Compositions and methods for inhibition of RHO kinase

Abstract

The present invention provides compositions and methods for treating a disease characterized by Rho-associated coiled-coil protein kinase (ROCK) activity in a subject by administering to the subject in need thereof an effective amount of laradazole or a derivative of laradazole, or a pharmaceutically acceptable salt thereof, in an amount and in a manner effective to inhibit Rho-associated ROCK activity in tissue.

Description

Field of the Invention

[0001] The present invention provides compositions, preparations and methods for treating and preventing diseases associated with Rho-associated coiled-coil kinase (ROCK) activity.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 308,330, filed on February 9, 2022, the entire contents of which are hereby incorporated by reference in their entirety.

[0004] Description of the electronically submitted text file

[0005] This application contains a sequence listing in XML format submitted electronically through Patent Center. The content of the XML copy was created on February 7, 2023, named "NMT-038PC_116031-5038.xml", and is 6,919 bytes in size. The sequence listing is incorporated herein by reference in its entirety. Background of the Invention

[0007] Rho-associated coiled-coil kinase (ROCK) is an effector of the RhoA small GTPase and plays a key role in promoting actomyosin cytoskeletal contractility downstream of RhoA and RhoC activation. ROCK kinases are essential for processes such as cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs, ROCK1 and ROCK2, have been implicated in various disease processes such as cardiovascular disease, vascular injury, fibrosis, inflammatory bowel disease, and cancer.

[0008] There is a need for pharmaceutical compositions and methods for inhibiting ROCK, and treating or preventing ROCK-related medical diseases. Summary of the invention

[0009] The present invention relates in part to compositions and methods useful for treating Rho-associated coiled-coil kinase (ROCK)-related diseases.

[0010] In various aspects, the present invention provides a method for treating a disease characterized by Rho-associated coiled-coil protein kinase (ROCK) activity in a subject. The method comprises administering to the subject an effective amount of larazotide or a larazotide derivative or a pharmaceutically acceptable salt thereof in an amount and manner effective to inhibit ROCK activity in an organ or tissue. In various embodiments, the organ or tissue is selected from the gastrointestinal tract, cancerous tissue, eye, respiratory tract, and vascular system.

[0011] In some embodiments, larizolide or its derivatives are applied to the gastrointestinal tract. In such embodiments, the subject may suffer from a disease selected from: cancer (e.g., GI cancers such as colorectal cancer or gastric cancer, or non-GI cancers), adenoma, inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), celiac disease, esophagitis, inflammatory liver disease, nephropathy, pancreatitis, hyperglycemia, diabetes, and lung or heart inflammation or fibrosis. In certain embodiments, the subject suffers from colorectal cancer or has a risk of colorectal cancer. For example, the subject's family history, genetic mutations, and / or health history can increase the risk of colorectal cancer in the subject.

[0012] In other embodiments, larizolide or derivatives are delivered directly to cancer tissue, which may be a primary tumor or a metastatic tumor. For example, the subject may suffer from a cancer selected from the following: lung cancer, breast cancer, kidney cancer, liver cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, melanoma, ovarian cancer, bone cancer, urothelial cancer (urothelial cancer), gastric cancer, head and neck cancer, glioblastoma, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, hormone-refractory prostate cancer and lymphoma. In some embodiments, the tissue is a metastatic cancer, such as metastatic melanoma. In some embodiments, the cancer is a sarcoma (sarcoma) or a carcinoma (carcinoma).

[0013] In other embodiments, larizolide or a derivative is administered to the eye. For example, the subject may have an eye disease selected from Sjogren's syndrome, dry eye syndrome, age-related macular degeneration (AMD), macular edema, diabetic retinopathy, and glaucoma.

[0014] In some embodiments, larizolide or a derivative is administered to the respiratory tract. For example, the subject may be suffering from a disease selected from asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, cystic fibrosis, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), emphysema, bronchitis, pneumonia, lung cancer, and respiratory tract infection.

[0015] In other embodiments, larizolide or a derivative is administered to the vasculature or systemically. For example, the subject may be suffering from a disease selected from cardiac fibrosis, cardiac hypertrophy, hypertension, pulmonary hypertension, angina pectoris, vasospastic angina, heart failure, atherosclerosis, arteriosclerosis, diabetes, pancreatitis, nephropathy (including renal fibrosis) and stroke (e.g., in prevention or stroke or stroke recovery).

[0016] In various embodiments, the larazotide derivative comprises one or more modifications that increase the ROCK inhibitor activity compared to larazotide. For example, in some embodiments, the larazotide derivative comprises at least one, at least two, at least three, at least four, at least five (d)-amino acids. In certain embodiments, each amino acid (except Gly) of the larazotide derivative is a (d)-amino acid, and the derivative is optionally retro-inversolarazotide.

[0017] Other aspects and embodiments of the invention will be apparent from the following detailed description and working examples.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 AB shows the assessment of paracellular permeability during anoxia / reoxygenation (A / R) injury in C2BBe1 enterocytes treated with larazol acetate (LA). Figure 1 A shows a schematic diagram of the timeline of TEER measurements in LA treatment and A / R injury. Figure 1 B shows that treatment with 10 mM LA significantly (p<0.001) increased TEER compared to untreated A / R injured cells.

[0020] Figure 2 Showing that hypoxia causes myosin to become phosphorylated, leading to internalization of the tight junction protein occludin. The figure shows how LA is predicted to prevent this phosphorylation.

[0021] Figure 3 Shown are the distribution of TJ proteins analyzed by immunoblotting and IF analysis of membrane and cytoplasmic fractions after 1 h of reoxygenation. Figure 3 The results showed that the transmembrane protein occludin was significantly (p<0.05) internalized in hypoxic injured cells compared to control cells. However, 10 mM LA significantly (p<0.05) increased occludin in the membrane compared to untreated hypoxic injured cells.

[0022] Figure 4Shown is an assessment of the localization of tight junction proteins and cytoskeletal proteins by immunofluorescence microscopy analysis at 1 hour oxygenation. The permeable support membrane was fixed and stained for ZO-1 (red), occludin (green) and F-actin (purple). The immunolocalization of ZO-1 and occludin was analyzed by Z stack 3-D analysis. Untreated hypoxic injured cells showed destruction of occludin, ZO-1 and F-actin, while cells treated with larazol acetate (LA) showed well-organized tight junction proteins and F-actin.

[0023] Figure 5 The pMLC-2 / MLC-2 ratio assessed by immunoblotting at the 1 hour reoxygenation time point is shown. The expression of pMLC-2 increased dramatically after A / R injury and was significantly reduced by pretreatment with 10 mM LA (##p<0.01).

