Composition for preventing or treating liver fibrosis containing triazole derivative as active ingredient

Abstract

The present invention relates to a pharmaceutical composition or a functional food composition for preventing or treating a liver disease selected from the group consisting of fatty hepatitis, liver fibrosis, and liver cirrhosis. The triazole derivative compound according to the present invention can reduce the accumulation of collagen in liver tissue and significantly inhibit the expression of inflammatory factors. Therefore, unlike conventional drugs that have only a simple lipid-lowering effect, the triazole derivative compound effectively prevents the progression of a series of serious liver diseases, from excessive fibrosis of liver tissue to tissue cirrhosis, resulting in a decrease in the number of hepatocytes and liver failure, thereby effectively serving as a composition for fundamentally treating liver fibrosis and liver cirrhosis.

Description

Technical Field

[0001] This invention relates to a method for inhibiting liver tissue inflammation, fibrosis, and the resulting liver cirrhosis using triazole derivatives. Background Technology

[0002] Chronic liver disease is a leading cause of death in adults in South Korea. It is a serious condition that, with persistent symptoms and progression to cirrhosis, can lead to liver cancer due to various complications that determine the prognosis of liver disease. Preventing the progression of chronic hepatitis to cirrhosis is one of the ultimate goals in treating liver diseases caused by multiple factors, including dyslipidemia, hepatitis B and C virus infection. Furthermore, since cirrhosis occurs during liver fibrosis, which is caused by various factors, effectively blocking and inhibiting the progression of liver fibrosis is the most fundamental and crucial step in preventing serious liver diseases such as liver cancer.

[0003] Liver fibrosis is caused by the excessive deposition of extracellular matrix (ECM) in liver tissue due to chronic intrahepatic inflammation. If this excessive ECM deposition persists, it eventually progresses to cirrhosis, accompanied by liver structural deformation and a decrease in the number of hepatocytes. Although research on therapeutic drugs that effectively block the process from steatohepatitis to liver fibrosis and then to cirrhosis is actively underway, no therapeutic candidate has yet been developed that effectively alleviates fibrosis and has a suitable safety profile for long-term administration.

[0004] For example, thiazolidinediones (TZDs), a PPAR-γ agonist, have been the most extensively studied drug for treating steatohepatitis by promoting adiponectin secretion and improving insulin resistance in adipose, muscle, and liver tissues. However, no significant improvement in fibrosis was observed when thiazolidinediones (TZDs) were administered, and most cases returned to their pre-treatment state after discontinuation of administration. Furthermore, thiazolidinediones (TZDs) have been reported to have side effects such as weight gain, bone loss, and an increased incidence of bladder cancer, and therefore development as a therapeutic agent has been discontinued. Metformin, developed as a biguanide-based oral antidiabetic drug and an AMPK activator, was expected to have an effect on steatohepatitis and liver fibrosis; however, no histological improvement in steatohepatitis was observed with metformin in clinical studies in adult and pediatric patients. Furthermore, statins, used to treat hyperlipidemia, have no significant effect on non-alcoholic fatty liver disease. Some studies have shown that statins can lower ALT levels, and that combined administration of statins with vitamins C and E can reduce steatosis on imaging. However, statins are not effective in improving fatty liver, inflammatory activity, and fibrosis in patients with steatohepatitis.

[0005] Therefore, there is an increasing need to develop a safe therapeutic agent that is suitable for long-term administration to treat chronic diseases while effectively blocking inflammation and fibrosis in liver tissue.

[0006] Numerous publications and patent documents have been referenced and cited in this specification. The disclosures of the cited publications and patent documents are incorporated herein by reference in their entirety to more clearly describe the state of the relevant art and the contents of this disclosure. Summary of the Invention

[0007] Technical issues

[0008] The inventors have conducted extensive research efforts to develop a pharmaceutical composition that effectively blocks the progression of a series of pathological stages leading to inflammation, fibrosis, and sclerosis in liver tissue. As a result, the inventors discovered that when the compound of Formula I was administered to subjects, the progression of fibrosis was significantly inhibited, while the expression of inflammatory factors and collagen in liver tissue was significantly reduced, thus completing this invention.

