PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING NONALCOHOLIC FATTY LIVER DISEASE, CONTAINING GPR119 LIGAND AS ACTIVE INGREDIENT

Abstract

The present invention pertains to a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, the composition containing G protein coupled receptor 119 (GPR119) ligand as an active ingredient. The pharmaceutical composition according to the present invention exhibits the excellent effects of improving lipid metabolism, reducing fat accumulation in liver tissues, and preventing histological damage caused by inflammation and fibrosis in liver tissues, and can thus be useful in preventing or treating non-alcoholic fatty liver disease.

Claims

1. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising a compound represented by a following chemical formula 1, pharmaceutically acceptable salts thereof, optical isomers thereof, hydrates or solvates thereof, or mixtures thereof as an effective ingredient: ##STR00004## wherein A is oxadiazole, dihydrooxazole, thiazole, or thiadiazole, the A is independently unsubstituted or substituted with at least one substituent selected from the group consisting of halogen, C1-C6 straight or branched-chain alkyl and C1-C6 alcohol, and the alkyl or alcohol group is independently unsubstituted or substituted with halogen or C1-C6 alkoxy group; B is pyridine, pyrimidine, pyrazine, or oxadiazole, the B is independently unsubstituted or substituted with at least one substituent selected from the group consisting of halogen, C1-C6 straight or branched-chain alkyl, C1-C6 alcohol, C1-C6 alkoxy and oxadiazole group, and the C1-C6 straight or branched-chain alkyl, C1-C6 alcohol, C1-C6 alkoxy or oxadiazole group is independently unsubstituted or substituted with halogen, C1-C6 alkyl or C1-C6 alkoxy group; and X is independently F, Cl, Br or I.

2. The pharmaceutical composition according to claim 1, wherein, in the above chemical formula 1, A is ##STR00005## and R1 to R3, R5 and R6 are each independently one or more substituents selected from the group consisting of hydrogen, halogen, C1-C6 straight or branched-chain alkyl and C1-C6 alcohol, and the alkyl or alcohol group is independently unsubstituted or substituted with halogen or C1-C6 alkoxy group.

3. The pharmaceutical composition according to claim 1, wherein, in the chemical formula 1, B is ##STR00006## and the R7 to R11 are independently substituted with one or more substituents selected from the group consisting of hydrogen, halogen, C1-C6 straight or branched-chain alkyl, C1-C6 alcohol, C1-C6 alkoxy and oxadiazole group, and the alkyl, alcohol, alkoxy or oxadiazole group is independently unsubstituted or substituted with halogen, C1-C6 alkyl or C1-C6 alkoxy group.

4. The pharmaceutical composition according to claim 1, wherein, in the chemical formula 1, X is F.

5. The pharmaceutical composition according to claim 1, wherein, in the chemical formula 1, A is oxadiazole substituted with C1-C6 straight or branched-chain alkyl, B is pyrimidine substituted with C1-C6 straight or branched-chain alkyl, and X is F.

6. The pharmaceutical composition according to claim 1, wherein the compound represented by the chemical formula 1 is 3-(4-(3-(1-(5-ethylpyrimidine-2-yl)piperidine-4-yl)propoxy)-2,6-difluorophenyl)-5-isopropyl-1,2,4-oxadiazole.

7. The pharmaceutical composition according to claim 1, wherein the non-alcoholic fatty liver disease is selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis and cirrhosis.

8. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits deposition of triglycerides in liver tissues.

9. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits infiltration of inflammatory cells in liver tissues.

10. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition inhibits fibrosis of liver tissues.

11. A method for preventing or treating non-alcoholic fatty liver disease, the method comprising administering a therapeutically effective amount of a compound represented by a following chemical formula 1, pharmaceutically acceptable salts thereof, optical isomers thereof, hydrates or solvates thereof, or mixtures thereof into a subject in need of treatment: ##STR00007## wherein A, B and X are the same as shown in claim 1.

12. A food composition for preventing or alleviating non-alcoholic fatty liver disease, which comprises a compound represented by a following chemical formula 1, pharmaceutically acceptable salts thereof, optical isomers thereof, hydrates or solvates thereof, or mixtures thereof as an effective ingredient: ##STR00008## wherein A, B, and X are the same as in claim 1.

13. A method for preventing or alleviating non-alcoholic fatty liver disease, the method comprising administering a food composition comprising a compound represented by a following chemical formula 1, pharmaceutically acceptable salts thereof, optical isomers thereof, hydrates or solvates thereof, or mixtures thereof into a subject in need of amelioration: ##STR00009## wherein A, B, and X are the same as in claim 1.

