Compositions and methods for inhibiting Rho kinase

Abstract

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

Description

[Technical field]

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

[0002] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 308,330, filed February 9, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] [Description of text file submitted electronically] This application contains a Sequence Listing in XML format that was submitted electronically via the Patent Center herewith. The contents of the XML copy created on February 7, 2023 is named "NMT-038PC_116031-5038.xml" and is 6919 bytes in size. The Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Rho-associated coiled-coil-containing kinases (ROCKs) are effectors of the RhoA small GTPase and play a key role in promoting contractility of the actomyosin cytoskeleton downstream of RhoA and RhoC activation. ROCK kinases are important in processes such as cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs, ROCK1 and ROCK2, are involved in various disease processes, including cardiovascular disease, vascular injury, fibrosis, inflammatory bowel disease, and cancer, among others.

[0005] There is a need for pharmaceutical compositions and methods for inhibiting ROCK, i.e., for the treatment or prevention of ROCK-associated disease states. Summary of the Invention

[0006] The present invention contemplates, in part, compositions and methods useful for treating disease conditions associated with Rho-associated coiled-coil kinase (ROCK).

[0007] In various aspects, the invention provides methods of treating a subject for a condition characterized by Rho-associated coiled-coil kinase (ROCK) activity. The methods comprise administering to the subject an effective amount of larazotide or a larazotide derivative, or a pharma- ceutically acceptable salt thereof, in an amount and manner effective to inhibit ROCK activity in the organ or tissue. In various embodiments, the organ or tissue is selected from the gastrointestinal tract, cancerous tissue, eye, respiratory tract, and vascular system.

[0008] In some embodiments, larazotide or a derivative thereof is administered to the gastrointestinal tract. In such embodiments, the subject may have a condition selected from cancer (e.g., GI cancer, such as colon or stomach cancer, or non-GI cancer), adenoma, inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), celiac disease, esophagitis, inflammatory liver disease, kidney disease, pancreatitis, hyperglycemia, diabetes, and lung or cardiac inflammation or fibrosis. In certain embodiments, the subject has or is at risk for developing colon cancer. For example, the subject's family history, genetic mutations, and / or health history may increase the subject's risk of colon cancer.

[0009] In other embodiments, larazotide or a derivative is delivered directly to the cancer tissue, which may be a primary tumor or a metastatic tumor. For example, the subject may be suffering from a cancer selected from lung cancer, breast cancer, kidney cancer, liver cancer, prostate cancer, cervical cancer, colon cancer, pancreatic cancer, melanoma, ovarian cancer, bone 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 or carcinoma.

[0010] In other embodiments, the larazotide or derivative is administered ophthalmically, for example, the subject may have an ophthalmic condition selected from Sjogren's syndrome, dry eye syndrome, age-related macular degeneration (AMD), macular edema, diabetic retinopathy, and glaucoma.

[0011] In some embodiments, the larazotide or derivative is administered to the respiratory tract. For example, the subject may have a condition 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 a respiratory infection.

[0012] In other embodiments, the larazotide or derivative is administered to the vascular system or systemically. For example, the subject may have a condition selected from myocardial fibrosis, cardiac hypertrophy, hypertension, pulmonary hypertension, angina, vasospastic angina, heart failure, atherosclerosis, arteriosclerosis, diabetes, pancreatitis, kidney disease (including renal fibrosis), and stroke (e.g., in stroke prevention or stroke reversal).

[0013] In various embodiments, the larazotide derivative comprises one or more modifications that enhance ROCK inhibitor activity relative to larazotide. For example, in embodiments, the larazotide derivative comprises at least one, at least two, at least three, at least four, or at least five (d)-amino acids. In certain embodiments, each amino acid (other than Gly) of the larazotide derivative is a (d)-amino acid, and the derivative is optionally retro-inverso larazotide.

[0014] Other aspects and embodiments of the present invention will become apparent from the following detailed description and examples. [Brief description of the drawings]

