Method for treating metabolic dysfunction-associated steatohepatitis through co-administration of CCR2 inhibitor and TGF-beta receptor inhibitor

Abstract

The present invention relates to a method for treating metabolic dysfunction-associated steatohepatitis through co-administration of a CCR2 inhibitor and a TGF-beta receptor inhibitor. Specifically, when administering a C-C chemokine receptor 2 (CCR2) inhibitor and a transforming growth factor beta (TGF-beta) receptor inhibitor alone or in combination to an animal model in which steatohepatitis was induced by diet, it has been identified that the TGF-beta receptor inhibitor alone significantly alleviated liver fibrosis but increased fat accumulation in liver tissue, and the CCR2 inhibitor alone inhibited inflammatory cell accumulation and fat synthesis in liver tissue but did not completely alleviate fibrosis, whereas, in the group to which these two substances were co-administered, liver fibrosis was significantly alleviated and, at the same time, inflammatory cells accumulated in liver tissue and fat accumulation were also inhibited, and ER stress and mitochondrial dysfunction were alleviated. Therefore, a combination formulation of the CCR2 inhibitor and the TGF-beta receptor inhibitor can be effectively used for the purpose of preventing, alleviating or treating metabolic liver diseases including metabolic dysfunction-associated steatohepatitis.

Description

Treatment of metabolic steatohepatitis using combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor

[0001] The present invention relates to a pharmaceutical composition, quasi-drug, and food composition for preventing, improving, or treating metabolic liver disease, which comprises as active ingredients a CCR2 (CC chemokine receptor 2) inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β (transforming growth factor beta) receptor inhibitor or a pharmaceutically acceptable salt thereof.

[0002] Fatty liver disease includes alcohol-induced fatty liver disease and metabolic liver disease such as hepatic steatosis, steatohepatitis, or liver fibrosis, which are the result of metabolic dysregulation.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a type of metabolic liver disease, unrelated to alcohol consumption, caused by fat accumulation in the liver. NAFLD encompasses a group of conditions, including simple steatosis, which simply involves excessive fat accumulation in hepatocytes, and non-alcoholic steatohepatitis (NASH), which is accompanied by hepatocyte necrosis, inflammation, and fibrosis.

[0004] Nonalcoholic steatohepatitis (NASH) occurs as nonalcoholic fatty liver disease (NAFLD) worsens. First, inflammatory cytokines are secreted when destroyed hepatocyte debris is engulfed by Kupffer cells and macrophages. These cytokines activate hepatic stellate cells, which regulate blood flow between the hepatic sinusoid endothelial cells and hepatocytes, synthesizing and secreting connective tissue components, including collagen, leading to fibrosis. As this process progresses, NASH progresses beyond simple steatosis, characterized by fatty liver cells, to a serious lesion characterized by ballooning, inflammation, and fibrosis.

[0005] As research into the causes and mechanisms of the disease progresses, the term "nonalcoholic fatty liver disease" (NAFLD) has been criticized for failing to reflect its heterogeneous nature and diverse course. Therefore, changes to the term and definition have been proposed. While obesity and metabolic syndrome are independent risk factors for NAFLD, a significant number of patients with NAFLD have a normal body mass index (BMI), meaning they are lean or non-obese. Reflecting this, in 2020, international liver experts from Europe, South America, North America, and Asia-Pacific issued a joint statement proposing to change the name of NAFLD to metabolic dysfunction associated fatty liver disease (MAFLD) (J Hepatol 2020;73:202-209), and at the European Association for the Study of the Liver (EASL 2023) Congress held in June 2023, the multinational liver association renamed NAFLD to metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD was defined as including patients with hepatic steatosis and at least one of the five cardiometabolic risk factors. In addition, nonalcoholic steatohepatitis (NASH) was decided to be replaced with metabolic dysfunction-associated steatohepatitis (MASH).

[0006] Meanwhile, signaling pathways that induce hepatocyte damage can originate in adipose tissue or the intestine and exacerbate local inflammatory mechanisms within the liver, including Kupffer cells, monocytes, and lymphocytes. While various signaling pathways are simultaneously activated, identifying the pathway most closely associated with disease progression is crucial for developing targeted therapies. In particular, the CCL2 / CCL5 ligand for CCR2 / 5 (CC chemokine receptor 2 / 5) is activated and upregulated in NASH. Furthermore, CCR2 / 5, a chemokine receptor, is expressed on monocytes, macrophages, Kupffer cells, hepatic stellate cells, natural killer cells, and T cells, playing a crucial role in the activation of hepatic stellate cells and the progression of fibrosis. Therefore, cenicriviroc (CVC), which acts on CCR2 / CCR5, has been shown to exhibit anti-inflammatory and antifibrotic effects in preclinical animal models. Based on this, a phase 3 clinical trial was conducted to observe the effect of treating liver fibrosis in patients with non-alcoholic steatohepatitis with advanced liver fibrosis, but it was reported that the effect was not as great as expected.

[0007] Furthermore, transforming growth factor-β (TGF-β) is a cytokine that promotes fibrosis in the liver by transdifferentiating hepatic stellate cells (HSCs) into myofibroblasts. Therefore, blocking TGF-β signaling in chronic liver diseases is known to be an ideal method for treating liver fibrosis. EW-7197, a TGF-β receptor inhibitor, is a highly potent, selective, and orally available inhibitor of the TGF-β receptor Activin receptor-like kinase 4 (AKL4) or Activin receptor-like kinase 5 (AKL5). In a previous study, the above EW-7197 was found to not only suppress liver damage in a liver fibrosis model induced by carbon tetrachloride (CCl4) administration and bile duct ligation, but also to have the effect of suppressing tissue fibrosis by regulating TGF-β / Smad and ROS signals in a model of renal fibrosis induced by unilateral ureteral obstruction (UUO) and lung fibrosis induced by bleomycin (BLM).

[0008] However, TGF-β receptor inhibitors have the side effect of promoting hepatic fat production in the process of suppressing liver fibrosis, so more effective treatment methods for nonalcoholic steatohepatitis are required.

[0009] Accordingly, the inventors of the present invention have made efforts to develop a more effective treatment method for metabolic liver disease, particularly non-alcoholic steatohepatitis, and have newly discovered that by co-administering a CCR2 inhibitor and a TGF-β receptor inhibitor, liver fibrosis can be suppressed, fat accumulation in the liver can be suppressed, and ER stress and mitochondrial dysfunction can be improved, thereby completing the present invention.

[0010] [Prior Art Literature]

[0011] [Non-patent literature]

[0012] Anstee et al. Cenicriviroc Lacked Efficacy to Treat Liver Fibrosis in Nonalcoholic Steatohepatitis: AURORA Phase III Randomized Study. Clinical Gastroenterology and Hepatology 2024;22:124-134

[0013] Existing CCR2 / CCR5 antagonists have been shown to be ineffective in treating liver fibrosis in patients with nonalcoholic steatohepatitis.

[0014] In addition, existing TGF-β (Transforming growth factor-β) receptor inhibitors have an anti-fibrotic effect, but they have the problem of increasing fat production in liver cells during the process of suppressing tissue fibrosis.

[0015] The purpose of the present invention is to solve the above problems, and to provide a pharmaceutical composition for preventing or treating metabolic liver disease, which comprises as active ingredients a CCR2 (CC chemokine receptor 2) inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof.

[0016] Another object of the present invention is to provide a quasi-drug for preventing or treating metabolic liver disease, comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients.

[0017] Another object of the present invention is to provide a food composition for preventing or improving metabolic liver disease, comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients.

[0018] Another object of the present invention is to provide a complex, mixed or combined preparation kit for treating metabolic liver disease, which comprises a preparation comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, in a complex, mixed or combined manner.

[0019] In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating metabolic liver disease, comprising as active ingredients a CCR2 (CC chemokine receptor 2) inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a pharmaceutical composition for use in preventing or treating metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a method for preventing or treating metabolic liver disease, comprising a step of administering a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; and a use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for preventing or treating metabolic liver disease.

[0020] In addition, the present invention provides a quasi-drug for preventing or treating metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a quasi-drug for use in preventing or treating metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; and a use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof for manufacturing a quasi-drug for preventing or treating metabolic liver disease.

[0021] In addition, the present invention provides a food composition for preventing or improving metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a food composition for use in preventing or improving metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a method for preventing or improving metabolic liver disease, comprising a step of administering a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; and a use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof for producing a food composition for preventing or improving metabolic liver disease.

[0022] In addition, the present invention provides a combination, mixture or combination kit for treating metabolic liver disease comprising a formulation comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, in a complex, mixed or combined manner; a combination, mixed or combination kit for use in treating metabolic liver disease comprising a formulation comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; as active ingredients, in a complex, mixed or combined manner; a method for treating metabolic liver disease, comprising a step of combining, mixing or combined administration of a formulation comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; as active ingredients, and a combination, mixed or combination kit for preparing a combination, mixed or combination kit for treating metabolic liver disease, comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; And the use of a formulation comprising a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient is provided.

