WO2020175597A1

WO2020175597A1 - Medicine for preventing or treating tissue fibrotic diseases

Info

Publication number: WO2020175597A1
Application number: PCT/JP2020/007901
Authority: WO
Inventors: 昇芦田; 周成宮; 良孝平山; 荒森　一朗
Original assignee: 国立大学法人京都大学; アステラス製薬株式会社
Priority date: 2019-02-28
Filing date: 2020-02-27
Publication date: 2020-09-03
Also published as: JP2022065212A

Abstract

Provided is a medicinal composition to be used for preventing and/or treating tissue fibrotic diseases. The medicinal composition according to the present invention, which comprises a PI3Kδ inhibitor as an active ingredient, exhibits excellent effects on tissue fibrosis in various organs and, therefore, can prevent and/or treat tissue fibrotic diseases. As an example of the PI3Kδ inhibitor, [(3S)-3-({6-[2-(difluoromethyl)-1H-benzoimidazol-1-yl]-2-(morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or a salt thereof may be cited.

Description

\¥0 2020/175597 1 卩 (: 17 2020 /007901 Clarification

Title of invention: Pharmaceutical field for prevention or treatment of diseases caused by tissue fibrosis

The present invention relates to a medicament for preventing or treating a disease caused by tissue fibrosis.

[0002] This application claims the priority of Japanese application No. 2019-035081, which is incorporated herein by reference.

Background technology

[0003] Tissue fibrosis is a bioadaptive reaction found in the healing process of damaged tissues, but when it occurs excessively, it destroys normal tissues and causes organ failure. It is believed that tissue fibrosis is mainly due to abnormal growth of connective tissue in the tissue and excessive deposition of extracellular matrix such as collagen produced by fibroblasts.

[0004] Scleroderma is a systemic disease centering on vascular disorders, mainly inflammatory and fibrotic changes. In addition to sclerosing lesions of the skin starting from the fingers, changes in various organs such as pulmonary fibrosis. Accompanied by. It is considered that various cytokines such as TGF-/S and IL-6 are involved in fibrosis (Non-patent Documents 1 and 2). In addition, the skin fat layer is reduced in the skin of patients with scleroderma, but in recent years, skin adipocyte precursor cells control skin myofibroblasts and fibrosis in scleroderma (Non-patent document 3). ), Sera of patients with scleroderma suppress the differentiation of adipose-induced stem cells into adipocytes, and the adipocyte markers perilipin and adiponectin (ad i ponect). It has been shown to reduce the expression of (in) and change it into myofibroblast-like cells (Non-patent document 4), and it is considered that recovery of skin adipose tissue may contribute to suppression of fibrosis. Adiponectin is one of the cytokines produced by adipocytes, and has various functions such as adipocyte differentiation promoting action, anti-inflammatory action, and anti-fibrotic action (Non-patent document 1, Non-patent document 5, and Non-patent document 6). However, it has been reported to be decreased in the blood of patients with scleroderma (Non-patent Document 7), which also suggests a relationship between the condition of scleroderma and adipose tissue. Thus, although the pathophysiology of scleroderma is becoming clear, it is still a cure. \¥02020/175597 2 卩 (: 171-1? 2020 /007901

There is only symptomatic treatment, and it is required to establish a fundamental treatment method.

[0005] As one of the mechanisms of tissue fibrosis, activation of Akt by phosphorylation of phosphatidylinositol-3-kinase (PI3K) leads to pulmonary fibrosis (Non-Patent Document 8) and renal fibrosis (Non-patent reference). Reference 9), it is disclosed that it is involved in skin fibrosis (Non-Patent Document 10). In addition, there is a report that a PI3K inhibitor can be used for the treatment of fibrosis, particularly idiopathic pulmonary fibrosis (Patent Document 1). Patent Document 2 ~

In 6, various types of diseases associated with immune disorders are listed as uses of PI3KS selective inhibitors, and examples thereof include use for fibrosis, scleroderma, etc. There is no example showing the therapeutic effect on fibrosis and scleroderma.

[0006] In Non-Patent Document 11, the PI3K/Akt/mT0R pathway is involved in scleroderma, and BEZ235, which inhibits PI3K/Akt/mTOR signaling activity and mTOR signaling activity, is reported in vitro and in vitro. It has been disclosed that in vivo experiments using a dermatosis model mouse (bleomycin induction model) showed anti-fibrotic activity.

[0007] Patent Document 7 discloses a compound having the following chemical formula as one of PI3K 8 (Phosphat i dy I i nos i to l-3-ki nase delta) selective inhibitors, [(3S)-3- ({6-[2-(Difluoromethyl)-1H-benzimidazol-1-yl]-2-(morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxane-4 -Yl)methanone (Table 222, Ex. A 293) (hereinafter sometimes referred to as Compound 1) is disclosed, and rejection in organ transplantation, allergic disease, autoimmune disease, blood fluid is disclosed. It has been shown that it can be used for the prevention/treatment of tumors. However, Patent Document 7 does not describe a disease caused by fibrosis. \¥0 2020/175597 3 卩(: 171? 2020 /007901

[Chemical 1]

Prior art documents

Non-patent literature

[0008] Non-Patent Document 1: Nature Reviews Rheumatology, 2014; 10, 706-719

Non-Patent Document 2: Rheumatol., 2012; 39, 1120.

Non-Patent Document 3: Nature Reviews Rheumatology, 2017; 13, 71-72 Non-Patent Document 4: Clin. Med., 2019; 8, 1256

Non-Patent Document 5 :Bi Lipid. Res., 2005; 46, 1369-1379

Non-Patent Document 6: Arthr i t i s. Res. Ther., 2012; 14, R229

Non-Patent Document 7: Exp. Dermatol., 2011; 20, 764-766.