[0009] Therefore, the object of the present invention is to provide a composition or functional food composition for the prevention or treatment of liver diseases selected from the group consisting of: fatty liver disease, liver fibrosis, and cirrhosis.

[0010] Another object of the present invention is to provide a method for screening compositions that inhibit liver fibrosis.

[0011] Other objects and advantages of the invention will become clearer from the following detailed description of the invention, the appended claims and the accompanying drawings.

[0012] Technical solution

[0013] In one aspect of the invention, the invention provides a composition for the prevention or treatment of liver diseases selected from the group consisting of: fatty liver disease, liver fibrosis, and cirrhosis, comprising a compound represented by formula I or a pharmaceutically acceptable salt thereof as an active ingredient:

[0014]

[0015] In this configuration, R1 and R2 are each independently hydrogen or C1-C3 alkyl, R1 and R2 are not both hydrogen, and R3 is C2-C4 alkyl.

[0016] The inventors have conducted extensive research efforts to develop a pharmaceutical composition that effectively blocks the progression of a series of pathological stages leading to inflammation, fibrosis, and sclerosis in liver tissue. As a result, the inventors have found that when the compound of Formula I is administered to subjects, the progression of fibrosis is significantly inhibited, while the expression of inflammatory factors and collagen in liver tissue is significantly reduced.

[0017] As used herein, the term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group, and the term "C1-C3 alkyl" refers to an alkyl group having 1 to 3 carbon atoms in an alkyl unit, and when a C1-C3 alkyl is substituted, the carbon atom of the substituent is not included.

[0018] In a particular embodiment of the invention, R1 is a C1 alkyl group, R2 is hydrogen, and R3 is a C3 alkyl group. More specifically, R3 is isopropyl.

[0019] The IUPAC name of the compound of formula I (where R1 is a C1 alkyl (methyl), R2 is hydrogen, and R3 is isopropyl) is “N-(4-(1H-1,2,3-triazol-1-yl)benzyl)-2-(2-isopropylphenyl)-5-methylpyrimidin-4-amine” (CAS No.: 1572414-83-5). This compound is known to possess ubiquitin-specific proteinase 1 (USP1) inhibitory activity and is therefore considered to have therapeutic potential for USP1-mediated diseases such as breast and ovarian cancer; however, its relationship with liver fibrosis remains unclear.

[0020] As used herein, the term "pharmaceutically acceptable salt" refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases include salts of alkali metals such as sodium, alkaline earth metals such as magnesium, ammonium, etc. Specifically, the pharmaceutically acceptable salt used in this invention is a sodium salt.

[0021] As used in this article, the term "steatohepatitis" refers to chronic inflammation of the liver tissue caused by the accumulation of fat within the liver. Steatohepatitis is a pathological condition distinct from hepatic steatosis, in which abnormal levels of fat are simply present in the liver tissue or hepatocytes over a prolonged period. The symptoms of steatohepatitis cannot be relieved or eliminated simply by lowering blood lipids; only by controlling activated inflammatory cytokines and reactive oxygen species can steatohepatitis be fundamentally treated.

[0022] As used in this article, the term "liver fibrosis" refers to a disease caused by excessive deposition of extracellular matrix due to chronic inflammation of the liver, resulting in the formation of excessive fibrous connective tissue in the liver tissue and impairing the normal structure and function of the liver.

[0023] As used in this article, the term "cirrhosis" refers to a pathological condition in which the liver tissue is unable to function properly due to the loss of normal liver tissue caused by prolonged inflammation and extracellular matrix deposition, and the replacement of lost liver tissue with scar tissue.