[0015] [Figure 1]Figure 1A and Figure 1B show the evaluation of paracellular permeability during anoxia / reoxygenation (A / R) injury in larazotide acetate (LA)-treated C2BBe1 intestinal cells. Figure 1A shows a schematic diagram showing the schedule of LA treatment and TEER measurement in A / R injury. Figure 1B shows that treatment with 10 mM LA significantly (p<0.001) increases TEER compared to untreated A / R injured cells. [Diagram 2] Anoxia phosphorylates myosin and internalizes the tight junction protein occludin. This schematic shows how LA would be predicted to prevent this phosphorylation. [Diagram 3] Distribution of TJ proteins analyzed by immunoblotting and IF analysis from membrane and cytoplasmic fractions after 1 h of reoxygenation. Figure 3 shows that the transmembrane protein occludin is significantly (p<0.05) internalized in anoxic injured cells compared to control cells. However, occludin was significantly (p<0.05) increased in the membrane by 10 mM LA compared to untreated anoxic injured cells. [Figure 4] Figure 1. Assessment of localization of tight junction proteins and cytoskeletal proteins by immunofluorescence microscopy at 1 hour of oxygenation. Permeable support membranes were fixed and stained with ZO-1 (red), occludin (green), and F-actin (purple). Immunolocalization of ZO-1 and occludin was analyzed by Z-stack 3D analysis. Occludin, ZO-1, and F-actin were disrupted in untreated anoxic injured cells, whereas tight junction proteins and F-actin were well organized in larazotide acetate (LA) treated cells. [Diagram 5] Figure 2 shows the ratio of pMLC-2 / MLC-2 assessed by immunoblotting at 1 h of reoxygenation. pMLC-2 expression was dramatically increased after A / R injury and was significantly decreased by pretreatment with 10 mM LA (##<0.01). [Figure 6]Figure 1 shows the regulation of phosphorylation of MLC that regulates the TJ barrier in intestinal epithelial cells. The TJ barrier is mainly determined by the phosphorylation level of regulatory light chain of myosin (MLC), which is regulated by two enzymes, myosin light chain kinase (MLCK) and rho-associated coiled-coil protein kinase (ROCK). Elevated intracellular Ca2+ 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). [Figure 7] Figure 1 shows that inhibition of MLCK and ROCK increases TEER compared to untreated A / R injured cells with or without larazotide. Treatment with Pep18 (MLCK inhibitor) alone or in combination with LA increased TEER. Fasudil (ROCK inhibitor) treated monolayers increased TEER. Treatment with fasudil and LA in combination increased TEER, but to a lesser extent than fasudil alone (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). [Figure 8] 1 shows possible mechanisms of regulation of phosphorylation of MLC, which regulates the paracellular actin myosin ring in intestinal epithelial cells. The TJ barrier is mainly determined by the phosphorylation level of regulatory light chain of myosin (MLC), which is regulated by two enzymes, myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). These mechanisms can be regulated by various cellular pathways, such as Ca2+ homeostasis, G protein-coupled receptor (GPCR), and growth factor (GF) pathways. [Figure 9] Figures 9A and 9B show various expression analyses using next-generation RNA-seq. Figure 9A shows a Venn diagram analysis of the quantity of differentially expressed genes (DEGs) identified. Figure 9B shows a hierarchical clustering of DEGs, which was used to estimate the expression patterns under different experimental conditions. [Figure 10]Figure 10 shows the Gene Ontology (GO) functional classification analysis of (DEGs) from various group combinations. It includes three major branches: biological process, cellular component, and molecular function. GO terms with padj<0.05 are significantly enriched. Figure 10 shows the 20 significantly enriched biological processes, cellular components, and molecular functions. [Figure 11] Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of enrichment of DEGs from various group combinations. KEGG pathway terms with padj<0.05 are significantly enriched. [Figure 12] Figure 1: Evaluation of paracellular permeability in C2BBe1 intestinal cells. Treatment with 10 mM LA significantly increased TEER compared to untreated A / R injured cells 1 hour after treatment. [Figure 13] Figure 1. Assessment of C2BBe1 intestinal cell proliferation. Treatment with 10 mM LA significantly increases proliferation compared to untreated cells. [Figure 14] Figures 14A and 14B show the evaluation of proliferation and migration of C2BBe1 intestinal cells. Figure 14A shows that 10 mM LA-treated cells in normal medium significantly increased wound healing at 48 hours. Figure 14B shows that no significantly different migration patterns were detected between 10 mM LA-treated and untreated cells in serum-free medium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention provides compositions and methods for treating a condition characterized by Rho-associated coiled-coil kinase (ROCK) activity in a subject in need of treatment by administering to the subject an effective amount of larazotide or a larazotide derivative, or a pharma- ceutically acceptable salt thereof, in an amount and manner effective to inhibit ROCK activity in the tissue.

[0017] The present disclosure shows that larazotide and larazotide derivatives, or pharma- ceutically acceptable salts thereof, inhibit rho-associated protein kinase (ROCK). Inhibition of ROCK can provide therapeutic benefit to subjects with pathologies associated with ROCK activity. ROCK and its downstream targets are involved in regulating the dynamics of the actin cytoskeleton, and thus are responsible for cell migration and motility. In addition, they are involved in diverse biological processes, such as cell junction integrity, cell cycle control, and cell apoptosis.

[0018] There are two isoforms of ROCK enzymes: ROCK1 and ROCK2. Both kinases contain an N-terminal catalytic kinase domain followed by a central coiled-coil domain containing a Rho-binding domain (RBD), and a C-terminal pleckstrin homology (PH) domain.

[0019] The peptide agent known as larazotide has the amino acid sequence Gly-Gly-Val-Leu-Val-Gln-Pro-Gly (SEQ ID NO: 1). Larazotide promotes tight junction integrity in epithelial and endothelial tissues, including the intestinal epithelium, and has been evaluated as a treatment for patients with celiac disease (CeD). According to certain aspects and embodiments, the present invention provides larazotide derivatives that, inter alia, have increased resistance to exoheptidase degradation, including aminopeptidase degradation. In various embodiments, the larazotide derivatives include one or more amino acid substitutions, deletions, and / or insertions with respect to a peptide having the amino acid sequence of SEQ ID NO: 1. Exemplary modifications are described in U.S. Patent No. 8,785,374, U.S. Patent No. 8,957,032, U.S. Patent No. 9,279,807, International Application No. PCT / US2019 / 19350, and International Application No. PCT / US21 / 27410, all of which are incorporated herein by reference.

[0020] In some embodiments, the peptide derivative of larazotide contains one or more (d) amino acids. For example, the larazotide derivative may contain one, two, three, four, or five (d) amino acids (i.e., D-form instead of L-form). In some embodiments, the larazotide derivative has the amino acid sequence Gly-Gly-Val-Leu-Val-Gln-(d)Pro-Gly (SEQ ID NO: 2). This peptide is referred to herein as "(d)-Pro" or (d)-Prolarazotide. In other embodiments, the larazotide derivative has the amino acid sequence Gly-Gly-(d)Val-(d)Leu-(d)Val-(d)Gln-(d)Pro-Gly (SEQ ID NO: 3). This peptide is referred to herein as "(d)-larazotide." As demonstrated herein, (d)-larazotide is surprisingly effective at promoting tight junction integrity at significantly lower and higher concentrations compared to larazotide (which exhibits a bell-shaped dose-response curve). This is a surprising observation because typically, substitution of L-amino acids with D-amino acids in peptide drugs results in loss of potency. That is, peptides with D-amino acids would be expected to bind with lower affinity to functional receptors compared to peptides with natural L-amino acids.

[0021] In various embodiments, the present invention uses larazotide derivatives that are more effective at substantially lower or higher doses than larazotide, especially when administered to the gastrointestinal tract.Accordingly, the pharmaceutical composition of the present invention may contain less than about 0.5 mg of larazotide derivative.For example, in some embodiments, the pharmaceutical composition contains less than about 0.4 mg of larazotide derivative, or less than about 0.3 mg of larazotide derivative, or less than about 0.25 mg of larazotide derivative, or less than about 0.2 mg of larazotide derivative, or less than about 0.15 mg of larazotide derivative, or less than about 0.1 mg of larazotide derivative, or less than about 50 μg of larazotide derivative, or less than about 25 μg of larazotide derivative. In some embodiments, the pharmaceutical composition contains from about 50 μg to about 400 μg or less of a larazotide derivative, or from about 50 μg to about 200 μg or less of a larazotide derivative, or from about 50 μg to about 150 μg or less of a larazotide derivative.

