Combination therapy including a JAK pathway inhibitor and a ROCK inhibitor

Abstract

The present application provides combination therapies comprising JAK inhibitors and ROCK inhibitors and methods of using same to treat disorders such as graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) and systemic sclerosis (scleroderma).

Description

[Technical field]

[0001] Disclosed herein are combination therapies comprising a JAK pathway inhibitor and a ROCK pathway inhibitor, and methods of using same to treat disorders such as graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD), and systemic sclerosis (scleroderma). [Background technology]

[0002] Protein kinases (PKs) are a group of enzymes that regulate a variety of important biological processes including, inter alia, cell proliferation, survival and differentiation, organogenesis and morphogenesis, neovascularization, tissue repair and regeneration. Protein kinases exert their physiological functions by catalyzing the phosphorylation of proteins (or substrates) and thereby regulating the cellular activity of the substrates in various biological contexts.

[0003] Protein kinases can be classified as receptor and non-receptor. Receptor tyrosine kinases (RTKs) have an extracellular portion, a transmembrane domain, and an intracellular portion, whereas non-receptor tyrosine kinases are entirely intracellular. The Janus kinase family of protein tyrosine kinases (JAKs) belongs to the non-receptor type of tyrosine kinases and includes the following family members: JAK1 (also known as Janus kinase-1), JAK2 (also known as Janus kinase-2), JAK3 (also known as Janus kinase, leukocyte; JAKL; L-JAK and Janus kinase-3) and TYK2 (also known as protein tyrosine kinase 2).

[0004] Pathways involving JAKs and signal transduction and activators of transcription (STATs) are involved in the signal transduction of a wide range of cytokines. Cytokines are small polypeptides or glycoproteins that stimulate biological responses in virtually all cell types. In general, cytokine receptors lack intrinsic tyrosine kinase activity, so receptor-associated kinases are required to propagate the phosphorylation cascade. JAKs perform this function. Cytokines bind to their receptors and cause receptor dimerization, which allows the JAKs to phosphorylate each other and specific tyrosine motifs within the cytokine receptor. STATs that recognize these phosphotyrosine motifs are recruited to the receptor and are then activated themselves by JAK-dependent tyrosine phosphorylation events. Upon activation, STATs dissociate from their receptors, dimerize, translocate to the nucleus, bind to specific DNA sites, and alter transcription (Scott, MJ, CJ Godshall, et al. (2002). "Jaks, STATs, Cytokines, and Sepsis." Clin Diagn Lab Immunol 9(6):1153-9).

[0005] The JAK family plays a role in the cytokine-dependent control of proliferation and function of cells involved in immune response. The JAK / STAT pathway, particularly all four members of the JAK family, are believed to play a role in the pathogenesis of asthmatic responses, chronic obstructive pulmonary disease, bronchitis, and other related inflammatory diseases of the lower airways. In addition, multiple cytokines that signal through JAK kinases have been linked to inflammatory diseases or conditions of the upper airways, such as those that affect the nose and paranasal sinuses (e.g., rhinitis and sinusitis), whether or not they are classical allergic reactions. The JAK / STAT pathway is also believed to play a role in inflammatory diseases / conditions of the eye, including, but not limited to, iritis, uveitis, scleritis, conjunctivitis, and chronic allergic reactions. Thus, inhibition of JAK kinases may play a beneficial role in the therapeutic treatment of these diseases.

[0006] Blocking signal transduction at the level of JAK kinases holds promise for the development of human cancer treatments. Inhibition of JAK kinases is also assumed to benefit patients suffering from skin immune disorders and skin sensitization, such as psoriasis. Thus, inhibitors of Janus kinases or related kinases are widely sought, and several publications have reported effective classes of compounds. For example, the selective JAK1 inhibitor itacitinib, {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, is described in U.S. Patent Application Publication Nos. 2011 / 0224190 and 2015 / 0065484; and the JAK1 / 2 inhibitor ruxolitinib ((R)-3-(4-(7 H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile) and INCB018424 (sold as the phosphate salt under the tradenames JAKAFI® and JAKAVI®) are reported in U.S. Pat. Nos. 7,598,257, 8,415,362, and 8,722,693, the disclosures of which are each incorporated herein by reference.

[0007] Rho-associated coiled-coil kinase family members, consisting of Rho-associated kinase 1 (ROCK1) and Rho-associated kinase 2 (ROCK2), are serine-threonine kinases that are activated by Rho GTPases. Rho-associated kinases are involved in a wide range of cellular processes, including inflammation (see, for example, Flynn, R., Blood, 2016, 127(17), 2144-54). The two ROCK isoforms are similar but are expressed and regulated differently in certain tissues. For example, ROCK1 is ubiquitously expressed at relatively high levels, whereas ROCK2 is preferentially expressed in the heart, brain, and skeletal muscle. The isoforms are also expressed in some tissues in a developmental stage-specific manner. Summary of the Invention

[0008] The present application relates, inter alia, to a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD), and systemic sclerosis (scleroderma) in a subject, comprising administering to the subject: (i) a Janus kinase (JAK) inhibitor; and (ii) a Rho-associated protein kinase (ROCK) inhibitor,

[0009] Other features, objects, and advantages of the invention will become apparent from the description and the claims. [Brief description of the drawings]

[0010] [Figure 1]

[0023] Figure 1 shows graft-versus-host disease (GVHD) scores in a mouse model of scleroderma-type chronic GVHD. Starting on day 21, mice were administered vehicle, itacitinib, belmosudil, or itacitinib + belmosudil. [Diagram 2] 1 shows the total GVHD burden in a mouse model of scleroderma-type chronic GVHD following administration of vehicle, itacitinib, belmosudil, or itacitinib+belmosudil. [Diagram 3]

[0033] Figure 2 shows graft-versus-host disease (GVHD) scores in a mouse model of scleroderma-type chronic GVHD. Starting on day 21, mice were administered vehicle, ruxolitinib, belmosudil, or ruxolitinib + belmosudil. [Figure 4] 1 shows the total GVHD burden in a mouse model of scleroderma-type chronic GVHD following administration of vehicle, ruxolitinib, belmosudil, or ruxolitinib + belmosudil. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present application relates to a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and sclerosis (scleroderma) in a subject, comprising administering to the subject: (i) a Janus kinase (JAK) inhibitor; and (ii) a Rho-associated protein kinase (ROCK) inhibitor.

[0012] In some embodiments, the present application provides a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and systemic sclerosis (scleroderma) in a subject, comprising administering to the subject: (i) a JAK1 / 2 inhibitor; and (ii) a ROCK inhibitor; and

[0013] In some embodiments, the present application provides a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and systemic sclerosis (scleroderma) in a subject, comprising administering to the subject:

[0014] (i) a selective JAK1 inhibitor; and (ii) a ROCK inhibitor; and

[0015] In some embodiments, the present application provides a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and systemic sclerosis (scleroderma) in a subject, comprising administering to the subject: (i) a JAK inhibitor; and (ii) a ROCK2 inhibitor,

[0016] In some embodiments, the present application provides a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and systemic sclerosis (scleroderma) in a subject, comprising administering to the subject: (i) a JAK1 / 2 inhibitor; and (ii) a ROCK2 inhibitor,

[0017] In some embodiments, the present application provides a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and systemic sclerosis (scleroderma) in a subject, comprising administering to the subject: (i) a selective JAK1 inhibitor; and (ii) a ROCK2 inhibitor,

[0018] The methods described herein utilize JAK pathway inhibitors (e.g., JAK1 / 2 inhibitors, selective JAK1 inhibitors, etc.) and ROCK inhibitors (e.g., ROCK2 inhibitors), either of which may be in the form of a pharma- ceutically acceptable salt.

[0019] JAK inhibitors I. JAK1 / 2 Inhibitors In some embodiments, the methods provided herein utilize a JAK1 / 2 inhibitor.

[0020] In some embodiments, the JAK1 / 2 inhibitor is baricitinib, or a pharma- ceutically acceptable salt thereof.

[0021] In some embodiments, the JAK1 / 2 inhibitor is ruxolitinib, or a pharma- ceutically acceptable salt thereof, (see, e.g., U.S. Pat. No. 7,598,257, the disclosure of which is incorporated by reference in its entirety herein). In some embodiments, the salt is ruxolitinib phosphate (see, e.g., U.S. Pat. No. 8,722,693, the disclosure of which is incorporated by reference in its entirety herein).

[0022] In some embodiments, the JAK1 / 2 inhibitor is ruxolitinib, in which one or more hydrogen atoms are replaced by deuterium atoms, or a pharmaceutically acceptable salt thereof.In some embodiments, the JAK1 / 2 inhibitor is any compound of U.S. Patent No. 9,249,149 (incorporated herein by reference in its entirety), or a pharmaceutically acceptable salt thereof.In some embodiments, the JAK1 / 2 inhibitor is CTP-543 (compound 111 having the following structure), or a pharmaceutically acceptable salt thereof. [ka]

[0023] In some embodiments, the JAK1 / 2 inhibitor is a compound of formula I: [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 is selected from H and D; Each R 2 is each R bonded to a common carbon 2 are independently selected from H and D, provided that Each R 3 is each R bonded to a common carbon 3 are independently selected from H and D, provided that R 4 is selected from H and D; Each R 5 are the same and are selected from H and D; and R 6 , R7 , and R 8 are each independently selected from H and D, with the proviso that R 1 is H, and each R 2 and each R 3 is H and R 4 is H and R 6 , R 7 , and R 8 If each of is H, then each R 5 is D.