Non-Patent Document 8: Int. 丄 Mol. Sc i ., 2018; 19, 778

Non-Patent Document 9: Bowl Cell. Mol. Med., 2017; 21, 1248-1259

Non-Patent Document 10: Invest. Dermatol., 2006; 126, 551-560.

Non-Patent Document 11 :丄 Dermatol Sc i ., 2014 Nov; 76(2): 104-111

Patent literature

[0009] Patent Document 1: International Publication WO2013/117503

Patent Document 2: Japanese Patent Laid-Open No. 2017-186378

Patent Document 3: International Publication WO2014/006572

Patent Document 4: International Publication WO2012/126901

Patent Document 5: International Publication WO2012/107465

Patent Document 6: International Publication WO2008/064018

Patent Document 7: International Publication WO2012/020762 \\0 2020/175597 4 (17 2020/007901 Summary of the invention

Problems to be Solved by the Invention

[0010] There is provided a pharmaceutical composition, which includes, for example, a compound having a 131<5 inhibitory action or a salt thereof as an active ingredient, and is useful for preventing and/or treating a disease caused by tissue fibrosis.

Means for solving the problem

[001 1] The present inventors have conducted extensive studies to solve the above problems, and as a result,

The present invention was completed for the first time by discovering that a pharmaceutical composition containing 1 or a salt thereof suppresses tissue fibrosis in skin, lung, esophagus, kidney and the like.

[0012] That is, the present invention consists of the following.

[0013] (1) A pharmaceutical composition for preventing and/or treating a disease caused by tissue fibrosis.

(2) A pharmaceutical composition for preventing and/or treating a disease due to tissue fibrosis, which comprises a compound having a 131<5 inhibitory action or a salt thereof as an active ingredient.

(3) The compound or its salt is [(33)-3-({6-[2-(difluoromethyl)-1 benzimidazol-1-yl]-2-(morpholin-4-yl)pyrimidine-4- Il}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or a salt thereof, the pharmaceutical composition according to the above (2).

(4) The pharmaceutical composition according to the above (2) or (3), which is a pharmaceutical composition which further comprises a pharmaceutically acceptable excipient. The pharmaceutical composition is [(33)-3-({6-[2-(difluoromethyl)-benzimidazol-1-yl]-2-(morpholin-4-yl)pyrimidine-4- Ill}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or a salt thereof is included in the preventive and/or therapeutic agent for diseases caused by tissue fibrosis.

(5) Tissue fibrosis is caused by lung, skin, heart, esophagus, stomach, intestine, kidney, liver, blood vessel, spleen, bone marrow, mammary gland, muscle, peritoneum, pleura, thyroid, lymph node, joint, bladder, trachea The pharmaceutical composition according to any one of (1) to (4) above, which is fibrosis in any one or more tissues selected from wall, adipose tissue and connective tissue. \¥0 2020/175597 5 卩 (: 171? 2020 /007901

(6) The pharmaceutical composition according to any one of (1) to (4) above, wherein the disease due to tissue fibrosis is a disease due to fibrosis associated with scleroderma and/or organ failure.

(7) The pharmaceutical composition according to any one of (1) to (4) above, wherein the disease caused by tissue fibrosis is scleroderma.

(8) Scleroderma and/or diseases caused by fibrosis associated with organ failure include pulmonary fibrosis, pulmonary hypertension, skin sclerosis, reflux esophagitis, scleroderma renal crisis, and diffuse skin sclerosing systemic sclerosis. The pharmaceutical composition according to (6) above, which is one or more diseases selected from the group consisting of illness, localized systemic scleroderma and autoimmune diseases.

(9) The pharmaceutical composition according to the above (6), wherein the disease caused by fibrosis associated with scleroderma and/or organ failure is pulmonary fibrosis or pulmonary hypertension.

(10) The pharmaceutical composition according to the above (7), wherein the active ingredient contained in the pharmaceutical composition has an action of suppressing reduction of the skin fat layer associated with scleroderma.

(11) The pharmaceutical composition according to any one of (1) to (4) above, wherein the active ingredient contained in the pharmaceutical composition has an action of inducing the differentiation of skin fibroblasts into adipocytes.

(12) [(33)-3-({6-[2-(difluoromethyl)- for manufacturing a pharmaceutical composition for preventing or treating a disease caused by tissue fibrosis]

Use of benzimidazol-1-yl]-2-(morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](dioxan-4-yl)methanone or a salt thereof.

(13) [(33)-3-({6-[2-(difluoromethyl)-benzimidazol-1-yl]-2-(morpholine-4 for the prevention or treatment of diseases caused by tissue fibrosis. -Ill) Pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or a salt thereof.

(14) [(3)-3-({6-[2-(difluoromethyl)-1benzimidazol-1-yl]-2-()) for use in the prevention or treatment of diseases caused by tissue fibrosis. Morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or a salt thereof.

(15) [(33)-3-({6-[2-(difluoromethyl)-1 benzimidazole-1-y \¥0 2020/175597 6 卩 (: 171-1? 2020 /007901

]]-2-(Morpholine-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl; |(oxan-4-yl)methanone or a salt thereof is administered to the subject. , A method for preventing or treating a disease caused by tissue fibrosis. The “subject” is a human or other animal in need of prevention or treatment thereof, and in one embodiment, a human in need of prevention or treatment thereof.

Effect of the invention

[0014] The pharmaceutical composition of the present invention showed an excellent therapeutic effect in a fibrosis model animal from the viewpoint of evaluation of skin score and fat layer ratio. Furthermore, histologically, for example, it showed an excellent improving effect on skin fibrosis, esophageal submucosal fibrosis, and renal glomerulosclerosis. It also showed an excellent improvement effect on the circumference of the right ventricle. Furthermore, it was shown that the pharmaceutical composition of the present invention was directly applied to fibroblasts of animal models of fibrosis and humans to differentiate into adipocytes. From this, it was confirmed that the pharmaceutical composition of the present invention exhibits an excellent effect on tissue fibrosis in various organs.