[0022] In other embodiments, the present invention contemplates pharmaceutical compositions containing greater than about 0.5 mg of a larazotide derivative that substantially avoid the reverse dose or "bell shaped" reaction observed with larazotide. For example, in some embodiments, the pharmaceutical compositions contain greater than about 0.6 mg of a larazotide derivative, or greater than about 0.75 mg of a larazotide derivative, or greater than about 1.0 mg of a larazotide derivative, or greater than about 1.25 mg of a larazotide derivative, or greater than about 1.5 mg of a larazotide derivative, or greater than about 2.0 mg of a larazotide derivative.

[0023] In some embodiments, the larazotide derivative (e.g., (d)-larazotide or (d)-Pro) is administered at about 0.5 mg. For example, the derivative may be more effective at a 0.5 mg dose than larazotide.

[0024] The present invention provides methods and compositions for treating conditions associated with ROCK activity in a subject in need of treatment by administering a composition comprising larazotide or a larazotide derivative to a tissue having ROCK activity, thereby inhibiting ROCK activity in the tissue. The terms "subject" and "patient" are used interchangeably herein and generally refer to a mammalian subject / patient. In various embodiments, the subject is a human subject. Thus, the composition may be formulated and / or delivered by various routes to inhibit ROCK activity in the desired organ or tissue. In various embodiments, a pharmaceutical composition comprising larazotide or a derivative thereof (or a salt thereof) is administered, for example, to the gastrointestinal (GI) tract (e.g., enteral delivery) or by parenteral, intranasal, buccal, ophthalmic or pulmonary delivery. Delivery may be local to the affected tissue or systemic.

[0025] In some embodiments, the peptide or pharmaceutical composition is administered to the gastrointestinal (GI) tract of the subject.

[0026] In some embodiments, the subject has an inflammatory condition or injury to the digestive 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), colon cancer, or stomach cancer. ROCK activity has been shown to be involved in 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, which may originate from any tissue, such as the skin, colon, breast, lung, brain, bone, pancreas, kidney, liver, bladder, ovary, testis, or prostate.

[0027] In other embodiments, the subject is afflicted with leukemia, myeloma, or lymphoma.

[0028] In some embodiments, the subject is at risk of developing colorectal cancer. For example, in some embodiments, the subject is at high risk of colorectal cancer due to the subject's family history, genetic mutations, and / or health history (e.g., a history of advanced adenomas or colorectal cancer). In some embodiments, the subject, or a member of the subject's family, has or has had familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), Peutz-Jeghers syndrome, or MUTYH-associated polyposis (MAP). In some embodiments, the subject has a mutation in one or more genes that increase the risk of colorectal cancer, including, but not limited to, APC, MLH1, MSH2, MSH6, PMS2, EPCAM, STK11 (LKB1), and MUTYH. For example, but not limited to, the subject may 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 subject is receiving chronic antibiotic use. For example, chronic antibiotic use includes the use of antibiotic therapy for at least 6 months, and may increase the risk of CRC.

[0029] In some embodiments, the subject has a ROCK mutation associated with the development of cancer, such as Val1309, Tyr405, Ser1126, Pro1193S for the ROCK1 isoform, or Thr431Asn, Asp601Val, and Lys1083Met for the ROCK2 isoform.

[0030] In some embodiments, the subject is undergoing or has undergone a cancer treatment selected from one or more of chemotherapy, radiation, resection, and immunotherapy and cancer immunotherapy agents. In embodiments, the cancer immunotherapy is a treatment with an immune checkpoint inhibitor or immune stimulatory ligand, including but not limited to inhibitors of programmed cell death ligand 1 (PD-L1, also known as B7-H1, CD274), programmed cell death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, IDO1, IDO2. T cell stimulatory ligands include but are not limited to agonists of CD28, OX-40, and ICOS. In certain embodiments, the subject is administered an immune checkpoint inhibitor selected from anti-CTLA-4, anti-PD-1, anti-PD-L1, and / or PD-L2 agents. In some embodiments, the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, pembrolizumab, and nivolumab.

[0031] In yet other embodiments, the subject suffers from inflammatory liver disease, kidney disease, pancreatitis, hyperglycemia, or lung or cardiac 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 due to hepatitis, obesity, diabetes, insulin resistance, hyperglyceridemia, chronic kidney disease, IgA nephropathy (also known as Berger's disease), abetalipoproteinemia, glycogen storage disease, Weber-Christian disease, Wolman disease, acute fatty liver of pregnancy, and lipodystrophy. See U.S. Patent Application Publication No. 2019 / 0358289 and U.S. Patent Application Publication No. 2021 / 0069286, which are incorporated herein by reference in their entireties.

[0032] In some embodiments, the subject has a condition associated with organ fibrosis, such as pulmonary fibrosis, myocardial fibrosis, renal fibrosis, or liver fibrosis. Knipe R., et al. The Rho Kinases: Critical Mediators of Multiple Profibrotic Processes and Rational Targets for New Therapies for Pulmonary Fibrosis, Pharmacol Rev. 2015 Jan; 67(1): 103-117. In some embodiments, the subject has a condition 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 infections.

[0033] In other embodiments, larazotide or a derivative is administered directly to the cancer tissue, including but not limited to, intratumoral administration, or via encapsulation or attachment to nanoparticles, or other means.ROCK enzymes function in various processes of cancer progression, such as tumor invasion and metastasis, proliferation, and apoptosis or survival, and affect both cancer and cancer-associated cells, such as fibroblasts and endothelial cells.

