Systemic sclerosis waveform protein variant protein activity inhibition therapeutic composition using STAT6 inhibitor

Abstract

The present invention relates to a therapeutic composition for inhibiting the activity of a systemic sclerosis waveform protein variant using a STAT6 inhibitor, and has been confirmed that the STAT6 inhibitor inhibits the expression of fibrotic T cells and M2 macrophages, which are immune cells associated with systemic sclerosis, increases the expression of Treg, and inhibits the expression of fibrotic T cells and M2 macrophages, which are immune cells associated with systemic sclerosis. It has been confirmed that the expression of TGF-beta, Col1a1, and alpha-SMA, which are fibrosis factors associated with systemic sclerosis, is inhibited. Moreover, it has been confirmed that the expression of cell surface waveform protein, which is a waveform protein variant associated with systemic sclerosis, is reduced in skin tissue and lung tissue, and it has been confirmed that the waveform protein variant can be regulated. In addition, the presence of pSTAT6-expressed CD8T cells is confirmed in fibrotic tissues, and the expression of pSTAT6-expressed CD8T positive cells is confirmed to be regulated by administration of an STAT6 inhibitor. The STAT6 inhibitor also has been confirmed to inhibit the tissue fibrosis of systemic sclerosis in which the antigen-specific disease activity is increased in an animal model with increased waveform protein-specific disease activity, and to inhibit the expression of CD8-positive TRM expressed by an IL-17 cytokine capable of inducing inflammation and fibrosis, which is a systemic sclerosis cause cell.

Description

Technical Field

[0001] The present invention relates to a therapeutic composition for inhibiting the activity of a variant vimentin protein in systemic sclerosis using a STAT6 inhibitor. Background Art

[0002] Although diseases caused by immune allergic reactions are increasing worldwide, the root causes of these diseases are still not fully understood. Currently, the treatment of myopathy caused by immune reactions is to alleviate or even reduce the various symptoms caused by the above diseases by administering immunosuppressants alone or in combination.

[0003] Immunosuppressants are a variety of substances used to reduce or block the host's ability to produce antibodies to antigens (humoral immune response) or to induce cellular immune response. In addition to organ transplantation, the above-mentioned immunosuppressants can also be effectively used in autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, as well as skin allergic reactions such as atopy and allergies. Excellent immunosuppressants should be able to regulate the imbalance of immune response, should ensure safety for the human body, and should have a low frequency of recurrence of the disease during long-term treatment.

[0004] Currently used immunosuppressants include cyclosporine A and FK506, which are derived from natural compounds with complex structures. They are not economical in terms of raw material supply due to high costs, and there is also the risk of various side effects caused by long-term administration. Therefore, there is an urgent need to develop new immunosuppressants that have low toxicity and can induce immune tolerance while being economically produced.

[0005] On the other hand, vimentin is a structural protein encoded by the VIM gene in the human body, and plays an important role in supporting and fixing the position of cell organs. The cytoskeleton of chondrocytes is mainly composed of actin microfilaments, tubulin microtubules and vimentin intermediate filaments. Although it is known that vimentin plays an important role in the structural formation of cells or tissues, in patients with autoimmune diseases or animal models. The protein expression level and filament network organization of vimentin are changed, and abnormal overexpression of vimentin is confirmed. It is reported that this is related to the damaged vimentin network. In addition, systemic sclerosis and tissue fibrosis occur due to repeated tissue damage and chronic inflammation associated with autoimmune diseases, and chronic inflammation is induced as the existing tissue recovery system is interrupted, thereby secreting inflammatory cytokines, chemokines and growth factors in immune cells. These factors induce the activation of fibroblasts and astrocytes, respectively inducing deformation toward myofibroblasts and activated astrocytes. Then, the activated myofibroblasts and astrocytes induce the production of structural elements of the extracellular substrate such as collagen fibers, and the excessive accumulation of the extracellular substrate causes the related tissues to become hardened, thereby inducing structural and functional damage. In this case, it is reported that cell surface vimentin is specifically expressed in activated myofibroblasts and astrocytes.

[0006] Therefore, there is a need to develop novel therapeutic agents in systemic sclerosis that can ameliorate myopathy by acting specifically on vimentin. Summary of the invention

[0007] Technical issues

[0008] An object of the present invention is to provide a pharmaceutical composition for preventing or treating systemic sclerosis, comprising a STAT6 (Signal transducer and activator of transcription 6) inhibitor as an active ingredient.

[0009] Another object of the present invention is to provide a food composition for preventing or improving systemic sclerosis, comprising a STAT6 inhibitor as an active ingredient.

[0010] Another object of the present invention is to provide a quasi-drug composition for preventing or treating systemic sclerosis, comprising a STAT6 inhibitor as an active ingredient.

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising a STAT6 inhibitor as an active ingredient for preventing or treating fibrosis caused by vimentin variant protein.

[0012] Another object of the present invention is to provide a method for treating systemic sclerosis, comprising the step of administering a STAT6 inhibitor to an individual in a pharmaceutically effective amount.