Brief description of the drawings

[0015] [Fig. 1] Fig. 1 shows IKK /S ^f m. ^x Sm22 a -Cre ^+/ _ mouse (hereinafter, in the specification or figures,

It may be described as "K0", "K0 mouse", riKK /S -KO mice. 3] is a graph showing the evaluation results by the skin score when Compound 1 which is one of PI3K 5 inhibitors is administered. The administration of Compound 1 suppressed the increase in skin score, and the symptom improving effect was obtained. (Example 1)

[Fig. 2] Fig. 2 is a diagram showing the results of confirming the ratio of the fat layer in the epidermis when the compound 1 was orally administered to K0 mice. (Example 1)

[FIG. 3] FIG. 3 is a photograph showing the results of confirming the proportion of the fat layer in the epidermis when the compound 1 was orally administered to K0 mice. (Example 1)

[FIG. 4] FIG. 4 is a diagram showing the results of evaluation by the circumference of the right ventricle, which reflects the degree of pulmonary hypertension when Compound 1 was orally administered to K0 mice. (Example 2)

[Fig. 5] Fig. 5 shows the effect of oral administration of Compound 1 to K0 mice on the fibrosis of the submucosa of the esophagus, which was stained with Masson Trichrome. \¥0 2020/175597 7 卩(: 171-1?2020/007901

It is a color organization chart, and the arrow shows a submucosal fibrotic focus. (Example 3)

[FIG. 6] FIG. 6 shows the improving effect on renal glomerulosclerosis when Compound 1 is orally administered to 1<0 mice.

It is a stained tissue chart, and the arrow shows a glomerulus. (Example 3)

[Fig. 7] Fig. 7 shows the degree of lung fibrosis when compound 1 was administered to a lung fibrosis model mouse (bleomycin-induced) by 831 (("○ 1 ^: 1; score evaluation results. It is a diagram (Example 4)

[Fig. 8] Fig. 8 shows the results obtained when Compound 1 was applied to skin fibroblasts of 1<0 mice.

FIG. 3 is a diagram showing the results of Western plot showing increased expression of H. (Example 5)

[FIG. 9] FIG. 9 shows compounds in human dermal fibroblasts.

FIG. 3 is a diagram showing the results of Western blot showing increased expression. (Example 6)

[Fig. 10] Fig. 10 shows the results obtained when Compound 1 was applied to skin fibroblasts of scleroderma patients.

It is a figure which shows the result of G. (Example 7)

MODE FOR CARRYING OUT THE INVENTION

[0016] Hereinafter, the present invention will be described in detail.

As used herein, the term “tissue fibrosis” refers to abnormal growth of connective tissue in a tissue. For example, it is the bioadaptive response seen in the healing process of damaged tissues. However, excessive accumulation of extracellular matrix such as collagen fiber (collagen) causes the tissue to harden, destroys normal tissue, and causes organ failure. The phenomenon that the structure becomes hard like this is also called hardening. In the present specification, “tissue fibrosis” is applied as long as fibrosis is observed in a tissue, at which site fibrosis is caused, which disease is caused by fibrosis, or which disease is caused. There is no particular limitation as to whether or not It is also called fibrosis rather than tissue fibrosis, and there is no particular limitation regarding fibrosis and hardening.

[0017] In the present specification, "disease due to tissue fibrosis" means autoimmune disease, collagen disease, skin disease, heart disease, respiratory system disease, esophageal disease, gastrointestinal disease, liver disease, renal disease. \¥0 2020/175597 8 卩 (: 171? 2020 /007901

, Cranial nerve diseases, eye diseases, bone marrow diseases, cancer, arteriosclerosis, obesity, diabetes and the like. Examples include scleroderma and fibrosis associated with organ failure. Tissue fibrosis in the present specification is not limited to these diseases as long as the tissue has fibrosis.

[0018] In the present specification, the tissue in which "tissue fibrosis" occurs is not particularly limited, and examples thereof include lung, skin, heart, esophagus, stomach, intestine, kidney, liver, blood vessel, and spleen. , Bone marrow, mammary gland, muscle, peritoneum, pleura, thyroid, lymph node, joint, bladder, tracheal wall, adipose tissue, connective tissue, and/or one or more types of tissue. In one aspect, it refers to fibrosis in any tissue of lung, skin and heart. In addition, the cause of tissue fibrosis and diseases associated with tissue fibrosis in the present specification are not particularly limited.

[0019] Scleroderma is classified into, for example, systemic scleroderma (system i c scleros i s: SSc) and localized scleroderma. Systemic scleroderma is a systemic disease centering on vascular disorders, mainly inflammatory and fibrotic changes, and in addition to sclerotic lesions of the skin starting from the fingers, lesions of various organs such as pulmonary fibrosis are also present. Accompany. Systemic scleroderma is a diffuse skin-curing systemic scleroderma (diffuse cut aneous SSc: dcSSc) in which skin fibrosis (hardening) extends to the forearm and trunk, and is localized to the fingers and face. It is classified as skin-curing systemic scleroderma (limited cutaneous SSc: IcSSc). Symptoms of systemic scleroderma include, for example, skin swelling, finger swelling, skin hardening, pigmentation and loss, and peripheral circulatory disorders such as Raynaud's phenomenon, finger ulcers, organ disorders such as pulmonary arterial hypertension. , Myocardial disorders such as heart fibrosis, gastrointestinal dysfunction, interstitial lung disease, and renal crisis.

