Pharmaceutical composition for preventing or treating metabolic diseases containing bone morphogenetic protein 10 as an active ingredient

Abstract

The present invention relates to a composition for preventing or treating metabolic diseases, which contains bone morphogenetic protein 10 (BMP10) as an active ingredient. It has been confirmed that BMP10-treated mouse embryonic mesenchymal cell line C3H10T1 / T2 cells promoted brown fat differentiation, and increased brown fat differentiation of stromal vascular fraction adipose stem cells isolated from subcutaneous adipose tissue. It has also been confirmed that BMP10-treated animal models of obesity induced weight loss, improved insulin resistance, and changes in blood lipid levels resulted. Therefore, the composition containing BMP10 as an active ingredient can be used as a preventive or therapeutic agent for metabolic diseases, including obesity, diabetes, and dyslipidemia.

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing or treating metabolic diseases, which contains bone morphogenetic protein 10 as an active ingredient. [Background technology]

[0002] Obesity is caused by an imbalance between energy intake and expenditure, and excess energy is converted into fat cells and stored in the body. Free fatty acids and cytokines secreted from accumulated fat cells induce insulin resistance and increase inflammatory responses, directly contributing to the onset of chronic diseases such as metabolic syndrome, diabetes, cardiovascular disease, and cancer.

[0003] To treat this type of obesity, various treatments have been introduced, including exercise, improving eating habits through dietary therapy, drug therapy, and surgery. As for the development of anti-obesity drugs to suppress double obesity, more than 100 types of drugs are on the market or in development in the United States, and the market size is expected to gradually expand.

[0004] Currently, obesity treatments can be broadly divided into drugs that affect appetite by acting on the central nervous system, and drugs that act on the gastrointestinal tract to inhibit absorption. Drugs that act on the central nervous system include drugs such as fenfluramine and dexfenfluramine, which inhibit the serotonin (5HT) nervous system through their respective mechanisms, drugs such as ephedrine and caffeine, which act through the noradrenergic nervous system, and more recently, drugs such as sibutramine, which inhibits obesity by simultaneously acting on the serotonin and noradrenergic nervous systems.

[0005] However, among the drugs currently in use, fenfluramine has recently been banned due to side effects such as primary pulmonary hypertension and cardiac valvular lesions, sibutramine has the side effect of increasing blood pressure, and orlistat has been reported to cause side effects such as gastrointestinal disorders, steatorrhea, fecal incontinence, and interference with the absorption of fat-soluble vitamins. Other synthetic chemical drugs also cause problems such as decreased blood pressure and lactic acidemia, making them unsuitable for use in patients with heart failure or kidney disease.

[0006] Since the 2000s, it has been reported that proteins belonging to the BMP protein family (BMP2, BMP4, BMP6, BMP7, and BMP9) have functions in adipose tissue differentiation in addition to bone formation. In particular, BMP8 and BMP9 have been reported to promote brown fat differentiation and increase energy metabolism through brown fat activation. However, several studies and reviews have investigated the bone differentiation-inducing functions of the 14 members of the BMP protein family. BMP2, BMP4, BMP6, BMP7, and BMP9 proteins have been shown to significantly increase alkaline phosphatase activity, which is important for bone differentiation, and animal experiments have also confirmed that these proteins promote bone formation. However, these studies have shown that BMP10 fails to increase alkaline phosphatase activity, and bone formation has not been observed in animal experiments. These results suggest that various BMP proteins, excluding BMP10, can promote brown fat differentiation, but at the same time promote bone differentiation, which may act as a side effect in the development of obesity and diabetes treatments.In fact, while it is easy to find papers and patents on the development of bone formation-promoting treatments using BMP2 and BMP7, as well as examples of commercial development, no examples have been found for obesity treatments using these proteins. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides a composition containing bone morphogenetic protein 10 as an active ingredient for preventing or treating metabolic diseases such as obesity, diabetes, and dyslipidemia. [Means for solving the problem]

[0008] The present invention provides a pharmaceutical composition for preventing or treating metabolic diseases, which contains bone morphogenetic protein 10 (BMP10) as an active ingredient.

