Use of PPAR agonists for improving 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 of Korean Patent Application No. 10-2022-0089073, and the entire contents of the said patent application are incorporated herein by reference. The present invention relates to the use of a novel pan-PPAR agonist for preventing, ameliorating or treating metabolic disorders.

Background Art

[0002] PPAR (Peroxisome proliferator-activated receptors) is a nuclear hormone receptor family protein that can control the transcription of genes having a PPAR-responsive regulatory element (PPRE) together with a Retinoid X receptor (RXR), and is classified into isoforms such as PPARα, PPARβ / δ, and PPARγ.

[0003] PPAR acts as a lipid sensor in cells and functions as a key factor in normal energy metabolism homeostasis. Since PPAR agonists can improve various energy metabolism homeostases such as obesity, lipid metabolism, glucose homeostasis, and insulin resistance, they are considered as major targets for metabolic disorders (see, for example, the literature [Corrales, Patricia, Antonio Vidal-Puig, and Gema Medina-Gomez. International Journal of Molecular Sciences 19.7 (2018): 2124.]).

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

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

[0006] PPARγ increases insulin sensitivity and glucose metabolism through promoting fatty acid influx, triglyceride formation, and lipid droplet storage, etc., and is activated by pioglitazone, rosiglitazone, etc. (Literature [Soccio, Raymond E., Eric R. Chen, and Mitchell A. Lazar. Cell metabolism 20.4 (2014): 573-591.]).

[0007] In addition, for the improvement of PPAR agonists, saroglitazar and tesaglitazar, which are dual agonists of PPARα and PPARγ (such as the literature [Rastogi, Ashu, et al. Acta Diabetologica 57.7 (2020): 809 - 818.]), elafibranor (GFT505), which is a dual agonist of PPARα and PPARβ / δ (such as the literature [Ratziu, Vlad, et al. Gastroenterology 150.5 (2016): 1147 - 1159.]), ranifibranor, ciglitazar, etc., which are pan - agonists of PPARα, PPARβ / δ, and PPARγ, have been developed (such as the literature [Lefere, Sander, et al. Journal of hepatology 73.4 (2020): 757 - 770.]). In fact, it is necessary to develop new PPAR agonists to improve the efficacy and side effects of conventional PPAR agonists.

Summary of the Invention

Problems to be Solved by the Invention

[0008] Therefore, the present inventor discovered a novel PPAR agonist that can improve metabolic disorders such as obesity, diabetes, and fatty liver disease, and confirmed that the PPAR agonist effectively improves various metabolic disorders in cell and animal models, thus completing the present invention.

[0009] An object of the present invention is to provide a use for preventing, improving, or treating metabolic disorders of a compound represented by Formula 1 or a salt thereof. Another object of the present invention is to provide a use of a compound represented by Formula 1 or a salt thereof as a PPAR agonist.

Means for Solving the Problems

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

Chemical Formula

[0011] The compound represented by the above 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 health functional food composition for improving metabolic health, which contains the compound represented by the above formula 1 or a salt thereof. The improvement of metabolic health can be one or more effects selected from the group consisting of improvement of obesity, reduction of blood glucose, 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, which contains the compound represented by the above formula 1 or a salt thereof.

[0014] In another aspect, the present invention provides a method for activating PPAR protein, which includes the step of treating the compound represented by the above formula 1 or a salt thereof with isolated cells. The PPAR protein can 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 applied to 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 for improving metabolic health, feed compositions, etc.

Brief Description of the Drawings

[0016]

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Modes for Carrying Out the Invention

[0017] Hereinafter, the present invention will be described more specifically. Each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. Also, it cannot be considered that the scope of the present invention is limited by the specific description described below.

[0018] Furthermore, those with ordinary knowledge in the art can recognize or confirm a number of equivalents to the specific aspects of the present invention described in this application using only ordinary experiments. It is intended that such equivalents be included in 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).

