Use of PPAR agonists to improve energy metabolism

Abstract

The present invention provides a pharmaceutical composition for preventing or treating metabolic diseases, comprising a novel pan-PPAR agonist compound or a salt thereof as an active ingredient. The said compound or a salt thereof is a pan-PPAR agonist and can be usefully applied to pharmaceutical compositions for preventing or treating metabolic diseases such as obesity, diabetes, fatty liver disease, dyslipidemia, cardiovascular diseases and / or metabolic syndrome, food for improving metabolic health, feed compositions and the like.

Description

[Technical Field]

[0001] The present invention claims the benefit of priority from Korean Patent Application No. 10-2022-0089073, the entire contents of which are incorporated herein by reference. The present invention relates to the use of novel pan-PPAR agonists for the prevention, amelioration or treatment of metabolic disorders. [Background technology]

[0002] PPARs (peroxisome proliferator-activated receptors) are nuclear hormone receptor family proteins that, together with retinoid X receptors (RXRs), regulate the transcription of genes containing PPAR-responsive regulatory elements (PPREs). They are classified into isotypes such as PPARα, PPARβ / δ, and PPARγ.

[0003] PPARs act as lipid sensors within cells and are key factors in normal energy metabolic homeostasis. PPAR agonists can improve various aspects of energy metabolic homeostasis, including obesity, lipid metabolism, glucose homeostasis, and insulin resistance, and are therefore considered a major target for metabolic disorders (see, for example, Corrales, Patricia, Antonio Vidal-Puig, and Gema Medina-Gomez. International Journal of Molecular Sciences 19.7(2018):2124.).

[0004] Specifically, PPARα is involved in increasing cellular fatty acid influx, esterification, and trafficking, regulates lipoprotein metabolism genes, and is activated by (8S)-hydroxyeicosatetraenoic acid, pemafibrate (K-877), fenofibrate, WY14643, etc. (References [Sasaki, Yusuke, et al. Scientific Reports 10.1 (2020): 1-10.], etc.).

[0005] PPARβ / δ increases mitochondrial function and the fatty acid desaturation pathway, promoting lipid and glucose utilization, and is activated by (13S)-hydroxyoctadecadienoic acid, GW501516, Serradelpal (MBX-8025), L-165041, etc. (see Li, Xiuli, et al. International Journal of Molecular Medicine 36.3(2015):767-775., etc.).

[0006] PPARγ increases insulin sensitivity and glucose metabolism through the influx of fatty acids, the formation of triglycerides, and the promotion of lipid droplet storage, and is activated by pioglitazone, rosiglitazone, and other drugs (see Soccio, Raymond E., Eric R. Chen, and Mitchell A. Lazar. Cell metabolism 20.4(2014):573-591.).

[0007] In addition, to improve PPAR agonists, PPARα and PPARγ dual agonists such as saroglitazar and tesaglitazar (e.g., Rastogi, Ashu, et al. Acta Diabetologica 57.7(2020):809-818.), PPARα and PPARβ / δ dual agonist elafibranor (GFT505) (e.g., Ratziu, Vlad, et al. Gastroenterology 150.5(2016):1147-1159.), and PPARα, PPARβ / δ, and PPARγ pan-agonists such as lanifibranor and ciglitazar have been developed (e.g., Lefere, Sander, et al. Journal of hepatology 73.4(2020):757-770.], etc.), the reality is that there is a need to develop new PPAR agonists to improve the efficacy and side effects of conventional PPAR agonists. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the present inventors have discovered novel PPAR agonists that can improve metabolic disorders such as obesity, diabetes, and fatty liver disease, and have confirmed that the PPAR agonists effectively improve various metabolic disorders in cells and animal models, thereby completing the present invention.

[0009] An object of the present invention is to provide use of a compound represented by formula 1 or a salt thereof for the prevention, amelioration or treatment of metabolic disorders. It is still another object of the present invention to provide use of the compound represented by formula 1 or a salt thereof as a PPAR agonist. [Means for solving the problem]

[0010] In one aspect, the present invention provides a pharmaceutical composition for preventing or treating a metabolic disease, comprising, as an active ingredient, a compound represented by the following formula 1 or a salt thereof: [ka]

[0011] The compound represented by formula 1 or a salt thereof can activate PPARα, PPARβ / δ and PPARγ in the body. The metabolic disease may be one or more selected from the group consisting of obesity, diabetes, fatty liver disease, metabolic syndrome, dyslipidemia, and cardiovascular disease.

