S1PR4-targeting composition for preventing or treating non-alcoholic steatohepatitis

Abstract

The present invention relates to a S1PR4-targeting composition for preventing or treating non-alcoholic steatohepatitis and, more particularly, to a pharmaceutical composition and a health functional food composition, both comprising a sphingolipid compound which serves as a functional inhibitor against S1PR4, showing prophylaxis and therapy of non-alcoholic steatohepatitis. The sphingolipid compound of the present invention is expected to be applied as a leading material effective for the prevention or treatment of non-alcoholic steatohepatitis (NASH) as it has the effect of reducing the infiltration of inflammatory cells into hepatic tissues and suppressing fibrosis and decreases a level of liver injury (ALT), inflammation in hepatic tissues, the expression of a fibrosis-related gene.

Claims

1. A method for treating nonalcoholic steatohepatitis (NASH) comprising administering to a subject a pharmaceutical composition comprising a compound selected from the group consisting of the following compounds, an optical isomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient: (1) 2-amino-2-(2-(3-decylisoxazol-5-yl)ethyl)propane-1,3-diol; (2) 2-amino-2-(2-(1-decyl-1H-1,2,3-triazol-4-yl)ethyl)propane-1,3-diol; (3) 2-amino-2((3-octylisoxazol-5-yl)ethynyl)propane-1,3-diol; (4) 2-amino-2-(2-(3-octylisoxazol-5-yl)ethyl)propane-1,3-diol; (5) 2-amino-2-(hydroxymethyl)-4-(3-octylisoxazol-5-yl)butyl dihydrogen phosphate; (6) 2-amino-2-((3-decylisoxazol-5-yl)ethynyl)propane-1,3-diol; (7) 2-amino-4-(3-decylisoxazol-5-yl)-2-(hydroxymethyl)butyl dihydrogen phosphate; (8) 2-amino-2-(2-(3-(4-hexylphenethyl)isoxazol-5-yl)ethyl)propane-1,3-diol; (9) 2-amino-2((3-dodecylisoxazol-5-yl)ethynyl)propane-1,3-diol; (10) 2-amino-2-(2-(3-dodecylisoxazol-5-yl)ethyl)propane-1,3-diol; (11) 2-amino-4-(3-dodecylisoxazol-5-yl)-2-(hydroxymethyl)butyl dihydrogen phosphate; (12) 2-amino-2-(2-(1-octyl-1H-1,2,3-triazol-4-yl)ethynyl)propane-1,3-diol; (13) 2-amino-2-(2-(1-octyl-1H-1,2,3-triazol-4-yl)ethyl)propane-1,3-diol; (14) 2-amino-24(1-decyl-1H-1,2,3-triazol-4-yl)ethynyl)propane-1,3-diol; (15) 2-amino-2-(2-(1-(4-hexylphenethyl)-1H-1,2,3-triazol-4-yl)ethyl)propane-1,3-diol; (16) 2-amino-2-(1-butyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol; (17) 2-amino-2-(3-dodecylisoxazol-5-yl)propane-1,3-diol; (18) (E)-2-amino-2-(2-(3-decylisoxazol-5-yl)vinyl)propane-1,3-diol; (19) (E)-2-amino-2-(1-butyl-1H-1,2,3-triazol-4-yl)propane-1,3-diol; (20) 2-amino-2-(2-(3-(8-phenyloctyl)-isoxazol-5-yl)ethyl)propane-1,3-diol; (21) 2-amino-2-(2-(1-(8-phenyloctyl)-1H-1,2,3-triazolebutyl-4-yl)propane-1,3-diol; (22)N-(2-(1-dodecyl-1H-1,2,3-triazol-4-yl)-1,3-dihydroxypropan-2-yl)acetamide; (23)N-(2-(3-dodecylisoxazol-5-yl)-1,3-dihydroxypropan-2-yl)acetamide; (24)N-(4-(1-decyl-1H-1,2,3-triazol-4-yl)-1-hydroxy-2-(hydroxymethyl)butan-2-yl)acetamide; (25)N-(4-(3-decylisoxazol-5-yl)-1-hydroxy-2-(hydroxymethyl)butan-2-yl)acetamide; and (26)N-(4-(1-(4-hexylphenethyl)-1H-1,2,3-triazol-4-yl)-1-hydroxy-2-(hydroxymethyl)butan-2-y1)acetamide.