Chemical formula

[0020] In the present invention, the salt of the compound represented by Formula 1 means a salt appropriate depending on the industrial application field of the composition containing the salt, for example, a salt that can be usually used in pharmaceuticals, quasi-drugs, foods, feeds, etc. The salts include, for example, inorganic ion salts such as sodium, potassium, calcium, magnesium, lithium, copper, manganese, zinc, iron, etc. of the compound represented by Formula 1, inorganic acid salts such as hydrochloric acid, phosphoric acid, sulfuric acid, and in addition, 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, acetylsalicylic acid, and amino acid salts such as lysine, arginine, guanidine, etc., but are not limited thereto.

[0021] In the present invention, a metabolic disease means a metabolic disorder characterized by abnormal metabolism that can occur due to excessive nutrient intake, congenital enzyme abnormalities, acquired endocrine abnormalities, or loss of function of tissues involved in metabolism. Prevention or treatment of metabolic diseases in the present invention includes effects such as prevention, prevention, delay, improvement, elimination, alleviation, reduction, improvement, treatment, etc. of the pathological conditions or symptoms of metabolic diseases, and is not limited only to complete suppression of the onset of metabolic diseases.

[0022] In the present invention, PPAR (Peroxisome proliferator-activated receptors) is a nuclear hormone receptor family protein including isotypes such as PPARα, PPARβ / δ, and PPARγ, which acts as an intracellular lipid sensor and is known to play a central role in energy metabolism and homeostasis regulation. The gene or protein sequences of human PPARα, PPARβ / δ, and PPARγ are known in the art.

[0023] In an embodiment of the present invention, it was confirmed that the compound represented by Formula 1 is a pan-PPAR agonist that can bind to PPARα, PPARβ / δ, and PPARγ and activate the transcription function of the PPAR protein (Figure 1). Therefore, the compound represented by Formula 1 or a salt thereof can be administered in vivo, activate PPAR, improve energy metabolism, and be usefully utilized for the prevention or treatment of metabolic diseases.

[0024] In one embodiment, the metabolic disease is obesity. Obesity is a state in which there is an excess of adipose tissue in the body, which can be caused by an energy imbalance when nutrients are overingested for a long time compared to the energy consumption. In addition, obesity can also occur due to specific gene mutations that cause problems in the function of the appetite center, endocrine diseases, the use of appetite-increasing drugs, etc. Obesity can metabolically cause various obesity complications such as diabetes, fatty liver, dyslipidemia, and cardiovascular diseases such as hypertension.

[0025] In an embodiment of the present invention, the compound represented by Formula 1 effectively inhibits lipid accumulation in adipocytes (Figures 3 and 4), and in a high-fat diet animal model, effects such as weight loss (Figure 6), abdominal fat reduction (Figures 7, 8), reduction in the weight of adipose tissue (Figure 9), and 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 usefully utilized for the prevention or treatment of obesity including fat reduction, weight loss, and obesity complications.

[0026] In one embodiment, the metabolic disease is diabetes. Diabetes mellitus is a type of metabolic disease in which the secretion amount of insulin is insufficient or it does not function normally, characterized by hyperglycemia, and hyperglycemia can cause various diabetic complications such as retinopathy, renal dysfunction, neuropathy, and cardiovascular diseases. Diabetes is classified into type 1 diabetes caused by abnormal insulin production and type 2 diabetes characterized by insulin resistance, etc.

[0027] In the examples of the present invention, the compound represented by formula 1 was confirmed to have effects such as improvement of blood glucose tolerance (Figure 10), improvement of insulin tolerance (Figure 11), and blood glucose reduction (Figure 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 progressive liver tissue lesions. The fatty liver disease in the present invention includes simple fatty liver in which only fat accumulates in the liver, steatohepatitis with inflammatory findings accompanied by hepatocyte damage (such as ballooning degeneration or fibrosis), and cirrhosis accompanied by steatohepatitis, including various sub-diseases depending on the degree of disease progression. In addition, depending on the cause of the onset of fatty liver disease, it includes alcoholic fatty liver disease and non-alcoholic fatty liver disease. The non-alcoholic fatty liver disease (NAFLD) includes non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease-related hepatic fibrosis and / or cirrhosis.

