Combination therapy comprising belumosudil and gusacitinib

Abstract

Disclosed herein are combinations of belumosudil and gusacitinib, and methods of treating chronic graft-versus-host disease (cGVHD) with belumosudil and gusacitinib.

Description

Attorney Docket No. 01183-0343-00PCT-KADCOMBINATION THERAPY COMPRISING BELUMOSUDIL AND GUSACITINIBCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to European Application No. 25175384.4, filed May 9, 2025, U.S. Provisional Application No. 63 / 780,639, filed March 31, 2025, U.S. Provisional Application No. 63 / 750,579, filed January 28, 2025, and U.S. Provisional Application No. 63 / 750,571, filed January 28, 2025.FIELD

[0002] The present disclosure relates to combinations of belumosudil and gusacitinib, and methods of treating chronic graft-versus-host disease (cGVHD) using belumosudil and gusacitinib.BACKGROUND

[0003] 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 the fibrosis that occurs because of aberrant tissue repair. Studies have shown that belumosudil down-regulates proinflammatory responses and also inhibits aberrant pro-fibrotic signaling. The FDA approved belumosudil (REZUROCK®) in 2021 for the treatment of chronic GVHD (cGVHD) after failure of at least two prior lines of systemic therapy.

[0004] Gusacitinib is an oral pan-Janus kinase (JAK) inhibitor, with similar binding affinity to JAK1, JAK2, JAK3, and TYK2. It also inhibits spleen tyrosine kinase (SYK). JAK inhibitors disrupt the JAK-STAT signaling pathway in lymphocytes and SYK inhibitors disrupt the signaling in B cells. Gusacitinib is currently in clinical trials for the treatment of the inflammatory skin conditions chronic hand eczema (CHE) and atopic dermatitis (AD).

[0005] Chronic graft-versus-host disease (cGVHD) is characterized by inflammation and progressive fibrosis that can affect multiple organs, including the skin, lungs, gastrointestinal tract, and liver. Patients with cGVHD, particularly those exhibiting fibrotic manifestations, face significant challenges due to the limited efficacy and safety of current treatments. Despite the use of corticosteroids and second-line agents, patients often experience only temporary relief, with fibrosis continuing to progress and impair quality of life. Fibrotic involvement in cGVHD is particularly resistant to current treatments, which are primarily anti-inflammatory and not designed to target the structural and cellular pathways central to fibrosis.Attorney Docket No. 01183-0343-00PCT-KAD

[0006] Combination therapy is an attractive option for disease treatment, particularly using therapeutic agents that target distinct pathways. Accordingly, provided herein are combinations of belumosudil and gusacitinib, and methods of treating cGVHD with belumosudil and gusacitinib. The combination of ROCK2 and JAK / SYK inhibition may therefore not only limit immune-related manifestations of cGVHD but could also benefit patients with difficult-to-treat fibrotic manifestations of cGVHD.SUMMARY

[0007] Described herein are combinations of belumosudil and gusacitinib, kits comprising belumosudil and gusacitinib, and methods of treating cGVHD using belumosudil and gusacitinib.

[0008] The present disclosure provides combinations including 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0009] Also provided are pharmaceutical compositions including 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In an embodiment, the pharmaceutical compositions are used in the treatment of cGVHD.

[0010] The present disclosure also provides methods for treating cGVHD in a human patient in need thereof, the methods including administering to the human patient a therapeutically effective amount of a combination comprising 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof. In an embodiment, the cGVHD is sclerodermatous cGVHD. In an embodiment, cGVHD is lung cGVHD. In an embodiment, the cGVHD is sclerodermatous cGVHD or lung cGVHD.

[0011] Also provided is the compound 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, for use in the treatment of cGVHD, wherein the compound is formulated for co-administration with 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.Attorney Docket No. 01183-0343-00PCT-KAD

[0012] The present disclosure also provides the compound 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, for use in the treatment of cGVHD, wherein the compound is co-administered with 2-[l-[4-[4-(4-hydroxypiperi din- l-yl)anilino]-5-oxo-6JH-pyrimido[4,5-d]pyridazin-2-yl]piperi din-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0013] Also provided is the compound 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, for use in the treatment of cGVHD, wherein the compound is co-administered with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

[0014] Further provided is the compound 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic hand eczema or atopic dermatitis, wherein the compound is co-administered with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy }-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

[0015] The present disclosure also provides methods of treating cGVHD, the methods including administering to a patient in need thereof a therapeutically effective amount of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, wherein the patient has previously received treatment with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy }-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof.

[0016] Also provided are methods of treating cGVHD, the methods including administering to a patient in need thereof a therapeutically effective amount of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, wherein the patient is receiving treatment with 2-{3-[4- (lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof.

[0017] Further provided are methods of treating cGVHD, the methods including administering to a patient in need thereof a therapeutically effective amount of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, wherein the patient is on stable treatment with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof.Attorney Docket No. 01183-0343-00PCT-KAD

[0018] Additionally provided are kits including: a first pharmaceutical composition compri sing 2- { 3 - [4-( 1 H-indazol-5 -ylamino)-2-quinazolinyl]phenoxy } -N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient; a second pharmaceutical composition comprising 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6H-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient; and instructions for administering the first and second pharmaceutical compositions.

[0019] The present disclosure can be understood more fully by reference to the detailed description and examples, which are intended to exemplify non-limiting embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0021] FIG. 1 shows a plot of percent survival of the animals in the study of Example 1.

[0022] FIG. 2A shows a plot of percent weight change in the body weight of the animals in the study of Example 1. The upper plot shows body weight change as compared to Day 0, the lower left inset shows calculated AUC from Day 0 until study conclusion on Day 56, and the lower right inset shows calculated AUC from Day 21 until study conclusion on Day 56.

[0023] FIG. 2B shows a plot of percent weight change in the body weight of the animals in the study of Example 1, with the death weight carried. The upper plot shows body weight change with death weight carried as compared to Day 0, the lower left inset shows calculated AUC from Day 0 until study conclusion on Day 56, and the lower right inset shows calculated AUC from Day 21 until study conclusion on Day 56.

[0024] FIG. 3 A shows a plot of the GVHD score with standard scoring in the animals in the study of Example 1. The upper plot shows GVHD score with standard scoring, the lower left inset shows calculated AUC from Day 0 until study conclusion on Day 56, and the lower right inset shows calculated AUC from Day 21 until study conclusion on Day 56.

[0025] FIG. 3B shows a plot of GVHD score with standard scoring and the death score carried for the animals in the study of Example 1. The upper plot shows GVHD score with standard scoring with death weight carried, the lower left inset shows calculated AUC from DayAttorney Docket No. 01183-0343-00PCT-KAD0 until study conclusion on Day 56, and the lower right inset shows calculated AUC from Day 21 until study conclusion on Day 56.

[0026] FIG. 4A shows a plot of the modified GVHD score in the animals in the study of Example 1. The upper plot shows scGVHD score, the lower left inset shows calculated AUC from Day 0 until study conclusion on Day 56, and the lower right inset shows calculated AUC from Day 21 until study conclusion on Day 56.

[0027] FIG. 4B shows a plot of the modified GVHD score with the death score carried for the animals in the study of Example 1. The upper plot shows scGVHD score with death weight carried, the lower left inset shows calculated AUC from Day 0 until study conclusion on Day 56, and the lower right inset shows calculated AUC from Day 21 until study conclusion on Day 56.

[0028] FIG. 5 A is a plot of percent survival of the animals in the study of Example 1 using the standard GVHD scale for progression-free survival.

[0029] FIG. 5B is a plot of percent survival of the animals in the study of Example 1 using the standard GVHD scale for progression-free survival with death included.

[0030] FIG. 5C is a plot of percent survival of the animals in the study of Example 1 using the modified GVHD scale (scGVHD) for progression-free survival.

[0031] FIG. 5D is a plot of percent survival of the animals in the study of Example 1 using the modified GVHD scale (scGVHD) for progression-free survival with death included.

[0032] FIG. 6A shows the levels of hydroxyproline in units of pg / mL in the right lung, skin, and liver samples from the animals in the study of Example 1.

[0033] FIG. 6B shows the levels of hydroxyproline in units of pg per organ (total) in the right lung, skin, and liver samples from the animals in the study of Example 1.

[0034] FIG. 6C shows the total protein level in units of pg / mL in the right lung, skin, and liver samples from the animals in the study of Example 1.

[0035] FIG. 6D shows the levels of hydroxyproline normalized to protein levels in the right lung, skin, and liver samples from the animals in the study of Example 1.

[0036] FIG. 7A is a plot of mouse skin sum histopathology scores for the animals submitted to histopathological analysis from the study of Example 1.

[0037] FIG. 7B is a plot of mouse skin histopathology scores for the animals submitted to histopathological analysis from the study of Example 1.

[0038] FIG. 7C is a plot of mouse skin dermal thickness measurements for the animals submitted to histopathological analysis from the study of Example 1.

[0039] FIG. 7D is a plot of mouse skin aSMA immunolabeling scores for the animals submitted to histopathological analysis from the study of Example 1.Attorney Docket No. 01183-0343-00PCT-KAD

[0040] FIG. 7E is a summary of the mouse skin group histopathology scores plotted in FIG.7 A, 7B, 7C, and 7D.

[0041] FIG. 8A is a plot of mouse lung sum histopathology scores for the animals submitted to histopathological analysis from the study of Example 1.

[0042] FIG. 8B is a plot of mouse lung histopathology scores for the animals submitted to histopathological analysis from the study of Example 1.

[0043] FIG. 8C is a plot of mouse lung average Ashcroft scores for the animals submitted to histopathological analysis from the study of Example 1.

[0044] FIG. 8D is a plot of mouse lung increased perivascular collagen scores (PSR) for the animals submitted to histopathological analysis from the study of Example 1.

[0045] FIG. 8E is a plot of mouse lung aSMA immunolabeling scores for the animals submitted to histopathological analysis from the study of Example 1.

[0046] FIG. 8F is a summary of the mouse lung group histopathology scores plotted in FIG.8A, 8B, 8C, 8D, and 8E.

[0047] FIG. 9A is a plot of mouse liver sum histopathology scores for the animals submitted to histopathological analysis from the study of Example 1.

[0048] FIG. 9B is a plot of mouse liver histopathology scores for the animals submitted to histopathological analysis from the study of Example 1.

[0049] FIG. 9C is a plot of mouse liver increased collagen scores (PSR) for the animals submitted to histopathological analysis from the study of Example 1.

[0050] FIG. 9D is a plot of mouse liver aSMA immunolabeling scores for the animals submitted to histopathological analysis from the study of Example 1.

[0051] FIG. 9E is a summary of the mouse liver group histopathology scores plotted in FIG.9 A, 9B, 9C, and 9D.

[0052] FIG. 9F, FIG. 9G, and FIG. 9H are a series of representative liver photomicrographs stained against alpha-smooth muscle actin (aSMA) for three of the animals submitted to histopathological analysis from the study of Example 1.

[0053] FIG. 10A shows the percentage of disease reduction in a subset of animals in the study of Example 1 using the standard GVHD scale.

[0054] FIG. 10B shows a plot of percent survival of a subset of the animals in the study of Example 1 using the standard GVHD scale for progression-free survival with death included.

[0055] FIG. 11 A shows plots of the lung sum histopathology score; lung infiltrate in mononuclear cells, perivascular; lung histiocytosis, alveolar; and lung alveolar fibrin / necroticAttorney Docket No. 01183-0343-00PCT-KADdebris data for a subset of animals submitted to histopathological analysis from the study of Example 1.

[0056] FIG. 1 IB shows plots of the lung Ashcroft score; lung increased collagen, perivascular (PSR) score; and lung aSMA parenchymal immunolabeling score data for a subset of animals submitted to histopathological analysis from the study of Example 1.DETAILED DESCRIPTIONDefinitions

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. To the extent any material incorporated herein by reference is inconsistent with the express content of this disclosure, the express content controls. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an”, and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes”, and “included”, is not limiting.

[0058] Reference in the specification to “some embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosure.

[0059] As used herein, ranges and amounts can be expressed as “about” a particular value or range. Accordingly, “about” includes the exact amount modified by the term as well as an amount that would be expected to be within experimental error, such as for example, within 15%, 10%, or 5%. For example, “about 200 mg” means “200 mg,” and also a range of mgs that is within experimental error, e.g., plus or minus 15%, 10%, or 5% of 200 mg. As used herein, the term “about” may be used to modify a range or a particular value. It is understood that use of the term “about” before a listing of numerical values or a range or values applies the term “about” to each of the listed numerical values or across the full range of values. For example, “about 50 mg, 100 mg, or 200 mg” is understood to mean the same as “about 50 mg, about 100Attorney Docket No. 01183-0343-00PCT-KADmg, or about 200 mg,” and “about 50-200 mg” is understood to mean the same as “about 50 mg to about 200 mg.”

[0060] “Administering” or “administered to” as used herein (for example, with reference to administration of a drug for treatment, such as belumosudil or gusacitinib, to a subject), refers to the act of prescribing medicine(s) containing the drug for the subject to take during treatment, the act of dispensing the medicine(s) to the subject, and / or the act of physically receiving or ingesting the medicine(s). Thus, the drug (for example, belumosudil or gusacitinib) can be “administered” by a physician or other medical professional who writes prescriptions for medicine(s); and / or by a pharmacist who fills said prescriptions and / or dispenses the medicine(s) to the subject; and / or by the patient or subject who ingests the medicine and / or his or her partner or caretaker.

[0061] As used herein, “belumosudil” refers to the compound having the chemical name 2-{3-[4-(U / -indazol-5-ylamino)-2-quinazolinyl]phenoxy}-A-(propan-2-yl) acetamide and the chemical structure shown below. 2-{3-[4-(U / -indazol-5-ylamino)-2-quinazolinyl]phenoxy}-A-(propan-2-yl) acetamide is also referred to herein as belumosudil free base.

[0062] Belumosudil is also known as KD025 and SLx-2119.

[0063] Reference herein to “belumosudil” refers to the compound in any form, as well as pharmaceutically acceptable salts thereof, unless the context clearly indicates otherwise. The term “belumosudil” refers to the compound belumosudil (for example, in the free base form, amorphous form, and / or crystalline form), to pharmaceutically acceptable salts of belumosudil, for example, the mesylate salt form as used in as REZUROCK®, and to any form of belumosudil that may be used in a formulation or pharmaceutical composition for administering the compound to a patient. The mesylate salt of belumosudil is presently marketed in the United States and other countries under the tradename REZUROCK® (Kadmon Pharmaceuticals) for the treatment of patients with cGVHD, in some instances after failure of at least two prior lines of systemic therapy. The active pharmaceutical ingredient of REZUROCK® is belumosudil mesylate salt with the molecular formula C27H28N6O5S, a molecular weight of 548.62 g / mol, and the chemical name 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide methanesulfonate (1 : 1).Attorney Docket No. 01183-0343-00PCT-KAD

[0064] Gusacitinib is an oral inhibitor of JAK1 JAK2, JAK3, TYK2, and SYK. As used herein, “gusacitinib” refers to the compound having the chemical name 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile and the chemical structure shown below.

