Use of Vermostil metabolites in the treatment of chronic graft-versus-host disease and thereof

Abstract

The present disclosure provides a metabolite of vermostil and the use of the metabolite in the treatment of patients having a ROCK2-mediated disease including cGVHD.

Description

Technical Field

[0001] The present disclosure relates to metabolites of vermostilbene and the use of said metabolites for treating autoimmune disorders in patients, including 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 potential serious complication after solid organ transplantation and allogeneic hematopoietic cell transplantation (alloHCT). cGVHD affects up to 70% of all alloHCT recipients and has an incidence rate of 20% - 50% in children. It is the main cause of non-relapse mortality more than 2 years after alloHCT. The estimated prevalence of cGVHD is 14,000 patients in the United States (as of 2016). (Presented at ASH 2019, Orlando, FL, December 7 - 10, 2019 by Bachier CR et al: Epidemiology and real-world treatment of chronic graft-versus-host disease post allogeneic hematopoietic cell transplantation: A US claims analysis.) (“Bachier et al.”).

[0003] Patients with cGVHD have substantial impairments in quality of life (QOL), as evaluated by the Lee Symptom Scale (LSS) that measures the impact of cGVHD on patient function and well-being. Only one-third of patients with cGVHD who initiate systemic treatment are reported to survive, achieve remission, and discontinue immunosuppressive therapy by 5 years. (Lee SJ et al: Success of immunosuppressive treatments in patients with chronic graft-versus-host disease. Biol Blood Marrow Transpl 24:555 - 562, 2018) (“Lee et al.”).

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

[0005] The first-line treatment for National Institutes of Health (NIH)-defined moderate to severe chronic graft-versus-host disease (cGVHD) is corticosteroids alone or corticosteroids in combination with sirolimus or calcineurin inhibitors. However, up to 70% of patients require additional treatment lines (Bachier CR et al). Furthermore, 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 emergence of targeted therapies. cGVHD is characterized by the overproduction of the pro-inflammatory cytokines IL-21 and IL-17, and the overactivation of follicular helper T cells and B cells, which leads to the overproduction of antibodies.

[0007] In 2017, the US Food and Drug Administration approved the Bruton tyrosine kinase inhibitor ibrutinib for the treatment of adults with cGVHD after the failure of one or more prior systemic therapies. In patients with cGVHD who are required to have either an erythematous rash of more than 25% total body surface area or an NIH mouse score of more than 4, a trial using ibrutinib reported an overall response rate (ORR) of 67% and a discontinuation rate due to treatment-emergent adverse events (TEAE) of 43%. (Waller EK, et al: Ibrutinib for chronic graft-versus-host disease after failure of prior therapy: 1-Year update of a phase 1b / 2 study. Biol Blood Marrow Transpl 25:2002-2007, 2019).

[0008] There remains an opportunity to test other treatment options for patients with cGVHD, including those who have failed treatment for 1 or more lines.

SUMMARY OF THE INVENTION

MEANS FOR SOLVING THE PROBLEM

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

[0010] The present disclosure further provides belumosudil metabolites in isolated form or pharmaceutically acceptable salts thereof, and their use for treating autoimmune disorders and in some embodiments for treating cGVHD. The present disclosure provides isolated or synthetic compounds including belumosudil glucuronide, O-demethylated belumosudil sulfate, monohydroxy belumosudil and / or belumosudil diol, or pharmaceutically acceptable salts thereof.

[0011] The present disclosure further provides a metabolic pathway of belumosudil for use in preparing a pharmaceutical for treating an autoimmune disease in a subject. The use of the belumosudil metabolites and / or the metabolic pathway of belumosudil disclosed herein is useful for preparing a protocol for the treatment of autoimmune diseases in a subject.

[0012] This embodiment can be more fully understood by reference to the detailed description and examples that are intended to illustrate, but not limit, the embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0014] Overview Belumosudil is an oral selective rho-associated coiled-coil containing protein kinase 2 (ROCK2) inhibitor. ROCK2 inhibition acts on the dysregulated adaptive immune system and fibrosis resulting from abnormal tissue repair. Belumosudil inhibits ROCK2 and ROCK1 at IC 50 values of approximately 100 nM and 3 μM, respectively. Belumosudil downregulated pro-inflammatory responses via modulation of STAT3 / STAT5 phosphorylation and a shift in the Th17 / Treg balance in ex vivo or in vitro human T cell assays. Belumosudil also inhibited abnormal profibrotic signaling in vitro. In vivo, belumosudil showed activity in an animal model of chronic GVHD.

[0015] The compound belumosudil has the chemical name: 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide. The compound belumosudil is also known as KD025. The mesylate salt of belumosudil is marketed as REZUROCK™ in the United States and other countries for the treatment of patients with chronic GVHD (cGVHD), optionally after failure of at least two prior lines of systemic therapy. The active pharmaceutical ingredient of REZUROCK™ has the molecular formula C 27 H28 It is bermosdil mesylate having N6O5S and having the chemical name 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamidomethanesulfonate (1:1).

[0016] The chemical structure of bermosdil mesylate is as follows.

Chemical formula

[0017] The chemical structure of the radiolabeled bermosdil used in the test of Example 1 has the following structure.

Chemical formula

[0018] The manufacturing methods of bermosdil and 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.

[0019] By controlling ROCK2 activity, bermosdil mediates signal transduction in immune cell function and the fibrotic pathway, thereby alleviating the effects caused by this wasting disease, such as inflammation and fibrotic changes in multiple tissues involving several organs including the lungs, hepatobiliary system, musculoskeletal system, gastrointestinal (GI) tract, and skin.

[0020] In vitro evaluation suggested that the metabolism of bermosdil mainly depends on cytochrome P450 CYP3A4 activity and the solubility of bermosdil is pH-dependent. Preclinical studies in mice, rats, rabbits, and dogs showed that bermosdil undergoes liver metabolism to form two major metabolites called KD025m1 (minor metabolite) and KD025m2 (major metabolite).

[0021] KD025m1 has the chemical name 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide and has the following structure: [Chemical formula] It has the following structure.