[0034] In some embodiments, the tissue is a primary cancer. Primary cancer refers to cancer cells at the site of origin that become clinically detectable, and may be a primary tumor. For example, the cancer may be stage I or stage II cancer. In some embodiments, the tissue is a primary cancer, and the cancer is selected from lung cancer, breast cancer, kidney cancer, liver cancer, prostate cancer, cervical cancer, colon cancer, pancreatic cancer, melanoma, ovarian cancer, bone 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.

[0035] In embodiments, the tissue is a metastatic cancer. "Metastasis" refers to the spread of cancer from a primary site to other locations in the body. Cancer cells can break off from a primary tumor, infiltrate lymphatic and blood vessels, circulate via the bloodstream, and grow at distant foci (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. In some embodiments, the tissue is a metastatic cancer, such as metastatic melanoma. In various embodiments, the patient is afflicted with a cancer that is a sarcoma or a carcinoma.

[0036] In some embodiments, the subject is undergoing or has undergone a cancer treatment selected from one or more of chemotherapy, radiation, resection, and immunotherapy and cancer immunotherapy agents. In embodiments, the cancer immunotherapy is a treatment with an immune checkpoint inhibitor or immune stimulatory ligand, including but not limited to inhibitors of programmed cell death ligand 1 (PD-L1, also known as B7-H1, CD274), programmed cell death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3, TIM3, IDO1, IDO2. T cell stimulatory ligands include but are not limited to agonists of CD28, OX-40, and ICOS. In certain embodiments, the subject is administered an immune checkpoint inhibitor selected from anti-CTLA-4, anti-PD-1, anti-PD-L1, and / or PD-L2 agents. In some embodiments, the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, pembrolizumab, and nivolumab.

[0037] In some embodiments, the larazotide or derivative is administered to the eye, such as to the surface of the eye, or by intravitreal injection.

[0038] In an embodiment, the subject suffers from Sjogren's syndrome (SS). Sjogren's syndrome is an autoimmune condition in which the moisture-producing glands in the body are affected, causing, for example, dry mouth (xerostomia), dry eye syndrome (e.g., chronic dry eye and / or xerophthalmia), and / or dry skin (e.g., xeroderma), as well as other symptoms. According to an embodiment of the present invention, Sjogren's syndrome may be primary Sjogren's syndrome or may be secondary Sjogren's syndrome, i.e., occurring in association with another autoimmune disease or connective tissue disease. The inflammation caused by SS gradually damages the glands and is characterized by lymphocytic infiltration within the glands, elevated levels of B cell activating factor (BAFF), and the production of autoantibodies (e.g., anti-SSA / Ro). See Nair JJ and Singh TP, Sjogren's syndrome: Review of the etiology pathophysiology & potential therapeutic interventions. J. Clin. Exp. Dent. 2017; 9(4): e584-9. In an exemplary embodiment, larazotide or a derivative is administered to the ocular surface for the treatment of primary Sjogren's syndrome or secondary Sjogren's syndrome.

[0039] In some embodiments, the subject suffers from glaucoma.ROCK inhibitors are promising treatment options, for example, for lowering intraocular pressure (IOP) in glaucoma.For example, in some embodiments, glaucoma is treated by administering larazotide or its derivative (or its salt) to the ocular surface.

[0040] 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 an eye disease that causes progressive blindness with age. Inflammation is involved in the pathogenesis of AMD, including choroidal neovascularization and geographic atrophy. In some embodiments, larazotide or a derivative thereof is administered intraocularly (e.g., intravitreal injection) to the back of the eye.

[0041] In some embodiments of the invention, larazotide or a derivative is administered to the respiratory tract. See U.S. Patent No. 10,723,763, U.S. Provisional Application No. 63 / 181,486, and International Application No. PCT / US21 / 2741, all of which are incorporated herein by reference. In embodiments, the subject has a condition 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 infection.

[0042] In embodiments, larazotide or a larazotide derivative is formulated for administration to the lungs as a solution aerosol or powder.

[0043] In other embodiments, larazotide or a larazotide derivative is administered to the nasal epithelium as a nasal solution or nasal emulsion.

[0044] In some embodiments, the larazotide or derivative is administered to the vascular system. For example, the larazotide or derivative can be administered systemically or applied locally via a catheter. For example, the subject may have a condition selected from myocardial fibrosis, cardiac hypertrophy, hypertension, pulmonary hypertension, angina, vasospastic angina, heart failure, atherosclerosis, arteriosclerosis, diabetes, pancreatitis, kidney disease (including renal fibrosis), and stroke (e.g., prevention of ischemic stroke or reversal of stroke).

[0045] The pharmaceutical compositions provided herein can be formulated for release in affected areas of the GI tract (e.g., stomach, small intestine and / or large intestine). In other embodiments, larazotide or derivatives are administered systemically (e.g., by intravenous or subcutaneous injection). In some embodiments, the peptide composition is administered to the lungs as a solution aerosol or powder. In some embodiments, the peptide composition is administered to the nasal epithelium as a nasal solution or nasal emulsion. In some embodiments, the peptide composition is administered to the oral cavity or esophagus as a liquid or orally disintegrating tablet. In some embodiments, the peptide composition is administered to the ocular surface or intraocularly.

[0046] The larazotide derivatives of the present invention can be administered in any suitable form, including salts. For example, peptides can be administered as acetate salts. Alternative salts can be used, including any pharma- ceutically 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. PH Stahl and CG Wermuth (eds.), Verlag, Zurich (Switzerland) 2002 (incorporated herein in their entirety by reference).

[0047] In various embodiments, the peptides are formulated as pharmaceutical compositions and can take the form of tablets, pills, pellets, capsules, capsules containing liquids, capsules containing multiparticulates, 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.

[0048] In some embodiments, the pharmaceutical composition is formulated as a composition suitable for parenteral administration. Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, subcutaneous or intraperitoneal injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, etc. They can also be prepared in the form of sterile solid compositions (e.g., lyophilized compositions) that can be dissolved or suspended in a sterile injection medium immediately before use. They may, for example, contain a suspending agent or a dispersing agent. In these embodiments, the composition may be effective for treating pathologies involving systemic inflammation or damaged or inflamed endothelial tissue.