[0024] In some embodiments, the JAK1 / 2 inhibitor is a compound of formula I selected from the following compounds 100-130 in the table below, 6 , R 7 , and R 8 and each R is H), or a pharma- ceutically acceptable salt thereof. In some embodiments, the inhibitor of JAK1 and / or JAK2 is a compound of formula I selected from the following compounds 200-231 in the table below, 6 , R 7 , and R 8 each is D), or a pharma- ceutically acceptable salt thereof. [Table 1-1] [Table 1-2]

[0025] In some embodiments, the JAK1 / 2 inhibitor is baricitinib in which one or more hydrogen atoms have been replaced with deuterium atoms, or a pharma- ceutically acceptable salt thereof. In some embodiments, the JAK1 and / or JAK2 inhibitor is any compound described in U.S. Patent No. 9,540,367, which is incorporated herein by reference in its entirety, or a pharma- ceutically acceptable salt thereof.

[0026] In some embodiments, the JAK1 / 2 inhibitor or a pharmaceutically acceptable salt thereof is oclacitinib, momelotinib, or brepositinib, or a pharmaceutically acceptable salt thereof.

[0027] II. Selective JAK1 inhibitors In some embodiments, the methods utilized a JAK1 inhibitor, hi some embodiments, the JAK1 inhibitor or a pharma- ceutically acceptable salt thereof is upadacitinib, filgotinib, or abrocitinib, or a pharma- ceutically acceptable salt thereof.

[0028] In some embodiments, the methods provided herein utilize selective JAK1 inhibitors. Selective JAK1 inhibitors are compounds that preferentially inhibit JAK1 activity over other Janus kinases. JAK1 plays a central role in many cytokine and growth factor signaling pathways that, when dysregulated, can result in or contribute to disease states. For example, IL-6 levels are elevated in rheumatoid arthritis, a disease in which IL-6 has been suggested to have deleterious effects (Fonesca, et al., Autoimmunity Reviews, 8:538-42, 2009). Because IL-6 signals at least in part through JAK1, IL-6 may indirectly provide potential clinical benefits via JAK1 inhibition (Guschin, et al. Embo J 14:1421, 1995; Smolen, et al. Lancet 371:987, 2008). In other autoimmune diseases and cancers, elevated systemic levels of inflammatory cytokines that activate JAK1 may also contribute to the disease and / or associated symptoms. Thus, patients with such diseases may benefit from inhibition of JAK1. A selective inhibitor of JAK1 may be beneficial while avoiding the unnecessary and potentially undesirable effects of inhibiting other JAK kinases. In addition, JAK1 inhibition is also being investigated in the role of CAR-T cell-induced cytokine storm release (CRS) (NCT04071366).

[0029] The selective JAK1 inhibitors described herein, or pharma- ceutically acceptable salts thereof, selectively inhibit JAK1 over one or more of JAK2, JAK3, and TYK2. In some embodiments, the selective JAK1 inhibitor preferentially inhibits JAK1 over JAK2 (e.g., JAK2 / JAK1 IC 50 In some embodiments, the selective JAK1 inhibitor or salt is about 10-fold selective for JAK1 over JAK2. In some embodiments, the selective JAK1 inhibitor or salt has an IC 50 The antibody is about 3-fold, about 5-fold, about 10-fold, about 15-fold, or about 20-fold selective for JAK1 over JAK2, as calculated by measuring the affinity of the antibody to JAK1 (see, e.g., Example A).

[0030] In some embodiments, the selective JAK1 inhibitor is a compound in Table 1 or a pharma- ceutically acceptable salt thereof. IC obtained by the method of Example A at 1 mM ATP 50 The values ​​are shown in Table 1. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10]

[0031] In some embodiments, the selective JAK1 inhibitor is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof.

[0032] In some embodiments, the selective JAK1 inhibitor is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate.

[0033] The synthesis and preparation of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile and its adipate salts can be found, for example, in U.S. Patent Application Publication No. 2011 / 0224190, filed March 9, 2011, U.S. Patent Application Publication No. 2013 / 0060026, filed September 6, 2012, and U.S. Patent Application Publication No. 2014 / 0256941, filed March 5, 2014, each of which is incorporated herein by reference in its entirety.

[0034] In some embodiments, the selective JAK1 inhibitor is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide, or a pharmaceutically acceptable salt thereof.

[0035] In some embodiments, the JAK1 inhibitor is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide phosphate.

[0036] The synthesis and preparation of 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide and its phosphate salts can be found, for example, in U.S. Patent Publication No. 2014 / 0343030, filed May 16, 2014, which is incorporated by reference in its entirety.

[0037] In some embodiments, the selective JAK1 inhibitor is ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile, or a pharma- ceutically acceptable salt thereof.

[0038] In some embodiments, the selective JAK1 inhibitor is ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile monohydrate.

[0039] The synthesis of ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile and characterization of its anhydrate and monohydrate forms can be found in U.S. Patent Publication No. 2014 / 0121198, filed October 31, 2013, and U.S. Patent Publication No. 2015 / 0344497, filed April 29, 2015, each of which is incorporated by reference in its entirety.

[0040] In some embodiments, the compounds in Table 1 are disclosed in U.S. Patent Publication No. 2011 / 0224190, filed March 9, 2011; U.S. Patent Application Publication No. 2014 / 0343030, filed May 16, 2014; U.S. Patent Application Publication No. 2014 / 0121198, filed October 31, 2013; U.S. Patent Application Publication No. 2010 / 0298334, filed May 21, 2010; U.S. Patent Application Publication No. 2011 / 0059951, filed August 31, 2010; U.S. Patent Application Publication No. 2011 / 0059951, filed November 18, 2011; No. 2012 / 0149681, filed on the same day, U.S. Patent Publication No. 2012 / 0149682, filed November 18, 2011, U.S. Patent Publication No. 2013 / 0018034, filed June 19, 2012, U.S. Patent Publication No. 2013 / 0045963, filed August 17, 2012, and U.S. Patent Publication No. 2014 / 0005166, filed May 17, 2013, each of which is incorporated herein by reference in its entirety.

[0041] In some embodiments, the selective JAK1 inhibitor is selected from the group consisting of U.S. Patent Application Publication No. 2011 / 0224190, filed March 9, 2011, U.S. Patent Application Publication No. 2014 / 0343030, filed May 16, 2014, U.S. Patent Application Publication No. 2014 / 0121198, filed October 31, 2013, U.S. Patent Application Publication No. 2010 / 0298334, filed May 21, 2010, U.S. Patent Application Publication No. 2011 / 0059951, filed August 31, 2010, U.S. Patent Application Publication No. 2011 / 0059951, filed November 18, 2011, and U.S. Patent Application Publication No. 2011 / 006366, filed May 18, 2011. No. 2012 / 0149681, filed Nov. 18, 2011, U.S. Patent Publication No. 2012 / 0149682, filed June 19, 2012, U.S. Patent Publication No. 2013 / 0018034, filed Aug. 17, 2012, U.S. Patent Publication No. 2013 / 0045963, filed Aug. 17, 2012, and U.S. Patent Publication No. 2014 / 0005166, filed May 17, 2013, or a pharma- ceutically acceptable salt thereof, each of which is incorporated herein by reference in its entirety.

[0042] In some embodiments, the selective JAK1 inhibitor is a compound of formula II [ka] or a pharma- ceutically acceptable salt thereof, X is N or CH; L is C(=O) or C(=O)NH; A is phenyl, pyridinyl, or pyrimidinyl, each of which is selected from one or two independently selected R 1 is optionally substituted with a group; and Each R 1 is independently fluoro or trifluoromethyl.

[0043] In some embodiments, the compound of formula II is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof.

[0044] In some embodiments, the compound of formula II is 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[4-fluoro-2-(trifluoromethyl)phenyl]piperidine-1-carboxamide or a pharma- ceutically acceptable salt thereof.

[0045] In some embodiments, the compound of formula II is [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile or a pharma- ceutically acceptable salt thereof.

[0046] In some embodiments, the selective JAK1 inhibitor is a compound of formula III: [ka] or a pharma- ceutically acceptable salt thereof, R 2 is C 1-6 Alkyl, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, or C 3-6 Cycloalkyl-C 1-3 alkyl, 1-6 Alkyl, C 3-6 Cycloalkyl and C 3-6 Cycloalkyl-C 1-3 Each alkyl is optionally substituted with 1, 2, or 3 substituents independently selected from fluoro, -CF3, and methyl; R 3 is H or methyl; R 4 is H, F, or Cl; R 5 is H or F; R 6 is H or F; R 7 is H or F; R 8 is H or methyl; R 9 is H or methyl; R 10 is H or methyl; and R 11 is H or methyl.

[0047] In some embodiments, the compound of formula III is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide, or a pharma- ceutically acceptable salt thereof.

[0048] In some embodiments, the selective JAK1 inhibitor is a compound of formula IV [ka] or a pharma- ceutically acceptable salt thereof, Cy 4 CN, OH, F, Cl, C 1-3 Alkyl, C 1-3 Haloalkyl, Cyano-C 1-3 Alkyl, HO-C 1-3 Alkyl, Amino, C 1-3 Alkylamino and di(C 1-3 a tetrahydro-2H-pyran ring optionally substituted with one or two groups independently selected from the group consisting of aryl, ... 1-3 Alkyl and di(C 1-3 Alkyl)amino is F, Cl, C 1-3 Alkylaminosulfonyl and C 1-3 optionally substituted with 1, 2, or 3 substituents independently selected from alkylsulfonyl; and R 12 is -CH2-OH, -CH(CH3)-OH, or -CH2-NHSO2CH3.

[0049] In some embodiments, the compound of formula IV is ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile, or a pharma- ceutically acceptable salt thereof.

