Prophylactic or therapeutic agent for severe pulmonary hypertension or connective tissue disease

Abstract

The present disclosure addresses the problem of providing a prophylactic or therapeutic agent for severe pulmonary hypertension or connective tissue disease.　The prophylactic or therapeutic agent for severe pulmonary hypertension or connective tissue disease contains a JAK1 inhibitor.

Description

Preventive or therapeutic drug for severe pulmonary hypertension or collagen disease

[0001] The present disclosure relates to a prophylactic or therapeutic agent for severe pulmonary hypertension or collagen disease, and a method for screening for a prophylactic or therapeutic agent for severe pulmonary hypertension or collagen disease.

[0002] Pulmonary hypertension (PH) is a group of progressive diseases with poor prognosis that cause cardiac and pulmonary dysfunction due to elevated pulmonary arterial blood pressure. According to the Nice Classification, PH is classified into the following groups: Group 1: pulmonary arterial hypertension (PAH); Group 1': pulmonary veno-occlusive disease (PVOD) and / or pulmonary capillary hemangiomatosis (PCH); Group 1'': persistent pulmonary hypertension of the newborn; Group 2: pulmonary hypertension associated with left heart disease; Group 3: pulmonary hypertension associated with lung disease and / or hypoxemia; Group 4: chronic thromboembolic pulmonary hypertension; and Group 5: pulmonary hypertension associated with an unknown multifactorial mechanism.

[0003] PAH (Nice Classification Group 1) is a rare and intractable disease designated by the Ministry of Health, Labour and Welfare of Japan. It is characterized by vascular remodeling (stenosis and obstruction) of the pulmonary arteries due to unknown causes, leading to elevated pulmonary arterial pressure and right heart failure. Traditionally, three types of treatments for PAH have been used: prostacyclin derivatives, which dilate pulmonary blood vessels; endothelin receptor antagonists, which prevent endothelin, which causes pulmonary vasoconstriction, from binding to smooth muscle; and phosphodiesterase 5 (PDE5) inhibitors, which inhibit the degradation of cyclic GMP, thereby relaxing vascular smooth muscle contraction. While existing treatments can dramatically improve the prognosis of relatively mild PAH, the prognosis for severe PAH remains poor (see, for example, Non-Patent Document 1). Furthermore, the present inventors have reported that the aromatic hydrocarbon receptor (AHR), a regulator of inflammatory signaling and a receptor for environmental pollutants, holds the key to the pathogenesis of PAH, and that AHR inhibition is effective in treating PAH (Non-Patent Document 2).

[0004] On the other hand, collagen diseases are systemic autoimmune diseases that primarily affect connective tissue and share a common pathological basis in abnormalities of the autoimmune response. These diseases include systemic lupus erythematosus, systemic sclerosis, polymyositis / dermatomyositis, Sjögren's syndrome, mixed connective tissue disease, antiphospholipid syndrome, and Behçet's disease. These collagen diseases, like PAH, are designated as intractable diseases by the Ministry of Health, Labor, and Welfare of Japan. PAH associated with collagen diseases (Connective Tissue Disease-Associated PAH; CTD-PAH), especially PAH associated with systemic sclerosis (SSc-PAH), is known to have a poor prognosis (Non-Patent Documents 3-5).

[0005] A. Ogawa et al., Long-term patient survival with idiopathic / heritable pulmonary arterial hypertension treated at a single center in Japan, Life Sciences 118, 414-419 (2014). doi: 10.1016 / j.lfs.2014.01.077.T. Masaki et al., Aryl hydrocarbon receptor is essential for the pathogenesis of pulmonary arterial hypertension. PNAS 118(2021), e2023899118, doi.org / 10.1073 / pnas.2023899118.Escribano-Subias, P., et al. Survival in pulmonary hypertension in Spain: insights from the Spanish registry. Eur Respir J 40, 596-603 (2012).Gall, H., et al. The Giessen Pulmonary Hypertension Registry: Survival in pulmonary hypertension subgroups. J Heart Lung Transplant 36, 957-967 (2017).Hurdman, J., et al. ASPIRE registry: assessing the Spectrum of Pulmonary hypertension Identified at a REferral centre. Eur Respir J 39, 945-955 (2012).

[0006] Although existing vasodilatory drugs have opened up the possibility of treating PH, including PAH, which was previously an incurable disease, their effectiveness in treating severe PH is beginning to show limitations. To expand treatment options and further improve therapeutic efficacy, there is a need for the development of drugs that can prevent or treat severe PH through mechanisms other than vasodilatory therapy. Furthermore, there are unmet medical needs in collagen diseases and the associated PAH.

[0007] Therefore, an object of the present disclosure is to provide a new drug capable of preventing or treating severe PH or collagen diseases. Another object of the present disclosure is to provide a method for screening candidate substances useful as drugs for preventing or treating severe PH or collagen diseases.

[0008] The present inventors conducted extensive research to solve the above-mentioned problems, and as a result, they obtained the findings (i) to (iii) below, and found that a JAK1 inhibitor can be used as a preventive or therapeutic agent for severe PH or connective tissue disease. The present disclosure was completed through further research based on these findings. (i) Prophylactic administration of a JAK1 inhibitor to a SuHx rat model that develops severe PAH suppresses the onset of severe PAH. (ii) Administration of a JAK1 inhibitor to a SuHx rat model that has been induced to develop severe PAH ameliorates the pathology of severe PAH. (iii) Administration of a JAK1 inhibitor to a mouse model of severe connective tissue disease PAH ameliorates the pathology of severe PAH. Furthermore, administration of a JAK1 inhibitor to a mouse model of severe connective tissue disease PAH ameliorates the anemia and thrombocytopenia commonly observed in systemic lupus erythematosus and further suppresses the systemic inflammatory pathology associated with connective tissue disease.

[0009] That is, one embodiment of the present disclosure provides the following inventions relating to technologies for preventing or treating severe PH or connective tissue diseases: Item 1-1. A preventive or therapeutic drug for severe pulmonary hypertension or connective tissue diseases, comprising a JAK1 inhibitor. Item 1-2. The preventive or therapeutic drug according to Item 1-1, wherein the JAK1 inhibitor is a selective JAK1 inhibitor. Item 1-3. The preventive or therapeutic drug according to Item 1-2, wherein the selective JAK1 inhibitor is at least one selected from the group consisting of ABT-317, upadacitinib, abrocitinib, filgotinib, itacitinib, brepositinib, londamositinib, GDC-4379, TUL-01101, ivalmacitinib, povorcitinib, solcitinib, and salts thereof. Item 1-4. The prophylactic or therapeutic drug according to Item 1-1, wherein the JAK1 inhibitor is at least one selected from the group consisting of tofacitinib, itacitinib, baricitinib, and salts thereof.Item 1-5. The prophylactic or therapeutic drug according to Item 1-1, wherein the JAK1 inhibitor is at least one selected from the group consisting of pyrazolothiazole compounds shown in the following (1) to (6), their salts, and solvates: (1) methyl [1-({6-[(2S)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (2) methyl [1-({6-[(2R)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (3) methyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (4) ethyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (5) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (6) N-(1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide. Item 1-6. The prophylactic or therapeutic drug according to any one of Items 1-1 to 1-5, used for the prophylaxis or treatment of severe pulmonary arterial hypertension, severe pulmonary atresia, or severe pulmonary hypertension associated with lung disease and / or hypoxemia. Item 1-7. The prophylactic or therapeutic drug according to any one of Items 1-1 to 1-5, used for the prophylaxis or treatment of at least one collagen disease selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, polymyositis / dermatomyositis, Sjogren's syndrome, mixed connective tissue disease, antiphospholipid syndrome, and Behcet's disease, or pulmonary hypertension associated with such a collagen disease.Item 1-8. A method for preventing or treating severe pulmonary hypertension or collagen vascular disease, comprising administering a JAK1 inhibitor to a person in need of such prevention or treatment. Item 1-9. Use of a JAK1 inhibitor for the manufacture of a drug for the prevention or treatment of severe pulmonary hypertension or collagen vascular disease. Item 1-10. A JAK1 inhibitor for use in the treatment of preventing or treating severe pulmonary hypertension or collagen vascular disease.

[0010] Furthermore, another embodiment of the present disclosure provides the following invention, which relates to a screening technology for a preventive or therapeutic agent for severe PH or connective tissue disease: Item 2. A method for screening test substances for candidate substances that may be effective in preventing or treating severe PH or connective tissue disease, the screening method comprising the steps of: measuring the JAK1 inhibitory activity of the test substances; and selecting test substances that have been confirmed to have JAK1 inhibitory activity as the candidate substances.

[0011] The present disclosure provides a drug for preventing or treating severe PH or collagen disease, and also enables screening of test substances for candidate substances that may be effective as a drug for preventing or treating severe PH or collagen disease.