[0012] In another aspect, the present invention provides a functional health food composition for improving metabolic health, comprising the compound represented by formula 1 or a salt thereof. The metabolic health improvement can be one or more effects selected from the group consisting of improvement of obesity, lowering of blood sugar, improvement of fatty liver, improvement of lipid metabolism, improvement of blood circulation, and improvement of metabolic syndrome.

[0013] In another aspect, the present invention provides a feed composition for improving metabolic health, comprising the compound represented by formula 1 or a salt thereof.

[0014] In another aspect, the present invention provides a method for activating a PPAR protein, comprising treating isolated cells with a compound represented by Formula 1 or a salt thereof. The PPAR protein may include one or more selected from the group consisting of PPARα, PPARβ / δ, and PPARγ. [Effects of the Invention]

[0015] The compound represented by Formula 1 according to the present invention or a salt thereof is a pan-PPAR agonist and can be usefully used in pharmaceutical compositions for the prevention or treatment of metabolic diseases such as obesity, diabetes, fatty liver disease, dyslipidemia, cardiovascular disease and / or metabolic syndrome, food or feed compositions for improving metabolic health, etc. [Brief explanation of the drawings]

[0016] [Figure 1]This shows the results of measuring changes in luciferase activity after drug treatment in HEK293 cells transfected with a Gal4-luciferase reporter system. [Figure 2] 1 shows the results of pull-down analysis of PPAR proteins using drug-conjugated CNBr-beads. [Figure 3] Photographs of Oil Red O staining of drug-treated differentiated adipocytes are shown (scale bar 100 μm, magnification 200×). [Figure 4] The figures show the results of quantifying lipid accumulation (left) and lipid droplet size distribution (right) in Oil Red O-stained adipocytes. [Figure 5] 1 shows an animal experiment schedule for evaluating the effects of drug administration. [Figure 6] The graph shows the results of measuring changes in body weight (left), body weight (middle), and food intake (right) in drug-administered high-fat diet mice. [Figure 7] This shows the results of micro-CT imaging of a drug-administered high-fat diet mouse (adipose tissue: red, muscle: yellow). [Figure 8] 1 shows the results of measuring adipose tissue and muscle volumes in drug-administered high-fat diet mice. [Figure 9] 1 shows the results of measuring organ weights in drug-administered high-fat diet mice. [Figure 10] 1 shows the results of an intraperitoneal glucose tolerance test (IP-GTT) in drug-administered high-fat diet mice. [Figure 11] 1 shows the results of an intraperitoneal insulin tolerance test (IP-ITT) in drug-administered high-fat diet mice. [Figure 12] 1 shows the results of serum glucose measurements in drug-administered high-fat diet mice. [Figure 13]1 shows the results of measuring serum lipids in drug-administered high-fat diet mice. [Figure 14] This shows H&E staining images of adipose tissue in high-fat diet mice administered with a drug (scale bar 100 μm, magnification 200×). [Figure 15] This shows the results of quantifying the area of ​​lipid droplets in adipose tissue in drug-administered high-fat diet mice. [Figure 16] This shows H&E, Sirius Red, and Oil Red O stained images of liver tissue from drug-administered high-fat diet mice (scale bar 100 μm, magnification 200X). [Figure 17] 1 shows the results of measuring the non-alcoholic fatty liver disease activity score (NAFLD activity score) in liver tissue of drug-administered high-fat diet mice. [Figure 18] 1 shows the results of measuring the area of ​​Oil Red O staining in liver tissue of drug-administered high-fat diet mice. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention will be described in more detail below. Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific description described below.

[0018] Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0019] In the present invention, the following compound represented by formula 1 is also named [4-(4-methoxyphenyl)-8-methyl-2-oxochromen-7-yl](2S)-3-(1H-indol-3-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoate (DTMB). [ka]

[0020] In the present invention, the salt of the compound represented by Formula 1 refers to a salt appropriate for the industrial application field of the composition containing the salt, for example, a salt that can be commonly used in pharmaceuticals, quasi-drugs, foods, feeds, etc. Examples of the salt include, but are not limited to, inorganic ion salts of the compound represented by Formula 1, such as sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, and iron, inorganic acid salts such as hydrochloric acid, phosphoric acid, and sulfuric acid, organic acid salts such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, orotic acid, and acetylsalicylic acid, and amino acid salts such as lysine, arginine, and guanidine.