[0050] ROCK inhibitors Examples of Rho-associated coiled-coil containing protein kinase inhibitors include, but are not limited to, (R)-trans-N-(pyridin-4-yl)-4-(1-aminoethyl)cyclohexanecarboxamide, (R)-(+)-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4-(1-aminoethyl)benzamide, as disclosed in WO98 / 06433 and WO00 / 09162; 1-(5-isoquinolinesulfonyl)homopiperazine, as disclosed in WO97 / 23222. Rho-associated coiled coils containing protein kinase inhibitors such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; Rho-associated coiled coils containing protein kinase inhibitors such as (1-benzylpyrrolidin-3-yl)-(1H-indazol-5-yl)amine disclosed in WO01 / 56988; protein kinase inhibitors such as (1-benzylpiperidin-4-yl)-(1H-indazol-5-yl)amine disclosed in WO02 / 100833. Rho-associated coiled coils containing protein kinase inhibitors, such as N-[2-(4-fluorophenyl)-6,7-dimethoxy-4-quinazolinyl]-N-(1H-indazol-5-yl)amine, as disclosed in WO02 / 076976; and proteases, such as N-4-(1H-indazol-5-yl)-6,7-dimethoxy-N-2-pyridin-4-yl-quinazoline-2,4-diamine, as disclosed in WO02 / 076977. Rho-associated coiled coils comprising a protein kinase inhibitor such as 4-methyl-5-(2-methyl-[1,4]diazepane-1-sulfonyl)isoquinoline as disclosed in WO99 / 64011; Rho-associated coiled coils comprising a protein kinase inhibitor such as (S)-(-)-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine as disclosed in WO2006 / 068208; and Rho-associated coiled coils comprising a protein kinase inhibitor such as 4-(3-amino-1-(isoquinolin-6-ylamino)-1-oxopropan-2-yl)benzyl 2,4-dimethylbenzoate as disclosed in WO2010 / 126626.The disclosures of International Patent Publication Nos. WO98 / 06433, WO00 / 09162, WO97 / 23222, WO01 / 56988, WO02 / 100833, WO02 / 076976, WO02 / 076977, WO99 / 64011, WO2006 / 068208, and WO2010 / 126626 are each incorporated by reference in their entirety into this specification.

[0051] In some embodiments, the Rho-associated coiled-coil-containing protein kinase inhibitor is ripasudil, or (S)-(−)-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, or a pharma- ceutically acceptable salt thereof.

[0052] In some embodiments, the Rho-associated coiled-coil-containing protein kinase inhibitor is netarsudil, or [4-[(1S)-1-(aminomethyl)-2-(isoquinolin-6-ylamino)-2-oxoethyl]phenyl]methyl 2,4-dimethylbenzoate, or a pharmaceutically acceptable salt thereof.

[0053] In one embodiment, the Rho-associated coiled-coil-containing protein kinase inhibitor is fasudil, or 5-(1,4-diazepane-1-sulfonyl)isoquinoline.

[0054] Additional Rho-associated coiled-coil-containing protein kinase inhibitors include, but are not limited to, 4-substrate piperidone derivatives such as those described in U.S. Pat. Nos. 7,572,913 and 6,140,333 (e.g., BA-1049, BA-1041, BA-1042, BA-1043, BA-1044, BA-1050, and BA-1051), Y27632 [(R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexane-carboxamide as the dihydrochloride salt], which inactivates both ROCK1 and ROCK2 (e.g., Ishizaki et al. al. Mol. Pharmacol. 2000, 57:976; and U.S. Pat. No. 4,997,834), Rho-associated coiled-coil-containing protein kinase inhibitors such as those described in U.S. Pat. No. 9,815,820 (e.g., belmosudil or SLx-2119), HA-1077 (e.g., Zhao et al. Neurol. Med. Chir., (2011), (Tokyo) 51:679-683), and azaindole-based rho kinase inhibitors such as 6-chloro-N4-(3,5-difluoro-4-{(3-methyl-1H-pyrrolo){2,3-b}pyridin-4-yl)oxy}-phenyl)pyrimidine-2,4-diamine (azaindolele) (see, e.g., Kast et al. Brit. J. Pharmacol. 2007, 152:1070-1080). The disclosures of U.S. Patent Nos. 4,997,834, 6,140,333, 7,572,913, and 9,815,820 are each incorporated herein by reference in their entirety.

[0055] In some embodiments, the Rho-associated coiled-coil containing protein kinase inhibitor is a Rho-associated coiled-coil kinase 2 inhibitor (ROCK2 inhibitor).

[0056] In some embodiments, the Rho-associated coiled-coil kinase 2 inhibitor is belmosudil (i.e., 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide), or a pharma- ceutically acceptable salt thereof.

[0057] How to use In some embodiments, the disease or disorder is graft-versus-host disease. In some embodiments, the graft-versus-host disease is chronic graft-versus-host disease. In some embodiments, the graft-versus-host disease is scleroderma-type chronic graft-versus-host disease. In some embodiments, the graft-versus-host disease is acute graft-versus-host disease.

[0058] Itacitinib, a selective JAK1 pathway inhibitor, is currently under clinical investigation in the treatment of both acute and chronic GVHD after HSCT. Chronic GVHD (cGVHD) can affect any organ in the body, most commonly the skin, liver, and intestine. Chronic GVHD can also affect the lungs, with clinical symptoms similar to those of BOS after lung transplantation. The correlation between pulmonary cGVHD and BOS after lung transplantation was first reported in 1995, when clinical data and tissues were collected from nine patients with pulmonary cGVHD and BOS after lung transplantation. Both groups had similar signs and symptoms, including progressive dyspnea and irreversible obstruction patterns, as well as similar outcomes and histology, including diffuse inflammation causing large bronchi dilation (Philit et al., Eur. Respir. J. 1995, 8:551-558).

[0059] Pulmonary cGVHD is initiated during the transplant process through the progression of normal tissue injury that triggers a tissue-damaging response characterized by the release of cytokines, Toll-like receptor agonists, neutrophils, platelets, and vascular inflammation (Cooke et al, Biol. Blood Marrow Transplant 2017,23:211-234). CD4 and CD8 cells and Th17 cells are recruited to the site, but thymic injury or dysfunction impairs the negative selection of these cells, allowing autoreactive T cell clones to persist. Furthermore, the essential immunosuppressive regimens used in cGVHD, including CNIs, suppress T reg Ultimately, the initial T cell response leads to the activation of a variety of innate and adaptive immune cells, including T, B, and NK cells, as well as APCs to the site, leading to upregulation of the proinflammatory cytokines TGFβ, PDGFα, TNFα, and IL17. This chronic inflammation and recruitment of fibroblasts culminates with collagen deposition and continued dysfunction and fibrosis of the target organ (Cooke et al., Biol. Blood Marrow Transplant 2017, 23:211-234).

[0060] Furthermore, in mouse models, it has been demonstrated that prophylactic and therapeutic administration of itacitinib resulted in improved GVHD scores at either 60 mg / kg or 120 mg / kg per day, indicating clinical efficacy in an alloreactive mouse model.The JAK inhibitors ruxolitinib (JAK1 / 2 inhibitor) and itacitinib have demonstrated clinical efficacy in acute GVHD (aGVHD); furthermore, ruxolitinib provided clinical efficacy in chronic GVHD (cGVHD).

[0061] In some embodiments, the disease or disorder is chronic pulmonary allograft dysfunction (CLAD). In some embodiments, the chronic pulmonary allograft dysfunction (CLAD) is bronchiolitis obliterans syndrome (BOS).

[0062] The diagnosis of post-lung transplant BOS may be made clinically and may be defined by a persistent decline in lung function as measured by FEV1. To diagnose post-lung transplant BOS, other causes of post-transplant weakness, such as acute rejection, infection, native pulmonary problems in the single-lung recipient, excessive weight gain in the recipient, anastomotic failure, respiratory muscle dysfunction, pleural effusion, or technical problems, such as erroneous measurements due to device malfunction, may be excluded as causes of lung graft dysfunction. (Meyer, KC, et al. Eur. Respir. J. 2014; 44: 1479-1503). The BOS classification scheme, an accepted grading system for post-lung transplant BOS, may be based on spirometric assessment of a persistent decline in FEV1 to 80% or less of baseline FEV1 after transplantation. The baseline is defined as the two best FEV1s (or FEV1s) at least 3 weeks apart after functional recovery and stabilization after lung transplantation. 25~75% ) values. The latest update provided below includes a new classification, Grade 0p, added to ensure early diagnosis of BOS after lung transplantation. (Meyer, KC, et al. Eur. Respir. J. 2014;44:1479-1503). Table 1 is adapted from Meyer, KC, et al. Eur. Respir. J. 2014;44:1479-1503, which is incorporated herein by reference in its entirety. FEV1: forced expiratory volume in 1 second. FEF 25~75% : Forced expiratory flow rate of 25-75% of forced vital capacity. [Table 3]

[0063] Many of the same biological principles associated with cGVHD are unique to BOS after lung transplantation. The main difference is that in cGVHD, the immune response is abnormal, causing transplanted stem cells to attack host tissues, whereas in post-lung transplant BOS, the immune response is physiologically normal, yet still results in poor patient outcomes. In post-lung transplant BOS, the inciting event of the alloreactive acute inflammatory phase (i.e., allogeneic lung transplant transplantation) is evident. However, it should be noted that, similar to cGVHD, patients who have acute post-transplant rejection episodes, events leading to graft tissue damage including CMV infection, as well as other tissue damage phenomena, e.g., GERD and cold ischemia time, are more likely to develop post-lung transplant BOS. (Meyer, KC, et al. Eur. Respir. J. 2014;44:1479-1503). The thymic dysfunction seen in cGVHD is not relevant to post-lung transplant BOS because alloreactive T cells are not negatively selected. Importantly, however, the role of early CD4, CD8, and Th17 cell infiltration, and subsequent recruitment of B cells, NK cells, and APCs, even though these are physiologically relevant, are all well documented in BOS after lung transplantation (Boehler and Estenne, Eur. Respir. J. 2003, 22:1007-1018; Gupta et al., Am. J. Respir. Cell Mol. Biol. 2017, 56:708-715; Fukami et al., Am. J. Transplant, 2012, 12:867-876; Hodge et al, J. Heart Lung Transplant, 2012, 31:888-895, Leonard et al., Am. J. Respir. Crit. Care Med. 2000, 161:1349-1354). As with cGVHD, the maintenance immunosuppressive regimen consisted of CNIs and T reg (Meyer, KC, et al. Eur. Respir. J. 2014;44:1479-1503). Finally, each of the known cytokines, such as TGFβ, PDGFα, TNFα, and IL-17, as well as cellular mediators of aberrant tissue repair, have been demonstrated to be present in BOS following lung transplantation.