[0012] These figures demonstrate the preventive effect of a JAK1 inhibitor on severe PAH using a SuHx rat model that develops severe PAH. Figure A shows the experimental procedure. Figure B shows the results of right ventricular systolic pressure (RVSP) measurements 5 weeks after SU5416 administration. Figure C shows the results of Fulton's coefficient measurements 5 weeks after SU5416 administration. Figure D shows a representative image of pulmonary artery remodeling using Elastica van Gieson staining of lung tissue sections 5 weeks after SU5416 administration. Figure E shows the results of measuring the pulmonary artery medial thickening index 5 weeks after SU5416 administration. In Figures A to E, "Normoxia" refers to the untreated control group housed under normoxia, "Vehicle" refers to the vehicle group, and "JAK1i" refers to the JAK1 inhibitor group administered ABT-317 at 3 mg / kg / day, 10 mg / kg / day, or 30 mg / kg / day. Significance tests were performed using one-way ANOVA and the Turkey-Kramer method. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. The following results demonstrate the preventive effect of a JAK1 inhibitor on severe PAH using a SuHx rat model. (A) Schematic diagram of the experimental procedure. (B) and (C) Western blotting results for CD79b protein expression in lungs 5 ​​weeks after SU5416 administration. (D) Fluorescent immunostaining of CD79b protein in lung tissue sections 5 weeks after SU5416 administration. In A–D, "Normoxia" refers to the untreated control group housed under normoxia, "Vehicle" refers to the vehicle group, and "JAK1i" refers to the JAK1-inhibited group administered ABT-317 at 30 mg / kg / day. Significance tests were performed using one-way ANOVA and the Turkey-Kramer method. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. The following results demonstrate the preventive effect of a JAK1 inhibitor on severe PAH using a SuHx rat model that develops severe PAH. (A) A diagram illustrates the experimental procedure. (B) and (C) show the results of Western blotting analysis of the protein expression level of phosphorylated STAT3 in lungs 5 ​​weeks after SU5416 administration.In A–C, "Normoxia" refers to the untreated control group housed under normoxia, "Vehicle" refers to the vehicle group, and "JAK1i" refers to the JAK1 inhibitor group administered ABT-317 at 30 mg / kg / day. Significance was tested by one-way ANOVA using the Turkey-Kramer method. * indicates P<0.05, ** indicates P<0.01. The results demonstrate the therapeutic efficacy of a JAK1 inhibitor for severe PAH in a SuHx rat model. (A) Schematic diagram of the experimental procedure. (B) Right ventricular systolic pressure (RVSP) was measured 8 weeks after SU5416 administration. (C) Fulton coefficient was measured 8 weeks after SU5416 administration. (D) Representative images of pulmonary artery remodeling in Elastica van Gieson-stained lung tissue sections 5 weeks after SU5416 administration. E shows the results of measuring the medial thickness index of the pulmonary artery 8 weeks after SU5416 administration. F shows the results of measuring the degree of splenomegaly (ratio of spleen weight to body weight) 8 weeks after SU5416 administration. In A–F, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibition group administered ABT-317 at 30 mg / kg / day. Significance was tested using Student's t-test. * indicates P<0.05, ** indicates P<0.01. The results show the therapeutic effect of a JAK1 inhibitor on severe PAH using a SuHx rat model with severe PAH. A shows the experimental procedure. B and C show the results of measuring CD79b protein expression levels by Western blotting using lungs 8 weeks after SU5416 administration. In A to C, "Normoxia" indicates the untreated control group raised under normal oxygen conditions, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibition group administered 30 mg / kg / day of ABT-317. Significance tests were performed using one-way ANOVA and the Turkey-Kramer method. * indicates P<0.05. These are the results of verifying the preventive effect of a JAK1 inhibitor on severe PAH using a SuHx rat model that develops severe PAH. A shows the experimental procedure.Panels B and C show the results of Western blotting analysis of phosphorylated STAT3 protein expression levels in lungs 8 weeks after SU5416 administration. In A–C, "Normoxia" refers to the untreated control group housed under normoxia, "Vehicle" refers to the vehicle group, and "JAK1i" refers to the JAK1-inhibited group administered ABT-317 at 30 mg / kg / day. Significance was tested by one-way ANOVA using the Turkey-Kramer method. * indicates P<0.05, ** indicates P<0.01. The results show the preventive effect of a JAK1 inhibitor on moderate PAH using a SuHx mouse model. Panel A shows the experimental procedure. Panel B shows the results of right ventricular systolic pressure (RVSP) measurements 3 weeks after SU5416 administration. Panel C shows the results of Fulton's index measurements 3 weeks after SU5416 administration. D is a representative image of pulmonary artery remodeling in an EVG-stained lung tissue section 3 weeks after SU5416 administration. E is the result of measuring the medial thickness index of the pulmonary artery 3 weeks after SU5416 administration. F is the result of measuring splenomegaly (spleen weight to body weight ratio) 3 weeks after SU5416 administration. In A–E, "Normoxia" indicates the untreated control group housed under normoxia, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibitor group administered ABT-317 at 30 mg / kg / day. Significance tests were performed using one-way ANOVA and the Turkey-Kramer method. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and **** indicates P<0.0001. These figures show the results of examining the therapeutic effect of a JAK1 inhibitor on severe connective tissue disease PAH using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. Figure A shows the experimental procedure. Figure B shows the results of measuring right ventricular systolic pressure (RVSP) three weeks after the start of the experiment. Figure C shows the results of measuring Fulton's coefficient three weeks after the start of the experiment. Figure D shows an image of EVG-stained lung tissue sections three weeks after the start of the experiment. Figure E shows the results of measuring the pulmonary artery medial thickening index three weeks after the start of the experiment. Figure F shows the results of measuring splenomegaly (spleen weight to body weight ratio) three weeks after the start of the experiment.In A to F, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1-inhibited group administered ABT-317 at 30 mg / kg / day. Significance tests for B, C, E, and F were performed using Student's t-test. * indicates P<0.05. The results show the therapeutic effect of a JAK1 inhibitor on severe connective tissue disease PAH using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. A shows the experimental procedure. B shows the results of measuring the number of activated B cells (GC B cells) in the lung by flow cytometry. C shows the results of measuring the number of activated B cells in the spleen by flow cytometry. In A to C, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1-inhibited group administered ABT-317 at 30 mg / kg / day. Significance tests were performed using Student's t-test. Continuation of Figure 9-1. Figure D shows the results of measuring plasma cell counts in the spleen by flow cytometry. Figure E shows the results of measuring plasma cell counts in mesenteric lymph nodes (MLNs) by flow cytometry. In Figures D and E, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibition group administered ABT-317 at 30 mg / kg / day. Significance was tested using Student's t-test. * indicates P<0.05, ** indicates P<0.01. The therapeutic effect of a JAK1 inhibitor on severe connective tissue disease PAH was evaluated using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. Figure A shows the experimental procedure. Figure B shows the results of measuring the expression levels of phosphorylated STAT3, STAT3, IL-6, and IL-1β in the lung by Western blotting. Figure C shows the results of quantifying the expression levels of phosphorylated STAT3, IL-6, and IL-1β in the lung by Western blotting. In A to D, "Reg1f / f" indicates the control group in which Regnase-1 f / f mice were fed a powdered normal diet, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibitor group administered ABT-317 at 30 mg / kg / day. In A, significance was tested using Student's t-test.In C, significance was tested by one-way ANOVA using the Turkey-Kramer method. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. The results show the therapeutic effect of long-term administration of a JAK1 inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. A shows the experimental procedure. B shows the results of survival rate over time. C shows the results of body weight over time. D shows the results of mean arterial pressure (MAP) measurements 6 weeks after the start of the experiment. E shows the results of right ventricular systolic pressure (RVSP) measurements 6 weeks after the start of the experiment. F shows the results of the ratio of right ventricular systolic pressure to systemic blood pressure (RVSP / MAP) 6 weeks after the start of the experiment. G shows the results of Fulton's coefficient (RV / LV+S) measurements 6 weeks after the start of the experiment. H shows the results of measuring splenomegaly (the ratio of spleen weight to body weight) 6 weeks after the start of the experiment. In A-H, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibition group administered ABT-317 at 30 mg / kg / day. In B-H, significance was tested using Student's t-test. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. The results of examining the therapeutic effect of long-term administration of a JAK1 inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. A shows the experimental procedure. B shows the results of measuring red blood cell (RBC) counts. C shows the results of measuring hemoglobin (HGB) concentrations. D shows the results of measuring hematocrit (HCT). E shows the results of measuring platelet (PLTC) counts. In A-E, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibition group administered 30 mg / kg / day of ABT-317. In B-E, significance was tested using Student's t-test. The results show the therapeutic effect of long-term administration of a JAK1 inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. A shows the experimental procedure. B shows an image of an EVG-stained lung tissue section.(C) shows the results of measuring the medial hyperplasia index of the pulmonary artery. In A-C, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1-inhibition group administered ABT-317 at 30 mg / kg / day. In C, significance was tested using Student's t-test. The results of examining the therapeutic effect of long-term administration of a JAK1 inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. (A) shows the experimental procedure. (B) shows the results of measuring the number of Th17 (CD4+, IL-17A+, IL-21+) cells by flow cytometry after gating on CD4+ T cells among live lung cells and CD45+ cells. (C) shows the results of measuring the number of Th17 cells in the spleen by flow cytometry. In A-C, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1-inhibition group administered ABT-317 at 30 mg / kg / day. Significance tests were performed using Student's t-test. The following figures show the results of examining the therapeutic effect of long-term administration of a JAK1 inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. (A) A diagram showing the experimental procedure. (B) Flow cytometry results for measuring the number of Th1 (CD4+, IFN-γ+) cells and Th2 (CD4+, IL-4+) cells after gating on CD4+ T cells among live lung cells and CD45+ cells. (C) Flow cytometry results for measuring the number of Th1 and Th2 cells in the spleen. In A-C, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1-inhibited group administered ABT-317 at 30 mg / kg / day. Significance tests were performed using Student's t-test. These figures show the results of examining the therapeutic effect of long-term administration of a JAK1 inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe collagen disease PAH. A is a diagram showing the experimental procedure. B is the result of measuring blood IgG1. C is the result of measuring blood IgG2a. D is the result of measuring blood IgG2b. E is the result of measuring blood IgA. F is the result of measuring blood IgM.In A–F, "Reg1f / f" indicates the control group, where Regnase-1 f / f mice were fed a powdered normal diet; "Vehicle" indicates the vehicle group; and "JAK1i" indicates the JAK1-inhibited group administered ABT-317 at 30 mg / kg / day. Significance tests were performed using one-way ANOVA and Bonferroni's multiple comparison method. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001 compared to the vehicle group; # indicates P<0.05, ## indicates P<0.01, and ### indicates P<0.001 compared to Reg1f / f. The results demonstrate the therapeutic effect of long-term administration of a JAK1 inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. A diagram illustrates the experimental procedure. B shows HE-stained images of liver and kidney tissue sections. "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibition group administered ABT-317 at 30 mg / kg / day. The results show the therapeutic effect of long-term administration of the JAK inhibitor tofacitinib using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. Figure A shows the experimental procedure. Figure B shows the results of body weight measurement over time. Figure C shows the results of mean arterial pressure (MAP) measurement 6 weeks after the start of the experiment. Figure D shows the results of right ventricular systolic pressure (RVSP) measurement 6 weeks after the start of the experiment. Figure E shows the results of the ratio of right ventricular systolic pressure to systemic blood pressure (RVSP / MAP) 6 weeks after the start of the experiment. Figure F shows the results of Fulton's coefficient (RV / LV+S) measurement 6 weeks after the start of the experiment. Figure G shows the results of splenomegaly (spleen weight to body weight ratio) measurement 6 weeks after the start of the experiment. In A to G, "Vehicle" indicates the vehicle group, and "JAKi" indicates the JAK inhibitor group administered tofacitinib at 30 mg / kg / day. In B to G, significance was tested using the Student t-test. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.The therapeutic effect of long-term administration of a JAK inhibitor was investigated using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. (A) A diagram showing the experimental procedure. (B) Red blood cell (RBC) count measurement results. (C) Hemoglobin concentration measurement results. (D) Hematocrit (HCT) measurement results. (E) Platelet (PLT) count measurement results. In A-E, "Vehicle" indicates the vehicle group, and "JAKi" indicates the JAK1-inhibited group administered tofacitinib at 30 mg / kg / day. In B-E, significance was tested using Student's t-test. The therapeutic effect of long-term administration of a JAK inhibitor was investigated using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. (A) A diagram showing the experimental procedure. (B) EVG-stained lung tissue sections. In A and B, "Vehicle" indicates the vehicle group, and "JAKi" indicates the JAK inhibitor group administered tofacitinib at 30 mg / kg / day. The results show the therapeutic effect of long-term administration of a JAK inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. A shows the experimental procedure. B shows the results of measuring the number of activated B cells (GC B cells) in the lung and spleen by flow cytometry. C shows the results of measuring plasma cells in the spleen and mesenteric lymph nodes (MLN) by flow cytometry. In A-C, "Vehicle" indicates the vehicle group, and "JAKi" indicates the JAK inhibitor group administered tofacitinib at 30 mg / kg / day. Significance tests were performed using Student's t-test. Significance tests were performed using Student's t-test. This figure shows the results of investigating the therapeutic effect of long-term administration of JAK1 or a JAK inhibitor with JAK1 inhibitory effects on severe connective tissue disease PAH using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. (A) A diagram showing the experimental procedure. (B) Western blotting results showing the expression levels of phosphorylated STAT3 and STAT3 in the lungs.(C) Western blotting results for quantification of phosphorylated STAT3 expression in the lungs. In A–C, "Reg1f / f" represents the control group (Regnase-1 f / f mice fed a powdered normal diet), "Vehicle" represents the vehicle group, "JAK1i" represents the JAK1-inhibited group administered ABT-317 at 30 mg / kg / day, and "JAKi" represents the JAK1-inhibited group administered tofacitinib at 30 mg / kg / day. In (C), significance was tested by one-way ANOVA using the Turkey-Kramer method. ** indicates P<0.01. The results demonstrate the therapeutic effect of long-term administration of a JAK1 inhibitor using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. (A) Schematic diagram of the experimental procedure. (B) Temporal changes in body weight. (C) Temporal changes in survival rate. In A to C, "Vehicle" indicates the vehicle group, and "JAK1i" indicates the JAK1 inhibition group administered 30 mg / kg / day of ABT-317. In B, significance was tested using two-way analysis of variance with the Bonferroni method. In C, significance was tested using the Log-rank (Mantel-Cox) test. ** indicates P<0.01, and *** indicates P<0.001.

