Bruudil metabolite and use thereof to treat chronic graft versus host disease

Abstract

The present disclosure provides a metabolite of Bruudil and the use of the metabolite in the treatment of a patient suffering from a disease mediated by ROCK2, including in cGVHD.

Description

Technical Field

[0001] The present disclosure relates to metabolites of Belumosudil and the use of said metabolites to treat autoimmune disorders in patients, including for the treatment of chronic graft-versus-host disease (cGVHD). Background Art

[0002] Chronic graft-versus-host disease (cGVHD) is an immune-mediated inflammatory and fibrotic disorder. It is a potentially serious complication after solid organ transplantation and allogeneic hematopoietic cell transplantation (alloHCT). cGVHD affects up to 70% of all alloHCT recipients, with an incidence of 20%-50% in children. It is the leading cause of non-relapse mortality more than 2 years after alloHCT. In the United States, the estimated prevalence of cGVHD is 14,000 patients (as of 2016). (Bachier CR et al.: Epidemiology and real-world treatment of chronic graft-versus-host disease post allogeneic hematopoietic cell transplantation: A USclaims analysis. Presented at ASH 2019, held in Orlando, FL, December 7-10, 2019) ("Bachier et al.").

[0003] As assessed by the Lee Symptom Scale (LSS) (which measures the impact of cGVHD on the function and health of patients), the quality of life (QOL) of cGVHD patients is severely impaired. It is reported that only one-third of patients with cGVHD and starting systemic treatment will survive, be in remission and stop immunosuppressive therapy before 5 years. (Lee SJ et al.: Success of immunosuppressive treatments in patients with chronic graft-versus-hostdisease.Biol Blood Marrow Transpl 24:555-562,2018) ("Lee et al.").

[0004] The pathophysiology of cGVHD can be divided into three stages: early inflammation caused by tissue damage, dysregulation of the adaptive immune system, and chronic inflammation and abnormal tissue repair with fibrosis.

[0005] The first-line therapy for moderate to severe chronic graft-versus-host disease (cGVHD) as defined by the National Institutes of Health (NIH) is corticosteroids alone or in combination with sirolimus or calcineurin inhibitors. However, up to 70% of patients require additional lines of therapy. (Bachier CR et al.). In addition, long-term use of corticosteroids is associated with significant side effects. (Lee et al.).

[0006] The management of cGVHD has continued to evolve with the advent of targeted therapies. cGVHD is characterized by overproduction of the proinflammatory cytokines IL-21 and IL-17 and overactivation of T follicular helper cells and B cells, which in turn leads to excessive antibody production.

[0007] In 2017, the U.S. Food and Drug Administration approved ibrutinib (a Bruton's tyrosine kinase inhibitor) for the treatment of adults with cGVHD after failure of one or more lines of systemic therapy. In patients with cGVHD requiring >25% body surface area erythematous rash or NIH oral score>4, a study using ibrutinib reported an overall response rate (ORR) of 67% and a discontinuation rate of 43% due to adverse events (TEAEs) during treatment. (Waller EK et al.: Ibrutinib for chronic graft-versus-host disease after failure of priortherapy: 1-Year update of a phase 1b / 2study. Biol Blood Marrow Transpl 25: 2002-2007, 2019).

[0008] There remains an opportunity to investigate additional treatment options for patients with cGVHD, including those who have failed ≥1 line of therapy. Summary of the invention

[0009] The present disclosure provides metabolites of berushudil and uses of the metabolites in treating ROCK2-mediated diseases in patients (including treating autoimmune disorders such as cGVHD). In one embodiment, the present disclosure includes uses of compound 2-(3-(4-(1H-indazole-5-ylamino)quinazolin-2-yl)phenoxy)acetamide in treating autoimmune disorders (in some embodiments, treating cGVHD).

[0010] The present disclosure further provides belushudil metabolites or pharmaceutically acceptable salts thereof in isolated form, and their use in treating autoimmune disorders (in some embodiments, treating cGVHD). The present disclosure provides an isolated or synthesized compound or a pharmaceutically acceptable salt thereof, the compound comprising belushudil glucuronide, O-dealkyl belushudil sulfate, monohydroxy belushudil and / or belushudil diol.

[0011] The present disclosure further provides a metabolic pathway of berushudil for use in preparing a medicament for treating an autoimmune disease in a subject. The use of the berushudil metabolites and / or metabolic pathways of berushudil disclosed herein can be used to prepare a regimen for treating an autoimmune disease in a subject.

[0012] A more complete understanding of the embodiments of the present invention may be obtained by reference to the detailed description and examples which are intended to illustrate non-limiting embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is shown after administration of a single 200 mg dose of belusudil followed by administration 15 minutes [ 14 C] - Graph of the mean PK profile of unlabeled berushudil following berushudil IV infusion. The inset shows the data on a log-linear scale.

[0014] Figure 2 is shown after administration of a single 200 mg dose of belusudil followed by administration 15 minutes [ 14 C]-After IV infusion of Berushudil[ 14 C] - Graph of the mean PK profile of berushudil. The inset shows the data on a log-linear scale.

[0015] Figure 3 The metabolic pathway of berushudil in humans is shown. DETAILED DESCRIPTION Overview

[0016] Berushudil is an oral selective inhibitor of rho-associated coiled-coil-containing protein kinase 2 (ROCK2). ROCK2 inhibition plays a role in the dysregulated adaptive immune system and fibrosis due to abnormal tissue repair. Berushudil has an IC of approximately 100 nM and 3 μM, respectively. 50 Value inhibits ROCK2 and ROCK1. Berushudil downregulates proinflammatory responses in ex vivo or in vitro human T cell assays by modulating STAT3 / STAT5 phosphorylation and altering the Th17 / Treg balance. In vitro, berushudil also inhibits abnormal profibrotic signaling. In vivo, berushudil exhibits activity in animal models of chronic GVHD.

[0017] The chemical name of the compound belushudil is as follows: 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide. The compound belushudil is also known as KD025. The mesylate salt of belushudil is marketed in the United States and other countries as REZUROCK TM REZUROCK is marketed for the treatment of patients with chronic GVHD (cGVHD) and, in some cases, for the treatment of patients with cGVHD after failure of at least two prior lines of systemic therapy. TM The active pharmaceutical ingredient is berushudil mesylate, which has the molecular formula C 27 H 28 N 6 O 5 S, with a molecular weight of 548.62 g / mol, and a chemical name of 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide methanesulfonate (1:1).

[0018] The chemical structure of Belushudil mesylate is as follows:

[0019] The chemical structure of radiolabeled berushedil as used in the study of Example 1 has the following structure:

[0020] Berushudil and processes for making the compound are described in the following U.S. Patents: U.S. Patent No. 8,357,693, U.S. Patent No. 9,815,820, U.S. Patent No. 10,183,931, and U.S. Patent No. 10,696,660.

[0021] By controlling ROCK2 activity, berushudil mediates signaling in immune cell function and fibrotic pathways, thereby alleviating the effects caused by this debilitating disease, such as inflammation of multiple tissues and fibrotic changes that may involve multiple organs including the lungs, hepatobiliary system, musculoskeletal system, gastrointestinal (GI) tract and skin.