[0024] Liver fibrosis and cirrhosis are not independent diseases with different causes, but rather a continuous progression from chronic hepatitis through fibrosis to cirrhosis. To date, various commercially available drugs for treating metabolic diseases, despite their lipid-lowering effects, have failed to effectively alleviate liver fibrosis and cirrhosis.

[0025] As used in this article, the term "prevention" refers to the suppression of the occurrence of a condition or disease in subjects who have never been diagnosed with such a condition or disease but may develop it.

[0026] As used herein, the term "treatment" means (a) inhibiting the development of a condition, disease, or symptom; (b) alleviating a condition, disease, or symptom; or (c) eliminating a condition, disease, or symptom. When the compositions of the present invention are administered to a subject, they inhibit the progression of symptoms or eliminate or alleviate symptoms by inhibiting the proliferation of skin fibroblasts and the expression of fibrotic factors, thereby reducing the production of excess fibrous tissue in the skin. Therefore, the compositions of the present invention can be used alone as a therapeutic composition for a disease, or can be administered in combination with other pharmacological ingredients as an adjunct to the treatment of a disease. Therefore, as used herein, the terms "treatment" or "therapeutic agent" encompass "therapeutic adjunct" or "therapeutic auxiliary agent".

[0027] As used herein, the terms “application” or “administration” refer to the direct administration of a therapeutically effective amount of the composition of the present invention to a subject, such that the same amount of the composition is formed in the subject’s body.

[0028] As used herein, the term "therapeutic effective amount" refers to a composition containing a pharmacological ingredient (e.g., ethyl pyruvate) in an amount sufficient to provide a therapeutic or preventative effect to a subject who will be administered the pharmaceutical composition of the present invention. Therefore, the term "therapeutic effective amount" encompasses "preventative effective amount".

[0029] As used herein, the terms “application” or “administration” refer to the direct administration of an effective amount of the composition of the present invention to a subject, such that the same amount of the composition is formed in the subject’s body.

[0030] As used herein, the term "subject" includes, but is not limited to, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the subject of this invention is a human.

[0031] In one specific embodiment of the present invention, the steatohepatitis to be prevented or treated by the composition of the present invention is non-alcoholic steatohepatitis.

[0032] In a specific embodiment of the invention, the composition of the invention reduces the levels of ALT (alanine aminotransferase) or AST (aspartate aminotransferase) in the blood. ALT and AST are aminotransferases present in hepatocytes; when hepatocytes are damaged, ALT and AST are released into the bloodstream, and their levels in the blood increase. Therefore, ALT and AST are used as indicators of liver function. According to the invention, compared with the control group, the composition of the invention significantly reduces the levels of AST and ALT in the blood by 55% and 30%, respectively, thereby significantly restoring liver function reduced due to fatty liver disease and liver fibrosis.

[0033] In one specific embodiment of the invention, the composition of the invention reduces the expression of CC motif chemokine ligand 2 (CCL2), transforming growth factor-β (TGF-β), and F4 / 80 in liver tissue.

[0034] As shown in the following examples, the composition of the present invention significantly inhibits the expression of CCL2, TGF-β and F4 / 80, which are major inflammatory factors. This demonstrates that the composition not only exhibits a simple and quantitative effect of reducing accumulated lipids, but also effectively blocks the progression of inflammation and the resulting tissue damage.

[0035] As used herein, the term "reduced expression" refers to a decrease in the expression level of a protein or gene that serves as a marker or cause of inflammation to the extent that pathological inflammation and fibrosis in liver tissue ceases, lessens, or improves, or that the risk of such inflammation is reduced. Specifically, it can indicate a state of at least a 20% reduction in expression level compared to a control group, more specifically at least 30%, and more specifically at least 40%.

[0036] When the compositions of the present invention are prepared into pharmaceutical compositions, the pharmaceutical compositions of the present invention contain a pharmaceutically acceptable carrier.