[0024] Figure 6 The authors show that MLC phosphorylation is regulated to modulate the TJ barrier of intestinal epithelial cells. The TJ barrier is primarily determined by the phosphorylation level of regulatory myosin light chain (MLC), which in turn is regulated by two enzymes, myosin light chain kinase (MLCK) and rho-associated coiled-coil protein kinase (ROCK). Intracellular Ca 2+ Increased levels stimulate MLCK activity. Enhanced Rho kinase (ROCK) activity also directly phosphorylates MLC and inhibits myosin light chain phosphatase (MLCP) activity by phosphorylating myosin phosphatase target subunit 1 (MYPT1).

[0025] Figure 7 The results show that inhibition of MLCK and ROCK with or without larezolide increased TEER compared to untreated A / R injured cells. Pep 18 (MLCK inhibitor) treatment alone or in combination with LA increased TEER. Monolayers treated with Fasudil (ROCK inhibitor) showed an increase in TEER. Combination of Fasudil and LA increased TEER, but to a lesser extent than Fasudil alone (* <p0.05,**p<0.01,***p<0.005,****p<0.0001)。

[0026] Figure 8 Possible mechanisms for regulating the phosphorylation of MLC to regulate the paracellular actin-myosin ring in intestinal epithelial cells are shown. The TJ barrier is mainly determined by the phosphorylation level of regulatory myosin light chain (MLC), which is regulated by two enzymes, myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). These mechanisms can be mediated by factors such as Ca 2+It regulates multiple cellular pathways including homeostasis, G protein-coupled receptor (GPCR) and growth factor (GF) pathways.

[0027] Fig. 9A -B shows differential expression analysis using next generation RNA sequencing. Fig. 9A A Venn diagram analysis of the number of differentially expressed genes (DEGs) identified is shown. Fig. 9B Hierarchical clustering of DEGs is shown for evaluating expression profiles under different experimental conditions.

[0028] Fig.10 Gene Ontology (GO) functional classification analysis of DEGs from different combinations of groups is shown. It includes three main branches: biological process, cellular component and molecular function. GO terms with padj < 0.05 are significantly enriched. Fig.10 Twenty significantly enriched biological processes, cellular components, and molecular functions were shown.

[0029] Fig.11 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showing the enrichment of DEGs from different combinations of groups. KEGG pathway terms with padj < 0.05 were significantly enriched.

[0030] Fig.12 Evaluation of paracellular permeability in C2BBel enterocytes is shown. Treatment with 10 mM LA significantly increased TEER 1 h after treatment compared to untreated A / R injured cells.

[0031] Fig.13 Evaluation of C2BBel enterocyte proliferation is shown. Treatment with 10 mM LA significantly increased proliferation compared to untreated cells.

[0032] Fig.14 AB shows the assessment of C2BBel enterocyte proliferation and migration. Fig.14 A shows that cells treated with 10 mM LA in regular culture medium showed significantly improved wound healing at 48 hours. Fig.14 B shows that no significantly different migration patterns were detected between cells treated with 10 mM LA and untreated cells in serum-free medium. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides compositions and methods for treating a disease characterized by Rho-associated coiled-coil protein kinase (ROCK) activity in a subject by administering to a subject in need thereof an effective amount of larizolide or a larizolide derivative or a pharmaceutically acceptable salt thereof in an amount and manner effective to inhibit ROCK activity in a tissue.

[0035] The present disclosure shows that larizolide and larizolide derivatives or pharmaceutically acceptable salts thereof inhibit rho-associated protein kinase (ROCK). Inhibition of ROCK can provide therapeutic benefits for subjects suffering from diseases associated with ROCK activity. ROCK and its downstream targets are involved in regulating actin cytoskeleton dynamics and are therefore responsible for cell migration and movement. In addition, they are also involved in a variety of biological processes, such as cell junction integrity, cell cycle control and apoptosis.

[0036] ROCK enzymes have two isoforms: ROCK1 and ROCK 2. Both kinases contain a catalytic kinase domain at the N-terminus, followed by a central coiled-coil protein domain including a Rho-binding domain (RBD), and a pleckstrin-homology (PH) domain at the C-terminus.

[0037] The peptide agent called larizolide has the amino acid sequence Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO: 1). Larizolide promotes the tight junction integrity of epithelial and endothelial tissues (including intestinal epithelium) and is being evaluated as a therapeutic agent for patients with celiac disease (CeD). According to certain aspects and embodiments, the present invention provides larizolide derivatives that confer enhanced resistance to exopeptidase degradation, including aminopeptidase degradation, etc. In various embodiments, the larizolide derivatives comprise one or more amino acid substitutions, deletions and / or insertions relative to a peptide having an amino acid sequence of SEQ ID NO: 1. Exemplary modifications are described in US 8,785,374, US 8,957,032, US 9,279,807, PCT / US2019 / 19350, and PCT / US21 / 27410 (all of which are incorporated herein by reference).

[0038] In some embodiments, the peptide derivative of larizoline contains one or more (d) amino acids. For example, the larizoline derivative can contain 1, 2, 3, 4 or 5 (d) amino acids (i.e., D configuration, as opposed to L configuration). In some embodiments, the larizoline derivative has an amino acid sequence of Gly-Gly-Val-Leu-Val-Gln-(d)Pro-Gly (SEQ ID NO: 2). The peptide is referred to herein as "(d)-Pro" or (d)-Pro larizoline. In other embodiments, the larizoline derivative has an amino acid sequence of Gly-Gly-(d)Val-(d)Leu-(d)Val-(d)Gln-(d)Pro-Gly (SEQ ID NO: 3). The peptide is referred to herein as "(d)-larizoline". As demonstrated herein, (d)-larizoline is surprisingly effective in promoting tight junction integrity at significantly lower concentrations and higher concentrations compared to larizoline (which shows a bell-shaped dose-response curve). This is a surprising result because typically substitution of L amino acids with D amino acids in peptide drugs results in a loss of potency. That is, peptides with D amino acids are expected to have lower binding affinity to receptor function than peptides with native L amino acids.

[0039] In various embodiments, compared with larizoline, the larizoline derivatives used in the present invention are more effective at significantly lower doses or high doses, especially for gastrointestinal administration. Therefore, the pharmaceutical composition of the present invention may contain less than about 0.5 mg of larizoline derivatives. For example, in some embodiments, the pharmaceutical composition contains about 0.4 mg or less of larizoline derivatives, or about 0.3 mg or less of larizoline derivatives, or about 0.25 mg or less of larizoline derivatives, or about 0.2 mg or less of larizoline derivatives, or about 0.15 mg or less of larizoline derivatives, or about 0.1 mg or less of larizoline derivatives, or about 50 μg or less of larizoline derivatives, or about 25 μg or less of larizoline derivatives. In some embodiments, the pharmaceutical composition contains about 50 μg to about 400 μg or less of a larizolide derivative, or about 50 μg to about 200 μg or less of a larizolide derivative, or about 50 μg to about 150 μg or less of a larizolide derivative.

