WO2015111971A1 - Pharmaceutical composition containing gpr119 ligand as active ingredient for preventing or treating non-alcoholic fatty liver disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing a G protein coupled receptor 119 (GPR119) ligand as an active ingredient for preventing or treating non-alcoholic fatty liver disease. More specifically, it was established that the GPR119 ligand, which has been developed as only an anti-diabetic medicine, exhibits an excellent effect in the treatment of non-alcoholic fatty liver, and the signaling pathway in liver cells therefor is different from the signaling pathway in the small intestine or pancreas exhibiting an anti-diabetic effect, and thus it was confirmed that the GPR119 ligand can be useful to treat non-alcoholic fatty liver.

Description

Pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease comprising GPR119 ligand as an active ingredient

The present invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising GPR119 (G protein coupled receptor 119) ligand as an active ingredient.

Fatty liver refers to a condition in which abnormal fat is accumulated in liver cells, and a medical condition refers to a pathological condition in which the neutral lipid content exceeds 5% of the total liver weight. In general, fatty liver can be divided into alcoholic fatty liver disease (ALD) caused by persistent and excessive drinking and nonalcoholic fatty liver with little alcohol intake but showing liver tissue similar to alcoholic fatty liver. have.

Alcoholic fatty liver is common in Korea, and it is caused by the ingestion of alcohol, which promotes fat synthesis in the liver and does not undergo normal energy metabolism, and can develop into hepatitis or cirrhosis depending on the degree of alcohol consumption.

Non-alcoholic fatty liver can be caused by the causes of obesity, diabetes, hyperlipidemia, drugs, etc., regardless of drinking, and simple fatty liver (hepatcellular inflammation) and hepatocellular inflammation that do not accompany an inflammatory response with progress. It refers to a wide range of diseases including non-alcoholic steatohepatitis (NASH), advanced fibrosis and cirrhosis.

Nonalcoholic fatty liver disease (NAFLD) is an increase in adult disease caused by high-fat and high-calorie diets in modern society, with 20-30% of the adult population in developed countries representing non-alcoholic fatty liver disease (NAFLD). Not only is 3% reported to be non-alcoholic steatohepatitis (NASH) patients, especially histological findings of hepatitis with fibrosis and inflammation are very high risk of developing cirrhosis, liver failure and liver cancer.

It has been found that fatty liver disease is closely related to obesity, insulin resistance, type 2 diabetes, etc., but the clear pathogenesis is currently under active research. It is thought to be the main pathogenesis of insulin resistance, which begins with accumulation and is associated with obesity and diabetes. It has also been reported that lipotoxicity caused by increased free fatty acid (FFA) or cholesterol in hepatocytes and increased inflammatory cytokines and their receptors play an important role in the progression from fatty liver to fatty hepatitis.

According to the 2009 National Bureau of Statistics, the mortality rate from liver disease is the highest among the OECD countries, with the highest incidence of liver cancer, resulting in high social and economic loss. The domestic market for specialty liver disease drugs was KRW 77.2 billion in 2003. In 2010, the company's growth reached 47.5 billion won, up 4% from the previous year, and steady growth is expected in the future. In addition, the global market for soy protectors was $ 6.2 billion in 2003, and is expected to grow by 10-35% in five years. The domestic dietary supplement market also grew from 1.2 trillion won in 2003 to 1.5 trillion won in 2004, and is expected to reach 2 trillion won in 2010. According to a survey by global consulting firm BCC Research, The fatty liver drug market is estimated at $ 8 billion annually.

There are two main types of treatments currently used for alcoholic or nonalcoholic fatty liver patients: 1) obesity treatment (orlistat), insulin resistance treatment (metformin, pioglitazone, rosiglitazone), hyperlipidemia treatment (clofibrate, gemfibrozil, bezafibrate, atorvastatin drugs to treat and improve fatty liver through the correction of risk factors, such as simvastatin, etc. 2) Hepatoprotective agents (ursodeoxycholic acid and taurine) as agents for the recovery of hepatocytes and liver function that are already damaged independently of risk factors for fatty liver. Examples include antioxidants (vitamine E) and nutritional supporters (lectin, betaine, N-acetylcystein). However, the above-mentioned conventionally used therapeutic agents are not essential drugs in terms of efficacy, and thus are not used as target effects, and thus, there are few agents that can cure pharmacological fatty liver to date. Therefore, it is expected that a huge ripple effect can be expected when developing an appropriate treatment.

The most important concept in modern drug development is the target protein, which means that its function can be changed by the action of therapeutic drugs and affect the development of the disease. Finding compounds that can act is the first basic step in drug development. However, to date, many candidates for non-alcoholic fatty liver treatment proceed based on the results of animal or cell experiments, and thus many cases have not been established clearly.In case of using natural products, the inherent chemical composition is unclear. There was a limit to development because there were many.

The human GPR119 gene is located on the X chromosome and consists of a single exon. Although the gene composition of the mouse or rat is different, the expressed protein has many similarities between humans and mice. As with the other G Protein coupled receptor family, the G protein with 7 transmembrane domains and intracellular interactions is not yet clear.