[0022] In vitro evaluations have shown that the metabolism of belushudil is primarily dependent on the activity of cytochrome P450 CYP3A4 and that the solubility of belushudil is pH dependent. Preclinical studies in mice, rats, rabbits, and dogs have shown that belushudil undergoes hepatic metabolism to form 2 major metabolites, termed KD025m1 (minor metabolite) and KD025m2 (major metabolite).

[0023] The chemical name of KD025m1 is 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide and has the following structure:

[0024] The chemical name of KD025m2 is 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetic acid and has the following structure:

[0025] KD025m1 and KD025m2 were quantified in clinical trials of the berushudil development program; exposure of the major metabolite, KD025m2, was 15% to 20% of that of the parent, while exposure values ​​of the minor, more active metabolite, KD025m1, were <5% of that of the parent.

[0026] Human mass balance studies using radiolabeled compounds are very important for understanding the pharmacokinetic (PK) properties of research or clinical drugs. Mass balance studies are performed to identify and quantify metabolites and to characterize elimination pathways. One method used to determine absolute bioavailability is to use microtrace doses.

[0027] Understanding the metabolic pathways of research drugs or clinical drugs is very important for determining the effects of molecules (including energy consumed and produced via metabolic pathways, as well as synthesized molecules and produced byproducts) on patients. Understanding metabolic pathways is also useful for providing information about enzymes involved in catalytic biological mechanisms. Therefore, in addition to this, metabolic pathways may prove to be particularly useful in determining drug-drug interactions (DDIs), developing dose modifications, and / or addressing potential side effects of drug administration. See, for example, Schueller, O., et al., "A Phase I Pharmacokinetic Drug Interaction Study of Belumosudil Coadministered With CYP3A4 Inhibitors and Inducers and Proton Pump Inhibitors," Clinical Pharmacology in Drug Development, 2022, 11 (7) 795-806 (using metabolite exposure in assessing drug-drug interactions).

[0028] The present disclosure provides the metabolic pathway of belushudil. Previously unknown metabolites were identified, including O- dealkyl belushudil sulfate and belushudil glucuronide metabolites (identified in plasma) and monohydroxy belushudil and belushudil diol metabolites (identified in feces). These compounds can also be used to treat ROCK2-mediated diseases (including cGVHD) and / or for developing methods including treatment for cGVHD, such as determining DDI, dosage modification and solving side effects. definition

[0029] As used herein, "about" includes the exact amount modified by the term about as well as the amount expected to be within experimental error (such as within 15%, 10% or 5%). For example, "about 200 mg" means "200 mg" as well as mg ranges within experimental error (e.g., plus or minus 15%, 10% or 5% of 200 mg). As used herein, the term "about" can be used to modify ranges as well as specific values.

[0030] "API" means "active pharmaceutical ingredient."

[0031] "Allogeneic hematopoietic stem cell transplantation (allo-HSCT)" (also called bone marrow transplantation or stem cell transplantation) or "allogeneic hematopoietic cell transplantation (allo-HCT)" refers to a procedure in which hematopoietic cells from a donor are transplanted into a recipient who is not an identical twin. The source of hematopoietic stem cells for allogeneic transplantation can be peripheral blood stem cells (PBSC) or bone marrow (BM). In some cases, umbilical cord blood can be used. The human leukocyte antigen (HLA) genes of the donor and recipient may match, such as brothers and sisters. The donor and recipient may be parents and children who are only half-matched (haploid matched).

[0032] When the term "Belusudil" is used herein, it should be understood that unless the context clearly indicates otherwise, the term can cover any form of the compound Belusudil and its pharmaceutically acceptable salts. The term "Belusudil" refers to both the compound Belusudil (e.g., free base form, amorphous form or crystalline form), and the pharmaceutically acceptable salts of Belusudil (e.g., as described in REZUROCK TM The mesylate form used in the invention refers to any form of berushudil that can be used in a formulation or pharmaceutical composition for administering the compound to a patient.

[0033] "Clinical endpoint" or "study endpoint" refers to an event or outcome in a clinical trial, which can be objectively measured to determine the outcome and potential beneficial effects of a drug or administration regimen designed in a clinical trial. Examples of clinical endpoints include the following. Overall response rate (ORR) is the percentage of people with partial response (PR) or complete response (CR) to treatment in a certain period of time in a study or treatment group. Failure-free survival (FFS) refers to the time from the first dose of belusudil to the failure event, or the interval between the beginning of belusudil and the addition of a new cGVHD therapy, the recurrence of the underlying disease or non-recurrent death (NRM). Overall survival (OS) means the length of time from the diagnosis date of the disease or the start of treatment. Duration of response (DOR) means from the initial response time (e.g., PR or CR) until the time recorded from the best response progress of cGVHD, from the initial response to the start of another systemic cGVHD therapy or the time of death. Time to the next treatment (TTNT) means the time to start subsequent systemic cGVHD therapy.

[0034] "Clinically recommended amount" or "clinically recommended dose" refers to the amount or dose of an API recommended and / or approved by a person skilled in the art of medicinal chemistry for administration to a patient to treat the disease state in question following clinical trials, e.g., as described in Examples 1 and 2 herein.

[0035] "CYP3A" refers to the CYP3A family of p-450 isozymes, including CYP3A4.

[0036] "Disorder" and "disease" are used interchangeably and synonymously herein to include within the full scope of either term any condition of a subject in need of treatment.

[0037] "Glucuronides" also known as glucuronides are any substance produced by the connection of glucuronic acid to another substance via a glycosidic bond. Glucuronides belong to the class of glycosides.

[0038] "Glucuronidation" refers to the conversion of a compound into a glucuronide. It is a method used by animals to aid in the excretion of toxic substances, drugs or other substances that cannot be used as an energy source. Glucuronidation involves the metabolism of the parent compound by UDP-glucuronosyltransferase (UGT) to a hydrophilic and negatively charged glucuronide that cannot be excreted from the cell without the help of efflux transporters. The elimination of the parent compound via glucuronidation in metabolically active cells is controlled by two driving forces: the formation of glucuronides by UGT enzymes, and the (polar) excretion of these glucurons by efflux transporters located on the cell surface in various drug disposal organs.

[0039] "Immunosuppressive therapy" (IST) refers to therapy that is usually administered for at least six months after allo-HSCT in an attempt to prevent GVHD. Examples of IST include sirolimus, prednisone, and calcineurin inhibitors (such as tacrolimus and cyclosporine).

[0040] As used herein, "isolated" is synonymous with "substantially isolated". As used herein, "substantially isolated" means that the compound has been separated from the in vivo environment in which the compound is formed via a metabolic pathway. For example, when the compound is formed by a human metabolic pathway, the compound is "isolated" when the compound is no longer contained in an organ or system of the human body but is eliminated, excreted ex vivo and / or otherwise removed from the body (e.g., via a blood sample containing the compound). "Substantially isolated" includes partial separation and substantial separation from components of the metabolic pathway. For example, partial separation can include a composition or substance enriched with one or more compounds disclosed herein, for example, containing at least about 5% by weight, and in some embodiments, about 10% or at least about 15% metabolites. "Substantially isolated" can include a composition containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% metabolites by weight.