14. A feed composition for preventing or alleviating non-alcoholic fatty liver disease, which comprises a compound represented by a following chemical formula 1, pharmaceutically acceptable salts thereof, optical isomers thereof, hydrates or solvates thereof, or mixtures thereof as an effective ingredient: ##STR00010## wherein A, B, and X are the same as in claim 1.

15. A use of a compound represented by a following chemical formula 1, pharmaceutically acceptable salts thereof, optical isomers thereof, hydrates or solvates thereof, or mixtures thereof in preventing or treating non-alcoholic fatty liver disease: ##STR00011## wherein A, B, and X are the same as in claim 1.

16. Use of a compound represented by a following chemical formula 1, pharmaceutically acceptable salts thereof, optical isomers thereof, hydrates or solvates thereof, or mixtures thereof in the manufacture of a medicament for preventing or treating non-alcoholic fatty liver disease: ##STR00012## wherein A, B, and X are the same as in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0129] FIG. 1 is a picture of a tissue sample showing an effect of reducing triglycerides and inhibiting infiltration of inflammatory cells by the compound of the present invention in a mouse model with induced non-alcoholic steatohepatitis.

[0130] FIG. 2 is an analysis graph showing an effect of inhibiting infiltration of inflammatory cells and inhibiting fibrosis by the compound of the present invention in a mouse model with induced non-alcoholic steatohepatitis (#, p<0.05 vs. Normal; * & **, p<0.05 &p<0.01 vs. Ob-NASH).

[0131] FIG. 3 is a picture of a tissue sample showing an effect of inhibiting fibrosis by the compound of the present invention in a mouse model with induced non-alcoholic steatohepatitis.

[0132] FIG. 4 is a view showing an effect of reducing AST and ALT by the compound of the present invention in a mouse model with induced non-alcoholic steatohepatitis (#, p<0.05 vs. Normal; *, p<0.05 vs. Ob-NASH).

[0133] FIG. 5 is a view showing results of measuring a concentration of proteins associated with inflammation and fibrosis in a mouse model with induced non-alcoholic steatohepatitis (###, p<0.001 vs. Normal; ** & ***, p<0.01 & p<0.001 vs. Ob-NASH).

[0134] FIG. 6 is a view showing results of measuring an expression level of genes associated with inflammation and fibrosis in a mouse model with induced non-alcoholic steatohepatitis.

[0135] FIG. 7 is a view showing results of evaluating a gene expression of GPR119 in cells at a time point before differentiating human monocytes into macrophages and after differentiation for 24 and 48 hours.

[0136] FIG. 8 is a view showing results of evaluating an effect on differentiation of human monocytes into macrophages according to treatment with the compound of the present invention.

[0137] FIG. 9 is a view showing results of evaluating an effect on activation of differentiated macrophages according to treatment with the compound of the present invention.

[0138] FIG. 10 is a picture of a tissue sample showing an effect of reducing triglycerides and inhibiting infiltration of inflammatory cells in liver tissues by the compound of the present invention in a mouse model with induced non-alcoholic steatohepatitis.

[0139] FIG. 11 is a view showing results of measuring a content of triglycerides in liver tissues in a mouse model with induced non-alcoholic steatohepatitis (** & ***, p<0.01 & p<0.001 vs. DIO-NASH).

[0140] FIG. 12 is a view showing a change in NAFLD activity score (NAS) before and after administration into the same individual in a mouse model with induced non-alcoholic steatohepatitis.

[0141] FIG. 13 is a picture of a tissue sample showing an effect of inhibiting fibrosis by the compound of the present invention in a mouse model with induced non-alcoholic steatohepatitis.

[0142] FIG. 14 is a view showing results of measuring a content of type I collagen in liver tissues in a mouse model with induced non-alcoholic steatohepatitis (* & ***, p<0.05 & p<0.001 vs. DIO-NASH).

[0143] FIG. 15 is a view showing results of measuring a concentration of AST and ALT in blood in a mouse model with induced non-alcoholic steatohepatitis (** & ***, p<0.01 &p<0.001 vs. DIO-NASH).

[0144] FIG. 16 is a view showing results of measuring a gene expression level of monocyte attractant, macrophage markers and fibrosis markers in liver tissues of a mouse model with induced non-alcoholic steatohepatitis (*, ** & ***, p<0.05, p<0.01 & p<0.001 vs. DIO-NASH).