[0040] In other embodiments, the present invention relates to pharmaceutical compositions containing more than about 0.5 mg of a larizolide derivative and substantially avoiding the inverse dose or "bell-shaped" response observed with larizolide. For example, in some embodiments, the pharmaceutical composition contains about 0.6 mg or more of a larizolide derivative, or about 0.75 mg or more of a larizolide derivative, or about 1.0 mg or more of a larizolide derivative, or about 1.25 mg or more of a larizolide derivative, or about 1.5 mg or more of a larizolide derivative, or about 2.0 mg or more of a larizolide derivative.

[0041] In some embodiments, the larizolide derivative (eg, (d)-larizolide or (d)-Pro) is administered at about 0.5 mg. For example, the derivative can be more effective than larizolide at a 0.5 mg dose.

[0042] The present invention provides methods and compositions for treating diseases related to ROCK activity in subjects in need thereof by applying a composition comprising larizoline or a larizoline derivative to a tissue having ROCK activity so as to inhibit ROCK activity in the tissue. The terms "subject" and "patient" are used interchangeably herein and generally refer to mammalian subjects / patients. In various embodiments, the subject is a human subject. Therefore, the composition can be formulated and / or delivered by various routes to inhibit ROCK activity in a target organ or tissue. In various embodiments, a pharmaceutical composition comprising larizoline or a derivative thereof (or its salt) is applied to, for example, the gastrointestinal tract (GI) (e.g., enteral delivery), or delivered parenterally, intranasally, in the cheek, in the eye, or in the lungs. Delivery can be local delivery to the affected tissue or systemic delivery.

[0043] In some embodiments, the peptide or pharmaceutical composition is administered to the gastrointestinal tract (GI) of a subject.

[0044] In some embodiments, the subject has an inflammatory disease or injury to the gastrointestinal tract, such as celiac disease, inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), environmental enteropathy, esophagitis, necrotizing enterocolitis, and intestinal ischemia. In some embodiments, the subject has an adenoma (e.g., advanced adenoma), colorectal cancer, or gastric cancer. ROCK activity has been shown to be associated with tumor development. Wei L., et al., Novel Insights into the Roles of Rho Kinase in Cancer , Arch Immunol Ther Exp (Warsz). 2016; 64: 259–278. In some embodiments, the subject has a cancer that can originate from any tissue, such as from the skin, colon, breast, lung, brain, bone, pancreas, kidney, liver, bladder, ovary, testis, or prostate.

[0045] In other embodiments, the subject has a leukemia, myeloma, or lymphoma.

[0046] In some embodiments, the subject has the risk of colorectal cancer. For example, in some embodiments, due to the family history, genetic mutation and / or health history (for example, a history of advanced adenoma or colorectal cancer) of the subject, the subject has an increased risk of colorectal cancer. In some embodiments, the subject or a family member of the subject suffers from or has suffered from familial adenomatous polyposis (FAP), hereditary non-polyposis colorectal cancer (HNPCC), Peutz-Jeghers syndrome or MUTYH associated polyposis (MAP). In some embodiments, the subject has one or more mutations in genes that increase the risk of colorectal cancer, such as, but not limited to, APC, MLH1, MSH2, MSH6, PMS2, EPCAM, STK11 (LKB1) and MUTYH. For example, without limitation, the experimenter can have one or more single nucleotide polymorphisms (SNPs) selected from rs6983267, rs4939827, rs3802842, rs16892766, rs10795668, rs4444235, rs10411210, rs6691170, rs4925386, rs3824999, rs647161, rs2423279, rs3217810 and rs59336. In some embodiments, the experimenter has experienced long-term antibiotic use. For example, long-term antibiotic use includes using the antibiotic regimen of at least 6 months, which can increase the risk of CRC.

[0047] In some embodiments, the subject has a ROCK mutation associated with cancer development. Exemplary such mutations include Val1309, Tyr405, Ser1126, Pro1193S relative to the ROCK1 isoform, or Thr431Asn, Asp601Val and Lys1083Met relative to the ROCK2 isoform.

[0048] In some embodiments, the subject is receiving or has received one or more cancer treatments selected from chemotherapy, radiotherapy, resection and immunotherapy and immuno-oncology agents. In some embodiments, cancer immunotherapy is the treatment of using immune checkpoint inhibitors or immunostimulatory ligands, including but not limited to inhibitors of the following molecules: programmed death ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, IDO1, IDO 2. T cell stimulating ligands include but are not limited to agonists of CD28, OX-40 and ICOS. In certain embodiments, the subject receives an immune checkpoint inhibitor selected from anti-CTLA-4, anti-PD-1, anti-PD-L1 and / or PD-L2 agents. In some embodiments, immune checkpoint inhibitors are selected from ipilimumab, tremelimumab, pembrolizumab and nivolumab.

[0049] In other embodiments, the subject suffers from inflammatory liver disease, nephropathy, pancreatitis, hyperglycemia or lung or heart inflammation or fibrosis.For example, in some embodiments, the subject suffers from fatty liver disease, including but not limited to NAFLD, NASH, alcoholic steatohepatitis (ASH), or fatty liver disease caused by hepatitis, obesity, diabetes, insulin resistance, hypertriglyceridemia, chronic kidney disease, IgA nephropathy (also referred to as Berger's disease), abetalipoproteinemia, glycogen storage disease, Weber-Christian disease, Wolmans disease, acute fatty liver of pregnancy and lipodystrophy.See US 2019 / 0358289 and US2021 / 0069286, which are incorporated herein by reference in their entirety.

[0050] In some embodiments, the subject has a disease involving organ fibrosis, such as pulmonary fibrosis, cardiac fibrosis, renal fibrosis, or liver fibrosis. Knipe R., et al. The Rho Kinases:Critical Mediators of Multiple ProfibroticProcessesandRational Targets for New Therapies for Pulmonary Fibrosis , Pharmacol Rev. 2015 Jan; 67(1): 103–117. In some embodiments, the subject suffers from a disease associated with pulmonary fibrosis, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), emphysema, bronchitis, asthma, pneumonia, and respiratory tract infection.

[0051] In other embodiments, larizoline or a derivative is administered directly to cancer tissue, including but not limited to intratumoral administration or by encapsulation or binding to nanoparticles or other means. ROCK enzymes play a role in multiple processes of cancer progression, such as tumor invasion and metastasis, proliferation and apoptosis or survival, and affect cancer and cancer-related cells, such as fibroblasts and endothelial cells.