[0013] 1. Definitions Unless otherwise specified, terms used herein have the meanings that are commonly understood by those skilled in the art of medicine, pharmacology, molecular biology, microbiology, organic chemistry, etc. When a term defined herein does not have the same meaning as commonly understood, the description in this specification takes precedence.

[0014] In the present disclosure, a "JAK1 inhibitor" refers to a component that suppresses signal transduction of the JAK-STAT pathway by inhibiting JAK (Janus kinase) 1. In addition, in the present disclosure, a "JAK1 selective inhibitor" refers to a JAK1 inhibitor that selectively acts on JAK1 in the JAK family and does not act on other JAKs.

[0015] In the present disclosure, "pulmonary hypertension (PH)" refers to a group of progressive diseases with poor prognosis that cause cardiac and pulmonary dysfunction due to elevated pulmonary artery blood pressure, specifically a condition in which the mean pulmonary artery pressure (PAP) measured at rest using right heart catheterization is 20 mmHg or higher. In one embodiment of the present disclosure, a diagnostic criterion for PH may be a mean pulmonary artery pressure of 25 mmHg or higher. According to the Nice Classification, PH is classified into the following groups: Group 1: pulmonary arterial hypertension (PAH); Group 1': pulmonary veno-occlusive disease (PVOD) and / or pulmonary capillary hemangiomatosis (PCH); Group 1'': persistent pulmonary hypertension of the newborn; Group 2: pulmonary hypertension associated with left heart disease; Group 3: pulmonary hypertension associated with lung disease and / or hypoxemia; Group 4: chronic thromboembolic pulmonary hypertension; and Group 5: pulmonary hypertension associated with an unknown multifactorial mechanism.

[0016] In the present disclosure, the severity of PH symptoms is classified according to the WHO Functional Classification of Pulmonary Hypertension, as shown in Table 1. In the present disclosure, "severe PH" refers to PH of grade III or IV according to the WHO Functional Classification of Pulmonary Hypertension.

[0017] In the present disclosure, "collagen vascular disease" refers to a systemic autoimmune disease whose primary lesion is in connective tissue and whose common pathological basis is an abnormal autoimmune response. Collagen vascular diseases include systemic lupus erythematosus, systemic sclerosis, polymyositis / dermatomyositis, Sjögren's syndrome, mixed connective tissue disease, antiphospholipid syndrome, Behçet's disease, etc., but the term "collagen vascular disease" in the present disclosure is not intended to be limited to these. Furthermore, in the present disclosure, "collagen vascular disease" also includes PH associated with collagen vascular disease. In the present disclosure, the severity of "PH associated with collagen vascular disease" is not limited to severe, and also includes PH classified as Class I or II according to the WHO Functional Classification of Pulmonary Hypertension.

[0018] In this disclosure, "prevention" refers to inhibiting the onset of or prolonging the time until onset of a disease or condition, and "treatment" refers to alleviating, ameliorating, or slowing the rate of progression of a disease or condition.

[0019] 2. Preventive or therapeutic drug for severe PH or collagen disease One embodiment of the present disclosure is a preventive or therapeutic drug for severe PH or collagen disease, which comprises a JAK1 inhibitor. The preventive or therapeutic drug of the present disclosure is described in detail below.

[0020] [Active ingredient] The preventive or therapeutic drug of the present disclosure uses a JAK1 inhibitor as an active ingredient. While no restrictive interpretation is desired regarding the mechanism of action of the use of a JAK1 inhibitor in the prevention or treatment of severe PH or collagen vascular disease, it is presumed that JAK1 inhibition suppresses the induction of phosphorylated STAT3 in the lung, thereby improving the pathological condition of severe PH or collagen vascular disease.

[0021] The JAK1 inhibitor used in the present disclosure may be a selective JAK1 inhibitor, or may be a JAK1 inhibitor that acts not only on JAK1 but also on other JAKs.

[0022] Examples of JAK1 selective inhibitors include ABT-317, upadacitinib (ABT-494, CAS: 1310726-60-3; an analogue of ABT-317), abrocitinib (PF-04965842, CAS: 1622902-68-4), filgotinib (GLPG0634, CAS: 1206161-97-8), itacitinib (INCB39110, CAS: 1334298-90-6), brepositinib (PF-06700841, CAS: 1883299-62-4), londamositinib (AZD-4604), Examples of such anti-cancer drugs include ivalmasitinib (SHR0302, CAS:1445987-21-2), povorcitinib (INCB054707, CAS:1637677-22-5), solcitinib (GSK2586184, CAS:1206163-45-2), and salts thereof.

[0023] Examples of JAK1 inhibitors that act on JAKs other than JAK1 include JAK Inhibitor I (CAS: 457081-03-7), tofacitinib (CP-690550, CAS: 477600-75-2), ruxolitinib (CAS: 941678-49-5), baricitinib (CAS: 1187594-09-7), delgocitinib (CAS: 1263774-59-9), oclacitinib (CAS: 1208319-26-9), momelotinib (CAS: 1056634-68-4), celdulatinib (PRT062070, PRT2070, CAS: 1198300-79-6), and peficitinib (ASP015K, JNJ-54781532, CAS: 944118-01-8), lestaurtinib (CAS: 111358-88-4), gandotinib (LY2784544, CAS: 1229236-86-5), BSK805 (CAS: 1942919-79-0), ZM39923 (CAS: 1021868-92-7), XL019 (CAS: 945755-56-6), lopsacitinib (PF-06826647, CAS: 2127109-84-4), SAR-20347 (CAS: 1450881-55-6), GDC046 (CAS: 1258292-64-6), JAK3-IN-6 (CAS: 1443235-95-7), decernotinib (VX-509, CAS: 944842-54-0), and salts thereof.