MODE FOR INVENTION

[0145] The features and advantages of the present invention as well as methods for achieving them will be apparent with reference to exemplary embodiments described in detail hereinafter. However, the present invention is not limited to the Examples disclosed hereinafter, but will be implemented in various different forms. Hereinafter, the following Examples will be suggested for better understanding of the present invention and are provided only for the purpose of completely illustrating the scope of the present invention to those skilled in the art, and thus the present invention will be defined only by the scope of the claims thereto.

<Example 1> Confirmation of Efficacy of GPR119 Ligand in Mouse Model with Induced Steatohepatitis Due to Supply of Special Diet

[0146] In order to confirm an effect of a GPR119 ligand compound according to the present invention on preventing non-alcoholic fatty liver disease, a following experiment was performed.

[0147] Preparation of Mouse Model with Non-Alcoholic Steatohepatitis

[0148] A six-week-old male ob/ob mouse with leptin deficiency was fed with a special diet containing high fat, high fructose, and high cholesterol for 10 weeks, and thus was induced to develop non-alcoholic steatohepatitis. A mixed form of diet was prepared so that a daily dose of (3-(4-(3-(1-(5-ethylpyrimidin-2-yl)piperidin-4-yl)propoxy)-2,6-difluorophenyl)-5-isopropyl-1,2,4-oxadiazole (hereinafter, “compound 1”) reaches 100 mg/kg/day in the compound represented by the chemical formula 1, and supplied to the mouse from a time point of starting the supply of the special diet for 10 weeks.

[0149] Histological Examination

[0150] For a histological examination, the prepared mouse model with induced non-alcoholic steatohepatitis was subjected to autopsy to fix an isolated liver tissue in 10% formalin and prepare a paraffin block, thereby obtaining a 2 μm-thick tissue section. To confirm infiltration of inflammatory cells, Hematoxylin and Eosin (HE) stain was performed using an automatic staining machine (Autostainner XL, Leica), and the results thereof are shown in FIGS. 1 and 2.

[0151] As understood from FIGS. 1 and 2, the ob/ob mouse (Ob-NASH) fed with the special diet for 10 weeks showed a remarkable increase in triglycerides lipid droplets in liver tissues compared to C57BL/6 mouse fed with a normal diet (Normal), and the infiltration of inflammatory cells was also confirmed. In contrast, it was confirmed that the infiltration of inflammatory cells is significantly inhibited when the compound 1 of the present invention is simultaneously supplied with the special diet (Ob-NASH+Compd 1).

[0152] Then, for evaluation of fibrosis, fibers in liver tissue were specifically stained by using Masson's Trichrome stain or Sirius Res stain, and the results thereof are shown in FIGS. 2 and 3.

[0153] As understood from FIGS. 2 and 3, it was confirmed that the ob/ob mouse fed with the special diet for 10 weeks shows purple-colored liver tissues as a whole with a remarkable progress of fibrosis unlike the C57BL/6 mouse fed with the normal diet, but fibrosis of liver tissues is significantly inhibited when the compound 1 of the present invention is simultaneously supplied with the special diet.

[0154] AST and ALT Measurement in Blood

[0155] The mouse model with induced non-alcoholic steatohepatitis was subjected to autopsy, after which plasma was separated therefrom, so as to quantify aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by using an automatic blood analyzer (Konelab 20i), and the results thereof are shown in FIG. 4.

[0156] As shown in FIG. 4, it was confirmed that the ob/ob mouse fed with the special diet for 10 weeks shows a significant increase in ALT and AST levels in blood, which is a marker of liver damage, due to damage to liver tissues unlike the C57BL/6 mouse fed with the normal diet. On the other hand, it was confirmed that ob/ob mouse is administered compound 1 of the present invention together with the special diet shows a significant inhibition of increase in ALT and AST, which means that hepatocytes damage is alleviated.

[0157] Confirmation of Concentration of Proteins Associated with Inflammation and Fibrosis

[0158] To evaluate an effect on the expression of inflammation-related genes and fibrosis-related proteins in liver tissues, the liver tissues obtained from the mouse model were put into RIPA buffer (Cell Signaling) and pulverized with TissueLyser II™ (Quiagen), after which a total protein concentration was quantified with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and a protein concentration of Mouse Ccl2 (=Mcp1; R&D Systems, MJE00) and Timp1 (R&D Systems, MTM100) in the liver tissues was quantified by using a commercially available ELISA kit, so that the results thereof are shown in FIG. 5.