[0052] In some embodiments, tissue is primary cancer.Primary cancer refers to a cancer cell that becomes clinically detectable at the origin site, and can be a primary tumor.For example, cancer can be Phase I or Phase II cancer.In some embodiments, tissue is primary cancer, and cancer is selected from lung cancer, breast cancer, kidney cancer, liver cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, melanoma, ovarian cancer, bone cancer, urothelial carcinoma, gastric cancer, head and neck cancer, glioblastoma, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, hormone refractory prostate cancer and lymphoma.

[0053] In some embodiments, the tissue is a metastatic cancer. "Metastasis" refers to the spread of cancer from the primary site to other parts of the body. Cancer cells can break away from the primary tumor, infiltrate lymphatics and blood vessels, circulate through the bloodstream, and grow (metastasize) at a distance in normal tissues in other parts of the body. Metastasis can be local or remote. In some embodiments, the tissue is a metastatic cancer, such as a metastatic melanoma. In various embodiments, the cancer suffered by the patient is a sarcoma or a carcinoma.

[0054] In some embodiments, the subject is receiving or has received one or more cancer treatments selected from chemotherapy, radiotherapy, resection and immunotherapy and immuno-oncology agents. In some embodiments, cancer immunotherapy is the treatment of using immune checkpoint inhibitors or immunostimulatory ligands, including but not limited to inhibitors of the following molecules: programmed death ligand 1 (PD-L1, also known as B7-H1, CD274), programmed death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, IDO1, IDO 2. T cell stimulating ligands include but are not limited to agonists of CD28, OX-40 and ICOS. In certain embodiments, the subject receives an immune checkpoint inhibitor selected from anti-CTLA-4, anti-PD-1, anti-PD-L1 and / or PD-L2 agents. In some embodiments, immune checkpoint inhibitors are selected from ipilimumab, tremelimumab, pembrolizumab and nivolumab.

[0055] In some embodiments, larizolide or a derivative is administered to the eye, such as to the surface of the eye, or by intravitreal injection.

[0056] In some embodiments, the subject has Sjogren's syndrome (SS). Sjogren's syndrome is an autoimmune disease in which the body's water-producing glands are affected, resulting in symptoms such as dry mouth (xerostomia), dry eye syndrome (e.g., chronic dry eyes and / or xerosis), and / or dry skin (e.g., xerosis), among others. According to some embodiments of the invention, Sjogren's syndrome may be primary Sjogren's syndrome or secondary Sjogren's syndrome, i.e., occurring in association with another autoimmune or connective tissue disease. The inflammation caused by SS gradually damages the glands and is characterized by lymphocyte infiltration within the glands, elevated levels of B cell activating factor (BAFF), and production of autoantibodies (e.g., anti-SSA / Ro). See Nair JJ and Singh TP, Sjogren'ssyndrome:Review of the etiology pathophysiology&potential therapeutic interventions .J.Clin.Exp.Dent.2017;9(4):e584-9. In some exemplary embodiments, larizolide or a derivative is administered to the ocular surface for the treatment of primary or secondary Sjogren's syndrome.

[0057] In some embodiments, the subject suffers from glaucoma. ROCK inhibitors are promising treatment options, such as reducing intraocular pressure (IOP) in glaucoma. For example, in some embodiments, glaucoma is treated by applying larazol or a derivative thereof (or a salt thereof) to the ocular surface.

[0058] In other embodiments, the subject suffers from an inflammatory eye disease, such as macular degeneration, macular edema or diabetic retinopathy. Age-related macular degeneration (AMD) is a blinding eye disease that gradually increases with age. Inflammation is involved in the pathogenesis of AMD, including choroidal neovascularization and geographic atrophy. In some embodiments, larizolide or its derivatives are administered to the back of the eye by intraocular administration (e.g., intravitreal injection).

[0059] In some embodiments of the invention, larizoline or a derivative is administered to the respiratory tract. See US10,723,763, U.S. Provisional Application No. 63 / 181,486 and PCT / US21 / 2741, all of which are incorporated herein by reference. In some embodiments, the subject suffers from a disease selected from chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, cystic fibrosis, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), emphysema, bronchitis, asthma, pneumonia, lung cancer and respiratory tract infection.

[0060] In some embodiments, larizolide or a larizolide derivative is formulated for administration to the lungs as a solution aerosol or powder.

[0061] In other embodiments, larizolide or a larizolide derivative is administered to the nasal epithelium as a nasal solution or nasal emulsion.

[0062] In some embodiments, larizoline or a derivative is administered to the vasculature. For example, larizoline or a derivative can be administered systemically, or applied topically by a catheter. For example, the subject may suffer from a disease selected from cardiac fibrosis, cardiac hypertrophy, hypertension, pulmonary hypertension, angina pectoris, vasospastic angina, heart failure, atherosclerosis, arteriosclerosis, diabetes, pancreatitis, nephropathy (including renal fibrosis) and stroke (e.g., in the prevention or ischemic stroke or stroke recovery).

[0063] The pharmaceutical compositions provided herein can be formulated to release in the diseased part of the GI (e.g., stomach, small intestine and / or large intestine). In other embodiments, larizolide or derivatives are systemically administered (e.g., intravenous or subcutaneous injection). In some embodiments, the peptide composition is applied to the lungs in the form of a solution aerosol or powder. In some embodiments, the peptide composition is applied to the nasal epithelium as a nasal solution or nasal emulsion. In some embodiments, the peptide composition is applied to the oral cavity or esophagus as a liquid or orally disintegrating tablet. In some embodiments, the peptide composition is applied to the ocular surface or intraocular.

[0064] The larazolid derivatives of the present invention can be administered in any suitable form including salts. For example, the peptide can be administered as an acetate. Alternative salts can be used, including any pharmaceutically acceptable salts, such as those listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. PHStahl and CG Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are incorporated herein by reference in their entirety.

[0065] In various embodiments, the peptides are formulated into pharmaceutical compositions, which can take the form of tablets, pills, pellets, capsules, liquid-containing capsules, multi-particulate-containing capsules, powders, solutions, emulsions, drops, suppositories, emulsions, aerosols, sprays, suspensions, delayed-release formulations, sustained release formulations, modified release formulations, controlled release formulations, or any other form suitable for use.

[0066] In some embodiments, the pharmaceutical composition is formulated into a composition suitable for parenteral administration. The dosage form suitable for parenteral administration (e.g., intravenous, intramuscular, subcutaneous or intraperitoneal injection and infusion) includes, for example, solutions, suspensions, dispersions, emulsions, etc. They can also be made into the form of sterile solid compositions (e.g., lyophilized compositions), which can be dissolved or suspended in a sterile injectable medium immediately before use. They can contain, for example, suspending agents or dispersants. In these embodiments, the composition can effectively treat diseases involving systemic inflammation or damaged or inflamed endothelial tissue.