[0024] Further, other examples of JAK1 inhibitors that can be used in the present disclosure include at least one selected from the group consisting of pyrazolothiazole compounds, salts thereof, and solvates thereof shown in the following (1) to (135): (1) methyl [1-({6-[(2S)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (2) methyl [1-({6-[(2R)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (3) methyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (4) ethyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (5) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (6) N-(1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (7) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)acetamide, (8) N-(1-{[6-{[(2S)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (9) N-(1-{[6-{[(2S)-3,(3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)acetamide, (10) N-(1-{[6-{[(1S,2S)-2-(difluoromethoxy)cyclopentyl](methyl)amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (11) N-(1-{[6-{[(2S)-3,3-dimethylbutan-2-yl]amino}-5-methyl-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)acetamide, (12) N-(1-{[6-{[(2S)-3,3-dimethylbutan-2-yl]amino}-5-methyl-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (13) N-[1-({6-[({1-[(difluoromethoxy)methyl]cyclopropyl}methyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (14) N-[1-({6-[({1-[(difluoromethoxy)methyl]cyclobutyl}methyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (15) N-[1-({6-[({1-[(difluoromethoxy)methyl]cyclopentyl}methyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (16) N-[1-({6-[({4-[(difluoromethoxy)methyl]tetrahydro-2H-pyran-4-yl}methyl)amino]-2-(pyrazolo[5,1-b][1,(17) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-5-methyl-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (18) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-5-methyl-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)acetamide, (19) N-(1-{[6-{[(1S,2S)-2-(difluoromethoxy)cyclopentyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (20) N-[1-({6-[(2,2-dimethylpropyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (21) N-(1-{[6-{[(2S)-1-(difluoromethoxy)propan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (22) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-5-methyl-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)-2,2-difluoroacetamide, (23) N-(1-{[6-{[(1S,2S)-2-methoxycyclopentyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (24) [6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,(3) thiazol-7-yl)pyrimidin-4-yl](4-hydroxypiperidin-1-yl)methanone, (25) 1-fluoro-2-methylpropan-2-yl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (26) methyl (1-{[6-{[(2S)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (27) [6-{[(2S)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl](4-hydroxypiperidin-1-yl)methanone, (28) methyl (1-{[6-{[(2R)-3-methylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (29) methyl (1-{[6-{[(2S)-3-methylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (30) propan-2-yl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (31) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)benzamide, (32) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)pyridine-3-carboxamide, (33) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl)carbonyl}piperidin-4-yl)pyridine-3-carboxamide(3) N-(1-{[6-{[(1S)-1-cyclopropylethyl](methyl)amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)thiophene-2-carboxamide, (34) N-(1-{[6-{[(1S)-1-cyclopropylethyl](methyl)amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (35) 1-cyclopropyl-3-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)urea, (36) [6-{[(1S)-1-cyclopropylethyl](methyl)amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl][4-(pyrimidin-2-ylamino)piperidin-1-yl]methanone, (37) N-(1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (38) N-[1-({6-[(1-methylcyclopropyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (39) N-[1-({6-[(1-methoxy-2-methylpropan-2-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (40) methyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (41) ethyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (42) propan-2-yl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,(1-b) [1,3] thiazol-7-yl) pyrimidin-4-yl] carbonyl} piperidin-4-yl) carbamate, (43) N-(1-{[6-{[(1S)-1-cyclopropylethyl] (methyl) amino}-2-(pyrazolo[5,1-b][1,3] thiazol-7-yl) pyrimidin-4-yl] carbonyl} piperidin-4-yl) benzamide, (44) N-(1-{[6-(cyclopropylmethoxy)-2-(pyrazolo[5,1-b][1,3] thiazol-7-yl) pyrimidin-4-yl] carbonyl} piperidin-4-yl) cyclopropanecarboxamide, (45) N-[1-({6-[(3,3-dimethylbutan-2-yl)oxy]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (46) N-(1-{[6-(cyclobutyloxy)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (47) N-(1-{[6-(cyclopentyloxy)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (48) N-(1-{[6-{[(1R)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (49) N-[1-({6-[(1-hydroxy-2-methylpropan-2-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (50) [6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl][4-(1,2-oxazol-3-ylamino)piperidin-1-yl]methanone, (51) [6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl][4-(1,2-oxazol-3-ylamino)piperidin-1-yl]methanone(3-thiazol-2-ylamino)piperidin-1-yl]methanone, (52) [6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]{4-[(3-methyl-1,2-oxazol-5-yl)amino]piperidin-1-yl}methanone, (53) N-(1-{[6-{[(2S)-1-hydroxy-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (54) [6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl](4-hydroxypiperidin-1-yl)methanone, (55) methyl (1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (56) propan-2-yl (1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (57) methyl [1-({6-[(1-hydroxy-2-methylpropan-2-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (58) [6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl](4-hydroxypiperidin-1-yl)methanone, (59) methyl (1-{[6-{[(2S)-1-hydroxy-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (60) Methyl [1-({6-[(3,3-dimethylbutan-2-yl)oxy]-2-(pyrazolo[5,1-b][1,(3) thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (61) methyl (1-{[6-(cyclobutyloxy)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (62) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)methanesulfonamide, (63) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanesulfonamide, (64) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)ethanesulfonamide, (65) N-(1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)-2-methylpropanamide, (66) N-(1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)-2,2-difluoroacetamide, (67) ethyl (1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (68) [6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl][4-(pyridin-2-ylamino)piperidin-1-yl]methanone, (69) ethyl (1-{[6-{[(1R)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (70) [6-(tert-butylamino)-2-(pyrazolo[5,(1-b) [1,3]thiazol-7-yl)pyrimidin-4-yl][4-(1,3-thiazol-2-ylamino)piperidin-1-yl]methanone, (71) [6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl][4-(pyrimidin-2-ylamino)piperidin-1-yl]methanone, (72) methyl [1-({6-[(2-methylbutan-2-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (73) (4-hydroxypiperidin-1-yl)[6-(pentan-3-ylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]methanone, (74) propyl (1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (75) propyl (1-{[6-{[(1R)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (76) propyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (77) propyl(1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (78) propyl(1-{[6-{[(2S)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (79) Methyl [1-({6-[(2,2-dimethylpropyl)amino]-2-(pyrazolo[5,1-b][1,(3) thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (80) 2-methoxyethyl (1-{[6-{[(1R)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (81) 2-methoxyethyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (82) 2-methoxyethyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (83) 2-methoxyethyl (1-{[6-{[(2S)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (84) N-(1-{[6-{[(1R)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)-2-methoxyacetamide, (85) N-(1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)-2-methoxyacetamide, (86) Methyl [1-({6-[{[1-(methoxymethyl)cyclopropyl]methyl}(methyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (87) 2,2-difluoroethyl (1-{[6-{[(1R)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (88) 2,2-difluoroethyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (89) 2,2-difluoroethyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (90) 2,2-difluoroethyl (1-{[6-{[(2S)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (91) tert-butyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (92) 2-methoxyethyl (1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (93) 2-(dimethylamino)ethyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (94) 2,2,2-trifluoroethyl (1-{[6-{[(1R)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (95) 2,2,2-trifluoroethyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (96) 2-methoxy-N-(1-{[6-(pentan-3-ylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl)carbonyl}piperidin-4-yl)carbamate(97) N-(1-{[6-(2-ethylbutoxy)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (98) methyl (1-{[6-(pentan-3-yloxy)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (99) methyl (1-{[6-(pentan-3-ylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (100) N-[1-({6-[(2-ethylbutyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]cyclopropanecarboxamide, (101) N-(1-{[6-(pentan-3-yloxy)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (102) methyl [1-({6-[methyl(pentan-3-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (103) methyl(1-{[6-{[(2R)-3,3-dimethylbutan-2-yl](methyl)amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (104) N-(1-{[6-(2-ethylbutoxy)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)propanamide, (105) N-(1-{[6-{[(2R)-3,3-dimethylbutan-2-yl](methyl)amino}-2-(pyrazolo[5,1-b][1,(106) N-[1-({6-[methyl(pentan-3-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]propanamide, (107) [6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]{4-[(3-fluoropyridin-2-yl)amino]piperidin-1-yl}methanone, (108) N-[1-({6-[(2-methoxy-2-methylpropyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]propanamide, (109) (4-hydroxypiperidin-1-yl){6-[methyl(pentan-3-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}methanone, (110) N-(1-{[6-{[(2R)-3-methylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)propanamide, (111) N-(1-{[6-{[(2R)-3-methylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (112) N-(1-{[6-{[(2S)-3-methylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (113) N-(1-{[6-(pentan-3-ylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (114) N-[1-({6-[(1-methylcyclopropyl)amino]-2-(pyrazolo[5,1-b][1,(115) [6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]propanamide, (116) 2-methyl-N-[1-({6-[(2-methylbutan-2-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]propanamide, (117) N-[1-({6-[(2S)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]propanamide, (118) methyl [1-({6-[tert-butyl(methyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (119) cyclopropyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (120) cyclopropyl (1-{[6-(tert-butylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (121) 2,2-difluoro-N-(1-{[6-{[(2R)-3-methylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)acetamide, (122) 3-[1-({6-[(2,2-dimethylpropyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]-1,1-dimethylurea, (123) 3-(1-{[6-{[(1R)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,(124) propan-2-yl [1-({6-[(3-methyloxetan-3-yl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (125) methyl [1-({6-[(dicyclopropylmethyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (126) methyl (1-{[6-phenoxy-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (127) tert-butyl 4-{[6-({4-[(methoxycarbonyl)amino]piperidin-1-yl}carbonyl)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]amino}piperidine-1-carboxylate, (128) methyl (1-{[6-(piperidin-4-ylamino)-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (129) methyl [1-({6-[(1-cyanocyclopropyl)amino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (130) methyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-5-methoxy-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (131) methyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-5-methoxy-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (132) Methyl [1-({6-[(2S)-butan-2-ylamino]-5-methoxy-2-(pyrazolo[5,1-b][1,(133) methyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-5-ethoxy-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (134) methyl (1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-5-ethoxy-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (135) methyl (1-{[2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)-6-(pyridin-3-yloxy)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate.

[0025] The salts of the pyrazolothiazole compounds shown in (1) to (135) above are not particularly limited, provided that they are pharmaceutically acceptable. Examples include salts of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; and salts of organic acids such as acetic acid, citric acid, tartaric acid, maleic acid, succinic acid, fumaric acid, p-toluenesulfonic acid, benzenesulfonic acid, and methanesulfonic acid. The solvates of the pyrazolothiazole compounds shown in (1) to (135) or their salts are not particularly limited, provided that they are pharmaceutically acceptable. Examples include hydrates and ethanolates. Methods for producing the pyrazolothiazole compounds shown in (1) to (135), their salts, and their solvates are described, for example, in WO 2016 / 35814 and WO 2017 / 150477, and these compounds can be produced according to known synthetic methods.

[0026] Among these pyrazolothiazole compounds, preferred examples include the pyrazolothiazole compounds shown in (1) to (6), their salts, and their solvates. Specific examples of more preferred JAK1 inhibitors include the compounds shown in (1'), (2'), (3'), (4), (5), and (6) below. (1') methyl [1-({6-[(2S)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate tosylate monohydrate, (2') methyl [1-({6-[(2R)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate tosylate monohydrate, (3') methyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate tosylate monohydrate, (4) ethyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (5) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (6) N-(1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide.

[0027] These JAK1 inhibitors may be used alone or in combination of two or more. In a preferred embodiment of the prophylactic or therapeutic agent of the present disclosure, a JAK1 selective inhibitor is used as the JAK1 inhibitor. In another preferred embodiment of the prophylactic or therapeutic agent of the present disclosure, ABT-317 and tofacitinib are used as the JAK1 inhibitor.

[0028] The prophylactic or therapeutic agent of the present disclosure is provided in a desired dosage form by combining a JAK1 inhibitor with a pharmaceutically acceptable carrier, additive, etc. Examples of pharmaceutically acceptable carriers or additives include sterile water, physiological saline, stabilizers, excipients, antioxidants, buffers, preservatives, surfactants, chelating agents, binders, etc.

[0029] Dosage forms of the prophylactic or therapeutic agent of the present disclosure include, for example, capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated agents, pellets, troches, sublingual agents, chewable agents, buccal agents, pastes, syrups, suspensions, elixirs, emulsions, etc. The content of the JAK1 inhibitor in the prophylactic or therapeutic agent of the present disclosure may be appropriately determined depending on the dosage, dosage form, etc.

[0030] [Target Animal] The target animal to which the prophylactic or therapeutic agent of the present disclosure is administered is not particularly limited and may be, for example, a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, goat, sheep, pig, cow, or monkey, but is preferably a human.

[0031] [Target Disease] Severe PH In one embodiment of the prophylactic or therapeutic agent of the present disclosure, the target disease for prevention or treatment is severe PH. That is, when the prophylactic or therapeutic agent of the present disclosure is used for the prevention of severe PH, the target disease is PH patients with grade I or II according to the WHO Pulmonary Hypertension Functional Classification, and when the prophylactic or therapeutic agent of the present disclosure is used for the treatment of severe PH, the target disease is PH patients with grade III or IV according to the WHO Pulmonary Hypertension Functional Classification.

[0032] An example of severe PH to be prevented or treated by the prophylactic or therapeutic drug of the present disclosure is severe PH with a mean pulmonary artery pressure (PAP) of 40 mmHg or more. Another example of severe PH to be prevented or treated by the prophylactic or therapeutic drug of the present disclosure is severe PH with a cardiac index of 2.5 L / min / m 2 Less than or equal to 2.2 L / min / m 2Further, another example of severe PH that can be prevented or treated by the prophylactic or therapeutic drug of the present disclosure is severe PH in which the mean value of pulmonary artery pressure is 40 mmHg or more and the cardiac index is 2.5 L / min / m or less. 2 Less than or equal to 2.2 L / min / m 2 Severe PH that meets the following criteria:

[0033] In the prophylactic or therapeutic agents of the present disclosure, the clinical classification of severe PH to be prevented or treated is not particularly limited, and may be classified into any of Groups 1, 1', 1'', 2, 3, 4, and 5 of the Nice classification.

[0034] A suitable example of severe PH to be prevented or treated by the prophylactic or therapeutic drug of the present disclosure is severe PAH (Group 1 of the Nice Classification).

[0035] Other suitable examples of severe PH to be prevented or treated by the prophylactic or therapeutic agent of the present disclosure include severe PVOD and / or severe PCH (Nice Classification Group 1'). Since the pulmonary veins are the primary site of lesions in PVOD and / or PCH, dilatation of the pulmonary arteries may cause pulmonary edema, making it difficult to use conventional PAH treatments. However, another embodiment of the present disclosure uses JAK1 inhibition as its mechanism of action, and is therefore effective in preventing or treating severe PVOD and / or PCH.

[0036] Furthermore, another suitable example of severe PH to be prevented or treated by the prophylactic or therapeutic agent of the present disclosure is severe PH associated with pulmonary disease and / or hypoxemia (Nice Classification Group 3). In pulmonary hypertension associated with pulmonary disease and / or hypoxemia, pulmonary ventilation function is reduced, and treatment is performed to reduce the amount of blood sent to the lungs in order to improve pulmonary gas exchange. Therefore, dilating the pulmonary artery can cause an imbalance in the ventilation-perfusion ratio, potentially leading to worsening of respiratory status, making the use of conventional PAH therapeutic agents difficult in many cases. However, in another embodiment of the present disclosure, the mechanism of action is JAK1 inhibition, and therefore the agent is effective in preventing or treating severe PH associated with pulmonary disease and / or hypoxemia.

[0037] Further, other suitable examples of severe PH to be prevented or treated by the prophylactic or therapeutic drug of the present disclosure include severe chronic thromboembolic pulmonary hypertension (Nice Classification Group 4) and severe persistent pulmonary hypertension of the newborn (Nice Classification Group 1'').