[0023] In the present invention, when RS102895 as a CCR2 (CC chemokine receptor 2) inhibitor and EW-7197 as a TGF-β receptor inhibitor were administered alone or in combination to an animal model in which steatohepatitis was induced by diet, EW-7197 alone significantly improved liver fibrosis, but increased fat accumulation in liver tissue, and the CCR2 inhibitor alone suppressed the accumulation of inflammatory cells and fat synthesis in liver tissue, but did not completely improve fibrosis, whereas in the group administered in combination with these two substances, liver fibrosis was significantly improved, and inflammatory cells accumulated in liver tissue and fat accumulation were also suppressed, and ER stress and mitochondrial dysfunction were improved. Therefore, the combination preparation of the CCR2 inhibitor and EW-7197 can be usefully used for the purpose of preventing, improving, or treating metabolic liver diseases including steatohepatitis associated with metabolic disorders.

[0024] Figure 1 is a diagram showing the inhibitory effect of liver fibrosis on a mouse hepatocyte cell line induced with fibrosis by TGF-β, following treatment with RS102895 as a CCR2 (CC chemokine receptor 2) inhibitor and / or EW-7197 as a TGF-β receptor inhibitor, as measured by protein expression of fibrosis markers (*p < 0.05 vs. CON; # p < 0.05 vs. TGF-β; † p < 0.05 vs. TGF-β+CCR2i).

[0025] Figure 2 shows the inhibitory effect of liver fibrosis on a mouse hepatocyte cell line induced with fibrosis by TGF-β, following treatment with RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor, as confirmed through protein expression of fibrosis markers and signals related thereto (*p < 0.05 vs. CON; # p < 0.05 vs. TGF-β; † p < 0.05 vs. TGF-β+CCR2i).

[0026] Figure 3 shows the inhibitory effect of liver fibrosis on human hepatic stellate cell lines induced with fibrosis by TGF-β, following treatment with RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor, as measured by protein expression of fibrosis markers (**p < 0.05 vs. CON; ## p < 0.05 vs. TGF-β; †† p < 0.05 vs. TGF-β+CCR2i).

[0027] Figure 4 is a diagram showing the inhibitory effect of liver fibrosis on human hepatic stellate cell lines induced with fibrosis by TGF-β, following treatment with RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor, as determined by protein expression of fibrosis markers and signals related thereto (*p < 0.05 vs. CON; # p < 0.05 vs. TGF-β; † p < 0.05 vs. TGF-β+CCR2i).

[0028] Figure 5 is a diagram showing the effect of inhibiting fat synthesis after treatment with RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor together with palmitic acid treatment or TGF-β treatment in a mouse hepatocyte cell line, confirmed through BODIPY staining.

[0029] Figure 6 shows the protein expression of fat accumulation factors and fat metabolism regulators after treatment with RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in a mouse hepatocyte cell line induced to undergo fibrosis by TGF-β (*p < 0.05 vs. CON; # p < 0.05 vs. TGF-β; † p < 0.05 vs. TGF-β+CCR2i).

[0030] Figure 7 is a diagram confirming the change in body weight according to administration of RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the MCD diet.

[0031] Figures 8 and 9 are diagrams confirming physiological changes according to administration of RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by MCD diet (Figure 8: *p < 0.05 vs. CON, Figure 9: *p < 0.05 vs. CON; # p < 0.05 vs. MCD; † p < 0.05 vs. MCD+CCR2i).

[0032] Figure 10 is a diagram showing the effect of inhibiting fatty liver by administering RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the MCD diet, as confirmed by H&E staining (*p < 0.05 vs. CON; # p < 0.05 vs. MCD).

[0033] Figure 11 is a diagram confirming the effect of inhibiting liver inflammation by administering RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the MCD diet (*p < 0.05 vs. CON; # p < 0.05 vs. MCD).

[0034] Figure 12 is a diagram showing the effect of inhibiting liver inflammation by administering RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the MCD diet, as confirmed through protein expression of inflammatory signals and related markers (*p < 0.05 vs. CON; # p < 0.05 vs. MCD).

[0035] Figure 13 is a diagram showing the effect of inhibiting liver inflammation by administering RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by an MCD diet, as confirmed through staining with F4 / 80, an inflammatory marker.

[0036] Figure 14 is a diagram showing the inhibitory effect of liver fibrosis on non-alcoholic steatohepatitis animal models induced by the MCD diet, following administration of RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor, using Masson's trichrome staining and Sirius red staining.

[0037] Figure 15 is a diagram showing the inhibitory effect of liver fibrosis on the mRNA expression of liver fibrosis markers by administering RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the MCD diet (*p < 0.05 vs. CON; # p < 0.05 vs. MCD).

[0038] Figure 16 is a diagram showing the inhibitory effect of liver fibrosis on the administration of RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the MCD diet, as confirmed through protein expression of liver fibrosis markers and signals related thereto.

[0039] Figures 17 and 18 are diagrams confirming physiological changes in blood following administration of RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in a non-alcoholic steatohepatitis animal model by GAN diet (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0040] Figures 19 and 20 are graphs showing the comparison of adipocyte size and fat mass (% of body weight) of inguinal white adipose tissue and H&E staining and triglyceride of liver tissue according to administration of RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in a non-alcoholic steatohepatitis animal model induced by a GAN diet (Figure 19: ns, not significant, **p < 0.01, ***p < 0.001, Figure 20: *p < 0.05, ****p < 0.0001).

[0041] Figure 21 is a diagram showing the inhibitory effect of liver fibrosis according to administration of RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in a non-alcoholic steatohepatitis animal model induced by a GAN diet, as confirmed by sirius red staining and hydroxyproline in liver tissue (*p < 0.05, ****p < 0.0001).

[0042] Figure 22 is a diagram confirming the effect of improving energy expenditure by administering RS102895 as a CCR2 inhibitor and EW-7197 as a TGF-β receptor inhibitor in a non-alcoholic steatohepatitis animal model induced by a GAN diet (*p < 0.05).

[0043] Figure 23 is a diagram confirming the effect of improving ER stress (endoplasmic reticulum stress) in liver tissue by administering RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by a GAN diet (*p < 0.05 vs. CON; # p < 0.05 vs. GAN).

[0044] Figure 24 is a diagram confirming the effect of improving mitochondrial abnormalities in liver tissue following administration of RS102895 as a CCR2 inhibitor and EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by a GAN diet (*p < 0.05 vs. CON; # p < 0.05 vs. GAN).

[0045] Figure 25 is a diagram confirming the effect of improving energy consumption by administering RS102895 as a CCR2 inhibitor and EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by a CDAHFD diet (*p < 0.05).

[0046] Figure 26 is a diagram confirming the effect of improving ER stress in liver tissue by administering RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in a non-alcoholic steatohepatitis animal model induced by a CDAHFD diet (*p < 0.05 vs. CON; # p < 0.05 vs. CDAHFD).

[0047] Figure 27 is a diagram confirming the effect of improving mitochondrial dysfunction in liver tissue following administration of RS102895 as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by a CDAHFD diet (*p < 0.05 vs. CON; # p < 0.05 vs. CDAHFD).

[0048] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the invention. These embodiments of the present invention are provided to more fully explain the invention to those of ordinary skill in the art. Therefore, the embodiments of the present invention may be modified in various ways, and the scope of the present invention is not limited to the embodiments described below.

[0049] Throughout the specification of the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0050] As used herein, “pharmaceutically acceptable” means that the contained ingredients do not significantly stimulate the organism and do not inhibit biological activity and properties.

[0051] As used herein, “pharmaceutically acceptable salt” refers to a salt having desirable biological activity, and may include, but is not limited to, inorganic acid salts (hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid), organic acid salts (acetic acid, oxalic acid, maleic acid, fumaric acid, succinic acid, benzoic acid, ascorbic acid, tannic acid, pamoic acid, alginic acid, triethylamine, cyclohexylamine, pyridine), alkali metal salts (sodium salt, potassium salt), alkaline earth metal salts (calcium salt), ammonium salts, or addition salt forms thereof.

[0052] In this specification, “prevention” means any act of suppressing the symptoms or progression of a specific disease (e.g., metabolic liver disease) by administering the composition of the present invention into the body.

[0053] In the present invention, “treatment” means any act of improving or beneficially changing the symptoms of a specific disease (e.g., metabolic liver disease) by administering the composition of the present invention into the body.