[0065] Gusacitinib is also known as ASN002, has a molecular formula of C24H28N8O2, and a molecular weight of 460.54 gm / mol. Reference herein to “gusacitinib” refers to the compound in any form, as well as pharmaceutically acceptable salts thereof, unless the context clearly indicates otherwise. The term “gusacitinib” refers to the compound gusacitinib (for example, in a free base form, an amorphous form, and / or crystalline form), to pharmaceutically acceptable salts of gusacitinib, for example, the hydrochloride salt form, and to any form of gusacitinib that may be used in a formulation or pharmaceutical composition for administering the compound to a patient.

[0066] “Pharmaceutically acceptable salts” refers to non-toxic, inorganic and organic acid addition salts of a compound, such as belumosudil or gusacitinib. In one embodiment, the pharmaceutically acceptable salt of belumosudil is the mesylate salt. In one embodiment, the pharmaceutically acceptable salt of gusacitinib is the hydrochloride salt.

[0067] As used herein, a “dose” refers to a specified amount of a drug, such as a compound described herein (for example, belumosudil or gusacitinib), taken at one time. For example, a dose of 200 mg of belumosudil refers to administering 200 mg of belumosudil to a subject at one time, for example as a tablet or capsule. Similarly, a dose of 40 mg of gusacitinib refers to administering 40 mg of gusacitinib to a subject at one time, for example as a tablet or capsule. The “dosage” refers to a specific amount, number, and frequency of doses over a specific period of time, such as one dose of 200 mg of belumosudil is administered to a subject twice a day. For example, a daily dosage of 400 mg of belumosudil refers to administering 400 mg of belumosudil to a subject, as either a single dose or as multiple doses, over the course of one dayAttorney Docket No. 01183-0343-00PCT-KAD(for example, belumosudil administered at a dose of 200 mg twice daily). Similarly, a daily dosage of 80 mg of gusacitinib refers to administering 80 mg of gusacitinib to a subject, as either a single dose or as multiple doses, over the course of one day (for example, gusacitinib administered at a dose of 40 mg twice daily).

[0068] In an embodiment, a dose of 200 mg of belumosudil refers to administering a dose equivalent to 200 mg of the free base to a subject at one time, for example as a tablet or capsule. In an embodiment, a dose of 40 mg of gusacitinib refers to administering a dose equivalent to 40 mg of the free base to a subject at one time, for example as a tablet. In an embodiment, a daily dosage of 400 mg of belumosudil refers to administering a dose equivalent to 400 mg of the free base to a subject, as either a single dose or as multiple doses, over the course of one day (for example, belumosudil administered at a dose equivalent to 200 mg of the free base twice daily). In an embodiment, a daily dosage of 80 mg of gusacitinib refers to administering a dose equivalent to 80 mg of the free base to a subject, as either a single dose or as multiple doses, over the course of one day (for example, gusacitinib administered at a dose equivalent to 40 mg of the free base twice daily).

[0069] As used herein, “therapeutically effective amount” refers to the amount of a drug, such as belumosudil or gusacitinib, that provides an intended therapeutic effect. A “therapeutically effective amount” of a drug (such as belumosudil or gusacitinib) means an amount which, when administered to a human for treating a disease (for example, cGVHD) is sufficient to effect treatment for the disease state being treated. As applied to cGVHD, “treating” or “treatment” includes (1) reducing the risk of developing the disease and / or inhibiting the disease, z.e., arresting or reducing the development of the disease or its clinical symptoms; and (2) relieving the disease, i.e., causing regression, reversal, or amelioration of the disease or reducing the number, frequency, duration, or severity of its clinical symptoms. The therapeutically effective amount of a drug may vary depending upon the health and physical condition of the subject to be treated, the extent of disease progression, the assessment of the medical situation, and other relevant factors.

[0070] As used herein, the terms “individual(s)”, “subject(s)”, and “patient(s)” mean any mammal and may be used interchangeably. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g., constant or intermittent) of a health care worker (e.g., a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker). In some embodiments, the patient is a human patient.Attorney Docket No. 01183-0343-00PCT-KAD

[0071] Although various features of the disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination.Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the disclosure may also be implemented in a single embodiment.Belumosudil

[0072] The combinations and methods of treating cGVHD, described herein include combinations with, and administration of, belumosudil.

[0073] Belumosudil is a ROCK2 inhibitor. Belumosudil binds to and inhibits the serine / threonine kinase activity of ROCK2 and to a lesser extent ROCK1. Belumosudil inhibits ROCK2 and ROCK1 with ICso values of approximately 100 nM and 3 pM, respectively. As such, belumosudil is useful in treating diseases, disorders, and conditions regulated by ROCK including autoimmune and fibrotic disorders. Belumosudil has been approved by the FDA for the treatment of cGVHD.

[0074] Belumosudil has the chemical name 2-{3-[4-(177-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-A-(propan-2-yl) acetamide and is represented by the chemical structure shown below.belumosudil

[0075] In some embodiments, belumosudil is provided as a pharmaceutically acceptable salt. In some embodiments, belumosudil is provided as the mesylate salt. In some embodiments, belumosudil is provided as the free base compound (i.e., not as a pharmaceutically acceptable salt).

[0076] In some embodiments, belumosudil is provided as a capsule or tablet for oral administration. In some embodiments, the capsule or tablet of belumosudil is provided for once daily or twice daily administration. In some embodiments, the capsule or tablet of belumosudil is provided for once daily administration. In some embodiments, the capsule or tablet of belumosudil is provided for twice daily administration.Attorney Docket No. 01183-0343-00PCT-KAD

[0077] In some embodiments, belumosudil is provided in a dose of from about 25 mg to about 200 mg belumosudil. In some embodiments, belumosudil is provided in a dose of about 50 mg, 100 mg, or 200 mg. In some embodiments, belumosudil is provided in a dose of about 50 mg. In some embodiments, belumosudil is provided in a dose of about 100 mg. In some embodiments, belumosudil is provided in a dose of about 200 mg.

[0078] In some embodiments, belumosudil is provided in a dose of from about 25 mg to about 200 mg belumosudil measured as the equivalent amount of free base. In some embodiments, belumosudil is provided in a dose equivalent to about 50 mg of the free base. In some embodiments, belumosudil is provided in a dose equivalent to about 100 mg of the free base. In an embodiment, belumosudil is provided in a dose equivalent to about 200 mg of free base.

[0079] In some embodiments, belumosudil is administered at a dosage of from about 25 mg to about 400 mg belumosudil daily, or at dosage equivalent to about 25 mg to about 400 mg of the free base daily. In some embodiments, belumosudil is administered at a daily dosage of about 50 mg, or a daily dosage equivalent to about 50 mg of the free base. In some embodiments, belumosudil is administered at a daily dosage of about 100 mg, or at a daily dosage equivalent to about 100 mg of the free base. In an embodiment, belumosudil is administered at a daily dosage of about 200 mg, or at a daily dosage equivalent to about 200 mg of free base. In an embodiment, belumosudil is administered at a daily dosage of about 400 mg, or at a daily dosage equivalent to about 400 mg of free base.

[0080] In some embodiments, belumosudil is provided as a capsule or tablet. In some embodiments, belumosudil is provided as a capsule or tablet comprising about 200 mg of belumosudil. In some embodiments, belumosudil is provided as a capsule or tablet comprising a dose equivalent to about 200 mg of the free base. In some embodiments, belumosudil is provided as a tablet comprising 200 mg of belumosudil. In some embodiments, belumosudil is provided as a tablet comprising a dose equivalent to 200 mg of the free base.Gusacitinib

[0081] The combinations and methods of treating cGVHD described herein include combinations with, and administration of, gusacitinib.

[0082] Gusacitinib inhibits JAK1 JAK2, JAK3, TYK2, and SYK, with ICso values of 5 to 46 nM in bioassays (Bissonette et al., British J. of Dermatology 2019, vol. 181, pp. 733-42;Jimenez et al., J. American Academy of Dermatology 2023, vol 89: pp. 235-42). A summary of the inhibitory data for gusacitinib is presented below (from Bissonnette 2019):Attorney Docket No. 01183-0343-00PCT-KAD

[0083] Gusacitinib has the chemical name 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile, and is represented by the chemical structure shown below.gusacitinib

[0084] In some embodiments, gusacitinib is provided as a pharmaceutically acceptable salt. In some embodiments, gusacitinib is provided as the hydrochloride salt. In some embodiments, gusacitinib is provided as the free base compound (i.e., not as a pharmaceutically acceptable salt).

[0085] In some embodiments, gusacitinib is provided as a capsule or tablet for oral administration. In some embodiments, the capsule or tablet of gusacitinib is provided for once daily or twice daily administration. In some embodiments, the capsule or tablet of gusacitinib is provided for once daily administration. In some embodiments, the capsule or tablet of gusacitinib is provided for twice daily administration.

[0086] In some embodiments, gusacitinib is provided in a dose of from about 5 mg to about 80 mg gusacitinib. In some embodiments, gusacitinib is provided in a dose of about 10 mg, 20 mg, 30 mg, 40 mg, or 80 mg. In some embodiments, gusacitinib is provided in a dose of about 40 mg or 80 mg. In some embodiments, gusacitinib is provided in a dose of about 10 mg. In some embodiments, gusacitinib is provided in a dose of about 20 mg. In some embodiments, gusacitinib is provided in a dose of about 30 mg. In some embodiments, gusacitinib is provided in a dose of about 40 mg. In some embodiments, gusacitinib is provided in a dose of about 80 mg.Attorney Docket No. 01183-0343-00PCT-KAD

[0087] In some embodiments, gusacitinib is provided in a dose of from about 5 mg to about 80 mg gusacitinib measured as the equivalent amount of free base. In some embodiments, gusacitinib is provided in a dose equivalent to about 5 mg to about 80 mg of the free base. In some embodiments, gusacitinib is provided in a dose equivalent to about 10 mg, 20 mg, 30 mg, 40 mg, or 80 mg of the free base. In some embodiments, gusacitinib is provided in a dose equivalent to about 10 mg of the free base. In some embodiments, gusacitinib is provided in a dose equivalent to about 20 mg of the free base. In some embodiments, gusacitinib is provided in a dose equivalent to about 30 mg of the free base. In some embodiments, gusacitinib is provided in a dose equivalent to about 40 mg of the free base. In some embodiments, gusacitinib is provided in a dose equivalent to about 80 mg of the free base.

[0088] In some embodiments, gusacitinib is administered at a dosage of from about 5 mg to about 80 mg gusacitinib daily, or at dosage equivalent to about 5 mg to about 80 mg of the free base daily. In some embodiments, gusacitinib is administered at a daily dosage of about 10 mg, or a daily dosage equivalent to about 10 mg of the free base. In some embodiments, gusacitinib is administered at a daily dosage of about 20 mg, or at a daily dosage equivalent to about 20 mg of the free base. In some embodiments, gusacitinib is administered at a daily dosage of about 30 mg, or at a daily dosage equivalent to about 30 mg of the free base. In an embodiment, gusacitinib is administered at a daily dosage of about 40 mg, or at a daily dosage equivalent to about 40 mg of free base. In an embodiment, gusacitinib is administered at a daily dosage of about 80 mg, or at a daily dosage equivalent to about 80 mg of free base.

[0089] In some embodiments, gusacitinib is provided as a capsule or tablet. In some embodiments, gusacitinib is provided as a tablet comprising about 10 mg of gusacitinib, or a dose equivalent to about 10 mg of free base. In some embodiments, gusacitinib is provided as a tablet comprising about 20 mg of gusacitinib, or a dose equivalent to about 20 mg of free base. In some embodiments, gusacitinib is provided as a tablet comprising about 30 mg of gusacitinib, or a dose equivalent to about 30 mg of free base. In some embodiments, gusacitinib is provided as a tablet comprising about 40 mg of gusacitinib, or a dose equivalent to about 40 mg of free base. In some embodiments, gusacitinib is provided as a tablet comprising about 80 mg of gusacitinib, or a dose equivalent to about 80 mg of free base.Combinations including Pharmaceutical Compositions thereof

[0090] In one aspect, provided herein are combinations including belumosudil and gusacitinib. In an embodiment, the combination comprises 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof,Attorney Docket No. 01183-0343-00PCT-KADand 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0091] In some embodiments, the combination of belumosudil and gusacitinib comprises about 25-200 mg of 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof. For example, in an embodiment, the combination comprises about 200 mg of belumosudil and about 40 mg of gusacitinib. In an embodiment, the combination comprises about 100 mg of belumosudil and about 40 mg of gusacitinib. In an embodiment, the combination comprises about 50 mg of belumosudil and about 40 mg of gusacitinib. In an embodiment, the combination comprises about 200 mg of belumosudil and about 80 mg of gusacitinib. In an embodiment, the combination comprises about 100 mg of belumosudil and about 80 mg of gusacitinib. In an embodiment, the combination comprises about 50 mg of belumosudil and about 80 mg of gusacitinib. In an embodiment, the combination comprises about 200 mg of belumosudil and about 30 mg of gusacitinib. In an embodiment, the combination comprises about 100 mg of belumosudil and about 30 mg of gusacitinib. In an embodiment, the combination comprises about 50 mg of belumosudil and about 30 mg of gusacitinib. In an embodiment, the combination comprises about 200 mg of belumosudil and about 20 mg of gusacitinib. In an embodiment, the combination comprises about 100 mg of belumosudil and about 20 mg of gusacitinib. In an embodiment, the combination comprises about 50 mg of belumosudil and about 20 mg of gusacitinib. In an embodiment, the combination comprises about 200 mg of belumosudil and about 10 mg of gusacitinib. In an embodiment, the combination comprises about 100 mg of belumosudil and about 10 mg of gusacitinib. In an embodiment, the combination comprises about 50 mg of belumosudil and about 10 mg of gusacitinib.

[0092] In an embodiment, the combination comprises about 200 mg of belumosudil free base and about 40 mg of gusacitinib free base. In an embodiment, the combination comprises about 100 mg of belumosudil free base and about 40 mg of gusacitinib free base. In an embodiment, the combination comprises about 50 mg of belumosudil free base and about 40 mg of gusacitinib free base. In an embodiment, the combination comprises about 200 mg of belumosudil free base and about 80 mg of gusacitinib free base. In an embodiment, the combination comprises about 100 mg of belumosudil free base and about 80 mg of gusacitinib free base. In an embodiment, the combination comprises about 50 mg of belumosudil free base and about 80 mg of gusacitinib free base. In an embodiment, the combination comprises about 200 mg of belumosudil free base and about 30 mg of gusacitinib free base. In an embodiment, the combination comprises about 100 mg of belumosudil free base and about 30 mg of gusacitinib free base. In an embodiment,Attorney Docket No. 01183-0343-00PCT-KADthe combination comprises about 50 mg of belumosudil free base and about 30 mg of gusacitinib free base. In an embodiment, the combination comprises about 200 mg of belumosudil free base and about 20 mg of gusacitinib free base. In an embodiment, the combination comprises about 100 mg of belumosudil free base and about 20 mg of gusacitinib free base. In an embodiment, the combination comprises about 50 mg of belumosudil free base and about 20 mg of gusacitinib free base. In an embodiment, the combination comprises about 200 mg of belumosudil free base and about 10 mg of gusacitinib free base. In an embodiment, the combination comprises about 100 mg of belumosudil free base and about 10 mg of gusacitinib free base. In an embodiment, the combination comprises about 50 mg of belumosudil free base and about 10 mg of gusacitinib free base.