[0029] In the examples of the present invention, the compound represented by Formula 1 was confirmed to have effects such as a decrease in liver weight increased by a high-fat diet (Figure 9), improvement of fatty liver findings in liver tissue (Figure 16), decrease in NAFLD activity score (Figure 17), and decrease in fat accumulation in liver tissue (Figure 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 a decrease in liver fat accumulation, improvement of liver inflammation, and improvement of hepatic fibrosis and / or cirrhosis.

[0030] In one embodiment, the metabolic disease is dyslipidemia. Dyslipidemia is a state in which the normal serum lipid concentration increases or decreases, characterized by an increase in total blood cholesterol, LDL cholesterol, and triglyceride concentration, or a decrease in HDL cholesterol concentration. Dyslipidemia can occur due to causes such as obesity and diabetes, or can occur when specific lipids in the blood are increased as a genetic factor. In addition, dyslipidemia is a major risk factor for cardiovascular disease.

[0031] In an embodiment of the present invention, it was confirmed that the compound represented by Formula 1 effectively reduced the LDL-cholesterol value 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 usefully utilized for the prevention or treatment of dyslipidemia including hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, cardiovascular diseases due to hyperlipidemia, xanthoma, lipoma, lipodystrophy, etc.

[0032] In one embodiment, the metabolic disease is a cardiovascular disease. Metabolic disorders such as dyslipidemia are major risk factors for cardiovascular diseases, and the compound represented by Formula 1 or a salt thereof can be usefully utilized for the prevention or treatment of cardiovascular diseases, for example, reducing the risk of cardiovascular diseases due to dyslipidemia. The cardiovascular diseases include, but are not limited to, arteriosclerosis, atherosclerosis, hypertension, myocardial infarction, ischemic heart disease, stroke, etc.

[0033] In one embodiment, the metabolic disease is metabolic syndrome. Metabolic syndrome is a name that represents various symptoms of metabolic diseases as a single cluster, and was first presented by the term metabolic syndrome X, and is characterized by the appearance of metabolic disorder symptoms such as obesity, hyperglycemia, hyperlipidemia, atherosclerosis, hypertension, etc. at the same time. Metabolic syndrome is said to be mainly caused by insulin resistance, and when metabolic syndrome worsens, the risk of developing metabolic diseases such as fatty liver disease and cardiovascular diseases may increase.

[0034] As described above, in the examples of the present invention, since the improvement effects on obesity, hyperglycemia, hyperlipidemia, etc. were confirmed in cell and animal models (Figures 2 to 18), the compound represented by Formula 1 or a salt thereof can be usefully utilized for the prevention or treatment of metabolic syndrome.

[0035] In addition to the compound represented by Formula 1 or a salt thereof as an active ingredient, the pharmaceutical composition of the present invention can contain one or more pharmaceutically acceptable carriers, excipients, diluents, solubilizers, and the like. 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. Examples of the solubilizers include poloxamer and labrasol, but are not limited thereto.

[0036] The pharmaceutical composition can exist in suitable and diverse dosage forms for oral or parenteral administration. Dosage forms for oral administration in the present invention can include tablets, pills, powders, powders for external use, granules, pellets, capsules, troches, lozenges, suspensions, emulsions, syrups, and elixirs, etc. Dosage forms for parenteral administration can include injections, suppositories, respiratory inhalants, aerosols, ointments, liquids, lotions, patches, powders for application, oils, creams, gels, etc., but are not limited thereto.