[0067] In some embodiments, the composition is applied to the subject by contacting the epithelial tissue or mucosal surface of the gastrointestinal tract. For example, the composition can be formulated for delivery to one or more of the stomach, small intestine and / or large intestine. By targeting the peptide release in diseased areas (e.g., duodenum, jejunum and ileum, transverse colon, descending colon, ascending colon, sigmoid colon and cecum), ROCK inhibition at any position of the GI can be achieved. The targeted delivery of peptides in the small intestine or large intestine can be achieved by coating beads or particles with peptides and delaying release coatings (preventing release in the stomach and degradation at or near the target location).

[0068] In some embodiments, larizolide or a larizolide derivative is administered in the form of a sustained release or controlled release formulation that releases about 0.5 to about 5 mg of larizolide or a derivative in the intestine. In certain embodiments, the controlled release formulation contains at least 0.5 or 1 mg of larizolide or a derivative.

[0069] In some embodiments, peptides (e.g., larizolide or larizolide derivatives) are formulated for sustained or improved or controlled delivery in one or more locations of the GI. For example, the present invention includes a sustained or controlled release formulation that can functionally release peptides in the small intestine and / or large intestine within a time of at least about 2 hours, or at least about 2.5 hours, or at least about 3 hours, or at least about 4 hours, or at least about 5 hours. In some embodiments, sustained release or controlled release compositions begin to release peptides within about 10 to about 30 minutes of exposure to simulated intestinal fluid, and the release of peptides exposed to simulated intestinal fluid lasts for at least about 180 minutes, or at least about 210 minutes, or at least about 240 minutes, or at least about 280 minutes. For example, a composition with different enteric polymer coatings and / or polymer coatings of different thicknesses can be used to prepare a release profile. In some embodiments, the present invention provides a composition comprising an effective amount of larizolide or a derivative (or its salt), which is contained in a biodegradable or erodible polymer matrix and further comprises an enteric coating. Preparations using biodegradable or erodible matrices are described in WO 2021 / 034629, which is incorporated herein by reference in its entirety. In addition, erodible polymer matrices may include polysaccharide matrices. In some embodiments, the matrix includes one or more of the following: cellulose, chitin, chitosan, alginate, amylose, pectin, callose, laminarin, golden algae laminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, xanthan gum, glucan, welangum, gellan gum, diutan gum, pullulan, hyaluronic acid and derivatives thereof. In a further embodiment, the matrix includes microcrystalline cellulose. In these embodiments, the composition utilizes a low effective dose of larizoline or a larizoline derivative (e.g., (d)-larizoline or (d)-Pro), while also minimizing any local accumulation of inactive fragments. Furthermore, the formulations in these embodiments have the benefit of treating a large surface area of ​​the GI tract with a small dose of peptide that is continuously deposited during transit.

[0070] In various embodiments, the pharmaceutical composition can be formulated to have a delayed release profile, i.e., the active ingredient is not released immediately after ingestion; instead, the release of the active ingredient is delayed until the peptide passes through the stomach and is located in the lower gastrointestinal tract; for example, for release in the small intestine (e.g., one or more of the duodenum, jejunum, ileum) or the large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, or sigmoid colon). In one embodiment, the pharmaceutical composition is formulated to have a delayed release profile, as described, for example, in U.S. Pat. No. 8,168,594, the entire contents of which are incorporated herein by reference.

[0071] For example, the peptide can be administered to at least the duodenum of a patient in the form of an oral dose delayed release composition containing the peptide. In such embodiments, the composition includes a first bead group having a coating that is stable in gastric fluid and unstable in intestinal fluid, thereby degrading and significantly releasing the peptide in the duodenum. The composition may also include a second bead group having a pH-dependent coating to affect the release of the peptide in the jejunum and / or ileum of the patient. For example, the second bead group may release the peptide about 30 minutes or about 45 minutes after the release of the beads that release the peptide in the duodenum. The oral dosage composition may be in the form of a capsule or tablet. In some embodiments, the pH-dependent coating is a 1:1 copolymer of methacrylic acid and ethyl acrylate, wherein the thickness of the layer determines the release characteristics of each bead. The beads may have one or more additional coatings, such as a base coating, a separating layer, and an outer coating layer. In these embodiments, the contents of the beads will be released in a more concentrated manner at the target location, but the properties of (d)-larezole or (d)-Pro will be more effective than larezole with such release characteristics.

[0072] In an exemplary oral dosage composition, an effective amount of a peptide (e.g., in acetate form) is provided in a first delayed-release particle capable of releasing the peptide in the duodenum of a patient, and in a second delayed-release particle capable of releasing the peptide in the jejunum of a patient. Each particle has a core particle, a coating comprising a peptide (e.g., (d)-larazolane or (d)-Pro) on the core particle, and a delayed-release coating (e.g., a 1:1 copolymer of acrylate and methacrylate) outside the coating comprising the peptide. The first delayed-release particle releases at least 70% of the peptide in the first delayed-release particle at about 60 minutes of exposure to a simulated intestinal fluid having a pH greater than 5; the second delayed-release particle releases at least 70% of the peptide at about 30 and about 90 minutes of exposure to a simulated intestinal fluid having a pH greater than 5.

[0073] Typically, delayed release coatings can degrade over time, regardless of the presence of pH and / or enzymes. Such coatings may include, for example, water-insoluble polymers. Therefore, their solubility is independent of pH. The term "independent of pH" as used herein refers to that the permeability of the polymer and its ability to release a drug ingredient do not vary with pH and / or only very slightly depend on pH. Such coatings can be used to prepare, for example, sustained release formulations. Suitable water-insoluble polymers include, but are not limited to, cellulose ethers, cellulose esters, or cellulose ether-esters, i.e., cellulose derivatives in which some hydroxyls on the cellulose backbone are substituted with alkyl groups and some hydroxyls are modified with alkanoyl groups. Examples include ethyl cellulose, acetyl cellulose, nitrocellulose, etc.

[0074] Other examples of polymers for constructing delayed-release coatings include, but are not limited to, lacquer, ester polymers of acrylic acid and / or methacrylic acid, polymers or copolymers of acrylate or methacrylate with low quaternary ammonium content, or mixtures thereof, etc. Examples of insoluble polymers include and Insoluble polymers include, for example, polyvinyl esters, polyvinyl acetals, polyacrylates, butadiene styrene copolymers, and the like.