[0038] The prophylactic or therapeutic agent of the present disclosure exhibits effects of suppressing systemic inflammatory conditions and anemia and thrombocytopenia, which are pathological conditions of systemic lupus erythematosus, and can also improve systemic conditions associated with collagen diseases. Therefore, in one embodiment of the prophylactic or therapeutic agent of the present disclosure, severe PH associated with collagen diseases can be prevented or treated. Severe PH associated with collagen diseases is not particularly limited, but includes severe PH associated with at least one collagen disease selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, polymyositis / dermatomyositis, Sjögren's syndrome, mixed connective tissue disease, antiphospholipid syndrome, and Behçet's disease. Among these, preferred examples include severe PH associated with systemic lupus erythematosus or systemic sclerosis. Clinical classifications of severe PH associated with collagen diseases include, for example, severe PAH, severe PVOD, and severe PCH, preferably severe PAH.

[0039] - Collagen Disease In another embodiment of the prophylactic or therapeutic agent of the present disclosure, the disease to be prevented or treated is a collagen disease. The type of collagen disease to be prevented or treated by the prophylactic or therapeutic agent of the present disclosure is not particularly limited, and examples include systemic lupus erythematosus, systemic sclerosis, polymyositis / dermatomyositis, Sjögren's syndrome, mixed connective tissue disease, antiphospholipid syndrome, and Behcet's disease. Among these collagen diseases, preferred examples include systemic lupus erythematosus and systemic sclerosis.

[0040] Furthermore, in the prophylactic or therapeutic drug of the present disclosure, the collagen disease to be prevented or treated may be PH associated with a collagen disease, such as PH associated with at least one collagen disease selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, polymyositis / dermatomyositis, Sjögren's syndrome, mixed connective tissue disease, antiphospholipid syndrome, and Behcet's disease; PH associated with systemic lupus erythematosus or systemic sclerosis;

[0041] The severity of the symptoms of PH associated with collagen vascular disease is not particularly limited and may be any of grades I to IV in the WHO Functional Classification of Pulmonary Hypertension, with a preferred example being severe (i.e., grades III or IV in the WHO Functional Classification of Pulmonary Hypertension). Severe PH associated with collagen vascular disease has traditionally had an extremely poor prognosis, and conventional preventive or therapeutic agents have had limitations. In contrast, the preventive or therapeutic agents disclosed herein have both the effect of improving systemic inflammatory conditions associated with collagen vascular disease and the effect of improving PH conditions, and therefore can exhibit excellent preventive or therapeutic effects even against severe PH associated with collagen vascular disease.

[0042] The clinical classification of PH associated with collagen disease is not particularly limited, and may be classified into any of Groups 1, 1', 1'', 2, 3, 4, and 5 of the Nice Classification. Preferred clinical classifications of PH associated with collagen disease include PAH, PVOD, and PCH, and more preferably PAH.

[0043] [Dosage and Administration] In the present disclosure, the method of administering the prophylactic or therapeutic agent is not particularly limited, and examples thereof include oral administration (intake), intravascular (intra-arterial or intravenous) injection, pulmonary administration (inhalation), continuous infusion, subcutaneous administration, intramuscular administration, enteral administration, intraperitoneal administration, topical administration, etc. A suitable example of the method of administering the prophylactic or therapeutic agent of the present disclosure is oral administration.

[0044] The dosage of the prophylactic or therapeutic agent of the present disclosure may be appropriately determined to provide an effective prophylactic or therapeutic amount depending on the age and body weight of the subject, the type and pathology of the target disease, the type of JAK1 inhibitor used, etc. For example, the daily adult dose of a JAK1 inhibitor may be appropriately set within the range of about 0.01 mg to about 5 g / adult, or about 1 mg to about 500 mg / adult. The prophylactic or therapeutic agent of the present disclosure may be administered once a day or in divided doses for one day only, or may be administered continuously with an interval of one day or several days between doses.

[0045] 3. Method for screening candidate substances that may be effective in preventing or treating severe PH or collagen disease Yet another embodiment of the present disclosure is a method for screening test substances for candidate substances that may be effective in preventing or treating severe PH or collagen disease, the screening method comprising the steps of: measuring the JAK1 inhibitory activity of the test substances; and selecting test substances that have been confirmed to have JAK1 inhibitory activity as the candidate substances.

[0046] The test substance is a substance to be confirmed for its preventive and / or therapeutic effect on severe pulmonary hypertension of Class III or IV according to the WHO Functional Classification of Pulmonary Hypertension, treatment-resistant pulmonary hypertension, or drug-induced pulmonary hypertension. Specific examples of the test substance include synthetic compounds, nucleic acids (e.g., antisense nucleic acids, cDNA, siRNA, etc.), peptides, proteins, organic compounds, inorganic compounds, cell extracts, cell culture supernatants, plant extracts, culture products, and mixtures thereof.

[0047] Measurement of JAK1 inhibitory activity can be performed by known techniques. Kits for measuring JAK1 inhibitory activity are commercially available, such as the "JAK1 (Janus Kinase 1) Assay Kit" (BPS Bioscience), and in the screening method of the present disclosure, JAK1 inhibitory activity may be measured using a commercially available kit. In measuring JAK1 inhibitory activity, it is desirable to use a component known to have JAK1 inhibitory activity as a positive control, and determine the presence or absence and degree of JAK1 inhibitory activity of the test substance by comparison with the positive control.

[0048] Test substances that demonstrate JAK1 inhibitory activity will be selected as candidate substances that may be used to prevent or treat severe PH or collagen diseases.

[0049] The types and clinical classifications of severe PH or collagen disease, which are the target diseases of the candidate substances, are as described in the section "2. Preventive or therapeutic drugs for severe PH or collagen disease" above.

[0050] Candidate substances selected by the screening method of the present disclosure can be further subjected to tests such as safety evaluations and clinical trials to determine their clinical effectiveness as drugs for preventing or treating severe PH or collagen diseases.

[0051] The present disclosure will be explained in more detail below by showing examples, but it should not be construed as being limited to these examples.

[0052] 1. Experimental Materials and Methods 1-1. Experimental Animals Experiments were performed using 6-week-old rats or 6- to 8-week-old mice. For rat experiments, Sprague-Dawley (SD) rats were purchased from Charles River Japan. For mouse experiments, C57BL / 6 / J mice (CLEA Japan) or Regnase-1 f / f; CD11cCre mice were used. For hypoxia, animals were continuously housed in a hypoxic chamber at 10% oxygen for 3 weeks, with the exception of opening the chamber for approximately 5 minutes twice weekly for cleaning. Hypoxic gas was continuously delivered to the hypoxic chamber at a flow rate of 7 L / min. All animals were maintained at 24 ± 1°C under a 12-hour light / dark cycle and provided with standard laboratory chow and water unless otherwise noted.

[0053] 1-2. Creation of a Severe PAH Rat Model The SuHx rat model (Abe et al., Circulation 121, 2747, 2010) was used as a severe PAH rat model. The SuHx rat model was created as previously reported. Six-week-old SD rats were subcutaneously administered 20 mg / kg of SU5416 (a VEGF receptor antagonist) and housed in a hypoxic chamber with 10% O2 for three weeks. They were then removed from the hypoxic chamber and housed under normoxic conditions for two or five weeks.

[0054] 1-3. Creation of a Moderate PAH Mouse Model The SuHx mouse model (Vitali et al. Pulm Circ. 4(4): 619-629, 2014) was used as a moderate PAH mouse model. The SuHx mouse model was created as previously reported. Eight-week-old C57BL / 6J mice were subcutaneously administered 20 mg / kg of SU5416 and housed in a hypoxic chamber with 10% O2 for 3 weeks. During the hypoxic challenge, 20 mg / kg of SU5416 was subcutaneously administered weekly.

[0055] 1-4. Generation of a Severe Connective Tissue Disease PAH Mouse Model For the severe connective tissue disease PAH mouse model, we used Regnase-1 knockout mice, Regnase-1 f / f; CD11cCre (Reg1CKO) mice (A. Yaku et al. Circulation. 46(13):1006-1022, 2022). The procedure for generating Reg1CKO mice is outlined below. First, we prepared Regnase-1 f / f mice, in which a floxed site was introduced into the ZC3H12A gene (encoding Regnase-1), and CD11-Cre mice, in which Cre, a DNA recombinase induced by the CD11c gene promoter, was introduced. Regnase-1 f / f mice were crossed with CD11cCre mice to generate Reg1f / w; CD11cCre mice. Next, Reg1CKO mice were generated by artificial insemination of Reg1f / w; CD11cCre mice and Regnase-1 f / f mice, followed by embryo transfer.

[0056] 1-5. Treatment experiment on PAH model animals using a JAK1 inhibitor ABT-317, a selective JAK1 inhibitor, was provided by Abbvie. ABT-317 was administered in the feed at 30 mg / kg / day. Tofacitinib (HY-40354, MedChemExpress) was also used as a JAK inhibitor with JAK1 inhibitory effects. Tofacitinib was administered in the feed at 30 mg / kg / day. The vehicle group was allowed to freely consume powdered diet (normal powdered diet).

[0057] 1-6. Hemodynamic Measurements Using a Right Heart Catheter Hemodynamic measurements using a right heart catheter in rats were performed as follows. First, anesthesia was induced in the rats with 3% inhaled isoflurane. After tracheotomy, anesthesia was maintained with 1.5-2% isoflurane inhalation while respiratory management was performed with a ventilator (VentElite; Harvard apparatus). Body temperature during the procedure was maintained at 37-38°C with a thermostatically controlled heat pad linked to a rectal temperature monitor. A tracheotomy was performed, and the rats were ventilated with a tidal volume of 8-10 μl / g at 60-80 breaths / min with an 18-gauge BD angiocath. TM A catheter was inserted into the right external jugular vein and advanced to the right ventricle to measure right ventricular pressure (RVP), and a polyethylene tube (PE50) was inserted into the right carotid artery to measure arterial pressure (AP).

[0058] Hemodynamic measurements using right heart catheterization in mice were performed as follows. First, mice were anesthetized with 1.5–2% isoflurane. During the procedure, body temperature was maintained at 37–38°C using a thermostatically controlled heat pad linked to a rectal temperature monitor. After tracheotomy, mice were ventilated with a mouse ventilator (VentElite; Harvard apparatus) at a tidal volume of 200–300 μL and 160–180 breaths / min. Measurements were performed under spontaneous breathing in Reg1CKO mice. Right ventricular pressure (RVP) was measured by inserting a polyethylene tube (SP-31) into the right external jugular vein and advancing it to the right ventricle. AP was measured by inserting a heat-stretched polyethylene tube (PE50) with a tapered tip into the right carotid artery. AP and RVP signals were detected by a pressure transducer (MLT0670; AD Instruments), relayed by a pressure amplifier (ML117; AD Instruments), continuously sampled by a Power Lab system (AD Instruments), and recorded on a computer using Chart software (AD Instruments). Heart rate was calculated from the peak systolic arterial pressure. Experiments were performed only when mean arterial pressure was 50 mmHg or higher and heart rate (HR) was between 200 and 400 beats / min (rats) or 350 and 600 beats / min (mice). HR below 200 beats / min (rats) or 350 beats / min (mice) was excluded from the measurements.