[0049] In some embodiments, the composition is administered to a subject by contacting it with epithelial tissue or mucosal surface of the gastrointestinal tract. For example, the composition can be formulated to be delivered to one or more of the stomach, small intestine, and / or large intestine. ROCK inhibition can be achieved anywhere in the GI tract by targeting peptide release in the affected area(s) (e.g., duodenum, jejunum and ileum, transverse colon, descending colon, ascending colon, sigmoid colon, and cecum). Targeted delivery of peptides in the small or large intestine can be achieved by coating beads or particles with the peptide to prevent release in the stomach and provide a delayed release coating that degrades at or near the target location(s).

[0050] In some embodiments, larazotide or a larazotide derivative is administered in a sustained or controlled release formulation that releases about 0.5 mg to about 5 mg of larazotide or a derivative in the intestine. In certain embodiments, the controlled release formulation contains at least 0.5 mg or 1 mg of larazotide or a derivative.

[0051] In some embodiments, the peptide (e.g., larazotide or a larazotide derivative) is formulated for sustained or modulated or controlled delivery at one or more locations in the GI. For example, the present invention contemplates sustained or controlled release formulations that can functionally release the peptide in the small and / or large intestine over at least about 2 hours, or over at least about 2.5 hours, or over at least about 3 hours, or over at least about 4 hours, or over at least about 5 hours. In some embodiments, the sustained or controlled release composition begins to release the peptide within about 10 to about 30 minutes of exposure to simulated intestinal fluid, and release of the peptide continues for at least about 180 minutes, or at least about 210 minutes, or at least about 240 minutes, or at least about 280 minutes after exposure to simulated intestinal fluid. Release profiles can be created, for example, using compositions with different enteric polymer coats and / or different thicknesses of the polymer coat. In some embodiments, the present invention provides a composition comprising an effective amount of larazotide or a derivative (or salt thereof) contained within a biodegradable or erodible polymer matrix, the composition further comprising an enteric coating. Formulations using biodegradable or erodible matrices are described in WO 2021 / 034629, which is incorporated herein by reference in its entirety. Additionally, the erodible polymer matrix may comprise a polysaccharide matrix. In some embodiments, the matrix comprises one or more of cellulose, chitin, chitosan, alginate, amylose, pectin, callose, laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan, xanthan gum, dextran, welan gum, gellan gum, diutan gum, pullulan, hyaluronic acid, and derivatives thereof. In further embodiments, the matrix comprises microcrystalline cellulose. In these embodiments, the compositions utilize low effective doses of larazotide or a larazotide derivative (eg, (d)-larazotide or (d)-Pro) while minimizing local accumulation of inactive fragments.Additionally, the formulations of these embodiments have the advantage of treating a large surface area of ​​the GI tract with a low dose of peptide that is continuously deposited during delivery.

[0052] In various embodiments, the pharmaceutical composition can be formulated to have a delayed release profile, i.e., not to release the active ingredient(s) immediately after ingestion, but rather to postpone release of the active ingredient(s) until the peptide has passed through the stomach and reached the lower part of the gastrointestinal tract, for example, for release in the small intestine (e.g., one or more of the duodenum, jejunum, ileum) or large intestine (e.g., one or more of the cecum, ascending colon, transverse colon, descending colon, or sigmoid colon). In embodiments, the pharmaceutical composition is formulated to have a delayed release profile, for example, as described in U.S. Patent No. 8,168,594, the entire contents of which are incorporated herein by reference.

[0053] For example, the peptide may be administered to at least the duodenum of a patient as an oral dosage delayed release composition containing the peptide. In such an embodiment, the composition includes a first population of beads having a coating that is stable in gastric fluids and unstable in intestinal fluids, thereby degrading and substantially releasing the peptide in the duodenum. The composition may further include a second population of beads having a pH-dependent coating that affects the release of the peptide in the jejunum and / or ileum of a patient. For example, the second population of beads may release the peptide about 30 minutes or about 45 minutes after the beads release the peptide in the duodenum. The oral dosage composition may be in the form of a capsule or tablet. The pH-dependent coating in some embodiments is a 1:1 copolymer of methacrylic acid and ethyl acrylate, with the thickness of the layer determining the release profile of each bead. The beads may have one or more additional coatings, such as a base coat, a separation layer, and an overcoat layer. In these embodiments, the contents of the beads are released in a more bolus manner at the target location, but the properties of (d)-larazotide or (d)-Pro are more effective than larazotide with such a release profile.

[0054] In an exemplary composition for oral administration, an effective amount of peptide (e.g., as acetate) is provided in a first delayed release particle capable of releasing the peptide in the duodenum of a patient, and a second delayed release particle capable of releasing the peptide in the jejunum of a patient. Each particle has a core particle, a coat containing a peptide (e.g., (d)-larazotide or (d)-Pro) covering the core particle, and a delayed release coating (e.g., a 1:1 copolymer of acrylate and methacrylate) outside the coat containing the peptide. The first delayed release particle releases at least 70% of the peptide in the first delayed release particle by exposure to simulated intestinal fluid having a pH of greater than 5 for about 60 minutes, while the second delayed release particle releases at least 70% of the peptide by exposure to simulated intestinal fluid having a pH of greater than 5 for about 30 minutes and about 90 minutes.

[0055] In general, delayed release coatings may degrade over time, regardless of pH and / or the presence of enzymes. Such coatings may, for example, include water-insoluble polymers. Therefore, their solubility is not dependent on pH. The term "pH-independent" as used herein means that the permeability of the polymer and its ability to release pharmaceutical ingredients are not dependent on pH and / or are only slightly dependent on pH. Such coatings may, for example, be used to prepare 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 of the hydroxyl groups on the cellulose backbone are replaced with alkyl groups and some are modified with alkanoyl groups. Examples include ethyl cellulose, acetyl cellulose, nitrocellulose, etc.