[0022] KD025m2 has the chemical name 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetic acid and has the following structure: [Chemical formula] It has the following structure.

[0023] KD025m1 and KD025m2 were quantified in clinical trials throughout the Velmosdil development program. The exposure of the major metabolite KD025m2 was 15% - 20% of the parent, while the exposure value of the minor, more active metabolite KD025m1 was less than 5% of the parent.

[0024] Human mass balance studies using radiolabeled compounds are important for understanding the pharmacokinetic (PK) properties of investigational or clinical drugs. Mass balance studies are conducted to identify and quantify metabolites and to determine the characteristics of elimination pathways. One approach for determining absolute bioavailability is the use of microtracer doses.

[0025] Understanding the metabolic pathways of investigational or clinical drugs is important in confirming the effects of the molecules on patients, including the energy consumed and generated through the metabolic pathways and the synthesized molecules and by-products generated. Understanding the metabolic pathways is also beneficial with respect to the enzymes involved in catalyzing biological mechanisms. Thus, metabolic pathways can be found to be useful, inter alia, in determining drug-drug interactions (DDIs), in developing dose modifications, and / or in addressing potential side effects from 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 (use of metabolite exposure in the assessment of drug-drug interactions).

[0026] The present disclosure provides the metabolic pathways of belumosudil. Previously unknown metabolites were identified, including O-demethylated sulfate belumosudil and belumosudil glucuronide metabolites (identified in plasma), and monohydroxy-belumosudil and belumosudil diol metabolites (identified in feces). These compounds may also be useful in the treatment of ROCK2-mediated diseases, including cGVHD, and / or in the development of treatments, including the treatment of cGVHD, for example, in determining DDIs, changing doses, and addressing side effects.

[0027] Definitions As used herein, "about" includes the exact amount modified by the term "about" and amounts expected to be within experimental error, such as within 15%, 10%, or 5%. For example, "about 200 mg" means "200 mg" and also means the range of mg within experimental error, such as ±15%, 10%, or 5% of 200 mg. When used herein, the term "about" can be used to modify ranges and specific values.

[0028] "API" means "active pharmaceutical ingredient".

[0029] "Allogeneic hematopoietic stem cell transplantation (allo-HSCT)" or "allogeneic hematopoietic cell transplantation (allo-HCT)", also referred to as bone marrow transplantation or stem cell transplantation, refers to a technique of transplanting donor-derived hematopoietic cells 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 situations, cord blood can be used. The donor and recipient can be matched by the human leukocyte antigen (HLA) gene, such as siblings. The donor and recipient can be a parent and child who are half-matched (haplotype-matched).

[0030] When the term "belumosudil" is used herein, it should be understood that this term can include any form of the compound belumosudil and its pharmaceutically acceptable salts, unless the context clearly indicates otherwise. The term "belumosudil" refers to both the compound belumosudil (e.g., in free base form, amorphous form, or crystalline form), the pharmaceutically acceptable salts of belumosudil, such as the mesylate form used as REZUROCK™, and any form of belumosudil that can be used in a formulation or pharmaceutical composition for administering the compound to a patient.

[0031] "Clinical endpoint" or "trial endpoint" refers to an event or outcome in a clinical trial that can be objectively measured to determine the outcome and potential beneficial effects of a drug or dosing protocol designed in a clinical trial. Examples of clinical endpoints include the following. Overall response rate (ORR) is the percentage of people in a test or treatment group who have a partial response (PR) or complete response (CR) to treatment within a certain period. Failure-free survival (FFS) means the time from the first dose of belumosudil to the event of failure, or the interval from the start of belumosudil to the addition of a new cGVHD therapy, recurrence of the underlying disease, or time to non-relapse mortality (NRM). Overall survival (OS) means the length of time from the date of diagnosis of the disease or the start of treatment. Duration of response (DOR) means the time from the point of initial response (e.g., PR or CR) to documented progression from the best response of cGVHD, to the start of additional systemic cGVHD therapy from the initial response, or to death. Time to next treatment (TTNT) means the time to the start of subsequent systemic cGVHD therapy.

[0032] "Clinically recommended amount" or "clinically recommended dosage" refers to the amount or dosage of an API that is recommended and / or approved for administration to a patient by a person skilled in the art of pharmaceutical chemistry for treating the disease state in question after a clinical trial, as described, for example, in Examples 1 and 2 of this specification.

[0033] "CYP3A" refers to the CYP3A family of p-450 isoenzymes, including CYP3A4.

[0034] "Disorder" and "disease" are used interchangeably and synonymously herein and include any condition of a subject in need of treatment within the full scope of either term.

[0035] "Glucuronide", also known as glucuronoside, is any substance produced by binding glucuronic acid to another substance via a glycosidic bond. Glucuronides belong to the glycosides.

[0036] "Glucuronidation" refers to the conversion of a compound to its glucuronide. This is a method used by animals to assist in the excretion of toxic substances, drugs, or other substances that cannot be used as an energy source. Glucuronidation involves the metabolism of a parent compound by UDP-glucuronosyltransferase (UGT) to a hydrophilic and negatively charged glucuronide that cannot exit the cell without the help of an efflux transporter. The elimination of the parent compound via glucuronidation in metabolically active cells is controlled by two driving forces: the formation of glucuronide by the UGT enzyme and the (polarized) excretion of these glucuronides by efflux transporters located on the cell surface of various drug disposition organs.

[0037] "Immunosuppressive therapy" (IST) refers to a therapy that is typically administered for at least 6 months after allogeneic HSCT in an attempt to prevent GVHD. Examples of IST include sirolimus, prednisone, and calcineurin inhibitors such as tacrolimus and cyclosporine.

[0038] As used herein, "isolated" is synonymous with "substantially isolated." As used herein, "substantially isolated" means that a compound is separated from the in vivo environment in which it is formed via a metabolic pathway. For example, if a compound is formed via a human metabolic pathway, the compound is "isolated" when it is no longer contained within an organ or system of the human body but has been removed from the body, excreted, and / or otherwise removed from the body, for example, by collection of a blood sample containing the compound. "Substantially isolated" includes both partial separation and substantial separation from the components of the metabolic pathway. For example, partial separation can include a composition or substance in which the compounds disclosed herein are concentrated, for example, a composition or substance containing at least about 5%, in some embodiments about 10%, or at least about 15% metabolite. "Substantially separated" 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% metabolite by weight.