2. A method for treating nonalcoholic steatohepatitis (NASH) comprising administering to a subject a pharmaceutical composition comprising a compound represented by the following Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient: ##STR00033##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

(2) FIG. 1 is a schematic diagram showing a mechanism for treating nonalcoholic steatohepatitis of a compound according to the present invention;

(3) FIG. 2 shows the result identifying the inhibitory effect of the sphingolipid compound SLB736 according to the present invention on the activity of NLRP3 inflammation-regulating complex;

(4) FIG. 3 shows the result identifying the inhibitory effect of the SLB736 compound according to the invention on S1PR4 expression in a mouse liver tissue model;

(5) FIG. 4 shows the result identifying the inhibitory effect of the SLB736 compound according to the present invention on S1PR4 expression in a mouse macrophage model;

(6) FIG. 5 shows the result identifying that the SLB736 compound of the present invention is not recycled to the cell membrane after being introduced into the cells;

(7) FIG. 6 shows the result identifying the inhibitory effect of reducing IL-β production of S1PR4 shRNA in mouse macrophages;

(8) FIG. 7 shows the result of microscopic examination for identifying the effect of SLB736 on the prevention of steatosis and lobular inflammation in liver tissue of a NASH-induced mouse model;

(9) FIG. 8 shows the result of microscopic examination for identifying the effect of SLB736 on the prevention of fibrosis in liver tissue of a NASH-induced mouse model;

(10) FIG. 9 shows the result of evaluation on the NASH prevention effect of SLB736 in liver tissue of the NASH-induced mouse model based on the CRN scoring system;

(11) FIG. 10 shows the result identifying the expression inhibition effects of inflammatory and fibrosis markers in liver tissue of the mouse model;

(12) FIG. 11 shows the result identifying the therapeutic effect of SLB736 in liver tissue of the mouse model (A—treatment of steatosis and lobular inflammation; B—treatment of fibrosis); and

(13) FIG. 12 shows the result identifying that lymphopenia does not appear in blood after administration of SLB736, compared to after administration of FTY720.

BEST MODE

(14) Hereinafter, the present invention will be described in more detail with reference to the following examples. However, it will be obvious to those skilled in the art that these examples are provided only for illustration of the present invention and should not be construed as limiting the scope of the present invention.

EXAMPLE 1

Selection of Sphingolipid Candidates

(15) Fifteen types of sphingolipid compounds structurally similar to myriocin and FTY720 (fingolimod), among about 600 types of sphingolipid compounds held by a sphingolipid material bank, were obtained in order to develop lead candidates for the development of nonalcoholic steatohepatitis (NASH) drugs.

(16) The result of measurement of agonistic activity of S1P receptors (sphingosine 1-phosphate receptors) using the GPCR activity measurement method of DiscoveRX Corp. showed that 2-amino-2-(2-(1-decyl)-1H-1,2,3-triazol-4-yl)ethyl)propane-1,3-diol (hereinafter referred to as “SLB736”) has an agonistic effect specific for S1PR4.

(17) ##STR00032##

EXAMPLE 2

Inhibitory Activity of NLRP3 Inflammation-Regulating Complex

(18) It is well known that the treatment of macrophages with lipopolysaccharide (LPS) results in activation of inflammation-regulating complex (inflammasome), thereby increasing IL-1β production (Mariathasan et al., Nat. Rev. Immunol. 2007; 17186029). In order to determine the inhibitory effect of the activity of inflammation-regulating complex (NLRP3 inflammasome), which is a representative inflammation-regulating protein, the IL-1(3 production of candidate substances upon the treatment of macrophages with LPS was measured and evaluated.

(19) First, 12 hours after treatment of Raw cells (mouse macrophage lines) with LPS (100 ng/ml, Sigma-Aldrich), the concentration of IL-1β in the medium was found to be markedly increased.

(20) The macrophages were treated with myriocin in different concentrations as a control group and were then treated with the same concentration of LPS for 24 hours. As a result, it was found that 10 μM of myriocin effectively reduced IL-1β production. Based on this, experiments were conducted to determine the effect of sphingolipid candidates on the inhibition of IL-1β.