[0064] There is no standard of care or consensus treatment algorithm for BOS therapy, and few high-quality randomized trials have demonstrated a clear benefit for patients with BOS (Meyer, KC, et al. Eur. Respir. J. 2014;44: 1479-1503).

[0065] Of note, four of five patients in the study with pulmonary cGVHD demonstrated an FEV1 response, defined by an increase in FEV1 of 10% or more. Additionally, a study examining the use of ruxolitinib in five pediatric patients (four evaluable) with pulmonary cGVHD demonstrated two responses, with one patient experiencing a 9% increase in FEV1. Four of the five patients were able to completely wean off steroids, with the last patient able to reduce his steroid requirements by more than 50% (Schoettler et al, Bone Marrow Transplantation, 2019, 54:1158-1160). In addition to ruxolitinib, itacitinib has also shown significant clinical activity in patients with aGVHD. A recent study evaluated the safety and efficacy of two doses of itacitinib, 200 mg (QD) and 300 mg (QD), in patients with treatment-naïve or steroid-refractory aGVHD.

[0066] In some embodiments, the subject is a lung transplant recipient (eg, a single lung transplant recipient or a double lung transplant recipient).

[0067] In some embodiments, the subject is a bilateral lung transplant recipient.

[0068] In some embodiments, the subject has grade 0, grade 0p, grade 1, grade 2, or grade 3 bronchiolitis obliterans syndrome as determined by the International Society of Heart and Lung Transplantation (ISHLT) criteria.

[0069] In some embodiments, the subject has bronchiolitis obliterans syndrome, grade 0p, grade 1, grade 2, or grade 3 as determined by the International Society of Heart and Lung Transplantation (ISHLT) criteria.

[0070] In some embodiments, the subject has Grade 1 or Grade 2 bronchiolitis obliterans syndrome as determined by the International Society of Heart and Lung Transplantation (ISHLT) criteria.

[0071] In some embodiments, the subject has a fractional decrease in FEV1 of less than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the post-transplant baseline FEV1. These values ​​can be used to define ranges, such as from about 50% to about 75%.

[0072] In some embodiments, the subject has a FEF of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or less. 25~75% These values ​​may be used to define a range, such as from about 60% to about 80%.

[0073] In some embodiments, the subject has Grade 0 bronchiolitis obliterans syndrome, where Grade 0 bronchiolitis obliterans syndrome is defined as a partial decrease in FEV1 to less than 100%, but greater than 90% of post-transplant baseline FEV1 and / or a decrease in baseline FEV1 to greater than 75% of post-transplant baseline. 25~75% It is defined as:

[0074] In some embodiments, the subject has bronchiolitis obliterans syndrome of grade 0p, which is characterized by a partial decrease in FEV1, with a baseline FEV1 of 81-90% of post-transplant baseline FEV1 and / or a baseline FEV1 of 75% or less of post-transplant baseline. 25~75% It is defined as:

[0075] In some embodiments, the subject has Grade 1 bronchiolitis obliterans syndrome, where Grade 1 bronchiolitis obliterans syndrome is defined as a partial decrease in FEV1 to 66-80% of baseline FEV1 after transplant.

[0076] In some embodiments, the subject has Grade 2 bronchiolitis obliterans syndrome, defined as a partial decrease in FEV1 to 51-65% of baseline FEV1 after transplant.

[0077] In some embodiments, the subject has Grade 3 bronchiolitis obliterans syndrome, defined as a partial decline in FEV1 to below 50% of baseline FEV1 post-transplant.

[0078] In some embodiments, treatment of bronchiolitis obliterans syndrome comprises an increase in FEV1 of greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% about 4 weeks, 8 weeks, 12 weeks, 3 months, 4 months, 5 months or 6 months after the first administration of a JAK inhibitor and a ROCK inhibitor described herein.

[0079] In some embodiments, treatment of bronchiolitis obliterans syndrome comprises an increase in FEV1 of greater than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% about 4 weeks, 8 weeks, 12 weeks, 3 months, 4 months, 5 months or 6 months after the first administration of a JAK inhibitor and a ROCK inhibitor described herein.

[0080] In some embodiments, treatment of bronchiolitis obliterans syndrome comprises an increase in FEV1 of about 10% or more about 12 weeks after the first administration of a JAK inhibitor and a ROCK inhibitor as described herein.

[0081] In some embodiments, treatment of bronchiolitis obliterans syndrome comprises an increase in FEV1 of about 10% or more 12 weeks after the first dose of a JAK inhibitor and a ROCK inhibitor as described herein.

[0082] In some embodiments, the application provides a method for reducing the risk of bronchiolitis obliterans syndrome in a subject, the method comprising administering to the subject a JAK inhibitor and a ROCK inhibitor described herein.

[0083] In some embodiments, the application provides a method of reducing a risk of lung retransplantation in a subject, the method comprising administering to the subject a JAK inhibitor and a ROCK inhibitor described herein.

[0084] In some embodiments, the present application provides a method for improving FEV1 in a subject with bronchiolitis obliterans syndrome, the method comprising administering to the subject a JAK inhibitor and a ROCK inhibitor described herein.

[0085] In some embodiments, the present application provides a method of improving quality of life in a subject with bronchiolitis obliterans syndrome, the method comprising administering to the subject a JAK inhibitor and a ROCK inhibitor described herein.

[0086] In some embodiments, the application provides a method of reducing mortality, reducing progressive bronchiolectasis, reducing organ failure, reducing decline in lung function, increasing recovery and stabilization after lung transplantation, reducing hospitalization, reducing healthcare utilization, and / or reducing the risk of retransplantation, as well as other potential benefits provided herein, in a subject, the method comprising administering to the subject a JAK inhibitor and a ROCK inhibitor as described herein.

[0087] In some embodiments, the application provides a method of reducing a risk of hospitalization in a subject, the method comprising administering a JAK inhibitor and a ROCK inhibitor as described herein to the subject, the subject (a) having been diagnosed with bronchiolitis obliterans syndrome; (b) having undergone a lung transplant within 1-5 years prior to administration of the JAK inhibitor and the ROCK inhibitor; and (c) not having a decline in FEV1 due to causes other than bronchiolitis obliterans syndrome.

[0088] In some embodiments, the application provides a method of treating non-transplant-associated bronchiolitis obliterans syndrome in a subject, the method comprising administering to the subject a JAK inhibitor and a ROCK inhibitor described herein.

[0089] In some embodiments, the disease or disorder is restrictive allograft syndrome (RAS).

[0090] In some embodiments, the disease or disorder is systemic sclerosis (scleroderma).

[0091] Medication and Administration In some embodiments, the JAK inhibitor is administered to a subject in a daily dose of about 1 mg to about 2000 mg, about 10 mg to about 2000 mg, or about 100 mg to about 2000 mg.

[0092] In some embodiments, the JAK inhibitor is administered to a subject at a dosage of about 50 mg to about 1200 mg on a free base basis. Thus, in some embodiments, the JAK inhibitor is administered to a subject at a dosage of about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 640 mg, about 660 mg, about 680 mg, about 690 mg, about 700 mg, about 710 mg, about 720 mg, about 730 mg, about 740 mg, about 750 mg, about 760 mg, about 770 mg, about 780 mg, about 790 mg, about 800 mg, about 810 mg, about 820 mg, about 830 mg, about 840 mg, about 850 mg, about 860 mg, about 870 mg, about 880 mg, about 890 mg, about 900 mg, about 910 mg, about 920 mg, about 930 mg, about 940 mg, about 950 mg, about 960 mg, about 970 mg, about 980 mg, about 990 mg, about 1000 mg, about 1010 mg, about 1020 mg, about 1030 mg, about 1040 mg, about 1050 mg, about 1060 mg, about g, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1125 mg, about 1150 mg, about 1175 mg, or about 1200 mg.

[0093] In some embodiments, the JAK inhibitor is administered to the subject in a daily dose of about 200 mg to about 1200 mg on a free base basis. In some embodiments, the JAK inhibitor is administered to the subject in a daily dose of about 200 mg to about 1200 mg on a free base basis on days 1 to 28 of a 28-day treatment cycle. The daily dose of about 200 mg to about 1200 mg may be administered, for example, twice daily in separate doses of about 100 mg to about 600 mg.

[0094] In some embodiments, the JAK inhibitor is administered to a subject in a daily dosage of about 100 mg to about 600 mg on a free base basis.

[0095] In some embodiments, the JAK inhibitor is administered to a subject in a daily dose of about 100 mg to about 600 mg, on a free base basis, on days 1 to 28 of a 28 day treatment cycle.

[0096] In some embodiments, the JAK inhibitor is administered to a subject once daily.

