Pharmaceutical composition for preventing or treating cholestatic liver disease comprising β-lapachone as an active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cholestatic liver disease, comprising β-lapachone as an effective ingredient, and can provide an effective preventive and therapeutic agent for cholestatic liver disease.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition for the prevention or treatment of cholestatic liver disease, with β-lapachone as the active ingredient. Background Technology

[0002] Cholestatic liver disease is a liver disease caused by impaired bile formation or flow. Cholestasis refers to all biochemical, physiological, and clinical changes resulting from the circulatory disturbance of bile, which is produced by the liver and circulates through the bile ducts and intestines.

[0003] Primary biliary cirrhosis (PBC), a representative symptom of cholestatic liver disease, is an autoimmune disease of the liver characterized by the slow, progressive destruction of the intralobular ducts and small bile ducts in the liver during the early stages of the disease. When these ducts are damaged, bile accumulates in the liver (cholestasis), which over time damages tissue, leading to scarring, fibrosis, and cirrhosis.

[0004] Besides its function of excreting waste products, bile plays a crucial role in aiding the absorption of fats and vitamins by transporting bile acids (salts) to the intestines. Bile is produced in hepatocytes and secreted into the bile ducts via bile canaliculi, then enters the enterohepatic circulation through the duodenum. If this bile secretion process is impaired, cholestasis occurs, and bile acids, bilirubin, and other bile components accumulate in hepatocytes and the blood. Alternatively, as the amount of bile transported to the intestines decreases, symptoms such as jaundice, pruritus, gray stools, steatorrhea, and malabsorption of vitamins may appear. Furthermore, if the condition worsens, it can develop into liver failure, potentially requiring a liver transplant.

[0005] Primary sclerosing cholangitis (PSC) is a long-term progressive disease of the liver and gallbladder, characterized by inflammation and fibrosis of the bile ducts that normally drain bile from the gallbladder. Symptoms of PSC may not appear early, and patients may experience signs and symptoms of liver disease, such as yellowing of the skin and eyes, itching, and abdominal pain.

[0006] Biliary duct damage occurring in pyelonephritis (PSC) narrows the biliary tree and obstructs bile flow into the intestines, potentially leading to cirrhosis and liver failure. PSC increases the risk of various cancers, such as liver cancer, gallbladder cancer, colorectal cancer, and cholangiocarcinoma. The underlying cause of PSC is unclear, but genetic susceptibility, immune system dysfunction, and gut microbiota abnormalities may play significant roles. Given the high proportion of patients with PSC also having inflammatory bowel disease (IBD), most commonly ulcerative colitis, the association between PSC and IBD is strong. Approximately 3%–7.5% of IBD patients also have PSC, and 80% of PSC patients have some form of IBD.

[0007] As is well known, post-cholestatic liver disease (PSC) typically occurs primarily in young adults in their 30s or 40s. Currently, there is no effective treatment for PSC; the most reliable treatment is liver transplantation, but there is still a risk of recurrence after transplantation. There are no PSC-targeting therapies approved by the US Food and Drug Administration (FDA). Some experts recommend ursodeoxycholic acid (UDCA), which has been shown to lower elevated liver enzyme levels in PSC patients and is effective against other cholestatic liver diseases. However, the effectiveness of UDCA in improving PSC symptoms and increasing patient survival is not yet clear. Although guidelines from the American Association for the Study of Liver Diseases (AAAD) and the American College of Gastroenterology do not recommend the use of UDCA to treat PSC, the European Association for the Study of the Liver (EASL) recommends the use of moderate doses (13 mg / kg-15 mg / kg) of UDCA. Therefore, liver transplantation is the only proven treatment for PSC to date, but not all patients are eligible for liver transplantation, so there is an urgent need to develop an effective treatment for PSC.

[0008] IBD is a chronic, relapsing inflammatory bowel disease of unknown cause, affecting 80% of patients with post-coital stenosis (PSC). It is best represented by ulcerative colitis (UC) and Crohn's disease (CD). The disease is characterized by repeated exacerbations and remissions, eventually leading to serious complications such as strictures and perforations. While previous reports indicated a lower incidence in Asian countries compared to Western countries, the global prevalence of IBD is now on the rise, with a higher growth rate in Asian countries than in Western countries. Unlike other chronic diseases, given the high risk of developing IBD in younger populations, very careful management from both social and economic perspectives is necessary.

[0009] On the other hand, β-Lapachone (BL) is a quinone compound that has long been obtained from the Lapacho tree and used for medicinal purposes. Both in vitro and in vivo studies have confirmed that BL promotes the conversion of NADH to NAD+ by accepting two electrons from NADH via the intracellular NQO1 (NAD(P)H: quinone oxidoreductase) enzyme, particularly in NQO1-knock-out cell studies. However, to date, there are no studies on the application of BL as an NAD+ promoter in cholestatic liver disease. Summary of the Invention

[0010] The problem the invention aims to solve

[0011] The purpose of this invention is to provide a pharmaceutical composition containing β-Lapachone as an active ingredient for the prevention or treatment of cholestatic liver disease.