[0039] The Lee Symptom Scale (LSS) summary score measures the impact on a patient's function and well-being. The Lee Symptom Scale is a 30-item scale developed to measure the 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.

[0040] A "treatment line" or "therapy line" describes the order or sequence in which different treatments are given to a patient as the patient's disease progresses. An initial treatment (first-choice treatment) may stop functioning or cease to function after a certain period. After both first-choice treatments have been discontinued, a second, different treatment (second-choice therapy) may be administered. Subsequent treatment lines may be administered if the second-choice therapy stops functioning or ceases to function. Some patients may be administered multiple treatment lines over the course of their disease.

[0041] The first-choice therapy for National Institutes of Health (NIH)-defined moderate to severe chronic graft-versus-host disease (cGVHD) can be corticosteroids alone or corticosteroids in combination with 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).

[0042] Examples of corticosteroid therapies for the treatment of cGVHD include, but are not limited to, prednisone, prednisolone, methylprednisolone, and budesonide. Examples of prior systemic therapies for treating cGVHD include, but are not limited to, prednisone, tacrolimus, extracorporeal photopheresis (ECP), sirolimus, ibrutinib, ruxolitinib, mycophenolate mofetil (MMF), rituximab, methotrexate (MTX), cyclosporine, imatinib, ixazomib, and ofatumumab.

[0043] "Metabolic pathway" refers to biochemical reactions (transformations) that convert one chemical species into another, such as anabolic or catabolic pathways. Anabolic pathways involve building larger molecules from smaller ones and are energy-requiring processes. Catabolic pathways involve the breakdown of larger molecules and often release energy.

[0044] "Metabolite" refers to intermediates or products produced in a metabolic pathway, including those produced via anabolic pathways (larger molecules are produced via catabolic pathways (smaller molecules or moieties)).

[0045] "Myeloablative transplantation" refers to a transplantation process that uses very high doses of chemotherapy or radiation prior to transplantation with autologous or allogeneic hematopoietic stem cells. Non-myeloablative transplantation, or reduced-intensity transplantation, involves the patient receiving less intensive chemotherapy prior to transplantation with allogeneic hematopoietic stem cells.

[0046] The "NIH Lung Symptom Score" or "NIH cGVHD Lung Score" is a score based on clinical symptoms in the range of 0 - 3. Score 0 is used for no symptoms, score 1 is used for shortness of breath with stairs, score 2 is used for shortness of breath on flat ground, and score 3 is used for shortness of breath at rest or shortness of breath requiring oxygen.

[0047] "Or" is used in an inclusive sense (equivalent to "and / or") unless the context requires otherwise.

[0048] As used herein, "patient" or "subject" includes animals or humans, in one embodiment humans.

[0049] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual containing a pharmaceutical agent. For example, a pharmaceutical composition can include a sterile aqueous solution or API formulated into an oral dosage form such as a tablet or capsule.

[0050] "Pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of the compounds provided herein. "Pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds in which the parent compound is modified by converting an existing acidic or basic moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts of the present invention include, for example, conventional non-toxic salts of the parent compounds formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or free base forms of these compounds with a stoichiometric amount of the appropriate base or acid, in water or an organic solvent, or in a mixture of the two, and generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0051] As used herein, "protocol" refers to the method or plan used to administer one or more APIs to a subject in need of treatment. The term "protocol" is intended to encompass the overall and detailed plan for a patient's care, as well as the individual or partial steps that are part of the overall plan. For example, a protocol can include the dosage used for each API that a patient is to receive (or is receiving), the combination of APIs that a patient is receiving, the timing and method of administration of each API (e.g., taking into account DDI, the effect of food, and the impact that different formulations or delivery modes can have on absorption and bioavailability), as well as the management of side effects, and the overall plan that includes the dosage, combination, timing and method of administration, and side effects considered together.

[0052] "Side effect" means a physiological response resulting from a treatment other than the desired effect. In certain embodiments, side effects include, but are not limited to, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, and myopathy. Side effects can be detected directly or indirectly. For example, an increase in serum aminotransferase levels can indicate hepatotoxicity or abnormal liver function. As another example, an increase in bilirubin can indicate hepatotoxicity or abnormal liver function.

[0053] "Steroid-refractory" (SR) cGVHD is defined as the progression of cGVHD during steroid or corticosteroid administration, in one embodiment, during prednisone administration.

[0054] A "therapeutically effective amount" of an API means an amount sufficient to effect the treatment of a disease state (e.g., cGVHD) when administered to a human. When applied to cGVHD in humans, "treating" or "treatment" includes (1) reducing the risk of developing cGVHD and / or inhibiting cGVHD, i.e., stopping or reducing the development of cGVHD or its clinical symptoms and (2) alleviating cGVHD, i.e., causing regression, recovery, or improvement of cGVHD, or reducing the number, frequency, duration, or severity of its clinical symptoms.

[0055] The therapeutically effective amount of an API can vary depending on the health and physical condition of the treated subject, the degree of disease progression, the evaluation of the medical situation, and other relevant factors. It is expected that the therapeutically effective amount can be within the range that can be determined through testing and with reference to clinical trial data and results, as described, for example, in Examples 1 and 2 of this specification and in the scientific literature.

[0056] Exemplary Embodiments The present disclosure provides vermostil and / or metabolites of the vermostil metabolic pathway.

[0057] In some embodiments, the present disclosure provides an isolated or synthetic compound comprising bermodzil glucuronide, O-desalkyl bermodzil sulfate, monohydroxy bermodzil, and / or bermodzil diol, or a pharmaceutically acceptable salt thereof.

[0058] The compounds and metabolic pathways provided by the present disclosure are useful for the treatment of ROCK2-mediated diseases in a patient, including the development of treatments and / or protocols for the treatment of autoimmune disorders such as chronic graft-versus-host disease (cGVHD) and / or the treatment of ROCK2-mediated diseases.