[0041] The Lee Symptom Scale (LSS) total score measures the impact on patient function and well-being. The Lee Symptom Scale is a 30-item scale developed to measure symptoms of cGVHD and is described in Lee SJ et al., Development and validation of a scale to measure symptoms of chronic graft-versus host disease. Biol Blood Marrow Transplant 2002; 8:444-452.

[0042] "Line of treatment" or "line of therapy" describes the order or sequence in which different therapies are given to a patient as his or her disease progresses. The initial treatment (first-line therapy) may not work or may stop working after a period of time. After the first-line therapy is discontinued, a second, different treatment (second-line therapy) may be given. When the second-line therapy does not work or stops working, a subsequent line of therapy may be given. Some patients may be given multiple lines of therapy during the course of their disease.

[0043] The first-line therapy for moderate to severe chronic graft-versus-host disease (cGVHD) defined by the National Institutes of Health (NIH) can be a single corticosteroid or a combination of corticosteroids and sirolimus or a calcineurin inhibitor. (Carpenter PA et al.: A phase II / III randomized, multicenter trial of prednisone / sirolimus versus prednisone / sirolimus / calcineurin inhibitor for the treatment of chronic graft-versus-host disease: BMT CTN 0801. Haematologica 103: 1915-1924, 2018).

[0044] The example of corticosteroid therapy for the treatment of cGVHD includes but is not limited to prednisone, prednisolone, methylprednisolone and budesonide. The example of previous systemic therapy for the treatment of cGVHD includes but is not limited to prednisone, tacrolimus, extracorporeal photopheresis (ECP), sirolimus, ibrutinib, ruxolitinib, mycophenolate mofetil (MMF), rituximab, methotrexate (MTX), cyclosporine, imatinib, ixazomib and ofatumumab.

[0045] "Metabolic pathway" refers to a biochemical reaction that converts (transforms) one chemical into another, such as an anabolic pathway or a catabolic pathway. Anabolic pathways involve the construction of larger molecules from smaller molecules, a process that requires energy. Catabolic pathways involve the breakdown of larger molecules, usually releasing energy.

[0046] "Metabolite" refers to an intermediate or product produced during a metabolic pathway, including those produced via anabolic pathways (larger molecules) or catabolic pathways (smaller molecules or moieties).

[0047] "Myeloablative transplant" refers to a transplant procedure that uses very high doses of chemotherapy or radiation before transplanting autologous or allogeneic hematopoietic stem cells. A nonmyeloablative transplant or reduced-intensity transplant involves the patient undergoing less intensive chemotherapy before transplanting allogeneic hematopoietic stem cells.

[0048] The “NIH Pulmonary Symptom Score” or “NIH cGVHD Pulmonary Score” is a score based on clinical symptoms ranging from 0 to 3. A score of 0 is used for no symptoms, 1 for symptoms of shortness of breath when climbing stairs, 2 for symptoms of shortness of breath on level ground, and 3 for shortness of breath at rest or requiring oxygen.

[0049] Unless the context requires otherwise, "or" is used in its inclusive sense (equivalent to "and / or").

[0050] As used herein, "patient" or "subject" includes animals or humans, and in one embodiment includes humans.

[0051] "Pharmaceutical composition" means a mixture of substances (including medicaments) suitable for administration to an individual. For example, a pharmaceutical composition may comprise a sterile aqueous solution or an API formulated in an oral dosage form such as a tablet or capsule.

[0052] "Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of the compounds provided herein. "Pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral acid or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids, etc. The pharmaceutically acceptable salts of the present invention include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent or in a mixture of water and an organic solvent; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0053] As used herein, "protocol" refers to a method or plan for administering one or more APIs to a subject in need of treatment. The term "protocol" is intended to cover the overall detailed care plan for the patient and the individual or partial steps that are part of the overall plan. For example, a regimen may include the dosage of each API that the patient will (or is) receiving, the combination of APIs that the patient receives, the administration schedule and method of each API (e.g., considering the effects that DDI, food effects, and different formulations or delivery methods may have on absorption and bioavailability) and the management of side effects, as well as an overall plan covering the dosage, combination, administration schedule and method and side effects considered together.

[0054] "Side effects" means physiological responses other than the desired effects attributable to a treatment. In certain embodiments, side effects include, but are not limited to, abnormal liver function tests, abnormal renal function, hepatotoxicity, nephrotoxicity, and myopathy. Side effects may be detected directly or indirectly. For example, elevated transaminase levels in serum may indicate hepatotoxicity or abnormal liver function. As another example, elevated bilirubin may indicate hepatotoxicity or abnormal liver function.

[0055] "Steroid-refractory" (SR) cGVHD is defined as cGVHD that progresses on the use of steroids or corticosteroids (in one embodiment, on the use of prednisone).

[0056] A "therapeutically effective amount" of an API means an amount sufficient to achieve treatment of the disease state being treated when administered to a human to treat a disease (e.g., cGVHD). As applied to human cGVHD, "treating" or "treatment" includes (1) reducing the risk of developing cGVHD and / or inhibiting cGVHD, i.e., preventing or reducing the development of cGVHD or its clinical symptoms; (2) alleviating cGVHD, i.e., causing regression, reversal or improvement of cGVHD or reducing the number, frequency, duration or severity of its clinical symptoms.

[0057] The therapeutically effective amount of the API may vary depending on the health and physical condition of the subject to be treated, the extent of disease progression, the assessment of the medical condition, and other relevant factors. It is expected that the therapeutically effective amount may fall within the range that can be determined by experimentation and by reference to clinical trial data and results as described, for example, in Examples 1 and 2 herein and in the scientific literature. Exemplary embodiments

[0058] The present disclosure provides metabolites of berushudil and / or metabolic pathways of berushudil.

[0059] In some embodiments, the present disclosure provides an isolated or synthesized compound or a pharmaceutically acceptable salt thereof, the compound comprising belushudil glucuronide, O-desalkyl belushudil sulfate, monohydroxy belushudil and / or belushudil diol.

[0060] The compounds and metabolic pathways provided by the present disclosure can be used to treat ROCK2-mediated diseases in patients, including in the treatment of autoimmune disorders such as chronic GVHD (cGVHD) and / or in the development of one or more treatments and / or regimens for treating ROCK2-mediated diseases.

[0061] In one embodiment, the disclosure includes the use of the compound 2-(3-(4-(lH-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide to treat autoimmune disorders (in some embodiments, to treat cGVHD).

[0062] In some embodiments, the present disclosure provides a compound comprising 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide for use in treating an autoimmune disorder in a subject, wherein the autoimmune disorder is cGVHD, acute GVHD (aGVHD), pulmonary fibrosis, idiopathic pulmonary fibrosis, moderate to severe psoriasis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, atopic dermatitis, or eczema.

