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 steatohepatitis, hepatic fibrosis and cirrhosis. A triazole derivative compound discovered in the present invention reduces collagen accumulation in liver tissue and significantly inhibits the expression of inflammatory factors. Thus, the triazole derivative compound effectively blocks the progression of a series of severe liver diseases, from excessive fibrogenesis in liver tissue through tissue hardening (cirrhosis) to a decrease in the number of hepatocytes and loss of liver function, unlike conventional drugs that have only a simple lipid-lowering effect. Accordingly, the triazole derivative compound may be useful as a composition for fundamental treatment of liver fibrosis and cirrhosis.

Claims

1. A method for preventing or treating a liver disease selected from the group consisting of steatohepatitis, hepatic fibrosis and cirrhosis, comprising administering to a subject in need thereof a composition comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: ##STR00004## wherein R.sub.1 and R.sub.2 are each independently hydrogen or C.sub.1-C.sub.3 alkyl, R.sub.1 and R.sub.2 are not simultaneously hydrogen, and R.sub.3 is C.sub.2-C.sub.4 alkyl.

2. The method according to claim 1, wherein R.sub.1 is C.sub.1 alkyl, R.sub.2 is hydrogen, and R.sub.3 is C.sub.3 alkyl.

3. The method according to claim 2, wherein R.sub.3 is an isopropyl group.

4. The method according to claim 1, wherein the steatohepatitis is non-alcoholic steatohepatitis.

5. The method according to claim 1, wherein the composition reduces a level of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in blood.

6. The method according to claim 1, wherein the composition reduces expression of C-C motif chemokine ligand 2 (CCL2), transforming growth factor-β (TGF-β) and F4/80 in liver tissue.

7. A method for ameliorating a liver disease selected from the group consisting of steatohepatitis, hepatic fibrosis and cirrhosis, comprising administering to a subject in need thereof a functional food composition comprising a compound represented by the following Formula 1 or a food-acceptable salt thereof as an active ingredient: ##STR00005## wherein R.sub.1 and R.sub.2 are each independently hydrogen or a C.sub.1-C.sub.3 alkyl, R.sub.1 and R.sub.2 are not simultaneously hydrogen, and R.sub.3 is a C.sub.2-C.sub.4 alkyl.

8. The method according to claim 7, wherein R.sub.1 is C.sub.1 alkyl, R.sub.2 is hydrogen, and R.sub.3 is C.sub.3 alkyl.

9. The method according to claim 8, wherein R.sub.3 is an isopropyl group.

10. A method for screening a composition for inhibiting hepatic fibrosis, comprising: (1) bringing a test substance into contact with a biological sample containing cells expressing ubiquitin specific protease 1 (USP1); and (2) measuring an activity or expression level of USP1 in the sample, wherein, when the activity or expression level of USP1 decreased, the test substance is determined as a composition for inhibiting hepatic fibrosis.

11. The method according to claim 10, wherein the biological sample contains hepatocytes or a culture thereof.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0058] FIG. 1 shows the results of analyzing the viability of LX-2 cells treated with 0, 1, 5, 10 and 25 μM of ML-323 and untreated control cells.

[0059] FIG. 2 shows the result of measuring the mRNA expression level of the fibrosis-related factor COL1A1 after treating LX-2 cells with ML323.

[0060] FIG. 3 shows the results of observing changes in liver size in a fatty liver mouse model after oral administration of ML-323.

[0061] FIG. 4 shows the results of measuring the AST and ALT levels in serum from a fatty liver mouse model after oral administration of ML-323.

[0062] FIGS. 5A and 5B show the results of observing changes in the expression of collagen (FIG. 5A) and inflammatory factors (FIG. 5B) in liver tissue from a fatty liver mouse model after oral administration of ML-323.

[0063] FIG. 6 shows the results of immunohistochemical staining for collagen, which indicate the fibrosis-ameliorating effect of ML-323 in the liver tissue of a fatty liver mouse model after oral administration of ML-323.

[0064] FIG. 7 shows the results of staining liver tissue with each of H&E, F4/80 and α-SMA after administering a vehicle and ML-323 to mice fed a non-alcoholic steatohepatitis-inducing diet for 8 weeks.

MODE FOR INVENTION

[0065] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention according to the subject matter of the present invention is not limited by these examples.

EXAMPLES

[0066] Experimental Methods

[0067] Cytotoxicity Measurement

[0068] Human hepatic stellate LX-2 cells were seeded in a 96-well plate with DMEM (Welgene) containing 2% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco), and cultured in an incubator for 24 hours at 37° C. under 5% CO.sub.2. Next, 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, and the absorbance at 450 nm was measured using a microplate reader.

[0069] Measurement of Cell Fibrosis

[0070] Human hepatic stellate LX-2 cells were seeded in a 6-well plate with DMEM (Welgene) containing 2% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco), and cultured in an incubator for 24 hours at 37° C. under 5% CO.sub.2. Thereafter, the medium was replaced with serum-free DMEM, and the cells were starved for 24 hours. Next, the cells were activated by treatment with TGF-β at a concentration of 2.5 ng/ml, and at the same time, the cells were treated with ML323 at concentrations of 0, 1, 10 and 25 μM. After 24 hours, the cells were harvested and used for gene expression analysis.

[0071] Animal Model

[0072] Twenty-four 6-week-old C57BL/6 mice were acclimated for 1 week and then divided into two groups. One group (12 mice) was fed a normal diet, and the other group (12 mice) was fed a high-fat diet for 9 weeks. After 9 weeks, each of the normal diet group and the high-fat diet group was subdivided into two groups, and each of vehicle and 50 mg/kg of ML323 was orally administered thereto twice a week for 7 weeks. Normal diet and high-fat diet were continued during oral administration.

