Novel Peptide and Its Use

Abstract

The present invention relates to a peptide derived from IF1 protein and its pharmaceutical use. The peptide suppresses appetite, inhibits lipid accumulation and differentiation of adipocytes in obese animal models, and promotes insulin secretion and improves glucose tolerance in diabetic animal models, and can therefore be useful for anti-obesity and anti-diabetes applications.

Description

Technical Field

[0001] The present invention relates to a novel peptide derived from a biological protein and its use for anti-obesity and anti-diabetes.

Background Art

[0002] Pharmaceuticals used for the treatment and prevention of diseases are classified into pharmaceuticals composed of chemical substances and biopharmaceuticals. Biopharmaceuticals mainly consist of protein pharmaceuticals composed of hormones, antibodies, etc., and are further composed of genetic substances, etc. Different from pharmaceuticals centered on chemical substances composed of low-molecular substances, since biopharmaceuticals are mostly high-molecular substances, various problems starting from their size occur. Typically, there are problems from the perspective of activity such as a decrease in accessibility to target substances according to the large size and autoimmune reactions, and industrial problems such as an increase in the complexity involved in production as the size increases, and the resulting increase in cost and time required. This is a phenomenon that occurs because, in addition to the active site that mainly affects the function of the protein, accessory structures exist as components. If the main active site of the protein is detected, the in-vivo function can be maintained while reducing the size of the protein pharmaceutical, and thus considerable improvements can be achieved from the perspectives of various side effects and costs of the protein pharmaceutical.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Under the above circumstances, the present inventors focused on the invention of a peptide derived from the IF1 (ATPase inhibitory factor 1) protein for improving the function of the IF1 protein expressed in vivo. As a result, a novel IF1-derived peptide showing an effect of improving metabolic disorders including obesity and glucose metabolism was discovered.

[0004] Therefore, an object of the present invention is to provide the novel peptide and its pharmaceutical use.

Means for Solving the Problems

[0005] To achieve the above object, one aspect of the present invention provides a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 2 derived from a biological protein.

[0006] IF1 (ATPase inhibitory factor 1) is known as a major protein that is expressed intracellularly, binds to ATPase present in the mitochondrial membrane, interferes with the rotational movement, and as a result, interferes with ATP synthesis / decomposition by the electron transport system, affecting intracellular energy regulation and mitochondrial homeostasis.

[0007] The present inventors conducted research to search for a novel functional peptide from the IF1 protein (SEQ ID NOs: 3 and 4), and discovered the peptides of SEQ ID NOs: 1 and 2 having anti-obesity and anti-diabetic activities.

[0008] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating obesity containing the peptide as an active ingredient.

[0009] The term "prevention" used in the present invention means all acts of suppressing the progression of a disease or delaying its onset by administering the pharmaceutical composition according to the present invention.

[0010] The term "treatment" used in the present invention means all acts of improving the symptoms of a disease or changing them beneficially by administering the pharmaceutical composition according to the present invention.

[0011] The present inventors administered a high-fat diet and a functional peptide together to mice (obesity prevention experiment) or administered a high-fat diet and a functional peptide together to mice induced with obesity (obesity treatment experiment) in order to confirm the physiological activity of a peptide derived from the IF1 protein (hereinafter referred to as a functional peptide).

[0012] When the administration results showed that co - administration of a high - fat diet and a functional peptide to mice suppressed weight gain compared to the control group (Figure 1C), and co - administration of a high - fat diet and a functional peptide to obese - induced mice suppressed weight gain and even led to weight loss at the end of the experiment (Figure 1A and 1B).

[0013] Therefore, the said composition can be usefully used for both the prevention and treatment of obesity.

[0014] In addition, the functional peptide suppresses lipid accumulation and differentiation of adipocytes, which is achieved by suppressing the expression of genes of PPARγ (peroxisome proliferator - activated receptor gamma), adiponectin, FABP4 (fatty acid - binding protein 4), C / EBPα (CCAAT / enhancer - binding protein alpha) and LPL (lipoprotein lipase), which are indicators of adipocyte differentiation in adipocytes.

