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WO2015111971A1 - Composition pharmaceutique contenant un ligand gpr119 comme principe actif pour prévenir ou traiter une stéatose hépatique non alcoolique - Google Patents

Composition pharmaceutique contenant un ligand gpr119 comme principe actif pour prévenir ou traiter une stéatose hépatique non alcoolique Download PDF

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WO2015111971A1
WO2015111971A1 PCT/KR2015/000766 KR2015000766W WO2015111971A1 WO 2015111971 A1 WO2015111971 A1 WO 2015111971A1 KR 2015000766 W KR2015000766 W KR 2015000766W WO 2015111971 A1 WO2015111971 A1 WO 2015111971A1
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gpr119
ligand
fatty liver
treatment
pharmaceutical composition
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PCT/KR2015/000766
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English (en)
Korean (ko)
Inventor
강건욱
이경
양진원
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동국대학교 산학협력단
서울대학교 산학협력단
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Priority claimed from KR1020150010771A external-priority patent/KR101665846B1/ko
Application filed by 동국대학교 산학협력단, 서울대학교 산학협력단 filed Critical 동국대학교 산학협력단
Priority to US15/302,228 priority Critical patent/US20170049773A1/en
Publication of WO2015111971A1 publication Critical patent/WO2015111971A1/fr
Priority to US16/039,781 priority patent/US20190008864A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • 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.
  • GPR119 G protein coupled receptor 119
  • Fatty liver refers to a condition in which abnormal fat is accumulated in liver cells
  • a medical condition refers to a pathological condition in which the neutral lipid content exceeds 5% of the total liver weight.
  • 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.
  • ALD alcoholic fatty liver disease
  • 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.
  • NASH non-alcoholic steatohepatitis
  • advanced fibrosis cirrhosis
  • Nonalcoholic fatty liver disease 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.
  • NASH non-alcoholic steatohepatitis
  • 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.
  • FFA free fatty acid
  • inflammatory cytokines and their receptors play an important role in the progression from fatty liver to fatty hepatitis.
  • 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.
  • 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.
  • alcoholic or nonalcoholic fatty liver patients 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).
  • 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 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.
  • GPR119 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).
  • ITT insulin tolerance test
  • GTT glucose tolerance test
  • 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.
  • N-acylathanolamine (NAE) such as OEA, PEA, LEA, etc.
  • 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.
  • MBX2982 and GSK1292263 are the most advanced state of the art phase 2 substances.
  • 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.
  • 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.
  • GPR119 G protein coupled receptor 119
  • 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).
  • the GPR119 ligand is characterized by inhibiting triglyceride accumulation in the liver.
  • the GPR119 ligand is characterized by increasing the activity of AMP-activated protein kinase (AMPK).
  • AMPK AMP-activated protein kinase
  • the GPR119 ligand is characterized in that it inhibits the activity of Sterol regulatory element binding protein-1c (SREBP-1c).
  • SREBP-1c Sterol regulatory element binding protein-1c
  • the GPR119 ligand is characterized by inhibiting the expression of fatty acid synthase (FAS).
  • FAS fatty acid synthase
  • the GPR119 ligand is characterized by inhibiting the expression of acetyl CoA carboxylase (ACC).
  • ACC acetyl CoA carboxylase
  • the GPR119 ligand is characterized by inhibiting the expression of Stearoyl-CoA desaturase (SCD).
  • SCD Stearoyl-CoA desaturase
  • 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 G protein coupled receptor 119
  • 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.
  • GPR119 receptor shows little expression in liver and that fatty acid content does not change in GPR119 gene-deficient mice.
  • 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.
  • FAS synthase
  • ACC acetyl CoA carboxylase
  • SCD Stearoyl-CoA desaturase
  • 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.
  • FIG. 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.
  • 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.
  • FIG. 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
  • 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.
  • 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.
  • GPR119 G protein coupled receptor 119
  • 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.
  • AMPK AMP-activated protein kinase
  • SREBP-1c Sterol regulatory element binding protein-1c
  • FAS fatty acid synthase
  • ACC acetyl CoA carboxylase
  • SCD Stearoyl-CoA desaturase
  • 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.
  • nonalcoholic fatty liver disease includes simple fatty liver, nonalcoholic steatohepatitis, liver fibrosis and cirrhosis.
  • 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.
  • 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.
  • 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.
  • 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.
  • base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.
  • 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.
  • 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.
  • the obesity induction diet used in the present invention is a high fat diet (HFD: 60% fat calorie, Research diets, D12492, USA).
  • mice 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.
  • 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.
  • 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).
  • 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 ⁇ m, stained with H & E, and observed with an optical microscope.
  • 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 ⁇ g of protein was electrophoresed on SDS polyacrylamide gel, the expression changes of FAS and SREBP-1c proteins were measured using Western blot.