[0009] The present invention also provides a health food for preventing or ameliorating metabolic diseases, which contains bone morphogenetic protein 10 (BMP10) as an active ingredient. [Effects of the Invention]

[0010] According to the present invention, it has been confirmed that brown adipose differentiation of mouse embryonic mesenchymal cell line C3H10T1 / T2 cells treated with bone morphogenetic protein 10 (BMP10) is promoted, and that brown adipose differentiation of stromal vascular fraction adipose stem cells isolated from subcutaneous adipose tissue is increased. Furthermore, weight loss, improvement in insulin resistance, and changes in blood lipid levels have been confirmed in an obese animal model induced by a high-fat diet. Therefore, a composition containing BMP10 as an active ingredient can be provided as a preventive or therapeutic agent for metabolic diseases including obesity, diabetes, and dyslipidemia. [Brief explanation of the drawings]

[0011] [Figure 1] This is an experimental process conducted to confirm the brown adipocyte differentiation ability of bone morphogenetic protein 10 (BMP10). [Figure 2] The results show that the degree of brown adipocyte differentiation was confirmed in cells 4 and 8 days after differentiation induction after BMP10 treatment of C3H10T1 / T2 cells, a mouse embryonic mesenchymal cell line in which adipocyte differentiation was induced. [Figure 3]These are Western blot results of examining the expression levels of brown fat markers in C3H10T1 / T2 cells, a mouse embryonic mesenchymal cell line induced to differentiate into adipocytes, treated with BMP7, BMP9, BMP10, and BMP11 on days 4 and 8 of differentiation induction. [Figure 4] The results were obtained by treating adipose stem cells from the stromal vascular fraction (SVF) isolated from mouse subcutaneous fat with BMP10, and then confirming the effect of BMP10 on brown adipose tissue transformation on the fourth day after differentiation induction. [Figure 5] This is the result of Western blot analysis of the expression levels of brown fat markers in adipose stem cells from the stromal vascular fraction (SVF) isolated from mouse subcutaneous fat, treated with BMP9 and BMP10, and then 4 days after differentiation induction. [Figure 6] These are the results of examining BMP10 expression levels in the heart and blood of mice that had been exercising for four weeks. [Figure 7] The results confirmed the effect of recombinant BMP10 in improving metabolic diseases in a high-fat diet-induced obesity model. Mice were fed a high-fat diet for 6 weeks, and recombinant BMP10 was administered intraperitoneally (1.0 mg / kg, ip, qd) once a week for 6 weeks. Changes in body weight, improvement of diabetes, and changes in blood lipids were then confirmed. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will now be described in more detail.

[0013] Bone morphogenetic protein 10 (BMP10) is a protein expressed in the heart and is known to play an important role in cardiac development during development. Its blood concentration rapidly decreases after birth and is hardly expressed in adulthood. The present inventors have confirmed that BMP10 has a promoting effect on adipose tissue differentiation, particularly brown adipose differentiation and browning of white adipose tissue, and have completed the present invention.

[0014] The present invention can provide a pharmaceutical composition for preventing or treating metabolic diseases, which contains bone morphogenetic protein 10 (BMP10) as an active ingredient.

[0015] The bone morphogenetic protein 10 (BMP10) is NCBI Entrez Gene number 27302.

[0016] The bone morphogenetic protein 10 induces brown adipocyte differentiation.

[0017] The bone morphogenetic protein 10 induces the transformation of white fat into brown fat.

[0018] Furthermore, the bone morphogenetic protein 10 increases the expression of Ucp1, a marker for brown adipose tissue formation.

[0019] The metabolic disease is selected from the group consisting of obesity, diabetes and dyslipidemia.

[0020] Brown fats are brown in color and are distinct from white adipose tissue, which is generally stored fat. They are composed of cells rich in mitochondria and remnants, have many sympathetic nerve fibers, and are highly metabolically active, especially in lipolysis and fatty acid oxidation.