[0015] Also, the functional peptide can suppress appetite, which can be achieved by suppressing the expression of AgRP (agouti - related protein) and NPY (neuropeptide Y), which are peptides that promote appetite in hypothalamic cells.

[0016] Therefore, the composition for preventing or treating obesity can exhibit an anti - obesity effect through suppression of adipocyte differentiation and suppression of appetite.

[0017] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating diabetes or diabetic complications, containing the functional peptide as an active ingredient.

[0018] In order to further confirm the physiological activity of the functional peptide, the present inventors fasted obese-induced mice and type 2 diabetic mice, and then injected a glucose solution to conduct a glucose tolerance test. As a result, it was confirmed that administration of the functional peptide decreased blood glucose levels (Figs. 4 and 5A, 5B) and increased insulin secretion (Fig. 5E).

[0019] Therefore, the composition for preventing or treating diabetes can exhibit an anti-diabetic effect through improving glucose tolerance and promoting insulin secretion.

[0020] Diabetic complications refer to complications such as myocardial infarction, stroke, retinopathy, and renal failure that appear due to damage to the blood vessel wall caused by a long-term hyperglycemic state. Since improvement of the hyperglycemic state can improve diabetic complications, the functional peptide of the present invention can be used for the prevention or treatment of diabetic complications.

[0021] In addition, the pharmaceutical composition of the present invention can be produced in a form further containing a suitable carrier, excipient, or diluent usually used in the production of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier.

[0022] Specifically, the pharmaceutical composition can be formulated and used in the form of an oral dosage form such as a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, etc., an external preparation, a suppository, and a sterile injection solution by ordinary methods.

[0023] In the present invention, carriers, excipients and diluents that may be included in the pharmaceutical composition 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. When formulating, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are commonly used.

[0024] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0025] Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used.

[0026] The pharmaceutical composition is a sterile aqueous or oily suspension and can be in the form of a sterile injectable preparation. This suspension can be formulated by techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension (such as a solution in 1,3 - butanediol) in a non - toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Also, sterile non - volatile oils are usually used as solvents or suspending media. For such purposes, any non - volatile oil containing synthetic mono - or diglycerides and having low irritation can be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in injectable preparations, as are pharmaceutically acceptable natural oils (such as olive oil or castor oil), especially their polyoxyethylated counterparts.

[0027] Parenteral administration of the pharmaceutical composition according to the present invention is particularly useful when the intended treatment is related to a site or organ where local application is accessible and the treatment is easily achievable. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water.

[0028] The content of the active ingredient contained in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be contained in an amount of 0.0001 - 50% by weight, more preferably 0.01 - 10% by weight, based on the total weight of the final composition.

[0029] Another aspect of the present invention provides a method for treating obesity, which includes administering to an individual in need of treatment the pharmaceutical composition for preventing or treating obesity, and a method for treating diabetes or diabetic complications, which includes administering to an individual in need of treatment the pharmaceutical composition for preventing or treating diabetes or diabetic complications.

[0030] Since the treatment method of the present invention uses the above-described pharmaceutical composition, the content common to both is omitted from the description in order to avoid excessive complexity of this specification.

[0031] As described above, the pharmaceutical composition can be administered orally or parenterally, and the dosage of the pharmaceutical composition can be determined by a medical professional.

Effects of the Invention

[0032] The present invention relates to a peptide derived from a biological protein and its pharmaceutical use. Since the peptide suppresses appetite in an obese animal model, inhibits lipid accumulation and differentiation of adipocytes, promotes insulin secretion in a diabetic animal model, and improves glucose tolerance, it can be usefully used for anti-obesity and anti-diabetic applications.

Brief Description of the Drawings

[0033]

Figure 1

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Mode for Carrying Out the Invention

[0034] Hereinafter, one or more specific examples will be described in more detail based on the examples. However, these examples are for illustrative purposes of one or more specific examples, and the scope of the present invention is not limited to these examples.