[0054] The present invention provides a pharmaceutical composition for preventing or treating metabolic liver disease, comprising as active ingredients a CCR2 (CC chemokine receptor 2) inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a pharmaceutical composition for use in preventing or treating metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a method for preventing or treating metabolic liver disease, comprising a step of administering a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; and a use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for preventing or treating metabolic liver disease.

[0055] In addition, the present invention provides a combination, mixture or combination kit for treating metabolic liver disease, comprising a formulation comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, in a complex, mixed or combined manner; a combination, mixture or combination kit for use in treating metabolic liver disease, comprising a formulation comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, in a complex, mixed or combined manner; a method for treating metabolic liver disease, comprising a step of administering a formulation comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, in a complex, mixed or combined manner; and a combination, mixture or combination kit for preparing a combination, mixture or combination kit for treating metabolic liver disease, comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; And the use of a formulation comprising a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient is provided.

[0056] In one embodiment of the present invention, the CCR2 inhibitor may have a structure of [Chemical Formula 1] as 1'-[2-[4-(Trifluoromethyl)phenyl]ethyl]-spiro[4H-3,1-benzoxazine-4,4'-piperidin]-2(1H)-one. In one embodiment of the present invention, the CCR2 inhibitor refers to RS102895.

[0057] [Chemical Formula 1]

[0058]

[0059] The CCR2 inhibitor of the present invention can be synthesized and used according to a known method, or can be purchased and used as a commercially available product.

[0060] In one embodiment of the present invention, the TGF-β receptor inhibitor may be N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine, which may have a structure represented by the following [Chemical Formula 2]. In one embodiment of the present invention, the TGF-β receptor inhibitor refers to EW-7197.

[0061] [Chemical Formula 2]

[0062]

[0063] The TGF-β receptor inhibitor of the present invention can be synthesized and used according to a known method, or can be purchased and used as a commercially available product.

[0064] The molar ratio of the CCR2 inhibitor and the TGF-β receptor inhibitor of the present invention may be 1:25 to 25:1, 1:24 to 24:1, 1:23 to 23:1, 1:22 to 22:1, 1:21 to 21:1, 1:20 to 20:1, 1:19 to 19:1, 1:18 to 18:1, 1:17 to 17:1, 1:16 to 16:1, 1:15 to 15:1, 1:14 to 14:1, 1:13 to 13:1, 1:12 to 12:1, 1:11 to 11:1, or 1:10 to 10:1.

[0065] In the present invention, metabolic liver disease may be non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatotic liver disease (MASLD). Specifically, the metabolic liver disease in the present invention may be non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), NAFLD-associated liver fibrosis, or metabolic dysfunction-associated steatohepatitis (MASH), and more specifically, may be non-alcoholic steatohepatitis or metabolic dysfunction-associated steatohepatitis. In the present invention, metabolic liver disease may be accompanied by lipidation, necrosis, inflammation, and fibrosis of hepatocytes. The composition of the present invention can treat the metabolic liver disease by reducing steatosis, fibrosis and steatohepatitis.

[0066] In addition, the pharmaceutical composition of the present invention may further include, or be used together with, a substance that has been previously recognized as having an effect of treating fatty liver disease, a effect of treating liver disease, a effect of protecting the liver, a effect of improving liver function, etc., in addition to the effective ingredient of the present invention, such as a substance used as a fatty liver disease treatment agent, a hepatocellular protection agent, a liver disease treatment agent, or an agent for improving liver function. When the pharmaceutical composition of the present invention is used together with a conventional agent for preventing or treating fatty liver, it may be administered simultaneously or sequentially, and is not affected by the number of times and order thereof.

[0067] The content of the CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and the TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof in the pharmaceutical composition of the present invention may be 0.001 wt% to 99.999 wt%, 0.01 wt% to 99.99 wt%, 0.1 wt% to 99.9 wt%, or 1 wt% to 90 wt%, but is not limited thereto, and the content of the CCR2 inhibitor or a pharmaceutically acceptable salt thereof and the TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof of the present invention may be appropriately adjusted and used depending on the subject to be administered, the mode of use, the method of use, the form of the preparation, etc.

[0068] In addition, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

[0069] The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, the concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent, or used in combination with surgery, hormone therapy, drug therapy, and biological response modifiers, and can be administered simultaneously, separately, or sequentially with the above agents, and can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0070] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and concomitantly administered drugs, and is generally 0.1 mg to 100 mg per kg of body weight, preferably 0.3 mg to 80 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0071] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time of administration, but can be appropriately selected by those skilled in the art.

[0072] Additionally, the pharmaceutical composition of the present invention may be delivered using pharmaceutically acceptable carriers such as colloidal suspensions, powders, saline solutions, lipids, liposomes, microspheres, or nano-spheres. These may be complexed with the carrier, linked to each other, or contained in the carrier, and may be delivered into / in a living body using carrier systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation agents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances, or fatty acids. In addition, pharmaceutically acceptable carriers may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, which are commonly used in formulations. In addition, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included in addition to the above ingredients.

[0073] In addition, when formulating the pharmaceutical composition of the present invention, it can be formulated using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing at least one excipient (starch, calcium carbonate, sucrose, lactose, or gelatin) and a lubricant (magnesium stearate, talc), etc. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and these liquid preparations can be prepared by mixing at least one diluent (water, liquid paraffin), an excipient, a wetting agent, a sweetener, a fragrance, a preservative, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc., and the non-aqueous solvents and suspensions may include vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable esters such as ethyl oleate, and the suppositories may include witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin, etc.

[0074] In one aspect of the present invention, the pharmaceutical composition for preventing or treating metabolic liver disease may be an oral pharmaceutical formulation. Specifically, the oral pharmaceutical formulation may include at least one selected from the group consisting of tablets, granules, pills, powders, capsules, and liquids.

[0075] In one embodiment of the present invention, preparations for oral administration may be in dosage unit forms, such as tablets, granules, pills, powders, capsules, or liquids, and in ampoules. These may be prepared by methods known per se, such as conventional mixing, granulation, confectionery, compounding, dissolving, or lyophilization.

[0076] The present invention also provides a quasi-drug for preventing or treating metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a quasi-drug for use in preventing or treating metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; and a use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof for manufacturing a quasi-drug for preventing or treating metabolic liver disease.

[0077] The present invention relates to a pharmaceutical composition for preventing or treating metabolic liver disease, comprising the CCR2 inhibitor or a pharmaceutically acceptable salt thereof; a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; and a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, and therefore the content thereof is cited, and only the unique composition of the over-the-counter drug is described below.

[0078] The term "quasi-drug" used in the present invention refers to products that are used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases of humans or animals, and have a milder effect than pharmaceutical products. For example, according to the Pharmaceutical Affairs Act, a quasi-drug is a product other than a product used for the purpose of pharmaceutical products, and may be a product used for the treatment or prevention of diseases of humans or animals, or a product that has a mild effect on the human body or does not act directly.

[0079] The above-mentioned quasi-drug composition may be manufactured in a dosage form selected from the group consisting of internal preparations converted from pharmaceuticals, such as body cleansers, disinfectants, detergents, kitchen detergents, cleaning agents, toothpastes, mouthwashes, wet tissues, detergents, soaps, hand washes, hair cleansers, hair softeners, humidifier fillers, masks, ointments and filter fillers, vitamin preparations, mineral preparations, tonics, digestive aids, and intestinal preparations. Specifically, the above-mentioned quasi-drug of the present invention may be an internal preparation converted from pharmaceuticals.

[0080] The present invention also provides a food composition for preventing or improving metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a food composition for use in preventing or improving metabolic liver disease, comprising as active ingredients a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; a method for preventing or improving metabolic liver disease, comprising a step of administering a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; and a use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof for producing a food composition for preventing or improving metabolic liver disease.

[0081] The present invention relates to a pharmaceutical composition for preventing or treating metabolic liver disease, comprising the CCR2 inhibitor or a pharmaceutically acceptable salt thereof; a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof; and a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, and therefore the content thereof is cited, and only the unique composition of the food composition will be described below.

[0082] The above food composition may be a functional food or a health functional food, and may be a health functional food composition for improving or preventing metabolic liver disease and additionally protecting the liver and improving liver function.

[0083] The food composition according to the present invention refers to a natural product or processed product containing one or more nutrients, and may mean, for example, a product that has undergone a certain degree of processing and has become directly edible, and typically includes all of foods, food additives, health functional foods, and beverages.

[0084] Examples of foods to which the CCR2 inhibitor or a pharmaceutically acceptable salt thereof of the present invention and the TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof can be added include various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. In addition, the foods in the present invention include, but are not limited to, special nutritional foods (e.g., formulated milk, infant food, baby food, etc.), processed meat products, fish products, tofu, starch jelly, noodles (e.g., ramen, noodles, etc.), health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g., snacks), processed dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickled vegetables, etc.), beverages (e.g., fruit and vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (e.g., ramen soup, etc.). The above food, beverage or food additive can be manufactured by a conventional manufacturing method.