[0013] Another object of the present invention is to provide a method for treating fibrosis caused by vimentin variant protein, comprising the step of administering a STAT6 inhibitor to an individual in a pharmaceutically effective amount.

[0014] Technical Solution

[0015] In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating systemic sclerosis, comprising a STAT6 inhibitor as an active ingredient.

[0016] Furthermore, the present invention provides a food composition for preventing or improving systemic sclerosis, comprising a STAT6 inhibitor as an effective ingredient.

[0017] Furthermore, the present invention provides a quasi-drug composition for preventing or treating systemic sclerosis, comprising a STAT6 inhibitor as an active ingredient.

[0018] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating fibrosis caused by vimentin variant protein, comprising a STAT6 inhibitor as an active ingredient.

[0019] Furthermore, the present invention provides a method for treating systemic sclerosis, comprising the step of administering a STAT6 inhibitor to an individual in a pharmaceutically effective amount.

[0020] Furthermore, the present invention provides a method for treating fibrosis caused by vimentin variant protein, comprising the step of administering a STAT6 inhibitor to an individual in a pharmaceutically effective amount.

[0021] Effects of the Invention

[0022] The present invention confirms that STAT6 inhibitors inhibit the expression of profibrotic T cells and M2 macrophages, which are immune cells associated with systemic sclerosis, increase the expression of regulatory T cells (Treg), and confirm the inhibition of transforming growth factor-β (TGF-β), type I collagen (Col1a1), and α-smooth muscle actin (α-SMA), which are fibrotic factors associated with systemic sclerosis. In addition, it was confirmed in a systemic sclerosis animal model that when STAT6 inhibitors were administered, the activity of M2 macrophages that may induce fibrosis in the blood of mice was inhibited, and the thickness of the dermis of the skin and pulmonary fibrosis were inhibited. In addition, it was confirmed that the expression of cell surface vimentin, which is a vimentin variant protein associated with systemic sclerosis, was reduced in skin tissue and lung tissue, thereby confirming that vimentin variant proteins can be regulated. In addition, the presence of pSTAT6-expressing CD8 T cells was confirmed in fibrotic tissues, and it was confirmed that the expression of pSTAT6-expressing CD8 T-positive cells was regulated by administering STAT6 inhibitors. In an animal model with increased vimentin-specific disease activity, inhibition of STAT6 also inhibited tissue fibrosis in systemic sclerosis activated by antigen-specific myopathy, and suppressed the expression of IL-17 cytokine-expressing CD8-positive TRMs, which are the causative cells of systemic sclerosis and may induce inflammatory hyperfibrosis, and can be effectively utilized in related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Diagram for confirming the regulation of immune cells in spleen cells following treatment with the STAT6 inhibitor of the present invention (Part A: confirmation of expression of profibrotic T cells, Part B: confirmation of expression of M2 macrophages, Part C: confirmation of expression of Tregs, Part D: quantification of the expression ratio of M2 / M1).

[0024] Figure 2 The figures show the expression of fibrotic factors in fibroblasts treated with the STAT6 inhibitor of the present invention (Part A: confirmation of TGF-β expression, Part B: confirmation of Col1a1 expression, Part C: confirmation of α-SMA expression).

[0025] Figure 3 This figure shows the confirmation by flow cytometry analysis that the activity of M2 in the blood is suppressed by administration of a STAT6 inhibitor in systemic sclerosis mice.

[0026] Figure 4 The diagram shows the confirmation of the dermal thickness following administration of a STAT6 inhibitor in systemic sclerosis mice by hematoxylin and eosin (H&E) (Part A: staining results, Part B: quantification of the staining results).

[0027] Figure 5 The figures show the confirmation of pulmonary fibrosis following administration of a STAT6 inhibitor in systemic sclerosis mice by hematoxylin and eosin (H&E) (Part A: staining results, Part B: quantification of staining results).

[0028] Figure 6 The figures show the expression of fibrotic factors and cell surface vimentin in skin tissues of systemic sclerosis mice confirmed by immunohistochemical staining following administration of a STAT6 inhibitor (Part A: staining results, Part B: quantification of staining results).

[0029] Figure 7 The diagram shows the expression of fibrotic factors and cell surface vimentin in lung tissues of systemic sclerosis mice following administration of a STAT6 inhibitor by immunohistochemical staining (Part A: staining results, Part B: quantification of staining results).

[0030] Figure 8 The figures show the expression of pSTAT6-positive cells and CD8-positive cells in the skin and lung tissues of systemic sclerosis mice confirmed by confocal microscopy following administration of a STAT6 inhibitor (A: confocal analysis results, B: quantification of the analysis results).

[0031] Fig. 9 Schematic diagram of the preparation of the vimentin antigen-specific systemic sclerosis overactivation animal model and the drug administration regimen.