[0097] In some embodiments, the JAK inhibitor is administered to a subject twice daily.

[0098] In some embodiments, the dose of JAK inhibitor is administered to the subject as one or more sustained release dosage forms.The sustained release dosage form of JAK inhibitor {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile or its pharma- ceutically acceptable salt (Table 1, compound 1) can be found in U.S. Patent Nos. 9,655,854 and 10,561,616, which are incorporated herein by reference.

[0099] In some embodiments, a dose of the JAK inhibitor is administered as one or more immediate release dosage forms.

[0100] In some embodiments, the ROCK inhibitor is administered to a subject in a daily dose of about 1 mg to about 2000 mg, about 10 mg to about 2000 mg, or about 100 mg to about 2000 mg.

[0101] In some embodiments, the ROCK inhibitor is administered to a subject in a daily amount of about 50 mg to about 1200 mg on a free base basis. Thus, in some embodiments, the ROCK inhibitor is administered in a daily amount of about 50 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 62 mg, about 66 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, about 100 mg, about 102 mg, about 104 mg, about 105 mg, about 106 mg, about 107 mg, about 108 mg, about 109 mg, about 110 mg, about 111 mg, about 112 mg, about 113 mg, about 114 mg, about 115 mg, about 116 mg, about 117 mg, The compound is administered to a subject in a daily amount of about 5 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1125 mg, about 1150 mg, about 1175 mg, or about 1200 mg.

[0102] In some embodiments, the ROCK inhibitor is administered to a subject in an amount of about 100 mg / kg to about 300 mg / kg on a free base basis.

[0103] In some embodiments, the ROCK inhibitor is administered to a subject in an amount of about 100 mg / kg to about 200 mg / kg on a free base basis.

[0104] In some embodiments, the ROCK inhibitor is administered to a subject in an amount of about 100 mg / kg on a free base basis.

[0105] In some embodiments, the ROCK inhibitor is administered to a subject in an amount of about 150 mg / kg on a free base basis.

[0106] In some embodiments, the ROCK inhibitor is administered to a subject in an amount of about 200 mg / kg on a free base basis.

[0107] In some embodiments, the ROCK inhibitor is administered to a subject in an amount of about 250 mg / kg on a free base basis.

[0108] In some embodiments, the ROCK inhibitor is administered to a subject in an amount of about 300 mg / kg on a free base basis.

[0109] In some embodiments, the ROCK inhibitor is administered to a subject once daily.

[0110] In some embodiments, the inhibitor of ROCK is administered to a subject twice daily.

[0111] In some embodiments, a dose of a ROCK inhibitor is administered to a subject in one or more sustained release dosage forms.

[0112] In some embodiments, a dose of a ROCK inhibitor is administered as one or more immediate release dosage forms.

[0113] In some embodiments, the JAK inhibitor and the ROCK inhibitor are administered simultaneously. In some embodiments, the JAK inhibitor and the ROCK inhibitor are administered sequentially. In some embodiments, the JAK inhibitor and the ROCK inhibitor are each administered once per day. In some embodiments, the JAK inhibitor and the ROCK inhibitor are each administered orally once per day. In some embodiments, the JAK inhibitor and the ROCK inhibitor are each administered twice per day. In some embodiments, the JAK inhibitor and the ROCK inhibitor are each administered orally twice per day.

[0114] In some embodiments, administration of a JAK inhibitor and a ROCK inhibitor results in amelioration of one or more symptoms of a disease or disorder provided herein.

[0115] In some embodiments, administration of the JAK inhibitor and the ROCK inhibitor results in an improvement in one or more symptoms of GVHD in the subject (e.g., as measured by a GVHD score) compared to the one or more symptoms prior to administration of the JAK inhibitor and the ROCK inhibitor.

[0116] In some embodiments, administration of a JAK inhibitor and a ROCK inhibitor reduces or halts the progression of one or more symptoms of a disease or disorder provided herein.

[0117] In some embodiments, administration of the JAK inhibitor and the ROCK inhibitor reduces or halts the progression of one or more symptoms of GVHD in the subject (e.g., as measured by a GVHD score) compared to the one or more symptoms prior to administration of the JAK inhibitor and the ROCK inhibitor.

[0118] In some embodiments, administration of a JAK inhibitor and a ROCK inhibitor results in a reduction in the subject's GVHD score compared to the subject's GVHD score prior to administration of the JAK inhibitor and the ROCK inhibitor. For example, in some embodiments, administration of a JAK inhibitor and a ROCK inhibitor reduces the subject's GVHD score from 4 to 3, 4 to 2, 4 to 1, 4 to 0, 3 to 2, 3 to 1, 3 to 0, 2 to 1, 2 to 0, or 1 to 0. Scleroderma GVHD score assessments are provided in Example 1, Table 3 herein.

[0119] In some embodiments, administration of a JAK inhibitor and a ROCK inhibitor results in an improvement in one or more of the subject's weight loss, the subject's posture, the subject's activity level, the subject's hair texture, and the subject's skin integrity as measured by a GVHD score compared to the subject's weight loss, the subject's posture, the subject's activity level, the subject's hair texture, and the subject's skin integrity prior to administration of the JAK inhibitor and a ROCK inhibitor.

[0120] In some embodiments, administration of the JAK inhibitor and the ROCK inhibitor results in an improvement in the subject's weight loss as measured by GVHD score compared to the subject's weight loss as measured by GVHD score prior to administration of the JAK inhibitor and the ROCK inhibitor. For example, in a subject who exhibited >15% weight loss prior to administration of the JAK inhibitor and the ROCK inhibitor (Grade 4 as measured by GVHD score), administration of the JAK inhibitor and the ROCK inhibitor may result in a reduction in the subject's weight loss of 10%-15% (Grade 3 as measured by GVHD score), 5%-10% (Grade 2 as measured by GVHD score), 0%-5% (Grade 1 as measured by GVHD score), or no weight loss (Grade 0 as measured by GVHD score).

[0121] In some embodiments, administration of the JAK inhibitor and the ROCK inhibitor results in an improvement in the subject's posture as measured by the GVHD score, compared to the subject's posture as measured by the GVHD score prior to administration of the JAK inhibitor and the ROCK inhibitor. For example, in a subject that exhibited severe hunched posture (Grade 3 as measured by the GVHD score) prior to administration of the JAK inhibitor and the ROCK inhibitor, administration of the JAK inhibitor and the ROCK inhibitor may improve the subject's posture to a moderate hunched posture (Grade 2 as measured by the GVHD score), a mild hunched posture (Grade 1 as measured by the GVHD score), or a normal posture (Grade 0 as measured by the GVHD score).

[0122] In some embodiments, administration of the JAK inhibitor and the ROCK inhibitor results in an improvement in the subject's activity as measured by GVHD score compared to the subject's activity as measured by GVHD score prior to administration of the JAK inhibitor and the ROCK inhibitor. For example, in a subject who exhibited immobility prior to administration of the JAK inhibitor and the ROCK inhibitor (Grade 3 as measured by the GVHD score), administration of the JAK inhibitor and the ROCK inhibitor may result in the subject's activity improving to ambulation slower and refusing to move when touched (Grade 2 as measured by the GVHD score), ambulation slower (Grade 1 as measured by the GVHD score), or normal activity (Grade 0 as measured by the GVHD score).

[0123] In some embodiments, administration of the JAK inhibitor and the ROCK inhibitor results in an improvement in the subject's hair texture as measured by a GVHD score, compared to the subject's hair texture as measured by a GVHD score prior to administration of the JAK inhibitor and the ROCK inhibitor. For example, complete hair loss or hair loss of >1 cm 2 Subjects showing an area of ​​hair loss >1 cm2 had hair loss (grade 4 as measured by GVHD score) prior to JAK and ROCK inhibitor treatment and then experienced hair loss >1 cm2 in a single area after JAK and ROCK inhibitor treatment. 2 alopecia (grade 3 as measured by GVHD score), single area of ​​alopecia <1cm 2The texture of a subject's hair may be improved to hair that is wavy with a small amount of hair loss (Grade 2 as measured by GVHD score), wavy hair with a small amount of hair loss (Grade 1 as measured by GVHD score), or normal hair texture (Grade 0 as measured by GVHD score).

[0124] In some embodiments, administration of the JAK inhibitor and the ROCK inhibitor results in an improvement in the skin integrity of the subject as measured by the GVHD score, compared to the hair texture of the subject prior to administration of the JAK inhibitor and the ROCK inhibitor as measured by the GVHD score. For example, in a subject exhibiting crusting or bleeding with multiple lesions (Grade 4 as measured by the GVHD score) prior to administration of the JAK inhibitor and the ROCK inhibitor, administration of the JAK inhibitor and the ROCK inhibitor may improve the skin integrity of the subject to crusting or bleeding with a single lesion (Grade 3 as measured by the GVHD score), peeling / skinning with a single lesion (Grade 2 as measured by the GVHD score), red / irritated skin lesions (Grade 1 as measured by the GVHD score), or normal skin integrity (Grade 0 as measured by the GVHD score).

[0125] In some embodiments, the present application provides a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and systemic sclerosis (scleroderma) in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharmaceutically acceptable salt thereof; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0126] In some embodiments, the application provides a method of treating graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof.

[0127] In some embodiments, the application provides a method of treating graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0128] In some embodiments, the application provides a method of treating chronic graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof.

[0129] In some embodiments, the application provides a method of treating chronic graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0130] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma-ceutically acceptable salt thereof; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma-ceutically acceptable salt thereof.

[0131] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0132] In some embodiments, the present application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject about 150 mg to about 250 mg of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof, on a free base basis; or 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof.