[0085] In the present invention, the term "functional food" refers to a food group that has been given added value by using physical, biochemical, or bioengineering techniques to enable the function of the food to function and express for a specific purpose, or a food that has been designed and processed so that the food composition's body-regulating function regarding biological defense rhythm regulation, disease prevention, and recovery, etc., is sufficiently expressed in the body. The functional food may include food-based acceptable food additives, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.

[0086] In the present invention, the term "beverage" refers to a general term for something consumed to quench thirst or enjoy a taste, and includes functional beverages. The beverage contains, in addition to the CCR2 inhibitor or a pharmaceutically acceptable salt thereof and the TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients in the indicated proportions as essential ingredients, other ingredients are not particularly limited, and, like conventional beverages, may contain various flavoring agents or natural carbohydrates as additional ingredients. Examples of the natural carbohydrates include conventional sugars such as monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; and polysaccharides, such as dextrin and cyclodextrin; and sugar alcohols, such as xylitol, sorbitol, and erythritol. In addition to the above-mentioned flavoring agents, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudiroside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 g to 20 g or 5 g to 12 g per 100 ml of the composition of the present invention. In addition, the composition of the present invention may additionally contain fruit pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage.

[0087] In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination. The proportion of these additives is not so critical, but may be selected from a range of 0 to 200,000 parts by weight per 100 parts by weight of the food composition of the present invention.

[0088] In the present invention, a functional beverage means a group of beverages that have added value by using physical, biochemical, or bioengineering techniques to make the beverage function for a specific purpose work and express the function of the beverage, or a beverage that has been designed and processed to sufficiently express the body's body-regulating function for regulating the biological defense rhythm, disease prevention, and recovery, etc., of the beverage composition.

[0089] The functional beverage above has no particular limitations on other ingredients other than containing the effective ingredient of the food composition of the present invention as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical beverage. Examples of the natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrates may generally be about 1 g to 20 g or 5 g to 12 g per 100 ml of the composition of the present invention.

[0090] In addition, in a food composition for improving or preventing metabolic liver disease, which comprises the CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and the TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, the content of the active ingredients may be comprised in an amount of 0.0001 wt% to 99.99 wt%, 0.001 wt% to 99.9% wt%, 0.01 wt% to 90 wt%, or 0.1 wt% to 50 wt% of the total food weight, and the beverage composition may be comprised in an amount of 0.0002 g to 5 g, or 0.03 g to 1 g, based on 100 ml.

[0091] In addition, the intake amount of the food composition for improving or preventing metabolic liver disease, which comprises the CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and the TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, may vary depending on the condition and body weight of the individual to be ingested, the degree of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art. For example, the food composition of the present invention may be administered at 0.0001 g / kg (amount of active ingredient / body weight) to 12 g / kg (amount of active ingredient / body weight) or 0.01 g / kg to 9 g / kg per day based on the active ingredient. The administration method may be once a day or divided into several times, and the number of administrations and the administration method do not limit the scope of the present invention in any way.

[0092] The composition of the present invention can be formulated in the same manner as the pharmaceutical composition described above and 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, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gum, ice cream, alcoholic beverages, vitamin complexes, health supplements, and the like.

[0093] Hereinafter, the present invention will be described in detail through examples and experimental examples. However, the following examples and experimental examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0094] <Example 1> Cell experiment

[0095] <1-1> Cell culture

[0096] Cell experiments were performed using AML12 mouse hepatocytes and LX-2 human hepatic stellate cells. AML12 cells were cultured in F-12 media (Corning, Arizona, USA), a 50:50 mixture of DMEM and Ham's F-12, which is used for culturing hepatocytes or prostate epithelial cells, supplemented with 5 ml of insulin (ITS), 40 ng / ml of dexamethasone, 1% penicillin streptomycin (anti-anti), and 10% fetal bovine serum (FBS). LX-2 cells were cultured in a medium containing high glucose DMEM (4500 mg / L D-glucose, L-glutamine, 110 mg / L sodium pyruvate, sodium bicarbonate) supplemented with 1% penicillin streptomycin and 10% fetal bovine serum.

[0097] <1-2> Confirmation of fibrosis of liver cells

[0098] Fibrosis was induced in hepatocytes with TGF-β (2 ng / ml), and the degree of fibrosis was confirmed after single or combined administration of a CCR2 inhibitor or a TGF-β receptor inhibitor.

[0099] Specifically, after administering 2 ng / ml of TGF-β to the cells of the above Example <1-1>, 0.5 μM EW-7197 (EW) as a TGF-β receptor inhibitor and / or 10 μM RS102895 (CCR2i) as a CCR2 inhibitor were administered or not, and cultured for 24 hours. The control group (Con) was not treated with either TGF-β or the sample. Thereafter, the cells were harvested, and the protein expression of α-SMA, collagen I (Col1), p-SMAD2, and p-SMAD3 as fibrosis markers was confirmed by Western blotting. For this, the harvested cells were lysed in RIPA buffer (Thermo, MA, USA) with protease inhibitors and phosphatase inhibitors (Roche, IN, USA) and incubated on ice for 30 minutes. The lysates were centrifuged at 13,000 rpm for 20 min at 4°C, and insoluble material was removed. Protein concentration was measured using BCA assay (Pierce Biotechnology). Equal amounts of cell lysates were loaded onto SDS-PAGE gels for electrophoresis and transferred to PVDF membranes (polyvinylidene difluoride, Immobilon-P, Merck KGaA, Darmstadt, Germany). The membranes were then blocked with 5% (w / v) skim milk in 1X TBST for 1 h at room temperature. After blocking, the membrane was treated with primary antibodies, anti-β-actin antibody (1:2000; Santa Cruz), anti-α-SMA antibody (1:1000; Santa Cruz), anti-collagen I antibody (1:1000; Santa Cruz), anti-p-SMAD2 antibody (1:1000; Cell signaling), and anti-p-SMAD3 antibody (1:1000; Cell signaling), respectively, and incubated overnight at 4°C.After washing several times with 1X TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (anti-mouse, rabbit, or goat, 1:2000; Cell signaling) in blocking buffer for 1 hour at room temperature (RT). The membrane was then washed, briefly incubated with Pico Enhanced Peroxidase Detection (EPD) Western reagent (Elpis Biotechnology) according to the manufacturer's procedure, and quantified using an image analyzer (ImageQuant™ LAS 500, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). β-Actin was used as an internal control. Densitometric measurements of protein intensity were quantified using Image J (National Institutes of Health).

[0100] In addition, to confirm the degree of fibrosis of hepatocytes, cells cultured for 24 hours were fixed with 4% paraformaldehyde and stained with α-SMA (SantaCruz, Dallas, Texas, USA), alexafluor 488 (Cell Signaling, Danvers, Massachusetts USA) antibodies and DAPI (abcam Cambridge, MA, USA). After mounting with a mounting solution (Fisher Scientific, Hampton, Rockingham, USA), the prepared samples were stained and photographed using a confocal microscope, and the degree of color development in the stained images was analyzed using the Image J program.

[0101] <1-3> Confirmation of fat synthesis and decomposition

[0102] Fat accumulation was induced in hepatocytes with palmitic acid, and fat synthesis and decomposition were confirmed after single or combined administration of a CCR2 inhibitor or a TGF-β receptor inhibitor.

[0103] Specifically, for palmitic acid, it was dissolved in 0.2% bovine serum albumin (BSA) at a concentration of 250 μM and treated by incubating the cells for 24 hours, and 0.5 μM EW-7197 (EW) or 10 μM RS102895 (CCR2i) was treated alone or in combination for 24 hours. Afterwards, BODIPY-Fatty acid solution (Sigma, 1 mg / mL stock DMSO solution was diluted with PBS 1X to obtain a final concentration of 1 μg / mL) was administered to the cells cultured in a 5% CO2, 37℃ incubator and incubated for 1 hour. After washing with PBS 1X, the stained cells were observed.

[0104] Additionally, fibrosis was induced with TGF-β (2 ng / ml) and BODIPY-fatty acid staining was performed using the same method as above after treatment with 0.5 μM EW-7197 (EW) or 10 μM RS102895 (CCR2i) alone or in combination for 24 hours.