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising β-Lapachone as an active ingredient for the prevention or treatment of cholestatic liver disease and inflammatory bowel disease.

[0013] means for solving problems

[0014] The present invention provides a pharmaceutical composition for the prevention or treatment of cholestatic liver disease comprising β-lapachone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0015] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of cholestatic liver disease comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] [Chemical Formula 1]

[0017]

[0018] The aforementioned cholestatic liver diseases can be selected from primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), benign reccurrent intrahepatic cholestasis, intrahepatic cholestasis of pregnancy (ICP), cholestasis caused by viral hepatitis, cholestasis caused by alcoholic hepatitis, drug-induced cholestasis, cholestasis during parenteral nutrition, cholestasis due to malignant tumors, cholestasis after liver transplantation, infectious cholestasis, and Alagille syndrome. More preferably, a pharmaceutical composition is provided for the prevention or treatment of primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), or progressive familial intrahepatic cholestasis (BENign Reccurrent Intrahepatic Cholestasis, Aragerry syndrome) or one or more of the groups consisting of primary sclerosing cholangitis (AS).

[0019] The aforementioned β-lapaquinone (BL) can be used in the form of pharmaceutically acceptable salts, which can be prepared as acid addition salts formed from pharmaceutically acceptable free acids, or as pharmaceutically acceptable metal salts using bases, but are not limited to these. For example, free acids can be inorganic acids and organic acids. Inorganic acids can include hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc., while organic acids can include citric acid, acetic acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, or aspartic acid, etc.

[0020] The pharmaceutical composition of the present invention for the prevention or treatment of cholestatic liver disease is characterized by particularly inhibiting fibrosis of bile duct cells and inhibiting inflammatory cytokines.

[0021] Furthermore, the composition is characterized in that it improves the value of one or more of the group consisting of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and bilirubin in the blood.

[0022] The pharmaceutical composition of the present invention for the prevention or treatment of cholestatic liver disease is characterized by its ability to inhibit fibrosis and inflammation, wherein the inhibition of fibrosis is the inhibition of at least one of the following fibrotic factors: type I collagen α1 (Col1α1), type IV collagen α1 (Col4α1), α-smooth muscle actin (α-SMA), fibronectin, transforming growth factor beta 1 (TGF-β1), type I collagen α2 (Col1α2), and transforming growth factor beta 2 (TGF-β2).

[0023] The inhibition of the aforementioned inflammation refers to the inhibition of at least one of the inflammatory cytokines selected from interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-18 (IL-18), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), and monocyte chemoattractant protein-1 (MCP-1).

[0024] Furthermore, the above-mentioned pharmaceutical composition for the prevention or treatment of cholestatic liver disease is characterized by its superior efficacy in cholestatic liver disease accompanied by inflammatory bowel disease (IBD), which may be Crohn's disease (CD) or ulcerative colitis (UC).

[0025] The composition of the present invention is characterized in that the composition further comprising ursodeoxycholic acid (UDCA) or obeticholic acid (hereinafter referred to as OCA) is more effective in treating cholestatic liver disease than when administered alone.

[0026] Based on the total weight of the pharmaceutical composition, 0.001% to 50% by weight of the above-mentioned β-lapaquinone may be added, more preferably 0.001% to 40% by weight of the above-mentioned β-lapaquinone may be added, and most preferably 0.001% to 30% by weight of the above-mentioned β-lapaquinone may be added.

[0027] The above-mentioned pharmaceutical compositions can be formulated into oral dosage forms, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, and aerosols, using conventional methods; external dosage forms; suppositories; and sterile injectable solutions. Examples of carriers, excipients, and diluents that can be included in the above-mentioned pharmaceutical compositions include lactose, dextran, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In the case of formulation, commonly used fillers, expanders, binders, humectants, disintegrants, surfactants, sweeteners, acidulants, and other diluents or excipients are used. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms are prepared by mixing at least one excipient with β-lapaquinone of the present invention, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, oral solutions, emulsions, syrups, etc. Besides commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as humectants, sweeteners, flavorings, preservatives, acidulants, etc. Dosage forms for non-oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, etc. Non-aqueous solvents and suspensions may use propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable esters (such as ethyl oleate). The base of suppositories can be made of semi-synthetic fatty acid esters (witepsol), polyethylene glycol, Tween-61, cocoa butter, lauryl ester, glycerin gelatin, etc.