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

[0038] The pharmaceutical composition can be administered in a pharmaceutically effective amount. Here, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The level of the effective amount can be determined according to factors including the patient's condition, weight, gender, age, health status, degree of the disease, sensitivity to the drug, administration time, administration route, excretion rate, treatment period, drugs used concomitantly, and other factors widely known in the medical field.

[0039] In addition to the compound represented by Formula 1 or a salt thereof, the pharmaceutical composition further contains one or more active ingredients having a prophylactic or therapeutic effect on metabolic diseases, and can be used as a combination agent for metabolic diseases.

[0040] The present invention also provides a method for preventing or treating metabolic diseases, which includes the step of administering to a patient a therapeutically effective amount of the compound represented by Formula 1 or a salt thereof. The present invention also provides a use of the compound represented by Formula 1 or a salt thereof for preventing or treating metabolic diseases. The present invention also provides a use of the compound represented by Formula 1 or a salt thereof for the manufacture of a medicament for preventing or treating metabolic diseases.

[0041] In the present invention, the food composition includes all forms of foods such as health foods, functional health foods, beverages, food additives, and food supplements, and preferably may be a functional health food. Here, the functional health food means a food manufactured and processed using raw materials and components having useful functions for the human body so that the biological regulation function can be efficiently exhibited in addition to nutritional supply.

[0042] The present invention also provides a method for improving metabolic health, which includes the step of ingesting the compound represented by Formula 1 or a salt thereof. The present invention also provides a use of the compound represented by Formula 1 or a salt thereof for improving metabolic health. The present invention also provides a use of the compound represented by Formula 1 or a salt thereof for the manufacture of a food for improving metabolic health. The feed composition in the present invention is a diet provided for ingestion by animals including mammals other than humans, and can exert a prophylactic, ameliorating or therapeutic effect on metabolic disorders of the animals.

[0043] The present invention also provides a method for improving metabolic health, which includes the step of causing an animal other than a human to ingest the compound represented by Formula 1 or a salt thereof. The present invention also provides a use of the compound represented by Formula 1 or a salt thereof for improving metabolic health. The present invention also provides the use of a compound represented by Formula 1 or a salt thereof for the production of a feed for improving the metabolic health of animals other than humans.

[0044] Hereinafter, the present invention will be described in more detail through examples. However, these embodiments are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these embodiments.

[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) (designated as "D6" in the drawings). WY14643, GW501516, and rosiglitazone were purchased from Sigma-Aldrich (St. Louis, MO, USA). The above compounds were dissolved in DMSO (dimethyl sulfoxide) and diluted in the culture medium before being used in the experiments. CNBr (Cyanogen bromide)-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 then purified for use. The His antibody was purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). IBMX (3-Isobutyl-1-methylxanthine), dexamethasone, bovine pancreas-derived insulin, and Oil Red O were purchased from Sigma-Aldrich (St. Louis, MO, USA). PRO-PREP (trademark application) 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 plasmid was transformed into E. coli (DE3, Rosetta), transfected into HEK293 cells, and used. With reference to known literatures such as [Kanno, Y. & Inouye, Y. J Toxicol Sci 35, 515 - 525 (2010).] and [Zhao, S. et al. J Nat Prod 79, 879 - 885 (2016).], luciferase assays were performed for three types of pcDNA5 - GAL4 and pGALRE - luc containing PPARα - LBD, PPARβ / δ - LBD or PPARγ - LBD. pProEX - PPAR and pET - 28a were used for the purification of His - tagged recombinant hPPAR - LBD protein.

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

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

[0049] 3. Luciferase Assay After 24 hours of drug treatment of the transfected HEK293 cells, the cells were collected and then resuspended in luciferase lysis buffer (Promega, Madison, Wisconsin, USA) and incubated on ice for 10 minutes. After removing the cell debris by centrifugation at 15,000 rpm for 10 minutes at 4°C, the supernatant was separated. Next, the Fluc (Firefly Luciferase) and Rluc (Renilla Luciferase) activities were measured using the Dual Luciferase Reporter Assay System (Promega, Madison, Wisconsin, USA). The ratio of Fluc to Rluc activity was defined as the PPAR's LBD (Ligand Binding Domain) activity.