[0035] Example 1: Isolation and Purification of Peptide Derived from IF1 The coding sequences of the IF1 protein and the IF1-derived peptide (hereinafter referred to as the functional peptide; SEQ ID NO: 1 or 2) were inserted into the pGEX-4T-1 and pet28a vectors, respectively. The vectors were transformed into the BL21(DE3) strain of Escherichia coli for expression, and a cell model that stably expresses IF1 and the functional peptide was established. After culturing the E. coli cells, 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) was treated to induce protein expression. Then, the solution obtained through centrifugation and sonication was separated by affinity chromatography to separate IF1 and the functional peptide, and purified by adjusting the salt concentration with PBS solution at pH 7.4 using Snakeskin dialysis tubing to increase the purity. GST used as the control group was produced using the pGEX-4T-1 vector instead of the pet28a vector.

[0036] The sequences of the IF1 protein and the functional peptide are shown in Table 1 below.

[0037]

Table 1

[0038] Example 2: Analysis of the anti-obesity effect of the functional peptide 2-1. Production of an obese mouse model Four-week-old male C57BL / 6J mice were purchased from ORIENT BIO and allowed to adapt for 1 week by providing a normal diet (Normal diet chow; ND). During this period, the temperature of the breeding space was maintained at 18 - 24 °C and the humidity at 50 - 60% throughout the experimental period, and free feeding was implemented during both the adaptation period and the experimental period. The mice that had undergone the adaptation period were divided into an obesity prevention experimental group and an obesity treatment experimental group and bred according to their respective purposes. The normal diet and the high-fat diet were manufactured and provided as shown in Table 2 below.

[0039]

Table 2

[0040] -Obesity prevention experimental group: Divided into a control group and an experimental group, and raised for 30 days under the following conditions respectively. Control group: High-fat diet (HFD) + PBS administration (n = 10) Experimental group: High-fat diet (HFD) + Functional peptide administration (n = 10) -Obesity treatment experimental group: After a 1-week adaptation period, a high-fat diet (see Table 1) was provided for 8 weeks to induce obesity, and then divided into a control group and an experimental group, and raised for 6 weeks under the following conditions respectively. Control group: High-fat diet (HFD) + PBS administration (n = 10) Experimental group: High-fat diet (HFD) + Functional peptide administration (n = 10)

[0041] 2-2. Anti-obesity effect of functional peptide During the experimental period, PBS and the peptide were administered once a day through intraperitoneal injection according to the experimental conditions of Example 2-1. The corresponding conditions are as follows. Control group: Intraperitoneal injection of PBS (volume corresponding to the following functional peptide solution) Experimental group: Intraperitoneal injection of functional peptide (5 mg / kg)

[0042] During the experimental period, the body weights of all mice were measured, and the weight changes due to the intake of the high-fat diet were observed. As a result, it was confirmed that the weight gain inhibitory effect appeared from the time when one week had passed after the peptide was administered in the functional peptide administration group of the obesity treatment experimental group, and such a weight gain inhibitory phenomenon continued until the end of the experiment (Figure 1A). As a result of comparing the body weights at the end point of the final experiment, the control group was rapidly induced to obesity by weight gain due to the high-fat diet, while the functional peptide administration group showed a weight loss effect instead (Figure 1B).

[0043] Even in the obesity prevention experimental group, during the entire experimental period, the weight change rate in the functional peptide administration group showed a low level (Figure 1C). It was confirmed that the food efficiency ratio (the ratio of weight gain to food intake, Food Efficiency Ratio) (Figure 1D) and the daily food intake also decreased in the functional peptide administration group (Figure 1E), thus confirming the effective anti-obesity efficacy of the functional peptide.

[0044] After the experiment, the mice in the obesity prevention and obesity treatment experimental groups were fasted for 12 hours, tissues were separated and stored, and serum was separated. As a result of analyzing the serum lipid index, a decrease in free fatty acids (Figure 2A) and triglycerides (Figure 2B) was confirmed in the functional peptide administration group compared to the control group.

[0045] Also, as a result of observing adipose tissue by H&E (Hematoxylin and Eosin) staining and immunostaining, it was confirmed that the area occupied by adipocytes decreased by 15% due to the administration of the functional peptide, indicating that the functional peptide prevents lipid accumulation (Figure 2C). The expression level of UCP-1 (uncoupling protein-1), which is a thermogenesis and energy homeostasis active factor in adipose tissue, also increased by 355% in the functional peptide administration group (Figure 2D).