(21) The result of treatment with the known sphingolipid derivative drug, FTY720, showed that FTY720 exhibits an inhibitory effect of IL-1β production similar to 10 μM myriocin at a concentration of 0.1 μM, which is a much lower concentration than myriocin. On the other hand, when treating with SLB736, the compound represented by Formula 2 according to the present invention, at 0.1 μM or 1.0 μM and then with LPS, it was found that the inhibitory effect of IL-1β production was excellent at each concentration to a significant extent to FTY720 (FIG. 2).

EXAMPLE 3

Identification of Effects of SLB736 on Inhibition of Expression of S1PR4 in Tissue and Cells

(22) In order to determine whether or not SLB736, the compound of the present invention, acts as a functional antagonist of S1PR4, changes in S1PR4 protein expression were observed after administration of candidate substances to nonalcoholic steatohepatitis (NASH) model mice and macrophages thereof.

(23) It is well known that the administration of a methionine choline-deficient diet (MCDD) to mice results in non-alcoholic steatohepatitis. The mice (C57BL/6N, Orient Bio, Inc., Korea) used in the experiments were divided into three groups, and MCDD alone, 1 mg/kg of MCDD and FTY720, and 1 mg/kg of MCDD and SLB736 were administered to each group for 6 weeks. Then, changes in the expression of S1PR4 protein in liver tissue of mice were measured through Western blotting. As a result, it was found that the expression of S1PR4 was significantly increased in the mice administered with MCDD alone compared to the control (con) mice not administered with MCDD, and the amount of S1PR4 protein was decreased in the mice administered with FTY720 or SLB736 in combination with MCDD (FIG. 3).

(24) In addition, Raw cells, which are mouse macrophage lines, were treated with a FTY720 or SLB736 substance, and were then treated with LPS, and S1PR4 expression was then measured. As a result, the expression of S1PR4 was significantly increased in cells treated only with LPS, compared to the control group (con) without any treatment, whereas treatment with FTY720 or SLB736 in combination with LPS was found to significantly reduce the expression level of S1PR4 (FIG. 4).

(25) This result demonstrates that the SLB736 compound of the present invention functions to reduce the expression level of S1PR4 protein.

EXAMPLE 4

Identification of Activity as Functional Antagonist of S1PR4

(26) Cell lines overexpressing S1PR4-EGFP were used to identify whether or not SLB736 and FTY720 actually internalize S1PR4 into cells and thereby act as functional antagonists.

(27) Specifically, the cell lines overexpressing S1PR4-EGFP were incubated in a cover glass allowing for microscopic observation and were then treated with a vehicle (dimethyl sulfoxide hydrochloric acid, 100 nM), S1P (100 nM; agonist, positive control group), FTY720 (100 nM) and SLB736 (1 μM). 30 minutes and 2 hours after treatment with each material, the cell lines were fixed with a fixing solution and observed with a fluorescence microscope. At this time, the cells were cultured after treatment with a protein synthesis inhibitor (cycloheximide) in order to inhibit the synthesis of new receptors, were first observed with a fluorescence microscope 30 minutes after each material treatment, and were then washed. Then, the cells were further cultured for 1 hour 30 minutes and were secondarily observed, and cell images were obtained.

(28) The results are shown in FIG. 5. It could be seen that S1PR4 was internalized in the medium treated with S1P, FTY720 and SLB736 after cell culture for 30 minutes, and in SlP (agonist)-treated cells after cell culture for 2 hours, S1PR4 was recycled to the cell membrane, whereas FTY720 and SLB736 were not recycled thereto. This indicates that SLB736 according to the present invention acts as a functional antagonist of S1PR4, like FTY720.

EXAMPLE 5

Identification of Inhibition of IL-1β Production by S1PR4 shRNA

(29) In addition, after infecting Raw cells, the macrophage line, with S1PR4 shRNA lentivirus, viable cells capable of inhibiting S1PR4 expression were selected. When treating the selected cells with LPS, changes in IL-1β production were measured and were compared with control groups (Vec, Vec+LPS) not infected with shRNA lentivirus.

(30) As a result, it could be seen from FIG. 6 that the IL-1β concentration was significantly reduced in the macrophage line infected with S1PR4 shRNA lentivirus (shS1PR4+LPS) compared to the control group (Vec+LPS).