[0133] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject 150 mg to about 250 mg of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0134] In some embodiments, the present application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject about 200 mg of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof, on a free base basis; or 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof.

[0135] In some embodiments, the present application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject about 200 mg of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0136] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof; and about 100 mg / kg to about 200 mg / kg, on a free base basis, of 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof.

[0137] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and about 100 mg / kg to about 200 mg / kg of 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof, on a free base basis.

[0138] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof; and about 150 mg / kg of 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof, on a free base basis.

[0139] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and about 150 mg / kg of 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof, on a free base basis.

[0140] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject about 150 mg to about 250 mg of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof; and about 100 mg / kg to about 200 mg / kg of 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof, on a free base basis.

[0141] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject about 150 mg to about 250 mg of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and about 100 mg / kg to about 200 mg / kg of 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof, on a free base basis.

[0142] In some embodiments, the present application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject about 200 mg of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile, or a pharma- ceutically acceptable salt thereof; and about 150 mg / kg of 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof, on a free base basis.

[0143] In some embodiments, the present application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject about 200 mg of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and about 150 mg / kg of 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof, on a free base basis.

[0144] In some embodiments, the present application provides a method of treating a disease or disorder selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and systemic sclerosis (scleroderma) in a subject, comprising administering to the subject (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile, or a pharmaceutically acceptable salt thereof; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0145] In some embodiments, the application provides a method of treating graft-versus-host disease in a subject, comprising administering to the subject (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile, or a pharma- ceutically acceptable salt thereof; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof.

[0146] In some embodiments, the application provides a method of treating graft-versus-host disease in a subject, comprising administering to the subject (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0147] In some embodiments, the application provides a method of treating chronic graft-versus-host disease in a subject, comprising administering to the subject (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile, or a pharma- ceutically acceptable salt thereof; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof.

[0148] In some embodiments, the application provides a method of treating chronic graft-versus-host disease in a subject, comprising administering to the subject (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0149] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile, or a pharma-ceutically acceptable salt thereof; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma-ceutically acceptable salt thereof.

[0150] In some embodiments, the application provides a method of treating scleroderma-type chronic graft-versus-host disease in a subject, comprising administering to the subject (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharmaceutically acceptable salt thereof.

[0151] It is intended that the embodiments described herein may be combined in any suitable combination as if the embodiments were multiple dependent claims (e.g., embodiments relating to JAK inhibitors and dosages thereof, embodiments relating to ROCK2 inhibitors and dosages thereof, embodiments relating to any salt form of the compounds disclosed herein, and embodiments relating to compositions and / or administration may be combined in any combination). All possible combinations have not been separately recited herein merely for the sake of brevity.

[0152] The compounds described herein may be asymmetric (e.g., have one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise specified. Compounds containing asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials are known in the art, for example, by resolution of racemic mixtures or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like may also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers in the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0153] In some embodiments, the compounds have the (R)-configuration. In some embodiments, the compounds have the (S)-configuration.

[0154] The resolution of racemic mixtures of compounds can be carried out by any of a number of methods known in the art. Exemplary methods include fractional recrystallization using chiral resolving acids that are optically active, salt-forming organic acids. Suitable resolving agents for fractional recrystallization are, for example, optically active acids such as D- and L-forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids such as β-camphorsulfonic acid. Other suitable resolving agents for fractional crystallization include stereoisomerically pure forms of α-methylbenzylamine (e.g., S- and R-forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

[0155] Resolution of racemic mixtures may be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by one skilled in the art.

[0156] The compounds described herein also include tautomeric forms. Tautomeric forms are obtained by the exchange of adjacent double and single bonds with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Exemplary prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy more than one position of a heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H-, and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically fixed in one form by appropriate substitution.

[0157] The compounds described herein may also include isotopically labeled compounds of the present disclosure. An "isotopically labeled" or "radiolabeled" compound is a compound of the present disclosure in which one or more atoms have been replaced, or substituted, with an atom having an atomic mass or mass number different from that normally found in nature (i.e., naturally occurring). Suitable radionuclides that can be incorporated into the compounds of the present disclosure include, but are not limited to: 2 H (also written as D for deuterium), 3 H (also written as T for tritium), 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 18 F, 35 S, 36 Cl, 82 Br, 75 Br, 76 Br, 77 Br, 123 I, 124 I, 125 I and 131 For example, one or more hydrogen atoms in the compounds of the present disclosure may be replaced with deuterium atoms (e.g., replacing -CD3 with -CH3, etc., as appropriate, C of the compounds of formula (I), (II), or (III) or Table 2). 1-6 One or more hydrogen atoms of the alkyl group may be replaced by deuterium atoms.

[0158] The term "compound" as used herein is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted, unless the name indicates a specific stereoisomer. Compounds herein that are identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms, unless otherwise specified.

[0159] In some embodiments, the compounds described herein and their salts are substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation may include, for example, a composition enriched in the compounds described herein. Substantial separation may include a composition that includes at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds described herein or their salts. Methods for isolating compounds and their salts are routine in the art.

[0160] All compounds and their pharma- ceutically acceptable salts may be found together with other substances such as water and solvents (e.g., hydrates and solvates) or may be isolated. When in the solid state, the compounds and their salts described herein may occur in various forms, for example, in the form of solvates, including hydrates. The compounds may be in any solid state form, such as polymorphs or solvates, so unless otherwise specified, references to compounds and their salts herein should be understood to include any solid state form of the compounds.

[0161] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0162] The present invention also includes pharma- ceutically acceptable salts of the compounds described herein. The term "pharma- ceutically acceptable salts" refers to derivatives of the disclosed compounds, where the parent compound is modified by converting an existing acid or base moiety into its salt form. Examples of pharma- ceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharma- ceutically acceptable salts of the present invention include non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharma- ceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two, generally with non-aqueous media such as ether, ethyl acetate, alcohol (e.g., methanol, ethanol, isopropanol, or butanol) or acetonitrile (MeCN) being preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17 th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19 and in Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). In some embodiments, the compounds described herein include N-oxide forms.

[0163] The terms "individual," "patient," and "subject" are used interchangeably and refer to any animal, including a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, most preferably a human.

[0164] The phrase "therapeutically effective amount" refers to that amount of an active compound or pharmaceutical agent that elicits the biological or medicinal response desired in a tissue, system, animal, individual, or human by a researcher, veterinarian, physician, or other clinician.

[0165] As used herein, the term "treating" or "treatment" refers to inhibiting a disease, e.g., inhibiting the disease, condition, or disorder in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder (i.e., halting further development of the pathology and / or symptomology), or ameliorating a disease, e.g., ameliorating a disease, condition, or disorder in an individual experiencing or exhibiting the pathology or symptomology of the disease, condition, or disorder (i.e., reversing the pathology and / or symptomology), e.g., reducing the severity of the disease.

[0166] In some embodiments, the compounds provided herein are useful for preventing or reducing the risk of developing any of the diseases referred to herein, e.g., for preventing or reducing the risk of developing a disease, condition, or disorder in an individual who may be predisposed to the disease, condition, or disorder, but who has not yet experienced or exhibited the symptoms or symptomology of the disease.

[0167] The terms "for example" and "such as," and their grammatical equivalents, are understood to be followed by the phrase "and without limitation," unless expressly stated otherwise.

[0168] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise.

[0169] As used herein, the term "about" means "approximately" (eg, plus or minus about 10% of the indicated value).

[0170] Pharmaceutical Preparations and Dosage Forms When used as a pharmaceutical, the compounds of the present disclosure may be administered in the form of pharmaceutical compositions. These compositions may be prepared in a manner well known in the pharmaceutical art and may be administered by various routes depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (including transdermal, epidermal, ocular, and mucosal delivery, including intranasal, vaginal, and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, or injection or infusion, or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration may be in the form of a single bolus dose or, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0171] The present disclosure also includes pharmaceutical compositions that include one or more of the disclosed compounds or pharma- ceutically acceptable salts thereof (e.g., one or more JAK pathway inhibitors and / or one or more ROCK inhibitors in combination with one or more pharma- ceutically acceptable carriers (excipients)) as an active ingredient. In some embodiments, the compositions are suitable for topical administration. In making the compositions of the present disclosure, the active ingredient is typically mixed with an excipient, diluted by the excipient, or enclosed in such a carrier, for example, in the form of a capsule, sachet, paper, or other container. When the excipient functions as a diluent, it can be a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or liquid vehicles), ointments containing up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

[0172] When preparing formulations, active compound can be milled to provide suitable particle size before being combined with other components.If active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh.If active compound is substantially water-soluble, its particle size can be adjusted by milling to provide substantially uniform distribution in formulation, for example, about 40 mesh.

[0173] The compounds of the present disclosure can be milled using known milling procedures, such as wet milling, to obtain a particle size suitable for tableting and other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the present disclosure can be prepared by processes known in the art, see, for example, the disclosure of International Application No. WO2002 / 000196.

[0174] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose.The formulation may further include lubricants such as talc, magnesium stearate, and mineral oil, wetting agents; emulsifying and suspending agents; preservatives such as methylbenzoate and propylhydroxybenzoate, sweeteners, and flavoring agents.The composition of the present disclosure can be formulated to provide quick, sustained, or delayed release of active ingredient after administration to patient by using procedures known in the art.

[0175] The compositions can be formulated in unit dosage form, each dosage containing from about 5 to about 1000 mg (1 g), more usually about 100 to about 500 mg, of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect, in association with a suitable pharmaceutical excipient.

[0176] In some embodiments, the compositions of the present disclosure contain about 5 to about 50 mg of active ingredient. One of skill in the art will understand that the compositions of this embodiment contain about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, or about 45 to about 50 mg of active ingredient.