[0093] In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is provided in a dose of from about 25 mg to about 200 mg of belumosudil measured as the equivalent amount of free base. For example, in some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is provided in a dose equivalent to about 50 mg of the free base. In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is provided in a dose equivalent to about 100 mg of the free base. In an embodiment of a combination of belumosudil and gusacitinib, the belumosudil is provided in a dose equivalent to about 200 mg of free base.

[0094] In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 10 mg. In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 20 mg. In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 30 mg. In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 40 mg. In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 80 mg.

[0095] In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 10 mg. In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 20 mg. In some embodiments of a combination ofAttorney Docket No. 01183-0343-00PCT-KADbelumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 30 mg. In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 40 mg. In some embodiments of a combination of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 80 mg.

[0096] In certain embodiments, the combination of belumosudil and gusacitinib comprises about 5-80 mg of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof. For example, in an embodiment, the combination comprises about 10 mg of gusacitinib, about 20 mg of gusacitinib, about 30 mg of gusacitinib, about 40 mg of gusacitinib, or about 80 mg of gusacitinib. In an embodiment, the combination comprises about 10 mg of gusacitinib free base, about 20 mg of gusacitinib free base, about 30 mg of gusacitinib free base, about 40 mg of gusacitinib free base, or about 80 mg of gusacitinib free base.

[0097] In an embodiment, the combination of belumosudil and gusacitinib is formulated for oral administration to a human patient. In certain embodiments, the combination of belumosudil and gusacitinib comprises one tablet or capsule. In some embodiments, the combination of belumosudil and gusacitinib comprises more than one tablet or capsule. For example, the combination comprises one tablet of belumosudil and one tablet of gusacitinib. In an embodiment, the combination comprises one capsule of belumosudil and one capsule of gusacitinib, or the combination comprises one tablet of belumosudil and one capsule of gusacitinib. Alternatively, the combination of belumosudil and gusacitinib is a single tablet that includes both belumosudil and gusacitinib. In an embodiment, the combination of belumosudil and gusacitinib is a single capsule that includes both belumosudil and gusacitinib.

[0098] In another aspect, provided herein are pharmaceutical compositions including belumosudil and gusacitinib. In an embodiment, the pharmaceutical compositions comprise a combination of belumosudil and gusacitinib. In some embodiments, the pharmaceutical composition comprises 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0099] In an embodiment, the pharmaceutical compositions including belumosudil and gusacitinib are for use in the treatment of chronic graft-versus-host disease (cGVHD). In anAttorney Docket No. 01183-0343-00PCT-KADembodiment, the pharmaceutical compositions including belumosudil and gusacitinib are for use in the manufacture of a medicament to treat cGVHD.

[0100] In some embodiments, the pharmaceutical composition including belumosudil and gusacitinib comprises about 50-200 mg of belumosudil or a pharmaceutically acceptable salt thereof. For example, the pharmaceutical composition including belumosudil and gusacitinib comprises about 5-80 mg of gusacitinib or a pharmaceutically acceptable salt thereof. In an embodiment, the pharmaceutical composition of belumosudil and gusacitinib comprises about 50-200 mg of 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

[0101] For example, in an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil and about 40 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil and about 40 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil and about 40 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil and about 80 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil and about 80 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil and about 80 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil and about 30 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil and about 30 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil and about 30 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil and about 20 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil and about 20 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil and about 20 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil and about 10 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil and about 10 mg of gusacitinib. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil and about 10 mg of gusacitinib.

[0102] In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil free base and about 40 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil free base and about 40 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 50Attorney Docket No. 01183-0343-00PCT-KADmg of belumosudil free base and about 40 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil free base and about 80 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil free base and about 80 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil free base and about 80 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil free base and about 30 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil free base and about 30 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil free base and about 30 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil free base and about 20 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil free base and about 20 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil free base and about 20 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 200 mg of belumosudil free base and about 10 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 100 mg of belumosudil free base and about 10 mg of gusacitinib free base. In an embodiment, the pharmaceutical composition comprises about 50 mg of belumosudil free base and about 10 mg of gusacitinib free base.

[0103] In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is provided in a dose of from about 25 mg to about 200 mg of belumosudil measured as the equivalent amount of free base. For example, in some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is provided in a dose equivalent to about 50 mg of the free base. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is provided in a dose equivalent to about 100 mg of the free base. In an embodiment of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is provided in a dose equivalent to about 200 mg of free base.

[0104] In certain embodiments, the pharmaceutical composition of belumosudil and gusacitinib comprises about 5-80 mg of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof. For example, in an embodiment, the pharmaceutical composition comprises about 10 mg of gusacitinib, about 20 mg of gusacitinib, about 30 mg of gusacitinib, about 40 mg of gusacitinib, or about 80 mg of gusacitinib.Attorney Docket No. 01183-0343-00PCT-KAD

[0105] In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 10 mg. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 20 mg. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 30 mg. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 40 mg. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered once daily at a dose of 200 mg and the gusacitinib is administered once daily at a dose of 80 mg.

[0106] In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 10 mg. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 20 mg. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 30 mg. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 40 mg. In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil is administered twice daily at a dose of 200 mg and the gusacitinib is administered at a daily dosage of 80 mg.

[0107] In some embodiments of a pharmaceutical composition of belumosudil and gusacitinib, the composition is administered with food. For example, in an embodiment, the pharmaceutical composition comprising belumosudil and gusacitinib is administered once daily with food. In an embodiment, the pharmaceutical composition comprising belumosudil and gusacitinib is administered twice daily with food. Alternatively, the composition is administered without food. In some embodiments, the pharmaceutical composition comprising belumosudil and gusacitinib is administered orally once daily with food. In additional embodiments, the pharmaceutical composition comprising belumosudil and gusacitinib is administered orally twice daily with food.Attorney Docket No. 01183-0343-00PCT-KAD

[0108] In an embodiment, the pharmaceutical composition of belumosudil and gusacitinib is formulated for oral administration to a human patient. In certain embodiments, the pharmaceutical composition of belumosudil and gusacitinib comprises a tablet or capsule. In some embodiments, the pharmaceutical composition of belumosudil and gusacitinib comprises more than one tablet or capsule. For example, the pharmaceutical composition comprises one tablet of belumosudil and one tablet of gusacitinib. In an embodiment, the pharmaceutical composition comprises one capsule of belumosudil and one capsule of gusacitinib, or one tablet of belumosudil and one capsule of gusacitinib. Alternatively, the pharmaceutical composition of belumosudil and gusacitinib is a single tablet that includes both belumosudil and gusacitinib. In an embodiment, the pharmaceutical composition of belumosudil and gusacitinib is a single capsule that includes both belumosudil and gusacitinib.

[0109] In some embodiments of a combination or a pharmaceutical composition of belumosudil and gusacitinib, the belumosudil or a pharmaceutically acceptable salt thereof is present in an amount greater than the amount of gusacitinib or a pharmaceutically acceptable salt thereof. In an embodiment, the ratio of belumosudil or a pharmaceutically acceptable salt thereof to gusacitinib or a pharmaceutically acceptable salt thereof ranges between 75-90:25-10. In an embodiment, the ratio of belumosudil or a pharmaceutically acceptable salt thereof to gusacitinib or a pharmaceutically acceptable salt thereof is about 80:20. In an embodiment, the ratio of belumosudil or a pharmaceutically acceptable salt thereof to gusacitinib or a pharmaceutically acceptable salt thereof is about 86:14. In an embodiment, the belumosudil :gusacitinib ratio ranges between 75-90:25-10. In an embodiment, the belumosudil :gusacitinib ratio is about 80:20. In an embodiment, the belumosudikgusacitinib ratio is about 86:14.Methods of Treatment

[0110] In one aspect, provided herein are methods of treating chronic graft-versus-host disease (cGVHD) in a human patient in need thereof comprising administering to the human patient a therapeutically effective amount of a combination comprising belumosudil and gusacitinib. In an embodiment, the combination comprises 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof. In an embodiment, the combination comprises belumosudil mesylate and gusacitinib hydrochloride. In an embodiment, the cGVHD is sclerodermatous cGVHD. In an embodiment, cGVHD is lung cGVHD. In an embodiment, the cGVHD is sclerodermatous cGVHD or lung cGVHD.Attorney Docket No. 01183-0343-00PCT-KAD

[0111] In some embodiments of the disclosed methods of treating cGVHD with a combination comprising belumosudil and gusacitinib, the belumosudil is administered at a daily dosage of up to about 400 mg or at a daily dosage of about 25-400 mg. In an embodiment, the belumosudil is administered at a dose of about 50 mg. In an embodiment, the belumosudil is administered at a dose of about 100 mg. In an embodiment, the belumosudil is administered at a dose of about 200 mg. In an embodiment, the belumosudil is administered at a dose of about 50 mg, 100 mg, or 200 mg.

[0112] In some embodiments of the disclosed methods of treating cGVHD with a combination comprising belumosudil and gusacitinib, the belumosudil is expressed as an equivalent to the belumosudil free base. For example, in some embodiments of methods of treating cGVHD with a combination of belumosudil and gusacitinib, the belumosudil is provided in a dose equivalent to about 50 mg of the free base. In some embodiments, the belumosudil is provided in a dose equivalent to about 100 mg of the free base. In an embodiment, the belumosudil is provided in a dose equivalent to about 200 mg of free base.

[0113] In some embodiments of the disclosed methods of treating cGVHD with a combination comprising belumosudil and gusacitinib, the gusacitinib is expressed as an equivalent to the gusacitinib free base. For example, in some embodiments of methods of treating cGVHD with a combination of belumosudil and gusacitinib, the gusacitinib is provided in a dose equivalent to about 10 mg of the free base. In some embodiments, the gusacitinib is provided in a dose equivalent to about 20 mg of the free base. In some embodiments, the gusacitinib is provided in a dose equivalent to about 30 mg of the free base. In an embodiment, the gusacitinib is provided in a dose equivalent to about 40 mg of free base. In an embodiment, the gusacitinib is provided in a dose equivalent to about 80 mg of free base.

[0114] In some embodiments of the disclosed methods of treating cGVHD with a combination of belumosudil and gusacitinib, the dose of belumosudil is administered to the human patient once or twice daily. In an embodiment, the dose of belumosudil is administered to the human patient once daily. In an alternative embodiment, the dose of belumosudil is administered to the human patient twice daily. In an embodiment, the belumosudil is administered orally. In an embodiment, the belumosudil is administered as a tablet. In an embodiment, the belumosudil is administered as a capsule. The belumosudil may be administered with or without food. For example, in an embodiment, the dose of belumosudil is administered to the human patient once daily with food. In an embodiment, the dose of belumosudil is administered to the human patient twice daily with food. In an embodiment, the dose of belumosudil is administered to the human patient once daily without food. In anAttorney Docket No. 01183-0343-00PCT-KADembodiment, the dose of belumosudil is administered to the human patient twice daily without food. In some embodiments, the dose of belumosudil is administered orally to the human patient once daily with food. In alternative embodiments, the dose of belumosudil is administered orally to the human patient twice daily with food.

[0115] In a further aspect of the disclosed methods of treating cGVHD with a combination of belumosudil and gusacitinib, the gusacitinib is administered to the human patient at a dosage of up to about 80 mg daily. In an embodiment, the gusacitinib is administered to the human patient at a dosage of up to about 80 mg. In some embodiments, the gusacitinib is administered to the human patient at a dosage of about 5-80 mg. In some embodiments, the gusacitinib is administered to the human patient at a dosage of about 5-80 mg daily. In an embodiment, the gusacitinib is administered to the human patient at a dose of about 10 mg, 20 mg, 30 mg, 40 mg, or 80 mg. In an embodiment, the gusacitinib is administered to the human patient at a daily dose of about 10 mg, 20 mg, 30 mg, 40 mg, or 80 mg. In an embodiment, the gusacitinib is administered to the human patient at a dose of about 10 mg. In an embodiment, the gusacitinib is administered to the human patient at a dose of about 20 mg. In an embodiment, the gusacitinib is administered to the human patient at a dose of about 30 mg. In an embodiment, the gusacitinib is administered to the human patient at a dose of about 40 mg. In an embodiment, the gusacitinib is administered to the human patient at a dose of about 80 mg.

[0116] In some embodiments, the gusacitinib is administered to the human patient at a dosage equivalent to a dose of up to about 80 mg of the free base daily. In an embodiment, the gusacitinib is administered to the human patient at a dosage equivalent to about 80 mg of the free base. In some embodiments, the gusacitinib is administered to the human patient at a dosage equivalent to about 5-80 mg of the free base. In some embodiments, the gusacitinib is administered to the human patient at a dosage equivalent to about 5-80 mg of the free base daily. In an embodiment, the gusacitinib is administered to the human patient at a dose equivalent to about 10 mg, 20 mg, 30 mg, 40 mg, or 80 mg of the free base. In an embodiment, the gusacitinib is administered to the human patient at a daily dose equivalent to about 10 mg, 20 mg, 30 mg, 40 mg, or 80 mg of the free base. In an embodiment, the gusacitinib is administered to the human patient at a dose equivalent to about 10 mg of the free base. In an embodiment, the gusacitinib is administered to the human patient at a dose equivalent to about 20 mg of the free base. In an embodiment, the gusacitinib is administered to the human patient at a dose equivalent to about 30 mg of the free base. In an embodiment, the gusacitinib is administered to the human patient at a dose equivalent to about 40 mg of the free base. In an embodiment, the gusacitinib is administered to the human patient at a dose equivalent to about 80 mg of the free base.Attorney Docket No. 01183-0343-00PCT-KAD

[0117] In one embodiment, the gusacitinib is administered to the human patient once a day or twice a day. In an embodiment, the gusacitinib is administered to the human patient once a day. In an alternative embodiment, the gusacitinib is administered to the human patient twice a day. In some embodiments, the gusacitinib is administered orally. In some embodiments, the gusacitinib is administered as a tablet. In some embodiments, the gusacitinib is administered as a capsule. The gusacitinib may be administered with or without food. For example, in an embodiment, the dose of gusacitinib is administered to the human patient once daily with food. In an embodiment, the dose of gusacitinib is administered to the human patient twice daily with food. In an embodiment, the dose of gusacitinib is administered to the human patient once daily without food. In an embodiment, the dose of gusacitinib is administered to the human patient twice daily without food. In some embodiments, the dose of gusacitinib is administered orally to the human patient once daily with food. In alternative embodiments, the dose of gusacitinib is administered orally to the human patient twice daily with food.

[0118] In some embodiments of the disclosed methods of treating cGVHD with a combination of belumosudil and gusacitinib, the combination is administered to the human patient with food. For example, in an embodiment, the combination is administered once daily with food. In an embodiment, the combination is administered twice daily with food.Alternatively, the combination is administered to the human patient without food. In some embodiments, the combination of belumosudil and gusacitinib is administered orally once daily with food. In additional embodiments, the combination comprising belumosudil and gusacitinib is administered orally twice daily with food.