[0056] Other examples of polymers that make up the delayed release coating include, but are not limited to, lacquers, acrylic and / or methacrylic ester polymers, acrylate or methacrylate polymers or copolymers with low quaternary ammonium content, or mixtures thereof, etc. Examples of insoluble polymers include EUDRAGIT RS™, EUDRAGIT RL™, and EUDRAGIT NE™. Insoluble polymers include, for example, polyvinyl esters, polyvinyl acetals, polyacrylic esters, butadiene styrene copolymers, etc.

[0057] Various types of enteric coatings are known for the delayed and substantial delivery of active agents to the GI tract. In some embodiments, the sustained release composition includes an enteric agent that is substantially stable in an acidic environment and substantially unstable in a near-neutral to alkaline environment. In embodiments, the sustained release coating contains an enteric agent that is substantially stable in gastric fluids. The enteric agent can be selected from, for example, a solution or dispersion of methacrylic acid copolymers, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, carboxymethylethylcellulose, and EUDRAGIT™ type polymers (poly(methacrylic acid), methyl methacrylate), hydroxypropylmethylcellulose acetate succinate, cellulose acetate trimellitate, shellac, or other suitable enteric coating polymers. EUDRAGIT™ type polymers include, for example, EUDRAGIT™ FS 30D, L 30 D-55, L 100-55, L 100, L 12.5, L 12.5 P, RL 30 D, RL PO, RL 100, RL 12.5, RS 30 D, RS PO, RS 100, RS 12.5, NE 30 D, NE 40 D, NM 30 D, S 100, S 12.5, and S 12.5 P. In some embodiments, one or more of EUDRAGIT® FS 30D, L 30D-55, L 100-55, L 100, L 12.5, L 12.5 P RL 30D, RL PO, RL 100, RL 12.5, RS 30D, RS PO, RS 100, RS 12.5, NE 30D, NE 40D, NM 30D, S 100, S 12.5, and S 12.5 P are used. The enteric agent may be a combination of the above solutions or dispersions. In some embodiments, the enteric agent is EUDRAGIT F30D, which comprises a copolymer of methyl acrylate, methyl methacrylate, and methacrylic acid. The copolymer has a ratio of free carbonyl groups to ester groups of about 1:10.

[0058] In some embodiments, the beads include an enteric coating that is substantially resistant to dissolution in simulated gastric fluid. The composition may remain essentially intact or essentially insoluble in gastric fluid. The stability of the gastroresistant coating may be pH dependent. For example, the enteric coating may prevent substantial release of the peptide in simulated gastric fluid and in simulated intestinal fluid having a pH of about 5.5. In some embodiments, the matrix provides sustained release of the peptide in simulated intestinal fluid having a pH of about 6 or higher, such as about 6.5 to about 7.0. Thus, the enteric coating is stable in simulated gastric fluid but unstable in simulated intestinal fluid having a pH above about 6.0. The enteric coating of such embodiments does not substantially release the peptide in the duodenum, but delays release until the composition enters the jejunum, and then provides sustained release in the jejunum and ileum.

[0059] In some embodiments, the composition is a capsule for oral delivery comprising a population of beads, the population of beads comprising an effective amount of larazotide or a derivative (e.g., (d)-larazotide or (d)-Pro or a salt thereof) contained within an erodible polymer matrix, the beads further comprising an enteric coating, which may comprise a copolymer of methyl acrylate, 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 be 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 provides less than about 15% release of the peptide after about 2 hours in simulated gastric fluid. Additionally, the composition provides less than about 25% release of the peptide after about 2 hours in simulated intestinal fluid having a pH of about 5.5. In various embodiments, the compositions release at least about 40% to no more than about 80% of the peptide after about 2 hours in simulated intestinal fluid having a pH of about 7.0. In various embodiments, 100% release in simulated intestinal fluid having a pH of about 7 is not reached until at least 3 hours, or in some embodiments, until at least about 3.5 hours or at least about 4 hours.

[0060] In some embodiments, the pharmaceutical composition comprises a coated tablet or coated beads or granules with a delayed release profile, for example as described in U.S. Patent No. 8,168,594, the entire contents of which are incorporated herein by reference. An exemplary enteric coating comprises a copolymer of acrylate and methacrylate, in some embodiments a 1:1 copolymer. Other fillers, binders and plasticizers, including those for seal coats or top coats, are described in U.S. Patent No. 8,168,594, the entire contents of which are incorporated herein by reference.

[0061] According to certain embodiments, the invention provides for the administration of the compositions described herein one or more times per day. For example, the compositions may be administered about once per day, about twice per day, or about three times per day. In various embodiments, the once-daily to three-times-daily regimen is continued for an extended period of time. In some embodiments, the compositions are administered daily. In other embodiments, the larazotide or larazotide derivative compositions are administered one to three times per week. In some embodiments, the regimen is continued for at least about one month, at least about two months, at least about four months, at least about six months, or at least about eight months. In some embodiments, the treatment is continued to slow or prevent progression of the disease or to reduce or ameliorate symptoms of a chronic disease. EXAMPLES

[0062] Example 1: Elucidation of the mechanism of action of larazotide acetate on intestinal barrier function This example provides results investigating the role of larazotide acetate in regulating MLC-2 phosphorylation during A / R injury. Results show that apically applied larazotide acetate to A / R-injured Caco-2BBe1 monolayers protected epithelial barrier function through regulating TJ proteins and stabilizing actin. Furthermore, larazotide acetate treatment attenuated the increase in MLC-2 phosphorylation during A / R injury and regulated epithelial barrier function.

[0063] A. Assessment of Barrier Function Pretreatment of human intestinal epithelial cells with larazotide acetate has been shown to tighten the intestinal barrier during anoxia / reoxygenation (A / R) injury. Because the actomyosin ring at parajunctions contracts in response to phosphorylation of myosin light chain 2 (MLC-2), which triggers internalization of TJ transmembrane proteins, this study evaluates whether larazotide acetate protects the TJ barrier during anoxia / reoxygenation (A / R) injury via inhibition of MLC-2 phosphorylation.