GPR119 receptors are known to be present mainly in beta cells of the pancreas, K-cells and L-cells, which are enteroendocrine cells of the small intestine. GPR119 activation in the pancreas is known to increase insulin secretion against external glucose stimulation by increasing intracellular adenylate cyclase as a second messenger. This insulin promoting action is a glucose-dependent response, and therefore, the GPR119 receptor has an advantage that there is no hypoglycemic effect of the existing diabetes treatment. Activation of GPR119 in the small intestine has been reported to promote the secretion of GLP-1, GLP-2, peptide YY in L-cells and the secretion of insulinotropic peptide (GIP) in K-cells. All are associated with a signal that promotes hypoglycemic activity and can predict the mechanism of antidiabetic efficacy. Indeed, the administration of GPR119 ligand in mice has been reported to effectively improve the insulin tolerance test (ITT) and the glucose tolerance test (GTT).

Endogenous GRP119 ligands include human lipid-like substances [N-acylathanolamine (NAE) such as OEA, PEA, LEA, etc.], and these have been reported to have affinity for receptors such as PPAR alpha and TRPV1 in addition to GPR119. Synthetic GPR119 ligands were prepared by major pharmaceutical companies such as Arena Pharmaceuticals and GlaxoSmithKline (GSK), as shown in the figure below. Most of these synthetic GPR119 ligands are expected to be the next generation of therapies for diabetes. In particular, MBX2982 and GSK1292263 are the most advanced state of the art phase 2 substances.

In the previous studies, the expression of GPR119 was rarely observed in the liver, and thus the efficacy of GPR119 ligand on fatty liver has not been evaluated to date (Odori S et al., Metabolism, in press). In addition, the fact that the amount of free fatty acids and triglycerides in the GPR119 gene-deficient mice was not high compared to normal mice was the reason for not evaluating the efficacy of the GPR119 receptor ligand on non-alcoholic fatty liver (Lan et al., 2009 J. Endocrinol.).

The present invention has been made to solve the above-mentioned problems in the prior art, by revealing that the ligand acting on the GPR119 receptor has a therapeutic effect on non-alcoholic fatty liver, GPR119 ligand to prevent or prevent non-alcoholic fatty liver disease It is suggested that the present invention can be suitably applied to treatment.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising GPR119 (G protein coupled receptor 119) ligand as an active ingredient.

In one embodiment of the invention, the GPR119 ligand is 4-((4- (1H-tetrazol-1-yl) phenoxy) methyl) -2- (1- (5-ethylpyrimidin-2-yl) piperidin-4- yl) thiazole (MBX2982) or 3-isopropyl-5- (4-(((6- (4- (methylsulfonyl) phenyl) pyridin-3-yl) oxy) methyl) piperidin-1-yl) -1,2, 4-oxadiazole (GSK1292263).

In another embodiment of the present invention, the GPR119 ligand is characterized by inhibiting triglyceride accumulation in the liver.

In another embodiment of the present invention, the GPR119 ligand is characterized by increasing the activity of AMP-activated protein kinase (AMPK).

In another embodiment, the GPR119 ligand is characterized in that it inhibits the activity of Sterol regulatory element binding protein-1c (SREBP-1c).

In another embodiment, the GPR119 ligand is characterized by inhibiting the expression of fatty acid synthase (FAS).

In another embodiment, the GPR119 ligand is characterized by inhibiting the expression of acetyl CoA carboxylase (ACC).

In another embodiment of the present invention, the GPR119 ligand is characterized by inhibiting the expression of Stearoyl-CoA desaturase (SCD).

In another embodiment of the present invention, the non-alcoholic fatty liver disease is characterized in that it is selected from the group consisting of simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis and cirrhosis.

The present invention provides a method for preventing or treating a non-alcoholic fatty liver disease, comprising administering a G protein 119 ligand to a subject.

The present invention provides a use of the G protein coupled receptor 119 (GPR119) ligand for the prevention or treatment of non-alcoholic fatty liver disease.

GPR119 Ligand, currently in clinical trials for the treatment of diabetes, is an important core drug of the future and is a huge investment in global pharmaceutical companies. However, there have been no studies on the association between GPR119 receptor and non-alcoholic fatty liver due to the fact that GPR119 receptor shows little expression in liver and that fatty acid content does not change in GPR119 gene-deficient mice.

In the present invention, the expression of GPR119 is increased in mouse liver and liver cell lines by treatment of two drugs (MBX2982, GSK1292263) currently being tested in phase 2 clinical trials with GPR119. Expression of synthase (FAS), acetyl CoA carboxylase (ACC), and Stearoyl-CoA desaturase (SCD) was inhibited, and the activity of SREBP-1c, a key transcription factor that regulates the expression of these fatty acid synthase systems, In addition, the eight-week high-fat diet revealed that the administration of both ligands completely inhibited fatty liver production.

Therefore, the GPR119 ligand is excellent in inhibiting fatty liver production, and thus can be effectively applied to the prevention or treatment of non-alcoholic fatty liver.

1 is a result confirming that the GPR119 receptor is upregulated by GPR119 ligand treatment in human liver cancer cells.

Figure 2 is a diagram showing the enzyme system involved in fatty liver production [Triglyceride (TG) synthesis].

Figure 3 shows the results confirming the SREBP-1c expression inhibitory effect by the GPR119 ligand.

Figure 4 is a result confirming the inhibitory effect of LXR reporter activity by GPR119 ligand.

5 is a result confirming the SCD-1 and FAS mRNA expression inhibitory effect by the GPR119 ligand.