[0028] The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, and the route of treatment. Determining the dosage based on these factors is within the capabilities of those skilled in the art, and the dosage is typically in the range of 0.01 mg / kg / day to about 500 mg / kg / day. A preferred dosage is 0.1 mg / kg / day to 200 mg / kg / day, and a more preferred dosage is 1 mg / kg / day to 200 mg / kg / day. Administration may be once daily or divided into several doses. The above dosages do not limit the scope of the invention in any way.

[0029] The pharmaceutical compositions of the present invention can be administered to mammals such as mice, livestock, and humans via various routes. All possible routes of administration are contemplated, such as oral, rectal or intravenous, intramuscular, subcutaneous, intradural or intracerebral injection, and topical application. The pharmaceutical compositions comprising β-lapaquinone of the present invention have virtually no toxicity or side effects, and are therefore safe for long-term use even for preventative purposes.

[0030] Furthermore, the present invention provides a health food containing β-lapachone as an active ingredient for the prevention or improvement of cholestatic liver disease.

[0031] This invention provides a health food containing β-lapachone as an active ingredient for the prevention or symptom improvement of cholestatic liver disease accompanied by inflammatory bowel disease. The aforementioned inflammatory bowel disease can be Crohn's disease or ulcerative colitis.

[0032] Health foods containing β-lapachone as an active ingredient for the prevention or improvement of cholestatic liver disease may also contain ursodeoxycholic acid (UDCA) or obeticholic acid (OCA).

[0033] Regarding the aforementioned health functional food, relative to the total weight of the whole food, 0.001% to 50% by weight of a composition containing β-lapaquinone (BL) may be added, more preferably 0.001% to 30% by weight of a composition containing β-lapaquinone (BL) may be added, and most preferably 0.001% to 10% by weight of a composition containing β-lapaquinone (BL) may be added.

[0034] The aforementioned health functional foods refer to foods prepared and processed into tablets, capsules, powders, pills, or liquids using functional raw materials or ingredients beneficial to the human body. Here, "functional" refers to the role of regulating nutrients in the structure and function of the human body, or achieving health benefits such as physiological effects. The health functional foods of this invention can be prepared using methods commonly used in the art, and commonly used raw materials and ingredients can be added during the preparation process.

[0035] The effects of the invention

[0036] This invention relates to a pharmaceutical composition containing β-lapaquinone as an active ingredient for the prevention or treatment of cholestatic liver disease, which can be used as an effective preventive and therapeutic agent for cholestatic liver disease.

[0037] Furthermore, it was confirmed that pharmaceutical compositions containing β-lapaquinone as an active ingredient for the prevention or treatment of cholestatic liver disease are effective in inhibiting fibrosis and inflammation, and thus can be used as effective preventive and therapeutic agents for cholestatic liver disease. Attached Figure Description

[0038] Figure 1 Images of the tissue surrounding the portal vein (PV) with or without β-lapaquinone (BL) in an animal model of DDC-induced cholestatic liver disease.

[0039] Figure 2 This paper illustrates the effect of β-lapaquinone (BL) on periportal collagen tissue in a DDC-induced cholestatic liver disease animal model. Part A shows images of periportal tissue stained with Sirius Red and Masson's trichrome, while Part B presents quantitative graphs of the images of tissue stained with Sirius Red and Masson's trichrome.

[0040] Figure 3 This study demonstrates the effect of β-lapaquinone (BL) on the expression of fibrosis-related genes in DDC-induced cholestatic liver disease mice (n=6).

[0041] Figure 4 The effects of β-lapaquinone (BL) on blood parameters (ALT, AST, ALP, and bilirubin) in DDC-induced cholestatic liver disease mice (n=6) are shown.

[0042] Figure 5 This study demonstrates the effect of β-lapaquinone (BL) on the expression of fibrosis-related genes in DDC-induced cholestatic liver disease mice (n=6).

[0043] Figure 6 This study demonstrates the effect of β-lapaquinone (BL) on the expression of inflammation-related genes in DDC-induced cholestatic liver disease mice (n=6).

[0044] Figure 7 The effects of β-lapaquinone (BL) on blood parameters (total bilirubin, ALP) in a DDC-induced cholestatic liver disease animal model are shown.

[0045] Figure 8 This study demonstrates the effects of β-lapaquinone (BL) on blood parameters AST, ALT, and ALP in a polyinosinic acid (Poly I:C)-induced cholestatic liver disease animal model.

[0046] Figure 9This study demonstrates the effect of β-lapaquinone (BL) on the survival rate of DSS-induced inflammatory bowel disease mice.

[0047] Figure 10 A graph showing the effect of β-lapaquinone on body weight change (A) and colitis score (B) in DSS-induced inflammatory bowel disease mice (n=10).

[0048] Figure 11 To show a comparison of intestinal length in DSS-induced inflammatory bowel disease mice, picture (A) and graph (B) representing the length.

[0049] Figure 12 A graph illustrating the effect of β-lapaquinone (BL) on the production of inflammatory cytokines in the intestinal tissue of DSS-induced inflammatory bowel disease mice.