[0032] Fig.10 The figure shows the confirmation of skin fibrosis and cell surface vimentin expression in lung tissue following treatment with STAT6 inhibitors in a vimentin antigen-specific systemic sclerosis overactivation animal model by hematoxylin and eosin staining and immunohistochemical staining (Part A: staining results, Part B: quantification of skin thickness, Part C: quantification of cell surface vimentin).

[0033] Fig.11 Figure 3. Confocal microscopy confirms the expression of CD8 TRMs in a vimentin antigen-specific systemic sclerosis hyperactivation animal model following treatment with STAT6 inhibitors. DETAILED DESCRIPTION

[0034] Best Mode for Carrying Out the Invention

[0035] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of technologies with distinct main themes in the technical field to which the present invention belongs will be omitted. Furthermore, in the process of describing the present invention, when it is determined that the specific description of the relevant known functions or structures may confuse the gist of the present invention, the detailed descriptions will be omitted. Furthermore, the terminology used in this specification is used to appropriately express the preferred embodiments of the present invention, and may vary depending on the intention of the user, the operator, or the management of the technical field to which the present invention belongs.

[0036] Therefore, these terms should be defined based on the overall content of this specification. Throughout the specification, when a part is mentioned to "comprise" a certain structural element, unless otherwise stated, it means that other structural elements may also be included, rather than excluding other structural elements.

[0037] The present invention provides a pharmaceutical composition for preventing or treating systemic sclerosis, comprising a STAT6 inhibitor as an active ingredient.

[0038] The term "prevention" used in the present invention refers to all actions to suppress the symptoms of a specific disease or delay the progression of the disease by administering the composition of the present invention.

[0039] The term "treatment" used in the present invention refers to any action to improve or change the symptoms of a specific disease to cure by administering the composition of the present invention.

[0040] In addition to the active ingredients, the pharmaceutical composition of the present invention may further include an adjuvant. The adjuvant may be any adjuvant known in the technical field to which the present invention belongs without limitation, and its effect may be increased by further including Freund's complete adjuvant or incomplete adjuvant.

[0041] The pharmaceutical composition of the present invention can be prepared in a form in which the active ingredient is mixed into a pharmaceutically acceptable carrier. Wherein, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. The pharmaceutically acceptable carrier that can be used in the pharmaceutical composition of the present invention can be lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto.

[0042] The pharmaceutical composition of the present invention can be formulated according to conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions for use.

[0043] In the case of formulation, fillers, extenders, binders, wetting agents, disintegrants, surfactants and other diluents or excipients can be used to prepare. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and the above-mentioned solid preparations can be prepared by mixing more than one excipient to the active ingredient, for example, mixed starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talcum powder can also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin as commonly used diluents, a variety of excipients can also be included, for example, wetting agents, sweeteners, aromatics, preservatives, etc. For parenteral administration, sterilized aqueous solutions, non-water-soluble solvents, suspensions, oils, freeze-dried preparations and suppositories are included. Non-water-soluble solvents and suspensions can use propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. As the base of the suppository, witepsol, Tween 61, cocoa butter, caraway resin, glycerin gelatin, etc. can be used.

[0044] The pharmaceutical composition of the present invention can be administered to an individual by a variety of routes. All modes of administration are contemplated, for example, administration can be performed by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0045] The dosage of the pharmaceutical composition of the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient contained in the above-mentioned pharmaceutical composition can be variously selected according to the object, and preferably, the pharmaceutical composition contains an active ingredient at a concentration of 0.01 to 5000 μg / ml. If the concentration is less than 0.01 μg / ml, the drug activity may not be shown, and if it is greater than 5000 μg / ml, it will show toxicity in the human body.

[0046] The "STAT6 (Signal transducer and activator of transcription 6)" of the present invention is a transcription factor of the term STAT series, and the proteins of the STAT series transduce signals from the receptor complex to the cell nucleus and activate gene expression. Similar to other STAT series proteins, STAT6 is also activated by growth factors and cytokines, and it is known that STAT6 is mainly activated by IL-4 and IL-13. The STAT6 signal transduction pathway is required for the development of Th2 cells and Th2 immune responses. It is known that the activation of STAT6 signal transduction is necessary for the function of macrophages and is also required for the activation of the M2 subtype of macrophages.

[0047] According to one embodiment of the present invention, the STAT6 inhibitor may be a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0048] Chemical formula 1

[0049]

[0050] According to one embodiment of the present invention, the above-mentioned STAT6 inhibitor may also include a substance that inhibits the gene expression of STAT6 by specifically binding to the STAT6 gene or a substance that inhibits the expression or activity of the STAT6 protein by specifically binding to the STAT6 protein. The above-mentioned substance that inhibits the expression of the STAT6 gene may be selected from the group consisting of antisense nucleotides (antisense nucleotids), small interfering ribonucleic acids (siRNA) and short hairpin ribonucleic acids (shRNA) that complementarily bind to the messenger ribonucleic acid of the STAT6 gene. The above-mentioned inhibitor of the expression or activity of the STAT6 protein may be selected from the group consisting of peptides, peptidomimetics, substrate analogs, nucleic acid aptamers and antibodies that complementarily bind to the STAT6 protein, but as long as it is a substance that inhibits the expression and activity of the STAT6 gene or protein, it is not limited.