[0075] Various types of enteric coatings are known for delayed but substantial delivery of active agents to the GI tract. In some embodiments, the sustained release composition comprises an enteric solvent that is substantially stable in an acidic environment and substantially unstable in a near neutral to alkaline environment. In one embodiment, the sustained release coating comprises an enteric solvent that is substantially stable in gastric fluid. The enteric solvent can be selected from, for example, methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, polyvinyl acetate phthalate, carboxymethyl ethyl cellulose and A solution or dispersion of a type polymer (poly(methacrylic acid, methyl methacrylate), hydroxypropyl methylcellulose acetate succinate, cellulose acetate trimellitate, shellac or other suitable enteric coating polymer. Type polymers include, for example FS 30D, L 30D-55, L 100-55, L 100, L 12,5, L 12,5P, RL 30D, RL PO, RL 100, RL 12,5, RS 30D, RS PO, RS100, RS12,5, NE 30D, NE 40D, NM 30D, S100, S12,5, and S12,5P. In some embodiments, the use of In some embodiments, the enteric solvent is EUDRAGIT F30D, which comprises a copolymer of methyl acrylate, methyl methacrylate and methacrylic acid. The ratio of free carbonyl groups to ester groups of the copolymer is about 1:10.

[0076] In some embodiments, the beads are included in an enteric coating that can resist dissolution in simulated gastric fluid. The composition remains intact in gastric fluid, or may be substantially insoluble. The stability of the gastric resistant coating may depend on pH. For example, the enteric coating may prevent the peptide from being released in large quantities in simulated gastric fluid and simulated intestinal fluid of pH about 5.5. In some embodiments, the matrix provides a sustained release of the peptide in simulated intestinal fluid of pH about 6 or higher (e.g., about 6.5 to about 7.0). Therefore, the enteric coating is stable in simulated gastric fluid, but unstable in simulated intestinal fluid of pH higher than about 6.0. In such an embodiment, the enteric coating does not release the peptide substantially in the duodenum, but delays release until the composition enters the jejunum, and then provides sustained release in the jejunum and ileum.

[0077] In some embodiments, the composition is a capsule for oral delivery comprising a bead population comprising an effective amount of larizolide or a derivative (e.g., (d)-larizolide or (d)-Pro or a salt thereof) contained in an erodible polymer matrix, the beads further comprising an enteric coating, which may comprise a copolymer of methacrylate, methyl methacrylate, and methacrylic acid. The ratio of free carbonyl groups to ester groups in the copolymer may be about 1:10 (e.g., EUDRAGIT F30D). In such embodiments, the enteric coating may account for about 20% to about 30% of the total weight of the composition. In some embodiments, the erodible matrix comprises microcrystalline cellulose. In some embodiments, the composition releases less than about 15% of the peptide after about 2 hours in simulated gastric fluid. In addition, the composition releases less than about 25% of the peptide after about 2 hours in simulated intestinal fluid at a pH of about 5.5. In various embodiments, the composition releases at least about 40% but no more than about 80% of the peptide after about 2 hours in simulated intestinal fluid at a pH of about 7.0. In various embodiments, 100% release is not achieved until at least three hours, or in some embodiments, until at least about 3.5 hours or at least about four hours in simulated intestinal fluid at a pH of about 7.

[0078] In some embodiments, the pharmaceutical composition is directed to a coated tablet or coated beads or granules having delayed release properties, as described, for example, in U.S. Pat. No. 8,168,594, the entire contents of which are hereby incorporated by reference. Exemplary enteric coatings include copolymers of acrylates and methacrylates, which in some embodiments are 1:1 copolymers. Other fillers, binders, and plasticizers, including plasticizers for seal coatings or top coatings, are described in US 8,168,594, which is hereby incorporated by reference.

[0079] According to certain embodiments, the present invention provides a composition as described herein that is administered once or more per day. For example, the composition can be administered about once a day, about twice a day, or about three times a day. In various embodiments, the regimen of once a day to three times a day lasts for an extended period of time. In some embodiments, the composition is administered every day. In other embodiments, larazolamid or larazolamid derivative compositions are administered 1 to 3 times per week. In some embodiments, the regimen lasts for at least about 1 month, at least about 2 months, at least about 4 months, at least about 6 months, or at least about 8 months. In some embodiments, treatment is continuous, to delay or prevent disease progression or to alleviate or improve the symptoms of chronic diseases. Example

[0080] Example 1: Elucidating the mechanism of action of larizolide acetate in intestinal barrier function

[0081] This example provides the results of a study on the effect of larizolide acetate on regulating MLC-2 phosphorylation during A / R injury. The results showed that apical larizolide acetate protected epithelial barrier function by regulating TJ proteins and actin stabilization on A / R-injured Caco-2BBe1 monolayer cells. In addition, larizolide acetate treatment also reduced the increased MLC-2 phosphorylation during A / R injury, thereby regulating epithelial barrier function.

[0082] A. Assessment of Barrier Function

[0083] Pretreatment of human intestinal epithelial cells with larazolidone acetate was shown to tighten the intestinal barrier during hypoxia / reoxygenation (A / R) injury. The prejunctional actomyosin ring contracts in response to phosphorylation of myosin light chain 2 (MLC-2), leading to internalization of TJ transmembrane proteins, so this study evaluated whether larazolidone acetate protects the TJ barrier during hypoxia / reoxygenation (A / R) injury by inhibiting MLC-2 phosphorylation.

[0084] Specifically, to assess barrier function, transepithelial electrical resistance (TEER) was measured in hypoxia / reoxygenation (A / R) injured C2BBe1 (Caco-2 brush border expressing Caco-2) cells treated with or without larazol acetate (LA). C2BBe1 monolayers were treated with LA (0.001, 0.1, 1, and 10 mM) and subjected to hypoxia for 2 h followed by reoxygenation with 21% O2. Figure 1A. To generate A / R injury, C2BBe1 cells were placed in a modular culture chamber (Billups-Rothenberg, San Diego, CA) and flushed with 95% N2 / 5% CO2 for 5 minutes. The modular culture chamber was then sealed and placed in an incubator for 2 hours. After 2 hours, the cells were removed from the modular incubator chamber and placed in a 21% O2 normal environment. TEER was measured to assess barrier function during A / R injury and recovery. TEER was measured on the basal and apical sides of the monolayer using a chopstick electrode set (WPI, LLC, Sarasota, FL) and a connected epithelial volt-ohmmeter 2 (WPI, LLC, Sarasota, FL).

[0085] The TEER of C2BBe1 monolayers treated with 10 mM LA was significantly increased compared to control cells (p<0.001). Figure 1 B. Increased TEER demonstrates that LA is able to close the “leaky” tight junction barrier induced by hypoxic injury.