[0063] In some embodiments, the present disclosure provides metabolites of berushudil and / or berushudil metabolic pathways for use in preparing a medicament for treating an autoimmune disease in a subject; in some embodiments, for preparing a regimen for treating an autoimmune disease in a subject.

[0064] In some embodiments, the present disclosure provides metabolites of berushudil and / or berushudil metabolic pathways for use in preparing a regimen that includes developing a unit dose (e.g., a clinically recommended unit dose) for administering an active agent to a subject; and / or includes a combination of active agents to be administered to the subject (e.g., taking into account drug-drug interactions); and / or taking into account the timing of administering one or more active agents to a subject; and / or for avoiding or minimizing one or more side effects caused by the therapeutic use of at least one active agent to a subject.

[0065] In some embodiments, the present disclosure provides compounds having the formula: wherein - represents a single bond connecting any available carbon atom within a bracketed portion of the molecule to one or more portions of an adjacent bracket; or a pharmaceutically acceptable salt thereof.

[0066] In some embodiments, the present disclosure provides an isolated compound that is berushudil glucuronide having the formula: or a pharmaceutically acceptable salt thereof; in some embodiments, the molecular weight of the berushudil glucuronide is 628.

[0067] In some embodiments, the present disclosure provides an isolated compound that is an O-dealkyl belusudil sulfate salt having the formula: or a pharmaceutically acceptable salt thereof; in some embodiments, wherein the molecular weight of the O-dealkyl belusudil sulfate is 433.

[0068] In some embodiments, the present disclosure provides an isolated compound which is monohydroxyberushedil having the formula: or a pharmaceutically acceptable salt thereof; in some embodiments, wherein the molecular weight of the monohydroxyberushedil is 468.

[0069] In some embodiments, the present disclosure provides an isolated compound that is belushidil diol having the formula: or a pharmaceutically acceptable salt thereof; in some embodiments, wherein the molecular weight of belushudil diol is 486.

[0070] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or use of the pharmaceutical composition for treating an autoimmune disorder in a subject, wherein the autoimmune disorder is chronic GVHD (cGVHD), acute GVHD (aGVHD), pulmonary fibrosis, idiopathic pulmonary fibrosis, moderate to severe psoriasis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, atopic dermatitis, or eczema.

[0071] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or the use of the pharmaceutical composition for treating an autoimmune disorder.

[0072] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or the use of the pharmaceutical composition for treating chronic GVHD (cGVHD).

[0073] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or the use of the pharmaceutical composition for treating acute GVHD (aGVHD).

[0074] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or use of the pharmaceutical composition for treating pulmonary fibrosis.

[0075] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or the use of the pharmaceutical composition for treating idiopathic pulmonary fibrosis.

[0076] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or the use of the pharmaceutical composition for treating moderate to severe psoriasis.

[0077] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or the use of the pharmaceutical composition for treating multiple sclerosis.

[0078] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or use of the pharmaceutical composition for treating systemic lupus erythematosus (SLE).

[0079] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or the use of the pharmaceutical composition for treating Crohn's disease.

[0080] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or the use of the pharmaceutical composition for treating atopic dermatitis.

[0081] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the aforementioned compounds, or use of the pharmaceutical composition for treating eczema.

[0082] In some embodiments, the subject receiving the treatment has undergone an allogeneic hematopoietic stem cell transplant as a matched HSCT. In some embodiments, the allogeneic hematopoietic stem cell transplant is a haploidentical HSCT.

[0083] In some embodiments, based on the patient's tolerance, continue berushedil treatment until active cGVHD symptoms subside or progress. The number of cycles and duration of treatment depend on the patient. In some embodiments, berushedil is administered to the patient in one or more 28-day cycles.

[0084] In some embodiments, the range of cycle number is 3 to 15. In some embodiments, the range of cycle number is 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, or 3 to 4. In some embodiments, the range of cycle number is 5 to 11. In some embodiments, the range of cycle number is 6 to 12. In some embodiments, the range of cycle number is 5 to 10, 5 to 9, or 5 to 8. In some embodiments, the range of cycle number is 5 to 7. In some embodiments, the range of cycle number is 5 to 6. In some embodiments, the number of cycles is 5. In some embodiments, the number of cycles is 6. In some embodiments, the number of cycles is 7. In some embodiments, the number of cycles is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0085] In some embodiments, the number of cycles ranges from 3 cycles to loss of response. In some embodiments, the number of cycles ranges from 4 cycles to loss of response. In some embodiments, the number of cycles ranges from 5 cycles to loss of response. In some embodiments, the number of cycles ranges from 6 cycles to loss of response. In some embodiments, the number of cycles ranges from 7 cycles to loss of response. In some embodiments, the number of cycles ranges from 8 cycles to loss of response. In some embodiments, the number of cycles is greater than 3, 4, 5, 10, 15, 20, 25, or 30, or until the desired response is achieved.

[0086] In some embodiments, the subject experiences an improvement defined by the Lee Symptom Scale (LSS). In some embodiments, the subject experiences a reduction in LSS score of at least 7 points. In some embodiments, the subject experiences a reduction in LSS score of at least 10 points. In some embodiments, the improvement is maintained in at least two consecutive evaluations. In some embodiments, the LSS score is evaluated at baseline and on the first day of each cycle starting from the first day of the second cycle.

[0087] In some embodiments, the subject has chronic graft-versus-host disease and has failed one to three prior lines of systemic therapy for chronic graft-versus-host disease. In some embodiments, the subject has chronic graft-versus-host disease and has failed at least two prior lines of systemic therapy for chronic graft-versus-host disease. In some embodiments, the subject has chronic graft-versus-host disease and has failed two to five prior lines of systemic therapy for chronic graft-versus-host disease. In some embodiments, the subject has failed at least one, at least two, at least three, at least four, or at least five prior lines of systemic therapy for chronic graft-versus-host disease.

[0088] In some embodiments, the subject experienced a complete response to the last treatment for graft-versus-host disease prior to berushudil. In some embodiments, the subject experienced a partial response to the last treatment for graft-versus-host disease prior to berushudil. In some embodiments, the subject experienced stable disease during the last graft-versus-host disease treatment prior to berushudil.

[0089] In some embodiments, a prior line of systemic therapy for chronic graft-versus-host disease has been discontinued.

[0090] In some embodiments, the prior line of systemic therapy is selected from prednisone, tacrolimus, ECP, sirolimus, ibrutinib, ruxolitinib, MMF, rituximab, MTX, cyclosporine, imatinib, ixazomib, and ofatumumab.

[0091] In some embodiments, the cGVHD is steroid refractory (SR) cGVHD.In some embodiments, the subject is refractory to the last line of treatment prior to berushudil treatment.