[0050] 4. Analysis of CNBr-Bead Conjugation After adding 100 mg of CNBr-activated Sepharose 4B to an e-tube, the beads were activated by rotating with 1 ml of 1 mM HCl at room temperature for 10 minutes. After 10 minutes, the solution was newly exchanged and this process was repeated. After removing the supernatant by centrifugation at 5,000 rpm for 1 minute, the sample was washed with coupling buffer, and this was repeated 3 times. The sample was separated with 20 mg of beads in another e-tube. The drug was added to each e-tube and rotated overnight at 4°C, at which time the concentration was measured to be 20 μM. The next day, the sample was washed 3 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 3 times with washing buffer, repeating in the order of washing buffer and Pull-down buffer. Next, the supernatant was centrifuged at 5,000 rpm for 1 minute and the supernatant was removed. 0.8 ml of Pull-down buffer and tablet protein were added to the pellet and rotated overnight at 4°C. Finally, the sample was washed 3 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 prepared through the RCSB Protein Data Bank. For additional docking analysis, the energies of D6 or PPAR ligands (WY14643, GW501516, and rosiglitazone) were minimized using open babel within the PyRx software.

[0052] 6. Differentiation of 3T3-L1 Adipocytes 3T3-L1 cells were seeded at 5×10 in a 12-well plate 4Seeded at a density of cells / well. The cells were cultured in DMEM containing 10% CS and 1% P / S until confluency was reached. After reaching 100% confluency, on the second day, DMEM medium containing 10% FBS and 1% P / S with MDI (0.5 mM IBMX, 1 μM dexamethasone, 1 μg / ml insulin) added was added to the cells. On the second day after MDI addition, the medium was replaced with 1 μg / ml insulin. Two days later, the medium was replaced with DMEM containing 10% FBS and 1% P / S, and the cells were treated with drugs. The medium was replaced with fresh DMEM containing 10% FBS and 1% P / S, and the drugs were added three times every two days. Finally, the medium was replaced with DMEM containing only the drug on serum starvation. Two days later, the differentiation of 3T3-L1 was completed.

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

[0054] 8. Western Blotting After treating differentiated 3T3-L1 cells with drugs, the cells were harvested and resuspended in PRO-PREP (trademark application) 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), the protein concentration was measured by Bradford assay using 1 mg / ml BSA. The same amount of protein was separated from SDS-polyacrylamide gels and blotted onto PVDF (polyvinylidene difluoride membrane) (Millipore, Billerica, MA, USA). After blocking with blocking solution (5% BSA in TTBS) at room temperature for 30 minutes, the samples were incubated with primary antibodies (anti-HSL, anti-ATGL, anti-Actin, etc.) overnight at 4°C. Next, the samples were incubated with secondary antibodies for 2 hours at room temperature, and the signals were detected with a LAS4000.

[0055] 9. Laboratory Animals The management and use of experimental animals were carried out with approval from 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 allowed to acclimatize for 2 weeks. The mice were randomly divided and orally gavaged with vehicle or 5 mg / kg / day of D6 or WY14643 for 13 weeks to induce obesity 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 after 16 weeks of age, the volumes of adipose tissue and muscle were measured with a Trimodality imaging system.

[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 after intraperitoneal glucose administration at 2 g / kg per body weight, a glucose tolerance test (GTT) was performed. Blood glucose levels were measured at 0, 15, 30, 45, 60, 90, and 120 minutes after tail vein injection. Mice aged 17 weeks or older were fasted for 3 hours, and after intraperitoneal insulin administration at 0.75 U / kg per body weight, an insulin tolerance test (ITT) was performed. Blood glucose levels were measured at 0, 15, 30, 45, 60, and 90 minutes after tail vein injection.