[0086] B. Assessment of tight junctions and MLC activity

[0087] like Figure 2 As shown, A / R injury induces phosphorylation of myosin light chain 2 (MLC-2) to internalize tight junction proteins. The aim of this experiment was to determine whether LA helps inhibit phosphorylated MLC-2 to protect the tight junction barrier during A / R injury. To this end, the localization of tight junction proteins, the structure of actin, and pMLC-2 were studied.

[0088] Specifically, the distribution of tight junction proteins and actin structures was evaluated using Western blotting and immunofluorescence microscopy analysis of membrane and cytoplasmic fractions. Mem-PER eukaryotic membrane protein extraction kit (ThermoScientific) was used to fractionate membrane and cytoplasmic compartments. The extracted protein was blotted with primary antibodies against ZO-1 and anti-occludens (Invitrogen). Monolayer cells fixed with cold methanol were stained with primary antibodies (ZO-1 and occludens) and phalloidin (F-actin) for immunofluorescence microscopy analysis. Olympus IX83 inverted electric microscope (Olympus, Tokyo, Japan) equipped with cellSens software was used to examine the stained monolayer cells. Western blot analysis was performed using a primary antibody (CST, Danvers, MA) for pMLC-2 and MLC-2 to evaluate pMLC-2 / MLC-2 expression.

[0089] Data show that during A / R injury, the tight junction protein occludin is internalized into the cytoplasm and ZO-1 is destroyed. Figure 3As shown, treatment with 10 mM LA prevented the disruption of tight junction proteins during A / R injury. F-actin structure was also disrupted during A / R injury and was protected by 10 mM LA. Figure 4 .also, Figure 5 It was shown that pMLC-2 was significantly increased by A / R injury, whereas treatment with 10 mM LA significantly reduced this phosphorylation. Taken together, the data suggest that LA protects the tight junction barrier during A / R injury by reducing pMLC-2.

[0090] LA treatment increased the tight junction barrier in human intestinal cells during A / R injury. The results showed that LA treatment protected tight junction proteins in C2BBe1 cells during A / R injury. Hypoxia-induced phosphorylated MLC-2 increased during A / R injury, while LA significantly reduced phosphorylated MLC-2.

[0091] LA treatment preserved the distribution of tight junction proteins in human intestinal epithelial cells during A / R injury. This enhancement of barrier function during A / R injury caused by LA treatment was caused by a decrease in phosphorylated MLC-2 and stabilization of actin.

[0092] C. Assessment of MLCK and ROCK activity

[0093] Figure 6 It was shown that phosphorylated myosin light chain (pMLC) is regulated by myosin light chain kinase (MLCK), myosin light chain phosphatase (MLCP) and Rho kinase (ROCK). The purpose of this example was to elucidate the role of LA in kinase activity by performing kinase inhibition experiments.

[0094] To assess barrier function, TEER was measured in A / R injured C2BBel cells treated with or without LA. C2BBel cell monolayers were treated with 10 mM LA, 1 μM Y-27632 dihydrochloride (ROCK inhibitor, ApexBio, Houston, TX), and 400 μM peptide 18 (MLCK inhibitor, Tocris Bioscience, Minneapolis, MN) and then subjected to 2 h of hypoxia followed by reoxygenation with 21% O2.

[0095] like Figure 7 As shown, treatment of A / R injured cells with peptide 18 alone or in combination with LA increased TEER values ​​compared to untreated A / R injured cells. In particular, combined treatment with peptide 18 and LA showed a synergistic effect in A / R injured cells. However, combined treatment with Y-27632 and LA did not show any additive or synergistic effect.

[0096] Based on the significant increase in TEER when LA and peptide 18 were used together in A / R-injured cells, it was speculated that LA acts on an independent pathway different from MLCK. TEER levels were similar in A / R-injured cells treated with LA and A / R-injured cells treated with LA and Y-27632. These data suggest that the reduction of MLC-2 phosphorylation by LA can be regulated by ROCK activity.

[0097] D. RNAseq analysis

[0098] Since the phosphorylation of MLC can be regulated by various cellular pathways, such as Figure 8 As shown, high-throughput RNAseq analysis was performed to elucidate the mechanism of action of LA. Specifically, C2BBe1 cell monolayers were treated with 10 mM LA, deprived of oxygen for 2 h, and then reoxygenated with 21% O2 for 1 h. Next-generation RNA sequencing and biostatistical analysis were used to evaluate cellular regulatory pathways.

[0099] Different expression analysis showed the presence of 12,999 clusters, of which 11,925 clusters were shared by control (CT), control and LA (CT+LA), hypoxia (Anox), and hypoxia and LA (Anox+LA). Fig. 9B These analyses also revealed the most significantly differentially expressed genes between CT vs. CT+LA and Anox vs. Anox+LA (shown in Table 1).

[0100] Table 1: Most significant DEGs in control compared with 3 h exposure to LA

[0101]

[0102] Further analysis of gene ontology annotations revealed that there were many key signaling pathways that were differentially expressed in cells treated with LA, including biological processes involved in establishing cell polarity, molecular functions regulating junctional architecture, and cellular components associated with epithelial repair (cell leading edge, ruffles, and apical junction complex). See Table 2.

[0103] Table 2: Most significant DEGs in hypoxic cells compared with cells exposed to LA for 3 h

[0104]

[0105] also, Fig.10 It was shown that Ras / Rho GTPase binding and protein serine / threonine kinase activities were differentially expressed in cells treated with LA. In addition, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed enrichment of target genes for "cell cycle", "adherens junction" and "Wnt signaling pathway". Fig.11 .

[0106] According to GO analysis, multiple translation processes were significantly increased in cells treated with LA compared to untreated cells. The analysis showed that LA has a key role in protein translation signaling. LA treatment was also associated with multiple junction pathways, such as cadherin binding and cell adhesion. KEEG pathway analysis showed that LA was closely associated with cell cycle, adherens junction, and translation pathways. Data including RNAseq analysis suggest that LA protects the tight junction barrier during A / R injury by regulating multiple cellular pathways including cell cycle, migration, and apical junction complex.

[0107] E. Evaluation of barrier function under normal conditions

[0108] This experiment evaluated the role of LA in paracellular permeability under normal conditions.

[0109] For barrier function analysis, TEER was measured in C2BBe1 cell monolayers treated with or without 10 mM LA. TEER was measured at the basal and apical sides of the monolayer using a chopstick electrode set (WPI, LLC, Sarasota, FL) connected to an Epithelial Voltaohmmeter 2 (WPI, LLC, Sarasota, FL).

[0110] Fig.12 The results shown show that treatment with 10 mM LA increased TEER in C2BBe1 cell monolayers, indicating that LA has a function to tighten paracellular pores under normal conditions and during A / R injury. LA has a function to tighten intercellular junctional structures under normal conditions.