[0021] In addition, brown adipose tissue transformation, in which white adipose tissue transforms into a tissue similar to brown adipose tissue, has been reported to increase energy consumption. Exercise methods and drugs that can induce brown adipose tissue differentiation and brown adipose tissue transformation are attracting attention as a treatment for metabolic diseases such as obesity and diabetes by increasing energy consumption.

[0022] The present invention is a technology that has confirmed that bone morphogenetic protein 10 (BMP10) can effectively induce brown adipocyte differentiation ability and brown adipose tissue transformation of white fat. According to one embodiment of the present invention, mouse embryonic mesenchymal stem cells that had been induced to differentiate into adipocytes were treated with BMP10 to confirm their brown adipocyte differentiation ability. As a result, as shown in Figure 2, an increase in cells differentiated into brown adipocytes was confirmed from day 4 after adipocyte differentiation was induced in the BMP10-treated cell group, and as shown in Figure 3, it was confirmed that the expression of Ucp1, a major marker of brown fat, was significantly increased in the BMP10-treated cells.

[0023] In another embodiment of the present invention, adipose stem cells were isolated from the stromal vascular fraction (SVF) of a mouse's subcutaneous fat, and the isolated adipose stem cells were treated with BMP10 to confirm the effect of BMP10 on inducing brown adipose formation. As a result, as shown in Figure 4, it was confirmed that brown adipose formation was increased in adipose stem cells derived from the stromal vascular fraction treated with BMP10 compared to the control group.

[0024] From the above results, a composition containing bone morphogenetic protein 10 (BMP10) as an active ingredient can induce brown adipocyte differentiation and brown adipose tissue transformation of white fat, converting it into brown fat with excellent lipolysis and fatty acid oxidation effects. Since effective fat reduction is induced through this brown adipose tissue transformation, BMP10 can be used as a composition for preventing or treating metabolic diseases.

[0025] The pharmaceutical composition is contained in an amount of 0.1 to 90 parts by weight per 100 parts by weight of the total pharmaceutical composition.

[0026] In one embodiment of the present invention, the pharmaceutical composition for preventing or treating metabolic diseases containing the bone morphogenetic protein 10 as an active ingredient can be prepared in any one of the dosage forms selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, infusions, and liquids by conventional methods.

[0027] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating obesity containing bone morphogenetic protein 10 as an active ingredient may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, leavening agents, lubricants, flavoring agents, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders, and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0028] Specifically, carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid formulations can be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Oral liquid formulations include suspensions, oral solutions, emulsions, syrups, etc., and may contain various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, in addition to the commonly used simple diluents of water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases include witepsol, macrogol, Tween 61, cocoa butter, lauric butter, and glycerogelatin.

[0029] According to one embodiment of the present invention, the pharmaceutical composition may be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes.

[0030] The desired dosage of the bone morphogenetic protein 10 varies depending on the condition and weight of the subject, the type and severity of the disease, the drug form, the route and duration of administration, and can be appropriately selected by those skilled in the art. According to one embodiment of the present invention, the daily dosage is, but is not limited to, 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, more specifically 0.1 to 100 mg / kg. The dosage may be administered once a day or in divided doses, and the scope of the present invention is not limited thereto.

[0031] In the present invention, the "subject" is a mammal, including a human, but is not limited to these examples.

[0032] The present invention can provide a health food for preventing or ameliorating metabolic diseases, which contains bone morphogenetic protein 10 (BMP10) as an active ingredient.

[0033] The health food is used in conjunction with other foods or food additives in addition to the osteogenic protein 10 and is used appropriately in a conventional manner. The amount of the active ingredient to be mixed is determined appropriately depending on the purpose of use, for example, prevention, health, or therapeutic treatment.

[0034] The effective dose of the compound contained in the health food can be used in accordance with the effective dose of the therapeutic agent, but in the case of long-term intake for the purpose of health and hygiene or health regulation, it is below the above range, and it is certain that the active ingredient can be used in an amount greater than the above range because there is no safety issue.

[0035] The types of the health foods are not particularly limited, and examples include meats, sausages, bread, chocolates, candies, snacks, sweets, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes.