[0051] According to an embodiment of the present invention, the systemic sclerosis may further include systemic sclerosis with increased disease activity due to vimentin.

[0052] According to one embodiment of the present invention, the STAT6 inhibitor can reduce skin thickness.

[0053] According to one embodiment of the present invention, the STAT6 inhibitor can inhibit tissue fibrosis induced by systemic sclerosis, and the tissue can be a tissue selected from the group consisting of skin, lung, liver, muscle, kidney, intestine and spleen, but is not limited thereto.

[0054] According to one embodiment of the present invention, the STAT6 inhibitor can regulate immune cells in tissues, spleen or blood.

[0055] According to one embodiment of the present invention, regulating the above immune cells may be reducing the expression of profibrotic T cells or M2 macrophages, reducing the expression ratio of M2 macrophages to M1 macrophages, or reducing the activity of M2 macrophages.

[0056] According to one embodiment of the present invention, regulating the above immune cells may be increasing the expression of Treg.

[0057] According to one embodiment of the present invention, regulating the above-mentioned immune cells may be reducing the expression of pSTAT6 positive cells and CD8 positive cells or IL-17 positive cells, CD103 positive cells and CD8 positive cells.

[0058] According to an embodiment of the present invention, the STAT6 inhibitor can reduce fibrosis factors, and the fibrosis factors can be factors selected from the group consisting of TGF-β, Col1a1, α-SMA and IL-17.

[0059] According to one embodiment of the present invention, the STAT6 inhibitor can inhibit the activity of vimentin variant protein in tissues, and the vimentin variant protein can be cell surface vimentin.

[0060] The "vimentin variant protein" of the present invention is a cell surface vimentin protein, which is a vimentin protein expressed on the cell surface when myofibroblasts and astrocytes of fibrotic tissue are activated. It is reported that the activated myofibroblasts and astrocytes secrete collagen or extracellular matrix in the tissue cells, causing the tissue to harden, thereby inducing tissue fibrosis. In addition, in an environment where inflammatory cytokines are activated and oxidative stress occurs, filamentous vimentin is deformed under the action of citrullination, phosphorylation, etc., and the deformed vimentin loses its function as a filament and acts as an autoantigen to increase the production of autoantibodies. In addition, the flagged vimentin or deformed vimentin can also act as an autoantigen to induce the activity of macrophages and the like.

[0061] Furthermore, the present invention provides a food composition for preventing or improving systemic sclerosis, comprising a STAT6 inhibitor as an effective ingredient.

[0062] The term "improvement" as used in the present invention refers to all actions that reduce a parameter associated with the state of treatment, such as the extent of symptoms.

[0063] In addition to the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, similar to conventional food compositions.

[0064] Examples of the above-mentioned natural carbohydrates include: monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as sorbitol, erythritol, etc. The above-mentioned flavoring agents can advantageously use natural flavoring agents (thaumatin), stevia extracts (such as rebaudioside A, glycyrrhizic acid, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be prepared in the same manner as the above-mentioned pharmaceutical composition to be used as a functional food, or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meats, chocolates, foods, biscuits, pizzas, instant noodles, other noodles, chewing gums, candies, ice creams, alcoholic beverages, vitamin complexes, and health supplementary foods.

[0065] In addition, in addition to the extract as an effective ingredient, the food composition may also include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may also include pulp for preparing natural fruit juices, fruit juice beverages, and vegetable beverages.

[0066] The functional food composition of the present invention can be prepared and processed into tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing or treating systemic sclerosis. In the present invention, "health functional food composition" refers to a food prepared and processed using raw materials or ingredients with useful functions for the human body according to Korean Law No. 6727 related to health functional food, and is ingested for the purpose of regulating nutrients or obtaining useful effects in health purposes such as physiological effects for the structure and function of the human body. The health functional food of the present invention may contain common food additives. If there are no other provisions, the suitability of food additives is determined by the general provisions of the Food Additive Code approved by the Korean Food and Drug Safety Administration and the usual test methods, etc., through the specifications and standards related to the relevant items. The items included in the above-mentioned "Food Additive Code" include, for example: chemical synthetics such as ketones, glycerin, calcium citrate, hydrochloric acid, cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, sorghum pigment, guar gum; mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar pigment preparations. For example, the health functional food in tablet form can be granulated by a common method by mixing the active ingredient of the present invention with a mixture of excipients, binders, disintegrants and other additives, and then a lubricant is added to press out the mixture, or the mixture can be directly pressed out. In addition, the health functional food in the form of tablets can also contain flavoring agents as needed. In the health functional food in the form of capsules, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients in a common hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The soft capsules can contain plasticizers such as glycerol or sorbitol, colorants, preservatives, etc. as needed. The health functional food in pill form can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. by existing known methods, and can be coated with white sugar or other coating agents as needed, or using substances such as starch and talcum powder on the surface coating. The health functional food in the form of granules can be prepared by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc. into granules by a conventionally known method, and can contain flavoring agents, flavoring agents, etc. as needed.