[0177] In some embodiments, the compositions of the present disclosure contain about 50 to about 500 mg of the active ingredient. One of skill in the art will appreciate that the compositions of this embodiment contain about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 350 to about 400, or about 450 to about 500 mg of the active ingredient.

[0178] In some embodiments, the compositions of the present disclosure contain about 500 to about 2000 mg of active ingredient. Those of skill in the art will appreciate that the compositions of this embodiment contain about 500 to about 550, about 550 to about 600, about 600 to about 650, about 650 to about 700, about 700 to about 750, about 750 to about 800, about 800 to about 850, about 850 to about 900, about 900 to about 950, about 950 to about 1000 mg, about 1000 mg to about 1100 mg, about 1100 mg to about 1200 mg, or about 1500 mg to about 2000 mg. mg, about 1200 mg to about 1300 mg, about 1300 mg to about 1400 mg, about 1400 mg to about 1500 mg, about 1500 mg to about 1600 mg, about 1600 mg to about 1700 mg, about 1700 mg to about 1800 mg, about 1800 mg to about 1900 mg, or about 1900 mg to about 2000 mg of active ingredient(s).

[0179] Similar dosages of the compounds described herein may be used in the methods and uses of the present disclosure.

[0180] The active compound may be effective over a wide dosage range and is generally administered in a pharma- ceutically effective amount. However, it will be understood that the amount of compound actually administered will usually be determined by the physician according to the relevant circumstances, including the condition being treated, the selected route of administration, the compound actually administered, the age, weight, and response of the individual patient, the severity of the patient's condition, and the like.

[0181] To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical excipients to form a solid preformulation composition containing a homogenous mixture of the compounds of the present disclosure. When these preformulation compositions are referred to as homogenates, the active ingredient is typically dispersed evenly throughout the composition, so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above, for example, containing about 0.1 to about 1000 mg of the active ingredient of the present disclosure.

[0182] The tablet or pill of the present disclosure can be coated or otherwise compounded to provide a dosage form that provides the advantage of long-term action.For example, the tablet or pill can include an inner dose and an outer dose component, the latter being in the form of an envelope on the former.The two components can be separated by an enteric layer that resists disintegration in the stomach and allows the inner component to pass intact into the duodenum or to be delayed in release.A variety of materials can be utilized as such enteric layer or coating, including a number of polymeric acids, and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

[0183] Liquid forms into which the compounds and compositions of the present disclosure may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and emulsions flavored with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0184] Compositions for inhalation or insufflation include liquids and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by oral or nasal respiratory route for local or systemic effect. Compositions may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered orally or nasally from a device that delivers the formulation in an appropriate manner.

[0185] Topical formulations may contain one or more conventional carriers. In some embodiments, ointments may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and the like. The carrier composition of creams may be based on water in combination with glycerol and one or more other components, for example, glycerol monostearate, PEG-glycerol monostearate, and cetylstearyl alcohol. Gels may be formulated using isopropyl alcohol and water, suitably in combination with other components, such as, for example, glycerol, hydroxyethylcellulose, and the like. In some embodiments, topical formulations include at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5% by weight of a compound of the present disclosure. Topical formulations may be suitably packaged in 100 g tubes, optionally accompanied by instructions for treating a selected indication, such as, for example, psoriasis or other skin conditions.

[0186] The amount of compound or composition administered to a patient will vary depending on what is being administered, the purpose of administration, such as prophylaxis or treatment, the condition of the patient, the method of administration, etc. In therapeutic applications, the compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. The effective dose will depend on the disease condition being treated and the judgment of the attending physician based on factors such as the severity of the disease, the age, weight, and general condition of the patient.

[0187] The compositions administered to a patient may be in the form of pharmaceutical compositions described above. These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. Aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparations being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations will typically be 3-11, more preferably 5-9, most preferably 7-8. It will be understood that the use of certain of the aforementioned excipients, carriers, or stabilizers will result in the formulation of pharmaceutical salts.

[0188] The therapeutic amount of a compound of the present disclosure may vary according to, for example, the particular application to be treated, the method of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound of the present disclosure in a pharmaceutical composition may vary depending on several factors, including the dosage, chemical properties (e.g., hydrophobicity), and route of administration. For example, a compound of the present disclosure may be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w / v of the compound for parenteral administration. Some typical dosage ranges are about 1 μg / kg to about 1 g / kg of body weight per day. In some embodiments, the dosage range is about 0.01 mg / kg to about 100 mg / kg of body weight per day. The dosage may depend on such variables as the type and extent of progression of the disease or disorder, the overall health of the particular patient, the relative bioefficacy of the compound selected, the formulation of excipients, and its route of administration. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0189] kit The present disclosure also includes, for example, useful pharmaceutical kits, comprising one or more containers containing a therapeutically effective amount of a pharmaceutical composition comprising a compound or any embodiment thereof. As would be readily apparent to those skilled in the art, such kits may further include one or more of a variety of conventional pharmaceutical kit components, such as a container containing one or more pharma-ceutically acceptable carriers, additional containers, etc. Instructions for use, either as an insert or label indicating the amount of components to be administered, guidelines for administration, and / or guidelines for mixing the components, may also be included in the kit. EXAMPLES

[0190] The present invention will be described in more detail by specific examples. The following examples are provided for illustrative purposes and are not intended to limit the present invention in any manner. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially the same results.

[0191] Example A. In vitro JAK kinase assay Selective JAK1 inhibitors that may be used in combination with the ROCK inhibitors described herein are tested for inhibitory activity of JAK targets according to the in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104 below. The catalytic domains of human JAK1 (amino acids 837-1142), JAK2 (amino acids 828-1132), and JAK3 (amino acids 781-1124) with N-terminal His tags are expressed in insect cells using baculovirus and purified. The catalytic activity of JAK1, JAK2, or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogeneous time-resolved fluorescence (HTRF). The IC of the compound for each kinase was determined in a 40 μL reaction containing enzyme, ATP, and 500 nM peptide in 50 mM Tris (pH 7.8) buffer containing 100 mM NaCl, 5 mM DTT, and 0.1 mg / mL (0.01%) BSA. 50 The IC of 1 mM was measured. 50 For the measurements, the ATP concentration in the reaction was 1 mM. The reaction was carried out at room temperature for 1 hour and then stopped with 20 μL of 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, MA). Binding to europium-labeled antibodies was carried out for 40 minutes and the HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, MA). The compounds in Table 1 were tested in this assay and showed the IC 50 It was shown that the value was

[0192] Example B: Cellular Assay JAK inhibitors that can be used in combination with ROCK inhibitors described herein are tested for inhibitory activity of JAK targets according to at least one of the following cellular assays.

[0193] To study the effect of compounds on T cell activation, mouse splenocytes were cultured at 2 × 10 per well in RPMI 1640, 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.05 mm beta-mercaptoethanol. 6 Cells are plated (in a 24-well plate format) and activated with CD3 / CD28 beads (Gibco, Walttham, Mass.). Compounds are added to the cells in DMSO / media (final concentration of 0.2% DMSO) and incubated for 72 hours at 37° C., 5% CO2. The effect of compound(s) is assessed by quantifying cytokine levels using a multispot assay system, proinflammatory panel 1 (IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, KC / GRO, IL-10, IL-1p70, and TNFα; Meso Scale Diagnostics [MSD], Rockville, Maryland).

[0194] Furthermore, the combined effect on T cell activation can be measured by mixed lymphocyte reaction (MLR) assay. Dendritic cells were generated by culturing bone marrow cells isolated from femurs and tibias of BALB / c mice in RPMI 1640, 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin and 25 ng / ml granulocyte-macrophage colony-stimulating factor (GM-CSF) at 37°C, 5% CO2 (3x10 per well). 6 Cells are incubated in a 6-well plate format. Allogeneic responder C57BL / 6 splenocytes are labeled with carboxyfluorescein succinimidyl ester (CFSE) dye and added to the cultures with compounds after 7 days. The effect of compounds on T cell activation is assessed by measuring cytokine secretion (MSD, Rockville, Maryland) and T cell proliferation by CFSE dilution by flow cytometry.

[0195] To study the effect of compounds on macrophages, mouse splenocytes were plated at 3x10 per well in RPMI 1640, 10% FBS, 100U / ml penicillin, 100μg / ml streptomycin and 50ng / ml macrophage colony-stimulating factor (M-CSF). 6 Cells were plated (6-well plate format) and incubated at 37° C., 5% CO2. On day 6, cells are treated with compounds. On day 7, macrophages are activated with 5 ng / mL lipopolysaccharide (LPS) and compound effects are assessed 24 hours later by measuring cytokines from the supernatant (MSD, Rockville, Maryland).

[0196] Certain compounds herein have been or may be evaluated for their activity in inhibiting T cell proliferation. Such assays may also be considered as proliferation assays with a second cytokine (i.e., JAK) and simplified assays of inhibition of immune suppression or immune activation. Below is a brief outline of how such experiments can be performed. Peripheral blood mononuclear cells (PBMCs) are prepared from human whole blood samples using Ficoll Hypaque separation, and T cells (fraction 2000) can be obtained from PBMCs by elutriation. Freshly isolated human T cells are cultured at 2×10 at 37° C. 6At a density of 1000 cells / ml, T cells can be maintained in culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin) for up to 2 days. For IL-2-stimulated cell proliferation assay, T cells are first treated with phytohemagglutinin (PHA) at a final concentration of 10 μg / ml for 72 hours. After one wash with PBS, 6000 cells / well are plated in 96-well plates and treated with different concentrations of compounds in culture medium in the presence of 100 U / ml human IL-2 (ProSpec-Tany TechnoGene; Rehovot, Israel). Plates are incubated at 37° C. for 72 hours and proliferation indicators are assessed using CellTiter-Glo Luminescent reagent according to the manufacturer's suggested protocol (Promega; Madison, WI).