[0020] Examples of the diseases due to tissue fibrosis other than scleroderma include liver fibrosis and inflammatory diseases that predispose it. For example, acute or chronic hepatitis, biliary tract disease and toxic liver damage, nephrosclerosis due to diabetic nephropathy, myelofibrosis, fibroid fibrosis, post-wound scar, post-angioplasty restenosis, arteriosclerosis, Aneurysm, arterial calcification, aortic valve stenosis, ischemic heart disease, cardiomyopathy, arthritis, mammary fibrosis, muscle fibrosis, retroperitoneal fibrosis, thyroid fibrosis, lymph node fibrosis, bladder fibrosis, Pleural line \\0 2020/175597 9 卩 (: 171-1? 2020 /007901

Fibrosis and chronic obstructive pulmonary disease (chronic obstructive plumonary di sease: COPD) To be

[0021] In the present specification, an active ingredient of a pharmaceutical composition applicable to the prevention and/or treatment of tissue fibrosis includes a PI3KS selective inhibitor. Specific examples of the PI3K S selective inhibitor as an active ingredient include Compound 1 or a salt thereof.

The present invention includes a pharmaceutically acceptable prodrug of Compound 1 or a salt thereof. The pharmaceutically acceptable prodrug is a compound having a group which can be converted into an amino group, a hydroxyl group, a carboxyl group or the like by solvolysis or under physiological conditions. Examples of groups that form prodrugs include the groups described in Prog. Med., 5, 2157-2161 (1985) and “Development of pharmaceuticals” (Hirokawa Shoten, 1990) Volume 7 Molecular Design 163-198. Can be mentioned.

[0023] The salt of Compound 1 is a pharmaceutically acceptable salt of Compound 1, and specifically, an acid addition salt (inorganic acid salt (hydrochloride, hydrobromide, hydrogen iodide, Acid salts, sulfates, phosphates, nitrates, etc.), organic acid salts (formic acid, propionic acid, acetate, trifluoroacetate, lactate, malonic acid, succinic acid, malic acid, mandelic acid, dibenzoyltartaric acid, Tartrate, ditoluoyl tartaric acid, oxalate, fumarate, maleate, benzoate, citrate, methanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, Glucuronate, gluconate, aspartic acid, glutamic acid, etc.) and the like.

[0024] Compound 1 or a salt thereof contained as an active ingredient in the pharmaceutical composition of the present invention has a function as a PI3K5 selective inhibitor. The compound 1 or a salt thereof can be produced by using the method described in Patent Document 7, a method obvious to those skilled in the art, or a modified method thereof.

[0025] In the present specification, the "PI3K S selective inhibitor" means that the PI3K S inhibitory activity is 10 times or more in IC ₅₀ value as compared to the PI3K a inhibitory activity, and in another embodiment, Double \¥0 2020/175597 10 卩 (: 171-1? 2020 /007901

As described above, still another embodiment means an inhibitor showing a strong activity with 100 times or more selectivity.

The amount of Compound 1 or a salt thereof contained in the pharmaceutical composition of the present invention varies depending on the route of administration, dosage form, administration site, kinds of excipients and additives, but is 0.01 to 100% by weight.

In one embodiment, the content can be 0.01 to 50% by weight.

[0027] The pharmaceutical composition of the present invention may also include various hydrates or solvates of Compound 1 or a salt thereof, and crystalline polymorphic substances. Examples of the solvate include solvates with water and alcoholic solvents (eg, ethanol).

[0028] Further, the pharmaceutical composition of the present invention includes all pharmaceutically acceptable compounds 1 or salts thereof labeled with one or more radioactive or non-radioactive isotopes. Examples of suitable isotopes used for the isotope labeling of the compound which is the active ingredient of the pharmaceutical composition of the present invention include hydrogen (and ³ H etc.), carbon ("0, ¹³ (: and ¹⁴ etc.)) , nitrogen (1 ³ 1 \ 1 and the like), oxygen ( ^'5 〇, ¹⁷ 0 and ¹⁸ 0, etc.), Ru isotopes of fluorine 〇 equality) is wrapped.

The compound, which is an isotope-labeled active ingredient of the pharmaceutical composition of the present invention, can be used in studies such as tissue distribution studies of drugs and/or substrates. For example, radioactive isotopes such as tritium () and carbon ¹⁴ ( ¹⁴⁰ ) can be used for this purpose because of the ease of labeling and the ease of detection.

[0030] Substitution with a heavier isotope, for example, substitution of hydrogen with deuterium (), has a therapeutic advantage due to improved metabolic stability (eg,

The increase in half-life at ○, decrease in required dose, decrease in drug interaction) may occur.

[0031] The pharmaceutical composition of the present invention is orally administered by tablets, pills, capsules, granules, powders, solutions, etc., or injections such as intravenous, intramuscular, etc., suppositories, transdermal solutions, It may be administered in any form of parenteral administration such as ointment, transdermal patch, transmucosal solution, transmucosal patch, and inhalant.

[0032] When the pharmaceutical composition of the present invention is a solid composition for oral administration, tablets, powders, granules and the like are used. In such solid compositions the active ingredient is mixed with at least one inert excipient. The composition is according to a conventional method. \\0 2020/175597 1 1 卩 (: 171-1? 2020 /007901

It may also contain an inert additive such as a disintegrant, a stabilizer, and a solubilizing agent. If necessary, the tablets or pills may be coated with sugar or a film of a gastric or enteric substance.

[0033] When the pharmaceutical composition of the present invention is a liquid composition for oral administration, it contains a pharmaceutically acceptable emulsion, solution, suspension, syrup or elixir, etc. It may contain a diluent used conventionally. The liquid composition may contain a solubilizing agent, a wetting agent, an auxiliary agent such as a suspending agent, a sweetening agent, a flavoring agent, an aromatic agent, and a preservative, in addition to the diluent.