[0159] As shown in FIG. 5, it was confirmed that the mouse fed with the special diet shows an increase in protein concentrations of Ccl2 (=Mcp1), in the liver tissues with regard to Ccl2 (=Mcp1), which is a factor for inducing inflammatory cells to the liver tissues, and Timp1, an endogenous inhibitor for enzymes degrading fibers deposited in the liver tissues, while protein levels of Ccl2 and Timp1 are significantly inhibited when the compound 1 of the present invention is administered together with the special diet.

[0160] Confirmation of Expression of Genes Associated with Inflammation and Fibrosis

[0161] To measure an expression level of inflammation-related genes and fibrosis-related genes in liver tissues, Trizol® (Invitrogen) was added to the liver tissues collected from the mouse model, after which a total RNA was extracted from the tissues according to the method provided by the manufacturer, and then cDNA was synthesized by using a reverse transcriptase. Then, a real-time polymerase chain reaction was performed in LightCycler®480 Instrument II (RocheLifeScience) equipment by using a reagent of SYBR Green I Master Mix (Roche, 04707516001), and the results thereof are shown in FIG. 6.

[0162] As shown in FIG. 6, it was confirmed that the mouse fed with the special diet for weeks shows an increase in expression of inflammation-related mouse genes (Ccl2, Lgals3, Tnfa) and fibrosis-related genes (Tgfb1, Col1a, Timp1, Timp2, Acta2), while the increase in expression of genes associated with inflammation and fibrosis is significantly inhibited when the compound 1 of the present invention is administered together with the special diet.

[0163] The above results suggest that the compound of the present invention has an excellent effect on non-alcoholic fatty liver disease by inhibiting damage, inflammation and fibrosis of liver tissues.

<Example 2> Confirmation of Inhibitory Effect on Differentiation of Human Monocytes and Activation of Differentiated Macrophages

[0164] To evaluate a direct anti-inflammatory effect of the compound according to the present invention, the effect on the differentiation of human monocytes and the activation thereof was confirmed.

[0165] First, as a result of evaluating a gene expression of GPR119 in cells at a time point before differentiating human monocytes (THP-1, ATCC® TIB-202™) into macrophages and after differentiation for 24 and 48 hours by using Phorbol 12-myristate 13-acetate (PMA), it was confirmed that an expression of GPR119 receptor increases with time as monocytes are differentiated into macrophages (FIG. 7).

[0166] Then, THP-1 monocytes were treated with PMA (50 ng/ml) for 48 hours to be differentiate into macrophages, while the compound 1 was co-treated, then replaced with serum-free medium, and then treated with lipopolysaccharides (LPS, 0.5 ng/ml) for four hours so as to quantity interleukin-1β (IL-1β, R&D Systems, DY201) secreted into the medium by using a commercialized ELISKA kit and evaluate an effect on the differentiation of monocytes, so that the results thereof are shown in FIG. 8.

[0167] As shown in FIG. 8, it was confirmed that IL-1β secretion caused by activation of macrophages is decreased in a concentration-dependent manner when treated with the compound of the present invention in a process of differentiating monocytes into macrophages.

[0168] In addition, after treating the differentiated macrophages with the compound 1 for 48 hours, the secreted IL-1β was quantified by the same method as above with regard to the activation of immune cells by LPS, so as to evaluate an effect on the activation of differentiated macrophages. Thus, the results thereof are shown in FIG. 9.

[0169] As shown in FIG. 9, it was confirmed that IL-1β secretion caused by activation of macrophages is decreased in a concentration-dependent manner even when the differentiated macrophages are treated with the compound of the present invention.

[0170] The above results suggest that the compound of the present invention has an excellent effect on preventing and treating non-alcoholic fatty liver disease by directly inhibiting the differentiation and activation of immune cells.

<Example 3> Confirmation of Efficacy of GPR119 Ligand in Mouse Model with Induced Steatohepatitis Due to Supply of Special Diet

[0171] In order to confirm an effect of a GPR119 ligand compound according to the present invention on treating non-alcoholic fatty liver disease, a following experiment was performed.