[0119] In additional aspects of the disclosed methods of treating cGVHD with a combination of belumosudil and gusacitinib, the belumosudil and gusacitinib are administered separately. For example, the belumosudil is administered orally twice daily and the gusacitinib is administered orally once daily at a time different than the administration of belumosudil. In an embodiment, the belumosudil and gusacitinib are administered at times that differ by at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In certain embodiments, the belumosudil and gusacitinib are administered at times that differ by no more than about 1 hour, no more than about 2 hours, no more than about 3 hours, no more than about 4 hours, no more than about 5 hours, no more than about 6 hours, no more than about 7 hours, no more than about 8 hours, no more than about 9 hours, no more than about 10 hours, no more than about 11 hours, or no more than about 12 hours. In some embodiments, the belumosudil and gusacitinib are administered separately and within 5 minutes of the other, within 10 minutes of the other,Attorney Docket No. 01183-0343-00PCT-KADwithin 15 minutes of the other, within 20 minutes of the other, within 25 minutes of the other, within 30 minutes of the other, within 35 minutes of the other, within 40 minutes of the other, within 45 minutes of the other, within 50 minutes of the other, within 55 minutes of the other, or within 60 minutes of the other. In some embodiments, the belumosudil and gusacitinib are administered separately and at times that differ by no more than about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, the belumosudil is administered on a different day than the gusacitinib. In alternative embodiments, the belumosudil is administered on the same day as the gusacitinib.

[0120] In an embodiment of the disclosed methods of treating cGVHD with a combination of belumosudil and gusacitinib, the belumosudil and gusacitinib are administered to the human patient sequentially. For example, the belumosudil is administered orally once daily, and the gusacitinib is administered orally once daily at a time after the administration of the belumosudil. In some embodiments, the gusacitinib is administered orally once daily, and the belumosudil is administered orally once daily at a time after the administration of the gusacitinib. In an embodiment, the belumosudil and gusacitinib are administered at times that differ by at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In certain embodiments, the belumosudil and gusacitinib are administered at times that differ by no more than about 1 hour, no more than about 2 hours, no more than about 3 hours, no more than about 4 hours, no more than about 5 hours, no more than about 6 hours, no more than about 7 hours, no more than about 8 hours, no more than about 9 hours, no more than about 10 hours, no more than about 11 hours, or no more than about 12 hours. In some embodiments, the belumosudil and gusacitinib are administered sequentially within 5 minutes of the other, within 10 minutes of the other, within 15 minutes of the other, within 20 minutes of the other, within 25 minutes of the other, within 30 minutes of the other, within 35 minutes of the other, within 40 minutes of the other, within 45 minutes of the other, within 50 minutes of the other, within 55 minutes of the other, or within 60 minutes of the other. In some embodiments, the belumosudil and gusacitinib are administered sequentially and at times that differ by no more than about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes. In some embodiments, the belumosudil is administered on a different day than the gusacitinib. In alternative embodiments, the belumosudil is administeredAttorney Docket No. 01183-0343-00PCT-KADon the same day as the gusacitinib. In some embodiments, the belumosudil is administered on one day and the gusacitinib is administered on the day after the belumosudil. In some embodiments, the gusacitinib is administered on one day and the belumosudil is administered on the day after the gusacitinib.

[0121] In some embodiments of the disclosed methods of treating cGVHD with a combination of belumosudil and gusacitinib, the belumosudil and gusacitinib are administered to the human patient simultaneously. For example, the belumosudil is administered orally once daily, and the gusacitinib is administered orally once daily at the same time as the administration of the belumosudil. In an embodiment, the belumosudil and gusacitinib are administered at times within 5 minutes of the other, within 10 minutes of the other, or within 15 minutes of the other. In some embodiments, the belumosudil and gusacitinib are administered times that differ by no more than about 5 minutes, about 10 minutes, or about 15 minutes. In some embodiments, the belumosudil is administered in the same tablet as the gusacitinib. In alternative embodiments, the belumosudil is administered in the same capsule as the gusacitinib. In some embodiments, the belumosudil and the gusacitinib are administered in separate tablets that are taken by the human patient at the same time.

[0122] Also provided herein are aspects of belumosudil for use in the treatment of cGVHD, wherein the compound is formulated for co-administration with gusacitinib. For example, in some embodiments, the belumosudil is formulated to be in the same tablet as the gusacitinib. In alternative embodiments, the belumosudil is formulated to be in the same capsule as the gusacitinib. In an embodiment, the belumosudil is formulated for administration separately, sequentially, or simultaneously with the gusacitinib. In an embodiment, the belumosudil is formulated to be in a different tablet than the gusacitinib. In alternative embodiments, the belumosudil is formulated to be in a different capsule than the gusacitinib.Kits / Article of Manufacture

[0123] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more of the methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic. In some embodiments, the kit comprises belumosudil and gusacitinib. In some embodiments, the kit comprises belumosudil. In some embodiments, the kit comprises gusacitinib.Attorney Docket No. 01183-0343-00PCT-KAD

[0124] A kit typically includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included. In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.

[0125] Also disclosed herein are pharmaceutical compositions for use in the methods described herein. In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for drugs, or the approved product insert. In one embodiment, compositions containing the combination of belumosudil and gusacitinib provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0126] In an embodiment, provided is a kit comprising a first pharmaceutical composition including belumosudil and at least one pharmaceutically acceptable excipient, a second pharmaceutical composition including gusacitinib and at least one pharmaceutically acceptable excipient, and instructions for administering the first and second pharmaceutical compositions. In an embodiment, the first pharmaceutical composition and the second pharmaceutical composition are in separate compartments of the kit. In certain embodiments, the instructions comprise instructions to administer the first pharmaceutical composition separately, sequentially, or simultaneously with the second pharmaceutical composition.

[0127] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein in their entirety by reference.Attorney Docket No. 01183-0343-00PCT-KAD

[0128] Embodiments. Exemplary, non-limiting embodiments include the following.

[0129] Embodiment 1. A combination comprising 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0130] Embodiment 2. The combination of Embodiment 1, comprising about 25-200 mg of 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

[0131] Embodiment 3. The combination of Embodiment 1 or 2, comprising about 5-80 mg, such as 40-80 mg, of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0132] Embodiment 4. The combination of any one of Embodiments 1-3, wherein the combination is formulated for oral administration to a human patient.

[0133] Embodiment 5. The combination of any one of Embodiments 1-4, wherein the combination comprises one tablet or capsule.

[0134] Embodiment 6. The combination of any one of Embodiments 1-4, wherein the combination comprises more than one tablet or capsule.

[0135] Embodiment 7. A pharmaceutical composition comprising 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0136] Embodiment 8. The pharmaceutical composition of Embodiment 7 for use in the treatment of chronic graft-versus-host disease (cGVHD).

[0137] Embodiment 9. The pharmaceutical composition of Embodiment 8, wherein the cGVHD is sclerodermatous cGVHD.

[0138] Embodiment 10. The pharmaceutical composition of Embodiment 8, wherein the cGVHD is lung cGVHD.

[0139] Embodiment 11. The pharmaceutical composition of any one of Embodiments 7-10, comprising about 25-200 mg of 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

[0140] Embodiment 12. The pharmaceutical composition of any one of Embodiments 7-11, comprising about 5-80 mg of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6JT-Attorney Docket No. 01183-0343-00PCT-KADpyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0141] Embodiment 13. The pharmaceutical composition of any one of Embodiments 7-12, formulated for oral administration to a human patient.

[0142] Embodiment 14. A method for treating cGVHD in a human patient in need thereof comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and (b) 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0143] Embodiment 15. The method of Embodiment 14, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, is administered to the human patient at a daily dosage equivalent to up to about 400 mg of the free base.

[0144] Embodiment 16. The method of Embodiment 14, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof, is administered to the human patient at a daily dosage equivalent to about 25-400 mg of the free base.

[0145] Embodiment 17. The method of any one of Embodiments 14-16, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof, is administered to the human patient at a dose equivalent to about 50 mg, 100 mg, or 200 mg of the free base.

[0146] Embodiment 18. The method of any one of Embodiments 15-17, wherein the dose is administered to the human patient once or twice daily.

[0147] Embodiment 19. The method of any one of Embodiments 15-17, wherein the dose is administered to the human patient once daily.

[0148] Embodiment 20. The method of any one of Embodiments 15-17, wherein the dose is administered to the human patient twice daily.

[0149] Embodiment 21. The method of any one of Embodiments 14-20, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, is administered orally.

[0150] Embodiment 22. The method of any one of Embodiments 14-21, wherein the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-Attorney Docket No. 01183-0343-00PCT-KADyl]acetonitrile or a pharmaceutically acceptable salt thereof, is administered to the human patient at a daily dosage equivalent to up to about 80 mg of the free base.

[0151] Embodiment 23. The method of any one of Embodiments 14-21, wherein the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, is administered to the human patient at a daily dosage equivalent to about 5-80 mg of the free base.

[0152] Embodiment 24. The method of any one of Embodiments 14-23, wherein the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, is administered to the human patient at a dose equivalent to about 10 mg, 20 mg, 30 mg, 40 mg, or 80 mg of the free base.

[0153] Embodiment 25. The method of any one of Embodiments 14-24, wherein the dose of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, is administered to the human patient once a day or twice a day.

[0154] Embodiment 26. The method of any one of Embodiments 14-25, wherein the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, is administered orally.

[0155] Embodiment 27. The method of any one of Embodiments 14-26, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, are administered separately.

[0156] Embodiment 28. The method of Embodiment 27, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, are administered sequentially.

[0157] Embodiment 29. The method of any one of Embodiments 14-26, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, are administered simultaneously.

[0158] Embodiment 30. The compound 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof,Attorney Docket No. 01183-0343-00PCT-KADfor use in the treatment of cGVHD, wherein the compound is formulated for co-administration with 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0159] Embodiment 31. The compound for use according to Embodiment 30, wherein the compound is administered separately, sequentially, or simultaneously with the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0160] Embodiment 32. The compound 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy }-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, for use in the treatment of cGVHD, wherein the compound is co-administered with 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0161] Embodiment 33. The compound for use according to Embodiment 32, wherein the compound is administered separately, sequentially, or simultaneously with the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

[0162] Embodiment 34. The compound 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, for use in the treatment of cGVHD, wherein the compound is co-administered with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy } -N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

[0163] Embodiment 35. The compound for use according to Embodiment 34, wherein the compound is administered separately, sequentially, or simultaneously with the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

[0164] Embodiment 36. The compound 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic hand eczema or atopic dermatitis, wherein the compound is co-administered with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}- N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

[0165] Embodiment 37. The compound for use according to Embodiment 36, wherein the compound is administered separately, sequentially, or simultaneously with the 2-{3-[4-(lH- indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.Attorney Docket No. 01183-0343-00PCT-KAD

[0166] Embodiment 38. A method for treating cGVHD comprising administering to a patient in need thereof a therapeutically effective amount of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, wherein the patient has previously received treatment with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof.

[0167] Embodiment 39. A method for treating cGVHD comprising administering to a patient in need thereof a therapeutically effective amount of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, wherein the patient is receiving treatment with 2-{3-[4-(1 H-indazol-5-ylamino)-2-quinazolinyl]phenoxy }-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof.

[0168] Embodiment 40. A method for treating cGVHD comprising administering to a patient in need thereof a therapeutically effective amount of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, wherein the patient is on stable treatment with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof.

[0169] Embodiment 41. The method of any one of Embodiments 14-29 or 38-40, or the compound for use of any one of Embodiments 30-37, wherein the cGVHD is sclerodermatous cGVHD or lung cGVHD.

[0170] Embodiment 42. The method of any one of Embodiments 14-29 or 38-40, or the compound for use of any one of Embodiments 30-37, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, are administered with food.

[0171] Embodiment 43. The method of any one of Embodiments 14-29 or 38-40, or the compound for use of any one of Embodiments 30-37, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, are administered without food.Attorney Docket No. 01183-0343-00PCT-KAD

[0172] Embodiment 44. A kit comprising: (i) a first pharmaceutical composition comprising 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient; (11) a second pharmaceutical composition comprising 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6H-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient; and (iii) instructions for administering the first and second pharmaceutical compositions.

[0173] Embodiment 45. The kit according to Embodiment 44, wherein the first pharmaceutical composition and the second pharmaceutical composition are in separate compartments.

[0174] Embodiment 46. The kit according to Embodiment 44, wherein the instructions comprise instructions to administer the first pharmaceutical composition separately, sequentially, or simultaneously with the second pharmaceutical composition.EXAMPLES

[0175] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

[0176] Example 1. Activity of Belumosudil and Gusacitinib in a Mouse Model of Sclerodermatous Chronic Graft-Versus-Host Disease (cGVHD).

[0177] The main objective of this study was to evaluate the effect of novel treatments on a murine model of sclerodermatous cGVHD.

[0178] Animals.

[0179] Recipient female C57B1 / 6 (CD45.1) mice (n = 102; 8 weeks) with an average starting body weight (± SEM) of 18.56 ± 0.09 g were obtained from Jackson Laboratories (Bar Harbor, Maine). Donor female LP / J mice (n = 144; 6-10 weeks) were obtained from Jackson Laboratories (Bar Harbor, Maine). Prior to the study start, the animals were observed daily in order to reject any that presented in poor condition.

[0180] Animals were fed with standard chow and water was provided ad libitum. Animals were randomized into nine (9) groups at the start of the study: one (1) group of six (6) animals and eight (8) groups of twelve (12) animals each.

[0181] Study Design.

[0182] Mice were randomized into one (1) group of six (6) and eight (8) groups of twelve (12). Sclerodermatous cGVHD was induced in Group 3-9 C57B1 / 6 (CD45.1) mice using a singleAttorney Docket No. 01183-0343-00PCT-KADacute dose of 8.5 Gy of total body irradiation (TBI) on Day -1. On Day 0, Group 3-9 C57B1 / 6 (CD45.1) recipients were given an intravenous (IV) injection of a combination of bone marrow (BM) cells and splenocytes in sterile PBS. Group 1 served as the Naive control group and did not receive either TBI or a cell transfer. Group 2 received an allogeneic cell transfer consisting of only bone marrow cells obtained from donor female LP / J (CD45.2) mice. Groups 3-9 received an allogeneic cell transfer consisting of bone marrow and splenic cells obtained from donor female LP / J (CD45.2) mice. The spleen cells were isolated using the Miltenyi GentleMACS Dissociators. Post-transplant, all animals were housed under standard environmental conditions and were maintained on the appropriate irradiated sterile rodent chow and sterile water ad libitum. Additionally, following TBI on Day -1 and up to Day 8, all animals were given supplemental high calorie highly palatable food in a dish on the bottom of the cage. Following transfer on Day 0, and then again as needed, all animals were given 1 mL / animal / day of supplemental fluids (warmed ringers solution) via subcutaneous injection. An additional 1 mL fluid was given in the pm on an as needed basis.

[0183] All animals were monitored daily to record weight change, survival, GVHD score (Table 2), and incidence of diarrhea and bloody stool. In addition, a second scoring system for sclerodermatous chronic GVHD (scGVHD) was used (Table 3). Animals that appeared to be in pain were administered Buprenorphine BID, as needed. Animals that were unable to right themselves, cold to the touch or moribund were euthanized. Animals requiring euthanasia were euthanized by CO2 inhalation and did not have collections performed. Animals that were found dead did not have sample collections performed.

[0184] On Day 20, animals in Groups 3-9 were redistributed into groups so that average scGVHD score was similar across Groups 3-9. Redistribution was based on the composite, 5-component scGVHD score described in Table 3. Animals were dosed with vehicle or test articles as detailed in Table 1, below. Dosing started on Day 21 and continued until Day 55. On Days 33, 36, 39, 42, and 45 all animals were photographed under isoflurane anesthesia (top-down picture, plain white background) to assess disease severity. Each photo was tagged with the corresponding animal’s number to aid identification. On Day 56, all surviving animals were sacrificed by CO2 inhalation and organs detailed in Table 1 were collected.