[0059] In one embodiment, the present disclosure includes the use of the compound 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide for treating cGVHD and, in some embodiments, for treating autoimmune disorders.

[0060] 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 graft-versus-host disease (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.

[0061] In some embodiments, the present disclosure provides a metabolite of bermodzil and / or the bermodzil metabolic pathway for use in preparing a pharmaceutical for treating an autoimmune disease in a subject and, in some embodiments, for use in preparing a protocol for treating an autoimmune disease in a subject.

[0062] In some embodiments, the present disclosure provides bermostatil and / or metabolites of the bermostatil metabolic pathway for use in preparing a protocol for developing unit doses (e.g., clinically recommended unit doses) of an active pharmaceutical for administration to a subject, and / or for use in an active pharmaceutical combination administered to a subject (e.g., taking into account drug-drug interactions), and / or for use in relation to the timing of administration of one or more active pharmaceuticals to a subject, and / or for use in avoiding or minimizing one or more side effects resulting from the therapeutic use of at least one active pharmaceutical in a subject.

[0063] In some embodiments, the present disclosure provides a compound having the formula:

Chemical formula

Chemical formula

[0064] In some embodiments, the present disclosure provides a compound having the formula:

Chemical formula

[0065] In some embodiments, the present disclosure provides a compound having the formula:

Chemical formula

[0066] In some embodiments, the present disclosure provides an isolated compound that is monohydroxybvermectin having the formula:

Chemical formula

[0067] In some embodiments, the present disclosure provides an isolated compound that is bvermectin diol having the formula:

Chemical formula

[0068] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of the pharmaceutical composition for treating an autoimmune disorder in a subject having chronic graft-versus-host disease (cGVHD), acute graft-versus-host disease (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.

[0069] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of the pharmaceutical composition for treating an autoimmune disorder.

[0070] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of the pharmaceutical composition for treating chronic graft-versus-host disease (cGVHD).

[0071] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of the pharmaceutical composition for treating acute graft-versus-host disease (aGVHD).

[0072] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of said pharmaceutical composition for treating pulmonary fibrosis.

[0073] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of said pharmaceutical composition for treating idiopathic pulmonary fibrosis.

[0074] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of said pharmaceutical composition for treating moderate to severe psoriasis.

[0075] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of said pharmaceutical composition for treating multiple sclerosis.

[0076] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of said pharmaceutical composition for treating systemic lupus erythematosus (SLE).

[0077] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of said pharmaceutical composition for treating Crohn's disease.

[0078] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of said pharmaceutical composition for treating atopic dermatitis.

[0079] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the foregoing compounds, or the use of said pharmaceutical composition for treating eczema.

[0080] In some embodiments, the subject undergoing the treatment has received an allogeneic hematopoietic stem cell transplantation that is a matched HSCT. In some embodiments, the allogeneic hematopoietic stem cell transplantation is a haploidentical HSCT.

[0081] In some embodiments, the belumosudil treatment is continued based on the patient's tolerance until the active cGVHD symptoms resolve or progress. The number of treatment cycles and the duration depend on the patient. In some embodiments, belumosudil is administered to the patient in one or more 28-day cycles.

[0082] In some embodiments, the number of cycles ranges from 3 to 15. In some embodiments, the number of cycles ranges from 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 number of cycles ranges from 5 to 11. In some embodiments, the number of cycles ranges from 6 to 12. In some embodiments, the number of cycles ranges from 5 to 10, 5 to 9, or 5 to 8. In some embodiments, the number of cycles ranges from 5 to 7. In some embodiments, the number of cycles ranges from 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.

[0083] In some embodiments, the number of cycles ranges from 3 cycles to loss of efficacy. In some embodiments, the number of cycles ranges from 4 cycles to loss of efficacy. In some embodiments, the number of cycles ranges from 5 cycles to loss of efficacy. In some embodiments, the number of cycles ranges from 6 cycles to loss of efficacy. In some embodiments, the number of cycles ranges from 7 cycles to loss of efficacy. In some embodiments, the number of cycles ranges from 8 cycles to loss of efficacy. In some embodiments, the number of cycles is greater than 3, 4, 5, 10, 15, 20, 25, or 30, or until the desired efficacy is achieved.

[0084] In some embodiments, the subject experiences improvement as defined by the Lille Symptom Scale (LSS). In some embodiments, the subject experiences at least a 7-point decrease in the LSS score. In some embodiments, the subject experiences at least a 10-point decrease in the LSS score. In some embodiments, the improvement is maintained over at least two consecutive evaluations. In some embodiments, the LSS score is evaluated on the first day of each cycle starting from the baseline and the first day of cycle 2.

[0085] In some embodiments, the subject has chronic graft-versus-host disease and has failed 1 to 3 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 2 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 2 to 5 prior lines of systemic therapy for chronic graft-versus-host disease. In some embodiments, the subject has failed at least 1, at least 2, at least 3, at least 4, or at least 5 prior lines of systemic therapy for chronic graft-versus-host disease.

[0086] In some embodiments, the subject experienced a complete response to the last treatment of graft-versus-host disease prior to belumosudil. In some embodiments, the subject experienced a partial response to the last treatment of graft-versus-host disease prior to belumosudil. In some embodiments, prior to belumosudil, a stable state with respect to the last treatment of graft-versus-host disease.

[0087] In some embodiments, the prior line of systemic therapy for chronic graft-versus-host disease has been discontinued.

[0088] In some embodiments, the prior line of systemic therapy is selected from the group consisting of prednisone, tacrolimus, ECP, sirolimus, ibrutinib, ruxolitinib, MMF, rituximab, MTX, cyclosporine, imatinib, ixazomib, and ofatumumab.

[0089] In some embodiments, the cGVHD is steroid-refractory (SR) cGVHD. In some embodiments, the subject is refractory to the last treatment line prior to belumosudil treatment.