[0172] Preparation of a Mouse Model with Non-Alcoholic Steatohepatitis and Study Design

[0173] A six-week-old male C57BL/6J mouse was fed with a special diet comprising high fat, high fructose, and high cholesterol for at least 26 weeks, and thus was induced to develop non-alcoholic steatohepatitis. It was confirmed that fatty liver and inflammatory fibrosis are induced through a biopsy of liver tissues three weeks before drug supply, and mice were evenly distributed into each group based on the area of collagen stained in the liver tissues. A feed was prepared by mixing the compounds according to the present invention in a special diet and supplied for additional eight weeks. According to the diet supplied to the mice, each of the groups was classified as a control group fed with a normal diet (Normal), a positive control group in which mice with induced non-alcoholic steatohepatitis due to the special diet were not supplied with a drug (DIO-NASH), and a group in which mice with induced non-alcoholic steatohepatitis were supplied with the compound 1 in an amount of 30 mg/kg/day (L) and 100 mg/mg/day (H) (Cmpd1 (L) and Cmpd1 (H), respectively).

[0174] Histological Examination

[0175] For a histological examination, the prepared C57BL/6J mouse model was subjected to autopsy to fix an isolated liver tissue in 10% formalin and prepare a paraffin block, thereby obtaining a 2 μm-thick tissue section. Then, Hematoxylin and Eosin (HE) stain was performed using an automatic staining machine (Autostainner XL, Leica), and the results thereof are shown in FIG. 10. In addition, the liver tissue isolated from the C57BL/6J mouse model was used to measure a content of triglycerides in the liver tissue by using a Triglyceride reagent (Roche Diagnostics, #22-045-795), and the results are shown in FIG. 11.

[0176] As understood from FIGS. 10 and 11, the mice fed with a special diet only (DIO-NASH) showed a remarkable increase in fat deposits and infiltration of inflammatory cells in liver tissues compared to the mice fed with a normal diet (Normal), while the DIO-NASH_Compd1 (H) group fed with the compound 1 of the present invention together with the special diet showed a significant decrease in fat deposits and infiltration of inflammatory cells.

[0177] In addition, as a result of calculating an NAFLD activity score (NAS) reflecting a ratio of fat cells, infiltration of inflammatory cells and damage to hepatocytes based on the above histological examination, before and after administration, the disease worsened in 50% of the DIO-NASH group, while no individual was deteriorated in case of administering the compound, and the disease was ameliorated in about 50% of the DIO-NASH_Compd1 group (FIG. 12).

[0178] Then, for the evaluation of fibrosis, the fibers of the liver tissues were specifically stained with Sirius Res stain in the mice of each group, and a content of type I collagen in the liver tissues was measured by an imaging analysis method after immunostaining, so that the results thereof are shown in FIGS. 13 and 14.

[0179] As shown in FIGS. 13 and 14, the mice fed with the special diet only (DIO-NASH) showed a remarkable progress of fibrosis in liver tissues compared to the mice fed with the normal diet (Normal), while the group fed with the compound 1 of the present invention together with the special diet (DIO-NASH_Compd1) showed a remarkable decrease in a cross-linking formation in fibers of the liver tissues and also showed a significant decrease in type I collagen.

[0180] AST and ALT Measurement in Blood

[0181] Each of the mouse groups was subjected to autopsy, after which plasma was separated therefrom, so as to quantity aspartate aminotransferase (AST) and alanine aminotransferase (ALT) by using an automatic blood analyzer (Konelab 20i). Thus, the results thereof are shown in FIG. 15.

[0182] As shown in FIG. 15, the group of mice fed with the special diet only (DIO-NASH) showed a significant increase in a concentration of ALT and AST in blood compared to the mice fed with the normal diet, while the group of mice fed with the compound 1 of the present invention together with the special diet (DIO-NASH_Compd1) showed a significantly inhibited increase in ALT and AST.

[0183] Confirmation of Change in Gene Expression

[0184] To analyze a change in total gene expression in liver tissues isolated from the above mouse model in eight weeks after drug administration, the isolated RNA fraction was used to generate a library by using NeoPrep (Illumina), and subjected to RNAseq profiling through NexSeq 500 (Illumina), after which bioinformatics analysis was performed and changes in major gene groups were classified for each function, so that the results thereof are shown in FIG. 16.

[0185] As shown in FIG. 16, the mice fed with the special diet only (DIO-NASH) showed an increase in the expression of monocyte attractants, macrophage markers and fibrosis markers in liver tissues compared to the mice fed with the normal diet (Normal), while the DIO-NASH_Compd1 group of mice fed with the compound 1 of the present invention together with the special diet showed a remarkable decrease in the above markers.

[0186] The above results suggest that the compound of the present invention has an effect on non-alcoholic fatty liver disease by inhibiting the damage, inflammation and fibrosis of liver tissues, and has a more excellent therapeutic effect by co-administering a DPPIV inhibitor.