Figure 6 shows the results confirming the inhibitory effect of SREBP-1C and FAS expression by GPR119 ligand when exposed to high sugar / high insulin.

Figure 7 is the result confirming the effect of GPR119 ligand on body weight and tissue fat accumulation by high fat diet.

Figure 8 is a result confirming the effect of GPR119 ligand on fatty liver formation, liver weight increase, blood total cholesterol, sugar, ALT level increase by high fat diet.

9 is a result confirming the effect of GPR119 ligand on the expression of SREBP-1C, FAS and GPR119 mRNA in the liver by a high-fat diet.

10 is a result confirming the association between AMPK activation and SREBP-1C activity by GPR119 ligand in HepG2 cells.

11 is a result confirming that the treatment of the PPR inhibitor H-89 does not affect the action of the GPR119 ligand on SREBP-1C.

12 is a schematic diagram showing the difference in the signaling system of anti-diabetic and anti-fatty effect of GPR119 ligand.

Figure 13 shows the results of confirming the effect of GPR119 ligand in choline deficiency, amino acid fixed high fat diet mouse fat liver model.

The present inventors have shown that the GPR119 ligand, which is being developed as an antidiabetic agent, has an excellent effect on the treatment of non-alcoholic fatty liver, and that the intracellular signal transduction system for this is different from the small and pancreatic signal transduction system showing antidiabetic effect By clarifying, the fact that the GPR119 ligand can be suitably used for the treatment of non-alcoholic fatty liver has been found and based on this, the present invention has been completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease comprising GPR119 (G protein coupled receptor 119) ligand as an active ingredient.

The GPR119 ligand is 4-((4- (1H-tetrazol-1-yl) phenoxy) methyl) -2- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) thiazole (MBX2982) or 3 -isopropyl-5- (4- (((6- (4- (methylsulfonyl) phenyl) pyridin-3-yl) oxy) methyl) piperidin-1-yl) -1,2,4-oxadiazole (GSK1292263) Preferred, but not limited to, may be commercially available or synthesized, any material that binds to the GPR119 receptor to increase the expression of the receptor.

The GPR119 ligand increases the activity of AMP-activated protein kinase (AMPK), inhibits the activity of Sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC) or By inhibiting the expression of Stearoyl-CoA desaturase (SCD), it is preferable to inhibit triglyceride accumulation in the liver, but is not limited thereto.

The non-alcoholic fatty liver disease refers to non-alcoholic fatty liver disease, including both primary and secondary non-alcoholic fatty liver disease, preferably resulting from primary hyperlipidemia, diabetes or obesity. For example, nonalcoholic fatty liver disease includes simple fatty liver, nonalcoholic steatohepatitis, liver fibrosis and cirrhosis.

The composition of the present invention may further contain at least one known active ingredient having a non-alcoholic fatty liver treatment effect with a GPR119 ligand.

The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. It may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like in the form of conventional formulations, external preparations, suppositories, and sterile injectable solutions. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be used. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.

Preferred dosages of the pharmaceutical compositions of the present invention depend on the condition and weight of the individual, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art. For preferred effects, the GPR119 ligand of the present invention may be administered in an amount of 0.1 mg / kg to 100 mg / kg, preferably in an amount of 1 to 30 mg / kg, and may be administered once or several times a day. It may be.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections. The pharmaceutical composition of the present invention is determined according to the type of drug that is the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient and the severity of the disease.

In addition, the pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug treatment and biological response modifiers for the prevention and treatment of non-alcoholic fatty liver disease.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[Materials and methods]

A. Animal Testing

Breeding of Experimental Animals

The obesity induction diet used in the present invention is a high fat diet (HFD: 60% fat calorie, Research diets, D12492, USA).

Six-week-old male C57BL / 6J mice (Central Laboratory Animals, Seoul) were adapted to the laboratory environment for 1 week with solid feed, and then randomly placed into the control group and the experimental group according to the randomized block design. Fatty liver animal models were established by weekly administration. Fatty liver production was confirmed by blood biochemical and histological analysis methods. Six weeks after the high-fat diet was administered to the mice, GPR119 ligand (10 mg / kg) was suspended in 40% PEG400 and administered orally once a day, five times a week, in parallel with the high-fat diet. Choline deficient, amino acid-fixed, high-fat dietary hepatitis model was prepared by ingesting a diet containing 60 KCal fat and 0.1% methionine for 4 weeks in 6-week-old male C57BL / 6J mice (Central test animal, Seoul). At the end of the experiment, the animals were fasted for at least 12 hours, and blood, liver, and visceral fat tissue (diplordial fat, peripheral kidney fat) were collected under anesthesia with diethyl ether and washed with 0.1 M phosphate buffer (pH 7.4). After that, the weight was measured. Blood collected from the abdominal aorta was centrifuged at 3000 x RPM for 20 minutes using an SST tube to separate serum.

Biochemical analysis of blood and liver tissue

Serum total cholesterol and glucose concentrations were measured in experimental animals bred for 12 weeks as follows. Serum total cholesterol and glucose concentration were measured twice each using a commercial measurement kit (Bio Clinical system). The amount of ALT (alanine aminotransferase) used in liver function index was measured using a commercial assay kit (Bio Clinical System, Korea).