[0059] 1-7. Morphological Analysis. After hemodynamic measurements using a right heart catheter, animals were euthanized by exsanguination after blood collection from the inferior vena cava. After euthanasia, saline was perfused through the right heart catheter, and blood was removed by incising the left atrium. The right lung was ligated and excised for protein expression analysis and FACS analysis. The remaining left lung was collected for histological analysis. The trachea was perfused with 4% paraformaldehyde (PFA) and fixed under conditions that distended the airway. The excised lung samples were fixed in 4% PFA overnight at 4°C, then replaced with PBS, embedded in paraffin, and sectioned at 4 μm thickness. The atria, aorta, and pulmonary artery were removed from the heart, and the right ventricle (RV) was separated from the left ventricle (LV) and septum. Tissue weight was measured, and the RV / (LV + septum) ratio, calculated as the Fulton coefficient, was used as a marker for right ventricular hypertrophy. Morphological analysis of pulmonary vessels was performed using Elastica van Gieson (EVG) staining and hematoxylin-eosin staining. Morphological analysis of the heart, liver, and kidneys was performed using Masson trichrome staining and hematoxylin-eosin staining. Images of each section were captured using a NanoZoomer (Hamamatsu Photonics). Morphological analysis was performed using lung sections from randomly selected animals in each experimental group. Blood vessels with diameters of 30 to 100 μm were selected and evaluated, with more than 40 blood vessels selected from each experimental group. For vascular analysis, an Aperio ImageScope (Leica) was used to calculate the pulmonary artery medial thickening index ((1 - pulmonary artery internal diameter / pulmonary artery external diameter) x 100%) from measurements of the external and internal diameters of the pulmonary artery.

[0060] Western blot analysis. Frozen rat or mouse lungs were homogenized (disrupted and lysed) in lysis buffer (50 mM HEPES, 100 mM sodium fluoride, 2 mM sodium orthovanadate, 4 mM EDTA, 1% Tween-20, 0.1% SDS, protease inhibitor cocktail Complete (Roche Applied Science)) using a Polytron homogenizer. After centrifugation, the supernatant was subjected to SDS-PAGE electrophoresis using standard methods. Blots were developed using the ECL system (GE Healthcare).

[0061] Western blot analysis was performed using the following antibodies: anti-p-STAT3-Tyr705 antibody (Clone D3A7, Cell Signaling Technology), anti-STAT3 antibody (Clone 79D7, Cell Signaling Technology), anti-IL-1β antibody (sc-7884, Santa Cruz), and anti-IL-6 antibody (Clone D5W4V, Cell Signaling). Anti-β-actin antibody (Clone 13E5, Cell Signaling) was also used to detect endogenous expression controls.

[0062] 1-9. Flow cytometry analysis. Mouse lung tissue was minced into approximately 1 mm pieces and incubated at 37°C for 30 minutes in DMEM medium (Nacalai Tesque) containing collagenase (1 mg / mL, Wako), dispase (0.5 mg / mL, Roche), and DNase 1 (0.02 mg / mL, Roche). The resulting tissue suspension was then subjected to cell dispersion using gentleMACS (Miltenyi). The cells were passed through a 70 μm mesh and the pellet was resuspended in PBS-F (PBS containing 2% fetal bovine serum (FBS)) and washed with PBS-F. Mouse spleen and mesenteric lymph nodes were passed through a 70 μm mesh and the pellet was resuspended in PBS-F and washed with PBS-F.Cells were preincubated with CD16 / 32 (clone 93, BioLegend) to block Fcγ receptors, and then stained for 30 minutes on ice with the following surface antigen antibodies: FITC-conjugated CD4 (clone RM4-5, BioLegend), FITC-conjugated CD95 (clone SA367H8, BioLegend), FITC-conjugated CD38 (clone 90, BioLegend), APC-conjugated CD279 (clone RMP1-30, BioLegend), APC-conjugated CD19 (clone 6D5, BioLegend), PE-conjugated CXCR5 (clone SPRCL5, Invitrogen), PE-conjugated GL-7 (clone GL7, BioLegend), and PE-conjugated CD267 (clone 8F10, BioLegend). BioLegend), PE / Cy7-conjugated B220 (clone RA3-6B2, BioLegend), PE / Cy7-conjugated CD62L (clone MEL-14, BioLegend), Alexa Fluor 647-conjugated CD11b (clone M1 / 70, BioLegend), APC / Cy7-conjugated CD44 (clone IM7, BioLegend), APC / Cy7-conjugated CD45 (clone 30-F11, BioLegend), APC / Cy7-conjugated CD19 (clone 6D5, BioLegend), BV421-conjugated B220 (clone RA3-6B2, BioLegend), BV421-conjugated CD3ε (clone 145-2C11, BioLegend), BV421-conjugated CD27 (clone LG.3A10, BioLegend), BV510-conjugated CD45 (clone 30-F11, BioLegend), BV510-conjugated CD138 (clone 281-2, BioLegend).Dead cells were removed by staining with 7-amino-actinomycin D (7-AAD, BioLegend).

[0063] For intracellular cytokine staining, cells were cultured for 4 hours at 37°C in IMDM medium (Nacalai Tesque) containing phorbol myristate acetate (0.5 μg / mL, Sigma-Aldrich), ionomycin (1 μg / mL, Sigma-Aldrich), and brefeldin A (20 μg / mL, Sigma-Aldrich). To remove dead cells, cells were stained with Zombie aqua (BioLegend) and then surface stained for CD4. Cells were then fixed with Fixation Buffer (Biolegend) and permeabilized with Intracellular Staining Permeabilization Wash Buffer (Biolegend). After washing, cells were stained with the following antibodies for 30 minutes on ice: PE-conjugated IFN-γ (BioLegend), APC-conjugated IL-4 (BioLegend), APC-conjugated IL-17A (eBioscience), and PE-conjugated IL-21 (eBioscience). Cells were analyzed using FACSverse (BD Biosciences) and FlowJo software (BD Biosciences).

[0064] Analysis of immunoglobulin levels in mouse plasma samples. Mouse immunoglobulin isotypes were measured using a highly sensitive immunoassay system based on electrochemiluminescence (MESO QuickPlex SQ 120, Meso Scale Discover: MSD). Mouse immunoglobulin levels were quantified from 10,000-fold diluted mouse plasma using the Mouse Isotyping Panel Kit (K15183B, MSD).

[0065] 1-11. Statistical analysis All data are shown as mean ± standard error. Significant differences between multiple groups were tested using one-way ANOVA and the Turkey-Kramer method. Differences between two groups were analyzed using the Student t-test. A p value of less than 0.05 was considered statistically significant. * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0066] 2. Experimental Results 2-1. Preventive Effect of JAK1 Inhibitors on Severe PAH in a Rat Model. The preventive effect of JAK1 inhibitors on severe PAH was examined using a SuHx rat model, which develops severe PAH. Severe PAH was induced in rats by administering SU5416 (0 wk) to the rats and then housing them under hypoxic conditions for 3 weeks followed by normoxic conditions for 2 weeks. The JAK1 inhibitor group (n = 7-9) received the JAK1 inhibitor ABT-317 (3, 10, or 30 mg / kg / day) in the diet for 3 weeks after SU5416 administration (0 wk), followed by a powdered regular diet for the next 2 weeks (Figure 1A). The vehicle group (n = 14) received a powdered regular diet throughout the entire 5-week period, starting with SU5416 administration (0 wk) (Figure 1A). Right ventricular systolic pressure was measured 5 weeks (5w) after SU5416 administration, and the rats were euthanized, the hearts and lungs were excised, and the tissue weights were measured and then subjected to tissue analysis.

[0067] Compared with the vehicle group, the JAK1 inhibitor group showed dose-dependent suppression of right ventricular systolic pressure, Fulton's coefficient, and pulmonary arterial medial thickness index. In particular, the JAK1 inhibitor group treated with 30 mg / kg / day of ABT-317 demonstrated significant suppression of pulmonary hypertension pathology (Fig. 1B–E). Western blotting analysis of lungs 5 ​​weeks after SU5416 administration revealed a significant increase in the protein expression of the B cell marker CD79b in the vehicle group compared with untreated control rats housed under normoxic conditions, whereas this increase was significantly suppressed in the JAK1 inhibitor group (Fig. 2B, C). Furthermore, the vehicle group showed a significant increase in CD79b-positive cells around pulmonary vessels 5 weeks after the start of the experiment, whereas this increase was suppressed in the JAK1 inhibitor group (Fig. 2D). Furthermore, Western blotting analysis of lungs 5 ​​weeks after SU5416 administration showed that the vehicle group had significantly increased phosphorylated STAT3 protein expression compared with untreated control rats housed under normoxic conditions, whereas this increase was significantly suppressed in the JAK1 inhibitor group (Fig. 3B, C). These results confirmed that JAK1 inhibitors are effective in preventing the onset of severe PAH.

[0068] 2-2. Therapeutic Effect of JAK1 Inhibitors on Severe PAH in a Rat Model. The therapeutic effect of JAK1 inhibitors on severe PAH was examined using a SuHx rat model with severe PAH. Rats were treated with SU5416 (0w) and housed under hypoxic conditions for 3 weeks, followed by normoxic conditions for 2 weeks to induce severe PAH, followed by an additional 3 weeks under normoxic conditions. The JAK1 inhibitor group (n = 5) received a powdered normal diet for 5 weeks after SU5416 administration (0w), followed by ABT-317 (30 mg / kg / day) in the diet for the next 3 weeks (Figure 4A). The vehicle group (n = 6) received a powdered normal diet for the entire 8-week period after SU5416 administration (0w) (Figure 4A). Right ventricular systolic pressure was measured 8 weeks after SU5416 administration (8w). The rats were euthanized, and the hearts and lungs were removed for tissue weight measurement and histological analysis.

[0069] The JAK1 inhibitor group showed significant reductions in right ventricular systolic pressure, Fulton's coefficient, pulmonary arterial medial thickness index, and splenomegaly (spleen weight to body weight ratio) compared with the vehicle group, indicating a significant suppression of PH pathology (Fig. 4B–F). Western blotting analysis of lungs 8 weeks after SU5416 administration revealed that CD79b protein expression was significantly elevated in the vehicle group compared with untreated control rats housed under normoxia, whereas this increase was significantly suppressed in the JAK1 inhibitor group (Fig. 5B, C). Furthermore, Western blotting analysis of lungs 8 weeks after SU5416 administration revealed that phosphorylated STAT3 protein expression was significantly elevated in the vehicle group compared with untreated control rats housed under normoxia, whereas this increase was significantly suppressed in the JAK1 inhibitor group (Fig. 6B, C). These results confirm that JAK1 inhibitors are effective in treating severe PAH.

[0070] 2-3. Preventive Effect of JAK1 Inhibitors on Moderate PAH in a Mouse Model. The preventive effect of JAK1 inhibitors on moderate PAH was examined using a SuHx mouse model, which develops moderate PAH. Mice were induced with moderate PAH by administering SU5416 (0 wk) and then housing them under hypoxic conditions for 3 weeks. The JAK1 inhibitor group (n = 6) received dietary ABT-317 (30 mg / kg / day) throughout the entire 3-week period, starting from the time of SU5416 administration (0 wk) (Fig. 7A). The vehicle group (n = 6) received a powdered normal diet throughout the entire 3-week period, starting from the time of SU5416 administration (0 wk) (Fig. 7A). Right ventricular systolic pressure was measured 3 weeks after SU5416 administration (3 wk). Mice were euthanized, and the hearts and lungs were removed. Their tissue weights were measured and subjected to histological analysis.