[0197] Example 1. Combination treatment with JAK1 and ROCK2 inhibitors in a mouse model of chronic graft-versus-host disease (GvHD) of the scleroderma type The effects of therapeutic doses of a selective JAK1 inhibitor (itacitinib), a ROCK2 inhibitor (belmosudil), and a combination of a selective JAK1 inhibitor and a ROCK2 inhibitor (itacitinib + belmosudil) were tested in a mouse model of scleroderma-type chronic GVHD.

[0198] Scleroderma-type chronic GVHD was induced in C57Bl / 6 mice using a single acute dose of 8.5 Gy total body irradiation on day -1. On day 0, recipient mice were intravenously injected with a combination of splenocytes and bone marrow cells from donor LP / J mice. Starting on day 21, animals were administered vehicle, itacitinib, belmosudil, or a combination of both itacitinib and belmosudil (PO, BID, see Table 2). All animals were monitored daily to record weight change, survival rate, and GVHD score (see Table 3). On days 33, 36, 39, 42, and 45, all animals were photographed under isoflurane anesthesia to assess disease severity. [Table 4] [Table 5]

[0199] Treatment with single agent itacitinib or belmosudil resulted in a reduction in GVHD scores when compared to vehicle-treated animals (see Figure 1). Combination therapy with itacitinib and belmosudil resulted in a significant reduction in GVHD scores when compared to vehicle-treated animals (see Figure 1). By AUC analysis, animals treated with the combination therapy showed significantly lower GVHD scores than vehicle, itacitinib-treated, and belmosudil-treated animals (see Figure 2). These results indicate that itacitinib + belmosudil combination therapy has potential synergistic effects in a mouse model of scleroderma-type chronic GVHD.

[0200] Example 2. Combined treatment with JAK1 / 2 and ROCK2 inhibitors in a mouse model of chronic graft-versus-host disease (GvHD) of the scleroderma type The effects of therapeutic doses of a JAK1 / 2 inhibitor (ruxolitinib), a ROCK2 inhibitor (belmosudil), and a combination of a JAK1 / 2 inhibitor and a ROCK2 inhibitor (ruxolitinib + belmosudil) were tested in a mouse model of scleroderma-type chronic GVHD.

[0201] Scleroderma chronic GVHD was induced in C57Bl / 6 mice using a single acute dose of 8.5 Gy total body irradiation on day -1. On day 0, recipient mice were intravenously injected with a combination of splenocytes and bone marrow cells from donor LP / J mice. Starting on day 21, animals were administered vehicle, ruxolitinib, belmosudil, or a combination of both ruxolitinib and belmosudil (PO, BID, see Table 4). All animals were monitored daily to record weight change, survival rate, and GVHD score (see Table 5). On days 33, 36, 39, 42, and 45, all animals were photographed under isoflurane anesthesia to assess disease severity. [Table 6] [Table 7]

[0202] Treatment with single-agent ruxolitinib resulted in a numerical reduction in GVHD scores when compared to vehicle-treated animals, whereas single-agent belmosudil provided no benefit (see FIG. 3). By AUC analysis, animals treated with combination therapy showed lower GVHD scores than vehicle, ruxolitinib, and belmosudil-treated animals (see FIG. 4). Combination therapy resulted in a numerically lower AUC disease burden compared to ruxolitinib or belmosudil monotherapy.

[0203] In addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications, and publications, is hereby incorporated by reference in its entirety.

Claims

1. A combination pharmaceutical comprising (i) a JAK inhibitor and (ii) a ROCK inhibitor for treating a disease or disorder in a subject selected from graft-versus-host disease (GVHD), restrictive allograft syndrome (RAS), chronic pulmonary allograft dysfunction (CLAD) (e.g., bronchiolitis obliterans syndrome (BOS)), and systemic sclerosis (scleroderma), The JAK inhibitor and the ROCK inhibitor are administered to the subject simultaneously or sequentially. The combination drug.

2. The pharmaceutical combination of claim 1 , wherein the JAK inhibitor is a selective JAK1 inhibitor.

3. 3. The pharmaceutical combination of claim 1 or 2, wherein the JAK inhibitor is selective for JAK1 over JAK2, JAK3 and TYK2.

4. The JAK inhibitor, {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[4-fluoro-2-(trifluoromethyl)phenyl]piperidine-1-carboxamide; [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile; 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide; ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile; 3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile; 3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile; 4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile; 4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile; [trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-3-(4-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperazin-1-yl)cyclobutyl]acetonitrile; {trans-3-(4-{[4-[(3-hydroxyazetidin-1-yl)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; {trans-3-(4-{[4-{[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; {trans-3-(4-{[4-{[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; 4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile; 5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide; 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide; 5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide; {1-(cis-4-{[6-(2-hydroxyethyl)-2-(trifluoromethyl)pyrimidin-4-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {1-(cis-4-{[4-[(ethylamino)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {1-(cis-4-{[4-(1-hydroxy-1-methylethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {1-(cis-4-{[4-{[(3R)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {1-(cis-4-{[4-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {trans-3-(4-{[4-({[(1S)-2-hydroxy-1-methylethyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; {trans-3-(4-{[4-({[(2R)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; {trans-3-(4-{[4-({[(2S)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; and {trans-3-(4-{[4-(2-hydroxyethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; The pharmaceutical combination according to any one of claims 1 to 3, which is selected from the group consisting of:

5. The pharmaceutical combination according to any one of claims 1 to 3, wherein the JAK inhibitor is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile or a pharma- ceutically acceptable salt thereof.

6. The pharmaceutical combination according to any one of claims 1 to 3, wherein the JAK inhibitor is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate.

7. The pharmaceutical combination of claim 1 , wherein the JAK inhibitor is a JAK1 / 2 inhibitor.

8. The pharmaceutical combination according to claim 1 or 7, wherein the JAK inhibitor is (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile, or a pharma- ceutically acceptable salt thereof.

9. The pharmaceutical combination according to claim 1 or 7, wherein the JAK inhibitor is (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate.

10. The pharmaceutical combination according to any one of claims 1 to 9, wherein the JAK inhibitor is administered to the subject at a dose of about 100 mg to about 300 mg.

11. The pharmaceutical combination of any one of claims 1 to 9, wherein the JAK inhibitor is administered to the subject at a dose of about 200 mg.

12. The combination pharmaceutical according to any one of claims 1 to 11, wherein the ROCK inhibitor is a ROCK2 inhibitor.

13. The pharmaceutical combination according to claim 12, wherein the ROCK2 inhibitor is 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof.

14. The pharmaceutical combination according to any one of claims 1 to 13, wherein the ROCK inhibitor is administered to the subject at a dose of about 200 mg / kg to about 300 mg / kg.

15. The pharmaceutical combination according to any one of claims 1 to 13, wherein the ROCK inhibitor is administered to the subject at a dose of about 200 mg / kg.

16. The pharmaceutical combination of any one of claims 1 to 15, wherein the JAK inhibitor is administered to the subject once daily.

17. The pharmaceutical combination of any one of claims 1 to 15, wherein the JAK inhibitor is orally administered to the subject once daily.

18. The pharmaceutical combination of any one of claims 1 to 15, wherein the JAK inhibitor is administered to the subject twice daily.

19. The pharmaceutical combination of any one of claims 1 to 15, wherein the JAK inhibitor is administered orally to the subject twice daily.

20. The pharmaceutical combination according to any one of claims 1 to 19, wherein the ROCK inhibitor is administered to the subject once a day.

21. The pharmaceutical combination according to any one of claims 1 to 19, wherein the ROCK inhibitor is orally administered to the subject once a day.

22. The pharmaceutical combination according to any one of claims 1 to 19, wherein the ROCK inhibitor is administered to the subject twice daily.

23. The pharmaceutical combination according to any one of claims 1 to 19, wherein the ROCK inhibitor is orally administered to the subject twice daily.

24. The pharmaceutical combination of any one of claims 1 to 15, wherein the JAK inhibitor and the ROCK inhibitor are each orally administered to the subject twice daily.

25. The pharmaceutical combination according to any one of claims 1 to 24, wherein the disease or disorder is graft-versus-host disease.

26. The pharmaceutical combination according to claim 25, wherein the graft-versus-host disease is chronic graft-versus-host disease.

27. The pharmaceutical combination according to claim 25, wherein the graft-versus-host disease is chronic graft-versus-host disease of the scleroderma type.

28. 28. The pharmaceutical combination of any one of claims 1 to 27, wherein administration of the JAK inhibitor and the ROCK inhibitor to the subject results in a decrease in the GVHD score of the subject compared to the GVHD score of the subject prior to administration of the JAK inhibitor and the ROCK inhibitor.

29. A combination pharmaceutical for treating scleroderma-type chronic graft-versus-host disease in a subject, comprising {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma-ceutically acceptable salt thereof, the subject is administered, simultaneously or sequentially, {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile adipate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof; The combination drug.

30. A combination pharmaceutical for treating scleroderma-type chronic graft-versus-host disease in a subject, comprising (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or pharma-ceutical acceptable salts thereof, the subject is administered, simultaneously or sequentially, (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate; and 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide, or a pharma- ceutically acceptable salt thereof; The combination drug.

31. The pharmaceutical combination of any one of claims 1 to 30, wherein the JAK inhibitor and the ROCK inhibitor are administered simultaneously.

32. The pharmaceutical combination of any one of claims 1 to 30, wherein the JAK inhibitor and the ROCK inhibitor are administered sequentially.

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