[0090] In some embodiments, the subject is undergoing concomitant corticosteroid therapy. In some embodiments, the concomitant corticosteroid therapy is selected from the group consisting of 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 one cycle of vermostil 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 one cycle of vermostil treatment. In some embodiments, the dosage of the concomitant corticosteroid therapy is reduced by 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 one cycle of vermostil treatment. In some embodiments, the concomitant corticosteroid therapy is discontinued after at least one cycle of vermostil treatment.

[0091] In some embodiments, the subject is undergoing concomitant calcineurin inhibitor therapy.

[0092] In some embodiments, the subject has involvement of at least four organs. In some embodiments, the subject has involvement of at least three organs. In some embodiments, the subject has involvement of at least two organs.

[0093] Compositions and Tablets According to another aspect, a pharmaceutical composition comprising a compound described herein as an active ingredient is disclosed. These pharmaceutical compositions also include an effective amount of at least one compound disclosed herein, or a pharmaceutically acceptable salt of said compound, and at least one pharmaceutically acceptable excipient.

[0094] The excipient is selected from conventional excipients known to those skilled in the art according to the desired pharmaceutical form and method of administration.

[0095] In these pharmaceutical compositions for oral, parenteral (including subcutaneous, intramuscular, intradermal and intravenous), transdermal, bronchial or nasal administration, the active ingredient of formula (I) or its salt can be mixed with conventional pharmaceutical excipients and administered to animals and humans in unit dosage forms for the prevention or treatment of the following disorders or diseases.

[0096] The pharmaceutical compositions described herein and / or the pharmaceutical compositions for use in the methods described herein may further comprise a pharmaceutically acceptable carrier / excipient. In some embodiments, a 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 delivery process of the API to the patient and / or to stabilize the API during transport for delivery to the patient. The term "acceptable" as used in this context means that the material is compatible with the other components of the formulation and does not cause unacceptable adverse side effects to the patient.

[0097] The pharmaceutical compositions contemplated herein include dosage forms suitable for oral, parenteral (including subcutaneous, intramuscular, intradermal and intravenous), transdermal, bronchial or nasal administration.

[0098] For solid pharmaceutical dosage forms for oral administration (capsules, tablets, pills, powders, granules, etc.), the API can be mixed with at least a pharmaceutically acceptable carrier comprising one or more pharmaceutically acceptable excipients.

[0099] In one embodiment, the compound is formulated into tablets for oral administration. Belsomra mesylate is a yellow powder that is substantially insoluble in water. Belsomra tablets can be prepared for oral administration. Each tablet contains 200 mg of the free base corresponding to 242.5 mg of belsomra mesylate. The tablets may also contain the following inactive ingredients: microcrystalline cellulose, hypromellose, croscarmellose sodium, colloidal silicon dioxide, and magnesium stearate. The tablet film consists 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 "KDM" debossed on one side and "200" debossed on the other side. The tablets are stored at room temperature, 20°C to 25°C (68°F to 77°F), with a deviation of 15°C to 30°C (59°F to 86°F) allowed.

[0100] The compounds and pharmaceutical compositions disclosed herein may be useful for inhibiting the ROCK1 and / or ROCK2 enzymes, preferentially ROCK2, and thus, among other indications, for the treatment of fibrotic disorders such as autoimmune disorders and / or 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, which are regulated by the ROCK enzymes.

[0101] The following abbreviations may be useful in considering the examples and descriptions herein.

[0102]

Table 1

Examples

[0103] Example 1: In healthy subjects 14 Phase I open-label two-part study on the assessment of the absolute bioavailability, mass balance, and metabolic profile of In this first-phase two-part study, after single administration of unlabeled oral tablets of vermoxizole (200 mg), radiolabeled intravenous (IV) microtracer injection of vermoxizole (100 μg), and radiolabeled oral capsules (200 mg) to subjects, the pharmacokinetics, mass balance, and metabolic profile of vermoxizole were evaluated. This study was conducted in two parts for the following purposes: (1) to determine the absolute oral bioavailability of vermoxizole (Part 1), (2) to determine the mass balance recovery after single oral administration of radiolabeled vermoxizole (Part 2), and (3) to perform metabolite profiling and structural elucidation of the identified metabolites (Part 2).

[0104] The absolute bioavailability was calculated as 63.7% based on the area under the plasma concentration-time curve from time 0 to infinity for the oral dose / plasma concentration-time curve from time 0 to infinity for the IV dose. Administration of the radiolabeled IV microtracer showed low extraction rates and distribution of vermoxizole into tissues. Since the majority of the total radioactivity was recovered in feces and the minimum amount was recovered in urine, it was suggested that renal excretion of vermoxizole was minimal.

[0105] Study Design This was a single-site non-randomized, non-blinded two-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 dosing day.

[0106] For the evaluation of absolute bioavailability (Part 1), subjects were administered a single oral dose of 200 mg of vermoxizole tablets (therapeutic dose), followed 1.75 hours later by a 15-minute IV infusion of 100 μg of 14 C]-vermoxizole containing (NMT) 37 kBq 14 C] (matched to the time at which the predicted maximum concentration [Cmax] occurred). To determine absolute bioavailability, a microtracer dose was selected since the need for an IV formulation with good stability was negated. Furthermore, due to the use of a very low dose, additional non-clinical studies, such as local tolerance studies and general toxicity studies, were not justified.

[0107] For mass balance and metabolic profiling studies (Part 2), after at least 7 days of washout from Part 1, subjects received a single oral dose of 200 mg 14 C]-containing belumosudil capsules. This radioactivity level was selected based on human dosimetry calculations performed by Public Health England that determined the NMT 9.8 MBq radioactive oral dose necessary to enable appropriate metabolite profiling of the main sample. Considering the microtracer doses from Part 1, the total committed effective dose per subject received in the study was 3 mSv, which is included in the International Commission on Radiological Protection guidelines for Category IIb studies. Furthermore, to remain within the radiation dose limits approved by the UK Radioactive Substances Committee, 14 the target-specific activity of 14 C]-belumosudil was set at 90% of the threshold radiation dose limit.