[0073] Measurement of Liver Weight Change

[0074] After the experimental period was over, mice were sacrificed, and liver tissues were harvested and weighed.

[0075] Measurement of Liver Function Indicators in Blood

[0076] After the experimental period was over, blood was collected from each mouse, and after 30 minutes, the supernatant (plasma) was separated from the blood by centrifugation at 2,000 rpm for 15 minutes. The separated plasma was analyzed by Seoul Clinical Laboratories (SCL) to measure the levels of AST and ALT, which are indicators of liver function.

[0077] Measurement of Expression of Hepatic Fibrosis Inducers

[0078] The tissue harvested from each mouse was added to 1 ml of Easy blue (Intron) and then minced using a homogenizer. 200 μl of chloroform was added to and mixed well with the tissue, and the mixture was incubated for 3 minutes and then centrifuged at 13,200 rpm for 15 minutes at 4° C. After centrifugation, only the supernatant was transferred to a fresh tube, and the same amount of isopropyl alcohol was added thereto and mixed well therewith. After incubation for 10 minutes, centrifugation was performed at 13,200 rpm for 10 minutes at 4° C. After removing the supernatant, the RNA pellet 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 pellet for 10 minutes, the RNA pellet was dissolved in RNAase-free water.

[0079] cDNA was synthesized from the extracted RNA using ReverTraAce RT master mix (TOYOBO), and then gene expression changes were measured using a real-time PCR system (ABI). The primer sets used are summarized in Table 1 below.

TABLE-US-00001 TABLE 1 Primer sequences used in real-time PCR Gene Direction Primer sequence COL1A1 Forward 5′-GCTTCACCTACAGCACCCTT-3′ Reverse 5′-GTCCGAATTCCTGGTCTGGG-3′ CCL2 Forward 5′-TAAAAAACCTGGATCGGAACCAA-3′ Reverse 5′-GCATTAGCTTCAGATTTACGGGT-3′ TGF-β Forward 5′-CCTGCAAGACCATCGACATG-3′ Reverse 5′-TGTTGTACAAAGCGAGCACC-3′ F4/80 Forward 5′-CGTCAGCCGATTTGCTATCT-3′ Reverse 5′-CGGACTCCGCAAAGTCTAAG- 3′

[0080] Immunohistochemical Analysis

[0081] The liver tissue harvested from each mouse was fixed in a 10% formalin solution, and a paraffin block and a slide were prepared using each liver tissue that had been fixed. The degree of fibrosis in liver tissue was measured using Sirius red solution staining and a-SMA immunostaining.

[0082] In addition, ten 6-week-old C57BL/6 mice were acclimated for 1 week and then fed CDAA (choline-deficient L-amino-defined) for 8 weeks to induce non-alcoholic fatty liver. Thereafter, the 10 mice were divided into two groups, to which a vehicle and 50 mg/kg of ML-323 were orally administered, respectively, and then liver tissues were stained with H&E, F4/80, and a-SMA.

[0083] Experimental Results

[0084] Cytotoxicity measurement (WST assay)

[0085] There was no significant difference in viability between cells treated with 0, 1, 5, 10, and 25 11M of ML323 and untreated cells (FIG. 1). Accordingly, it was confirmed that ML323 did not exhibit cytotoxicity at all the treatment concentrations.

[0086] Measurement of Cell Fibrosis

[0087] It was confirmed that the mRNA expression of COL1A1 encoding collagen normally increased in the group treated with TGF-B compared to the control group not treated with TGF-B. It was confirmed that, when cells were treated with TGF-β, the mRNA expression of COL1A1 in the cells treated with ML323 was significantly lower than in the cells not treated with ML323 (FIG. 2). Thereby, it was confirmed that the composition of the present invention efficiently controls hepatic fibrosis in a hepatic fibrosis cell model.

[0088] Measurement of Liver Weight Change

[0089] It was confirmed that, in the case of the group fed the normal diet, there was no significant difference in liver weight or size between the ML323-admistered group and the control group, but in the case of the group fed the high-fat diet, the accumulation of fat in liver tissue in the ML323-administered group significantly decreased compared to that in the control group, and the liver weight also decreased in the ML323-administered group (FIG. 3).

[0090] Measurement of Liver Function Indicators in Blood

[0091] It was confirmed that the levels of AST and ALT in the blood from the ML323-administerd mice in the high-fat diet group significantly decreased by 40% and 70%, respectively, compared to those in the vehicle-administered control group, suggesting that the compound of the present invention significantly restores reduced liver function (FIG. 4).

[0092] Measurement of Expression of Hepatic Fibrosis Inducers

[0093] It was observed that the mRNA expression level of the COL1A1 gene encoding type 1 collagen greatly decreased in the liver tissue from the ML323-administered mice in the high-fat diet group (FIG. 5A), and the mRNA expression levels of the inflammation-related genes CCL2, TGF-0 and F4/80 also significantly decreased compared to those in the control group (FIG. 5B), suggesting that the composition of the present invention efficiently controls inflammation and fibrosis of liver tissue.

[0094] Immunohistochemical Analysis

[0095] As a result of Sirius red staining and a-SMA immunostaining, it was confirmed that hepatic fibrosis significantly decreased in the liver tissue from the ML323-administered mice (FIG. 6). In addition, as a result of H&E staining, f4/80 immunostaining and a-SMA immunostaining, it was observed that inflammation and hepatic fibrosis significantly decreased in the liver tissue from the ML323-administered group in the CDAA diet group (FIG. 7). From these results, it was confirmed that the composition of the present invention exhibits the effect of histologically ameliorating actual hepatic fibrosis.

[0096] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.