[0046] As a result of observing through immunostaining of adipose tissue obtained from the obesity treatment experimental group, similar to the results of the obesity prevention experimental group, it was observed that the expression level of UCP-1 increased by 263% (Figure 2E), confirming that the anti-obesity effect of the functional peptide includes lipid accumulation and activation of energy metabolism.

[0047] 2-3. Analysis of the mechanism of action of the anti-obesity effect of the functional peptide To differentiate 3T3-L1 adipocytes into fat, cells were cultured for a total of 8 days, including 2 days in a primary differentiation medium (DMEM, 10% FBS (Fetal Bovine Serum), 1% Antibiotics, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 10 μg / ml Insulin) and 6 days in a secondary differentiation medium (DMEM, 10% FBS, 1% Antibiotics, 10 μg / ml Insulin) to induce differentiation. At this time, each substance (control group: PBS, experimental group: 100 nM of the functional peptide) was added to the medium during the differentiation period for use.

[0048] As a result of this experiment, it was confirmed that the gene levels of PPARγ (peroxisome proliferator-activated receptor gamma), adiponectin, FABP4 (fatty acid-binding protein 4), C / EBPα (CCAAT / enhancer-binding protein alpha), and LPL (lipoprotein lipase), which are indicators of adipogenesis, all decreased in the functional peptide-treated group, indicating that the functional peptide regulates adipogenesis and exhibits an anti-obesity effect (Figure 3A).

[0049] In addition, to confirm the appetite-regulating effect of the functional peptide in mHypoE-N41 hypothalamic cells, hypothalamic cells were treated with PBS (control group) and 100 nM of a mouse-derived functional peptide (experimental group) for 24 hours. After the experiment, the cells were collected and the gene levels of Agrp (agouti-related peptide) and NPY (neuropeptide Y), which are known to promote appetite, were observed. The results showed that the Agrp gene decreased by 50.2% and the NPY gene decreased by 48.8% compared to the control group, confirming that the functional peptide has an appetite-suppressing effect (Figure 3B).

[0050] Similarly, the hypothalamic cells were treated with PBS (control group) and 100 nM of human-derived functional peptide (experimental group) for 24 hours, and the gene levels of appetite-related factors were observed. As a result, the Agrp gene decreased by 47.5% compared to the control group, and the NPY gene decreased by 59.6%, confirming the appetite-suppressing effect of the human peptide (Figure 3C).

[0051] Through the results of this example, the anti-obesity effect of the functional peptide was finally verified.

[0052] Example 3: Analysis of the effects of functional peptides related to glucose metabolism 3-1. Breeding of type 2 diabetic mouse (db / db) model To confirm the effects of functional peptides related to glucose metabolism, type 2 diabetic mice (db / db) were used. The db / db model corresponding to type 2 diabetic mice is an animal model in which a gene mutation is induced in the leptin receptor responsible for diet regulation, and it is a representative model of diabetes in which the insulin signaling pathway and satiety regulation are impossible and obesity is induced.

[0053] Male C57BLKS / 6J-db / m+ (Hetero mutation) and C57BLKS / 6J-db / db (Homo mutation) mice at 8 weeks of age were purchased from Central Lab and provided with a normal diet (Normal diet chow; ND) for 1 week to allow for adaptation. During this period, the temperature of the breeding space was maintained at 18 - 24 °C and the humidity was maintained at 50 - 60% throughout the experimental period, and free feeding was implemented during both the adaptation period and the experimental period.

[0054] 3-2. Analysis of the glucose metabolism improvement effect of functional peptides The mice that had undergone the adaptation period were divided into a control group and an experimental group and bred under the following conditions for 44 days. The normal diet was manufactured and provided as shown in Table 1 above. db / m+ control group: normal diet (ND) + intraperitoneal administration of PBS (n = 10) db / db control group: normal diet (ND) + intraperitoneal administration of PBS (n = 10) db / db experimental group: normal diet (ND) + intraperitoneal administration of functional peptide (5 mg / kg) (n = 10)

[0055] After the 44-day experimental period ended, the mice were fasted for 14 hours, then euthanized, and thereafter dissected to collect tissues for further analysis. Also, blood was collected, serum was separated by centrifugation, and analyzed.