Hematoxylin and eosin staining

The liver tissues collected above were fixed with 10% neutral formalin solution, and the tissues were embedded with paraffin after the usual fixation and dehydration procedures. The embedded tissues were tissue sections with a thickness of 4 μm, stained with H & E, and observed with an optical microscope.

Confirmation of Protein Expression in Tissues by Western Blot Analysis

In the mortar, a certain amount of liver tissue was homogenized with liquid nitrogen and cell lysis buffer, and then the tissue solution was transferred to a new tube and vortexed. After centrifugation at 14,000 rpm and 4 ° C. for 20 minutes, the middle layer was taken and protein was quantified by the Bradford method. After 30 μg of protein was electrophoresed on SDS polyacrylamide gel, the expression changes of FAS and SREBP-1c proteins were measured using Western blot.

Isolation and Identification of RNA in Tissues Using the Trizol Method

Tissue was ground by adding 1 mL of trizol solution per 0.1 g of liver tissue, and then centrifuged at 4 ° C. and 12,000 × g for 10 minutes. The supernatant was transferred to a new tube, 200 μl of chloroform was added, and vortexed. After transferring the supernatant to a new tube, isopropanol and supernatant were added in a 1: 1 ratio. Shake vigorously for 15 seconds and then leave at room temperature for 10 minutes, centrifuge at 12,000 × g, 4 ° C for 10 minutes, remove supernatant, add 1 ml of 70% ethanol to the remaining precipitate, and then add 7,500 × g, 4 Centrifuge for 5 minutes at < RTI ID = 0.0 > After ethanol was removed, the tube containing the RNA precipitate was dried at room temperature for 5 minutes, and the RNA pellet was dissolved using nuclease free water. Measure the concentration of RNA samples extracted at 260 nm and 280 nm using UV / VIS spectrometer (Nanodrop, Thermo, USA), synthesize cDNA using RT kit, and use SCD-1 using Real time PCR. And mRNA expression change of the FAS was confirmed.

MRNA expression analysis using Real time-PCR (Real time-polymerase chain reaction)

CDNA was synthesized by reverse transcription using cDNA synthesis PCR kit on RNA samples extracted from liver tissue. CDNA obtained through reverse transcription as a template (template) and the 5 'and 3' flanking sequence of the gene cDNA to be amplified using the primers of the following [Table 1] real time PCR (mini-opticon, bio -rad, USA) was performed to check the expression level of mRNA.

Table 1 gene primer Sequence (5'-3 ') Annealing Temperature (℃) SEQ ID NO: PCR product (bp) GPR119 F TGCAGCTGCCTCTGTCCTCA 64.2 One 252 bp R GCACAGGAGAGGGTCAGCAC 61.8 2 β-actin F CCACAGCTGAGAGGGAAATC 58.2 3 193bp R AAGGAAGGCTGGAAAAGAGC 58.5 4

B. Isolation and Culture of Hepatocytes from Mice

Hepatocytes were isolated from male C57BL / 6 mice (C57BL / 6 mice, central laboratory animals, Seoul) according to the method of Seglen et al. (Seglen et al., Exp Cell Res., 82, pp 391-398, 1973). C57BL / 6 mice were anesthetized, abdominal incisions were made, intubated into the portal vein, and a mixture of 5% CO ₂ and 95% O ₂ was blown, and Hank's balanced salt solution without calcium and magnesium ions at 37 ° C (Ca ²⁺ , Mg ²⁺ -free Hank's balanced salt solution was flowed through the tube to remove liver blood. Thereafter, a 0.1% type IV collagenase (collagenase type IV, Sigma, USA) solution was flowed. After removing the liver tissue from the body, hepatic cell suspension was prepared, centrifuged at 50 × g for 2 minutes, the precipitated hepatocytes were taken, washed twice with a balanced salt solution of calcium and magnesium ions, and then collagen type 1 (collagen type). I, Sigma, USA) 10% (v / v) fetal bovine serum (FBS; fetal bovineserum, GibcoBRL), 10-8M insulin, 50 U / ml penicillin and 50 μg / ml streptomycin Williams culture medium (Sigma) added (WME; WilliamsMedium E, GibcoBRL, USA) was used as a culture medium so that the number of cells was 5 × 10 ⁶ cells / ml, and then cultured at 5% CO ₂ and 37 ° C. After 1 hour of incubation, the cells were washed with Hank's balanced salt solution (HBSS) containing Ca ²⁺ and Mg ²⁺ to remove non-adherent hepatocytes and replaced with fresh culture solution at 5% CO ₂ and 37 ° C. After incubating for 4 hours, the medium was changed to a culture medium without adding FBS, followed by incubation for 18 hours, and used for the experiment.

C. Human Hepatocyte Line (HepG2) Culture

HepG2, a human hepatocyte cell line (HepG2, ATCC, USA) was dispensed in 6-well plates and 1% penicillin-streptomycin (Hyclone, USA), 10% fetal bovine serum (Hyclone, USA) The added DMEM medium was used to incubate in a confluent state at 37 ℃, 5% CO ₂ incubator. Hepatocytes grown in the confluent state were changed to a culture medium without FBS instead of a culture medium containing 10%, followed by incubation for 18 hours, and used for the experiment.