[0071] Compared with the vehicle group, the JAK1 inhibitor group had significantly lower right ventricular systolic pressure, Fulton's coefficient, pulmonary arterial medial thickness index, and splenomegaly (spleen weight to body weight ratio), demonstrating a significant suppression of pulmonary hypertension pathology (Fig. 7B-F). These results demonstrate that JAK1 inhibitors are effective in suppressing the onset of PAH in both rats and mice, confirming that JAK1 inhibitors are effective in preventing or ameliorating PAH pathology regardless of species.

[0072] 2-4. Therapeutic effect of JAK1 inhibitors on a mouse model of severe connective tissue disease PAH Regnase-1 (a protein encoded by the Zc3h12a gene) is an RNA-binding protein that degrades the mRNA of inflammatory cytokines such as IL-6. It has been reported that mice with systemic Regnase-1 knockout exhibit an autoimmune disease phenotype and exhibit inflammation in the lungs (K. Matsuhisa et al., Nature 2009. 458, 1185-90). Reg1f / f; CD11cCre (Reg1CKO) mice are a conditional knockout of Regnase-1 in which Regnase-1 is lacking in innate immune cells (myeloid cells including macrophages and dendritic cells). It has been reported that these mice not only spontaneously develop autoimmune diseases, but also develop smooth muscle hyperplasia and plexiform lesions in the pulmonary arteries, resulting in the pathology of severe collagen vascular disease-related PAH (A. Yaku et al. Circulation. 46(13):1006-1022, 2022).

[0073] In this study, we investigated the therapeutic efficacy of a JAK1 inhibitor in severe connective tissue disease PAH using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. In the JAK1 inhibition group (n = 4–8), 7-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were administered ABT-317 (30 mg / kg / day) in their diet for 3 weeks (Fig. 8A). In the vehicle group (n = 5–8), 7-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed a powdered normal diet for 3 weeks (Fig. 8A). Right ventricular systolic pressure was measured 3 weeks after the start of the experiment (3w), and the mice were euthanized. The hearts and lungs were excised, tissue weights were measured, and the tissues were subjected to histological analysis.

[0074] Compared with the vehicle group, the JAK1 inhibitor group showed a tendency toward suppression of right ventricular systolic pressure, and significant decreases in Fulton's coefficient, pulmonary arterial media thickness index, and splenomegaly (spleen weight to body weight ratio) were observed (Fig. 8B, C, E, F). EVG staining of lung tissue revealed significant inflammatory cell infiltration throughout the lung sections, particularly in the trachea and perivascular areas, in the vehicle group, whereas inflammatory cell infiltration was significantly suppressed in the JAK1 inhibitor group (Fig. 8D). Furthermore, flow cytometric analysis of immune cells in the lung, spleen, and mesenteric lymph nodes (MLNs) revealed significant decreases in activated B cells (GC B cells) and plasma cells in the JAK1 inhibitor group compared with the vehicle group (Fig. 9B–E). Furthermore, Western blotting analysis of lungs from Reg1CKO mice at 10 weeks of age revealed that the vehicle group exhibited significant induction of phosphorylated STAT3 (pSTAT3), which indicates activation of inflammatory cytokine signaling, whereas the JAK1 inhibition group significantly suppressed the induction of phosphorylated STAT3 (Fig. 10B, C). Furthermore, the JAK1 inhibition group also significantly suppressed the increase in IL-6 and IL-1β protein expression (Fig. 10B, C). These results confirm that JAK1 inhibitors are effective in treating severe connective tissue disease PAH.

[0075] 2-5. Therapeutic Effects of Long-Term Administration of a JAK1 Inhibitor in a Severe Connective Tissue Disease-Induced PAH Mouse Model. We investigated the suppression of PAH pathology by long-term administration of a JAK1 inhibitor. In the JAK1 inhibition group (n = 6), 6-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed ABT-317 (30 mg / kg / day) in their diet for 6 weeks (Fig. 11A). In the vehicle group (n = 6), 6-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed a powdered normal diet for 6 weeks (Fig. 11A). Right ventricular systolic pressure was measured 6 weeks after the start of the experiment (6 weeks). Mice were euthanized, and the hearts and lungs were excised, weighed, and subjected to histological analysis.

[0076] Six weeks after the start of the experiment, mice in the vehicle group showed signs of sagging fur, significant weight loss, and a hunched posture, whereas mice in the JAK1 inhibition group showed healthy fur and were active and active. Furthermore, the JAK1 inhibition group showed a significantly increased survival rate and significantly reduced weight loss compared to the vehicle group (Fig. 11B, C).

[0077] Six weeks after the start of the experiment, systemic blood pressure was significantly reduced in the vehicle group (Fig. 11D). In contrast, systemic blood pressure normalized and right ventricular systolic pressure appeared to increase in the JAK1 inhibition group (Fig. 11D, E). However, when comparing the ratio of right ventricular systolic pressure to systemic blood pressure, the JAK1 inhibition group showed a significant decrease compared with the vehicle group (Fig. 11F). Reflecting this, the JAK1 inhibition group also showed a tendency toward improvement in right heart hypertrophy (Fig. 11G). Furthermore, the JAK1 inhibition group also showed a significant improvement in splenomegaly (spleen weight to body weight ratio) (Fig. 11H).

[0078] Furthermore, 6 weeks after the start of the experiment, the vehicle group showed decreased red blood cell counts, platelet counts, hemoglobin concentration, and hematocrit values, indicating anemia and thrombocytopenia, whereas the JAK1 inhibition group showed significant improvement in anemia and thrombocytopenia (Figure 12B-E).

[0079] Furthermore, EVG staining of lung tissue revealed significant inflammatory cell infiltration throughout the lung sections, particularly in the trachea and perivascular areas, in the vehicle group, whereas inflammatory cell infiltration was significantly suppressed in the JAK1 inhibition group (Fig. 13B). The medial hyperplasia index of the pulmonary artery was also significantly reduced in the JAK1 inhibition group (Fig. 13C). Furthermore, flow cytometry analysis of immune cells in the lung, spleen, and mesenteric lymph nodes (MLNs) revealed significant decreases in activated B cells (GC B cells) and plasma cells in the JAK1 inhibition group compared with the vehicle group (Fig. 14B, C). Furthermore, the vehicle group exhibited a significant increase in Th1 and Th17 cells, which are helper T cells involved in tissue inflammation, whereas the JAK1 inhibition group significantly suppressed these increases (Fig. 15B, C).

[0080] Furthermore, blood immunoglobulin levels were significantly elevated in the vehicle group, whereas blood levels of IgG1, IgG2a, IgG2b, and IgA were significantly decreased in the JAK1 inhibition group (Fig. 16B–E). Furthermore, significant inflammatory cell infiltration was observed in the liver perisinusoids in the vehicle group, whereas this infiltration was significantly suppressed in the JAK1 inhibition group (Fig. 17B). Furthermore, abnormalities in glomerular structure and immune cell infiltration were observed in the kidney in the vehicle group, whereas almost no abnormal glomeruli were observed in the JAK1 inhibition group (Fig. 17B).

[0081] These results demonstrate that administering a JAK1 inhibitor to Reg1CKO mice improves anemia and thrombocytopenia (common pathologies of systemic lupus erythematosus) and also suppresses the systemic inflammation associated with collagen diseases. These findings demonstrate that JAK1 inhibitors are effective in treating not only severe PH but also collagen diseases.

[0082] Furthermore, we investigated the long-term suppression of PAH pathology using tofacitinib as a JAK1 inhibitor. In the JAK inhibitor group (n = 5), 6-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed tofacitinib (30 mg / kg / day) in their diet for 6 weeks (Fig. 18A). In the vehicle group (n = 5), 6-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed a powdered normal diet for 6 weeks (Fig. 18A). Right ventricular systolic pressure was measured 6 weeks after the start of the experiment (6 weeks). Mice were euthanized, and the hearts and lungs were excised, weighed, and subjected to histological analysis.

[0083] Six weeks after the start of the experiment, mice in the vehicle group showed signs of sagging fur, significant weight loss, and a hunched posture, whereas mice in the JAK inhibitor group showed healthy fur and were active and active. Furthermore, weight loss was significantly suppressed in the JAK inhibitor group compared to the vehicle group (Fig. 18B).

[0084] Six weeks after the start of the experiment, systemic blood pressure was significantly reduced in the vehicle group (Fig. 18C). In contrast, systemic blood pressure returned to normal and right ventricular systolic pressure tended to decrease in the JAK inhibition group (Fig. 18C, D). The ratio of right ventricular systolic pressure to systemic blood pressure was significantly reduced in the JAK inhibition group compared with the vehicle group (Fig. 18E). Reflecting this, right heart hypertrophy was significantly suppressed in the JAK inhibition group (Fig. 18F). Furthermore, splenomegaly (spleen weight to body weight ratio) was significantly improved in the JAK inhibition group (Fig. 18G).

[0085] Furthermore, 6 weeks after the start of the experiment, the vehicle group showed decreased red blood cell counts, hemoglobin concentrations, and hematocrit values, indicating anemia, whereas the JAK1 inhibition group showed significant improvement in anemia and thrombocytopenia (Figure 19B-E).

[0086] Furthermore, EVG staining of lung tissue revealed significant inflammatory cell infiltration throughout the lung sections, particularly in the trachea and perivascular areas, in the vehicle group, whereas inflammatory cell infiltration was significantly suppressed in the JAK inhibition group (Fig. 21B). Medial thickening of the pulmonary artery and obstructive vascular remodeling were also reduced in the JAK inhibition group (Fig. 21B). Furthermore, flow cytometric analysis of immune cells in the lung, spleen, and mesenteric lymph nodes (MLNs) revealed significant decreases in activated B cells (GC B cells) and plasma cells in the JAK inhibition group compared with the vehicle group (Fig. 21B,C).

[0087] 2-6. Mechanism of suppression of PH pathology by JAK inhibitors In the experiment described above in "2-5. Therapeutic effect of long-term administration of a JAK1 inhibitor on a severe connective tissue disease PAH mouse model," we analyzed the expression levels of phosphorylated STAT3 and STAT3 in the lungs of Reg1CKO mice at 12 weeks of age by Western blotting. While the vehicle group showed significant induction of phosphorylated STAT3 (pSTAT3), which indicates activation of inflammatory cytokine signaling, the JAK1-inhibited group treated with ABT-317 showed significant suppression of phosphorylated STAT3 (Figure 22B, C). Furthermore, the JAK1-inhibited group treated with tofacitinib also showed a tendency for suppression of phosphorylated STAT3 compared to the vehicle group.

[0088] Previous studies of JAK knockdown in cancer cells have reported that only knockdown of JAK1 inhibited the induction of phosphorylated STAT3 (Song et al. Mol Cancer Ther; 10(3); 481-94, 2011). Based on this previous study and the finding that administration of ABT-317 or tofacitinib inhibited the induction of phosphorylated STAT3 in the lungs of a mouse model of severe connective tissue disease PAH, it is reasonably inferred that the therapeutic effects of ABT-317 or tofacitinib on severe connective tissue disease PAH are due to the inhibition of phosphorylated STAT3 induction in the lungs by JAK1 inhibition. Therefore, drugs other than ABT-317 and tofacitinib that can inhibit JAK1 may also be able to suppress the pathology of severe PH or connective tissue disease.

[0089] 2-7. Effect of Long-Term Administration of a JAK1 Inhibitor on Survival in a Mouse Model of Severe Connective Tissue Disease PAH We investigated the effect of long-term administration of a JAK1 inhibitor on survival in Reg1CKO mice. In the JAK1 inhibition group (n = 15), 6-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were administered ABT-317 (30 mg / kg / day) in their diet for 14 weeks. In the vehicle group (n = 15), 6-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed a powdered normal diet for 14 weeks.