[0057] 11. Tissue Examination Mouse tissues were fixed with 10% formalin solution (Sigma-Aldrich, St. Louis, MO, USA), paraffin-embedded tissues were sectioned, and hematoxylin and eosin (H&E) staining and Sirius Red staining (IHC world, Woodstock, USA) were performed. Fibrosis and vesicular fat in liver tissue were observed using Sirius Red and Oil Red O staining methods. The NAFLD activation score was evaluated according to the NASH Clinical Research Network Scoring System with reference to literature [Kleiner, D. E. et al. Hepatology 41, 1313 - 1321, doi:10.1002 / hep.20701 (2005).] and others.

[0058] 12. Serum Analysis Mouse blood was obtained by cardiac puncture and incubated at room temperature for 30 minutes. Next, the blood sample was centrifuged at 3,000 rpm for 30 minutes at 4°C, and the supernatant was collected for analysis.

[0059] <Example> Example 1. Confirmation of the Effect of a Pan-PPAR Agonist To confirm whether the compound of Formula 1 is a PPAR agonist, Gal4 trans-activation luciferase assay was performed. Specifically, HEK293 cells were transfected with the Gal4-luciferase reporter system for each of PPARα, PPARβ / δ and PPARγ, harvested after 24-hour drug treatment, and then analyzed by Dual-Luciferase assay. The relative luciferase activity was compared by the Fluc activity against the Rluc activity. WY14643, GW501516 and rosiglitazone are representative PPARα, PPARδ and PPARγ agonists respectively, corresponding to the positive control group. As a result of the experiment, the compound of Formula 1 increased the relative luciferase activity against all of PPARα, PPARβ / δ and PPARγ in a dose-dependent manner (Figure 1).

[0060] On the other hand, to confirm whether the compound of Formula 1 binds directly to the PPAR protein, CNBr-bead conjugation analysis was performed. Specifically, the compound of Formula 1 (D6) and each PPAR agonist (WY14643, GW501516 and rosiglitazone) were coupled with CNBr and reacted with His-tagged recombinant human PPAR-Ligand Binding Domain (hPPAR-LBD), and then their binding to PPAR was confirmed by Western blotting. The free drug not conjugated to CNBr ("Free") acted as a competitor to 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 did not bind as strongly to all three PPAR subtypes as the PPAR agonists used as the positive control group. To further confirm the results of the above experiments, in-silico analysis was performed to predict the binding position of the compound of Formula 1 within the LBD (ligand-binding domain) of PPAR and the binding energy between PPAR LBD and the compound of Formula 1. As a result, the binding energy of the compound of Formula 1 of the present invention was confirmed to be at a level similar to that of a typical agonist of PPAR.

[0062] Example 2. Confirmation of the Effect on Energy Metabolism such as Lipid Accumulation in Adipocytes To confirm the effect of the compound of Formula 1 on adipocytes, a differentiation experiment of 3T3-L1 adipocytes was conducted. During the differentiation of the compound of Formula 1 (D6), rosiglitazone (Ro), or both, after treatment (drug concentration: 25 μM), metabolic changes such as the accumulation amount of lipid droplets in adipocytes were observed through Oil Red O staining (Figure 3). As a result, it was confirmed that the lipid droplets in adipocytes treated with the compound of Formula 1 alone were similar to those in the DMSO-treated group, which was the negative control group, and the lipid accumulation increased by rosiglitazone treatment decreased when the compound of Formula 1 was co-treated (the left side of Figure 3 and Figure 4). In particular, when rosiglitazone was treated alone, the size of small lipid droplets decreased and the size of large lipid droplets increased, but when the compound of Formula 1 was co-treated, it was confirmed that the size distribution of lipid droplets was effectively decreased (the right side of Figure 4).