[0037] Examples of pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc. In addition to the above-mentioned components, the pharmaceutical compositions of the present invention may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and reagents are described in detail in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0038] The pharmaceutical compositions of the present invention can be administered orally or parenterally. Specifically, they can be administered parenterally. More specifically, they can be administered subcutaneously or transdermally.

[0039] The appropriate dosage of the pharmaceutical composition of the present invention can vary depending on various factors such as preparation method, route of administration, patient's age, weight, sex, pathological condition, diet, administration time, route of administration, excretion rate, and response sensitivity. The preferred dosage range of the pharmaceutical composition of the present invention is 0.001 to 100 mg / kg for adults.

[0040] The pharmaceutical compositions of the present invention can be prepared into unit dosage forms or into multi-dose containers by formulation with pharmaceutically acceptable carriers and / or excipients, according to methods readily performed by those skilled in the art. Here, the dosage form of the pharmaceutical composition can be a solution, suspension, syrup, or emulsion of the pharmaceutical composition in an oil or aqueous medium, or an extract, powder, granule, tablet, or capsule containing the pharmaceutical composition, and may also contain dispersants or stabilizers.

[0041] In another aspect of the invention, a functional food composition is provided for improving liver diseases selected from the group consisting of fatty liver disease, liver fibrosis, and cirrhosis, comprising a compound represented by formula I or a food-acceptable salt thereof as an active ingredient:

[0042]

[0043] In this configuration, R1 and R2 are each independently hydrogen or C1-C3 alkyl, R1 and R2 are not both hydrogen, and R3 is C2-C4 alkyl.

[0044] Since the compound of formula I used in this invention and the liver disease improved by using the compound have already been described above, their description will be omitted to avoid excessive repetition.

[0045] As used herein, the term "food-acceptable salt" refers to a salt in the form of a salt composed of cations and anions bound together by electrostatic attraction, which can be used in food compositions, and specific examples include the aforementioned examples of "pharmaceutically acceptable salts".

[0046] When the compositions of the present invention are prepared into food compositions, they may contain not only the compounds of the present invention as active ingredients, but also carbohydrates, flavorings, and seasonings commonly added in food preparation. Examples of carbohydrates include, but are not limited to, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of seasonings include natural seasonings [kiwifruit protein, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic seasonings (saccharin, aspartame, etc.). For example, when the food compositions of the present invention are made into beverages, in addition to pine bark extract, which is an active ingredient of the present invention, they may also contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, eucommia extract, jujube extract, licorice extract, etc.

[0047] In another aspect, the present invention provides a method for screening compositions that inhibit liver fibrosis, the method comprising the following steps:

[0048] (1) Contact the test substance with a biological sample containing cells expressing ubiquitin-specific protease 1 (USP1); and

[0049] (2) Measure the activity or expression level of USP1 in the sample.

[0050] Specifically, when the activity or expression level of USP1 decreases, the test substance is identified as a composition that inhibits liver fibrosis.

[0051] The correlation between inflammatory fibrotic diseases (such as liver fibrosis) and the USP1 enzyme is unclear. In this invention, the inventors, through observation of reported USP1-inhibiting compounds of this invention inhibiting liver fibrosis, have demonstrated for the first time that the USP1 enzyme can serve as a therapeutic target for liver fibrosis.

[0052] According to the screening method of the present invention, the test substance is first contacted with a biological sample containing cells expressing USP1. As used herein, the term "biological sample" is any sample containing USP1-expressing cells obtained from mammals (including humans), and includes, but is not limited to, tissues, organs, cells, or cell cultures. Specifically, biological samples include hepatocytes or cultures thereof.