[0067] Furthermore, the present invention provides a quasi-drug composition for preventing or treating systemic sclerosis, comprising a STAT6 inhibitor as an active ingredient.

[0068] The term "quasi-drug" used in the present invention refers to an item used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing myopathy in humans or animals, and its effect is milder than that of a drug. For example, according to the Korean Pharmaceutical Affairs Act, a quasi-drug refers to a product used in the treatment or prevention of human / animal diseases other than an item used for the purpose of a drug, including products that have a mild effect on the human body or do not directly act on the human body.

[0069] The above-mentioned quasi-drug composition of the present invention can be prepared in a dosage form selected from the group consisting of shower gel, disinfectant cleaner, detergent, kitchen cleaner, cleanser, toothpaste, mouthwash, wet wipes, detergent, soap, hand soap, shampoo, softener, humidifier filler, facial mask, ointment and filter filler, but is not limited thereto.

[0070] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating fibrosis caused by vimentin variant protein, comprising a STAT6 inhibitor as an active ingredient.

[0071] Furthermore, the present invention provides a method for treating systemic sclerosis, comprising the step of administering a STAT6 inhibitor to an individual in a pharmaceutically effective amount.

[0072] The treatment method of the present invention includes the step of administering a compound of the above-mentioned chemical formula 1 or a pharmaceutically acceptable salt thereof to an individual in a therapeutically effective amount. Preferably, the specific therapeutically effective amount for a specific individual is applied differently according to various factors including the type and degree of the reaction to be achieved, the specific composition including whether other preparations are used according to the situation, the age, weight, half health status, gender and diet of the individual, the administration time, the route of administration and the secretion rate of the composition, the treatment period, the drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. Based on the amount of the pharmaceutical composition of the present invention, the daily dosage can be 0.0001 to 100 mg / kg, preferably, 0.01 to 100 mg / kg, and can be administered 1 to 6 times a day. However, it is obvious to a person skilled in the art of the present invention that the dosage or dosage of each active ingredient should be such that side effects are not caused by excessively high content of the active ingredient. Therefore, preferably, the effective amount of the composition suitable for the purpose of the present invention should be determined after considering the above matters.

[0073] The above individuals can be applied to any mammals, including not only humans and primates, but also domestic animals such as cows, pigs, sheep, horses, dogs and cats.

[0074] The compound represented by the chemical formula 1 of the present invention or a pharmaceutically acceptable salt thereof can be administered to mammals such as mice, rats, livestock, humans, etc. through various routes. All administration methods can be envisioned, for example, administration can be performed by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine or intracerebroventricular injection.

[0075] Furthermore, the present invention provides a method for treating fibrosis caused by vimentin variant protein, comprising the step of administering a STAT6 inhibitor to an individual in a pharmaceutically effective amount. DETAILED DESCRIPTION

[0077] The present invention is described in more detail below by way of examples. However, it is obvious to those skilled in the art that these examples are only used to describe the present invention in more detail, and the scope of the present invention is not limited to these examples.

[0078] Example 1: Confirmation of regulation of fibrosis-related immune cells by inhibiting STAT6

[0079] In order to confirm the inhibitory effect of the present invention on STAT6 related to systemic sclerosis, the regulatory effect of fibrosis-related immune cells was confirmed. Specifically, spleen single cells of B6 mice were obtained and treated with 10ng / ml transforming growth factor β (TGF-β) and 10mM STAT6 inhibitor (YM-341619, Chemical Formula 1), cultured for 72 hours and analyzed by flow cytometry for the expression of profibrotic T cells, M2 macrophages and Tregs associated with systemic sclerosis. In addition, the regulation of immune cells was confirmed by confirming the expression ratio of M2 macrophages and M1 macrophages. The untreated control group (Nil) and the TGF-β alone treatment group were used as the control group.

[0080] Chemical formula 1

[0081]

[0082] Results, such as Figure 1 As shown in Figure 2, in the group treated with TGF-β alone, the expression of profibrotic T cells and M2 macrophages increased compared with the Nil group, but in the group treated with STAT6 inhibitor, the increased expression of profibrotic T cells and M2 macrophages was confirmed to be significantly reduced ( Figure 1 In addition, it was confirmed that the expression of Tregs in the group treated with STAT6 inhibitors was significantly increased compared with the group treated with TGF-β alone ( Figure 1In addition, it was confirmed that the expression ratio of M2 / M1 increased in the TGF-β alone treatment group compared with the Nil group, but the expression ratio of M2 / M1 obtained by TGF-β was significantly reduced by the treatment of STAT6 inhibitor ( Figure 1 D), thus confirming that STAT6 inhibitors regulate immune cells associated with systemic sclerosis.