D. Western blot

Sodium dodecylsulfate-polyacrylamidegel electrophoresis (SDS-PAGE) is performed using a gel electrophoresis device (Mighty Small SE 250, Hoefer Scientific Instruments, San Francisco) according to Lammmli UK, Nature 227, 680-685, 1970 It was. Cell lysis fractions were diluted in sample dilution buffer [63 mM Tris (pH.6.8), 10% glycerol, 2% SDS, 0.0013% bromophenol blue, 5% β-mercaptoethanol] and then 8%, 10 Electrophoresis was performed in electrode buffer (containing 15 g of Tris, 72 g of glycerin, 5 g of SDS) in an electrode buffer. The electrophoresis gel was subjected to nitrocellulose membrane for 3 hours at 40 mAmps in a transfer buffer solution (25 mM Tris, 192 mM glycerin, 20% v / v methanol (pH.8.3)] using a transfer electrophoresis device. The protein was transferred to anti-fattyacid synthase (FAS), anti-GPR119, anti-SRBP-1c, anti-phosphate AMPK-α, anti-phosphate ACC, and anti-phosphate SREBP-1c, respectively, as primary antibodies. After reacting with the nitrocellulose membrane, horseradish peroxidase-conjugated goat anti-rabbit IgG (horseradish peroxidase-conjugated goat anti-rabbit IgG) and horseradish peroxidase-conjugated goat anti-sheep Anti-mouse IgG was reacted for 1 hour and developed using an ECL detection system (ECL chemiluminecence system, Amersham, Gaithersberg, MA.) The homogeneity of the protein content in the sample was anti-β-actin (anti-β). -actin) antibody, anti-Lamin A / C. Changes in protein expression in the blot The plots were derived using densitometry, and the densitometry scans were performed using an Image Scan & Analysis System (Alpha-Innotech Co.), each lane using the AlphaEase ^™ version 5.5 software. Calculated by subtracting the strength of.

E. Isolation and Identification of Cellular RNA Using the Trizol Method

Tissues were pulverized by adding 1 mL of trizol solution to the cells, and then centrifuged at 12,000 × g for 10 minutes at 4 ° C. The supernatant was transferred to a new tube, 200 μl of chloroform was added, and vortexed. After transferring the supernatant to a new tube, isopropanol and supernatant were added in a 1: 1 ratio. Shake vigorously for 15 seconds and leave at room temperature for 10 minutes, centrifuge at 12,000 xg, 4 ° C for 10 minutes, remove supernatant, add 1 ml of 70% ethanol to the remaining precipitate, and then at 7,500 xg, 4 ° C Centrifuge for 5 minutes. After ethanol was removed, the tube containing the RNA precipitate was dried at room temperature for 5 minutes, and the RNA pellet was dissolved using nuclease free water. Measure the concentration of RNA samples extracted at 260 nm and 280 nm using UV / VIS spectrometer (Nanodrop, Thermo, USA), synthesize cDNA using RT kit, and use SCD-1 using Real time PCR. And mRNA expression change of the FAS was confirmed.

F. Analysis of Intracellular mRNA Expression Using Real Time-PCR (Real Time-polymerase Chain Reaction)

CDNA was synthesized by reverse transcription using a cDNA synthesis PCR kit on RNA samples extracted from cells. CDNA obtained through reverse transcription as a template (template) and the 5 'and 3' flanking sequence of the gene cDNA to be amplified using the primers of the following [Table 2] real time PCR (mini-opticon, bio -rad, USA) was performed to check the expression level of mRNA.

TABLE 2 gene primer Sequence (5'-3 ') Annealing Temperature (℃) SEQ ID NO: PCR product (bp) SCD1 F GCTGCTCGGATCACTAGTGAA 61.1 5 281 bp R TTCTGCTATCAGTCTGTCCAG 65.4 6 FAS F AGTACACACCCAAGGCCAAG 65 7 125 bp R GGATACTTTCCCGTCGCATA 62 8 β-actin F GATGAGATTGGCATGGCTTT 61 9 102 bp R GTCACCTTCACCGTTCCAGT 65 10

EXAMPLE

Example 1 Increasing Receptor Expression by GPR199 Ligand in Human Hepatocytes

Since little GPR119 intrahepatic expression was observed in previous studies, the function of GPR119 ligand on fatty liver was not evaluated (Odori S et al., Metabolism, in press).

Accordingly, the present inventors confirmed the expression change of GPR119 in HepG2 (human liver cancer cell line) cells by treatment with two selective ligands of GPR119 (MBX2982, GSK1292263).

To this end, 3 μM of GPR119 ligands (MBX-2982, GSK-1292263A) were treated in a time-dependent manner to human hepatocytes (HepG2) cultured as in the methods of B and C. At this time, the treatment conditions of the GPR119 ligand in each experimental group are as shown in the following [Table 3] and [Table 4], the expression change of the GPR119 protein was measured using the Western blot method of the D.

TABLE 3 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 1 hour treatment with 3 μM of GPR119 ligand (MBX-2982) Group 3 GPR119 ligand (MBX-2982) 3 μM-containing culture solution 3 hours treatment 4th group GPR119 ligand (MBX-2982) 3 μM-containing culture solution 6 hours treatment 5 groups 9 hours treatment of culture medium containing 3 μM of GPR119 ligand (MBX-2982)

Table 4 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 0.5 hour treatment with 3 μM of GPR119 ligand (GSK-1292263A) Group 3 1 hour treatment with 3 μM of GPR119 ligand (GSK-1292263A) 4th group 3 hours treatment with 3 μM of GPR119 ligand (GSK-1292263A) 5 groups GPR119 ligand (GSK-1292263A) 3 μM-containing culture solution 6 hours treatment 6 groups 9 hours treatment with 3 μM of GPR119 ligand (GSK-1292263A)

As a result, both selective ligands of GPR119 increased GPR119 expression in HepG2 cells (FIG. 1).