[0053] The term "test substance" as used in relation to the screening method of this invention refers to an unknown substance used for screening to examine whether it affects the activity or expression level of USP1 at the gene or protein level. Examples of test substances include, but are not limited to, compounds, nucleotides, antibodies, antisense RNA, small interfering RNA (siRNA), and natural product extracts. Subsequently, the expression level or activity of USP1 in a biological sample treated with the test substance is measured. The measurement of expression level can be performed by various immunoassay methods known in the art, or by various gene detection methods using specially designed primers or probes targeting genes with known gene sequences. As a result of the measurement, when the expression level or activity of USP1 decreases, the test substance can be identified as a composition for inhibiting liver fibrosis.

[0054] As used herein, the term "reduced expression level or activity" refers to a decrease in the expression level or unique in vivo function of USP1 to a level that allows liver function to improve to measurable levels by significantly inhibiting USP1-induced tissue fibrosis. Decreased activity includes not only simple functional reduction but also a final decrease in activity due to reduced stability. Specifically, the term "reduced expression level or activity" can refer to a state where the activity or expression level is reduced by at least 20%, more specifically at least 40%, or even more specifically at least 60% compared to the control group.

[0055] According to another aspect of the present invention, the present invention provides a method for preventing, treating or improving liver diseases selected from the group consisting of fatty liver, liver fibrosis and cirrhosis, the method comprising the step of administering to a subject a composition comprising a compound represented by formula I or a food-acceptable salt thereof as an active ingredient.

[0056]

[0057] Since the compound of formula I used in this invention and the liver disease improved by using the compound have already been described above, their description will be omitted to avoid excessive repetition.

[0058] Beneficial effects

[0059] The features and advantages of this invention are summarized as follows:

[0060] (a) The present invention provides pharmaceutical compositions or functional food compositions for the prevention or treatment of liver diseases selected from the group consisting of: fatty liver disease, liver fibrosis and cirrhosis.

[0061] (b) The triazole-derived compounds discovered in this invention can reduce the accumulation of collagen in liver tissue and significantly inhibit the expression of inflammatory factors. Therefore, these triazole-derived compounds effectively prevent the progression of a range of serious liver diseases, from excessive liver fibrosis to cirrhosis, to reduced hepatocyte count and liver failure, unlike conventional drugs which only have a simple lipid-lowering effect. Therefore, these triazole-derived compounds can be used as compositions for the fundamental treatment of liver fibrosis and cirrhosis. Attached Figure Description

[0062] Figure 1 The results of the analysis of the viability of LX-2 cells treated with 0, 1, 5, 10 and 25 μM ML-323 and untreated control cells are shown.

[0063] Figure 2 The results show the measurement of mRNA expression levels of the fibrosis-associated factor COL1A1 after LX-2 cells were treated with ML323.

[0064] Figure 3 The results of observing changes in liver size after oral administration of ML-323 to a mouse model of fatty liver.

[0065] Figure 4 The results show the measurement of serum AST and ALT levels in a mouse model of fatty liver after oral administration of ML-323.

[0066] Figure 5 shows the collagen content in the liver tissue of a mouse model of fatty liver after oral administration of ML-323. Figure 5a ) and inflammatory factors ( Figure 5b Expressing the observed changes.

[0067] Figure 6 Immunohistochemical staining results of collagen were shown, indicating that ML-323 improved fibrosis in the liver tissue of a mouse model of fatty liver after oral administration.

[0068] Figure 7 The results of staining liver tissue with each of H&E, F4 / 80 and α-SMA are shown after administering the vehicle and ML-323 to mice fed a non-alcoholic steatohepatitis-induced diet for 8 weeks.

[0069] Implementation of the invention

[0070] The invention will now be described in more detail with reference to embodiments. These embodiments are merely illustrative of the invention and it will be apparent to those skilled in the art that the scope of the invention is not limited to these embodiments. Example

[0071] Test methods

[0072] Cytotoxicity measurement

[0073] Human hepatic stellate LX-2 cells were seeded in 96-well DMEM (Welgene) plates containing 2% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) and cultured at 37°C and 5% CO2 for 24 hours. Then, the cells were treated with ML323 at concentrations of 0, 1, 5, 10, and 25 μM. After 48 hours, 10 μl of EZ-Cytox was added to the cell culture medium and reacted for 30 minutes. The absorbance at 450 nm was measured using a microplate reader.