[0063] Example 3. Confirmation of the Effect on Energy Metabolism in High-Fat Diet Mice To confirm the effect of the compound of Formula 1 on suppressing metabolic diseases through animal experiments, after mice were adapted for 2 weeks, obesity was induced by ingestion of a 60% high-fat diet (HFD), and a vehicle, the compound of Formula 1 (D6) at 5 mg / kg / day, or WY14643 was orally administered for 13 weeks. At the 10th week of drug administration, experiments such as IP-GTT, IP-ITT, and micro-CT were conducted (Figure 5). As a result of the experiment, in high-fat diet mice, when the compound of Formula 1 was administered (HFD-D6), there was no significant change in the diet intake compared to the control group (HFD-V), but the body weight gain rate was significantly decreased (Figure 6).

[0064] Furthermore, as a result of micro-CT imaging and quantification of high-fat diet-fed mice, the volume of adipose tissue in the mice administered with the compound of Formula 1 was significantly decreased compared to the control group (Figs. 7 and 8). In addition, as a result of measuring the weights of the organs of high-fat diet-fed mice, in the mice (HFD-D6) administered with the compound of Formula 1, the weights of epididymal white adipose tissue (eWAT) and the liver were decreased compared to the mice (HFD-V) not administered with the drug (Fig. 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 conducted. As a result of the experiment, in the high-fat diet-fed mice (HFD-D6) administered with the compound of Formula 1, glucose tolerance and insulin tolerance were improved, and it was confirmed that there was an improvement effect on metabolic diseases such as diabetes-related symptoms (Figs. 10 and 11). As a result of the analysis of mouse serum components, in the mice administered with the compound of Formula 1, blood glucose and blood lipids (LDLC) were significantly decreased, and it was confirmed that there was an improvement effect on metabolic diseases such as diabetes and dyslipidemia (Figs. 12 and 13).

[0066] Example 4. Histological Observation of Adipose Tissue in High-Fat Diet Mice To observe the effect of the compound of Formula 1 on metabolic diseases, a histological examination of adipose tissue in high-fat diet-fed mice was performed. As a result of H&E staining of mouse adipose tissue, in the high-fat diet-fed mice (HFD-D6) administered with the compound of Formula 1, the size of adipocytes was decreased (Fig. 14). As a result of quantitatively measuring the size distribution of adipocytes, in inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT) and brown adipose tissue (BAT), the effect of decreasing the size of adipocytes was confirmed (Fig. 15).

[0067] Example 5. Histological Observation of Liver Tissue in High-Fat Diet Mice After administration of the compound of Formula 1, to evaluate the effect on fatty liver-related symptoms, H&E, Sirius Red and Oil Red O staining were performed on the liver tissues of mice fed a high-fat diet for 13 weeks. As a result of the experiment, in the liver tissues of high-fat diet mice (HFD-V), fat accumulation and ballooning of cells (arrow) were observed, and in high-fat diet mice administered with the compound of Formula 1 (HFD-D6), histological findings of fatty liver-related diseases decreased (Figure 16).

[0068] More specifically, as a result of evaluating the NAFLD activity score through H&E images of liver tissues, it was confirmed that high-fat diet mice administered with the compound of Formula 1 (HFD-D6) showed a better improvement effect on NAFLD disease indicators than high-fat diet mice administered with WY14643, which is a positive control group (HFD-WY) (Figure 17). Also, as a result of quantitatively evaluating the level of lipid accumulation in the liver through Oil Red O, it was confirmed that in high-fat diet mice administered with the compound of Formula 1 (HFD-D6), the level of lipid droplet accumulation decreased (Figure 18).

[0069] From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical idea and essential features. In this regard, the embodiments described above should be understood as being exemplary in all respects and not restrictive. The scope of the present invention should be construed to include all changes or modified forms derived from the meaning and scope of the following claims and their equivalent concepts, rather than the above detailed description.

Claims

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

5. The composition according to claim 4, wherein the improvement of metabolic health is one or more selected from the group consisting of improvement of obesity, reduction of blood glucose, 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. 【Chemical 1】

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