[0036] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[0037] Example 1: Induction of brown fat differentiation by BMP protein

[0038] To evaluate the brown fat differentiation potential of proteins belonging to the BMP group, including BMP10, we evaluated their brown fat cell differentiation potential using C3H10T1 / T2 cells, a mouse embryonic mesenchymal cell line, or stem cells from the stromal vascular fraction isolated from subcutaneous adipose tissue, using the process shown in Figure 1.

[0039] BMP treatment was initiated 3 days before induction of brown fat differentiation and continued until the end of differentiation. To induce brown fat differentiation, cells were treated with a brown fat differentiation cocktail consisting of 20 nM insulin, 1 nM T3, 5 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 0.125 μM indomethacin, and 1 μM rosiglitazone from day 0 (D0) to 8 days. The degree of differentiation and the expression of brown fat markers at each stage were monitored by sampling on days 4 (D4) and 8 (D8) after differentiation induction.

[0040] Example 2: Confirmation of the promoting effect of BMP10 on brown adipocyte differentiation

[0041] To confirm the ability of BMP10 to induce brown adipocyte differentiation, the cells induced to differentiate as in Example 1 on days 4 and 8 were stained with Oil Red O to confirm the state of differentiation into brown adipocytes.

[0042] As a result, as shown in Figure 2, it was confirmed that the BMP10-treated cell group differentiated into brown fat cells more rapidly from day 4 of differentiation induction compared to the control group, and even at the final stage, day 8, brown fat differentiation was promoted in the BMP10-treated cell group compared to the control group.

[0043] These results confirmed that BMP10 promotes brown adipocyte differentiation from the early stage and exhibits brown adipocyte differentiation potential.

[0044] Example 3: Confirmation of induction of adipogenesis marker expression by BMP10

[0045] To further confirm the ability of BMP10 to promote brown fat differentiation, as confirmed in previous experiments, changes in the expression of brown fat differentiation markers were confirmed by Western blotting.

[0046] Total proteins of cells on day 4 and day 8 after brown adipose differentiation was induced as in Example 1 were separated, and changes in expression levels were confirmed using antibodies against each marker.

[0047] As a result, as shown in Figure 3, we were able to confirm that the expression of each marker increased in BMP10-treated cells compared to the control group from day 4. In particular, we were able to confirm that the expression of Ucp1, a major marker of brown fat, increased only in BMP10-treated cells.

[0048] These results confirmed that BMP10 effectively induces differentiation into brown adipocytes.

[0049] Example 4: Confirmation of the brown adipose tissue transformation effect of BMP10 in stromal vascular fraction isolated from subcutaneous adipose tissue

[0050] Recently, it has been reported that brown adipose tissue transformation, in which white adipose tissue transforms into a tissue similar to brown adipose tissue during exercise or in a low-temperature environment, increases energy consumption. To confirm whether the brown adipose tissue differentiation-inducing ability of BMP10 confirmed in previous experiments could also be used in the treatment of metabolic diseases, adipose stem cells were isolated from the stromal vascular fraction (SVF) of mouse subcutaneous fat and the brown adipose tissue-inducing effect of BMP10 was confirmed using these adipose stem cells.

[0051] Brown fat differentiation was induced through the process shown in Figure 1. Previous experiments confirmed that BMP10 increased brown fat differentiation from day 4, so we compared the brown fat differentiation induced by BMP10 with that of the control group.

[0052] As a result, as shown in Figure 4, it was confirmed that brown adipose tissue transformation of SVF-derived adipose stem cells on day 4 was promoted by BMP10 compared to the control group.

[0053] These results confirmed that BMP10 not only promotes brown fat differentiation but also promotes the brown fat conversion of white adipocytes.

[0054] <Example 5> Confirmation of the expression level of BMP10-induced brown adipose tissue markers in the stromal vascular fraction isolated from subcutaneous adipose tissue

[0055] To further confirm the brown adipose tissue induction ability of BMP10 confirmed in previous experiments, BMP9 and BMP10 were treated with stromal vascular fraction isolated from subcutaneous adipose tissue, and changes in the expression levels of brown adipose tissue markers were confirmed by real-time quantitative PCR.