  • 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 ⁇ l of chloroform was added, and vortexed. After transferring the supernatant to a new tube, isopropanol and supernatant were added in a 1: 1 ratio.
  • 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.
  • 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.
  • 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.
  • collagenase type IV (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).
  • FBS fetal bovine serum
  • WME WilliamsMedium E, GibcoBRL, USA
  • HBSS Hank's balanced salt solution
  • 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 °C, 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.
  • Electrophoresis was performed using 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- ⁇ , anti-phosphate ACC, and anti-phosphate SREBP-1c, respectively, as primary antibodies.
  • FAS anti-fattyacid synthase
  • anti-GPR119 anti-GPR119
  • anti-SRBP-1c anti-phosphate AMPK- ⁇
  • anti-phosphate ACC anti-phosphate ACC
  • anti-phosphate SREBP-1c anti-phosphate SREBP-1c
  • 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.)
  • ECL detection system ECL chemiluminecence system, Amersham, Gaithersberg, MA.
  • the homogeneity of the protein content in the sample was anti- ⁇ -actin (anti- ⁇ ). -actin) antibody, anti-Lamin A / C.
  • 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 ⁇ l 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.
  • 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.
  • CDNA was synthesized by reverse transcription using a cDNA synthesis PCR kit on RNA samples extracted from cells.
  • 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).
  • 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.
  • HepG2 human hepatocytes
  • 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 4 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 0.5 hour treatment with 3 ⁇ M of GPR119 ligand (GSK-1292263A) Group 3 1 hour treatment with 3 ⁇ M of GPR119 ligand (GSK-1292263A) 4th group 3 hours treatment with 3 ⁇ M of GPR119 ligand (GSK-1292263A) 5 groups GPR119 ligand (GSK-1292263A) 3 ⁇ M-containing culture solution 6 hours treatment 6 groups 9 hours treatment with 3 ⁇ M of GPR119 ligand (GSK-1292263A)
  • both selective ligands of GPR119 increased GPR119 expression in HepG2 cells (FIG. 1).
  • ACC Acety-CoA carboxylase
  • Fatty acid synthase Fatty acid synthase
  • SCD Stearoyl-CoA desaturase
  • T0901317 N- (2,2, which is a well known Liver X receptor (LXR) ligand, SREBPP-1c activation signal
  • 2-Trifluoroethyl 2-Trifluoroethyl
  • 2-Trifluoroethyl 2-Trifluoroethyl
  • 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 ⁇ M T0901317 was treated for 9 hours.
  • 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 ⁇ M T0901317 Group 3 Treatment of culture containing 10 ⁇ M T0901317 and 0.3 ⁇ M of GPR119 ligand 4th group Treatment of culture containing 10 ⁇ M T0901317 and 1 ⁇ M of GPR119 ligand 5 groups Treatment of cultures containing 10 ⁇ M T0901317 and 3 ⁇ M of GPR119 ligand 6 groups Treatment of cultures containing 10 ⁇ M T0901317 and 10 ⁇ M GPR119 ligand
  • Table 6 Human Hepatocyte Line (HepG2) division Treatment method Group 1 Normal culture treatment Group 2 Treatment of culture solution containing 10 ⁇ M T0901317 Group 3 Treatment of culture containing 10 ⁇ M T0901317 and 0.1 ⁇ M of GPR119 ligand 4th group Treatment of culture containing 10 ⁇ M T0901317 and 0.3 ⁇ M of GPR119 ligand 5 groups Treatment of culture containing 10 ⁇ M T0901317 and 1 ⁇ M of GPR119 ligand 6 groups Treatment of cultures containing 10 ⁇ M T0901317 and 3 ⁇ M of GPR119 ligand
  • 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 ⁇ M 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.
  • 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.
  • 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 ⁇ M of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 1 ⁇ M of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 3 ⁇ M of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing 10 ⁇ M of GPR119 ligand
  • HepG2 Human Hepatocyte Line
  • 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.
  • liver tissue was extracted, H & E staining was performed to observe the degree of fat accumulation.
  • 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 ⁇ M of GPR119 ligand 4th group Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing AMPK inhibitors and 3 ⁇ M of GPR119 ligand 5 groups Treatment of hyperglycemia-containing and hyperinsulin cultures and cultures containing PKA inhibitors and 3 ⁇ M of GPR119 ligand
  • SREBP-1C has been reported to be inactive by phosphorylation in Ser-372 region by AMP-activated protein kinase (AMPK).
  • AMPK AMP-activated protein kinase
  • 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.
  • HepG2 human hepatocytes
  • 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 ⁇ M of GPR119 ligand (MBX-2982) Group 3 GPR119 ligand (MBX-2982) 3 ⁇ M-containing culture solution 3 hours treatment 4th group GPR119 ligand (MBX-2982) 3 ⁇ M-containing culture solution 6 hours treatment 5 groups 9 hours treatment of culture medium containing 3 ⁇ M 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 ⁇ M of GPR119 ligand (GSK-1292263A) Group 3 1 hour treatment with 3 ⁇ M of GPR119 ligand (GSK-1292263A) 4th group 3 hours treatment with 3 ⁇ M of GPR119 ligand (GSK-1292263A) 5 groups GPR119 ligand (GSK-1292263A) 3 ⁇ M-containing culture solution 6 hours treatment 6 groups 9 hours treatment with 3 ⁇ M of GPR119 ligand (GSK-1292263A)
  • 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.
  • 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.
  • GLP-1 and insulin secretion by a cAMP-increasing action of the known GPR119 ligand activate the AMPK in liver cells, thereby showing a fatty liver inhibitory effect have.
  • the GPR 119 ligand inhibits not only non-alcoholic fatty liver but also fatty liver disease, which is an advanced form of the disease.
  • 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.
  • 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.
  • 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.
  • FAS synthase
  • ACC acetyl CoA carboxylase
  • SCD Stearoyl-CoA desaturase
  • 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.

