to 4, wherein the inhibitor is selected from the group consisting of C3 exoenzyme, C3 transferase based, Rhosin, CCG-1423, CCG-203971, YS-49 monohydrate, YS-49, cerivastatin sodium, cerivastatin, Z62954982, Y16, MLS-573151, HA-100, HL07, DDO-5701, DDO-5713, DDO-5714, DDO-5715, DDO-5716, ML-7, MLCK18 and CT-04, and pharma- ceutically acceptable salts, solvates or prodrugs thereof.

9. The method of claim 8, wherein the inhibitor inhibits the RhoA / ROCK pathway by ribosylating the RhoA protein.

10. The method of claim 9 , wherein the inhibitor is C3 exoenzyme.

11. Use of a RhoA / ROCK pathway inhibitor in the treatment of a disease or disorder associated with muscle fibrosis.

12. The use according to claim 11, wherein the disease or disorder associated with muscle fibrosis is a muscular dystrophy.

13. 13. The use according to claim 11 or 12, wherein the disease or disorder associated with muscle fibrosis is a dystrophinopathy.

14. 14. The use according to any one of claims 11 to 13, wherein the disease or disorder associated with muscle fibrosis is selected from the group consisting of Duchenne muscular dystrophy, Becker muscular dystrophy, recessive and dominant limb-girdle muscular dystrophy, myotonic dystrophy type I, myotonic dystrophy type II, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy, inclusion body myositis, distal myopathy with dystrophic changes and myofibrillar myopathy.

15. The use according to any one of claims 11 to 14, wherein the inhibitor inhibits the RhoA / ROCK pathway by directly inhibiting ROCK1 and / or ROCK2.

16. The inhibitors include fasudil, Y27632, Y39983, Wf-536, AR-13324, AR-12286, AMA0076, PG324, azabenzimidazole-aminofurazan, DE-104, olefins, isoquinolines, indazoles, pyridine alkene derivatives, H-1152P, ROKα inhibitors, XD-4000, HMN-1152, 4-(1-aminoalkyl) -N-(4-pyridyl)cyclohexane-carboxamide, quinazoline, ripasudil, VAS-012, Ki-23095, BA-2017, BA-215, BA-285, BA-1037, BA-210, Rhostatin, ROCK-IN-1-preclinical, ROCK inhibitor-2, ROCK2-IN-5, ROCK2-IN-2, ROCK-IN-2 (azaindole 1, TC-S 7001), Chroman 1, Cotosudil, SAR407899 hydrochloride, SAR407899, hydroxyfasudil, ripasudil, ripasudil free base, belmosudil mesylate, belmosudil, sobesudil, RKI-1447, H-1152, GSK269962A, Rho-Kinase-IN-1, SB-772077B dihydrochloride, HSD1590, BDP5 290, ZINC00881524, berosudil, LX7101, GSK-25, GSK180736A, CRT0066854, Y-27632, CMPD101, thiazovivin, SR-3677, and GSK429286A, and pharma- ceutically acceptable salts, solvates or prodrugs thereof.

17. The inhibitor is a compound of formula (1) or a pharma- ceutically acceptable salt, solvate or prodrug thereof, 【Chemistry 2】 During the ceremony, A is one or more R １ is a monocyclic 5- to 7-membered heterocycloalkyl ring optionally substituted with Each R １ are independently １～６ Alkyl, Hydroxy-C １～６ Alkyl, halo-C １～６ Alkyl and -C(O)OR ２ is selected from R ２ is C １～６ is alkyl, Optionally, the compound of formula (1) is fasudil.

18. 15. The use according to any one of claims 11 to 14, wherein the inhibitor is selected from the group consisting of C3 exoenzyme, C3 transferase based, Rhosin, CCG-1423, CCG-203971, YS-49 monohydrate, YS-49, cerivastatin sodium, cerivastatin, Z62954982, Y16, MLS-573151, HA-100, HL07, DDO-5701, DDO-5713, DDO-5714, DDO-5715, DDO-5716, ML-7, MLCK18 and CT-04, and pharma- ceutically acceptable salts, solvates or prodrugs thereof.

19. The use according to claim 18, wherein the inhibitor inhibits the RhoA / ROCK pathway by ribosylating the RhoA protein.

20. 20. The use according to claim 19, wherein the inhibitor is C3 exoenzyme.

Images