[0074] Assay for cell fibrosis

[0075] Human hepatic stellate LX-2 cells were seeded in 6-well plates of DMEM (Welgene) containing 2% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco) and cultured at 37°C and 5% CO2 for 24 hours. Afterward, the medium was replaced with serum-free DMEM, and the cells were starved for 24 hours. Next, the cells were activated by treatment with 2.5 ng / ml TGF-β, and simultaneously treated with 0, 1, 10, and 25 μM ML323. After 24 hours, the cells were harvested and used for gene expression analysis.

[0076] animal models

[0077] Twenty-four 6-week-old C57BL / 6 mice were acclimatized for one week and then divided into two groups. One group (n=12) was fed a normal diet, and the other group (n=12) was fed a high-fat diet for 9 weeks. After 9 weeks, both the normal diet group and the high-fat diet group were further subdivided into two groups, and the mice were given the solvent and 50 mg / kg ML323 orally twice a week for 7 weeks. During the oral administration period, both the normal diet and the high-fat diet continued.

[0078] Measurement of liver weight change

[0079] Mice were euthanized at the end of the experimental period, and liver tissue was collected and weighed.

[0080] Measurement of liver function indicators in blood

[0081] At the end of the experimental period, blood was collected from each mouse. After 30 minutes, the blood was centrifuged at 2,000 rpm for 15 minutes to separate the supernatant (plasma) from the blood. The separated plasma was analyzed by the Seoul Clinical Laboratory (SCL) to measure AST and ALT levels as indicators of liver function.

[0082] Determination of expression of liver fibrosis inducers

[0083] Tissue harvested from each mouse was added to 1 ml of Easy Blue (Intron) and homogenized. 200 μl of chloroform was added to the tissue and mixed thoroughly. The mixture was incubated for 3 minutes, then centrifuged at 13,200 rpm for 15 minutes at 4°C. After centrifugation, only the supernatant was transferred to a new tube, and an equal volume of isopropanol was added and mixed thoroughly. After incubation for 10 minutes, the tube was centrifuged at 13,200 rpm for 10 minutes at 4°C. After removing the supernatant, the RNA precipitate was washed with 70% ethanol and centrifuged at 13,200 rpm for 5 minutes at 4°C. After removing the supernatant and drying the RNA precipitate for 10 minutes, the RNA precipitate was dissolved in RNase-free water.

[0084] cDNA was synthesized from extracted RNA using the ReverTraAce RT reaction mixture (master mix) (TOYOBO), and gene expression changes were then measured using a real-time PCR system (ABI). The primer sets used are summarized in Table 1 below.

[0085] Table 1 Primer sequences used for real-time PCR

[0086]

[0087]

[0088] Immunohistochemical analysis

[0089] Liver tissue collected from each mouse was fixed in 10% formalin solution, and paraffin blocks and slides were prepared using each fixed liver tissue. The degree of fibrosis in the liver tissue was measured using Sirius red staining and α-SMA immunostaining.

[0090] In addition, ten 6-week-old C57BL / 6 mice were acclimatized for one week and then fed CDAA (cholinergic L-amino acid) for eight weeks to induce non-alcoholic fatty liver disease. Subsequently, the ten mice were divided into two groups and administered the solvent orally and ML-323 (50 mg / kg) to the other group, respectively. The liver tissue was then stained with H&E, F4 / 80, and α-SMA.

[0091] Experimental results

[0092] Cytotoxicity assay (WST assay)

[0093] There was no significant difference in viability between cells treated with 0, 1, 5, 10, and 25 μM ML323 and untreated cells. Figure 1 Therefore, it was confirmed that ML323 did not exhibit cytotoxicity at all treatment concentrations.