When the pharmaceutical composition of the present invention is an injection, it may contain a sterile aqueous or non-aqueous solvent, a suspension, or an emulsion. Examples of the aqueous solvent include distilled water for injection and physiological saline. Examples of non-aqueous solvents include plant oils and alcohols. Such a composition may further contain a tonicity agent, a preservative, a wetting agent, an emulsifying agent, a dispersing agent, a stabilizing agent, or a solubilizing agent. These are sterilized by, for example, filtration through a bacteria-retaining filter, addition of a sterilizing agent, or irradiation. In addition, these can also be used by producing a sterile solid composition and dissolving or suspending it in sterile water or a sterile solvent for injection before use.

[0035] When the pharmaceutical composition of the present invention is an external preparation, it may also include an ointment, a plaster, a cream, a jelly, a poultice, a spray, a lotion and the like. Contains commonly used ointment bases, lotion bases, aqueous or non-aqueous liquids, suspensions, emulsions and the like.

[0036] When the pharmaceutical composition of the present invention is a transmucosal agent such as an inhalant or a nasal agent, a solid, liquid or semisolid one is used and can be produced according to a conventionally known method. For example, known excipients, and further, regulators, preservatives, surfactants, lubricants, stabilizers, thickeners and the like may be appropriately added. For administration, a device can be used as appropriate. For example, using a known device or nebulizer such as a metered dose inhaler, the compound may be dispensed alone or as a powder in a formulated mixture, or as a solution or suspension in combination with a pharmaceutically acceptable carrier. \¥0 2020/175597 12 卩 (: 171? 2020 /007901

Can be given. The dry powder inhaler and the like may be those for single or multiple administration, and dry powder or powder-containing capsules can be used.

[0037] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0038] When the pharmaceutical composition of the present invention is orally administered, the daily dose is about 0.0001 to 100 mg/kg of body weight, preferably 0.01 to 30 mg/kg, and more preferably 0.1. A suitable dose is 03 to 10 mg/kg, which can be administered once or in 1 to 4 divided doses. When the pharmaceutical composition of the present invention is administered intravenously, a suitable daily dose is about 0.00001-10 mg/kg of body weight, and the daily dose should be administered once or in multiple doses. You can In addition, as a transmucosal agent, about 0.0001 to 100 mg/kg of body weight can be administered once to multiple times a day. The dose is appropriately determined according to each case in consideration of symptoms, age, sex and the like.

The compound as an active ingredient contained in the pharmaceutical composition of the present invention is compound 1 or a salt thereof, as well as a compound as an active ingredient for use in the known prevention and/or treatment of tissue fibrosis. May be administered simultaneously, or separately and continuously, or at desired time intervals. The preparation for co-administration may be a combination drug or may be separately formulated.

[0040] In the present specification, the method for evaluating the efficacy of a compound as an active ingredient for use in the prevention and/or treatment of tissue fibrosis includes evaluation for the prevention and/or treatment of tissue fibrosis. However, the evaluation system described in International Publication WO2014/069597 can be applied.

[0041] As a method for analyzing fibrosis, the degree of fibrosis is decreased or suppressed, increased or accelerated, or there is no change such as increase or decrease, and the condition is measured and evaluated. Say. For example, using a non-human model animal that strongly reflects the pathological condition of human scleroderma as described in International Publication WO2014/069597, visual observation, histological prayer, biochemical analysis, image analysis, etc. It can be performed and can be appropriately determined by those skilled in the art. For example, a five-point score can be evaluated for the face and frontal area, occipital area, occipital area, left and right hindlimbs, and caudal area.Specifically, the skin score is (○: no lesion, 1: erosion, 2: wide range). Erosion 3: Ulcer \¥0 2020/175597 13 卩 (: 171-1? 2020 /007901

Therefore, it is possible to calculate the evaluation value such as 4: moderate ulcer, 5: extensive and serious ulcer).

[0042] Of the diseases caused by fibrosis, for example, the test for scleroderma can be applied to the test for scleroderma patients in addition to the above-described measurement of fibrosis. For example, autoantibody test, skin biopsy, visceral test, etc. are performed. Examples of autoantibodies specific to scleroderma include antinuclear antibodies, particularly anti-antibodies 70 antibodies (anti-topoisomerase 1 antibody), anti-centromere antibodies, anti-1^18 polymerase III antibodies, anti-nucleolar antibodies, etc. Is

It can be detected by the method. As the antibody and kit used for these, the above-mentioned commercially available products and the like can be appropriately used. For skin biopsy, visceral examination, etc., the usual method is used. [0043] For diseases due to fibrosis, further enlargement of the right ventricle, pulmonary hypertension, 8 and / or 1 ^{"infiltration} of cells, 108 cells and / or 1 in the ^spleen" to the skin lesions cell activation such as, or , Expression of activated/3-potency tenin in living fibroblasts derived from the non-human animal, antigen-presenting ability when co-cultured with dendritic cells (activation of Ding cells),

Thus, the preventive and/or therapeutic effect of scleroderma can be evaluated.

Example

[0044] In the following examples,

Compound 1 ([(35) -3-({6-[2-(difluoromethyl)-

Benzimidazole-1-yl]-2-(morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxan-4-yl)methanone) has a therapeutic effect on fibrosis. confirmed.

[0045] (Example 1) Effect of Compound 1 on scleroderma-like symptoms in fibrosis model mice

1. Preparation of fibrosis model mouse

The fibrosis model mouse shown in Example 1 described in International Publication ^2014/069597 was produced. Specifically, it was produced by the following method.