[0021] In the present invention, metabolic disease refers to a metabolic disorder characterized by abnormal metabolism that can occur due to excessive nutritional intake, congenital enzyme abnormalities, acquired endocrine abnormalities, or loss of function of tissues involved in metabolism, etc. Prevention or treatment of metabolic disease in the present invention includes effects such as preventing, preventing, delaying, improving, resolving, alleviating, mitigating, improving, and treating the pathology or symptoms of metabolic disease, and is not limited to complete suppression of the onset of metabolic disease.

[0022] In the present invention, PPARs (Peroxisome proliferator-activated receptors) are nuclear hormone receptor family proteins including isotypes such as PPARα, PPARβ / δ, and PPARγ, which are known to act as intracellular lipid sensors and play a key role in regulating energy metabolism and homeostasis. The gene or protein sequences of human PPARα, PPARβ / δ, and PPARγ are known in the art.

[0023] In the examples of the present invention, the compound represented by formula 1 was confirmed to be a pan-PPAR agonist that can bind to PPARα, PPARβ / δ, and PPARγ and activate the transcription function of the PPAR proteins (Figure 1). Therefore, the compound represented by formula 1 or a salt thereof can be administered to the body to activate PPARs, improve energy metabolism, and be useful for the prevention or treatment of metabolic diseases.

[0024] In one embodiment, the metabolic disease is obesity. Obesity is a condition in which the body has excess adipose tissue and can be caused by an energy imbalance resulting from prolonged excessive intake of nutrients relative to energy expenditure. Obesity can also occur due to problems with appetite center function caused by certain genetic mutations, endocrine disorders, or the use of appetite-stimulating drugs. Obesity can lead to a variety of metabolic complications, including diabetes, fatty liver, and dyslipidemia, as well as cardiovascular diseases such as hypertension.

[0025] In the examples of the present invention, the compound represented by formula 1 effectively suppresses fat accumulation in adipocytes (Figures 3 and 4), and in a high-fat diet animal model, effects such as weight loss (Figure 6), abdominal fat loss (Figures 7 and 8), adipose tissue weight loss (Figure 9), and a reduction in the area of ​​lipid droplets in adipose tissue (Figure 15) were confirmed. Therefore, the compound represented by formula 1 or a salt thereof can be useful for fat loss, weight loss, and the prevention or treatment of obesity, including obesity complications.

[0026] In one embodiment, the metabolic disease is diabetes. Diabetes mellitus is a type of metabolic disease characterized by insufficient insulin secretion or impaired insulin function, and is characterized by hyperglycemia, which can lead to numerous diabetic complications such as retinopathy, renal dysfunction, neuropathy, and cardiovascular disease. Diabetes is classified into type 1 diabetes, which is caused by abnormal insulin production, and type 2 diabetes, which is characterized by insulin resistance.

[0027] In the examples of the present invention, the compound represented by formula 1 was confirmed to have effects such as improving blood glucose tolerance (FIG. 10), improving insulin tolerance (FIG. 11), and lowering blood glucose (FIG. 12) in a high-fat diet animal model. Therefore, the compound represented by formula 1 or a salt thereof can be usefully utilized for the prevention or treatment of diabetes, including hyperglycemia, insulin resistance, impaired fasting glucose (IFG), impaired glucose tolerance (IGT), hyperinsulinemia, and diabetic complications.

[0028] In one embodiment, the metabolic disease is fatty liver disease. Fatty liver disease is a comprehensive concept that includes a state in which fat accumulates in hepatocytes and the resulting liver tissue lesions. In the present invention, fatty liver disease includes various subdiseases depending on the stage of disease progression, from simple fatty liver, which is a state in which only fat accumulates in the liver, to steatohepatitis, which shows inflammatory findings accompanied by hepatocellular damage (such as balloon degeneration or fibrosis), and cirrhosis accompanied by steatohepatitis. Furthermore, depending on the cause of fatty liver disease, it includes alcoholic fatty liver disease and nonalcoholic fatty liver disease. Nonalcoholic fatty liver disease (NAFLD) includes nonalcoholic fatty liver, nonalcoholic steatohepatitis (NASH), and nonalcoholic fatty liver disease-associated liver fibrosis and / or cirrhosis.