[0050] Figure 13 To demonstrate the effect of β-lapaquinone (BL) on the expression of fibrosis-related mRNA in the LX-2 hepatic stellate cell model.

[0051] Figure 14 A graph illustrating the effects of β-lapaquinone on the expression of inflammatory cytokine proteins IL-1β and TNF-α in the macrophage cell line Raw264.7.

[0052] Figure 15 A graph illustrating the effects of β-lapaquinone (BL) on the expression of inflammatory cytokines IL-1β and IL-18 proteins in peripheral blood mononuclear cells (PBMCs). Detailed Implementation

[0053] In the following text, unless otherwise stated, β-lapaquinone is a concept that includes β-lapaquinone itself and its pharmaceutically acceptable salts.

[0054] The causes of cholestasis are varied, including various drug side effects, infections, tumors, bile duct tumors, cysts, bile duct stones, strictures, and physical pressure on the bile ducts. Based on etiology, cholestatic liver diseases include primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), benign reccurrent intrahepatic cholestasis, intrahepatic cholestasis of pregnancy (ICP), cholestasis caused by viral hepatitis, cholestasis caused by alcoholic hepatitis, drug-induced cholestasis, and parenteral nutrition therapy. Cholestasis due to nutrition, cholestasis due to malignant tumors, cholestasis after liver transplantation, infectious cholestasis, and Alagille syndrome (AS).

[0055] Preferred embodiments of the invention will be described in detail below. However, the invention is not limited to the embodiments described herein and may be embodied in other forms, but is provided so that the content described herein will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

[0056] <Example 1. Construction of a mouse model of cholestatic liver disease>

[0057] 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) induces the accumulation of protoporphyrin in the liver by inhibiting the activity of ferrochelatase, an enzyme that inserts Fe into protoporphyrin IX to generate heme. Due to its hydrophobic nature, protoporphyrin can only be excreted from the liver via bile, leading to its accumulation and crystallization in the bile ducts, thereby inducing inflammation and fibrosis both inside and outside the bile ducts. Because it induces cholestasis, inflammation, and fibrosis in liver tissue through the above mechanism, the DDC diet-induced animal model is one of the most commonly used rodent models of cholestatic liver disease.

[0058] A DDC-induced cholestatic liver disease mouse model was established using 8-week-old male C57BL / 6 mice (Samtako, Korea) via methods such as enzyme-linked immunosorbent assay (ELISA Pose). In short, cholestatic liver disease was induced by feeding a standard rodent diet supplemented with 0.1% (w / w) DDC for 7 or 14 days. A 12-hour light-dark cycle was maintained, and the mice had free access to water. The standard rodent diet consisted of medium wheat, wheat, corn and corn gluten meal, and soybean oil (14% of protein). All animal-related procedures were reviewed and approved by the Animal Protection Agency and the Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB, KRIBB-AEC-20165).

[0059] <Example 2. Preventive effect of β-lapaquinone on lesions in a mouse model of cholestatic liver disease>

[0060] 2.1 Histological analysis of a mouse model of cholestatic liver disease

[0061] Prolonged supply of DDCs disrupts the bile duct epithelium by generating porphyrin plugs within the duct, thereby inducing ductal obstruction. The toxic accumulation of bile in the bile duct leads to activation of bile duct cells and ductular reaction proliferation, which is positive in Sirius Red and Masson's trichrome staining.

[0062] Tissue staining in animal models of cholestatic liver disease was performed using H&E staining (basal tissue staining), Sirius red staining (collagen staining), and Manson's trichrome staining (collagen, cytoplasm, myofibrils, etc.). Tissue staining was performed by skilled researchers.

[0063] After feeding mice with a diet containing 0.1% DDC for 14 days, tissue staining was performed to detect periportal inflammation, fibrosis, and adhesion molecules, and the results are shown in Figure 1.

[0064] To confirm the preventive effect of β-Lapachone (BL) on cholestatic liver disease, mice were fed diets at doses of 0 mg / kg (DDC solvent (DDC-vehicle)), 40 mg / kg (DDC-BL40), and 80 mg / kg (DDC-BL80) for 17 days prior to DDC treatment. From day 3 onwards, DDC was administered with the diet for 14 days. After 14 days of DDC treatment, the mice were sacrificed, and the liver was removed via the portal vein (PV) and bile duct. Peripheral lesions were confirmed using hematologic and endoscopic techniques and presented. Figure 1 .