Known

11122 mouse 〇丨㊀门〇㊀ (2003), ¥ 〇 1.300, 卩. 329-332) and heterozygous mouse

11122 «-○ 6 mice were obtained. The mouse,

.. Mating with the mouse,

11122 \¥0 2020/175597 14 卩 (: 171? 2020 /007901

"6 tenths ^/ -mouse (hereinafter, "1 <0 mouse"

311122

A mouse (hereinafter, "1^mouse") was obtained. 1<0 mice were born at a rate according to Mendelian's law, and their growth such as change in body weight was not different from that of town mice. All of the <<O mice obtained in the present invention were obtained by mating mice with a pure animal strain 0578176 as a genetic background.

[0046] 2. Evaluation of skin score by oral administration

Compound

) Solution) and suspend from 0.003 to 0.1

A preparation containing Compound 1 of Above 1. <16 weeks of age of 0 mice during the 30 weeks of age, formulation 10 comprising a compound 1> 1 produced in that orally administered 111_ _9, from 0.03 to 1> 11 _{9 9} compound 1 was administered twice a day. Oral administration of 10 11 117 was orally twice a day only for the solvent! <0 mice were defined as 6 ₁ (^6 groups. Experiments were conducted using 10 cases in each group.

[0047] The skin score is a 5-step score (0: no lesion, 1: erosion, 2: extensive erosion, 3: ulceration, 4: on the face and frontal region, occipital region, right and left hind limbs, and tail region. It was calculated as the total value of 5): moderate ulcer and 5: widespread and serious ulcer). The therapeutic effect was evaluated by calculating the value obtained by subtracting the score before administration from the score after administration of the preparation containing Compound 1.

[0048] In this example, erosion refers to a defect extending to the epidermal layer, and ulcer refers to a defect extending to the dermal layer. As a result, Compound 1 compared to administration group 6 ₁ Rei_16 group, 0.03 1 ^ / 1 ^ are suppressed increase in skin score from low dose, the symptoms of scleroderma-like is suppressed (Fig. 1).

[0049] 3. Evaluation of percentage of fat layer by oral administration

In generalized scleroderma, collagen fibers (collagen) are increased in the dermis and subcutaneous adipose tissue of the skin, and there is a finding that the subcutaneous adipose tissue is replaced by new collagen fibers. Therefore, as in 2 above, the monohydrochloride of Compound 1 was suspended in a solvent (0.5% !^ solution) to give 0.003 to 0.1

0.03 to 1111 by oral administration of the compound 1-containing preparation twice a day between the ages of 16 to 30 weeks in 1<0 mice, _{9 9} of Compound 1 were administered. After euthanasia, collect face and head skin \¥0 2020/175597 15 卩(: 171? 2020/007901

The skin pathological sections were stained with Masson-Trichrome. The stained image was digitally photographed under a microscope, and the ratio of the fat layer in the epidermis was measured using image analysis software 1! 11 ₃ 96". The solvent was orally administered 10 11 117 twice a day! <0 mice were set as ▽6^0 4 groups. The experiment was conducted using 10 cases in each group. As a result, the proportion of the fat layer in the epidermis was 0.3 in the compound 1-administered group compared with the 611 _1 (^6 group.

The rate was high in the treated group (Figs. 2 and 3).

[0050] From these results, it was expected that Compound 1 has an inhibitory effect on skin ulcer of scleroderma, an inhibitory effect on reduction of fat layer, and a concomitant normalizing effect on skin tissue.

(Example 2) Effect of Compound 1 on pulmonary hypertension in fibrosis model mice

Scleroderma may be associated with pulmonary fibrosis and pulmonary hypertension, resulting in dilation of the right ventricle and hypertrophy of the right ventricular wall. Therefore, Compound 1 monohydrochloride was suspended in a solvent (0.5% solution), and 0.003 to 0.1

Of the fibrosis model mouse prepared in 1 of Example 1 (○ <0 mouse) from 16 weeks to 30 weeks of age, and the preparation containing Compound 1 was administered twice a day. by oral administration in 11117, the compound was administered 1 0.0 3-1 111 _{9 9.} After euthanasia, the heart was removed. Cardiac pathology sections were prepared, stained with hematoxylin and eosin (), and the perimeter of the right ventricle was measured using 1111396” software (unit:

The effect of compound 1 was confirmed. 1<0 mice that were orally administered with the solvent alone twice a day and with 10 11 117 were defined as V 6^ 0 4 group. The experiment was conducted using 10 cases in each group. As a result, the right ventricular circumference was decreased in the compound 1-administered group as compared to the 611 _1 (^6 group (Fig. 4). These results indicate that compound 1 is effective for pulmonary hypertension. Was expected

(Example 3) Effect of Compound 1 on fibrosis in fibrosis model mouse Compound

Suspended in

A formulation containing Compound 1 was prepared. To a fibrosis model mouse prepared in 1 of Example 1 (0 <0 mouse), a formulation containing Compound 1 was orally administered twice a day from 16 weeks to 30 weeks of age at 10 11117. , 0.1 to 5 111 _{9 9} Compound 1 was administered to the esophageal mucosa \¥02020/175597 16 卩 (: 171-1? 2020 /007901

Fibrosis of the lower layer and renal glomerulosclerosis were confirmed. The group to which only the solvent was administered was defined as the Vehicle group. As a result, fibrosis in the submucosa of the esophagus (Fig. 5) and renal glomerulosclerosis (Fig. 6) were suppressed in the compound 1-administered group as compared to the Vehicle group. These results indicate that Compound 1 inhibits not only the skin but also the fibrosis of the submucosa of the esophagus and renal glomerulosclerosis.

Example 4 Compound in Pulmonary Fibrosis Model Mouse (Bleomycin Induced)

1 effect

1. Preparation of bleomycin-induced pulmonary fibrosis model mouse

C57BL/6J mice were anesthetized with sodium pentobarbital, and bleomycinin physiological saline solution (1.2 mg/mL) was administered intratracheally once at 50 ML/head using Microsprayer (registered trademark) (Penn-Century). did.