[0105] In addition, to confirm protein expression, the samples were processed in the same manner as above, and then for cell recovery, the recovered cells were lysed in RIPA buffer (Thermo, MA, USA) with protease inhibitors and phosphatase inhibitors (Roche, IN, USA) and incubated on ice for 30 minutes. The lysate was centrifuged at 13,000 rpm for 20 minutes at 4°C, and insoluble material was removed. Protein concentration was measured using BCA assay (Pierce Biotechnology). Equal amounts of cell lysates were loaded onto SDS-PAGE gels for electrophoresis and transferred to PVDF membranes (polyvinylidene difluoride, Immobilon-P, Merck KGaA, Darmstadt, Germany). The membranes were then blocked with 5% (w / v) skim milk in 1X TBST for 1 hour at room temperature. After blocking, the membrane was treated with primary antibodies such as anti-β-actin antibody (1:2000; Santa Cruz), anti-adipophilin antibody (1:1000; Santa Cruz), and anti-SREBP1c antibody (1:1000; Santa Cruz), respectively, and incubated overnight at 4°C. After washing several times with 1X TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (anti-mouse or rabbit, 1:2000; Cell signaling) in blocking buffer for 1 h at room temperature (RT). The membrane was then washed, briefly incubated with Pico Enhanced Peroxidase Detection (EPD) Western reagent (Elpis Biotechnology) according to the manufacturer's procedure, and quantified using an image analyzer (ImageQuant™ LAS 500, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). β-actin was used as an internal control.Protein intensity concentration measurements were quantified using Image J (National Institutes of Health).

[0106] <Example 2> Production of an animal model of steatohepatitis and co-administration of a CCR2 inhibitor and a TGF-β receptor inhibitor

[0107] <2-1> Non-alcoholic steatohepatitis model by MCD diet (preventive effect)

[0108] Eight-week-old male C57BL / 6 mice, weighing 20–25 g, were purchased from Daehan Biolink (Chungbuk). The mice underwent a one-week acclimation period under room temperature (25±2°C), 60±5% humidity, and a 12-h light / dark cycle. The mice were fed a regular diet (Research Diets, RD) and had free access to water. Afterwards, the mice were randomly divided into five groups according to the feed or drug administered. The five groups were a group fed a regular feed (Con), a group fed a methionine-choline deficient diet (MCD) (MCD), a group orally administered 10 mg / kg of RS102895 as a CCR2 inhibitor to the MCD feed (MCD+CCR2i), a group fed an MCD feed and orally administered 5 mg / kg of EW-7197 as a TGF-β receptor inhibitor, and a group orally administered 10 mg / kg of RS102895 and 5 mg / kg of EW-7197 to the MCD feed (MCD+CCR2i+EW) (n=10).

[0109] The drug was administered five days a week for six weeks. Body weights were measured weekly during the experimental period. After six weeks, the mice were sacrificed after fasting for at least eight hours. Blood and liver tissue were collected and stored at -80°C. All experiments were performed with the approval of the Animal Experiment Ethics Committee of Yonsei University Wonju College of Medicine (YWC-220121-1).

[0110] <2-2> Non-alcoholic steatohepatitis model by GAN diet (therapeutic effect)

[0111] Six-week-old C57BL / 6J mice were purchased from Daehan Biolink (Chungbuk). The mice underwent an acclimation period of two weeks under room temperature (25±2°C), 60±5% humidity, and a 12-h light / dark cycle. Sterilized, regular food (Research Diets, RD) and water were provided ad libitum. After the adaptation period, the mice were randomly divided into five groups according to the feed or drug administered. The five groups were a group fed regular feed (Regular diet), a group fed Gan (Gubra-Amylin non-alcoholic steatohepatitis (NASH))-diet, a group fed Gan-diet while orally administered RS102895 as a CCR2 inhibitor (GAN diet+CCR2i), a group fed Gan-diet while orally administered EW7197 as a TGF-β receptor inhibitor (GAN diet+EW7197), and a group fed Gan-diet while orally administered a combination of RS102895 and EW7197 (GAN diet+CCR2i+EW7197) (n=10).

[0112] To establish a NASH (Non-alcoholic steatohepatitis) model, sugar water (drinking water) was supplied along with a Gan-diet for a total of 32 weeks. The sugar water was composed of 23.1 g / L fructose and 18.9 g / L glucose. Specifically, 8-week-old mice were fed a Gan-diet along with sugar water for 12 weeks to establish an initial NASH model, and then RS102895 and EW7197 were administered orally, alone or in combination, for 20 weeks. RS102895 was administered orally directly into the stomach at 10 mg / kg daily for 5 days a week. EW-7197 was administered orally directly into the stomach at 20 mg / kg daily for 5 days a week. Body weights were measured weekly, and after 32 weeks, mice were sacrificed after fasting for more than 8 hours. Blood was collected by cardiac puncture, and liver tissue was extracted and stored at -80°C. All experiments were performed with the approval of the Animal Experiment Ethics Committee of Yonsei University Wonju College of Medicine (YWC-211007-1).

[0113] <2-3> Non-alcoholic steatohepatitis model by CDAHFD diet (therapeutic effect)

[0114] Six-week-old C57BL / 6J mice were purchased from Daehan Biolink (Chungbuk). The mice underwent an acclimation period of two weeks under room temperature (25±2°C), 60±5% humidity, and a 12-h light / dark cycle. The mice were fed sterilized regular diet (Research Diets, RD) and water ad libitum. After the adaptation period, the mice were randomly divided into five groups according to the diet or drug administered. The five groups were: a group fed regular diet (Regular diet), a group fed CDAHFD (A choline-deficient, L-amino acid-defined, high-fat diet)-diet, a group fed CDAHFD-diet while orally administered RS102895 as a CCR2 inhibitor (CDAHFD diet+CCR2i), a group fed CDAHFD-diet while orally administered EW7197 as a TGF-β receptor inhibitor (CDAHFD-diet+EW7197), and a group fed CDAHFD-diet while orally administered RS102895 and EW7197 together (CDAHFD diet+CCR2i+EW7197) (n=10).

[0115] To establish a NASH model, the CDAHFD-diet was administered for a total of 12 weeks. Specifically, 8-week-old mice were fed the CDAHFD-diet for 6 weeks to establish an initial NASH model, and then RS102895 and EW7197 were administered orally, alone or in combination, for 6 weeks. RS102895 was administered orally directly into the stomach at 10 mg / kg daily for 5 days a week. EW-7197 was administered orally directly into the stomach at 20 mg / kg daily for 5 days a week. Body weights were measured weekly, and after 12 weeks, the mice were sacrificed after fasting for more than 8 hours. Blood was collected by cardiac puncture, and liver tissue was extracted and stored at -80°C. All experiments were performed with the approval of the Animal Experiment Ethics Committee of Yonsei University Wonju College of Medicine (YWC-231221-1).

[0116] <2-4> Confirmation of physiological changes in animal models of fatty liver disease

[0117] To investigate physiological changes during co-administration of a CCR2 inhibitor (RS102895) and a TGF-β receptor inhibitor (EW-7197), liver weight, serum TG, T-CHO, AST, ALT, and γ-GT concentrations were measured. In addition, the expression of hydroxyprolin, a fibrosis marker, was confirmed in liver tissue.

[0118] At the end of the experiment, blood samples were collected and serum TG, T-CHO, AST, ALT, and γ-GT concentrations were measured. Blood glucose, triglyceride, cholesterol, AST, and ALT concentrations were measured using ELISA kti purchased from Asan. Liver γGT was measured using γ-Glutamyltransferase (GGT) Activity Colorimetric Assay Kit (Mak089) from Sigma, and hydroxyproline was measured using Hydroxyproline Assay Kit ELISA kit (STA-675) from Cell Biolabs. In addition, hydroxyproline expression was confirmed in the extracted liver tissue.

[0119] Alternatively, serum AST, ALT, triglyceride (TG), total glucose, and total cholesterol concentrations were measured using ELISA kits purchased from Asanpharm. γ-GT was measured using Cayman's Item No. 702210, free fatty acid, HDL, and LDL were measured using DoGenBio's DG-FFA100 and DG-CHO100, adiponectin was measured using Invitrogen's KMP0041, and insulin was measured using Abcam's ab285341. Hydroxyproline was measured in serum and liver tissue using Cell Biolabs' STA-675 ELISA kit.

[0120] <2-5> Observation of liver tissue lesions in animal models of fatty liver disease

[0121] Liver tissue staining was performed to confirm the inhibitory effect of combined administration of a CCR2 inhibitor (RS102895) and a TGF-β receptor inhibitor (EW-7197) on fat accumulation in the liver and fibrosis of the liver tissue. The extracted liver tissue was fixed with 10% formaldehyde, washed with running water for more than 12 hours, and then washed in 60% ethanol for 1 hour, 70% ethanol for 1 hour, and 100% ethanol for 1 hour each. Then, a clearing process in xylene was performed three times for 1 hour each, and an infiltration process in paraffin was performed twice for 1 hour each. The paraffin block was then cut into sections of approximately 4 μm thickness. After deparaffinization, the sections were stained with hematoxylin and eosin (H&E staining), and blood vessels and lipid droplets in the liver tissue were identified.