[0083] Example 2: Confirmation of inhibition of fibrosis factor expression by inhibition of STAT6

[0084] In order to confirm the inhibitory effect of the present invention on STAT6 related to systemic sclerosis, the inhibition of fibrogenic factors of fibroblasts was confirmed. Specifically, human dermal fibroblast strains were treated with 10ng / ml of TGF-β and 10nM of STAT6 inhibitor and cultured for 24 hours. Then, the expression of TGF-β, Col1a1 (Collagen, type I, alpha 1) and α-SMA (alpha smooth muscle actin) as fibrogenic factors was confirmed by real time polymerase chain reaction (real time PCR).

[0085] Results, such as Figure 2 As shown, it was confirmed that the expression of fibrogenic factors TGF-β, Col1a1, and α-SMA in fibroblasts was significantly increased in the group treated with TGF-β alone compared with the Nil group, but the increased expression of fibrogenic factors was significantly reduced when the STAT6 inhibitor was treated.

[0086] Example 3: Confirmation of the regulation of M2 macrophage activity following inhibition of STAT6 in an animal model of systemic sclerosis

[0087] In order to confirm whether the STAT6 inhibitor of the present invention improves systemic sclerosis, an animal model of systemic sclerosis was prepared. Specifically, after removing the hair of the 2×2 cm long and wide area on the back of the neck of B6 mice with scissors (clipper), 100 μl of phosphate buffered saline (PBS) containing 50 μg of Bleomycin (BLM) was subcutaneously injected every day for 2 weeks to induce systemic sclerosis. One week after the start of BLM administration, 200 μl of distilled water (DW) containing a STAT6 inhibitor was orally administered at a concentration of 10 mg / kg every day. Then, blood from mice in the first and fifth weeks of inducing systemic sclerosis was obtained, and the activity of M2 macrophages that can induce fibrosis was analyzed by flow cytometry from the obtained blood. Wild-type mice (WT) and the solvent (Vehicle) group that was not treated with drugs after inducing systemic sclerosis were used as the control group.

[0088] Results, such as Figure 3 As shown, it was confirmed that the activity of M2 macrophages that can induce fibrosis was significantly increased in the solvent group compared with wild-type mice, but the increased activity of M2 macrophages that can induce fibrosis was significantly reduced in the group administered with STAT6 inhibitors, thus confirming that STAT6 inhibitors can improve systemic sclerosis.

[0089] Example 4: Confirmation of inhibition of tissue fibrosis following inhibition of STAT6 in an animal model of systemic sclerosis

[0090] 4-1: Confirmation of inhibition of skin fibrosis

[0091] In order to confirm whether the STAT6 inhibitor of the present invention improves systemic sclerosis, the inhibition of skin fibrosis was confirmed. Specifically, the mice of Example 3 above were euthanized at the end of the experiment, and the skin tissue was obtained and stained with hematoxylin and eosin (H&E), and the skin inflammation reaction and fibrosis inhibition effect were confirmed by confirming the skin thickness.

[0092] Results, such as Figure 4 As shown, it was confirmed that the thickness of the dermis layer was significantly increased in the vehicle group compared with the wild-type mice, but the increased thickness of the dermis layer was significantly reduced by administration of the STAT6 inhibitor.

[0093] 4-2: Confirmation of the inhibitory effect on pulmonary fibrosis

[0094] To confirm whether the STAT6 inhibitor of the present invention is effective in pulmonary fibrosis in systemic sclerosis, lung tissue was obtained from the mice euthanized in Example 4-1 above, and hematoxylin and eosin staining was performed to confirm the histological score of pulmonary fibrosis.

[0095] Results, such as Figure 5 As shown, it was confirmed that the histological score was significantly increased in the vehicle group due to increased inflammation and fibrosis in the lung compared with wild-type mice, but the increased histological score was significantly reduced when the STAT6 inhibitor was administered.

[0096] Example 5: Confirmation of inhibition of expression of fibrotic factors and cell surface vimentin in systemic sclerosis

[0097] 5-1: Confirmation of inhibition of the expression of fibrotic factors and cell surface vimentin in skin tissue

[0098] Vimentin has been reported to play an important role in cell interactions and immune system function in autoimmune diseases. Deformed or native forms of vimentin have been implicated in inflammatory responses and autoimmunity. Disease pathogenesis related (Autoimmun Rev.2018Sep;17(9):926-934.doi :10.1016 / j.autrev.2018.04.004.Epub2018Jul 17.), in systemic sclerosis, the effect of STAT6 inhibitors on the expression of cell surface vimentin (CSV) was also confirmed. Specifically, the expression of α-SMA, Col1, IL-17, which are fibrosis factors, and CSV-positive cells were confirmed by immunohistochemical staining in the skin tissue obtained in Example 4-1.