[0185] The details of the study design are shown in Table 1.Attorney Docket No. 01183-0343-00PCT-KADTable 1. Study Design.&Total number = 102 animals; *Belumosudil administered for AM dose followed by vehicle for PM dose.

[0186] Disease Induction.

[0187] All recipient animals received a total body irradiation dose of 8.5 Gy on Day -1 as detailed in Table 1. On Day 0, Group 2 animals received a bone marrow (BM) transplant containing only bone marrow cells obtained from donor LP / J (CD45.2) mice in sterile PBS via the tail vein (200 pL) according to Table 1. On Day 0, Group 3-9 animals received a bone marrow (BM) transplant containing bone marrow cells in combination with splenocytes obtained from donor LP / J (CD45.2) mice in sterile PBS via the tail vein (200 pL) according to Table 1.Attorney Docket No. 01183-0343-00PCT-KADThe spleen cells were isolated using the Miltenyi Gentlemacs Dissociators. The BM cells were isolated using standard flushing practices (femur and tibia) and counted. Post-transplant, all recipient animals were housed under standard environmental conditions and were maintained on the appropriate rodent chow and sterile water ad libitum.

[0188] Vehicle. The vehicle was 0.4% methylcellulose (MC).

[0189] Belumosudil. Belumosudil was formulated with vehicle (0.4% MC).& &

[0190] *Belumosudil was administered to animals for the AM dose, 0.4% MC vehicle was administered for PM dose. For Groups 4 and 5, belumosudil was formulated alone for the first / AM dose.

[0191] Gusacitinib. Gusacitinib was formulated with vehicle (0.4% MC).Attorney Docket No. 01183-0343-00PCT-KAD&

[0192] Ruxolitinib. Ruxolitinib was formulated with vehicle (0.4% MC).

[0193] Animals in Group 1 were not administered any treatments throughout the study. Animals in Groups 2-9 were administered vehicle or test article by oral gavage (PO) with dosing beginning on Day 21 and administered twice per day (BID), until Day 55 as follows. Animals in Groups 2, 3, 6, and 7 received vehicle, gusacitinib or ruxolitinib as indicated in Table 1 for both BID doses. Animals in Groups 4, and 5 were administered belumosudil for the first / AM dose and vehicle for the last / PM dose each day of dosing. Animals in Groups 8 and 9 were administered gusacitinib and belumosudil for the first / AM dose and gusacitinib alone for the last / PM dose each day of dosing.

[0194] In-Life Monitoring and GVHD Assessment.

[0195] Animals were observed daily in order to assess possible differences among treatment groups and / or possible toxicity resulting from the treatments. Daily readouts were weight change, survival, standard GVHD score (Table 2), modified GVHD score (Table 3), incidence of diarrhea, and incidence of bloody stool. No incidence of diarrhea or bloody stool were observed.

[0196] A clinical score for GVHD was obtained daily for the entire study duration as assessed by a standard scoring system (Table 2) and modified scoring system (Table 3). The standard and modified GVHD score was based on 5 criteria: percentage of weight change, posture (hunching), activity, fur texture, and skin integrity. The standard scoring system has aAttorney Docket No. 01183-0343-00PCT-KADmaximum index = 10 while the modified GVHD scoring system has a maximum index = 18. The overall score was reported, and the scores for each individual parameter were recorded.

[0197] On Days 33, 36, 39, 42, and 45 all animals were photographed under isoflurane anesthesia (top-down picture, plain white background) to assess disease severity.

[0198] Table 2: Standard GVHD Scoring Protocol< > < >

[0199] Table 3: Modified Scoring Protocol for scGVHD> < > < > < >< > > >Attorney Docket No. 01183-0343-00PCT-KAD

[0200] Supportive Care and Euthanasia Criteria.

[0201] Following TBI on Day -1 up to Day 8, all animals were given supplemental high calorie highly palatable food in a dish on the bottom of the cage. Following transfer on Day 0, and then again as needed, all animals were given 1 mL / animal / day of supplemental fluids (warmed ringers solution) via subcutaneous injection. An additional 1 mL fluid was given in the pm on an as needed basis. Any animal exceeding 15% body weight loss was given highly palatable soft food and 1 mL supplemental fluids (warmed ringers solution) via subcutaneous injection once per day. Any animal exceeding 20% body weight loss was given 1 mL supplemental fluids (warmed ringers solution) via subcutaneous injection twice per day.Animals that appeared to be in pain were administered Buprenorphine BID, as needed. Animals that were unable to right themselves, cold to the touch, moribund, or in excess of 30% weight loss were euthanized. Animals requiring euthanasia were euthanized by CO2 inhalation and did not have organs collected.

[0202] Sacrifice and Sample Collection.

[0203] On Day 56, all surviving animals were sacrificed by ketamine and xylazine overdose and organs detailed in Table 1 were collected.

[0204] Blood. Prior to sacrifice, ~0.2 mL of blood was collected from all animals by retro-orbital bleed into K2EDTA tubes. The blood was centrifuged, and the plasma collected, and stored at -80°C.

[0205] Lung. The lungs were removed and whole lung and right lung were weighed. The right lung was tied off on the right bronchus, excised below the tie, snap frozen in liquid nitrogen and stored at -80°C. The left lung was insufflated with 10% Neutral Buffered Formalin (NBF), tied off on the trachea to maintain insufflation, fixed in 10% NBF for 24 hours, then moved to PBS prior to processing for histopathology.

[0206] Skin. Prior to collection, skin was shaved. A 1x1 cm portion of the skin (from the back of the animal, between the shoulders, closer to head) was excised, trimmed of any excess fat and / or connective tissue, sandwiched between foam in a tissue cassette, and placed in formalin. After 24 hours, the skin was transferred to PBS for storage until subsequent histological analyses. A second 1 cm xl cm portion of the skin (from the back of the animal, between the shoulders, just below the first piece) was excised, trimmed of any excess fat and / or connective tissue, weighed, flash frozen, and stored at -80°C. If a visible lesion was present, it was collected in a way that captured part of it in both fixed and frozen sections.

[0207] Spleen. The spleen was excised and trimmed of any excess connective tissue. The spleen was then weighed, flash frozen, and stored at -80°C.Attorney Docket No. 01183-0343-00PCT-KAD

[0208] Liver. The liver was excised and trimmed of any excess connective tissue. The whole liver was then weighed and cut into two pieces (left lateral lobe and remaining).

[0209] Survival and Progression Free Survival (PFS).

[0210] Animal deaths were evaluated during the course of the study. In this model, animal deaths are commonly attributable to severe disease. In this study, 37 animals were either found dead or were euthanized due to either euthanasia criteria or moribundity. Progression-free survival was defined as an increase in GVHD score of less than or equal to 2 in comparison to Day 21 GVHD score (standard or modified). A difference in GVHD score of 3 or higher relative to Day 21 meant that the animal no longer is in progression-free survival and had begun disease progression. In graphs that included progression-free survival and death, death would indicate disease progression in addition to above criteria. In death weight carried or death score carried graphs, once an animal was either euthanized or found dead, the final weight or composite GVHD score was included on every day forward until study conclusion.

[0211] Histopathology & Immunohistochemistry.

[0212] Fixed lung, skin, and liver samples were embedded in paraffin and sectioned at 4 microns prior to staining. One block contained liver and lung; a second block contained skin. One slide per block was sectioned and stained with either hematoxylin and eosin (H&E), Picrosirius Red (PSR), or aSMA using immunohistochemistry. The lung was evaluated by Ashcroft score, and dermal thickness measurements were performed for skin. All slides were evaluated and scored with light microscopy by a board-certified veterinary pathologist.

[0213] Hydroxyproline / BCA Assays (lung, skin, liver).

[0214] The frozen right lung, skin, and liver samples were homogenized in distilled water to make a 100 mg / mL homogenate. Homogenate was directly aliquoted for use in both the hydroxyproline assay (Chondrex, Catalog number 6017), BCA Assay (Pierce, Catalog number: 23225), and remainder. Once aliquoted, samples were frozen at -80°C. Homogenate was thawed for use just prior to running each assay. Assays were run in duplicate.

[0215] Outcome Evaluation.

[0216] Study endpoints were survival, progression-free survival, body weight change, standard GVHD score, modified GVHD score, hydroxyproline and total protein content (lung, skin, and liver), histopathology, and immunochemistry.

[0217] Statistical Analyses.

[0218] Weight and composite GVHD data were evaluated using one-way ANOVA with Dunnett’s multiple comparisons test to compare all groups to the vehicle control group (Group 3). Survival and PFS data were evaluated using Log-rank (Mantel-Cox) pairwise comparisonsAttorney Docket No. 01183-0343-00PCT-KADbetween each group and the vehicle-control group (Group 3) with no adjustment for multiple comparisons. All statistical analyses were performed using GraphPad Prism 10.2.0 (La Jolla, CA). Statistical significance was achieved when p<0.05.

[0219] Results.

[0220] Survival. Survival was tracked for all animals during the course of the study and the percent survival is shown in Figure 1 (FIG. 1). Animal survival was monitored on a daily basis for the duration of the study and percent survival was plotted. Overall comparison of survival curves was done using Log-rank (Mantel-Cox) to compare all groups to the vehicle-control group (Group 3) with no adjustment for multiple comparisons. *p<0.05; n = 6-12 per group.

[0221] All naive animals (Group 1) survived until the conclusion of the study. A 50% survival was observed in vehicle-treated animals that received a cell transfer of bone marrow only (Group 2) as well as in diseased animals treated with vehicle (Group 3). Diseased animals treated with 100 mg / kg belumosudil (Group 4) had 66.67% survival. Diseased animals treated with 150 mg / kg belumosudil (Group 5) had 75% survival. Diseased animals treated with gusacitinib (Group 6) had 58.33% survival. Diseased animals treated with ruxolitinib (Group 7) had 90.90% survival. Diseased animals treated with a combination of 100 mg / kg belumosudil and gusacitinib (Group 8) had 83.33% survival. Diseased animals treated with a combination of 150 mg / kg belumosudil and gusacitinib (Group 9) had 25% survival. Survival was significantly higher in naive animals relative to diseased animals treated with vehicle (p<0.05). A statistically significant increase in survival was observed in animals treated with ruxolitinib compared to diseased animals treated with vehicle (p<0.05).

[0222] Body Weight. GVHD disease induction prevented normal weight gain in all diseased animals. Survival past Day 14 indicates successful engraftment of donor cells. Percent weight change is plotted in Figure 2A (FIG. 2A) and percent weight change with death weight carried forward is plotted in Figure 2B (FIG. 2B).

[0223] Animals were weighed daily, and body weight change as compared to Day 0 was calculated. The body weight change is shown in FIG. 2A, upper plot. The AUC was calculated from Day 0 until study conclusion on Day 56 as shown in the lower left inset of FIG. 2A, or from Day 21 until study conclusion on Day 56 as shown in the lower right inset of FIG. 2A, using the trapezoidal rule. Statistical significance between groups was determined by one-way ANOVA with Dunnett’s multiple comparisons test used to compare all groups to the vehiclecontrol group (Group 3). Data is presented as mean ± SEM; n = 6-12 per group.

[0224] In FIG. 2B upper plot, the data is shown with body weight with which an animal was found dead or was euthanized carried forward for the duration of the study, that is, as percentAttorney Docket No. 01183-0343-00PCT-KADweight change with death weight carried. The AUC was calculated from Day 0 until study conclusion on Day 56 as shown in the lower left inset of FIG. 2B, and from Day 21 until study conclusion on Day 56 as shown in the lower right inset of FIG. 2B, using the trapezoidal rule. Statistical significance between groups was determined by one-way ANOVA with Dunnett’s multiple comparisons test used to compare all groups to the vehicle-control group (Group 3). Data is presented as mean ± SEM; n = 6-12 per group.

[0225] Naive animals (Group 1) displayed normal increases in weight throughout the study. By AUC analysis of weight change data from Day 0 to Day 56 or Day 21 to Day 56, no significant differences in percent weight change were observed between groups (see Figure 2A).

[0226] In order to account for changes in average weight due to deaths in the study which contribute to survivor bias, death weight was carried out until Day 56 and is presented in Figure 2B. By AUC analysis of data from Day 0 to Day 56 or Day 21 to Day 56, no significant differences in percent weight change with death weight carried were observed between groups. While not found to be statistically significant, the following trends were observed. Animals treated with either 100 mg / kg belumosudil (Group 4), 150 mg / kg belumosudil (Group 5), ruxolitinib (Group 7), or a combination of 100 mg / kg belumosudil and gusacitinib (Group 8) displayed less weight loss than diseased animals treated with vehicle (Group 3).

[0227] GVHD Score - Standard Scale. GVHD disease induction (cell transfer of splenocytes and bone marrow cells) induced disease in all animals in Groups 3-9 as assessed using the multiparameter GVHD scoring systems shown in Table 2. Survival of animals past Day 14 verified successful engraftment of the transplanted cells. Standard composite GVHD scores are displayed in Figure 3 A (FIG. 3 A). Animals were scored daily according to the protocol shown in Table 2.

[0228] FIG. 3 A upper plot shows the standard GVHD score for the animals in the study. The AUC was calculated from Day 0 until study conclusion on Day 56 as shown in the lower left inset of FIG. 3 A, and from Day 21 until study conclusion on Day 56 as shown in the lower right inset of FIG. 3 A, using the trapezoidal rule. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test to compare all groups to the vehicle-control group (Group 3). Data is presented as mean ± SEM. ***p<0.001; ****p<0.0001; n = 6-12 per group.

[0229] By AUC analysis, as shown in Figure 3 A, animals that received bone marrow only cell transfer and were treated with vehicle (Group 2) had significantly lower standard GVHD scores than diseased animals treated with vehicle (Group 3) when analyzed from either Day 0 to Day 56 (p<0.0001) or from Day 21 to Day 56 (p<0.001).Attorney Docket No. 01183-0343-00PCT-KAD

[0230] In order to account for changes in GVHD score due to deaths in the study which contribute to survivor bias, death score was carried out until Day 56 and is presented in Figure 3B (FIG. 3B). Animals were scored daily according to the protocol shown in Table 2 and death score was carried out until Day 56.

[0231] FIG. 3B upper plot shows GVHD scores with which an animal was found dead or was euthanized carried forward for the duration of the study, that is, the GVHD score with death score carried. The AUC was calculated from Day 0 until study conclusion on Day 56 as shown in the lower left inset of FIG. 3B, and from Day 21 until study conclusion on Day 56 as shown in the lower right inset of FIG. 3B, using the trapezoidal rule. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons to compare all groups to the vehicle-control group (Group 3). *p<0.05; **p<0.01. Data is presented as mean ± SEM; n = 6-12 per group.

[0232] By AUC analysis with death scores carried, as shown in Figure 3B, significantly lower standard GVHD scores were observed when analyzed from either Day 0 to Day 56 (p<0.01) or Day 21 to Day 56 (p<0.05) in vehicle-treated animals that received only bone marrow cell transfer (Group 2) compared to vehicle-treated diseased animals (Group 3).

[0233] GVHD Score - Modified Scale. GVHD disease induction (cell transfer of splenocytes and bone marrow cells) induced disease in all animals in Groups 3-9 as assessed using a modified multi-parameter GVHD scoring systems shown in Table 3, for sclerodermatous GVHD. Survival of animals past Day 14 verified successful engraftment of the transplanted cells. Modified composite GVHD scores are displayed in Figure 4A (FIG. 4A). Animals were scored daily according to the protocol shown in Table 3.