[0065] like Figure 1 As shown, compared to the normal group (standard diet (SD)), the DDC-treated group (DDC solvent) as the control group had increased lesions in the portal vein (PV) and bile ducts of mice (indicated by arrows). Furthermore, compared to the control group treated with the solvent (vehicle) (DDC-vehicle), the DDC-treated mice with DDC-induced cholestatic liver disease (DDC-BL40, DDC-BL80) showed a significant reduction in lesions around the portal vein (PV) and bile ducts (indicated by arrows). Moreover, compared to the 40 mg / kg (BL40) β-lapaquinone (BL) treatment group, the 80 mg / kg (BL80) β-lapaquinone (BL) treatment group showed a significant reduction in lesions, and β-lapaquinone (BL) concentration-dependently reduced the lesions in the portal vein and bile ducts of DDC-treated mice.

[0066] These results confirm that β-lapaquinone treatment has a preventive effect in the context of cholestatic liver disease, such as reducing portal vein and bile duct lesions.

[0067] 2.2 Effect of β-lapaquinone on collagen levels in a mouse model of cholestatic liver disease

[0068] The liver removed in Example 2.1 above was examined using Sirius red and Manson's trichrome staining to identify the tissue, surrounding connective tissue, and other features, and was then visualized. Figure 2 Part A.

[0069] like Figure 2 As shown in Part A, Sirius red and Manson's trichrome staining results confirmed that DDC led to increased connective tissue formation, while β-lapaquinone treatment inhibited this fibrosis. Figure 2 Part B is about quantification through digital images. Figure 2 The stained portion of part A.

[0070] The results above indicate that β-lapaquinone treatment has a preventive effect on fibrosis caused by increased connective tissue in the context of cholestatic liver disease.

[0071] For statistical processing, a two-tailed Student's t-test was used to analyze the results obtained from independent experiments (mean ± standard deviation (SEM)) across all embodiments. For differences, p < 0.05 was considered significant. In the case of survival comparison experiments, the log-rank (Mantel-Cox) test was used, and the results were graphically analyzed using Kaplan-Meier survival analysis, i.e., the product-limit method (Prism, version 5.0, GraphPad software).

[0072] 2.3 To confirm the effect of β-lapaquinone on the transcriptional level of fibrosis-related genes (mRNA) in a mouse model of cholestatic liver disease.

[0073] In the cholestatic liver disease mouse model, fibrosis was induced by DDC damage to the bile ducts, which confirmed the increased transcriptional levels of key factors in the development of fibrosis, including Col1α1 (Collagen Type I Alpha 1), Col4α1 (Collagen Type IV Alpha 1), α-SMA (Alpha-Smooth Muscle Actin), fibronectin, TGF-β1 (Transforming growth factor beta 1), Col1α2 (Collagen Type I Alpha 2), and TGF-β2 (Transforming growth factor beta 2).

[0074] The expression of the aforementioned factors in liver tissue obtained from the cholestatic liver disease mouse model of Example 2.1 was performed using a real-time polymerase chain reaction (PCR) method according to the following steps. Total ribonucleic acid (RNA) was extracted from liver samples using Tri-RNA Reagent (Favrogen Biotech Corp., Nong-Ke Rd, Taiwan, China) and following Favrogen's instructions. PrimeScript, which removes genomic DNA, was then used. TM RT kit (PrimeScript) TM The RT reagent kit with gDNA Eraser (TAKARA Korea Biomedical Inc., Seoul, 08506, Korea) was used to reverse transcribe DNA into complementary deoxyribonucleic acid (cDNA). Quantitative polymerase chain reaction (PCR) was performed using TB Green. TM Premix Ex Taq TM The assay was performed using Tli RNase HPlus, ROX plus (Boril Korea Biotechnology Co., Ltd., Seoul, South Korea), and QuantStudio 5 Real-Time PCR Instrument (Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences used are shown in Table 1 below.

[0075] [Table 1]

[0076]

[0077] In DDC-induced cholestatic liver disease mice treated with β-lapaquinone, the transcriptional levels of fibrosis-related genes Col1α1 (Collagen Type I Alpha1), Col4α1 (Collagen Type IV Alpha 1), α-SMA (Alpha-Smooth Muscle Actin), fibronectin, TGF-β1 (Transforming growth factor beta 1), and TGF-β2 (Transforming growth factor beta 2), which are associated with the progression of cholestatic liver disease, were confirmed. Expression levels were compared with those of mice treated with a vehicle as a control group. Figure 3 .like Figure 3 Part A to Figure 3 As shown in section F, consistent with the aforementioned results regarding reduced portal vein lesions and fibrosis, the transcriptional levels of Col1α1, Col4α1, α-SMA, fibronectin, and TGF-β1 were significantly reduced in DDC-induced cholestatic liver disease mice (DDC-BL40, DDC-BL80) treated with β-lapaquinone (BL) compared to the control mice (DDC-vehicle) treated with the solvent (vehicle). These results in DDC-induced cholestatic liver disease mice indicate that the reduction in portal vein lesions and fibrosis occurs at the expression level of fibrosis-related genes, and that the pathological changes occurring during cholestatic liver disease induction are inhibited by β-lapaquinone (BL) pretreatment.