EXAMPLE 6

Identification of Effects of SLB736 on Prevention of NASH Onset and Treatment of NASH

(31) In order to identify the effect of the SLB736 compound on the prevention of onset of non-alcoholic steatohepatitis (NASH), an animal model administered with MCDD for 6 weeks was used.

(32) Generally known characteristic microscopic findings of NASH include steatosis, ballooning degeneration of hepatocytes, lobular inflammation, perisinusoidal fibrosis and the like. In 2005, the NASH clinical research network (CRN) designed a detailed grading system for lesions corresponding to nonalcoholic steatohepatitis and thus proposed the nonalcoholic fatty liver disease activity score (NAS), which is widely used in various research due to the excellent reproducibility thereof. Accordingly, the present inventors have scored the grade of steatosis, location of steatosis, lobular inflammation and the extent of fibrosis, from the results of liver tissue observation of animal models, based on the CRN classification system. Based on this, the effect of SLB736, which is the target compound in the present invention, on the prevention or progression of non-alcoholic steatohepatitis caused by MCDD was evaluated.

(33) Specifically, eight-week-old mice (C57BL6/N, Orient Bio Inc., Korea) were divided into two groups, the control group was fed only with MCDD for 6 weeks to induce the onset of NASH, and the experimental group was fed with SLB736 in a dose of 1 mg/kg/day for six weeks in combination with MCDD, liver tissue was collected from each mouse and was observed with a microscope, and the degree of steatosis, lobular inflammation and fibrosis was observed. The result showed that the degree of steatosis and lobular inflammation (see black triangle in FIG. 7) were significantly reduced in mice administered in combination with SLB736, and the degree of fibrosis was also significantly reduced in the SLB736 administration group.

(34) In order to determine this quantitatively, evaluation was conducted according to the NAS system of the CRN. The result showed that, as shown in FIG. 9, the degree of steatosis, lobular inflammation and the degree of fibrosis were all significantly alleviated compared to the control group.

(35) In addition, the expression levels of TGF-β, TNF-α and MCP-1 were measured in order to identify the effects of the SLB736 compound on the expression of inflammatory markers and fibrosis markers in liver tissue. Specifically, RNA was extracted from liver tissue of the animal model and mRNA was measured using Q-PCR. The result showed that the expression of all of TGF-β, TNF-α and MCP-1 was reduced to a significant level in mice administered with SLB736 (FIG. 10).

(36) This shows that the compound SLB736 of the present invention is effective in preventing nonalcoholic steatohepatitis (NASH).

(37) A NASH-induced animal model obtained by administering MCDD to 8-week-old mice (C57BL6/N, Orient Bio Inc., Korea) for 4 weeks was used in order to identify the effect of the SLB736 compound on the treatment of non-alcoholic steatohepatitis (NASH). Specifically, the NASH-induced model mice were divided into two groups, the control group was further fed with MCDD for 4 weeks, the experimental group was fed with SLB736 at a dose of 5 mg/kg/day for 4 weeks in combination with MCDD, and liver tissue was collected from each mouse and observed with a microscope to determine the degree of steatosis, lobular inflammation and fibrosis.

(38) The result showed that the control group that had been continuously fed with MCDD was found to have steatosis, lobular inflammation and fibrosis in liver tissues due to the administration of MCDD, indicating the onset of NASH. On the other hand, the mice administered with SLB736, in combination with a general diet, exhibited a considerable reduction in the degree of steatosis and lobular inflammation (FIG. 11), and the SLB736 administration group also exhibited a considerable reduction in the degree of fibrosis (FIG. 11).

EXAMPLE 7

Comparison of Lymphopenia in Blood Between Administration of SLB736 and FTY720

(39) FTY720, which acts as a functional antagonist of S1PR, has been reported to cause the serious side effect, lymphopenia in the blood. Therefore, when the SLB736 of the present invention was administered, changes of leukocytes and lymphocytes in the blood were measured to determine the possibility of side effects. On the day of autopsy, blood was collected from the posterior vein using a syringe and whole blood was refrigerated in an EDTA-2K CBC bottle. The leukocytes and lymphocytes in the whole blood were measured using an automated analyzer.

(40) As shown in FIG. 12, the result showed that the mice administered with SLB736 had no reduction of lymphocytes and leukocytes in blood, which means that administration of FTY720 causes no side effects.