[0029] In the examples of the present invention, the compound represented by formula 1 was confirmed to have the effects of reducing liver weight increased by a high-fat diet (FIG. 9), improving fatty liver findings in liver tissue (FIG. 16), reducing NAFLD activity score (FIG. 17), and reducing fat accumulation in liver tissue (FIG. 18) in a high-fat diet animal model. Therefore, the compound represented by formula 1 or a salt thereof can be usefully utilized for the prevention or treatment of fatty liver disease, including reducing liver fat accumulation, ameliorating liver inflammation, and ameliorating liver fibrosis and / or cirrhosis.

[0030] In one embodiment, the metabolic disorder is dyslipidemia. Dyslipidemia is a condition in which normal serum lipid levels are increased or decreased, characterized by increased blood total cholesterol, LDL cholesterol, and triglyceride levels, or decreased HDL cholesterol levels. Dyslipidemia can be caused by obesity, diabetes, etc., or by genetic factors that increase specific lipids in the blood. Dyslipidemia is also a major risk factor for cardiovascular disease.

[0031] In the examples of the present invention, the compound represented by formula 1 was confirmed to effectively reduce LDL-cholesterol levels increased by a high-fat diet in a high-fat diet animal model (Figure 13). Therefore, the compound represented by formula 1 or a salt thereof can be useful for preventing or treating dyslipidemia, including hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, cardiovascular disease caused by hyperlipidemia, xanthomas, lipomas, and lipodystrophy.

[0032] In one embodiment, the metabolic disease is cardiovascular disease. Metabolic disorders such as dyslipidemia are major risk factors for cardiovascular disease, and the compound represented by formula 1 or a salt thereof can be useful for preventing or treating cardiovascular disease, for example, reducing the risk of cardiovascular disease caused by dyslipidemia. Cardiovascular diseases include, but are not limited to, arteriosclerosis, atherosclerosis, hypertension, myocardial infarction, ischemic heart disease, and stroke.

[0033] In one embodiment, the metabolic disease is metabolic syndrome. Metabolic syndrome is a term used to describe a group of various metabolic disease symptoms, and was first proposed as metabolic syndrome X. It is characterized by the simultaneous appearance of metabolic disorder symptoms such as obesity, hyperglycemia, hyperlipidemia, atherosclerosis, and hypertension. Insulin resistance is said to be the core cause of metabolic syndrome, and exacerbation of metabolic syndrome can increase the risk of developing metabolic diseases such as fatty liver disease and cardiovascular disease.

[0034] As described above, in the examples of the present invention, the ameliorative effects on obesity, hyperglycemia, hyperlipidemia, etc. were confirmed in cells and animal models (FIGS. 2 to 18). Therefore, the compound represented by formula 1 or a salt thereof can be usefully used for the prevention or treatment of metabolic syndrome.

[0035] In addition to the compound of Formula 1 or a salt thereof as an active ingredient, the pharmaceutical composition of the present invention may contain one or more pharmaceutically acceptable carriers, excipients, diluents, solubilizers, etc. Examples of the carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and examples of the solubilizers include, but are not limited to, poloxamer and Labrasol.

[0036] The pharmaceutical composition may be in a variety of dosage forms suitable for oral or parenteral administration. Oral dosage forms of the present invention may include, but are not limited to, tablets, pills, powders, granules, pellets, capsules, troches, lozenges, suspensions, emulsions, syrups, and elixirs. Parenteral dosage forms of the present invention may include, but are not limited to, injections, suppositories, respiratory inhalants, aerosols, ointments, liquids, lotions, patches, topical powders, oils, creams, gels, and the like.

[0037] The pharmaceutical composition may be administered orally or parenterally depending on the dosage form. Parenteral administration may include, but is not limited to, subcutaneous administration, intradermal administration, transdermal administration, hair administration, intraperitoneal administration, rectal administration, intravenous administration, intramuscular administration, thoracic administration, etc.

[0038] The pharmaceutical composition may be administered in a pharmaceutically effective amount, which means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the level of the effective amount may be determined depending on factors including the patient's condition, weight, sex, age, health condition, severity of the disease, sensitivity to the drug, administration time, administration route, excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0039] The pharmaceutical composition may further contain one or more active ingredients having efficacy in preventing or treating metabolic diseases in addition to the compound represented by Formula 1 or a salt thereof, and may be used as a combination drug for metabolic diseases.

[0040] The present invention also provides a method for preventing or treating a metabolic disease, which comprises administering to a patient a therapeutically effective amount of a compound represented by formula 1 or a salt thereof. The present invention also provides use of a compound represented by formula 1 or a salt thereof for the prevention or treatment of a metabolic disease. The present invention also provides use of a compound represented by formula 1 or a salt thereof for the manufacture of a medicament for the prevention or treatment of a metabolic disease.