[0092] In some embodiments, the subject is receiving concomitant corticosteroid therapy. In some embodiments, the concomitant corticosteroid therapy is selected from prednisone, prednisolone, methylprednisolone and budesonide. In some embodiments, the concomitant corticosteroid therapy is prednisone. In some embodiments, the dosage of the concomitant corticosteroid therapy is reduced after at least 1 cycle of Belushudil treatment. In some embodiments, the dosage of the concomitant corticosteroid therapy is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% or at least about 70% after at least 1 cycle of Belushudil treatment. In some embodiments, the dose of concomitant corticosteroid therapy is reduced by at least about 10% to about 70%, about 15% to about 65%, about 20% to about 60%, about 30% to about 60%, about 35% to about 60%, about 40% to about 60%, or about 45% to about 55% after at least 1 cycle of belushudil treatment. In some embodiments, concomitant corticosteroid therapy is discontinued after at least 1 cycle of belushudil treatment.

[0093] In some embodiments, the subject is receiving concomitant calcineurin inhibitor therapy.

[0094] In some embodiments, the subject has at least 4 organs affected. In some embodiments, the subject has at least 3 organs affected. In some embodiments, the subject has at least 2 organs affected. Composition and tablets

[0095] According to another aspect, pharmaceutical compositions comprising the compounds described herein as active ingredients are disclosed. These pharmaceutical compositions contain an effective dose of at least one compound disclosed herein or a pharmaceutically acceptable salt of said compound, and also at least one pharmaceutically acceptable excipient.

[0096] The excipients are selected according to the desired pharmaceutical form and the method of administration from among the customary excipients known to those skilled in the art.

[0097] In these pharmaceutical compositions for oral, parenteral (including subcutaneous, intramuscular, intradermal and intravenous), transdermal, bronchial or nasal administration, the active ingredient of the above-mentioned formula (I) or its salt in a mixture with conventional pharmaceutical excipients can be administered to animals and humans in a unit administration form to prevent or treat the following disorders or diseases.

[0098] The pharmaceutical compositions described herein and / or used in the methods described herein may further comprise a pharmaceutically acceptable carrier / excipient. In some embodiments, the pharmaceutically acceptable carrier means a pharmaceutically acceptable substrate, material, composition or vehicle, such as a diluent, solid filler, excipient or manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate or zinc stearate, or stearic acid), to assist in the process of delivering the API to the patient and / or to stabilize the API during transportation to the patient. The term "acceptable" as used in this sense means that the material is compatible with the other ingredients of the formulation and does not produce intolerable side effects that are harmful to the patient.

[0099] Pharmaceutical compositions contemplated herein include suitable dosage forms for oral, parenteral (including subcutaneous, intramuscular, intradermal and intravenous), transdermal, bronchial or nasal administration.

[0100] In solid pharmaceutical dosage forms for oral administration (capsules, tablets, pills, powders, granules, etc.), the API may be mixed with at least one pharmaceutically acceptable carrier (including one or more pharmaceutically acceptable excipients).

[0101] In one embodiment, the compound is formulated into tablets for oral administration. Belushudil mesylate is a yellow powder that is almost insoluble in water. Belushudil tablets can be prepared for oral administration. Each tablet contains 200 mg of free base, equivalent to 242.5 mg of belushudil mesylate. The tablets may also contain the following inactive ingredients: microcrystalline cellulose, hypromellose, cross-linked carboxymethyl cellulose sodium, colloidal silicon dioxide, and magnesium stearate. The tablet film is composed of polyvinyl alcohol, polyethylene glycol, talc, titanium dioxide, and yellow iron oxide. Each 200 mg tablet is a light yellow film-coated oval tablet with a concave pattern "KDM" on one side and a concave pattern "200" on the other side. The tablets are stored at room temperature 20°C to 25°C (68°F to 77°F); short-term travel allows 15°C to 30°C (59°F to 86°F).

[0102] The compounds and pharmaceutical compositions disclosed herein are useful for inhibiting ROCK1 and / or ROCK2 enzymes (preferably ROCK2), and are therefore useful for treating diseases regulated by ROCK enzymes, such as autoimmune disorders and / or fibrotic disorders, including, among other indications, GVHD (chronic and acute), pulmonary fibrosis, idiopathic pulmonary fibrosis, moderate to severe psoriasis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, dermatitis (e.g., atopic dermatitis), and eczema.

[0103] The following abbreviations may be helpful in considering the embodiments and descriptions herein. abbreviation AE Adverse Events AMS Accelerator mass spectrometry alloHCT Allogeneic hematopoietic cell transplantation BID Twice a day (twice daily) BM marrow cGVHD Chronic graft-versus-host disease CMV Cytomegalovirus CR Complete reaction DDI Drug-drug interactions DOR Reaction duration EOI End of infusion FFS Failure-free survival HLA Human leukocyte antigen IST Immunosuppressive therapy IV Intravenous LSC Liquid scintillation counting LSS Lee Symptom Scale NMT No more than ORR Overall response rate OS Overall survival PBSC Peripheral blood stem cells PPI Proton pump inhibitors PR Partial reaction QOL Quality of Life SD Standard Deviation SR Steroid refractory TEAE Adverse events during treatment TTNT Time until next treatment QD every day Example Example 1: About [ 14 A Phase I, open-label, 2-part study evaluating the absolute bioavailability, mass balance, and metabolic profiling of berushudil in healthy subjects

[0104] In this phase 1, two-part study, the pharmacokinetics, mass balance, and metabolic profile of belusudil were evaluated after subjects were administered a single dose of unlabeled belusudil oral tablets (200 mg), radiolabeled belusudil intravenous (IV) microtrace infusion (100 μg), and radiolabeled oral capsules (200 mg). The study was conducted in 2 parts with the following objectives: (1) determine the absolute bioavailability of belusudil (Part 1), (2) determine the mass balance recovery after a single oral dose of radiolabeled belusudil (Part 2), and (3) perform metabolite profiling and structural elucidation of identified metabolites (Part 2).

[0105] The absolute bioavailability based on the area under the plasma concentration-time curve from time 0 to infinity for the oral dose / area under the plasma concentration-time curve from time 0 to infinity for the IV dose was calculated to be 63.7%. Radiolabeled IV micro-trace administration demonstrated low extraction rates and partitioning of belushudil in tissues. Most of the total radioactivity was recovered in the feces, with a small amount recovered in the urine, indicating minimal renal elimination of belushudil. Study Design

[0106] This was a single-center, randomized, open-label, 2-part study in 5 healthy male subjects aged 30 to 65 years. All oral doses were administered after an overnight fast and a standard breakfast on the day of dosing.

[0107] For the absolute bioavailability evaluation (Part 1), subjects were administered a single oral dose of 200 mg berushidil tablets (therapeutic dose) followed 1.75 hours later by a 15-minute IV infusion containing no more than (NMT) 37 kBq [ 14 C] 100 μg [ 14 C]-berushedil (timed to occur at the expected maximum concentration [Cmax]). To determine the absolute bioavailability, a micro-trace dose was chosen because it does not require an IV formulation with good stability. In addition, due to the use of very low doses, further nonclinical studies such as local tolerance and general toxicology studies were not warranted.