[0099] Results, such as Figure 6 As shown, compared with wild-type mice, the expression of α-SMA, Col1, and IL-17, which are fibrosis factors in skin tissue, and the number of CSV-positive cells were significantly increased in the solvent group due to increased skin fibrosis, but when the STAT6 inhibitor was administered, the increased expression of α-SMA, Col1, and IL-17 and the number of CSV-positive cells were significantly reduced.

[0100] 5-2: Confirmation of inhibition of the expression of fibrotic factors and cell surface vimentin in lung tissue

[0101] It was confirmed whether the STAT6 inhibitor of the present invention inhibits cell surface vimentin in tissues. Specifically, the expression of fibrosis factors α-SMA, Col1, IL-17, and CSV-positive cells were confirmed by immunohistochemical staining in the lung tissues obtained in Example 4-2.

[0102] Results, such as Figure 7As shown, it was confirmed that the expression of α-SMA, Col1, and IL-17, which are fibrosis factors in skin tissue, and the number of CSV-positive cells were significantly increased in the solvent group due to the increase in lung fibrosis compared with wild-type mice, but when the STAT6 inhibitor was administered, the increased expression of α-SMA, Col1, and IL-17 and the number of CSV-positive cells were significantly reduced.

[0103] Example 6: Confirmation of inhibition of STAT6 expression in CD8 T cells

[0104] In systemic sclerosis, STAT6 is a major transcription factor of CD8 T cells. It has recently been known that in systemic sclerosis, the fibrotic response and inflammatory response of fibroblasts are increased due to the continuous survival of resident memory CD8 T cells in tissues. Therefore, it was confirmed whether the expression of pSTAT6-positive and CD8-positive T cells, which are active forms of STAT6, is regulated by treating with the STAT6 inhibitor of the present invention. Specifically, pSTAT6 and CD8-positive T cells were analyzed by confocal microscopy in the skin and lung tissues obtained in Example 4 above.

[0105] Results, such as Figure 8 As shown, it was confirmed that the expression of pSTAT6 and CD8-positive T cells in the skin and lung tissues of the group treated with STAT6 inhibitor (STAT6 inhibitor) was significantly reduced compared with the solvent group as a systemic sclerosis animal model, thereby confirming that the treatment with STAT6 inhibitor reduced the expression of active STAT6 and CD8-positive T cells.

[0106] Example 7: Confirmation of STAT6 inhibition effect in a vimentin antigen-specific systemic sclerosis animal model

[0107] 7-1: Confirmation of the effect of inhibiting skin and tissue fibrosis

[0108] In systemic sclerosis, vimentin increases the disease activity of systemic sclerosis, so the effect of STAT6 inhibitors was evaluated in a vimentin antigen-specific systemic sclerosis animal model. Specifically, in 8-week-old SKG mice, vimentin protein was used as an antigen, and after immunization, 100 μl of Belomycin (BLM) was subcutaneously injected 6 times within 2 weeks to induce systemic sclerosis. Then, in the second week of inducing systemic sclerosis, after secondary immunization with vimentin protein, the STAT6 inhibitor of the present invention was subcutaneously injected at a concentration of 10 mg / kg 3 times a week until the fifth week of the experiment. Then, at the end of the experiment, the mice were euthanized, and the skin and lung tissues were obtained. The thickness of the skin dermis was confirmed by hematoxylin and eosin staining, and the expression of CSV in the lung tissue was confirmed by immunohistochemical staining. The specific experimental process is as follows Fig. 9 As shown, a vimentin antigen-specific systemic sclerosis animal model (vim SSc group) in which disease activity was increased by vimentin antigen was used as a control group.

[0109] Results, such as Fig.10 As shown, it was confirmed that the thickness of the skin dermis layer in the group treated with STAT6 inhibitors was significantly reduced compared with the vim SSc group. In addition, it was confirmed that the expression of CSV in lung tissue was significantly reduced, thus confirming that the STAT6 inhibitor of the present invention can improve systemic sclerosis in which the disease activity is increased by vimentin antigen.

[0110] 7-2: Confirmation of regulatory IL-17 expressing cells

[0111] It is known that in systemic sclerosis, CD8 TRM persists in tissues and accelerates skin inflammation and fibrosis. Therefore, it is confirmed whether the STAT6 inhibitor of the present invention inhibits the expression of CD8 TRM. Specifically, in the skin tissue of the mice euthanized in the above Example 7-1, DAPI, anti-CD4 FITC, anti-CD103-PE, anti-IL-17-APC and anti-IFNγ-APC were used for staining and CD103 and CD8 positive cells expressing IL-17 were confirmed by confocal microscopy. In addition, CD103 and CD8 positive cells expressing IFNγ were confirmed.

[0112] Results, such as Fig.11 As shown, it was confirmed that the IL-17-expressing CD103 and CD8-positive cells (CD8TRM), which are the causative cells of systemic sclerosis, were significantly reduced in the group treated with STAT6 inhibitors compared with the vim SSc group. In addition, there was no change in IFNγ-expressing CD103 and CD8-positive cells, which are immune regulatory cells.