[0041] In the present invention, the food composition includes all types of food such as health foods, functional health foods, beverages, food additives, and food supplements, and is preferably a functional health food. Here, functional health foods refer to foods that are produced and processed using raw materials or ingredients that have functional properties useful for the human body, so as to efficiently exert a bioregulatory function in addition to providing nutrients.

[0042] The present invention also provides a method for improving metabolic health, comprising ingesting a compound of formula 1 or a salt thereof. The present invention also provides a use of a compound represented by formula 1 or a salt thereof for improving metabolic health. The present invention also provides the use of a compound of formula 1 or a salt thereof for the manufacture of a food product for improving metabolic health. The feed composition of the present invention is a diet provided for ingestion by animals, including mammals other than humans, and is effective in preventing, improving or treating metabolic disorders in the animals.

[0043] The present invention also provides a method for improving metabolic health, comprising administering to a non-human animal a compound of formula 1 or a salt thereof. The present invention also provides a use of a compound represented by formula 1 or a salt thereof for improving metabolic health. The present invention also provides the use of a compound of formula 1 or a salt thereof for the manufacture of a feed for improving the metabolic health of a non-human animal.

[0044] The present invention will be described in more detail with reference to the following examples. However, these examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0045] Materials and Methods 1. Compounds and Reagents The compound of the present invention represented by Formula 1 was synthesized by DAEJUNG Chemicals & Metals (Gyeonggi-do, Korea) (referred to as "D6" in the drawings). WY14643, GW501516, and rosiglitazone were purchased from Sigma-Aldrich (St. Louis, MO, USA). The compounds were dissolved in DMSO (dimethyl sulfoxide) and diluted in culture medium before use. Cyanogen bromide (CNBr)-activated Sepharose 4B was purchased from Sigma-Aldrich (St. Louis, MO, USA). His-tagged recombinant human PPAR-LBD (ligand binding domain) was expressed in E. coli (DE3, Rosetta) and purified before use. His antibody was purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). IBMX (3-Isobutyl-1-methylxanthine), dexamethasone, bovine pancreatic insulin, and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). PRO-PREP™ Protein Extraction Solution was purchased from iNtRON Biotechnology (iNtRON Biotech., Korea). Actin, HSL (Hormone Sensitive Lipase), and ATGL (Adipose Triglyceride Lipase) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Comet Assay Kits, 96-well, were purchased from Cell Biolabs (San Diego, CA, USA).

[0046] The plasmids were transformed into E. coli (DE3, Rosetta) and transfected into HEK293 cells. Luciferase analysis was performed on three types of pcDNA5-GAL4 and pGALRE-luc containing PPARα-LBD, PPARβ / δ-LBD, or PPARγ-LBD, according to published literature, such as [Kanno, Y. & Inouye, YJ Toxicol Sci 35, 515-525 (2010)] and [Zhao, S. et al. J Nat Prod 79, 879-885 (2016)]. pProEX-PPAR and pET-28a were used to purify His-tagged recombinant hPPAR-LBD proteins.

[0047] 2. Cell Culture and Transfection The HEK293 cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (Fetal Bovine Serum) and 1% fenicillin-streptomycin (P / S). Mouse preadipocyte 3T3-L1 cell line was cultured in DMEM supplemented with 10% goat serum (CS) and 1% P / S. For 3T3-L1 differentiation, 10% FBS and 1% P / S were used. The cells were cultured in a humidified incubator at 37°C with a 5% CO2 atmosphere.

[0048] For gene transfection, HEK293 cells were microporated using an MP-100 microporator (Invitrogen, Carlsbad, CA, USA).

[0049] 3. Luciferase assay Transfected HEK293 cells were treated with drugs for 24 hours, then harvested and resuspended in luciferase lysis buffer (Promega, Madison, Wisconsin, USA) and incubated on ice for 10 minutes. Cell debris was removed by centrifugation at 15,000 rpm for 10 minutes at 4°C, and the supernatant was separated. Fluc (Firefly Luciferase) and Rluc (Renilla Luciferase) activities were then measured using the Dual Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA). The ratio of Fluc to Rluc activity was defined as the PPAR LBD (Ligand Binding Domain) activity.