[0108] For the mass balance and metabolic profiling study (Part 2), after a washout of at least 7 days from Part 1, subjects received a single oral dose of NMT containing 9.8 MBq [14 200mg of C 14 C]-Berushudil capsules. This level of radioactivity was selected based on human dosimetry calculations performed by Public Health England, which determined that an oral dose of NMT 9.8MBq of radioactivity was necessary to allow adequate metabolite profiling of the primary sample. Taking into account the microtracer dose in Part 1, the total agreed effective dose received by the subjects in the study was 3mSv, which falls within the International Commission on Radiological Protection Guidelines for Category IIb studies. In addition, in order to remain within the limits of radioactive doses approved by the UK Administration of Radioactive Substances Advisory Committee, [ 14 C]-Target-specific activity of berushudil was set at 90% of the threshold radioactivity dosing limit. Pharmacokinetic sample collection and analysis Part 1: Blood sampling.

[0109] Blood samples for oral berushudil PK analysis were collected in tripotassium ethylenediaminetetraacetic acid tubes before and 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, and 48 hours after dosing. Blood samples for analysis of IV [ 14 [C]-Belusudil PK and total radioactivity. All samples were processed to plasma within 30 minutes of collection by centrifugation at 2000 g for 10 minutes at 4°C and then frozen at -70°C or below prior to analysis. Part 2 Blood sampling.

[0110] Whole blood samples (for total radioactivity) and plasma samples (for metabolite spectrum analysis and identification) were collected before and after administration at 1, 4, 8, 12, 24, 48, 72, 96, 120, 144 and 168 hours. Blood samples were collected at the same time points for plasma berushudil, KD025m1, KD025m2 and total radioactivity analysis, wherein additional samples were collected at 0.5, 1.5, 2, 3, 5, 6, 8, 10 and 36 hours after administration. Whole blood samples for total radioactivity were collected in tripotassium ethylenediaminetetraacetic acid tubes and stored at 2°C to 8°C to be analyzed. Blood samples for plasma belusudil, KD025m1, KD025m2 and total radioactivity quantification and metabolite profiling / identification were processed similarly to Part 1 and stored frozen at -20°C (belusudil, KD025m1, KD025m2 and total radioactivity samples) or -70°C (metabolite profiling samples). Part 2 Urine and feces sampling

[0111] Urine for total radioactivity and metabolite spectrum analysis and identification was collected before dosing (the first void of the day), and from 0 to 6, 6 to 12 and 12 to 24 hours after dosing, and then collected every day (24 hours) until the 8th day / discarded. Urine samples were collected according to intervals and stored at 2°C to 8°C for analysis. Feces were collected from admission until before dosing and then collected every day (24 hours) until discarded. Fecal samples were collected according to time intervals and stored at -20°C for analysis. Analytical methods

[0112] Part 1. Berushudil plasma samples were prepared for analysis using a protein precipitation method and quantified using a validated liquid chromatography-tandem mass spectrometry (LC-MS / MS) method with a lower limit of quantification (LLOQ) of 10 ng / mL.

[0113] The total [ 14 C]-Belusudil concentration. Prior to AMS analysis, selected samples were analyzed by liquid scintillation counting (LSC) to ensure that the radioactivity level would not saturate the AMS. Selected samples were diluted with commercial plasma to ensure that the concentration fell within the acceptable range for AMS analysis. Samples were burned to CO 2 It was then converted to graphite by reduction to graphite. The graphite targets were then analyzed by AMS (LLOQ for individual subjects was 0.00489, 0.00557, 0.00485, 0.00402, and 0.00372 ng equivalent / mL). 14 C data are based on pre-dose [ 14 C]-Berushudil concentrations were background corrected.

[0114] The quantification was performed by high performance liquid chromatography coupled with accelerated mass spectrometry (HPLC AMS). 14 C]-berushudil concentration. 19 Radiolabeled belusudil in plasma was prepared for sample analysis using a protein precipitation method and subsequently subjected to HPLC. The corresponding concentrations of [ 14 C]-elution fractions of Berushudil.

[0115] Part 2. As in Part 1, berushudil, KD025m1 and KD025m2 plasma concentrations were quantified by LC-MS / MS.

[0116] Total radioactive whole blood samples, plasma samples and urine samples were prepared for LSC by mixing with scintillation liquid. In addition, aliquots of whole blood were dissolved with AquiGest tissue dissolving liquid, and plasma samples were mixed with 10mM phosphate buffered saline. Urine and feces samples were collected by subject at time intervals before analysis. In addition, feces samples were burned using automatic sample oxidant before LSC. Radioactivity (2300TR, 2900TR and 3100TR Tri-Carb, scintillation counter; PerkinElmer, Waltham, Massachusetts) in all samples was measured using LSC with automatic external standard quenching correction, and samples were counted for 4 minutes. LLOQ was considered to be twice the background value of the same type of sample (1.39 [urine], 23.9 [feces], 35.5 [whole blood] and 30.9 [plasma] ng equivalent [free alkali] / g or mL).

[0117] In order to determine the most appropriate sampling pool for metabolite identification / analysis, plasma, urine and feces samples were collected based on the mass balance data obtained as described above. Plasma samples representing at least 80% of the total radioactivity in plasma were collected by time point, and plasma samples were collected across subjects before dosing and at 4 and 8 hours. Urine samples were collected from individual subjects and across subjects before dosing and at intervals of 0 to 6 hours, from 0 to 24 hours. Feces samples representing at least 80% of the total radioactivity in plasma were collected by time point, and feces samples were collected across subjects at intervals of 0 to 24 and 24 to 48 hours.

[0118] Quantification by LC-MS / MS 14C]-berushedil concentration. The chemical structures of those radioactive components accounting for >10% of the circulating radioactivity in plasma and >10% of the dose in urine and feces were then identified. Radioactive components representing <10% of the circulating radioactivity in plasma and <10% of the dose in urine and feces were also identified. Metabolite identification was then performed using accurate mass positive and negative ion full scan and positive ion product ion analysis. Samples were screened for the presence of: berushedil, metabolites KD025m1, KD025m2, O-dealkylated berushedil, any potential metabolites resulting from O-dealkylation and hydroxylation, and stage 2 conjugated metabolites. Any identified components were compared with [ 14 C] radiochromatograms were compared to identify metabolites. Pre-dose plasma and urine pools were used to correct for endogenous components during metabolite identification. Pharmacokinetic evaluation

[0119] The total plasma radioactivity, berushedil, [ 14 C]-PK parameters of berushedil (part 1 only) and KD025m1 and KD025m2 (part 2 only) (Certara, Princeton, NJ). PK parameter estimates were used to calculate absolute bioavailability and metabolite to parent ratios.

[0120] The PK parameter calculated in this study was the absolute bioavailability of berushudil (as measured by the area under the dose-adjusted plasma concentration-time curve (AUC) from time 0 to infinity for oral doses). inf,口服 ) / AUC from time 0 to infinity for the IV dose (AUC inf,IV )); mass balance recovery of total radioactivity in all excreta (as assessed by amount eliminated [Ae], %Ae, cumulative Ae, and cumulative %Ae); oral berushedil, KD025m 1 and KD025m 2 and various PK parameters of total radioactivity; IV[ 14 C]-PK parameters of berushudil; [ 14 C] Pathways and rates of elimination (plasma and excreta); total radioactivity whole blood:plasma ratios; and structural identification of metabolites in plasma and excreta.