Example 2. Inhibition of SREBP-1c Activity and Fatty Acid Synthetic Enzyme Expression by GPR199 Ligand in Human Hepatocytes and Primary Cultured Mouse Hepatocytes

As shown in Figure 2, the synthesis of fatty acids involved in causing fatty liver is involved in the increased expression of a series of enzymes, of which Acety-CoA carboxylase (ACC), Fatty acid synthase (FAS) and Stearoyl-CoA desaturase (SCD) expression is regulated by the transcription factor Sterol regulatory element binding protein-1c (SREBP-1c), and activation of SREBP-1c promotes the accumulation of triglycerides in the liver by expressing the enzyme system.

2-1. Confirmation of Expression Changes of SREBP-1c Following T0901317 Treatment

Therefore, the present inventors, in order to confirm the effect of inhibiting fat accumulation in the liver by GPR119 ligand treatment in the liver cell line, T0901317 (N- (2,2, which is a well known Liver X receptor (LXR) ligand, SREBPP-1c activation signal) 2-Trifluoroethyl) -N- [4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl] benzenesulfonamide) was used.

To this end, a culture solution containing GPR119 ligands (MBX-2982, GSK-1292263A) is added to the hepatocytes isolated and cultured as in the method of B, and the human hepatocytes (HepG2) cultured as in the method of C. After 30 minutes, 10 μM T0901317 was treated for 9 hours. At this time, the treatment conditions of the GPR119 ligand in each experimental group are as shown in the following [Table 5] and [Table 6], and the expression change of the SREBP-1c protein was measured using the Western blot method of D.

Table 5 Primary cultured hepatocytes division Treatment method Group 1 Normal culture treatment Group 2 Treatment of culture solution containing 10 μM T0901317 Group 3 Treatment of culture containing 10 μM T0901317 and 0.3 μM of GPR119 ligand 4th group Treatment of culture containing 10 μM T0901317 and 1 μM of GPR119 ligand 5 groups Treatment of cultures containing 10 μM T0901317 and 3 μM of GPR119 ligand 6 groups Treatment of cultures containing 10 μM T0901317 and 10 μM GPR119 ligand

Table 6 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 Treatment of culture solution containing 10 μM T0901317 Group 3 Treatment of culture containing 10 μM T0901317 and 0.1 μM of GPR119 ligand 4th group Treatment of culture containing 10 μM T0901317 and 0.3 μM of GPR119 ligand 5 groups Treatment of culture containing 10 μM T0901317 and 1 μM of GPR119 ligand 6 groups Treatment of cultures containing 10 μM T0901317 and 3 μM of GPR119 ligand

As a result, expression of SREBP-1c in HepG2 and primary hepatocytes treated with T0901317, a Liver X receptor (LXR) ligand, which is well known as an activation signal of SREBP-1c, was treated alone. Although this increased significantly, it was confirmed that when treated with two GPR119 ligand concentration-dependently inhibited (Fig. 3).

In addition, even when the LXR activity was evaluated by a specific reporter gene assay, it was confirmed that when the two GPR119 ligands were treated together, the activity was inhibited (FIG. 4).

2-2. Changes in mRNA Expression of SCD-1 and FAS after T0901317 Treatment

A culture solution containing GPR119 ligand (MBX-2982, GSK-1292263A) was added to the human liver cell line (HepG2) cultured as in the method of C. After 30 minutes, 10 μM T0901317 was treated for 9 hours. At this time, the treatment conditions of GPR119 ligand in each experimental group are as shown in [Table 7] below, and the change of SCD-1 and FAS mRNA expression was as in the method of E. RNA isolation and cDNA synthesis were performed in real time- MRNA was measured using the PCR method.

TABLE 7 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 Treatment of culture solution containing 10 μM T0901317 Group 3 Treatment of culture containing 10 μM T0901317 and 0.1 μM of GPR119 ligand 4th group Treatment of culture containing 10 μM T0901317 and 0.3 μM of GPR119 ligand 5 groups Treatment of culture containing 10 μM T0901317 and 1 μM of GPR119 ligand 6 groups Treatment of cultures containing 10 μM T0901317 and 3 μM of GPR119 ligand

As a result, mRNA expression of SCD-1 and FAS, enzymes involved in the synthesis of triglycerides in the liver, was increased by treatment of T0901317, and mRNA expression of SCD-1 and FAS increased by treatment with two GPR119 ligands. It was confirmed that all are suppressed (Fig. 5).

2-3. Changes in SREBP-1c and FAS Expression Following High Sugar / High Insulin Exposure

In order to confirm the changes in SREBP-1c and FAS expression following high sugar / high insulin exposure, which is well known as a signal for actual non-alcoholic fatty liver other than LXR ligand exposure, high sugar / high insulin was exposed to HepG2 and primary cultured human hepatocytes. In, two GPR119 ligands were treated.