[0234] FIG. 4A upper plot shows the modified scoring GVHD score, that is, the scGVHD score, for the animals in the study. The AUC was calculated from Day 0 until study conclusion on Day 56 as shown in the lower left inset of FIG. 4A, and from Day 21 until study conclusion on Day 56 as shown in the lower right inset of FIG. 4A, using the trapezoidal rule. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test to compare all groups to the vehicle-control group. Data is presented as mean ± SEM. *p<0.05; **p<0.01; n = 6-12 per group.

[0235] Although not statistically significant, the following trends were observed. Naive animals (Group 1) displayed lower scGVHD scores when compared to vehicle-treated diseased animals (Group 3). By AUC analysis of data, vehicle-treated animals that received bone marrow only cell transfer (Group 2) had significantly lower scGVHD scores when analyzed from eitherAttorney Docket No. 01183-0343-00PCT-KADDay 0 to Day 56 (p<0.01) or from Day 21 to Day 56 (p<0.05) relative to diseased animals treated with vehicle (Group 3).

[0236] In order to account for changes in average scGVHD score due to deaths in the study which contribute to survivor bias, death score was carried out until Day 56 and is presented in Figure 4B. Animals were scored daily according to the protocol shown in Table 3 and death score was carried out until Day 56. Figure 4B upper plot shows scGVHD scores with death score carried. The AUC was calculated from Day 0 until study conclusion on Day 56 as shown in the lower left inset of FIG. 4B, and from Day 21 until study conclusion on Day 56 as shown in the lower right inset of FIG. 4B, using the trapezoidal rule. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test to compare all groups to the vehicle-control group. *p<0.05. Data is presented as mean ± SEM; n = 6-12 per group.

[0237] With death score carried, naive animals displayed significantly lower scGVHD scores when AUC was analyzed from Day 21 to Day 56 compared to diseased animals treated with vehicle (Group 3; p<0.05). While not found to be statistically significant, the following trends were observed. Animals treated with either 100 mg / kg belumosudil (Group 4), 150 mg / kg belumosudil (Group 5), ruxolitinib (Group 7), or a combination of 100 mg / kg belumosudil and gusacitinib (Group 8) displayed lower scGVHD scores than diseased animals treated with vehicle (Group 3).

[0238] Progression-Free Survival. Progression free survival (PFS) was tracked for all animals during the course of the study and the percent progression free survival was plotted, as shown in Figure 5A (FIG. 5A, standard GVHD scale), Figure 5B (FIG. 5B, standard GVHD scale, death included), Figure 5C (FIG. 5C, modified scGVHD scale), and Figure 5D (FIG. 5D, modified scGVHD scale, death included). Progression free survival was defined as an increase in GVHD score of less than or equal to 2 in comparison to Day 21 GVHD score (standard or modified). Disease progression was defined as an increase in GVHD score of greater than 2 in comparison to Day 21 GVHD score (standard or modified). Animals that died without going into defined disease progression were not counted as going into disease progression (Figures 5A and 5C). In the death included graphs (Figures 5B and 5D), an animal that did not undergo disease progression, though was either euthanized or found dead, was counted as going into disease progression.

[0239] FIG. 5 A shows the Progression-Free Survival (Standard GVHD Scale) for the study animals. Progression free survival based on a standard GVHD score was tracked for the duration of the study and percent progression free survival was plotted. Overall comparison of survivalAttorney Docket No. 01183-0343-00PCT-KADcurves was done using Logrank (Mantel-Cox) to compare all groups to the vehicle-control group (Group 3) with no adjustment for multiple comparisons, n = 6-12 per group.

[0240] FIG. 5B shows the Progression-Free Survival (Standard GVHD Scale, Death Included) for the study animals. Progression free survival based on a standard GVHD score was tracked for the duration of the study and percent progression free survival with death included was plotted. Overall comparison of survival curves was done using Log-rank (Mantel-Cox) to compare all groups to the vehicle-control group (Group 3) with no adjustment for multiple comparisons, n = 6-12 per group.

[0241] FIG. 5C shows Progression-Free Survival (Modified GVHD Scale) for the study animals. Progression free survival based on a modified GVHD score was tracked for the duration of the study and percent progression free survival was plotted. Overall comparison of survival curves was done using Log-rank (Mantel-Cox) to compare all groups to the vehiclecontrol group (Group 3) with no adjustment for multiple comparisons. **p<0.01; n = 6-12 per group.

[0242] FIG. 5D shows Progression-Free Survival (Modified GVHD Scale, Death Included) for the study animals. Progression free survival based on a modified GVHD score was tracked for the duration of the study and percent progression free survival with death included was plotted. Overall comparison of survival curves was done using Log-rank (Mantel-Cox) to compare all groups to the vehicle-control group (Group 3) with no adjustment for multiple comparisons. **p<0.01; n = 6-12 per group.

[0243] Early in the study, a number of animals across groups were observed to show a thinning hair phenotype, earlier than what is typically associated with an induced GVHD phenotype, in which GVHD scores typically start to increase between Day 14-21. This can occur due to overgrooming behavior, which has been described to occur in some C57BL / 6 mice from Jackson Laboratories.

[0244] Due to the development of skin integrity and fur texture scores, 16.67% progression free survival was observed in naive animals (Group 1) using the standard GVHD scale, with and without death included. Animals that received a cell transfer of bone marrow only and vehicle treatment (Group 2) had 33.33% progression-free survival with and without death included. Analysis using the standard GVHD scoring scale showed that 33.33% of vehicle treated, diseased animals (Group 3), 25% of 150 mg / kg belumosudil treated animals (Group 5), 8.33% of gusacitinib treated animals (Group 6), and 45.45% of ruxolitinib treated animals (Group 7) did not display progressive disease by Day 56 with and without death included. Treatment with 100 mg / kg belumosudil (Group 4) resulted in 25% progression-free survival and 16.67%Attorney Docket No. 01183-0343-00PCT-KADprogression-free survival with death included. Animals treated with a combination of 100 mg / kg belumosudil + gusacitinib (Group 8) demonstrated 33.33% progression-free survival and 16.67% progression-free survival with death included. In animals treated with a combination of 150 mg / kg belumosudil and gusacitinib (Group 9), 8.33% of animals displayed progression-free survival by Day 56 and 0% displayed progression-free survival by Day 56 with death included. No significant differences in progression free survival were observed between groups when using the standard GVHD scale.

[0245] Analysis using the modified GVHD scale showed that 100% of naive animals (Group 1) displayed progression-free survival by Day 56 with and without death included. Animals that received only bone marrow cell transfer with vehicle treatment (Group 2) had 58.33% progression-free survival with and without death included. In diseased animals, 25% of animals treated with vehicle (Group 3), 0% of animals treated with 100 mg / kg belumosudil (Group 4), 16.67% of animals treated with 150 mg / kg belumosudil (Group 5), 16.67% of animals treated with gusacitinib (Group 6), 9.1% of animals treated with ruxolitinib (Group 7), and 0% of animals treated with a combination of 150 mg / kg belumosudil and gusacitinib (Group 9) displayed progression-free survival by Day 56 with and without death included. Animals treated with a combination of 100 mg / kg belumosudil and gusacitinib (Group 8) had 33.33% progression-free survival without and 25% progression-free survival with death included. Using the modified GVHD scale, naive animals demonstrated significantly higher progression-free survival compared to vehicle-treated diseased animals with and without death included (p<0.01).

[0246] Hydroxyproline and Total Protein Analysis. Frozen right lung, skin, and liver samples were analyzed for levels of hydroxyproline and total protein. Hydroxyproline levels are displayed as pg / mL and pg per organ (total) and displayed in Figures 6A and 6B. Total protein levels and hydroxyproline levels, normalized to protein levels, are displayed in Figures 6C and 6D.

[0247] FIG. 6A shows the hydroxyproline levels in the lung, skin, and liver in units of pg / mL for the study animals. FIG. 6B shows the hydroxyproline levels in the lung, skin, and liver in units of pg per organ (total) for the study animals. The frozen right lung, skin, and liver samples were assessed for levels of hydroxyproline and displayed as pg / mL in FIG. 6A and pg per organ (total) in FIG. 6B. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test to compare all groups to the vehicle-control group (Group 3).**p<0.01. Data is presented as mean ± SEM; n = 6-12 per group.

[0248] FIG. 6C shows the total protein levels in the lung, skin, and liver in units of pg / mL for the study animals. FIG. 6D shows the hydroxyproline levels in the lung, skin, and liverAttorney Docket No. 01183-0343-00PCT-KADnormalized to the protein levels of FIG. 6C, for the study animals. The frozen right lung, skin, and liver samples were assessed for levels of total protein, and are shown in FIG. 6C. Levels of hydroxyproline are displayed as pg / mg total protein in FIG. 6D. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test to compare all groups to the vehicle-control group (Group 3). ****p<0.0001. Data is presented as mean ± SEM; n = 6-12 per group.

[0249] Vehicle-treated diseased animals (Group 3) had significantly lower levels of skin hydroxyproline (pg / mL; p<0.01) and normalized skin hydroxyproline (pg / mg protein; p<0.0001) compared to animals that received only bone marrow cell transfer and vehicle treatment (Group 2). Significantly lower total skin hydroxyproline levels (pg / cm2skin) were observed in diseased animals treated with vehicle (Group 3) relative to naive animals (Group 1; p<0.01). No significant differences in hydroxyproline levels were observed between groups in either the lung or liver samples. No significant differences were observed in total protein between groups in either lung, skin, or liver samples.

[0250] As described in more detail below, lung, skin, and liver samples were embedded in paraffin and sectioned at 4 microns prior to staining. One block contained liver and lung; a second block contained skin. One slide per block was sectioned and stained with hematoxylin and eosin (H&E), Picrosirius Red (PSR), and aSMA using immunohistochemistry. The lung was evaluated by Ashcroft score, and dermal thickness measurements were performed for skin. All slides were evaluated and scored with light microscopy by a board-certified veterinary pathologist.

[0251] Histopathology Evaluation of Skin, Lung, and Liver. The development of histopathologic lesions in the skin, lung, and liver from the animals studied was evaluated. Efficacy of treatment with belumosudil, gusacitinib or a combination, or with ruxolitinib was evaluated in the reduction of lesion severity.

[0252] Skin, left lung, and left lateral liver lobes from sixty-five mice, fixed in 10% neutral buffered formalin, were submitted for evaluation. Skin samples were bisected with both halves embedded together in one block. Left lungs were embedded in the longitudinal plane and liver was trimmed / embedded along the long axis of lobe, with liver and lung embedded together in one block. One hundred thirty (130) individual tissue blocks were generated. Three slides per block were sectioned at approximately 4 pm and stained with hematoxylin and eosin (H&E) or by routine histochemical methods for picrosirius red (PSR). Immunohistochemical staining (IHC) targeted to alpha-smooth muscle actin (aSMA) was conducted on a Leica Bond RX automated Stainer using chromogenic methods. No antigen retrieval was performed. PrimaryAttorney Docket No. 01183-0343-00PCT-KADantibody (Abeam ab 124964 rabbit monoclonal [EPR5368]) was performed at room temperature for 30 minutes. For skin samples, primary antibody concentration was 1:4000 and for lung / liver, 1 : 1000. A secondary antibody was used to link the primary binding to an anti-rabbit polymer detection system with visualization via diaminobenzidine (DAB) application; hematoxylin counterstain was used to visualize nuclei.

[0253] Lesions in all organs were assigned a severity score based on the extent of findings on an ordinal range of 0-5, where 0=not present / within normal limits, l=minimal, 2=mild, 3=moderate, 4=marked, 5=severe. Evaluation / scoring per organ and stain are outlined in Table 4.

[0254] Table 4. Histopathology Evaluation by Organ and Stain&

[0255] Severity Scores (Skin, Lung, Liver H&E; Lung and Liver PSR):

[0256] Grade 0: Within normal limits.

[0257] Grade 1: Minimal; <10% of tissue compartment affected or minimal diffuse change.

[0258] Grade 2: Mild; 10-25% of tissue compartment affected or mild diffuse change.

[0259] Grade 3: Moderate; 26-50% of tissue compartment affected.

[0260] Grade 4: Marked; 51-75% of tissue compartment affected.

[0261] Grade 5: Severe; >75% of tissue compartment affected.

[0262] For skin, an additional score was used to capture very small lesions in a large tissue area:

[0263] Grade 0.5: Very minimal; <1% of tissue compartment affected; focal.

[0264] For liver and lung, perivascular mononuclear cell infiltrates were specifically scored according to the following scale:

[0265] Grade 0: Within normal limits.

[0266] Grade 1: Minimal; <5 vascular profiles have perivascular cuffs.

[0267] Grade 2: Mild; 5-9 vascular profiles have perivascular cuffs.

[0268] Grade 3: Moderate; 10-14 vascular profiles have perivascular cuffs.Attorney Docket No. 01183-0343-00PCT-KAD

[0269] Grade 4: Marked; 15-19 vascular profiles have perivascular cuffs.

[0270] Grade 5: Severe; > 20 vascular profiles have perivascular cuffs.

[0271] Histologic feature scores were added together to obtain a sum score. Lung range: 0-15; liver range: 0-15; skin range: 0-30.

[0272] Skin: Dermal Thickness Measurements (PSR). Using PSR-stained slides, the dermis was measured (pm) in five areas across the tissue, with a mean calculated for each sample. Measurements were performed from epidermal basement membrane to the most superficial border of dermal collagen with subcuticular adipose. These measurements were only performed in areas of non-tangentially sectioned tissue, free of histologic artifacts, and with an intact epidermis.

[0273] Lung: Ashcroft Scores. PSR-stained slides were scored according to the modified Ashcroft scale. Five representative, 200x microscopic fields per sample were scored. All scores were averaged to obtain a mean score for each animal.

[0274] Modified Ashcroft Scale:

[0275] Grade 0 = Normal lung; no fibrotic burden.

[0276] Grade 1 = Minimally detectable thickening of alveolar walls; isolated septae <3x normal thickness.

[0277] Grade 2 = Mild thickening of alveolar walls; clear fibrotic changes is isolated septae >3x normal thickness.

[0278] Grade 3 = Moderate contiguous thickening of walls; septae predominately >3x normal thickness.

[0279] Grade 4 = Single fibrotic masses totaling less than 10% of the microscopic field.

[0280] Grade 5 = Confluent fibrotic masses between 10-50% of the microscopic field.

[0281] Grade 6 = Large contiguous fibrotic masses consolidating more than 50% of the microscopic field.

[0282] Grade 7 = Severe distortion of structure and large fibrous areas; alveoli nearly obliterated with up to five air bubbles.

[0283] Grade 8 = Complete obliteration of microscopic field with fibrotic masses.

[0284] Skin, Lung, Liver: aSMA Immunolabel Scoring. aSMA immunolabeling abundance was scored according to the following scheme. In the skin, dermal and subcuticular immunolabeling of spindle cells was scored. In the liver and lung, parenchymal immunolabeling was scored (perivascular collagen changes were assessed with PSR).

[0285] Grade 0: Within normal limits (includes normal constituent blood vessel walls).Attorney Docket No. 01183-0343-00PCT-KAD

[0286] Grade 1: Minimal; <10% of tissue compartment affected; typically a single site in skin and lung.