[0050] 4. Analysis of CNBr-bead Conjugation 100 mg of CNBr-activated Sepharose 4B was added to an e-tube and activated with 1 ml of 1 mM HCl by rotation at room temperature for 10 minutes. After 10 minutes, the solution was replaced with a new one and the process was repeated. The supernatant was removed by centrifugation at 5,000 rpm for 1 minute, and the sample was washed with coupling buffer. This process was repeated three times. The sample was separated with 20 mg of beads in another e-tube. Drug was added to each e-tube and rotated overnight at 4°C. The concentration was measured to be 20 μM. The next day, the sample was washed three times with coupling buffer. After washing, the sample was blocked with 1 ml of blocking buffer at room temperature for 2 hours. After blocking, the sample was washed three times with washing buffer, followed by washing buffer and pull-down buffer. The supernatant was then centrifuged at 5,000 rpm for 1 minute and removed. 0.8 ml of pull-down buffer and tablet protein was added to the pellet and rotated overnight at 4°C. Finally, the sample was washed three times with pull-down buffer. The supernatant was completely removed, and the pellet was detected by Western blotting.

[0051] 5. In Silico Docking Analysis The crystal structures of the LBDs of the human PPAR family (PPARα: 4BCR, PPARβ / δ: 5U46, PPARγ: 5YCP) were obtained from the RCSB Protein Data Bank. For further docking analysis, the energy of D6 or PPAR ligands (WY14643, GW501516, and rosiglitazone) was minimized using open babel in PyRx software.

[0052] 6.3T3-L1 adipocyte differentiation 3T3-L1 cells were plated in a 12-well plate at 5 × 10 4The cells were seeded at a density of 1000 cells / well. They were cultured in DMEM containing 10% CS and 1% P / S until confluency was reached. Two days after reaching 100% confluency, DMEM containing 10% FBS and 1% P / S supplemented with MDI (0.5 mM IBMX, 1 μM dexamethasone, 1 μg / ml insulin) was added to the cells. Two days after MDI addition, the medium was replaced with 1 μg / ml insulin. After two days, the medium was replaced with DMEM containing 10% FBS and 1% P / S, and the drugs were added three times every two days. Finally, the medium was replaced with serum-free DMEM containing only the drug. After two days, 3T3-L1 differentiation was complete.

[0053] 7.Oil Red O staining After differentiation of 3T3-L1 cells in 12-well plates was completed, the cells were fixed with 4% formaldehyde and stained with Oil Red O solution. Photographs of the stained samples were taken under a microscope (Nikon Eclipse Ti-s). The Oil Red O staining agent in the samples was dissolved in isopropanol and detected by a spectrophotometer at 492 nm. The size distribution of lipid droplets was measured using ImageJ. OCT-embedded liver samples were stained according to the Roy Ellis protocol.

[0054] 8. Western Blotting After drug treatment, differentiated 3T3-L1 cells were harvested and resuspended in PRO-PREP™ Protein Extraction Solution (iNtRON Biotechnology, Korea). The samples were incubated on ice for 10 minutes, vortexed for 2 seconds, and then incubated on ice for another 10 minutes. After centrifugation (15,000 rpm, 20 minutes, 4°C), protein concentrations were measured by Bradford assay using 1 mg / ml BSA. Equal amounts of protein were separated from SDS-polyacrylamide gels and blotted onto PVDF (polyvinylidene difluoride membrane) (Millipore, Billerica, MA, USA). After blocking for 30 minutes with blocking solution (5% BSA in TTBS) at room temperature, the cells were incubated overnight at 4°C with primary antibodies (anti-HSL, anti-ATGL, anti-Actin, etc.). Next, the sections were incubated with secondary antibodies for 2 hours at room temperature, and the signals were detected using the LAS4000.

[0055] 9. Experimental Animals The care and use of experimental animals was approved by the Animal Experiment Ethics Committee of Pohang University of Science and Technology. C57BL / 6J mice were purchased from Japan SLC, Inc. Fifty 4-week-old male C57 / BL / 6J mice were adapted for 2 weeks. Mice were randomly assigned to receive vehicle or 5 mg / kg / day of D6 or WY14643 orally for 13 weeks, and obesity was induced with a 60% high-fat diet (#D12492, Research Diets, NJ, USA). Body weight and food intake were measured weekly from 6 weeks of age, and adipose tissue and muscle volumes were measured using a trimodality imaging system after 16 weeks of age.