To this end, a culture solution containing high sugar (glucose 30 mM) was added to human hepatocytes (HepG2) and GPR119 ligand was treated in a concentration-dependent manner. After 30 minutes, 200 nM insulin (Insulin) was treated for 24 hours. At this time, the treatment conditions of the GPR119 ligand in each experimental group are as shown in the following [Table 8] and [Table 9], and the expression change of the protein was measured using the Western blot method of D.

Table 8 Primary cultured hepatocytes division Treatment method Group 1 Normal culture treatment Group 2 High blood sugar content and high insulin culture Group 3 Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 0.3 μM of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 1 μM of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 3 μM of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 10 μM of GPR119 ligand

Table 9 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 High blood sugar content and high insulin culture Group 3 Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 1 μM of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 3 μM of GPR119 ligand

As a result, it was confirmed that the expression of SREBP-1c and the amount of nuclear transfer type (active) SREBP-1C in each cell were significantly increased when exposed to HepG2 and primary cultured human hepatocytes. It was confirmed that the expression level of SREBP-1c was completely inhibited when the MBX2982, which is a GPR119 ligand, was treated (FIG. 6). In addition, it was confirmed that the increased FAS expression by high sugar / high insulin exposure in HepG2 cells was also inhibited by MBX2982 treatment (FIG. 6).

Example 3 Efficacy of GPR199 Ligand in a Fatty Liver Induction Model by High Fat Diet

In order to confirm whether GPR119 ligands actually inhibit the production of fatty liver, the effect of GPR119 ligand was confirmed in a fatty liver induction model by a high fat diet based on the animal experiment method of A.

Specifically, the high-fat diet was administered for 4 weeks, and two drugs were orally administered once every two days at a 10 mg / kg dose under the additional high-fat diet for 4 weeks, and the specific experimental schedule is described in the upper part of FIG. 7. As shown.

3-1. Check the weight loss effect

We observed whether weight loss is induced by GPR119 ligand treatment in a high fat diet induction model.

As a result, a significant weight gain was observed in the actual high-fat diet group, and a significant weight loss was observed in the group treated with the GPR119 ligand (FIG. 7). In addition, it was confirmed that the fat weight (tissue) in the tissue was significantly reduced in the MBX2982 treatment group.

3-2. Confirmation of fat accumulation through H & E staining

After liver tissue was extracted, H & E staining was performed to observe the degree of fat accumulation.

As a result, in the liver of the high-fat diet group, fat accumulation was observed in almost all hepatocytes, while a significant improvement was observed in the GPR119 ligand-administered group (FIG. 8). In addition, it was confirmed that increased liver weight, total cholesterol, blood glucose, and ALT levels in the high-fat diet group were also significantly improved in the GPR119 ligand-administered group, thereby demonstrating the anti-fat efficacy of the GPR119 ligand (FIG. 8).

3-3. Confirmation of SREBP-1C and FAS Expression in Liver Tissue

The expression of SREBP-1C and FAS in liver tissues of the fatty liver induction model by the high fat diet was confirmed.

As a result, it was confirmed that in the group administered with the GPR119 ligand, there was a tendency to suppress the expression of SREBP-1C and the expression of FAS as compared to the high-fat diet group (FIG. 9).

In addition, when the two GRP119 ligands were administered to mice, an increase in the actual amount of GPR119 mRNA was observed. Especially, when MBX2982 was administered, the expression of GPR119 mRNA was increased by 7 times compared to the control group.

Example 4 Action of GPR119 Ligand as AMPK Activator

Based on these results, we infer that activation of SREBP-1C by GPR119 ligand is not related to the activation of cAMP / PKA (cyclic AMP / Protein kinase A), which is known as the secondary signaling system of GPR119. This was confirmed by confirming that the treatment of H-89 did not affect the action of the GPR119 ligand on SREBP-1C (Fig. 11).

To this end, the following experiments were performed. Specifically, a saline solution containing high sugar (glucose 30 mM) was added to human hepatocytes (HepG2), followed by treatment with an AMPK inhibitor, followed by treatment with 3 μM of GPR119 ligand 30 minutes later. After 30 minutes, 200 nM insulin (Insulin) was treated and incubated for 24 hours. At this time, the treatment conditions of the GPR119 ligand in each experimental group are as shown in the following [Table 10], and the expression change of the protein was measured using the Western blot method of D.

Table 10 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 High blood sugar content and high insulin culture Group 3 Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 3 μM of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing AMPK inhibitors and 3 μM of GPR119 ligand 5 groups Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing PKA inhibitors and 3 μM of GPR119 ligand

Recently, SREBP-1C has been reported to be inactive by phosphorylation in Ser-372 region by AMP-activated protein kinase (AMPK). Thus, the present inventors evaluated the effects of GPR119 ligands on AMPK activation and SREBP-1C phosphorylation in liver cells.

To this end, 3 μM of GPR119 ligands (MBX-2982, GSK-1292263A) were treated in a time-dependent manner to human hepatocytes (HepG2) cultured as in the methods of B and C. At this time, the treatment conditions of GPR119 ligand in each experimental group are as shown in the following [Table 11] and [Table 12], and the expression change of the protein was measured using the Western blot method of D.