[0121] The PK population included all subjects who received at least 1 dose of berushedil in Part 1 or Part 2 and provided sufficient data for at least 1 PK characteristic, provided that the subjects met the following criteria: (1) no missing samples at key time points, (2) no protocol deviations that affected PK endpoints, and (3) no adverse events (AEs) (e.g., vomiting or infusion site reactions) indicating that the entire dose was not delivered to the subject.

[0122] The mass balance population included all subjects who received at least 1 dose of berushudil in Part 2 with evaluable total radioactivity concentrations in urine / feces samples and had no protocol deviations that could have affected the mass balance analysis. Safety Assessment

[0123] Safety was assessed throughout the study by AEs, vital signs, electrocardiograms, physical examinations, and clinical laboratory tests.The safety population included all subjects who received at least 1 dose of study drug (oral or IV) in any part of the study. result Subject Disposition

[0124] Five male subjects were recruited and administered to the subjects in both Part 1 and Part 2. All subjects completed the study and were included in the PK population and mass balance population. Baseline demographics are summarized in Table 1. The subjects were between 41 and 64 years old (mean age, 53.0 years old), and most subjects were Caucasians. All subjects were within the protocol-defined reference range for body mass index. Table 1: Subject Demographics Pharmacokinetic evaluation Part 1: Absolute bioavailability of berushudil.

[0125] A single 200 mg oral dose of belusudil followed by 15 minutes [ 14 The mean PK profile of [C]-berushudil IV infusion is shown in Figure 2 (unlabeled berushedil) and Figure 3 ([ 14 C]-berushudil). In part 1 of the study, berushudil, [ 14 The PK parameters of [C]-berushudil and total radioactivity calculated are presented in Table 2 . Table 2: Summary of berushudil and total radiopharmacokinetic parameters in Parts 1 and 2

[0126] For IV[ 14 C]-berushudil infusion reached a maximum radiolabeled concentration at EOI and then declined in a biphasic manner. Radiolabeled concentrations were quantifiable until the final sampling time point (46 hours after EOI), and the terminal half-life was calculated for all subjects (geometric mean, 5.9 hours).

[0127] Peak berushedil concentrations following oral administration were reached 3 hours after oral administration and then declined in a biphasic manner over 48 hours. The terminal half-life was reported for all 5 subjects (geometric mean, 5.3 hours). inf,oral / AUC inf,IV ) is calculated as 63.7%. Part 2: Mass balance and excretion.

[0128] Most of the radioactivity (88.5%) was recovered in excreta, with 84.6% recovered in feces and <5% of the radiolabeled dose recovered in urine. Approximately 27.4% and 3.8% of the total radioactivity were recovered in feces and urine, respectively, within the first 24 hours after dosing. The range of total radioactivity concentrations in whole blood:plasma was 0.53 to 1.16. The PK parameters of belusudil and total radioactivity in Part 2 are summarized above in Table 2. Part 2: Metabolite identification and profiling. An attempt was made to structurally identify the radioactive components that accounted for 10% of the circulating radioactivity in plasma and >10% of the dose in feces. The belusudil metabolites identified in plasma, urine and feces are presented in Table 3. There was no single radioactive component that accounted for >10% of the dose in urine. In plasma, belusudil accounted for 64% of the total plasma radioactivity; KD025m2 and the previously unidentified second phase metabolite O-desalkyl belusudil sulfate combined were 11.5% of the total plasma radioactivity. Another metabolite, belusudil glucuronide, which has not yet been measured in clinical studies, accounted for 15% of the total plasma radioactivity. In feces, belusudil was 30% and KD025m2 and O-desalkyl belusudil sulfate combined accounted for 35% of the total sample radioactivity. Previously unidentified belusudil diol (belusudil +2O, +2H) and monohydroxy belusudil were 10% and 11% in feces, respectively. Table 3. Metabolite identification in plasma, urine and feces NA, component not represented or below the threshold required for metabolite identification; %ROI, mean % contribution of this region of interest to total sample radioactivity (across subjects). P, U, and F represent HPLC eluates in plasma, urine, and feces samples, respectively. Bold indicates >10% of circulating radioactivity in at least 1 plasma sample or >10% of dose in at least 1 fecal sample. a Molecular weights are monoisotopic and refer to the unlabeled component. b Nominal retention times of accurate mass extracted ion chromatograms of plasma or fecal samples. c For c-eluting fractions, %ROI refers to the total contribution of all fractions.

[0129] The chemical structures of the metabolite KD025m1 of berushudil discovered in this study and previously unidentified metabolites can be shown as follows:

[0130] In general, when taken as a 200 mg tablet then [ 14 C]-Belusudil micro-tracking infusion (Part 1) and 200 mg [ 14 C] Berushudil capsules (Part 2) When administered to healthy subjects, the safety profile of Berushudil was as expected. There were no deaths, serious AEs, AEs of severe intensity, or AEs that led to subject withdrawal in any part of this study. One treatment-emergent AE (mild, possibly related rash) was recorded in Part 1, while no treatment-emergent AEs were reported in Part 2. The event resolved spontaneously 2.5 hours after occurrence. No clinically significant laboratory, vital sign, electrocardiogram, or physical examination findings were reported after administration in any part of the study.

[0131] This study evaluated the absorption, metabolism, and excretion properties of berushedil in humans following oral doses of unlabeled tablets and radiolabeled capsules as well as IV microtracing of radiolabeled berushedil in healthy male subjects. As described in the Methods section, the microtracing approach was employed to accurately characterize the IV PK profile without encountering dose-dependent kinetics or conducting additional toxicology studies with the IV formulation. The use of a highly sensitive AMS method allows for precise measurement of [ 14 C]-belusudil to allow maximum data acquisition, while quantification of unlabeled belusudil occurred by standard HPLC-MS / MS. 14 C] tag, as it can be easily introduced into metabolically stable positions in the berushedil skeleton, thus enabling mass balance determination and metabolite profiling. 14 The synthesis and isolation of [C]-berushudil relies on the same chemical processes used in the production of berushudil.

[0132] In Part 1, after oral administration of a commercially available 200 mg berushudil tablet, the dose-adjusted AUC was estimated. inf,口服 / AUC inf,IV The absolute bioavailability of is 63.7%. 14 C]-berushedil, the total clearance after IV administration was lower than typical hepatic blood flow rates, suggesting a low extraction ratio (approximately equal to 0.3). The apparent volume of distribution was higher than total body water, suggesting that [ 14 C]-Berushudil is distributed into tissues. The half-life values ​​after IV and oral administration are comparable, approximately equal to 5 to 6 hours, and are similar to those observed in earlier clinical trials.