[0056] Specifically, to evaluate the efficacy of the functional peptide in improving glucose metabolism, the effects of improving glucose tolerance in obesity-induced glucose intolerance and gene mutation-induced glucose intolerance were confirmed through a glucose tolerance test.

[0057] First, at the end of the experiment, the obesity-preventing mouse model of Example 2-1 was fasted for 12 hours, and a 20% glucose solution was injected intraperitoneally to observe the change in blood glucose level. At this time, the substance corresponding to each group was injected as in Example 3-1 30 minutes before the injection of the glucose solution. As a result of confirming the change in blood glucose level, it was found that the blood glucose level decreased in the functional peptide-administered group from 30 minutes after glucose injection. As a result of quantifying the overall blood glucose change amount (blood glucose value × time; GLUAUC) as well, a decrease in blood glucose value due to the functional peptide was confirmed (Figure 4), and it was confirmed that the functional peptide has an effect of improving glucose metabolism.

[0058] The diabetic mouse model of Example 3-1 was fasted for 12 hours, and a 20% glucose solution was injected intraperitoneally to observe the change in blood glucose level. At this time, the substance corresponding to each group was injected as in Example 3-1 30 minutes before the injection of the glucose solution. As a result of confirming the change in blood glucose level, it was found that the blood glucose level decreased significantly in the functional peptide-administered group compared to the db / db control group at 60 minutes, 90 minutes, and 120 minutes after glucose injection. Also, as a result of quantifying the overall blood glucose change amount (blood glucose value × time; GLUAUC) as well, a decrease in blood glucose value due to the functional peptide was confirmed (Figure 5B), and it was confirmed that the functional peptide has an effect of improving glucose metabolism.

[0059] To observe the changes and disorders of hormone secretion starting from diabetes, the tissues isolated from the diabetic mouse model of Example 3-1 were analyzed, and the weight of the pancreas, which plays a major role in hormone secretion, and the blood concentration of insulin hormone, which induces sugar absorption, were specifically examined.

[0060] As a result of the examination, it was confirmed that both the weight of the pancreas (Figure 5C) and the value corrected by body weight (Figure 5D) increased in the functional peptide administration group, and it was confirmed that the functional peptide had a positive effect on the hormone secretion system. In addition, it was shown that the blood insulin secretion increased in the functional peptide administration group, and it was found that the hormone secretion was promoted by the functional peptide (Figure 5E). Overall, it was finally verified that the functional peptide had a positive effect on metabolism.

Claims

**Claim 1**: A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2. **Claim 2** A pharmaceutical composition for preventing or treating obesity, comprising the peptide according to Claim 1 as an active ingredient. **Claim 3** The pharmaceutical composition for preventing or treating obesity according to Claim 2, wherein the composition suppresses appetite. **Claim 4** The pharmaceutical composition for preventing or treating obesity according to Claim 3, wherein the appetite suppression is caused by suppression of AgRP (agouti-related protein) or NPY (neuropeptide Y) peptide. **Claim 5** The pharmaceutical composition for preventing or treating obesity according to Claim 2, wherein the composition suppresses lipid accumulation and differentiation of adipocytes. **Claim 6** The pharmaceutical composition for preventing or treating obesity according to Claim 2, wherein the composition increases the expression of the UCP-1 (uncoupling protein-1) gene that activates adipose tissue thermogenesis and energy homeostasis. **Claim 7** A pharmaceutical composition for preventing or treating diabetes or diabetic complications, comprising the peptide according to Claim 1 as an active ingredient. **Claim 8** The pharmaceutical composition for preventing or treating diabetes or diabetic complications according to Claim 7, wherein the composition promotes insulin secretion. **Claim 9** The pharmaceutical composition for preventing or treating diabetes or diabetic complications according to Claim 7, wherein the composition improves glucose tolerance.

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