[0287] Grade 2: Mild; 10-25% of the tissue compartment affected.

[0288] Grade 3: Moderate; 26-50% of the tissue compartment affected.

[0289] Grade 4: Marked; 51-75% of tissue compartment affected.

[0290] Grade 5: Severe; >75% of tissue compartment affected.

[0291] Histopathology Evaluation Statistical Analysis. Data are presented as mean ± standard error of the mean (SEM). Semi-quantitative sum and component histopathology scores were analyzed (GraphPad Prism) by non-parametric ANOVA (Kruskal -Wallis H test) with Dunn’s multiple comparison post-hoc tests; dermal measurements were analyzed by one-way ANOVA with post-hoc Tukey tests as appropriate. Significance was set at p<0.05 for all tests.

[0292] Histopathology Evaluation Results.

[0293] Skin. Morphologic Pathology (H&E)

[0294] The association of histologic findings in the skin with findings expected for the model were complicated by the presence of unexpected histologic lesions in the skin of control animals not receiving cell transfers (Group 1), and by the variability of disease severity typical of the model. One or a few animals from Groups 4-8 did exhibit more extreme but expected examples of GVHD-associated epidermal and dermal lesions, although most animals receiving cell transfers had relatively mild disease, which was primarily mild epidermal hyperplasia, minimal dermal inflammation, and minimal / mild dermal fibroplasia / fibrosis.

[0295] Expected findings in this GVHD model included epidermal hyperplasia with foci of erosion / ulceration and serocellular crust formation, an interface pattern of epidermal mononuclear cell inflammation, mixed dermal and subcuticular inflammation, and dermal fibroplasia or fibrosis, including dermal thickening. Serocellular crusts were characterized by the accumulation of proteinaceous fluid, necrotic debris, and degenerative neutrophils on the epidermal surface, with or without epidermal necrosis, which was typically seen in short segments either partial thickness (erosion) or full thickness, sometimes extending into the subcutis (ulceration). Epidermal hyperplasia was characterized by thickening of the epidermis by increased nucleated keratinocyte layers, up to 12 cells thick, although for most samples, hyperplasic epidermis was generally up to 5 cells thick. Mononuclear cell (lymphocyte) infiltration in the superficial dermis rarely extended into the epidermis or follicular epithelium, accompanied by necrotic / apoptotic keratinocytes in the interface-pattern lesion. In most samples, mononuclear cell infiltration was largely seen in the dermis, with inconsistent association with regions of hyperplastic epidermis and areas of dermal fibroplasia, which wasAttorney Docket No. 01183-0343-00PCT-KADcharacterized by increased numbers of hypertrophied fibroblasts embedded in amphophilic extracellular matrix (including collagen bundles) arranged in parallel, distorting normal dermal collagen organization; dermal fibrosis, which was characterized by increased density collagen bundles and / or thickness of the dermis was also assessed with thickness measurements.

[0296] Due to the presence of histologic lesions in the skin of control mice (Group 1), there was no cell transfer-associated increase in skin sum scores (Figure 7A). Additionally, only treatment with belumosudil (150 mg / kg) + gusacitinib (Group 9) was associated with lower sum scores compared to the vehicle control (Group 3), with no statistical differences seen. Among-group trends were consistent through most component histopathology scores (Figure 7B), with low scores in control mice (Groups 2 and 3) limiting the ability to see efficacy with treatments.

[0297] Figure 7A (FIG. 7A) and Figure 7B (FIG. 7B) are plots of representative skin histopathology scores for the animals in the study. FIG. 7A: Sum Histopathology Score. FIG.7B: Histopathology Scores. Group mean ± standard error of the mean (SEM). Histologic findings were more severe than expected in control mice not receiving cell transfers (Group 1). Due to the low level of disease seen in vehicle treated mice, no efficacy was seen with test article treatment. P values are presented in the summary of data shown in Figure 7E (FIG. 7E).

[0298] Skin. Fibrosis / Dermal Thickness (PSR).

[0299] In areas without overt histologic findings, the dermis was stained as expected with PSR, with red-stained collagen fibers loosely arranged. Areas with histologic findings on H&E (namely areas with fibroplasia or fibrosis) exhibited densely packed, red-stained collagen fibers which variably increased the thickness of the dermis.

[0300] Dermis from mice not receiving cell transfers (Group 1) was thicker than expected (209.2 ± 4.9 pm), with thinner dermal measurements recorded for vehicle-dosed mice receiving cell transfers (Group 2: 165.8 ± 10.5 pm; Group 3: 170.8 ± 7.0 pm). Animals dosed with test articles did not exhibit a reduction in mean dermal thickness compared to vehicle-dosed controls (Fig. 7C), with the exception of belumosudil (150 mg / kg) + gusacitinib (Group 9; 162.1 ± 20.1 pm).

[0301] Figure 7C (FIG. 7C) is a plot of the dermal thickness measurements. Group mean ± SEM. Dermal thickness was measured in 5 sites and averaged per animal. There was no GVHD-associated increase in dermal thickness with this model. Test article efficacy was not seen with this low level of disease induction. Data analyzed by one-way ANOVA with post-hoc Tukey tests. P values are presented in the summary of data shown in FIG. 7E.

[0302] Skin. aSMA Immunolabeling Scores.Attorney Docket No. 01183-0343-00PCT-KAD

[0303] aSMA IHC produced moderate to strong cytoplasmic immunolabeling of small caliber blood vessels of the dermis and occasional larger caliber blood vessels (tunica media) within the subcutis. This appearance was considered “baseline” abundance of aSMA expression in the skin. Increased aSMA immunolabeling was observed as spindle cells oriented parallel to collagen fibers within the dermis or oriented around follicular profiles. Occasionally, immunolabeling of epithelial cells of the follicles or epidermis was also observed (primarily in more severely lesioned samples), but this pattern was not specifically scored.

[0304] aSMA immunolabeling scores are shown in Figure 7D (FIG. 7D) and were relatively similar to histology (FIG. 7A and FIG. 7B) and dermal collagen (FIG. 7C) findings, with no GVHD-associated induction in dermal expression seen between the control mice (Group 1) and those receiving cell transfers with the vehicle (Groups 2, 3). There were no significant differences in mean aSMA immunolabeling scores with treatments (Groups 4-9) compared to the vehicle control (Group 3). For individual animals, skin sum scores positively corresponded with higher aSMA immunolabeling scores.

[0305] FIG. 7D is a plot of the aSMA immunolabeling scores. Group mean ± SEM. There was no GVHD-associated increase in dermal aSMA expression in this model. Test article efficacy was not seen with this low level of disease induction. Data analyzed by non-parametric one-way ANOVA. P values are presented in the summary of data shown in FIG. 7E.

[0306] Lung. Morphologic Pathology (H&E).

[0307] Histologic findings in pulmonary tissues were consistent for those expected in the chronic GVHD model. The primary finding was infiltration of mononuclear cells (lymphocytes dominant, with fewer plasma cells and macrophages; rare neutrophils seen) in perivascular and peribronchiolar zones. Mononuclear cell infiltrates were typically seen surrounding the largest caliber blood vessels, with more severely affected samples exhibiting dense cuffing (up to 15 cells thick) from large to small caliber blood vessels. Extension into the adjacent interstitium was rarely observed. Increased numbers of alveolar macrophages were also consistently seen, sometimes in areas with perivascular infiltrates, often along the alveoli of the lung periphery / lobe tips. Sporadically, fibrin and necrotic debris were observed in alveolar spaces, typically accompanied by increased alveolar macrophages.

[0308] Figure 8A (FIG. 8A) and Figure 8B (FIG. 8B) are plots of representative lung histopathology scores for the animals in the study. FIG. 8A: Sum Histopathology Score. FIG.8B: Histopathology Scores. Group mean ± SEM. Sum scores shown in FIG. 8 A, primarily driven by perivascular mononuclear infiltrate scores (FIG. 8B) were significantly higher in animals given splenocyte + bone marrow cell transfers compared to no cell transfers.Attorney Docket No. 01183-0343-00PCT-KADBelumosudil, 100 or 150 mg / kg, with or without gusacitinib, 25 mg / kg, was associated with lowered scores. Treatment with ruxolitinib also lowered sum (FIG. 8A) and mononuclear infiltrate (FIG. 8B) scores. Data analyzed by non-parametric one-way ANOVA with post-hoc Dunn’s multiple comparisons tests. * indicates p<0.05. ** indicates p<0.01. P values are presented in the summary of data shown in Figure 8F (FIG. 8F).

[0309] Splenocyte and bone marrow cell transfers (Group 3) were associated with a significant induction of lung sum scores (FIG. 8 A), driven primarily by perivascular mononuclear cell infiltrate scores (FIG. 8B), as compared to control mice (Group 1; p=0.03 and p=0.006, respectively). Sum scores were minimally reduced by treatment with belumosudil at 150 mg / kg (Group 5), but better reductions were seen with ruxolitinib (60 mg / kg; Group 7) and belumosudil in combination with gusacitinib (Groups 8 and 9). Although these comparisons did not reach statistical significance, a trend (p=0.10) was seen with vehicle (Group 3) compared to belumosudil, 100 mg / kg + gusacitinib, 25 mg / kg (Group 8). When comparing perivascular infiltrate scores (FIG. 8B) separately, this comparison was statistically significant (Group 3 vs. Group 8, p=0.01). Alveolar histiocytes and alveolar fibrin were minimally affected by treatments.

[0310] Lung. Fibrosis (Ashcroft and PSR Scores).

[0311] Figure 8C (FIG. 8C) is a plot of representative lung average Ashcraft scores, Figure 8D (FIG. 8D) is a plot of representative lung increased perivascular collagen scores (PSR), and Figure 8E (FIG. 8E) is a plot of representative lung aSMA immunolabeling scores. Group mean ± SEM. The score range for Ashcroft (FIG. 8C) is 0-8. The score range for perivascular collagen (FIG. 8D) and aSMA immunolabeling (FIG. 8E) is 0-5. Mildly increased Ashcroft scores (FIG.8C) and perivascular collagen scores (FIG. 8D) were induced by splenocyte + bone marrow cell transfers compared to no cell transfers, with lowered scores associated with ruxolitinib or belumosudil + gusacitinib treatment. aSMA immunolabeling induction was very minimal with only slight effects of test article treatment on mean scores. Data analyzed by non-parametric one-way ANOVA with post-hoc Dunn’s multiple comparisons tests. ** indicates p<0.01. P values are presented in the summary of data shown in FIG. 8F.

[0312] Overt interstitial fibrosis was not observed in this study. Interstitial thickening by collagen (identified as red-stained fibers with PSR) was typically focal and mild (up to Ashcroft grade 3), with rare sites which qualify as fibrous mass formation (Ashcroft grade 4) only observed in areas adjacent or possibly overlapping with perivascular cuffs. Alteration in the organization, along with a slight increase in the abundance, of perivascular collagen was seen inAttorney Docket No. 01183-0343-00PCT-KADareas of perivascular mononuclear cell cuffing; this feature is not specifically addressed by Ashcroft scores and was scored separately (FIG. 8D).

[0313] Average Ashcroft scores (FIG. 8C) were significantly higher in animals receiving cell transfers (Group 3) compared to controls (Group 1; p=0.008). Treatment with any test article or combination was associated with a lowering of average Ashcroft score, but not comparisons to the vehicle (Group 3) reached statistical significance. Similarly, increased perivascular collagen scores (FIG. 8D) were increased with cell transfers (Groups 2 and 3) compared to no transfer (Group 1), although statistical significance was not achieved. Belumosudil alone (150 mg / kg; Group 5) or in combination with gusacitinib (Groups 8 and 9), or ruxolitinib (Group 7) were associated with lowered mean perivascular collagen scores compared to vehicle treatment (Group 3).

[0314] Lung. Parenchymal aSMA Immunolabeling Scores.

[0315] IHC for aSMA in lung produced expected moderate to strong intensity immunolabeling in areas of smooth muscle supporting bronchioles and blood vessels of the lung in all samples. Increased aSMA expression within the lung parenchyma (alveolar walls / interstitium) was variably seen, characterized by focal expression, often in areas with visible but minimal severity interstitial thickening and / or alveolar histiocytosis. There were minimal differences in mean aSMA immunolabeling scores (FIG. 8E) among all groups, including controls (Group 1). The biological significance of a single site of increased aSMA immunolabeling (score 1) compared to no observed increased immunolabeling (score 0) is not completely clear.

[0316] Liver. Morphologic Pathology (H&E).

[0317] Figure 9A (FIG. 9A) and Figure 9B (FIG. 9B) are plots of representative liver histopathology scores for the animals in the study. FIG. 9A: Sum Histopathology Score. FIG.9B: Histopathology Scores. Group mean ± SEM. Sum scores (Fig. 9A) were primarily driven by perivascular infiltrate scores (Fig. 9B), which were increased with the GVHD model (Group 3) compared to no cell transfer (Group 1). Animals receiving belumosudil alone (Groups 4, 5) or in combination with gusacitinib (Groups 8, 9) had slightly lowered scores. Data analyzed by nonparametric one-way ANOVA with post-hoc Dunn’s multiple comparisons tests. P values are presented in the summary of data shown in Figure 9E (FIG. 9E).

[0318] Findings associated with the model in liver samples were perivascular mononuclear cell infiltrates, single cell hepatocyte necrosis, and hepatocyte vacuolation. Mononuclear cell infiltrates were similar to those seen in the lung, although less severe, forming cuffs and aggregates adjacent to central veins and portal tracts. These infiltrates were also associated withAttorney Docket No. 01183-0343-00PCT-KADincreased collagen deposition (see Fibrosis section, below) in perivascular zones. Single cell necrosis was characterized by eosinophilia and rounding of hepatocytes (separation from hepatic cords), with pyknosis sometimes observed; inflammatory response was generally absent in areas with necrotic cells. Hepatocyte vacuolation was characterized by the presence of one or few round non-stained vacuoles, consistent with lipid, within hepatocytes. These vacuoles were distinguished from glycogen vacuolation (not scored), which imparted a less well-defined nonstained vacuolar appearance to hepatocytes. Random subacute inflammation was seen in most samples across the study; this is a background finding in mice that did not significantly vary in severity with the model or treatment and was excluded from the sum score.

[0319] A slight, non-significant increase in sum liver scored (FIG. 9A) was seen with splenocyte and bone marrow transfer (Group 3) compared to control mice (Group 1). Slight reductions in mean sum scores were seen with belumosudil administered alone (Groups 4 and 5) or in combination with gusacitinib (Groups 8 and 9), although no comparisons were statistically significant. Trends in the sum score were driven primarily by mononuclear cell infiltrate scores (FIG. 9B), which were best lowered in mice receiving cell transfers by belumosudil + gusacitinib treatment (Groups 8 and 9).

[0320] Liver. Fibrosis (PSR Scores).

[0321] Figure 9C (FIG. 9C) is a plot of representative liver increased perivascular collagen scores (PSR) and Figure 9D (FIG. 9D) is a plot of representative liver aSMA immunolabeling scores. Group mean ± SEM. A slight increase in collagen (FIG. 9C) was observed in PSR-stained slides from mice receiving cell transfers (Groups 2, 3), with a reduction in collagen scores at the 150 mg / kg dose level of belumosudil, with or without gusacitinib (Groups 5 and 9). aSMA expression (FIG. 9D) was significantly increased with cell transfers (Group 3), although dosing of test compounds minimally affected aSMA immunolabeling abundance. Data analyzed by non-parametric one-way ANOVA with post-hoc Dunn’s multiple comparisons tests. * indicates p<0.05. P values are presented in the summary of data shown in FIG. 9E.