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Abstract

La présente invention concerne une composition pharmaceutique contenant un ligand de récepteur couplé aux protéines G 119 (GPR119) comme principe actif pour prévenir ou traiter une stéatose hépatique non alcoolique. Plus précisément, il a été établi que le ligand GPR119, qui a été développé uniquement comme médicament antidiabétique, présente un excellent effet dans le traitement de stéatose hépatique non alcoolique, et la voie de signalisation dans des cellules hépatiques est différente de la voie de signalisation dans l'intestin grêle ou le pancréas présentant un effet antidiabétique, et il a été ainsi confirmé que le ligand GPR119 peut être utile pour traiter une stéatose hépatique non alcoolique.
PCT/KR2015/000766 2014-01-23 2015-01-23 Composition pharmaceutique contenant un ligand gpr119 comme principe actif pour prévenir ou traiter une stéatose hépatique non alcoolique WO2015111971A1 (fr)

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US16/039,781 US20190008864A1 (en) 2014-01-23 2018-07-19 Pharmaceutical composition containing gpr119 ligand as effective ingredient for preventing or treating non-alcoholic steatohepatitis

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US9895370B2 (en) * 2015-11-19 2018-02-20 Pharmedix.Co., Ltd Pharmaceutical composition for preventing or treating of cirrhosis of liver comprising G protein coupled receptor 119 ligand as an active ingredient
CN112805002A (zh) * 2018-09-12 2021-05-14 东亚St株式会社 用于预防或治疗非酒精性脂肪肝病的含有gpr119配体作为活性成分的药物组合物

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9895370B2 (en) * 2015-11-19 2018-02-20 Pharmedix.Co., Ltd Pharmaceutical composition for preventing or treating of cirrhosis of liver comprising G protein coupled receptor 119 ligand as an active ingredient
CN112805002A (zh) * 2018-09-12 2021-05-14 东亚St株式会社 用于预防或治疗非酒精性脂肪肝病的含有gpr119配体作为活性成分的药物组合物
US12115159B2 (en) 2018-09-12 2024-10-15 Dong-A St Co., Ltd. Pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease, containing GPR119 ligand as active ingredient

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