[0056] As a result, as shown in Figure 5, it was confirmed that the expression of brown adipose tissue markers was significantly increased in the experimental group treated with BMP10 compared to the control group. In particular, it was confirmed that the expression of Ucp1, a key marker of brown adipose tissue, was significantly increased with BMP10 compared to BMP9.

[0057] These results confirmed that the effect of BMP10 on brown adipose tissue formation was superior to that of other BMP groups.

[0058] <Example 6> Confirmation of BMP10 expression level in mice subjected to 4 weeks of exercise

[0059] According to a recent report, irisin, secreted from muscles during exercise, promotes the transformation of white adipose tissue into brown adipose tissue. Based on this, we investigated whether the expression and secretion of BMP10 in the heart also increases in an environment that promotes the transformation of white adipose tissue into brown adipose tissue.

[0060] To this end, changes in BMP10 expression were confirmed in the heart and blood of 57Bl / 6J mice (12 weeks old, male) that underwent a 4-week endurance test using treadmill exercise, using real-time quantitative PCR and ELISA, respectively.

[0061] As a result, as shown in Figure 6, BMP10 mRNA expression increased in the hearts of mice that had exercised for four weeks, and an increase in BMP10 protein was also confirmed in the blood of the mice.

[0062] These results confirm that BMP10, which increases in blood during exercise, is secreted into the blood and promotes brown fat differentiation and brown adipose tissue formation.

[0063] <Example 7> Confirmation of the metabolic disease-ameliorating effect of recombinant BMP10 in a high-fat diet-induced obesity model

[0064] To confirm the efficacy of the promotion of brown fat differentiation and brown fat conversion of white fat confirmed in previous experiments, and the increase in BMP10 in the heart and blood due to exercise, in improving metabolic diseases, we confirmed the efficacy of improving obesity and diabetes in a high-fat diet-induced obese mouse model.

[0065] Mice were fed a high-fat diet for 6 weeks, and recombinant BMP10 was administered intraperitoneally (1.0 mg / kg, ip, qd) once a week for 6 weeks, and changes in body weight were monitored.

[0066] As a result, as shown in Figure 7, a tendency for a decrease in total body fat was observed after 6 weeks of recombinant BMP10 administration, and the increase in total body fat compared to before administration was also lower than in the control group. However, no change in food intake was observed during the recombinant BMP10 administration period compared to the control group.

[0067] Furthermore, the diabetes-improving efficacy of BMP10 was confirmed to significantly lower fasting blood glucose levels. A glucose tolerance test showed no difference in glucose tolerance between the recombinant BMP10-administered group and the control group, but the group secreted significantly less insulin than the control group, confirming that BMP10 has the effect of improving insulin resistance.

[0068] Finally, changes in blood lipids after six weeks of administration were examined, and it was found that blood triglycerides showed a tendency to decrease, and total cholesterol was significantly reduced.

[0069] These results confirm that BMP10 is effective in improving obesity, diabetes, and dyslipidemia.

[0070] Although the present invention has been described in detail above, it is obvious to those skilled in the art that the specific details are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the claims and their equivalents.

Claims

1. A pharmaceutical composition for preventing or treating a metabolic disease, comprising bone morphogenetic protein 10 (BMP10) as an active ingredient, A pharmaceutical composition for preventing or treating a metabolic disease, wherein the metabolic disease is selected from the group consisting of obesity, diabetes, and dyslipidemia.

2. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the bone morphogenetic protein 10 induces brown adipocyte differentiation.

3. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the bone morphogenetic protein 10 induces brown fat conversion from white fat.

4. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the bone morphogenetic protein 10 increases the expression of Ucp1, a marker for brown adipose tissue formation.

5. The pharmaceutical composition for preventing or treating metabolic diseases according to claim 1, wherein the pharmaceutical composition is contained in an amount of 0.1 to 90 parts by weight per 100 parts by weight of the total pharmaceutical composition.

6. A health food for preventing or improving metabolic diseases, comprising bone morphogenetic protein 10 (BMP10) as an active ingredient, A health food for preventing or improving a metabolic disease, wherein the metabolic disease is selected from the group consisting of obesity, diabetes, and dyslipidemia.

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