[0078] Example 3. Confirmation of the effect of β-lapaquinone in a mouse model of cholestatic liver disease.

[0079] To confirm the effect of β-lapaquinone (BL) on the pathogenesis of cholestatic liver disease, the characteristics of the animal model were confirmed by simultaneously administering 1% DDC and β-lapaquinone to mice.

[0080] 3.1 Confirm the effect on blood indicators (ALT, AST, ALP, bilirubin)

[0081] Eight-week-old male C57BL / 6 mice (Samtako, Korea) were divided into six groups and fed the following diets: a normal vehicle group, a control group (DDC), DDC + β-lapaquinone 20 mg / kg, DDC + β-lapaquinone 40 mg / kg, DDC + β-lapaquinone 80 mg / kg, DDC + β-lapaquinone 100 mg / kg, a positive control group (DDC + ursodeoxycholic acid 100 mg / kg), and DDC + obeticholic acid 30 mg / kg (for a total of 7 days). After 7 days of treatment, the mice were sacrificed, and blood was collected from the heart. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin were analyzed and are shown below. Figure 4 .

[0082] like Figure 4 As shown, DDC treatment significantly increased the levels of ALT, AST, ALP, and bilirubin in the blood, while β-lapaquinone (BL) inhibited the increases in ALT, AST, ALP, and bilirubin with DDC treatment in a concentration-dependent manner, demonstrating superior efficacy compared to ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) used as comparative substances.

[0083] 3.2 Confirming the impact on the transcriptional levels of fibrosis-related genes

[0084] The cholestatic liver disease mouse model of Example 3.1 was sacrificed, and after liver tissue was removed, the mRNA level of fibrosis-related genes was measured using a method similar to that of Example 2.3.

[0085] In a mouse model of cholestatic liver disease, fibrosis was induced by DDC damage to the bile ducts. This led to the confirmation of the transcriptional levels of Col1α1 (Collagen Type I Alpha 1), TGF-β1 (Transforming growth factor beta 1), and Col1α2 (Collagen Type I Alpha 2), key factors in fibrosis development, and their levels were then analyzed. Figure 5 .

[0086] like Figure 5As shown, DDC significantly increased the transcriptional levels of fibrosis-related genes Col1α1, TGF-β1, and Col1α2, while β-lapaquinone (BL) inhibited the transcriptional levels of each of Col1α1, TGF-β1, and Col1α2 in a concentration-dependent manner.

[0087] 3.3 Confirming the impact on the transcriptional levels of inflammation-related genes

[0088] In cholestatic liver disease, portal inflammation and intrahepatic bile duct damage develop chronically, leading to cholestasis and liver fibrosis. The levels of inflammatory cytokine gene mRNA in liver tissue of a mouse model of cholestatic liver disease obtained using the same method as in Example 3.1 above were measured and illustrated. Figure 6 The primer sequences used for gene expression analysis are shown in Table 2 below.

[0089] [Table 2]

[0090]

[0091] like Figure 6 As shown, DDC significantly increased the transcriptional levels of inflammation-related genes TNFα and IL-1β, while β-lapaquinone (BL) inhibited the transcriptional levels of both TNFα and IL-1β.

[0092] <Example 4. Confirmation of the therapeutic effect of β-lapaquinone in a mouse model induced by cholestatic liver disease>

[0093] The therapeutic effect of β-lapaquinone was confirmed in a mouse model of cholestatic liver disease. Eight-week-old male C57BL / 6 mice (Samtako, Korea) were divided into six groups. Except for the control group, each group was fed a diet containing 0.1% DDC for 3 days. For the next four days, the mice were fed the following diets for 7 days: normal control, DDC control (solvent), DDC + β-lapaquinone 20 mg / kg, DDC + β-lapaquinone 40 mg / kg, DDC + β-lapaquinone 80 mg / kg, and a positive control (DDC + obeticholic acid 30 mg / kg). After 7 days of feeding, the mice were sacrificed, and blood was collected from the heart. The blood bilirubin and ALP levels were analyzed and presented. Figure 7 .

[0094] like Figure 7 As shown, DDC treatment significantly increased the levels of bilirubin and ALP in the blood, while β-lapaquinone DDC inhibited bilirubin and ALP in a concentration-dependent manner. Furthermore, it demonstrated superior efficacy compared to ursodeoxycholic acid (UDCA) and obeticholic acid (OCA), which were used as comparative agents.

[0095] <Example 5. Confirmation of the therapeutic effect of β-lapaquinone in a mouse model of primary biliary liver disease (PBC)>

[0096] In cholestatic liver disease, primary biliary cirrhosis (PBC) can be induced into a similar state using viral RNA mimics and the Toll-like receptor polyinosinic-polycytidylic acid (Poly I:C).