[0322] Increased deposition of red-stained collagen fibers was observed primarily in perivascular zones of samples with mononuclear infiltrates. Minimally increased collagen staining was also seen along sinusoids extending out from blood vessels in animals receiving cell transfers. Although scores were low overall, increased collagen scores (FIG. 9C) were significantly higher in animals administered cell transfer (Group 2) compared to no transfer (Group 1; p=0.008), although the comparison with splenocyte + bone marrow transfers (Group 3) was not statistically significant. Treatment with belumosudil, particularly at 150 mg / kg (Group 5) or belumosudil (150 mg / kg) with gusacitinib (25mg / kg) (Group 9) was associatedAttorney Docket No. 01183-0343-00PCT-KADwith lowered collagen scores (FIG. 9D) compared to the vehicle (Group 3), although no comparisons were statistically significant.

[0323] Liver. Parenchymal aSMA Immunolabeling Scores.

[0324] Figures 9F, 9G, and 9H (FIG. 9F, FIG. 9G, and FIG. 9H) are a series of representative liver photomicrographs at lOOx magnification stained by immunohistochemical staining targeted to alpha-smooth muscle actin (aSMA). FIG. 9F: Score 0; Group 1, Animal 1. FIG. 9G: Score 1; Group 2, Animal 9. FIG. 9H: Score 2; Group 3, Animal 33.

[0325] FIG. 9F has Score 0: within normal limits. The captured aSMA immunolabeling (brown stain; open arrowhead) is seen within the tunica media of larger caliber vascular structures, such as this portal vein (P). FIG. 9G has Score 1 : minimal. Very thin tracts of aSMA immunolabeling (black arrows) are seen along sinusoids. FIG. 9H has Score 2: mild. Increased sinusoidal aSMA immunolabeling (black arrows) is accompanied by a focal increase within a portal tract (open arrow). For all images in Figures 9F, 9G, and 9H, P: portal vein / tract, C: central vein, open arrowhead: non-scored structural / smooth muscle aSMA expression.

[0326] IHC for aSMA produced moderate to strong granular immunolabeling of smooth muscle within the tunica media of blood vessel profiles, including within some central veins and in portal tracts (see FIG. 9F). Increased aSMA immunolabeling was seen in occasional foci, extending along sinusoids (see FIG. 9G) and less frequently, extending out from portal tracts, particularly those with mononuclear cell infiltrates (see FIG. 9H). Although the increase in mean aSMA immunolabeling scores (FIG. 9D) was modest, animals receiving splenocyte and bone marrow transfers (Group 3) had significantly higher scores than control mice (Group 1; p=0.02). Very slight lowering of scores was seen with ruxolitinib (Group 7) and belumosudil at 150 mg / kg + gusacitinib (Group 9).

[0327] Histopathologic Evaluation Conclusions. Administration of splenocyte + bone marrow cell transfers from LP / J donor mice to irradiated C57B1 / 6 mice in a model of chronic GVHD was associated with the development of expected histologic lesions in the lung and liver in this 56-day study; findings in the skin were less consistent with the GVHD model and efficacy with compound treatment was not observed. Perivascular mononuclear cell infiltrates were the primary lung and liver findings, with accompanying minimal to mild increases in perivascular collagen and parenchymal aSMA expression. Very modest increases in pulmonary interstitial collagen deposition (as assessed with average Ashcroft scores) and hepatic sinusoidal collagen deposition, were also seen in the lung and liver.Attorney Docket No. 01183-0343-00PCT-KAD

[0328] No significant differences in skin sum histopathology scores, skin component histopathology scores, dermal thickness measurements, or skin aSMA immunolabeling scores were observed between groups.

[0329] Sum histopathology scores of the lung (p<0.05), lung component score (p<0.01; Infiltrate, mononuclear cells, perivascular), as well as Ashcroft scores (p<0.01) were significantly higher in the lung samples of diseased animals treated with vehicle (Group 3) compared to naive animals (Group 1). Additionally, diseased animals treated with a combination of 100 mg / kg belumosudil and 25 mg / kg gusacitinib displayed significantly lower lung component score (p<0.05; Infiltrate, mononuclear cells, perivascular) than diseased, vehicle treated animals (Group 3). No significant differences in perivascular collagen were observed between groups, though was notably elevated in diseased animals. No significant differences in aSMA immunolabeling scores in the lung were observed between groups. In liver samples, a significant increase in aSMA immunolabeling scores was observed in diseased, vehicle-treated animals (p<0.05; Group 3) compared to naive animals (Group 1). Animals receiving a BM only cell transfer and treated with vehicle (Group 2) displayed significantly higher collagen scores (p<0.05) in liver samples than naive animals (Group 1). No significant differences in sum histopathology scores of the liver or component scores were observed between groups.

[0330] Dosed alone, gusacitinib (25 mg / kg) produced minimal changes in assessed features in the skin, liver, and lung compared to the vehicle. Ruxolitinib (60 mg / kg) treatment improved lung mononuclear cell infiltrates, perivascular collagen scores, and Ashcroft scores. Ruxolitinib also lowered aSMA immunolabeling scores in comparison to the vehicle. Belumosudil dosed at 150 mg / kg alone, or at either 100 or 150 mg / kg in combination with gusacitinib (25 mg / kg) produced the most consistent reductions in lung and liver lesion severity as compared to the vehicle, including lowered perivascular mononuclear cell infiltrate scores, lung Ashcroft and perivascular collagen scores, and liver increased collagen scores.

[0331] Additional Study Conclusions.

[0332] Survival was significantly higher in naive animals relative to diseased animals treated with vehicle. A statistically significant increase in survival was observed in animals treated with ruxolitinib compared to diseased animals treated with vehicle.

[0333] By AUC analysis of weight change data with and without death weight carried, either from Day 0 to Day 56 or Day 21 to Day 56, no significant differences were observed between groups. While not found to be statistically significant, the following trends were observed. Animals treated with either 100 mg / kg belumosudil, 150 mg / kg belumosudil, ruxolitinib, or aAttorney Docket No. 01183-0343-00PCT-KADcombination of 100 mg / kg belumosudil and gusacitinib displayed less weight loss than diseased animals treated with vehicle when death weight was carried forward.

[0334] No incidence of diarrhea or bloody stool were observed in this study.

[0335] By AUC analysis of composite standard GVHD scores with and without death score carried, either from Day 0 to Day 56 or Day 21 to Day 56, animals that received bone marrow only cell transfer and were treated with vehicle had significantly lower standard GVHD scores than diseased animals treated with vehicle.

[0336] By AUC analysis of composite scGVHD scores, vehicle-treated animals that received bone marrow only cell transfer had significantly lower scGVHD scores when analyzed from either Day 0 to Day 56 or from Day 21 to Day 56 relative to diseased animals treated with vehicle. With death score carried, naive animals displayed significantly lower scGVHD scores when AUC was analyzed from Day 21 to Day 56 compared to diseased animals treated with vehicle. While not found to be statistically significant, the following trends were observed. Animals treated with either 100 mg / kg belumosudil, 150 mg / kg belumosudil, ruxolitinib, or a combination of 100 mg / kg belumosudil and gusacitinib displayed lower scGVHD scores than diseased animals treated with vehicle.

[0337] No significant differences in progression free survival, regardless of death inclusion, were observed between groups when using the standard GVHD scale. Using the modified GVHD scale, naive animals demonstrated significantly higher progression-free survival compared to vehicle-treated diseased animals with and without death included.

[0338] Vehicle-treated diseased animals had significantly lower levels of skin hydroxyproline (pg / mL) and normalized skin hydroxyproline (pg / mg protein) compared to animals that received only bone marrow cell transfer and vehicle treatment. Significantly lower total skin hydroxyproline levels (pg / cm2skin) were observed in diseased animals treated with vehicle relative to naive animals. No significant differences in hydroxyproline levels were observed between diseased groups in either the lung or liver samples. No significant differences were observed in total protein between groups in either lung, skin, or liver samples.

[0339] Example 2. Detailed Analysis of Certain Results from the Study of Example 1.

[0340] Additional analysis of the data from the study of Example 1 concluded that the reduced GVHD score for mice treated with a combination of belumosudil and gusacitinib might indicate an additive effect on progression-free survival compared to belumosudil alone, gusacitinib alone, and ruxolitinib alone. Figure 10A (FIG. 10A) is a plot of the data from Example 1 showing the percent disease reduction for the subset using the standard GVHD scale,Attorney Docket No. 01183-0343-00PCT-KADand Figure 10B (FIG. 1OB) is the progression-free survival plot with death included from FIG.5C for the subset, using the standard GVHD scale.

[0341] The combination of belumosudil and gusacitinib shows overall lower lung biopsy scores compared to ruxolitinib or belumosudil alone, as indicated in FIG. 8A. Mice treated with the combination of belumosudil and gusacitinib had the lowest lung mean scores. The data suggests that a combination of belumosudil and gusacitinib could enhance the response rates and prolong the treatment failure free survival, without having a significant impact on safety in patients with severe, refractory or recurrent, active cGVHD with fibrotic manifestations.

[0342] A comparison of histopathology images in the lung additionally supports the utility of the combination of belumosudil and gusacitinib.

[0343] Additional analysis of the lung histopathology data from the study of Example 1 as shown in FIG. 8F allowed for replotting the data as shown in Figure 11 A (FIG. 11 A) and Figure 1 IB (FIG. 1 IB). Figure 11 A shows plots of the lung sum histopathology score; lung infiltrate in mononuclear cells, perivascular; lung histiocytosis, alveolar; and lung alveolar fibrin / necrotic debris data for Groups 1, 3, 4, and 8, with the indicated p values. Figure 1 IB shows plots of the lung Ashcroft score; lung increased collagen, perivascular (PSR) score; and lung aSMA parenchymal immunolabeling score for Groups 1, 3, 4, and 8, with the indicated p values.

[0344] In view of the data from the study of Example 1, a combination of belumosudil and gusacitinib could enhance the response rate and prolong the failure free survival, without impacting safety, in patients with cGVHD, including severe, refractory or recurrent, active cGVHD with fibrotic manifestations.Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the disclosure. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the disclosed methods and uses. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein in their entirety by reference. To the extent any material incorporated herein by reference is inconsistent with the express content of this disclosure, the express content controls.

Claims

Attorney Docket No. 01183-0343-00PCT-KADCLAIMS1. A combination comprising 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6H-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

2. The combination of claim 1, comprising:(a) about 25-200 mg of 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof;(b) about 5-80 mg of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6JT-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof; or(c) about 25-200 mg of 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and about 5-80 mg of 2-[l-[4-[4-(4-hydroxypiperi din- l-yl)anilino]-5-oxo-6JH-pyrimido[4,5-d]pyridazin-2-yl]piperi din-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

3. The combination of any one of claims 1-2, wherein the combination is formulated for oral administration to a human patient, particularly wherein the combination comprises one tablet or capsule, or wherein the combination comprises more than one tablet or capsule.

4. A pharmaceutical composition comprising 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition of claim 4 for use in the treatment of chronic graft-versus-host disease (cGVHD), particularly wherein the cGVHD comprises sclerodermatous cGVHD or lung cGVHD.

6. The pharmaceutical composition of any one of claims 4-5, comprising(a) about 25-200 mg of 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof;Attorney Docket No. 01183-0343-00PCT-KAD(b) about 5-80 mg of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6JT-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof; or(c) about 25-200 mg of 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and about 5-80 mg of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition of any one of claims 4-6, formulated for oral administration to a human patient.

8. A method for treating chronic graft-versus-host disease (cGVHD) in a human patient in need thereof comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and (b) 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

9. The method of claim 8, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof is administered to the human patient at:(a) a daily dosage equivalent to up to about 400 mg of the free base;(b) a daily dosage equivalent to about 25-400 mg of the free base; and / or(c) a dose equivalent to about 50 mg, 100 mg, or 200 mg of the free base.

10. The method of claim 9, wherein the dose is administered to the human patient once or twice daily, particularly wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}- N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof is administered orally.

11. The method of any one of claims 8-10, wherein the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, is administered to the human patient at:(a) a daily dosage equivalent to up to about 80 mg of the free base;Attorney Docket No. 01183-0343-00PCT-KAD(b) a daily dosage equivalent to about 5-80 mg of the free base; and / or(c) a dose equivalent to about 10 mg, 20 mg, 30 mg, 40 mg, or 80 mg of the free base.

12. The method of any one of claims 8-11, wherein the dose of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, is administered to the human patient once a day or twice a day, particularly wherein the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof is administered orally.

13. The method of any one of claims 8-12, wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, are administered separately, sequentially, or simultaneously.

14. The compound 2- { 3 - [4-( 1 H-indazol-5-ylamino)-2-quinazolinyl]phenoxy }-N-( propan-2 -yl) acetamide or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic graft-versus-host disease (cGVHD), wherein the compound is formulated for co-administration with 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, particularly wherein the compound is administered separately, sequentially, or simultaneously with the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

15. The compound 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic graft-versus-host disease (cGVHD), wherein the compound is co-administered with 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, particularly wherein the compound is administered separately, sequentially, or simultaneously with the 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

16. The compound 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, for useAttorney Docket No. 01183-0343-00PCT-KADin the treatment of chronic graft-versus-host disease (cGVHD), wherein the compound is coadministered with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, particularly wherein the compound is administered separately, sequentially, or simultaneously with the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

17. The compound 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic hand eczema or atopic dermatitis, wherein the compound is co¬ administered with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, particularly wherein the compound is administered separately, sequentially, or simultaneously with the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof.

18. A method of treating chronic graft-versus-host disease (cGVHD) comprising administering to a patient in need thereof a therapeutically effective amount of 2-[l-[4-[4-(4-hydroxypiperidin-l-yl)anilino]-5-oxo-6J / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, wherein:(a) the patient has previously received treatment with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof;(b) the patient is receiving treatment with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof; or(c) the patient is on stable treatment with 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide, or a pharmaceutically acceptable salt thereof.

19. The method of any one of claims 8-13 or 18, or the compound for use of any one of claims 14-17, wherein the cGVHD comprises sclerodermatous cGVHD or lung cGVHD, particularly wherein the 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and the 2-[l-[4-[4-(4-Attorney Docket No. 01183-0343-00PCT-KADhydroxypiperidin-l-yl)anilino]-5-oxo-6 / / -pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, are administered with food, or are administered without food.

20. A kit comprising:(i) a first pharmaceutical composition comprising 2- { 3 -[4-( 1 H-indazol-5-ylamino)- 2-quinazolinyl]phenoxy}-N-(propan-2-yl) acetamide or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient;(ii) a second pharmaceutical composition comprising 2-[l-[4-[4-(4-hydroxypiperidin- l-yl)anilino]-5-oxo-6Z7-pyrimido[4,5-d]pyridazin-2-yl]piperidin-4-yl]acetonitrile or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient; and(iii) instructions for administering the first and second pharmaceutical compositions,particularly wherein the first pharmaceutical composition and the second pharmaceutical composition are in separate compartments, and / or wherein the instructions comprise instructions to administer the first pharmaceutical composition separately, sequentially, or simultaneously with the second pharmaceutical composition.

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