[0090] Four weeks after the start of the experiment, mice in the vehicle group began to lose weight, but the JAK1 inhibition group prevented this loss and showed a significant weight gain compared to the vehicle group from 12 weeks of age (Fig. 23B).Furthermore, the JAK1 inhibition group showed a significantly increased survival rate compared to the vehicle group (Fig. 23C).

[0091] 3. Experimental Examples The following describes experiments to verify the preventive or therapeutic effects of each compound shown in Table 2 on severe PH and collagen diseases.

[0092] 3-1. Preventive Effects of the Compounds Shown in Table 2 on Severe PAH in a Rat Model The following experiment was conducted to verify the preventive effects of JAK1 inhibitors (pyrazolothiazole compounds A–F) on severe PAH in a SuHx rat model, which develops severe PAH. Severe PAH was induced in rats by administering SU5416 (0 wt.) to rats and housing them under hypoxic conditions for three weeks, followed by normoxic conditions for two weeks. In the JAK1 inhibitor group, rats were administered pyrazolothiazole compounds A, B, C, D, E, F, upadacinib, itacitinib, or baricitinib (0.01 mg / kg / day, 0.1 mg / kg / day, 1 mg / kg / day, 10 mg / kg / day, or 100 mg / kg / day) in their diet for three weeks after SU5416 administration (0 wt.), followed by a powdered normal diet for the next two weeks. The vehicle group was given powdered normal chow for the entire 5-week period starting from SU5416 administration (0w). Five weeks after SU5416 administration (5w), right ventricular systolic pressure was measured, and the rats were euthanized, their hearts and lungs were removed, and their tissue weights were measured before being subjected to histological analysis.

[0093] Comparison between the JAK1 inhibition group and the vehicle group confirmed that administration of each compound listed in Table 2 suppressed right ventricular systolic pressure, Fulton's coefficient, and pulmonary artery medial thickening index, thereby suppressing the pathology of pulmonary hypertension. Furthermore, tissue analysis confirmed that the JAK1 inhibition group administered each compound listed in Table 2 suppressed the increase in CD79b-positive cells around pulmonary blood vessels 5 weeks after the start of the experiment. Furthermore, Western blotting analysis of the lungs 5 ​​weeks after SU5416 administration confirmed that the increase in phosphorylated STAT3 protein expression was suppressed in the JAK1 inhibition group administered each compound listed in Table 2. Thus, these experimental results confirm that each compound listed in Table 2 is effective in suppressing the onset of severe PAH.

[0094] 3-2. Therapeutic Effects of the Compounds Shown in Table 2 on a Severe PAH Rat Model The following experiment was conducted to evaluate the therapeutic effects of JAK1 inhibitors (pyrazolothiazole compounds A–F) on severe PAH using a SuHx rat model with severe PAH. Rats were administered SU5416 (0 wk) and housed under hypoxic conditions for 3 weeks, followed by normoxic conditions for 2 weeks to induce severe PAH, followed by an additional 3 weeks under normoxic conditions. The JAK1 inhibitor group received a powdered normal diet for 5 weeks after SU5416 administration (0 wk), followed by a 3-week diet containing pyrazolothiazole compounds A, B, C, D, E, F, upadacinib, itacitinib, or baricitinib (0.01 mg / kg / day, 0.1 mg / kg / day, 1 mg / kg / day, 10 mg / kg / day, or 100 mg / kg / day). The vehicle group was given a powdered normal diet for the entire 8-week period starting from SU5416 administration (0w). Right ventricular systolic pressure was measured 8 weeks after SU5416 administration (8w), and the rats were euthanized, their hearts and lungs were removed, and their tissue weights were measured before being subjected to histological analysis.

[0095] Comparison between the JAK1 inhibition group and the vehicle group confirmed that administration of each compound listed in Table 2 reduced right ventricular systolic pressure, Fulton's coefficient, pulmonary artery medial thickening index, and splenomegaly, thereby suppressing PH pathology. Analysis of lung tissue by Western blotting 8 weeks after SU5416 administration confirmed that the JAK1 inhibition group administered each compound listed in Table 2 suppressed the increase in CD79b protein expression and also suppressed phosphorylated STAT3 protein expression. These experimental results confirm that each compound listed in Table 2 is effective in treating severe PAH.

[0096] 3-3. Therapeutic Effect of Each Compound in Table 2 on Severe Connective Tissue Disease PAH Mouse Model. The following experiment was conducted to evaluate the therapeutic effect of JAK1 inhibitors (each compound listed in Table 2) on severe connective tissue disease PAH using Reg1f / f; CD11cCre (Reg1CKO) mice, a mouse model of severe connective tissue disease PAH. In the JAK1 inhibitor group, 7-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed pyrazolothiazole compounds A, B, C, D, E, F, upadacinib, itacitinib, or baricitinib (0.01 mg / kg / day, 0.1 mg / kg / day, 1 mg / kg / day, 10 mg / kg / day, or 100 mg / kg / day) in their diet for 3 weeks. In the vehicle group, 7-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed a powdered normal diet for 3 weeks. Three weeks (3w) after the start of the experiment, right ventricular systolic pressure is measured, the mice are euthanized, the hearts and lungs are excised, the tissue weights are measured, and the tissues are subjected to tissue analysis.

[0097] Comparison of the JAK1 inhibition group and the vehicle group confirmed that administration of each compound listed in Table 2 reduced right ventricular systolic pressure, Fulton's coefficient, pulmonary arterial media thickness index, and splenomegaly (spleen weight to body weight ratio). EVG staining of the lungs confirmed that inflammatory cell infiltration was suppressed in the JAK1 inhibition group administered each compound listed in Table 2. Furthermore, flow cytometry analysis of immune cells in the lung, spleen, and mesenteric lymph node (MLN) confirmed that the JAK1 inhibition group administered each compound listed in Table 2 showed a significant reduction in activated B cells (GC B cells) and plasma cells compared to the vehicle group. Furthermore, Western blotting analysis of lungs from 10-week-old Reg1CKO mice confirmed that the induction of phosphorylated STAT3 was suppressed in the JAK1 inhibition group administered each compound listed in Table 2. These experimental results confirm that each compound listed in Table 2 is effective in treating severe collagen vascular disease PAH.

[0098] 3-4. Therapeutic Effects of Long-Term Administration of Each Compound in Table 2 in a Mouse Model of Severe Connective Tissue Disease PAH The following experiment was conducted to verify the suppression of PAH pathology by long-term administration of a JAK1 inhibitor. In the JAK1 inhibitor group, 6-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed pyrazolothiazole compounds A, B, C, D, E, F, upadacinib, itacitinib, or baricitinib (0.01 mg / kg / day, 0.1 mg / kg / day, 1 mg / kg / day, 10 mg / kg / day, or 100 mg / kg / day) in their diet for 6 weeks. In the vehicle group, 6-week-old Reg1f / f; CD11cCre (Reg1CKO) mice were fed a powdered normal diet for 6 weeks. Six weeks (6w) after the start of the experiment, right ventricular systolic pressure is measured, the mice are euthanized, the hearts and lungs are excised, the tissue weights are measured, and the tissues are subjected to tissue analysis.

[0099] Comparison between the JAK1 inhibition group and the vehicle group confirmed that administration of each compound listed in Table 2 normalized systemic blood pressure, reduced the ratio of right ventricular systolic pressure to systemic blood pressure, improved right ventricular hypertrophy, and improved splenomegaly (ratio of spleen weight to body weight). Furthermore, administration of each compound listed in Table 2 confirmed improvement of anemia and thrombocytopenia. EVG staining images of the lung confirmed that inflammatory cell infiltration and medial hyperplasia index of the pulmonary artery were suppressed in the JAK1 inhibition group administered with each compound listed in Table 2. Furthermore, analysis of immune cells by flow cytometry in the lung, spleen, and mesenteric lymph node (MLN) confirmed that activated B cells (GC B cells) and plasma cells were reduced, and the increase in Th1 cells and Th17 cells was also suppressed in the JAK1 inhibition group administered with each compound listed in Table 2. Furthermore, it was confirmed that the blood concentrations of IgG1, IgG2a, IgG2b, and IgA were reduced and no abnormalities in the glomerular structure of the kidney were observed in the JAK1 inhibition group administered with each compound shown in Table 2. In other words, these experimental results confirm that each compound shown in Table 2 is effective in treating severe PH and collagen diseases.

[0100] The present disclosure is not limited in any way to the description of the embodiments and examples of the invention. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of the documents and the like shown in this specification are hereby incorporated by reference in their entirety.

Claims

1. Drugs for the prevention or treatment of severe pulmonary hypertension or collagen diseases, including JAK1 inhibitors.

2. The preventive or therapeutic agent according to claim 1, wherein the JAK1 inhibitor is a selective JAK1 inhibitor.

3. The prophylactic or therapeutic drug according to claim 2, wherein the JAK1 selective inhibitor is at least one selected from the group consisting of ABT-317, upadacitinib, abrocitinib, filgotinib, itacitinib, brepositinib, londamositinib, GDC-4379, TUL-01101, ivalmacitinib, povorcitinib, solcitinib, and salts thereof.

4. The preventive or therapeutic drug according to claim 1, wherein the JAK1 inhibitor is at least one selected from the group consisting of tofacitinib, itacitinib, baricitinib, and salts thereof.

5. The prophylactic or therapeutic drug according to claim 1, wherein the JAK1 inhibitor is at least one selected from the group consisting of pyrazolothiazole compounds shown in the following (1) to (6), their salts, and solvates: (1) methyl [1-({6-[(2S)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (2) methyl [1-({6-[(2R)-butan-2-ylamino]-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl}carbonyl)piperidin-4-yl]carbamate, (3) methyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (4) ethyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate, (5) N-(1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide, (6) N-(1-{[6-{[(2R)-3,3-dimethylbutan-2-yl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)cyclopropanecarboxamide.

6. The prophylactic or therapeutic drug according to claim 1 or 2, which is used for the prevention or treatment of severe pulmonary arterial hypertension, severe pulmonary atresia, or severe pulmonary hypertension associated with lung disease and / or hypoxemia.

7. The prophylactic or therapeutic drug according to claim 1 or 2, which is used for the prevention or treatment of at least one collagen disease selected from the group consisting of systemic lupus erythematosus, systemic sclerosis, polymyositis / dermatomyositis, Sjogren's syndrome, mixed connective tissue disease, antiphospholipid syndrome, and Behcet's disease, or pulmonary hypertension associated with such collagen disease.

8. A method for preventing or treating severe pulmonary hypertension or collagen disease, comprising administering a JAK1 inhibitor to a person in need of such prevention or treatment.

9. Use of a JAK1 inhibitor for the manufacture of a medicament for the prevention or treatment of severe pulmonary hypertension or collagen disease.

10. A JAK1 inhibitor used in the treatment of severe pulmonary hypertension or collagen disease for the prevention or treatment of the disease.

11. A method for screening test substances for candidate substances that may be effective in preventing or treating severe pulmonary hypertension or collagen disease, comprising the steps of: measuring the JAK1 inhibitory activity of the test substances; and selecting test substances that have been confirmed to have JAK1 inhibitory activity as the candidate substances.

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