[0097] Eight-week-old male C57BL / 6 mice (Samtako, Korea) were divided into six groups. Except for the control group, each experimental group received Poly I:C intraperitoneally twice weekly for eight weeks at a dose of 5 mg / kg. Subsequently, the mice were fed a diet for eight weeks with a control group (solvent), β-lapaquinone 40 mg / kg, β-lapaquinone 80 mg / kg, and ursodeoxycholic acid 100 mg / kg and obeticholic acid 30 mg / kg as positive controls. The experiment was terminated at week 16. Mice were sacrificed, and blood was collected from the heart for analysis of AST, ALT, and ALP levels, which are shown below. Figure 8 .

[0098] like Figure 8 As shown, the levels of AST, ALT, and ALP in the blood increased due to Poly I:C, while β-lapaquinone (BL) inhibited the increase in AST, ALT, and ALP caused by Poly I:C. In the control group, β-lapaquinone (BL) showed superior efficacy against the increases in AST, ALT, and ALP caused by DDC compared to the comparative substances ursodeoxycholic acid (UDCA) and obeticholic acid (OCA).

[0099] <Example 6. Confirmation of the efficacy of β-lapaquinone in an inflammatory bowel disease-induced mouse model>

[0100] Since approximately 80% of patients with cholestatic liver disease also have inflammatory bowel disease (IBD), the inventors confirmed the efficacy of β-lapaquinone (BL) in a mouse model of IBD.

[0101] 6.1 Effects on survival rate, body weight, and colitis score

[0102] A mouse model of acute colitis induced by dextran sodium sulfate (DSS) was established using 8-week-old female C57BL / 6 mice (Samtako, Korea).

[0103] Eight-week-old female C57BL / 6 mice (Samtako, Korea) were divided into groups (10 in total). Each experimental group was fed a standard rodent diet supplemented with 3% (w / w) DSS in their drinking water for the first 5 days. The control group (solvent), β-lapaquinone 40 mg / kg and β-lapaquinone 80 mg / kg were orally administered for 14 days.

[0104] All animal-related procedures are reviewed and approved by the Hanyang University Animal Protection Office and the Use Committee. Survival rates over time are as follows: Figure 9 As shown, the rate of change in body weight of mice in each experimental group was measured daily and presented. Figure 10 Part A involved daily measurements of fecal occult blood, total bleeding loss, and fecal concentration in each rectum. The colitis score for each experimental group was determined by two skilled researchers unfamiliar with the treatment group. Measurements included the animal's weight loss, bloody stools, and fecal consistency, which were then converted into scores as shown in Table 3. The scores were summed and presented in Table 3. Figure 10 Part B.

[0105] [Table 3]

[0106]

[0107] like Figure 9 As shown, 70% of DSS-induced IBD mice died on day 14 of the experiment, while 50% died in the β-lapaquinone 40 mg / kg group and only 20% died in the β-lapaquinone 80 mg / kg group. Moreover, as... Figure 10 As shown in Part A, DSS-induced IBD mice experienced weight loss by day 8 of the experiment, decreasing to 75% of baseline. In the β-lapaquinone (BL) treatment group, weight loss due to DSS was observed, and in particular, the β-lapaquinone 80 mg / kg group recovered up to 90% of their initial body weight. Furthermore, as... Figure 10 As shown in Part B, it was confirmed that the clinical colitis score of DSS-induced IBD mice decreased in a concentration-dependent manner with increasing β-lapaquinone (BL) treatment concentration.

[0108] 6.2 Effects on intestinal length restoration and inflammation suppression

[0109] Using the same method as in Example 6.1 above, the experimental groups were divided into three groups: a DSS-untreated group (control), a DSS-treated group, and a DSS + β-lapaquinone 80 mg / kg treatment group. After the experiment, the mice were sacrificed, their intestines were removed, and the accurate length of the intestines was measured using a vernier caliper and shown. Figure 11 Part A and Figure 11 Part B. Furthermore, using real-time polymerase chain reaction (RT-PCR) as standard experimental procedures, the mRNA levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-18 (IL-18), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), and monocyte chemoattractant protein-1 (MCP-1), which are inflammatory cytokines in intestinal tissue, were analyzed and presented as follows. Figure 12 .

[0110] like Figure 11 Part A and Figure 11 As shown in Part B, DSS significantly shortened the intestinal length in mice, which could be restored by treatment with β-lapaquinone (BL).

[0111] Suppression of inflammation can include the inhibition of inflammatory cytokines and / or the suppression of cytokine gene expression. For example, Figure 12 As shown, DSS significantly increased the mRNA levels of inflammatory cytokines IL-1β, IL-6, IL-18, INF-γ, TNF-α, TNF-β, and MCP-1 in mouse intestinal tissue, while treatment with β-lapaquinone (BL) significantly reduced the levels of these inflammatory cytokines increased by DSS.