[0056] 10. Intraperitoneal glucose tolerance test (IP-GTT) and insulin tolerance test (IP-ITT) Mice aged 16 weeks or older were fasted for 16 hours, and then intraperitoneally administered glucose at 2 g / kg body weight. A glucose tolerance test (GTT) was performed. Blood glucose levels were measured 0, 15, 30, 45, 60, 90, and 120 minutes after the tail vein injection. Mice aged 17 weeks or older were fasted for 3 hours, and then intraperitoneally administered insulin at 0.75 U / kg body weight, followed by an insulin tolerance test (ITT). Blood glucose levels were measured 0, 15, 30, 45, 60, and 90 minutes after tail vein injection.

[0057] 11. Histological examination Mouse tissues were fixed in 10% formalin solution (Sigma-Aldrich, St. Louis, MO, USA), and paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) and Sirius Red (IHC world, Woodstock, USA). Liver fibrosis and vesicular fat were observed using Sirius Red and Oil Red O staining. NAFLD activation scores were assessed according to the NASH Clinical Research Network Scoring System, as described in Kleiner, DE et al. Hepatology 41, 1313-1321, doi:10.1002 / hep.20701(2005).

[0058] 12. Serum analysis Mouse blood was obtained by cardiac puncture and incubated at room temperature for 30 minutes, after which the blood samples were centrifuged at 3,000 rpm for 30 minutes at 4°C, and the supernatants were collected and analyzed.

[0059] <Example> Example 1. Confirmation of pan-PPAR agonist effect To confirm whether the compounds of Formula 1 are PPAR agonists, a Gal4 transactivation luciferase assay was performed. Specifically, HEK293 cells were transfected with the Gal4-luciferase reporter systems of PPARα, PPARβ / δ, and PPARγ, respectively, and then treated with drugs for 24 hours. After harvesting, the cells were analyzed by a dual-luciferase assay. Relative luciferase activity was compared between Rluc and Fluc activity. WY14643, GW501516, and rosiglitazone are representative PPARα, PPARδ, and PPARγ agonists, respectively, and served as positive controls. The results of the experiment showed that the compound of formula 1 increased the relative luciferase activity of all of PPARα, PPARβ / δ, and PPARγ in a dose-dependent manner (FIG. 1).

[0060] To confirm whether the Formula 1 compound directly binds to PPAR proteins, we performed a CNBr-bead conjugation assay. Specifically, Formula 1 compound (D6) and each PPAR agonist (WY14643, GW501516, and rosiglitazone) were reacted with CNBr-coupled, His-tagged recombinant human PPAR-Ligand Binding Domain (hPPAR-LBD), and their binding to PPAR was confirmed by Western blotting. Free drug ("Free"), not conjugated to CNBr, acted as a competitor with the CNBr-coupled drug. When the CNBr-coupled drug that had reacted with hPPAR-LBD was mixed with the free drug, the band disappeared.

[0061] As a result of the experiment, it was confirmed that the compound of formula 1 binds to all three PPAR subtypes as strongly as the PPAR agonist used as a positive control. To further support the results of the above experiments, in-silico analysis was performed to predict the binding site of the compound of Formula 1 in the PPAR LBD (ligand-binding domain) and the binding energy between the PPAR LBD and the compound of Formula 1. As a result, it was confirmed that the binding energy of the compound of Formula 1 of the present invention is at a level similar to that of representative agonists of PPAR.

[0062] Example 2: Confirmation of effects on energy metabolism such as lipid accumulation in adipocytes To confirm the effect of Formula 1 compound on adipocytes, we conducted a differentiation experiment of 3T3-L1 adipocytes. After treatment with Formula 1 compound (D6), rosiglitazone (Ro), or both (drug concentration: 25 μM) during differentiation, metabolic changes, such as lipid droplet accumulation in adipocytes, were observed using Oil Red O staining (Figure 3). Lipid droplets in adipocytes treated with Formula 1 compound alone were similar to those in the negative control DMSO-treated group. The increased lipid accumulation induced by rosiglitazone treatment was reduced when Formula 1 compound was co-treated (Figures 3 and 4, left). In particular, treatment with rosiglitazone alone reduced the size of small lipid droplets and increased the size of large lipid droplets, whereas treatment with Formula 1 compound effectively reduced the size distribution of lipid droplets (Figure 4, right).