[0122] Additionally, to confirm fibrosis, the tissue around the blood vessels was stained using Picrosirius red and Masson'strichrome staining, and then fibrosis was confirmed using a microscope equipped with a camera with 400x magnification (Pulnix, Sunnyvale, CA, USA).

[0123] <2-6> Confirmation of fibrosis and fat synthesis and decomposition in liver tissue of animal models of fatty liver disease

[0124] In order to confirm the inhibitory effect of co-administration of a CCR2 inhibitor (RS102895) and a TGF-β receptor inhibitor (EW-7197) on fat accumulation in liver tissue and fibrosis in liver tissue, the expression of fibrosis markers, fat metabolism regulatory factors, and related signaling factors in liver tissue was confirmed. Western blotting was performed using the extracted liver tissue. To confirm the expression of α-SMA, Col1, Fibronectin, p-SMAD2, p-SMAD3, smad 2 / 3, and CTGF proteins as fibrosis markers in liver tissue, anti-β-actin antibody, anti-α-SMA antibody, anti-Col1 antibody, anti-Fibronectin antibody, anti-p-SMAD2 antibody, anti-p-SMAD3, anti-SMAD2 / 3 antibody, and anti-CTGF antibody were used as primary antibodies.

[0125] <2-7> Confirmation of fibrosis and inflammation in liver tissue of an animal model of fatty liver disease

[0126] Liver tissue staining was performed to confirm the inflammatory response in liver tissue when a CCR2 inhibitor (RS102895) and a TGF-β receptor inhibitor (EW-7197) were co-administered. The extracted liver tissue was fixed with 10% formaldehyde, washed with running water for more than 12 hours, and then washed in 60% ethanol for 1 hour, 70% ethanol for 1 hour, and 100% ethanol for 1 hour each. Then, the tissue was cleared three times in xylene for 1 hour each and infiltrated twice in paraffin for 1 hour each. After that, the paraffin block was cut into sections of approximately 4 ㎛ thickness. The sections were deparaffinized and stained with hematoxylin and eosin (H&E staining), and blood vessels and lipid droplets in the liver tissue were identified. Additionally, to confirm fibrosis, the tissue around the blood vessels was stained using Picrosirius red and Masson'strichrome staining, and then fibrosis was confirmed using a microscope equipped with a camera with 400x magnification (Pulnix, Sunnyvale, CA, USA).

[0127] Meanwhile, in nonalcoholic steatohepatitis, macrophage activation and infiltration increase, which plays a key role in the progression of hepatic inflammation. Kupffer cells and infiltrated monocyte-derived macrophages can be identified through F4 / 80 staining. Therefore, to identify F4 / 80 as an inflammatory marker in liver tissue, the extracted liver tissue was fixed in 10% formaldehyde and then stained with F4 / 80 antibody and DAPI. After mounting with a mounting solution and completing the preparation, the stained samples were photographed using a confocal microscope, and the degree of color development in the stained images was analyzed using the ImageJ program.

[0128] <2-8> Confirmation of energy consumption in animal models of fatty liver disease

[0129] To determine energy expenditure when co-administered with a CCR2 inhibitor (RS102895) and a TGF-β receptor inhibitor (EW-7197), animal models were acclimated to the environment in the Comprehensive Lab Animal Monitoring System (CLAMS) for 2 days, and then energy expenditure was measured.

[0130] <2-9> Confirmation of ER stress (endoplasmic reticulum stress) in an animal model of fatty liver disease

[0131] To confirm the effect of co-administration of a CCR2 inhibitor (RS102895) and a TGF-β receptor inhibitor (EW-7197) on improving ER stress in liver tissue, the expression of ER stress markers in liver tissue was examined. Western blotting was performed using excised liver tissue. To confirm the expression of Bip, ATF4, and CHOP proteins as ER stress markers in liver tissue, anti-β-actin antibody, anti-Bip antibody, anti-ATF4 antibody, and anti-CHOP antibody were used as primary antibodies.

[0132] <2-10> Confirmation of mitochondrial dysfunction in an animal model of fatty liver disease

[0133] To confirm the effect of co-administration of a CCR2 inhibitor (RS102895) and a TGF-β receptor inhibitor (EW-7197) on improving mitochondrial dysfunction in liver tissue, the extracted liver tissue was fixed in a mixed solution of 2% glutaraldehyde and 2% paraformaldehyde and then observed using a transmission electron microscope (TEM).

[0134] <2-11> Statistical analysis

[0135] All data are expressed as mean ± standard deviation. Statistical analyses included one-way ANOVA and Tukey's post hoc test for multiple comparisons, and were performed using SPSS Statistics software (version 20.0; IBM Corp., Armonk, NY, USA). Statistical significance was set at P < 0.05.

[0136] <Experimental Example 1> Confirmation of the effect of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in a mouse hepatocyte cell line or a human hepatic stem cell line.

[0137] <1-1> Evaluation of the inhibitory efficacy of CCR2 inhibitor and TGF-β receptor inhibitor on TGF-β-induced fibrosis by single or combined administration in mouse hepatocyte cell lines

[0138] In order to determine the inhibitory effect of TGF-β-induced fibrosis by single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor, a mouse hepatocyte cell line was treated with TGF-β using the same method as described in Example <1-2> above to induce intracellular fibrosis, and then the degree of fibrosis inhibition by treatment with RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor was evaluated.

[0139] As a result, as shown in Fig. 1, the expression of α-SMA was significantly reduced in the CCR2 inhibitor alone treatment and the TGF-β receptor inhibitor alone treatment, and the degree was the most significant in the TGF-β receptor inhibitor, and the expression of α-SMA was reduced as much as in the TGF-β receptor inhibitor alone treatment in the combination treatment group.

[0140] <1-2> Changes in fibrosis markers and signal expression by single or combined administration of CCR2 inhibitors and TGF-β receptor inhibitors in mouse hepatocyte cell lines

[0141] In order to investigate the inhibitory effect of TGF-β on fibrosis development by single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor, mouse hepatocyte cell lines were treated with TGF-β using the same method as described in Example <1-2> above to induce intracellular fibrosis, and then fibrosis indices and changes in signal expression related thereto were confirmed according to treatment with RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor.

[0142] As a result, as shown in Fig. 2, it was confirmed that the expression of a-SMA, Collagen type I, CTGF, and related signals pSMAD2 and pSMAD3 increased by TGF-β, while the expression of smad7 decreased, while their expression was significantly restored by combined treatment with a CCR2 inhibitor and a TGF-β receptor inhibitor. Treatment with a TGF-β receptor inhibitor alone showed changes as significant as the combined treatment, but treatment with a CCR2 inhibitor alone suppressed fibrosis indices and only reduced pSMAD3.

[0143] <1-3> Evaluation of the inhibitory efficacy of CCR2 inhibitor and TGF-β receptor inhibitor on TGF-β-induced fibrosis in human hepatic stem cell lines by single or combined administration

[0144] Intracellular fibrosis was induced by treating human hepatic stromal cell lines with TGF-β using the same method as described in the above Example <1-2>, and then the degree of fibrosis inhibition according to treatment with RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor was evaluated.

[0145] As a result, as shown in Fig. 3, the expression of α-SMA activated by TGF-β treatment in human hepatic stromal cell lines was not reduced by treatment with a CCR2 inhibitor alone, but the expression of α-SMA was significantly reduced by treatment with a TGF-β receptor inhibitor alone and by combined treatment with a CCR2 inhibitor and a TGF-β receptor inhibitor.

[0146] <1-4> Changes in fibrosis markers and signal expression by single or combined administration of CCR2 inhibitors and TGF-β receptor inhibitors in human hepatic stromal cell lines

[0147] In order to investigate the inhibitory effect of TGF-β on fibrosis development by single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor, human hepatic stromal cell lines were treated with TGF-β using the same method as described in Example <1-2> above to induce intracellular fibrosis, and then fibrosis indices and changes in signal expression related thereto were confirmed according to treatment with RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor.

[0148] As a result, as shown in Fig. 4, the expression of a-SMA, Collagen type I, CTGF, and related signals pSMAD2 and pSMAD3 increased by TGF-β, and their expression was significantly restored by combined treatment with a CCR2 inhibitor and a TGF-β receptor inhibitor. In addition, in human hepatic stellate cell lines, a CCR2 inhibitor did not inhibit fibrosis, but single treatment with a TGF-β receptor inhibitor and combined treatment with a CCR2 inhibitor and a TGF-β receptor inhibitor significantly suppressed indices related to fibrosis.

[0149] <1-5> Evaluation of the efficacy of CCR2 inhibitors and TGF-β receptor inhibitors in suppressing fat accumulation in mouse hepatocyte cell lines by single or combined administration.