[0112] <Example 7. Confirmation of the antifibrotic effect of β-lapaquinone at the cellular level>

[0113] 7.1 Effects of β-lapaquinone on fibronectin and α-SMA activity in the LX-2 hepatic stellate cell line model

[0114] Focusing on the cirrhosis induced by bile duct inflammation and fibrosis in cholestatic liver disease, this study employed the method described by Takaaki Higashi et al. (2017) using the hepatic stellate LX-2 cell line model. The LX-2 cell line was passaged in DMEM medium containing 10% FBS, 2 mM glutamine, 100 U penicillin, and 0.1 mg / ml streptomycin. The LX-2 cell line was then cultured at 16 × 10⁻⁶ cells / mL. 4 Cells / well were added to 6-well plates at a density of 1 ng / ml TGF-β1, 1 ng / ml TGF-β1 + 0.5 μM β-lapaquinone, or 1 ng / ml TGF-β1 + 1 μM β-lapaquinone and cultured for 24 h in DMEM. The protein expression levels of fibrosis-related genes fibronectin and α-SMA in the obtained cells were then measured by Western blotting according to conventional methods known in the art.

[0115] like Figure 13 As shown, when the LX-2 cell line, which served as the control group, was treated with TGF-β1, the protein expression levels of fibrosis-associated fibronectin and α-smooth muscle actin (α-SMA) increased compared to the control group. However, when β-lapaquinone was administered, the increased levels of fibronectin and α-SMA due to TGF-β1 decreased in a concentration-dependent manner.

[0116] 7.2 Effects of β-lapaquinone on the expression of inflammatory cytokine proteins in the macrophage line Raw264.7 cell model

[0117] Macrophage line Raw264.7 (ATCC TIB-71, Manassas, Virginia, USA) was passaged in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% (penicillin / streptomycin).

[0118] To induce inflammation, RAW264.7 cells were suspended in DMEM containing 2% fetal bovine serum and then seeded in 12-well plates until the cell count reached 4 × 10⁶ cells / well. 5 / ml, and then incubated at 37°C in a 5% CO2 incubator for 24 hours. When changing to fresh medium, treat with appropriate concentrations of the substance, lipopolysaccharide (LPS) (100 ng / ml), and 0.2 μM, 0.5 μM, 1 μM, and 2 μM β-lapaquinone, and after 24 hours of incubation, treat with adenosine triphosphate (ATP) (2.5 mM) and incubate again for 30 minutes, then collect the supernatant. Cytokines (IL-1β, TNF-α) released by Raw264.7 macrophages were quantified using various Invitrogen cell-free supernatant-ELISA kits (Invitrogen, Billerica, MA, USA) according to the manufacturer's instructions, and are shown below. Figure 14 .

[0119] like Figure 14 As shown, when Raw264.7 cells were treated with LPS and ATP, the expression of inflammatory-related proteins IL-1β and TNF-α increased compared to the control group. However, when β-lapaquinone (BL) was administered, the expression of IL-1β and TNF-α proteins, which were increased due to LPS and ATP, decreased in a β-lapaquinone concentration-dependent manner.

[0120] 7.3 Effects of β-lapaquinone on the secretion of inflammatory cytokines IL-1β and IL-18 in peripheral blood mononuclear cells (PMBCs)

[0121] Focusing on the pathogenesis of cholestatic liver disease mediated by inflammatory cytokines, a peripheral blood mononuclear cell (PBMC) model was utilized by modifying the method of Boyum et al. (1968). PBMCs isolated from healthy 8-week-old male C57BL / 6 mice (Samtako, Korea) were primary cultured and maintained in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM glutamine, 100 U penicillin, and 0.1 mg / ml streptomycin. Stable PBMCs were treated with 200 ng / mL LPS, ATP, and 0 μM, 0.5 μM, 1 μM, and 2 μM β-lapaquinone for 4 hours. The expression levels of IL-1β and IL-18 were then confirmed, and the results are presented. Figure 15 .

[0122] like Figure 15As shown, when peripheral blood mononuclear cells were treated with LPS and ATP, the expression levels of inflammatory proteins IL-1β and IL-18 increased compared to the control group. However, when β-lapaquinone (BL) was administered, the expression levels of IL-1β and IL-18, which were increased due to LPS and ATP, decreased in a β-lapaquinone (BL) concentration-dependent manner.

Claims

1. Use of β-lapachone or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for preventing or treating cholestatic liver disease, characterized in that: The cholestatic liver disease is primary biliary cirrhosis and / or primary sclerosing cholangitis.

2. The use according to claim 1, characterized in that The cholestatic liver disease is associated with inflammatory bowel disease.

3. The use according to claim 2, characterized in that: The inflammatory bowel disease is Crohn's disease or ulcerative colitis.

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