[0063] Example 3. Confirmation of the effect on energy metabolism in high-fat-fed mice To confirm the metabolic disease-suppressing effects of Formula 1 compound in animal experiments, mice were acclimated for 2 weeks, then fed a 60% high-fat diet (HFD) to induce obesity. Then, vehicle or 5 mg / kg / day of Formula 1 compound (D6) or WY14643 was orally administered for 13 weeks. After 10 weeks of drug administration, IP-GTT, IP-ITT, microCT, and other experiments were performed (Figure 5). As a result of the experiment, when high-fat diet mice were administered the compound of formula 1 (HFD-D6), there was no significant change in food intake compared to the control group (HFD-V), but the rate of weight gain was significantly reduced (Figure 6).

[0064] Furthermore, micro-CT imaging and quantification of high-fat diet mice revealed that the volume of adipose tissue in mice administered with the compound of formula 1 was significantly reduced compared to the control group (FIGS. 7 and 8). Furthermore, when the organ weights of high-fat diet mice were measured, the weights of epididymal white adipose tissue (eWAT) and liver were reduced in mice administered with the compound of formula 1 (HFD-D6) compared to mice not administered with the drug (HFD-V) (Figure 9).

[0065] To confirm the effect of the compound of formula 1 on energy metabolism, a glucose tolerance test (GTT) and an insulin tolerance test (ITT) were performed. As a result of the experiment, it was confirmed that the compound of formula 1 improved glucose tolerance and insulin resistance in high-fat diet mice (HFD-D6) administered with the compound of formula 1, and had an effect of improving metabolic diseases such as diabetes-related symptoms (Figures 10 and 11). Analysis of mouse serum components revealed that blood glucose and blood lipids (LDLC) were significantly reduced in mice administered with the compound of formula 1, confirming the effectiveness of improving metabolic diseases such as diabetes and dyslipidemia (Figures 12 and 13).

[0066] Example 4. Histological observation of adipose tissue in high-fat-fed mice To observe the effect of the compound of formula 1 on metabolic diseases, histological examination was carried out on adipose tissue in high-fat diet mice. H&E staining of mouse adipose tissue showed that adipocyte size was reduced in high-fat diet mice (HFD-D6) administered with the compound of formula 1 (Figure 14). Quantitative measurement of adipocyte size distribution confirmed a reduction in adipocyte size in inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT) (Figure 15).

[0067] Example 5. Histological observation of liver tissue in high-fat-fed mice After administration of the compound of formula 1, to evaluate the effect on fatty liver-related symptoms, H&D, Sirius Red and Oil Red O staining was performed on liver tissues from mice that had been fed a high-fat diet for 13 weeks. As a result of the experiment, fat accumulation and cellular ballooning (arrows) were observed in the liver tissue of high-fat diet mice (HFD-V), while histological findings of fatty liver-related disease were reduced in high-fat diet mice (HFD-D6) administered with the compound of formula 1 (Figure 16).

[0068] More specifically, the NAFLD activity score was evaluated using H&E images of liver tissue, and it was confirmed that high-fat-fed mice (HFD-D6) treated with Formula 1 compound showed a greater improvement in NAFLD disease indicators than the positive control group, high-fat-fed mice (HFD-WY) treated with WY14643 (Figure 17).In addition, the lipid accumulation level in the liver was quantitatively evaluated using Oil Red O, and it was confirmed that the lipid droplet accumulation level was reduced in high-fat-fed mice (HFD-D6) treated with Formula 1 compound (Figure 18).

[0069] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics. In this regard, the above-described embodiments should be understood to be illustrative in all respects and not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and equivalent concepts thereof, rather than the above detailed description.

Claims

1. A pharmaceutical composition for preventing or treating a metabolic disease, comprising a compound represented by the following formula 1 or a salt thereof as an active ingredient: 【Chemistry 1】

2. The composition according to claim 1, wherein the compound represented by formula 1 or a salt thereof activates PPARα, PPARβ / δ, and PPARγ.

3. 2. The composition according to claim 1, wherein the metabolic disease is one or more selected from the group consisting of obesity, diabetes, fatty liver disease, dyslipidemia, cardiovascular disease and metabolic syndrome.

4. A food composition for improving metabolic health, comprising a compound represented by the following formula 1 or a salt thereof: 【Chemistry 1】

5. The composition of claim 4, wherein the metabolic health improvement is one or more selected from the group consisting of improvement of obesity, lowering of blood sugar, improvement of fatty liver, improvement of lipid metabolism, improvement of blood circulation, and improvement of metabolic syndrome.

6. A feed composition for improving metabolic health, comprising a compound represented by the following formula 1 or a salt thereof: 【Chemistry 1】

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