[0064] Specifically, to assess barrier function, transepithelial electrical resistance (TEER) was measured in anoxia / reoxygenation (A / R) injured C2BBe1 (Caco-2 brush border-Caco-2 expressing) cells treated or not with larazotide acetate (LA). C2BBe1 monolayers were treated with LA (0.001 mM, 0.1 mM, 1 mM, and 10 mM), anoxia for 2 h, and then reoxygenated with 21% O2. See Figure 1A. To produce A / R injury, C2BBe1 cells were placed in a modular incubator chamber (Billups-Rothenberg, San Diego, CA) and flushed with 95% N2 / 5% CO2 for 5 min. The modular chamber was then airtight and placed in an incubator for 2 h. After 2 h, the cells were removed from the modular incubator chamber and placed in a normal environment of 21% O2. To assess barrier function during A / R injury and recovery, TEER was measured using a Chopstick Electrode Set (WPI, LLC, Sarasota, FL) attached to an Epithelial Volt Ohm Meter2 (WPI, LLC, Sarasota, FL) on the basolateral and apical sides of the monolayer.

[0065] The resulting TEER was shown to be significantly increased (p<0.001) in C2BBe1 monolayers treated with 10 mM LA compared to control cells, see Figure 1B. The increase in TEER is evidence of the ability of LA to close the "leaky" tight junction barrier induced by anoxic injury.

[0066] B. Assessment of tight junction and MLC activity As shown in Figure 2, 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 examined.

[0067] Specifically, the distribution of tight junction proteins and actin structures was assessed using Western blots of membrane and cytoplasmic fractions and immunofluorescence microscopy. Membrane and cytoplasmic compartments were fractionated using the Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific). Extracted proteins were blotted with anti-ZO-1 and anti-occludin primary antibodies (Invitrogen). Monolayers fixed with cold methanol were stained with primary antibodies (ZO-1 and occludin) and phalloidin (F-actin) and subjected to immunofluorescence microscopy. Stained monolayers were examined with an Olympus IX83 Inverted Motorized Microscope (Olympus Corporation, Tokyo, Japan) equipped with cellSens software. pMLC-2 / MLC-2 expression was assessed by Western blot analysis using primary antibodies against pMLC-2 and MLC-2 (CST, Danvers, MA).

[0068] The data indicated that the tight junction protein occludin was internalized into the cytoplasm and ZO-1 was disrupted during A / R injury. However, as shown in Figure 3, 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 but was protected by 10 mM LA; see Figure 4. Furthermore, Figure 5 shows that pMLC-2 was significantly increased by A / R injury and this phosphorylation was significantly reduced by treatment with 10 mM LA. Taken together, the data suggest that LA protects the tight junction barrier during A / R injury by decreasing pMLC-2.

[0069] LA treatment strengthened the tight junction barrier in human intestinal cells during A / R injury. LA treatment was shown to protect tight junction proteins in C2BBe1 cells during A / R injury. Anoxia-induced phosphorylated MLC-2 and phosphorylated MLC-2, which were increased during A / R injury, were significantly reduced by LA.

[0070] LA treatment protected the distribution of tight junction proteins in human intestinal epithelial cells during A / R injury. This enhanced barrier function during A / R injury induced by LA treatment was mediated by a decrease in phosphorylated MLC-2 through actin stabilization.

[0071] C. Assessment of MLCK and ROCK activity Figure 6 shows 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 clarify the role of LA in kinase activity by performing kinase inhibition experiments.

[0072] To assess barrier function, TEER was measured in A / R-injured C2BBe1 cells treated or not with LA. C2BBe1 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), then made anoxic for 2 h and reoxygenated with 21% O2.

[0073] As shown in Figure 7, peptide 18 alone or with LA treatment in A / R injured cells increased the TEER value compared to untreated A / R injured cells. Notably, the combined treatment of peptide 18 with LA showed a synergistic effect in A / R injured cells. However, Y-27632 combined with LA showed no additive or synergistic effect.

[0074] The TEER was significantly increased when LA and peptide 18 were applied together to A / R injured cells, suggesting that LA acts through a pathway other than MLCK. The TEER levels of A / R injured cells treated with LA and those treated with LA and Y-27632 were similar. These data indicate that the decrease in phosphorylation of MLC-2 by LA may be regulated by ROCK activity.

[0075] D.RNAseq analysis Since MLC phosphorylation can be regulated by various cellular pathways as shown in Figure 8, we performed high-throughput RNAseq analysis to elucidate the mechanism of action of LA. Specifically, C2BBe1 monolayers were treated with 10 mM LA, anoxic for 2 h, and then reoxygenated with 21% O2 for 1 h. Next-generation RNA sequencing and biostatistical analysis were used to evaluate the cellular regulatory pathways.

[0076] Various expression analyses revealed the presence of 12999 clusters, of which 11925 were shared between control (CT), control with LA (CT+LA), anoxic (Anox), and anoxic with LA (Anox+LA) groups (see Figure 9B). These analyses also show the most significantly differentially expressed genes between CT and CT+LA and between Anox and Anox+LA (shown in Table 1).

[0077] [Table 1]

[0078] Further analysis of gene ontology annotations revealed several important signaling pathways that were differentially expressed in LA-treated cells, including biological processes involved in the establishment of cell polarity, molecular functions controlling junctional structure, and cellular components related to epithelial repair (cell tips, ruffles, and apical junctional complexes) (see Table 2 ).

[0079] [Table 2]

[0080] Furthermore, Figure 10 shows that Ras / Rho GTPase binding and protein serine / threonine kinase activity are differentially expressed in LA-treated cells. Also, pathway enrichment analysis of Kyoto Encyclopedia of Genes and Genomes revealed that target genes related to "cell cycle", "adherens junction" and "Wnt signaling pathway" were enriched. See Figure 11.

[0081] Based on GO analysis, various translation processes were significantly increased in LA-treated cells compared to untreated cells. The analysis indicated that LA has an important function in protein translation signaling. LA treatment was also associated with various junction pathways, such as cadherin junction and cell adhesion. KEEG pathway analysis showed that LA was closely associated with cell cycle, adherens junction, and translation pathways. The data, including RNAseq analysis, suggest that LA protects the tight junction barrier during A / R injury by regulating various cellular pathways, including cell cycle, migration, and apical junction complex.