[0150] In order to determine the efficacy of inhibiting fat accumulation by single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor, fat accumulation was induced with palmitic acid in a mouse hepatocyte cell line using the same method as described in Example <1-3> above, and the degree of fat accumulation inhibition by treatment with RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor was evaluated.

[0151] As a result, as shown in Figure 5, the group treated with the TGF-β receptor inhibitor alone showed increased lipogenesis. Palmitic acid stimulation increased lipogenesis, and CCR2 inhibitor alone treatment significantly inhibited lipogenesis, but TGF-β receptor inhibitor alone treatment did not inhibit lipogenesis induced by palmitic acid. However, combined treatment with the CCR2 inhibitor and TGF-β receptor inhibitor significantly reduced lipogenesis.

[0152] <1-6> Confirmation of the mechanism of lipogenesis following single administration of a TGF-β receptor inhibitor in a mouse hepatocyte cell line

[0153] Fat accumulation was induced in a mouse hepatocyte cell line with palmitic acid using the same method as described in the above Example <1-3>, and the mechanism of fat synthesis according to treatment with EW-7197 as a TGF-β receptor inhibitor was confirmed by Western blotting.

[0154] As a result, as shown in Fig. 6, the expression of adipophilin (ADRP), an indicator of fat accumulation, and the phosphorylation level of AMPK involved in fatty acid metabolism were evaluated, and the expression of adipophilin was increased by the TGF-β receptor inhibitor, and AMPK activity inhibited by TGF-β was not improved. However, it was confirmed that the phosphorylation of AMPK significantly increased and ADRP expression decreased with the combined treatment of a CCR2 inhibitor and a TGF-β receptor inhibitor.

[0155] <Experimental Example 2> Evaluation of the preventive efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis.

[0156] <2-1> Body weight changes due to single or combined administration of CCR2 inhibitor and TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the MCD diet

[0157] In order to investigate the preventive efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed an MCD diet using the same method as described in Example <2-1> above.

[0158] As a result, as shown in Fig. 7, the characteristic of steatohepatitis caused by the MCD diet was weight loss, and there was no difference in weight change between the disease group and the drug treatment group.

[0159] <2-2> Physiological changes caused by single or combined administration of CCR2 inhibitor and TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the MCD diet

[0160] In order to determine the preventive efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed an MCD diet using the same method as described in Example <2-1>, and physiological changes were confirmed using the same method as described in Example <2-4>.

[0161] As a result, as shown in Fig. 8, the characteristics of the steatohepatitis model by the MCD diet are a decrease in blood total cholesterol (T-CHO) and triglyceride (TG) levels, and the blood T-CHO and TG levels by single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor did not show any difference from the disease group.

[0162] In addition, as shown in Fig. 9, the increased levels of serum AST, ALT, and γ-GT in the non-alcoholic steatohepatitis model were significantly reduced by administration of a TGF-β receptor inhibitor alone or a combination of a CCR2 inhibitor and a TGF-β receptor inhibitor.

[0163] <2-3> Inhibitory effect of CCR2 inhibitor and TGF-β receptor inhibitor on fatty liver by single or combined administration in an animal model of non-alcoholic steatohepatitis induced by MCD diet

[0164] In order to investigate the preventive efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed an MCD diet using the same method as described in Example <2-1>, and liver tissue lesions were observed using the same method as described in Example <2-5>.

[0165] As a result, as shown in Figure 10, fat accumulation occurred in the liver tissue of the nonalcoholic steatohepatitis animal model, and neutral fat levels increased. However, in the group administered a CCR2 inhibitor alone or a CCR2 inhibitor and a TGF-β receptor inhibitor in combination, fat accumulation in the liver tissue was suppressed, and neutral fat levels in the liver tissue also significantly decreased.

[0166] <2-4> Inflammatory inhibition effect of CCR2 inhibitor and TGF-β receptor inhibitor alone or in combination in an animal model of non-alcoholic steatohepatitis induced by MCD diet

[0167] In order to determine the preventive efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed an MCD diet using the same method as described in Example <2-1>, and inflammation in liver tissue was confirmed using the same method as described in Example <2-7>.

[0168] As a result, as shown in Fig. 11, when non-alcoholic steatohepatitis occurs, the expression of CCR2, MCP1, and CD68 in liver tissue increases, whereas the expression of these factors was suppressed in the CCR2 inhibitor alone, the TGF-β receptor inhibitor alone, and their combination groups. In addition to liver tissue staining, mRNA expression of CCR2, MCP1, and CD68 was also significantly suppressed in the CCR2 inhibitor and TGF-β receptor inhibitor alone or combination groups.

[0169] In addition, as shown in Fig. 12, after extracting proteins from liver tissue, inflammatory signals and related markers were identified within the proteins, and it was confirmed that the expression of pNFkB, pIkB / IkB, and CCR2, MCP1, which were increased in the disease group, was significantly reduced by single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor. In particular, the protein expression of IL1β was significantly reduced in the TGF-β receptor inhibitor alone, and in the CCR2 inhibitor and TGF-β receptor inhibitor combination group.

[0170] In addition, as shown in Fig. 13, when the inflammation marker F4 / 80 was confirmed in the liver tissue, the increased F4 / 80 positive area in the disease group was reduced by single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor, and was more significantly reduced in the group administered with a combination of a CCR2 inhibitor and a TGF-β receptor inhibitor.

[0171] <2-5> Inhibitory effect of CCR2 inhibitor and TGF-β receptor inhibitor on liver fibrosis by single or combined administration in an animal model of non-alcoholic steatohepatitis induced by MCD diet

[0172] In order to investigate the preventive efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed an MCD diet using the same method as described in Example <2-1>, and fibrosis in liver tissue was confirmed using the same method as described in Examples <2-4> to <2-6>.

[0173] As a result, as shown in Fig. 14, when Masson'strichrome and Picrosirius red staining were performed on liver tissue, increased fibrosis (MTS-blue, Picrosirius red-red) in fatty liver was reduced in the CCR2 inhibitor, TGF-β receptor inhibitor monotherapy group and their combination group, and the degree of inhibition was significantly higher in the TGF-β receptor inhibitor monotherapy group and combination group.

[0174] In addition, as shown in Fig. 15, the mRNA expression levels of TGF-β and Fibronectin (FN), which are fibrosis indicators, in liver tissue were confirmed, and it was confirmed that the expression of TGF-β and FN increased by MCD was significantly suppressed by administration of a CCR2 inhibitor, a TGF-β receptor inhibitor alone, or a combination thereof.

[0175] In addition, as shown in Fig. 16, the results of confirming the fibrosis index and related signals showed that the expression of a-SMA, Collagen type I, CTGF, and fibrosis-related signals pSMAD2 and pSMAD3 increased in the disease group, and that the expression of these was significantly suppressed by single and combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor.

[0176] <2-6> Physiological changes caused by single or combined administration of CCR2 inhibitor and TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by GAN diet

[0177] In order to investigate the therapeutic efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed a GAN diet using the same method as described in Example <2-2>, and physiological changes were confirmed using the same method as described in Example <2-4>.

[0178] As a result, as shown in Figure 17, the insulin level increased by the GAN diet showed a significant decrease by CCR2 inhibitor alone and by combined administration of CCR2 inhibitor and TGF-β receptor inhibitor, and blood sugar level was significantly decreased only in the combined administration group. On the other hand, the adiponectin level, which was significantly decreased in the disease group, was significantly increased in the CCR2 inhibitor and TGF-β receptor inhibitor alone or combined administration groups.

[0179] In addition, as shown in Fig. 18, in the disease group, increases in blood AST, ALT, free fatty acid (FFA), T-CHO, ALT, triglyceride (TG), and γ-GT were observed due to fat accumulation and liver damage, but the increased levels of AST, ALT, FFA, and T-CHO were significantly reduced only in the group treated with a CCR2 inhibitor and a TGF-β receptor inhibitor combination. TG levels were significantly reduced in the group treated with a TGF-β receptor inhibitor alone and the group treated with a CCR2 inhibitor and a TGF-β receptor inhibitor combination. ALT and γ-GT levels were significantly reduced in the group treated with a CCR2 inhibitor alone and the group treated with a CCR2 inhibitor and a TGF-β receptor inhibitor combination.

[0180] <2-7> Inhibitory effect of CCR2 inhibitor and TGF-β receptor inhibitor on fatty liver by single or combined administration in an animal model of non-alcoholic steatohepatitis induced by GAN diet

[0181] In order to investigate the therapeutic efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed a GAN diet using the same method as described in Example <2-2>, and liver tissue lesions were observed using the same method as described in Example <2-5>.