[0133] In part 2, 200 mg [ 14 After administration of [C]-berushedil capsules, most of the total radioactivity (85%) was recovered in the feces and <5% was recovered in the urine, indicating minimal renal elimination of berushedil. Based on the absolute bioavailability of 63.7% in Part 1 and the low recovery of radioactivity in urine, [ 14 C]-Berushudil and related metabolites are likely to be primarily eliminated through the bile and / or intestine. The total radioactivity whole blood:plasma ratios ranged from 0.53 to 1.16, indicating that there was little preferential distribution of total radioactivity into the cellular fraction of whole blood.

[0134] The concentrations of berushedil, KD025m2, and KD025m1 present in the total circulating radioactivity in plasma in Part 2 suggest that other circulating metabolites may be present in plasma. In addition, the half-life of the total radioactivity is longer than that of berushedil (geometric means of 18.0 and 5.3 hours, respectively), which further supports the presence of other metabolites with longer elimination periods. Indeed, using standard LC-MS / MS metabolite profiling techniques, metabolites glucuronide and sulfate derivatives other than berushedil diol were identified in humans for the first time in this study. The metabolic pathway of berushedil in humans was developed through this study and is presented in Figure 3 middle.

[0135] In addition to the parent, the major metabolite KD025m2, and the active minor metabolite KD025m1, other metabolites were identified in plasma as O-desalkyl belusudil sulfate and belusudil glucuronide. In feces, the parent, KD025m2, O-desalkyl belusudil sulfate, monohydroxy belusudil, and belusudil diol were identified as metabolites accounting for >10% of the radioactive dose. No metabolites accounting for >10% of the dose were found in urine. Example 2: In vitro studies and inhibition of ROCK1 and ROCK2 enzymatic activity by berushudil and metabolites

[0136] The effects of berushudil and the major metabolites (KD025m1 and KD025m2) in inhibiting ROCK2 enzymatic activity were evaluated in enzymes purified from cell supernatants or tissue homogenates.

[0137] Full-length rat ROCK1 and ROCK2 proteins were isolated from various tumor cell lines or tissues by immunoadsorption to 24-well microtiter plates pre-coated with antibodies specific for the various isozymes. Reactions were initiated by the addition of S6 kinase substrate peptide ([γ33P]ATP) and study drug and incubated at 37°C for 20 minutes. Reactions were terminated by the addition of phosphoric acid and radioactivity was quantified using standard methods.

[0138] The results showed that berushudil inhibited ROCK2 enzymatic activity with an inhibitory effect approximately 30-fold higher than that of ROCK1; i.e., IC 50 Its inhibitory effect on ROCK1 is less than one-thirtieth.

[0139] The results further show the inhibitory activity of belusudil metabolites. Table 4 shows the order of inhibitory effects of belusudil (KD025), KD025m1 and KD025m2 on the ROCK enzyme. Fasudil and Y-27362 are included as reference ROCK inhibitors. Metabolite KD025m1 inhibits ROCK2 with a comparable Ki value to belusudil, while the second metabolite KD025m2 inhibits ROCK2 by about one-sixth. Table 4. Inhibition of ROCK 2 by berushudil and its metabolites KD025m1 and KD025m2

[0140] Although the present invention has been described in considerable detail by way of illustration and example for purposes of clarity and understanding, the description and example should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. A compound comprising 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide for use in treating an autoimmune disorder in a subject, wherein the autoimmune disorder is chronic graft-versus-host disease (cGVHD), acute GVHD (aGVHD), pulmonary fibrosis, idiopathic pulmonary fibrosis, moderate to severe psoriasis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, atopic dermatitis, or eczema.

2. The compound for use according to claim 1, wherein the autoimmune disorder is chronic GVHD (cGVHD).

3. An isolated or synthesized compound comprising belushudil glucuronide, O-desalkyl belushudil sulfate, monohydroxy belushudil or belushudil diol or a pharmaceutically acceptable salt thereof.

4. The compound according to claim 3, having the following formula: wherein - represents a single bond connecting any available carbon atom within a bracketed portion of the molecule to one or more portions of an adjacent bracket; or a pharmaceutically acceptable salt thereof.

5. The isolated compound according to claim 4, which is berushudil glucuronide having the formula: or a pharmaceutically acceptable salt thereof.

6. The compound according to claim 5, characterized in that Its molecular weight is 628.

7. The isolated compound according to claim 4, which is an O-dealkyl belusudil sulfate having the formula: or a pharmaceutically acceptable salt thereof.

8. The compound according to claim 7, characterized in that Its molecular weight is 433.

9. The isolated compound according to claim 4, which is monohydroxyberushidil having the formula: or a pharmaceutically acceptable salt thereof.

10. The compound according to claim 9, characterized in that Its molecular weight is 468.

11. The isolated compound according to claim 4, which is belushudil diol having the formula: or a pharmaceutically acceptable salt thereof.

12. The compound according to claim 11, characterized in that Its molecular weight is 486.

13. The isolated compound according to any one of claims 3 to 12, characterized in that Its molecular weight or retention time is shown in Table 3, or a pharmaceutically acceptable salt thereof.

14. A compound according to any one of claims 3 to 13 for use in treating an autoimmune disease in a subject.

15. The compound for use according to claim 14, wherein the autoimmune disease is chronic GVHD (cGVHD).

16. A pharmaceutical composition comprising a compound according to any one of claims 3 to 13 and a pharmaceutically acceptable carrier.

17. Use of a compound according to any one of claims 3 to 13 or a pharmaceutical composition according to claim 16 for treating an autoimmune disorder in a subject, wherein the autoimmune disorder is chronic GVHD (cGVHD), acute GVHD (aGVHD), pulmonary fibrosis, idiopathic pulmonary fibrosis, moderate to severe psoriasis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), Crohn's disease, atopic dermatitis or eczema.

18. The use according to claim 17, wherein the autoimmune disorder is pulmonary fibrosis or chronic GVHD (cGVHD).

19. Use of a compound according to any one of claims 3 to 13 or a metabolic pathway of berushudil in the preparation of a medicament for treating an autoimmune disease in a subject.

20. Use of a compound according to any one of claims 3 to 13 or a metabolic pathway of berushedil in the preparation of a regimen for treating an autoimmune disease in a subject.

21. A compound comprising 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide or 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetic acid for use in the preparation of a medicament for treating an autoimmune disorder in a subject.

22. A compound comprising 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide or 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetic acid for use in the preparation of a regimen for treating an autoimmune disease in a subject.

23. The use according to any one of claims 19 to 22, comprising preparing a unit dose of the active agent for administration to a subject taking into account drug-drug interactions.

24. The use according to any one of claims 19 to 22, comprising the preparation of a medicament for treating a disorder in a subject, wherein the medicament contains a combination of active agents to be administered to the subject.

25. The use according to any one of claims 20 or 22, comprising preparing a regimen for the timing of administering one or more active agents to a subject.

26. The use according to any one of claims 20 or 22, comprising preparing a regimen for avoiding one or more side effects caused by the therapeutic use of at least one active agent to a subject.

27. The use according to any one of claims 23 to 26, wherein the active agent is berushudil.

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