[0054] 2. Effect of Compound 1 on bleomycin-induced pulmonary fibrosis model mouse

Compound 1 monohydrochloride was suspended in a solvent (0.5% MC solution) to give a preparation containing 0.01 to 0.5 mg/mL of Compound 1. The bleomycin-induced pulmonary fibrosis model mouse prepared in 1. of the present Example was orally administered with a formulation containing Compound 1 at 10 mL/kg for 21 days including the day of bleomycin administration. Compound 1 at 5 mg/kg was administered twice daily. Dexamethasone (Dex: 0.25 mg/kg) as a control (positive control) was orally administered once daily. In addition, the vehicle group was a group in which 10 mL/kg of the solvent alone was orally administered twice a day to bleomycin-administered mice for 21 days. In addition, mice not administered with bleomycin were used as a control group. Control group 14 cases, Vehicle group 14 cases, Compound 1 0.1 mg/kg administration group 13 cases, 0.5 mg/kg administration group 14 cases, 1 mg/kg administration group 12 cases, 5 mg/kg administration group 13 cases, Dex group 11 Experiments were conducted using examples. Mice were euthanized on day 22 after bleomycin administration, lungs were removed and fixed with formalin, and tissue sections were stained with Masson's Trichrome to show the degree of fibrosis. Ashcroft score (Ashcroft TA et a 1., J Clin Pathol, 19 88; 41 (4), 467-70) was calculated to evaluate lung tissue fibrosis. The Ashcroft score is obtained by dividing the degree of fibrosis of the lung into 0 to 8 grades in each field under a microscope in a lung tissue sample, and scoring the score from 20 to 8 fields in each section. \¥02020/175597 17 卩 (: 171-1? 2020 /007901

The median value was used as the score of each individual. As a result, the compound 1 at 5 mg/kg was shown to have the same pulmonary fibrosis inhibitory effect as the positive control dexamethasone. It was considered that Compound 1 could be expected to be effective in diseases involving fibrosis of the lung, such as interstitial pneumonia associated with scleroderma and idiopathic pulmonary fibrosis (Fig. 7).

Example 5 Effect of Compound 1 on Adipocyte Differentiation in Skin Fibroblasts of Fibrosis Model Mice

In this example, the effect of Compound 1 on skin fibroblasts in a fibrosis model mouse was examined.

Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS) containing skin fibroblasts (2×10 ⁵ cells) prepared from the dorsal skin of the fibrosis model mouse (K0 mouse) prepared in 1. of Example 1 (DMEM, Fujifilm manufactured by Wako Pure Chemical Industries, Ltd.) 5% C0 ₂ atmosphere at, were cultured in 6-well plates at 37 ° C. Five days later, 0.5 mM isobuty l-methy Ixanthine (manufactured by Sigma-Aldrich), 1 fxU Dexamethasone (manufactured by Sigma-Aldrich), recombinant human insulin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (

1 Mg / ml), 2 M Rosig I i tazone ( manufactured by Fuji Film Wako Pure Chemical Industries) was added DMEM containing 10% FBS and 10nM Compound 1, 5% C0 ₂ atmosphere, and cultured at 37 ° C for 〇 After 2 days, change the medium to DMEM medium containing recombinant human insulin (1 yu,g/ml) _% 10% FBS and 10 nM compound 1, and after 2 days, add 10% FBS and 10 nM compound 1. The medium was replaced with the containing DMEM medium. Two days later, cells were collected, and the differentiation state of adipocytes was confirmed by Western blot using an anti-PPAR^ antibody (manufactured by CeU Signaling Technology) and an anti-FABP4 antibody (manufactured by CeU Signaling Technology). In addition, /S-actin was used as a control. As shown in Fig. 8, in the fibrosis model mouse, the expression of adipocyte markers PPAR^ and FABP4 was increased by compound 1, and compound 1 was directly applied to dermal fibroblasts and It was found to differentiate into.

(Example 6) Effect of Compound 1 on adipocyte differentiation in human skin fibroblasts

In this example, the effect of Compound 1 on adipocyte differentiation was observed in human dermal fibroblasts. \¥02020/175597 18 卩 (: 171-1? 2020 /007901

I checked if I could see it.

From human normal skin by adjusting the skin fibroblasts (2X10 ⁵ cells), Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) (DMEM, Fujifilm manufactured by Wako Pure Chemical Industries, Ltd.) 5% C0 ₂ atmosphere at The cells were cultured at 37°C. 7 days later, 0.5mM i sobuty l-methy Ixa nth i ne (Sigma-Aldrich), 1 yu _< M Dexamethasone (Sigma-Aldrich), recombinant human Insulin (Fujifilm Wako Pure Chemical Industries) (lyLtg / ml), 2JLLM Rosiglitazone (manufactured by Fuji Film Wako Pure chemical Industries) was added DMEM containing 10% FBS and 10 nM compound 1, 5% C0 ₂ atmosphere, were cultured at 37 ° C. Two days later, the medium was replaced with DMEM having the same composition as above. Two days later, the medium was replaced with a DMEM medium containing recombin human insulin (1 yu, g/ml) _% 10% FBS and 10 nM compound 1. Two days later, cells were collected and the adipocyte differentiation state was confirmed by Western blot using an anti-PerUipin antibody (CeU Signaling Technology). GAPDH was used as a control. As shown in Fig. 9, compound 1 increased the expression of PerUipin, a marker for adipocytes, and it was found that compound 1 also induces differentiation of human dermal fibroblasts into adipocytes.