Table 11 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 1 hour treatment with 3 μM of GPR119 ligand (MBX-2982) Group 3 GPR119 ligand (MBX-2982) 3 μM-containing culture solution 3 hours treatment 4th group GPR119 ligand (MBX-2982) 3 μM-containing culture solution 6 hours treatment 5 groups 9 hours treatment of culture medium containing 3 μM of GPR119 ligand (MBX-2982)

Table 12 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 0.5 hour treatment with 3 μM of GPR119 ligand (GSK-1292263A) Group 3 1 hour treatment with 3 μM of GPR119 ligand (GSK-1292263A) 4th group 3 hours treatment with 3 μM of GPR119 ligand (GSK-1292263A) 5 groups GPR119 ligand (GSK-1292263A) 3 μM-containing culture solution 6 hours treatment 6 groups 9 hours treatment with 3 μM of GPR119 ligand (GSK-1292263A)

As a result, it was confirmed that both GPR119 ligands strongly activated AMPK (AMPK phosphorylation and ACC phosphorylation) in liver cells, and increased Ser-372 phosphorylation of SREBP-1C (FIG. 10). Furthermore, it was confirmed that when treated with compound C, an inhibitor of AMPK, inhibition of SREBP-1C expression by MBX2982, a GPR119 ligand, was restored (FIG. 10).

Example 5 Efficacy of GPR199 Ligand in Choline Deficiency, Amino Acid Fixed High Fat Fatty Hepatitis Model

To determine whether GPR119 ligands are effective against nonalcoholic steatohepatitis, the effect of GPR119 ligand was confirmed in a choline deficiency, amino acid-fixed high-fat dietary hepatitis model based on the animal test method of A.

Specifically, the high-fat diet was administered for 4 weeks, and MBX2982 was orally administered once every 2 days at a 10 mg / kg dose under additional high-fat diet conditions for 4 weeks, and the specific experimental schedule was described in the upper part of FIG. 7. same.

After liver tissue was extracted, H & E staining was performed to observe the degree of fat accumulation. As a result, administration of GPR 119 ligand inhibited fat accumulation in the mouse nonalcoholic steatohepatitis model of the amino acid fixed high fat diet (FIG. 13). In addition, mRNA expression of MCP-1 and Pro-IL-1beta, which are representative inflammatory markers of hepatitis, was confirmed. As a result, administration of GPR 119 ligand suppressed mRNA expression of MCP-1 and Pro-IL-1beta. (FIG. 13).

In this way, GLP-1 and insulin secretion by a cAMP-increasing action of the known GPR119 ligand, a separate action, these ligands activate the AMPK in liver cells, thereby showing a fatty liver inhibitory effect have. It can also be seen that the GPR 119 ligand inhibits not only non-alcoholic fatty liver but also fatty liver disease, which is an advanced form of the disease.

Therefore, a key technique of the present invention is that the GPR119 receptor increases its expression in the liver by ligand exposure, inhibits the expression of fatty acids and triglyceride synthase by ligand treatment, and has the effect of treating fatty liver. At this time, the pharmacological mechanism is involved in the activation of AMPK, unlike the PKA signal activation according to the conventional cAMP increase, which is shown in the schematic diagram in FIG. As shown in FIG. 12, there is a clear difference between the GPR119 ligand fatty liver suppression signaling system and the antidiabetic signaling system.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

In the present invention, the expression of GPR119 is increased in mouse liver and liver cell lines by treatment of two drugs (MBX2982, GSK1292263) currently being tested in phase 2 clinical trials with GPR119. Expression of synthase (FAS), acetyl CoA carboxylase (ACC), and Stearoyl-CoA desaturase (SCD) was inhibited, and the activity of SREBP-1c, a key transcription factor that regulates the expression of these fatty acid synthase systems, In addition, the eight-week high-fat diet revealed that the administration of both ligands completely inhibited fatty liver production.

Therefore, the GPR119 ligand is excellent in inhibiting fatty liver production, and thus can be effectively applied to the prevention or treatment of non-alcoholic fatty liver.

Claims (11)

A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising a GPR119 (G protein coupled receptor 119) ligand as an active ingredient. The method of claim 1, wherein the GPR119 ligand is 4-((4- (1H-tetrazol-1-yl) phenoxy) methyl) -2- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) thiazole (MBX2982) or 3-isopropyl-5- (4-(((6- (4- (methylsulfonyl) phenyl) pyridin-3-yl) oxy) methyl) piperidin-1-yl) -1,2,4- Pharmaceutical composition, characterized in that oxadiazole (GSK1292263). The pharmaceutical composition of claim 1, wherein the GPR119 ligand inhibits triglyceride accumulation in the liver. The pharmaceutical composition of claim 1, wherein the GPR119 ligand increases the activity of AMP-activated protein kinase (AMPK). The pharmaceutical composition of claim 1, wherein the GPR119 ligand inhibits the activity of Sterol regulatory element binding protein-1c (SREBP-1c). The pharmaceutical composition of claim 1, wherein the GPR119 ligand inhibits expression of fatty acid synthase (FAS). The pharmaceutical composition of claim 1, wherein the GPR119 ligand inhibits the expression of acetyl CoA carboxylase (ACC). The pharmaceutical composition of claim 1, wherein the GPR119 ligand inhibits the expression of Stearoyl-CoA desaturase (SCD). The pharmaceutical composition of 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. A method of preventing or treating a non-alcoholic fatty liver disease, comprising administering a GPR119 (G protein coupled receptor 119) ligand to a subject. Use of GPR119 (G protein coupled receptor 119) ligand for the prevention or treatment of non-alcoholic fatty liver disease.

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