[0182] As a result, as shown in Figure 19, the increased body fat in the disease group did not show any change in the single administration group, but it was confirmed that the cell size in body fat and white adipose tissue decreased in the CCR2 inhibitor and TGF-β receptor inhibitor combination administration group.

[0183] Furthermore, as shown in Figure 20, fat accumulated in the liver tissue of the diseased group, and the neutral fat level in the liver tissue also significantly increased. Furthermore, while the monotherapy group of CCR2 inhibitor and TGF-β receptor inhibitor did not significantly suppress the increased neutral fat level, the combination treatment group significantly suppressed the accumulation of fat in the liver tissue.

[0184] In addition, as shown in Figure 21, the level of hydroxyproline, a fibrosis indicator, increased in the disease group, while it was decreased by both single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor, and the degree of decrease was most significantly in the TGF-β receptor inhibitor single administration group and the CCR2 inhibitor and TGF-β receptor inhibitor combined administration group.

[0185] <2-8> Changes in energy expenditure by single or combined administration of CCR2 inhibitors and TGF-β receptor inhibitors in an animal model of non-alcoholic steatohepatitis induced by the GAN diet

[0186] In order to investigate the therapeutic efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed a GAN diet using the same method as described in Example <2-2>, and energy consumption was confirmed using the same method as described in Example <2-8>.

[0187] As a result, as shown in Figure 22, it was confirmed that energy consumption was improved in the group receiving combined administration of CCR2 inhibitor and TGF-β receptor inhibitor compared to the disease group.

[0188] <2-9> Improvement of ER stress and mitochondrial dysfunction by single or combined administration of CCR2 inhibitor and TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by GAN diet

[0189] The endoplasmic reticulum (ER) is a crucial intracellular organelle responsible for protein synthesis, folding, and modification. It plays a crucial role in maintaining calcium homeostasis and the biosynthesis of sterols, carbohydrates, and lipids, and is abundant in hepatocytes. ER stress is a condition that disrupts ER homeostasis and is caused by excessive accumulation of lipids or denatured proteins, which activates the unfolded protein response (UPR). ER stress-induced signaling pathways are known to induce apoptosis, lipotoxicity, inflammation, and insulin resistance observed in patients with fatty liver disease, and are also known to stimulate oxidative stress, mitochondrial dysfunction, and Kupffer cell-mediated inflammation (Myeong Jun Song, "Myeong Jun Song", Korean J Med. 2021;96(2):92-100).

[0190] Accordingly, in order to investigate the therapeutic efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in a non-alcoholic steatohepatitis animal model, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to a non-alcoholic steatohepatitis animal model fed a GAN diet using the same method as described in Example <2-2>, and ER stress and mitochondrial dysfunction changes were confirmed using the same method as described in Examples <2-9> and <2-10>.

[0191] As a result, as shown in Fig. 23, it was confirmed that the protein expression of ER stress markers Bip, ATF4, and CHOP increased in the disease group, while it was decreased by single or combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor, and the degree of decrease was most significantly in the group administered with a CCR2 inhibitor and a TGF-β receptor inhibitor.

[0192] In addition, as shown in Figure 24, mitochondrial fission occurred in the liver tissue of the disease group, whereas restoration of mitochondrial morphology was observed in the group receiving combination therapy with a CCR2 inhibitor and a TGF-β receptor inhibitor.

[0193] <2-10> Changes in energy expenditure by single or combined administration of CCR2 inhibitor and TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the CDAHFD diet

[0194] In order to investigate the therapeutic efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed a CDAHFD diet using the same method as described in Example <2-3>, and energy consumption was confirmed using the same method as described in Example <2-8>.

[0195] As a result, as shown in Figure 25, it was confirmed that energy consumption was improved in the group receiving combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor compared to the disease group.

[0196] <2-11> Improvement of ER stress and mitochondrial dysfunction by single or combined administration of CCR2 inhibitor and TGF-β receptor inhibitor in an animal model of non-alcoholic steatohepatitis induced by the CDAHFD diet

[0197] In order to investigate the therapeutic efficacy of combined administration of a CCR2 inhibitor and a TGF-β receptor inhibitor in an animal model of nonalcoholic steatohepatitis, RS102895 (CCR2i) as a CCR2 inhibitor and / or EW-7197 as a TGF-β receptor inhibitor were administered to an animal model of nonalcoholic steatohepatitis fed a CDAHFD diet using the same method as described in Example <2-3>, and ER stress and mitochondrial dysfunction changes were confirmed using the same method as described in Examples <2-9> and <2-10>.

[0198] As a result, as shown in Fig. 26, the protein expression of ER stress markers Bip, ATF4, and CHOP was increased in the disease group, while it was confirmed that it was decreased by single or combined administration of CCR2 inhibitor and TGF-β receptor inhibitor, and the degree of decrease was most significantly in the group administered with CCR2 inhibitor and TGF-β receptor inhibitor combination.

[0199] Additionally, as shown in Figure 27, mitochondrial fragmentation occurred in the liver tissue of the disease group, whereas recovery of mitochondrial morphology was observed in the group receiving combination therapy with a CCR2 inhibitor and a TGF-β receptor inhibitor.

[0200] By co-administering the CCR2 (CC chemokine receptor 2) inhibitor and the TGF-β (Transforming Growth Factor beta) receptor inhibitor of the present invention to an animal model in which steatohepatitis was induced by diet, liver fibrosis was significantly improved, and inflammatory cells and fat accumulation accumulated in liver tissue were also suppressed, and ER stress and mitochondrial dysfunction were improved. Therefore, the combined preparation of the CCR2 inhibitor and the TGF-β receptor inhibitor can be usefully used for the purpose of preventing, improving, or treating metabolic liver diseases, including steatohepatitis related to metabolic disorders.

Claims

1. A pharmaceutical composition for preventing or treating metabolic liver disease, comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients.

2. A pharmaceutical composition for preventing or treating metabolic liver disease, wherein the CCR2 inhibitor in claim 1 is 1'-[2-[4-(trifluoromethyl)phenyl]ethyl]-spiro[4H-3,1-benzoxazine-4,4'-piperidin]-2(1H)-one (1'-[2-[4-(Trifluoromethyl)phenyl]ethyl]-spiro[4H-3,1-benzoxazine-4,4'-piperidin]-2(1H)-one; RS102895).

3. A pharmaceutical composition for preventing or treating metabolic liver disease, wherein the TGF-β receptor inhibitor in claim 1 is N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine (N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine; EW-7197).

4. A pharmaceutical composition for preventing or treating metabolic liver disease, wherein the molar ratio of the CCR2 inhibitor and the TGF-β receptor inhibitor in paragraph 1 is 1:25 to 25:

1.

5. A pharmaceutical composition for preventing or treating metabolic liver disease, wherein the metabolic liver disease in claim 1 is non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatotic liver disease (MASLD).

6. A pharmaceutical composition for preventing or treating metabolic liver disease, wherein the metabolic liver disease in claim 5 is at least one selected from the group consisting of non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), NAFLD-associated liver fibrosis, and metabolic dysfunction-associated steatohepatitis (MASH).

7. In claim 1, the pharmaceutical composition for preventing or treating metabolic liver disease reduces steatosis, liver fibrosis, and steatohepatitis.

8. A pharmaceutical composition for preventing or treating metabolic liver disease according to claim 1, which improves energy expenditure, endoplasmic reticulum stress, and mitochondrial abnormalities.

9. A pharmaceutical composition for preventing or treating metabolic liver disease, according to claim 1, which is included in an oral pharmaceutical preparation.

10. A pharmaceutical composition for preventing or treating metabolic liver disease, wherein the oral pharmaceutical preparation in claim 9 is formulated as one or more selected from the group consisting of tablets, granules, pills, powders, capsules, and liquids.

11. A quasi-drug for preventing or treating metabolic liver disease, comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients.

12. A food composition for preventing or improving metabolic liver disease, comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients.

13. A combination, mixture or combination kit for treating metabolic liver disease, comprising a preparation comprising a CCR2 inhibitor or a pharmaceutically acceptable salt thereof; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof as active ingredients, in combination, mixed or in combination.

14. A method for preventing or treating metabolic liver disease, comprising a step of administering a complex, mixture or combination of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof.

15. Use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for preventing or treating metabolic liver disease; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof.

16. Use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof for manufacturing an over-the-counter drug for preventing or treating metabolic liver disease; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof.

17. Use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof for producing a food composition for preventing or improving metabolic liver disease; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof.

18. Use of a CCR2 inhibitor or a pharmaceutically acceptable salt thereof for preparing a combination, mixed or combined drug kit for treating metabolic liver disease; and a TGF-β receptor inhibitor or a pharmaceutically acceptable salt thereof.

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