[0108] Pharmacokinetic Sample Collection and Analysis Part 1 Blood Sampling Blood samples for oral belumosudil PK analysis were collected in tripotassium ethylenediaminetetraacetate tubes pre-dose and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, and 48 hours post-dose. Blood samples for 14 C]-belumosudil PK analysis and total radioactivity were collected at -0.25, -0.16, -0.08, 0, 0.08, 0.16, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 22, 34, and 46 hours after end of infusion (EOI). All samples were processed to plasma by centrifugation at 2000 g for 10 minutes at 4°C within 30 minutes of collection and then frozen at -70°C or below prior to analysis.

[0109] Part 2 Blood Sampling Total radioactivity whole blood samples and plasma samples for metabolite profiling and identification were collected before dosing and at 1, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours after dosing. Blood samples for plasma vermostil, KD025m1, KD025m2, and total radioactivity analysis were collected at the same time points, and additional samples were collected at 0.5, 1.5, 2, 3, 5, 6, 8, 10, and 36 hours after dosing. Total radioactivity whole blood samples were collected into tripotassium ethylenediaminetetraacetate tubes and stored at 2 - 8°C until analysis. Plasma vermostil, KD025m1, KD025m2, and blood samples for total radioactivity quantification and metabolite profiling / identification were processed as in Part 1 and stored frozen at -20°C (vermostil, KD025m1, KD025m2, and total radioactivity samples) or -70°C (metabolite profiling samples).

[0110] Part 2 Urine and fecal sample collection. Urine for total radioactivity and metabolite profiling and identification was collected before dosing (first void of the day), pooled at 0 - 6, 6 - 12, and 12 - 24 hours after dosing, and subsequently collected daily (24 hours) until day 8 / discharge. Urine samples were pooled at intervals and stored at 2 - 8°C until analysis. Feces were collected from inpatients until before dosing and then daily (24 hours) until discharge. Fecal samples were pooled at time intervals and stored at -20°C until analysis.

[0111] Analytical methods Part 1. Plasma vermostil samples were prepared for analysis using the protein precipitation method and quantified using a validated liquid chromatography - tandem mass spectrometry (LC - MS / MS) method at a lower limit of quantification (LLOQ) of 10 ng / mL.

[0112] Total 14The 14 C]-vermosdil concentration was determined by accelerator mass spectrometry (AMS). Prior to AMS analysis, the selected samples were analyzed by liquid scintillation counting (LSC) to confirm that the radioactivity level did not saturate the AMS. The selected samples were diluted with commercially available plasma to ensure that the concentration was within the acceptable range of AMS analysis. The samples were converted to graphite by combustion to CO2 followed by reduction to graphite. The graphite target was then analyzed by AMS (LLOQ of 0.00489, 0.00557, 0.00485, 0.00402, and 0.00372 ng equivalent / mL for individual subjects). All 14 C data were background corrected based on the

[0113] 14 C]-vermosdil concentration was quantified by high performance liquid chromatography / accelerator mass spectrometry (HPLC AMS) method. 19 For sample analysis using the protein precipitation method, radiolabeled vermosdil in plasma was prepared followed by HPLC. 14 The eluate fraction corresponding to

[0114] C]-vermosdil was collected prior to AMS analysis (LLOQ of 1.55 pg equivalent / mL).

[0115] ​Total radioactivity whole blood, plasma, and urine samples were prepared for LSC by mixing with scintillation fluid. Additionally, aliquots of whole blood were solubilized with AquiGest tissue solubilizer, and plasma samples were mixed with 10 mM phosphate buffered saline. Urine and fecal samples were pooled for each subject according to the time intervals before analysis. Furthermore, fecal samples were combusted using an automated sample oxidizer prior to LSC. The radioactivity of all samples was measured using an LSC equipped with automatic external standard quench correction (Tri-Carb 2300TR, 2900TR, and 3100TR, Scintillation Counters; PerkinElmer, Waltham, Massachusetts), and the samples were counted for 4 minutes. The LLOQ was considered to be twice the background value of samples of the same type (1.39 [urine], 23.9 [feces], 35.5 [whole blood], and 30.9 [plasma] ng equivalent [free base] / g or mL).

[0116] To determine the most appropriate sampling pools for metabolite identification / profiling, plasma, urine, and fecal samples were pooled based on the mass balance data obtained as described above. Plasma samples were pooled for each individual subject until a time point representing at least 80% of the total plasma radioactivity, and pooled across subjects at pre-dose and 4 and 8 hours time points. Urine samples were pooled for each individual subject up to 0 - 24 hours, and pooled across subjects at pre-dose and 0 - 6 hour intervals. Fecal samples were pooled for each individual subject according to the time point representing at least 80% of the total plasma radioactivity, and pooled across subjects at 0 - 24 hour and 24 - 48 hour intervals.

[0117] 14 ​The concentration of 14 C]-vermosdil was quantified by LC-MS / MS. Subsequently, the chemical structures of the radioactive components that accounted for more than 10% of the circulating radioactivity in plasma and more than 10% of the dose in urine and feces were identified. Radioactive components representing less than 10% of the circulating radioactivity in plasma and less than 10% of the dose in urine and feces were also identified. Then, metabolite identification was performed using accurate mass positive and negative ion full scan and positive ion product ion analysis. Samples were screened for the presence of vermosdil, metabolite KD025m1, KD025m2, O-demethylated vermosdil, any potential metabolites resulting from O-demethylation and hydroxylation, and phase II conjugate metabolites. The identified components were 14 compared with the

[0118] radiochromatogram to identify metabolites. Pre-dose plasma pools and urine pools were used to correct for endogenous components during metabolite identification.

[0118] Pharmacokinetic evaluation Plasma total radioactivity, vermosdil, 14 C]-vermosdil (only part 1), and PK parameters for KD025m1 and KD025m2 (only part 2) were calculated using the standard non-compartmental method in Phoenix WinNonlin version 8.0 (Certara, Princeton, New Jersey). Absolute bioavailability and metabolite-to-parent ratios were calculated using the PK parameter estimates.