[0111] F. Assessment of Proliferation and Migration

[0112] This experiment evaluated the role of LA in proliferation and migration. Specifically, the proliferation of C2BBe1 cells that were untreated or treated with LA (10 mM) was assessed using a CCK-8 assay kit (Dojindo Molecular Technologies, Rockville, MD) according to the manufacturer's instructions. Cells seeded in 96-well culture plates were treated with 10 mM LA every other day. Viable cells were assessed using a CCK-8 detection kit for five consecutive days. C2BBe1 cells were plated in 3-well culture inserts (ibidiGmbH, Germany) and after reaching confluence, the insert was removed to form a "wound" in the cell monolayer to evaluate migration. 10 mM LA was added to serum-free and regular culture medium to evaluate migration and proliferation. Images were taken at 0, 4, 8, 24 and 48 hours using an Axio Vert A1 microscope (Carl Zeiss AG, Oberkochen, Germany), and wound closure was analyzed by measuring migration distance using ImageJ software.

[0113] Fig.13 showed that treatment with 10 mM LA in C2BBe significantly increased proliferation measured by CCK8. Cells treated with 10 mM LA showed a significant increase in proliferation compared to untreated C2BBe1 cells. In addition, Fig.14 It was shown that the addition of LA did not significantly alter migration, as can be seen in serum-free medium conditions.

[0114] The results showed that LA promoted cell proliferation but not cell migration. This LA-induced proliferation may promote the repair mechanism of intestinal damage.

Claims

1. A method of treating a disease characterized by Rho-associated coiled-coil kinase (ROCK) activity in a subject, the method comprising: An effective amount of larizolide or a larizolide derivative or a pharmaceutically acceptable salt thereof is administered to the subject in an amount and manner effective to inhibit ROCK activity in the tissue.

2. The method of claim 1, wherein the tissue is the gastrointestinal tract.

3. The method of claim 2, wherein the condition is selected from cancer, adenoma, celiac disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, environmental enteropathy, esophagitis, necrotizing enterocolitis, intestinal ischemia, inflammatory liver disease, kidney disease, pancreatitis, hyperglycemia, and pulmonary or cardiac inflammation or fibrosis.

4. The method of claim 3, wherein the cancer originates from any tissue, optionally wherein the cancer originates from the skin, colon, breast, lung, brain, bone, pancreas, kidney, liver, bladder, ovary, testis, or prostate.

5. The method of claim 3, wherein the cancer is optionally colorectal cancer, leukemia, myeloma, or lymphoma.

6. The method of claim 1, wherein the subject is at risk for colorectal cancer.

7. The method of claim 6, wherein the subject's family history, genetic mutation, and / or health history increases the risk of colorectal cancer.

8. The method of claim 7, wherein a family member of the subject suffers from or has suffered from familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), Poitz-Jegger syndrome, or MUTYH-associated polyposis (MAP).

9. The method of claim 7, wherein the subject has a mutation that increases the risk of colorectal cancer in one or more genes selected from APC, MLH1, MSH2, MSH6, PMS2, EPCAM, STK11 (LKB1), and MUTYH.

10. according to the method described in any one in claim 6 to 9, wherein the experimenter has one or more single nucleotide polymorphisms (SNPs) selected from rs6983267, rs4939827, rs3802842, rs16892766, rs10795668, rs4444235, rs10411210, rs6691170, rs4925386, rs3824999, rs647161, rs2423279, rs3217810 and rs59336.

11. The method of claim 1, wherein the tissue is a cancer, optionally a primary cancer or a metastatic cancer.

12. The method of claim 11, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, kidney cancer, liver cancer, prostate cancer, cervical cancer, colorectal cancer, pancreatic cancer, melanoma, ovarian cancer, bone cancer, urothelial carcinoma, gastric cancer, head and neck cancer, glioblastoma, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, hormone-refractory prostate cancer, and lymphoma.

13. The method of claim 11 or 12, wherein the tissue is a metastatic cancer.

14. The method of claim 13, wherein the patient has metastatic melanoma.

15. The method of any one of claims 11 to 14, wherein the cancer is a sarcoma or a carcinoma.

16. The method according to any one of claims 11 to 15, wherein the subject is receiving or has received cancer treatment selected from one or more of chemotherapy, radiotherapy, resection and immunotherapy and immuno-oncology agents, optionally wherein the cancer immunotherapy is treatment with an immune checkpoint inhibitor.

17. The method according to any one of claims 11 to 16, wherein the larizolide or larizolide derivative is formulated for intratumoral administration.

18. The method of claim 1, wherein the tissue is an eye.

19. The method of claim 18, wherein the larizolide or larizolide derivative is formulated for ocular administration, optionally to the ocular surface, or intraocular administration to the back of the eye.

20. The method of claim 1, wherein the larizolide or larizolide derivative is formulated for administration to the nasal and / or sinus cavities.

21. The method of claim 1, wherein the larizolide or larizolide derivative is formulated for administration to the ear canal.

22. The method of claim 1, wherein the tissue is the respiratory tract and the subject optionally has a disease selected from asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, cystic fibrosis, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), emphysema, bronchitis, pneumonia, lung cancer, and respiratory tract infection.

23. The method of claim 22, wherein the larizolide or larizolide derivative is formulated for administration to the lungs as a solution aerosol or powder.

24. The method of claim 1, wherein the tissue is vasculature and the subject optionally has a disease selected from the group consisting of cardiac fibrosis, cardiac hypertrophy, hypertension, pulmonary hypertension, angina, vasospastic angina, heart failure, and stroke.

25. The method of claim 24, wherein the larizolide or larizolide derivative is formulated for pulmonary administration.

26. The method of any one of claims 1 to 25, wherein the subject has a ROCK mutation selected from one or more of the following: Val1309, Tyr405, Ser1126, Pro1193S relative to the ROCK1 isoform, and / or Thr431Asn, Asp601Val and Lys1083Met relative to the ROCK2 isoform.

27. The method according to any one of the preceding claims, wherein a larizolide derivative or a pharmaceutically acceptable salt thereof is administered to the subject.

28. The method of claim 27, wherein the larizolide derivative comprises one or more modifications that increase ROCK inhibitor activity compared to larizolide.

29. The method of claim 27, wherein the larizolide derivative comprises at least one, at least two, at least three, at least four, at least five D-amino acids.

30. The method of claim 29, wherein each amino acid (except Gly) of the larisolade derivative is a D-amino acid, and the derivative is optionally retro-inverso larisolade.

31. The method according to any one of the preceding claims, wherein the larizolide or larizolide derivative is administered topically to a tissue.

32. A pharmaceutical composition comprising an effective amount of a peptide having the amino acid sequence Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof for inhibiting ROCK activity in a tissue, and a pharmaceutically acceptable carrier.

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