[0113] Therefore, the present invention confirms that STAT6 inhibitors inhibit the expression of profibrotic T cells and M2 macrophages, which are immune cells associated with systemic sclerosis, increase the expression of Treg, and confirm the inhibition of the expression of TGF-β, Col1a1 and α-SMA, which are fibrotic factors associated with systemic sclerosis. In addition, it was confirmed in a systemic sclerosis animal model that when STAT6 inhibitors were administered, the activity of M2 macrophages that can induce fibrosis in the blood of mice was suppressed, and the thickness of the dermis of the skin and pulmonary fibrosis were suppressed. In addition, in skin tissue and lung tissue, it was confirmed that the expression of cell surface vimentin, which is a vimentin variant protein associated with systemic sclerosis, was reduced, and it was confirmed that vimentin variant proteins can be regulated. In addition, the presence of pSTAT6-expressing CD8 T cells was confirmed in fibrotic tissues, and it was confirmed that the expression of pSTAT6-expressing CD8 T-positive cells was regulated by administering STAT6 inhibitors. In an animal model of vimentin-specific disease activity increase, it was also confirmed that STAT6 inhibitors inhibited tissue fibrosis in systemic sclerosis with antigen-specific disease activity increase, and suppressed the expression of IL-17 cytokine-expressing CD8-positive TRMs, which are the causative cells of systemic sclerosis and can induce inflammation and fibrosis.

Claims

1. A pharmaceutical composition for preventing or treating systemic sclerosis, characterized in that: Contains a STAT6 inhibitor as an active ingredient.

2. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 1, characterized in that: The STAT6 inhibitor is a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: Chemical formula 1:

3. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 1, characterized in that: The above-mentioned STAT6 inhibitors also include substances that inhibit STAT6 gene expression by specifically binding to the STAT6 gene or substances that inhibit the expression or activity of the STAT6 protein by specifically binding to the STAT6 protein.

4. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 3, characterized in that: The substance that inhibits the expression of the STAT6 gene is selected from the group consisting of antisense nucleotides, small interfering RNAs and short hairpin RNAs that complementarily bind to the messenger RNA of the STAT6 gene.

5. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 3, characterized in that: The substance that inhibits the expression or activity of STAT6 protein is selected from the group consisting of peptides, peptidomimetics, substrate analogs, nucleic acid aptamers and antibodies that complementarily bind to STAT6 protein.

6. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 1, characterized in that: The above-mentioned systemic sclerosis also includes systemic sclerosis in which disease activity is increased by vimentin.

7. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 1, characterized in that: The above STAT6 inhibitors reduce skin thickness.

8. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 1, characterized in that: The above-mentioned STAT6 inhibitors inhibit tissue fibrosis induced by systemic sclerosis.

9. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 8, characterized in that: The above-mentioned tissue is a tissue selected from the group consisting of skin, lung, liver, muscle, kidney, intestine and spleen.

10. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 1, characterized in that: The above-mentioned STAT6 inhibitors regulate immune cells in tissues, spleen or blood.

11. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 10, characterized in that: Regulating the above immune cells is to reduce the expression of profibrotic T cells or M2 macrophages.

12. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 10, characterized in that: The above immune cells are regulated to reduce the expression ratio of M2 macrophages to M1 macrophages.

13. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 10, characterized in that: Regulating the above immune cells is to reduce the activity of M2 macrophages.

14. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 10, characterized in that: The above immune cells are regulated to increase the expression of Treg.

15. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 10, characterized in that: Regulating the above immune cells is to reduce the expression of pSTAT6 positive cells and CD8 positive cells or to reduce the expression of IL-17 positive cells, CD103 positive cells and CD8 positive cells.

16. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 1, characterized in that: The above STAT6 inhibitors reduce fibrotic factors.

17. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 16, characterized in that: The fibrosis factor is a factor selected from the group consisting of TGF-β, Col1a1, α-SMA and IL-17.

18. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 1, characterized in that: The above STAT6 inhibitors inhibit the activity of vimentin variant proteins in tissues.

19. The pharmaceutical composition for preventing or treating systemic sclerosis according to claim 18, characterized in that: The above-mentioned vimentin variant protein is cell surface vimentin.

20. A food composition for preventing or improving systemic sclerosis, characterized in that: Contains a STAT6 inhibitor as an active ingredient.

21. A quasi-drug composition for preventing or treating systemic sclerosis, characterized in that: Contains a STAT6 inhibitor as an active ingredient.

22. A pharmaceutical composition for preventing or treating fibrosis caused by vimentin variant protein, characterized in that: Contains a STAT6 inhibitor as an active ingredient.

23. A method for treating systemic sclerosis, characterized in that: The method comprises the step of administering to the individual a pharmaceutically effective amount of a STAT6 inhibitor.

24. A method for treating fibrosis caused by vimentin variant protein, characterized in that: The method comprises the step of administering to the individual a pharmaceutically effective amount of a STAT6 inhibitor.

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