[0119] The PK parameters calculated for this study were the absolute bioavailability of vermosdil (oral dose (AUC inf、経口 ) is the area under the dose-adjusted plasma concentration-time curve (AUC) from 0 to infinity / IV dose (AUC inf、IV ) is evaluated by the AUC from 0 to infinity), the mass balance recovery rate of total radioactivity in all excreta (evaluated by amount eliminated [Ae], %Ae, cumulative Ae, and cumulative %Ae), oral vermosdil, KD025m 1 and KD025m 2 , and various PK parameters of total radioactivity, IV 14PK parameters of 14 C] Excretion route and rate (plasma and excreta), total radioactivity whole blood: plasma ratio, and structural identification of metabolites in plasma and excreta were determined.

[0120] The PK population included all subjects in either Part 1 or Part 2 who received at least one dose of

[0121]

[0122] Safety Evaluation Safety was evaluated throughout the study by AE, vital signs, electrocardiogram, physical examination, and clinical laboratory tests. The safety population included all subjects who received at least one dose of the study drug (oral or IV) in any part of the study.

[0123] Results Subject Disposition Five male subjects were enrolled and dosed in both Part 1 and Part 2. All subjects completed the study and were included in both the PK and mass balance populations. Baseline demographics are summarized in Table 1. The subjects were 41 to 64 years old (mean age 53.0 years), and the majority of the subjects were white. All subjects were within the protocol-defined reference range for body mass index.

[0124]

Table 2

[0125] ​Pharmacokinetic Evaluation Part 1: Absolute Bioavailability of Vermostil Following a single 200 mg oral dose of Vermostil and subsequent 15 minutes of 14 The mean PK profiles for [C]-Vermostil IV infusion are shown in Figure 2 (unlabeled Vermostil) and Figure 3 ( 14 [C]-Vermostil). In Part 1 of the study, the PK parameters calculated for Vermostil, 14 [C]-Vermostil and total radioactivity are shown in Table 2.

[0126]

Table 3

[0127] IV 14 For [C]-Vermostil infusion, the maximum radiolabeled concentration was achieved at EOI and then decreased biphasically. The radiolabeled concentration remained 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).

[0128] The peak concentration of Vermostil after oral administration was reached 3 hours after oral dosing and then decreased biphasically over 48 hours. The terminal half-life was reported for all 5 subjects (geometric mean, 5.3 hours). The absolute bioavailability (AUC inf、経口 / AUC inf、IV ) was calculated to be 63.7%.

[0129] Part 2: Mass Balance and Excretion Most of the radioactivity (88.5%) was recovered in the excreta, 84.6% in the feces, and less than 5% of the radiolabeled dose was recovered in the urine. Within the first 24 hours after dosing, approximately 27.4% and 3.8% of the total radioactivity were recovered in the feces and urine, respectively. Whole blood: plasma total radioactivity concentration ranged from 0.53 to 1.16. The PK parameters for Vermostil and total radioactivity in Part 2 are summarized in Table 2 above. Part 2: Identification and Profiling of Metabolites. We attempted to identify the radioactive components that accounted for 10% of the circulating radioactivity in plasma and more than 10% of the dose in feces. The identified vermoxil metabolites in plasma, urine, and feces are shown in Table 3. The single radioactive component present in urine did not account for more than 10% of the dose. In plasma, vermoxil accounted for 64% of the total plasma radioactivity, and KD025m2 and O-demethylated sulfate vermoxil, a hitherto unidentified phase II metabolite, together accounted for 11.5% of the plasma radioactivity. Another metabolite not measured in the clinical trial, vermoxil glucuronide, accounted for 15% of the total plasma radioactivity. In feces, vermoxil was 30%, and KD025m2 and O-demethylated vermoxil sulfate together accounted for 35% of the total sample radioactivity. Vermoxil diol (vermoxil + 2O, + 2H) and monohydroxy vermoxil, hitherto unidentified, were 10% and 11% in feces, respectively.

[0130]

Table 4

[0131] The chemical structures of the vermoxil metabolites KD025m1 discovered in this study and metabolites not previously identified can be shown as follows:

Chem.

Chem.

[0132] Overall, the safety profile of vermoxil was as a 200 mg tablet, followed by 14 C]-vermoxil microtracer injection (Part 1), and 200 mg 14As expected when administered to healthy subjects, as [[Part 2]] of this trial, there were no deaths, serious AEs, AEs of severe intensity, or AEs leading to subject withdrawal in any part of this trial. One AE that developed under one treatment was recorded in [[Part 1]] (mild and probably related rash), and no AEs that developed under treatment were reported in [[Part 2]]. The event resolved spontaneously 2.5 hours after onset. No clinically significant laboratory findings, vital signs, electrocardiograms, or physical examination findings were reported after administration in any part of the trial.

[0133] In this study, the absorption, metabolism, and excretion characteristics of vermoxil in humans after oral administration of unlabeled tablets and radiolabeled capsules, and the IV microtracer of radiolabeled vermoxil in healthy male subjects were evaluated. As described in the Methods section, a microtracer approach was employed to accurately characterize the IV PK profile without encountering dose-dependent kinetics or performing additional toxicity studies with the IV formulation. The use of the highly sensitive AMS method 14 enabled maximum data acquisition by allowing accurate measurement of [[C]]-vermoxil, while quantification of unlabeled vermoxil was performed by standard HPLC-MS / MS. 14 The [[C]]-label could be easily introduced at a metabolically stable position on the main chain of vermoxil and was selected to ensure appropriate mass balance determination and metabolite profiling. 14 The synthesis and isolation of [[C]]-vermoxil relied on the same chemical process used in the manufacture of vermoxil.

[0134] In [[Part 1]], the absolute bioavailability based on dose-adjusted AUC inf、経口 / AUC inf、IV was estimated to be 63.7% after oral administration of a commercially available 200 mg vermoxil tablet. After IV injection of 100 μg of 14 [[C]]-vermoxil, the total clearance after IV administration was lower than typical hepatic blood flow and showed a low extraction ratio (≈0.3). The apparent volume of distribution was higher than total body water,14 The distribution of [C]-vermosidil into tissues was demonstrated. The half-life values after IV and oral administrations were comparable at about 5 - 6 hours, similar to those observed in previous clinical trials.