[0094] Assay for cell fibrosis

[0095] This study confirmed that, compared with the untreated control group, the mRNA expression of COL1A1, encoding collagen, was normally increased in the TGF-β-treated group. It also confirmed that when cells were treated with TGF-β, the mRNA expression of COL1A1 in ML323-treated cells was significantly lower than that in untreated cells. Figure 2 This demonstrates that the composition of the present invention effectively controls liver fibrosis in a liver fibrosis cell model.

[0096] Measurement of liver weight change

[0097] It was confirmed that, under normal dietary conditions, there was no significant difference in liver weight or size between the ML323 administration group and the control group. However, under high-fat dietary conditions, the ML323 administration group showed significantly less fat accumulation in the liver tissue compared to the control group, and the liver weight was also reduced in the ML323 administration group. Figure 3 ).

[0098] Measurement of liver function indicators in blood

[0099] It has been confirmed that, compared with the solvent-treated control group, the levels of AST and ALT in the blood of mice treated with ML323 in the high-fat diet group were significantly reduced by 40% and 70%, respectively, indicating that the compound of the present invention significantly restores reduced liver function. Figure 4 ).

[0100] Determination of expression of liver fibrosis inducers

[0101] Observations showed that in the high-fat diet group, the mRNA expression level of the COL1A1 gene encoding type 1 collagen in the liver tissue of ML323-treated mice was significantly reduced. Figure 5a Furthermore, the mRNA expression levels of inflammation-related genes CCL2, TGF-β, and F4 / 80 were significantly lower compared to the control group. Figure 5b This indicates that the composition of the present invention effectively controls inflammation and fibrosis in liver tissue.

[0102] Immunohistochemical analysis

[0103] The results of Sirius red staining and α-SMA immunostaining confirmed that liver fibrosis was significantly reduced in the liver tissue of mice administered ML323. Figure 6 Furthermore, H&E staining, f4 / 80 immunostaining, and α-SMA immunostaining results showed that inflammation and liver fibrosis in the liver tissue of the CDAA diet group were significantly reduced in the ML323 administration group. Figure 7 These results confirm that the composition of the present invention has a histological effect in improving actual liver fibrosis.

[0104] Although the invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is merely a description of preferred embodiments and does not limit the scope of the invention. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents. <110> Yonsei University Industry-Academia Collaboration Group <120> Compositions containing triazole derivatives as active ingredients for the prevention or treatment of liver fibrosis <130> POPB214297PCTCN <150> KR 10-2020-0097358 <151> 2020-08-04 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A1 F primer <400> 1 gcttcaccta cagcaccctt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COL1A1 R primers <400> 2 gtccgaattc ctggtctggg 20 <210> 3 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> CCL2 F primers <400> 3 taaaaaacct ggatcggaac caa 23 <210> 4 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> CCL2 R primers <400> 4 gcattagctt cagatttacg ggt 23 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta F primers <400> 5 cctgcaagac catcgacatg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TGF-beta R primers <400> 6 tgttgtacaa agcgagcacc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F4 / 80 F primers <400> 7 cgtcagccga tttgctatct 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F4 / 80 R primer <400> 8 cggactccgc aaagtctaag 20

Claims

1. Use of a composition for the preparation of a medicament for the prevention or treatment of liver diseases selected from the group consisting of: fatty liver disease, liver fibrosis, and cirrhosis, said composition comprising a compound represented by formula I or a pharmaceutically acceptable salt thereof as an active ingredient: in, R1 is a C1 alkyl group, R2 is hydrogen, and R3 is isopropyl.

2. The use as described in claim 1, wherein, The fatty liver disease mentioned refers to non-alcoholic fatty liver disease.

3. The use as described in claim 1, wherein, The composition reduces the levels of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in the blood.

4. The use as described in claim 1, wherein, The composition reduces the expression of CC motif chemokine ligand 2 (CCL2), transforming growth factor-β (TGF-β), and F4 / 80 in liver tissue.

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