(Example 7) Effect of Compound 1 on adipocyte differentiation in skin fibroblasts of scleroderma patients

In this Example, it was confirmed whether the action of Compound 1 on adipocyte differentiation was also observed in skin fibroblasts of scleroderma patients.

Skin fibroblasts (2X10 ⁵ cells) were prepared from the skin of a scleroderma patient, and 5%C0 was added in Dulbecco's modified Eagle medium (DMEM, FUJIFILM Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS). Culture was performed at 37°C in ₂ atmospheres. After 7 days, 0.5 mM isobutyl-methyl xanthine (Sigma-Aldrich), 1 fxU Dexamethasone (Sigma-Aldrich), recombinant human Insulin (Fujifilm Wako Pure Chemical Industries) (1 M9/ml), 2 ^ M Rosiglitazone (manufactured by Fuji Film Wako Pure chemical Industries) was added DMEM containing 10% FBS and 10 nM compound 1, 5% C0 ₂ atmosphere, were cultured at 37 ° C. Two days later, the medium was replaced with DMEM having the same composition as above. Two days later, recomb i \¥02020/175597 19 卩 (: 171-1? 2020 /007901

The medium was replaced with DME M medium containing nant human insulin (1 yu, g/ml) _% 10% FBS and 10 nM compound 1. Two days later, the cells were collected, and the differentiation state of adipocytes was confirmed by Western blot using an anti-PerUipin antibody (manufactured by CeU Signaling Technology). GAPHD was used as a control. As shown in Fig. 10, the expression of PerUipin, which is a marker for adipocytes, was increased by Compound 1, and it was revealed that Compound 1 also induces differentiation of dermal fibroblasts of scleroderma patients into adipocytes.

Industrial availability

[0058] As described in detail above, the pharmaceutical composition of the present invention showed excellent therapeutic effect in the fibrosis model animal from the viewpoints of skin score and evaluation of fat layer ratio. Furthermore, histologically, it showed an excellent improving effect on, for example, skin fibrosis, lower esophageal fibrosis, and renal glomerulosclerosis. It also showed an excellent effect on the perimeter of the right ventricle. From this, it was confirmed that the pharmaceutical composition of the present invention has an excellent effect on tissue fibrosis in various organs and has industrial applicability.

Claims

\¥0 2020/175597 20 卩 (: 17 2020 /007901

The scope of the claims

[Claim 1] A pharmaceutical composition for preventing and/or treating a disease caused by tissue fibrosis.

[Claim 2]

A pharmaceutical composition for preventing and/or treating a disease caused by tissue fibrosis, which comprises a compound having an inhibitory action or a salt thereof as an active ingredient.

[Claim 3] The compound or salt thereof is [(33)-3-({6-[2-(difluoromethyl)-

The benzimidazol-1-yl]-2-(morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or a salt thereof. Pharmaceutical composition.

[Claim 4] The pharmaceutical composition according to claim 2 or 3, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[Claim 5] When the tissue fibrosis is lung, skin, heart, esophagus, stomach, intestine, kidney, liver, blood vessel, spleen, bone marrow, mammary gland, muscle, peritoneum, pleura, thyroid gland, lymph node, joint, bladder The pharmaceutical composition according to any one of claims 1 to 4, which has fibrosis in any one or a plurality of tissues selected from the trachea wall, adipose tissue and connective tissue.

[Claim 6] The pharmaceutical composition according to any one of claims 1 to 4, wherein the disease caused by tissue fibrosis is a disease caused by fibrosis associated with scleroderma and/or organ failure.

[Claim 7] The pharmaceutical composition according to any one of claims 1 to 4, wherein the disease caused by tissue fibrosis is scleroderma.

[Claim 8] A disease caused by fibrosis associated with scleroderma and/or organ failure is pulmonary fibrosis, pulmonary hypertension, skin sclerosis, reflux esophagitis, scleroderma renal crisis, diffuse skin sclerosis type whole body 7. The pharmaceutical composition according to claim 6, which is one or more kinds of diseases selected from scleroderma sclerosis, localized systemic scleroderma and autoimmune diseases.

[Claim 9] The pharmaceutical composition according to claim 6, wherein the disease due to fibrosis associated with scleroderma and/or organ failure is pulmonary fibrosis or pulmonary hypertension.

[Claim 10] The pharmaceutical composition according to claim 7, wherein the active ingredient contained in the pharmaceutical composition has an action of suppressing the reduction of the skin fat layer associated with scleroderma. \¥0 2020/175597 21 卩 (: 171? 2020 /007901

[Claim 11] The pharmaceutical composition according to any one of claims 1 to 4, wherein the active ingredient contained in the pharmaceutical composition has an action of inducing the differentiation of skin fibroblasts into adipocytes.

[Claim 12] [(33)-3-({6-[2-(difluoromethyl)-benzimidazol-1-yl]] for the manufacture of a pharmaceutical composition for preventing or treating a disease caused by tissue fibrosis. Use of 2-(morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or a salt thereof.

[Claim 13] For prevention or treatment of a disease caused by tissue fibrosis, [(35)-3-({6-[2

-(Difluoromethyl)-benzimidazol-1-yl]-2-(morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or its salt use.

[Claim 14] [(35)-3-({6-[2-(difluoromethyl)-1 benzimidazol-1-yl]-2-] for use in the prevention or treatment of a disease caused by tissue fibrosis. (Morpholine-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl; |(oxan-4-yl)methanone or a salt thereof.

[Claim 15] [(33)-3-({6-[2-(difluoromethyl)-

Benzimidazol-1-yl]-2-(morpholin-4-yl)pyrimidin-4-yl}amino)pyrrolidin-1-yl](oxan-4-yl)methanone or an effective amount of its salt is administered to the subject A method for preventing or treating a disease caused by tissue fibrosis.