[0082] E. Assessment of barrier function under normal conditions In this experiment, the role of LA in paracellular permeability under normal conditions was evaluated.

[0083] For barrier function assays, TEER was measured in monolayers of C2BBe1 cells treated or not with 10 mM LA using a chopstick electrode set (WPI, LLC, Sarasota, FL) attached to an Epithelial Volt Ohm Meter2 (WPI, LLC, Sarasota, FL) on the basolateral and apical sides of the monolayer.

[0084] The results shown in Figure 12 show that treatment with 10 mM LA increased TEER in C2BBe1 monolayers, demonstrating that LA has the function of tightening paracellular pores not only during A / R injury but also under normal conditions. LA has the function of tightening intercellular junction structures under normal conditions.

[0085] F. Assessment of Proliferation and Migration In this experiment, the role of LA in proliferation and migration was evaluated. Specifically, proliferation of untreated or LA-treated (10 mM) C2BBe1 cells was assessed with 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 with a CCK-8 assay kit for 5 consecutive days. Migration was assessed by seeding C2BBe1 cells in 3-well culture inserts (ibidi GmbH, Gräfelfing, Germany) and removing the inserts after reaching confluence to create a "wound" in the cell monolayer. 10 mM LA was added to serum-free and normal medium to assess migration and proliferation, respectively. Images were taken with an Axio Vert A1 microscope (Carl Zeiss AG, Oberkochen, Germany) after 0, 4, 8, 24 and 48 h, and wound closure was analyzed by measuring migration distance with ImageJ software.

[0086] Figure 13 shows that 10 mM LA treatment in C2BBe significantly increased proliferation as measured by CCK8. Proliferation of 10 mM LA-treated cells was significantly increased compared to untreated C2BBe1 cells. Furthermore, Figure 14 shows that the addition of LA did not significantly alter migration as evidenced by serum-free media conditions.

[0087] The results suggest that LA promotes cell proliferation but not cell migration, and this LA-induced proliferation may facilitate repair mechanisms from intestinal injury.

Claims

1. A pharmaceutical composition for preventing or treating a subject for a condition characterized by Rho-associated coiled-coil kinase (ROCK) activity, comprising larazotide or a larazotide derivative, or a pharmaceutically acceptable salt thereof, in an amount and manner effective to inhibit ROCK activity in tissue.

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

3. 3. The pharmaceutical composition of claim 2, wherein the pathology 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. 4. The pharmaceutical composition of claim 3, wherein the cancer originates from the skin, colon, breast, lung, brain, bone, pancreas, kidney, liver, bladder, ovary, testis, or prostate.

5. The pharmaceutical composition of claim 3 , wherein the cancer is colon cancer, leukemia, myeloma, or lymphoma.

6. The pharmaceutical composition of claim 1 , wherein the subject is at risk of developing colon cancer.

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

8. 8. The pharmaceutical composition of claim 7, wherein a family member of the subject has or has had familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (HNPCC), Peutz-Jeghers syndrome, or MUTYH-associated polyposis (MAP).

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

10. 10. The pharmaceutical composition of any one of claims 6 to 9, wherein the subject 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 pharmaceutical composition of claim 1 , wherein the tissue is a primary cancer or a metastatic cancer.

12. 12. The pharmaceutical composition of claim 11, wherein the primary or metastatic 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 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.

13. The pharmaceutical composition according to claim 11 or 12, wherein the tissue is a metastatic cancer.

14. The pharmaceutical composition of claim 13, wherein the subject has metastatic melanoma.

15. The pharmaceutical composition of claim 11 , wherein the cancer is a sarcoma or a carcinoma.

16. 12. The pharmaceutical composition of claim 11, wherein the subject is undergoing or has undergone cancer treatment selected from one or more of chemotherapy, radiation, resection, and immunotherapy and cancer immunotherapy agents.

17. 12. The pharmaceutical composition of claim 11, wherein the larazotide or larazotide derivative is formulated for intratumoral administration.

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

19. 19. The pharmaceutical composition of claim 18, wherein the larazotide or larazotide derivative is formulated for ophthalmic administration.

20. 10. The pharmaceutical composition of claim 1, wherein the larazotide or larazotide derivative is formulated for administration to the nasal cavity and / or paranasal sinuses.

21. 10. The pharmaceutical composition of claim 1, wherein the larazotide or larazotide derivative is formulated for administration to the ear canal.

22. 10. The pharmaceutical composition of claim 1, wherein the tissue is respiratory and the subject has a condition 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 a respiratory infection.

23. 23. The pharmaceutical composition of claim 22, wherein the larazotide or larazotide derivative is formulated for administration to the lung as a solution aerosol or powder.

24. 2. The pharmaceutical composition of claim 1, wherein the tissue is the vasculature and the subject has a condition selected from myocardial fibrosis, cardiac hypertrophy, hypertension, pulmonary hypertension, angina, vasospastic angina, heart failure, and stroke.

25. 25. The pharmaceutical composition of claim 24, wherein the larazotide or larazotide derivative is formulated for pulmonary administration.

26. 2. The pharmaceutical composition of claim 1, wherein the subject has a ROCK mutation selected from one or more of Val1309, Tyr405, Ser1126, Pro1193S for the ROCK1 isoform, and / or Thr431Asn, Asp601Val, and Lys1083Met for the ROCK2 isoform.

27. The pharmaceutical composition of claim 1, comprising a larazotide derivative or a pharmaceutically acceptable salt thereof.

28. 28. The pharmaceutical composition of claim 27, wherein the larazotide derivative comprises one or more modifications that enhance ROCK inhibitor activity compared to larazotide.

29. 28. The pharmaceutical composition of claim 27, wherein the larazotide derivative comprises at least one, at least two, at least three, at least four, or at least five D-amino acids.

30. 30. The pharmaceutical composition of claim 29, wherein each amino acid (other than Gly) of the larazotide derivative is a D-amino acid, and the derivative is retro-inverso larazotide.

31. The pharmaceutical composition of claim 1 , which is for local administration to said tissue.

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

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