[0135] After administration of the 200 mg 14 capsules of [C]-vermosidil, most of the total radioactivity was recovered in feces (85%), and less than 5% was recovered in urine, suggesting minimal renal excretion of vermosidil. Based on the 63.7% absolute bioavailability in Part 1 and the low recovery rate of radioactivity in urine, 14 the main clearance pathways of [C]-vermosidil and related metabolites are probably the bile and / or intestine. The total radioactivity whole blood:plasma ratio ranged from 0.53 - 1.16, indicating little or no preferential distribution of total radioactivity into the cellular components of whole blood. The concentrations of vermosidil, KD025m2, and KD025m1 present in the total circulating radioactivity in plasma in Part 2 suggested the possible presence of other circulating metabolites in plasma. Furthermore, the half-life of total radioactivity was longer than that of vermosidil (geometric mean, 18.0 and 5.3 hours, respectively), further supporting the presence of other metabolites with a longer elimination phase. Indeed, through this study using standard LC-MS / MS metabolite profiling techniques, in addition to vermosidil diol, metabolite glucuronide and sulfate derivatives were identified for the first time in humans. The human metabolic pathway of vermosidil was developed through this trial and is shown in Figure 3.

[0136] In addition to the parent, the major metabolite KD025m2, and the active minor metabolite KD025m1, other metabolites were identified in plasma as O-demethylated sulfate vermosidil and vermosidil glucuronide. In feces, the parent KD025m2, O-demethylated sulfate vermosidil, monohydroxy-vermosidil, and vermosidil diol were identified as metabolites accounting for >10% of the radioactive dose. Metabolites in urine were not found to account for >10% of the dose.

[0137] Example 2: In vitro tests with belumosudil and metabolites and inhibition of ROCK1 and ROCK2 enzyme activities The effects of belumosudil and the major metabolites, KD025m1 and KD025m2, on the inhibition of ROCK2 enzyme activity were evaluated with an enzyme purified from cell supernatants or tissue homogenates.

[0138] 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 each isozyme. The reaction was initiated by the addition of S6 kinase substrate peptide, [γ33P]ATP, and the test drug, and incubated at 37 °C for 20 minutes. The reaction was stopped by the addition of phosphoric acid, and radioactivity was quantified using standard methods.

[0139] The results showed that belumosudil inhibited ROCK2 enzyme activity with an inhibition approximately 30-fold greater compared to ROCK1, i.e., the IC 50 was more than 30-fold lower than the inhibitory effect on ROCK1.

[0140] The results further demonstrated the inhibitory activity of belumosudil metabolites. Table 4 shows the order of the inhibitory effects of belumosudil (KD025), KD025m1, and KD025m2 on ROCK enzymes. Fasudil and Y-27362 were included as reference ROCK inhibitors. The metabolite KD025m1 inhibited ROCK2 with a Ki value comparable to that of belumosudil, but the inhibition of ROCK2 by the second metabolite KD025m2 was approximately 6-fold lower.

[0141] [Table 5]

[0142] Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific documents cited herein are hereby expressly incorporated by reference in their entirety into this specification.

Claims

**Claim 1** A compound comprising 2-(3-(4-(1H-indazol-5-ylamino)quinazolin-2-yl)phenoxy)acetamide for use in treating autoimmune disorders 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. **Claim 2** The compound for use according to claim 1, wherein the autoimmune disorder is chronic GVHD (cGVHD). **Claim 3** An isolated or synthetic compound comprising barmothiazyl glucuronide, O-demethylated barmothiazyl sulfate, monohydroxy barmothiazyl or barmothiazyl diol or a pharmaceutically acceptable salt thereof. **Claim 4** Formula: 【Chemical 1】 【Chemical 2】 (wherein ____ represents a single bond connecting any available carbon atom within the bracketed portion of the molecule to the portion or portions adjacent to said brackets), the compound according to claim 3, or a pharmaceutically acceptable salt thereof. **Claim 5** Formula: 【Chemical Formula 3】 The isolated compound according to claim 4 or a pharmaceutically acceptable salt thereof, which is barmothiazyl glucuronide having the formula. **Claim 6** The compound according to claim 5, characterized by having a molecular weight of 628. **Claim 7** Formula: 【Chemical Formula 4】 The isolated compound according to claim 4 or a pharmaceutically acceptable salt thereof, which is O-demethylated barmothiazyl sulfate having the formula. **Claim 8** The compound according to claim 7, characterized by having a molecular weight of 433. **Claim 9** Formula: 【Chemical Formula 5】 The isolated compound according to claim 4 or a pharmaceutically acceptable salt thereof, which is monohydroxy barmothiazyl having the formula. **Claim 10** The compound according to claim 9, characterized by having a molecular weight of 468. **Claim 11** Formula: 【Chemical Formula 6】 The isolated compound according to claim 4 or a pharmaceutically acceptable salt thereof, which is barmothiazyl diol having the formula. **Claim 12** The compound according to claim 11, characterized by having a molecular weight of 486. **Claim 13** The isolated compound according to any one of claims 3 to 12 or a pharmaceutically acceptable salt thereof, characterized by having a molecular weight or retention time shown in Table 3. **Claim 14** The compound according to any one of claims 3 to 13 for use in treating autoimmune diseases in a subject. **Claim 15** A 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 the 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. 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 the metabolic pathway of vermostil for preparing 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 the metabolic pathway of vermostil for preparing a protocol 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 preparing 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 preparing a protocol for treating an autoimmune disease in a subject.

23. Use according to any one of claims 19 to 22, comprising preparing a unit dosage for administration of an active pharmaceutical to a subject taking into account drug-drug interactions.

24. Use according to any one of claims 19 to 22, comprising preparing a medicament for treating a disorder in a subject, wherein the medicament comprises a combination of active pharmaceuticals to be administered to the subject.

25. Use according to claim 20 or 22, comprising preparing a protocol regarding the timing of administration of one or more active pharmaceuticals to a subject.

26. The use according to claim 20 or 22, comprising the preparation of a protocol for avoiding one or more side effects resulting from the therapeutic use of at least one active pharmaceutical on a subject.

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

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