peptide

Abstract

To provide peptides, GLP-1 secretion promoters, pharmaceutical compositions, food compositions, methods for suppressing appetite, methods for suppressing blood glucose elevation, and methods for improving obesity. [Solution] A peptide consisting of the amino acid sequence VLVNA (SEQ ID NO: 1), the amino acid sequence EKLTEWT (SEQ ID NO: 2), or the amino acid sequence VLLPDEVSG (SEQ ID NO: 3).

Description

Technical Field

[0001] The present invention relates to a peptide, a GLP-1 secretion promoter, a pharmaceutical composition, a food composition, a method for suppressing appetite, a method for suppressing an increase in blood glucose level, and a method for improving obesity.

Background Art

[0002] Glucagon-like peptide 1 (GLP-1), which is a type of incretin, is a hormone involved in maintaining glucose homeostasis. When the blood concentration of GLP-1 increases, insulin secretion is promoted. Therefore, GLP-1 has attracted attention for various effects such as appetite suppression, suppression of an increase in blood glucose level, and improvement of obesity.

[0003] GLP-1 and ghrelin have been reported as hormones that control appetite, and the usefulness of substances that control their secretion amounts is expected. Patent Document 1 discloses a peptide having a specific amino acid sequence that has an action of suppressing ghrelin secretion.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a peptide, a GLP-1 secretion promoter, a pharmaceutical composition, a food composition, a method for suppressing appetite, a method for suppressing an increase in blood glucose level, and a method for improving obesity.

Means for Solving the Problems

[0006] As a result of intensive studies, the present inventors have found that a specific peptide has an action of promoting the secretion of GLP-1. The present invention includes the following aspects [1] to

[10] . [1] Amino acid sequence VLVNA (SEQ ID NO: 1), Amino acid sequence EKLTEWT (SEQ ID NO: 2) or A peptide consisting of the amino acid sequence VLLPDEVSG (SEQ ID NO: 3). [2] A GLP-1 secretagogue containing the peptide described in [1]. [3] A pharmaceutical composition containing the peptide described in [1]. [4] The pharmaceutical composition according to [3] for the purpose of suppressing appetite, suppressing the rise in blood glucose levels, and / or improving obesity. [5] A food composition containing the peptide described in [1]. [6] The food composition according to [5], wherein the peptide is added. [7] The food composition according to [5] or [6] for the purpose of suppressing appetite, suppressing the rise in blood glucose levels, and / or improving obesity. [8] A method for suppressing appetite, comprising the step of administering the peptide described in [1]. A method for suppressing an increase in blood glucose levels, comprising the step of administering the peptide described in [9] [1].

[10] A method for improving obesity, comprising the step of administering the peptide described in [1]. [Effects of the Invention]

[0007] The present invention provides novel peptides, GLP-1 secretion promoters, pharmaceutical compositions, food compositions, and appetite suppression methods. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the results of the example. [Modes for carrying out the invention]

[0009] [peptide] [GLP-1 secretion promoter] The peptide of the present invention consists of the amino acid sequence VLVNA (SEQ ID NO: 1), the amino acid sequence EKLTEWT (SEQ ID NO: 2), or the amino acid sequence VLLPDEVSG (SEQ ID NO: 3). The peptide consisting of the amino acid sequence of Sequence ID No. 1 above consists of 5 amino acid residues. The peptide consisting of the amino acid sequence of Sequence ID No. 2 above consists of 7 amino acid residues. The peptide consisting of the amino acid sequence of Sequence ID No. 3 above consists of 9 amino acid residues.

[0010] The amino acids that make up the peptide can be L-isomers, D-isomers, or DL-isomers (if the amino acids are a mixture of D-isomers and L-isomers, this includes racemic mixtures and amino acids with an excess of either enantiomer). The peptide is preferably composed of only L-isomers or only D-isomers, and more preferably composed of only L-isomers.

[0011] Peptides can be modified. The amino terminus (N-terminus) of a peptide may be a free amino group (NH2-) or may have modifications such as an acetyl group (CH3CO-). The carboxyl terminus (C-terminus) of a peptide may be a free carboxyl group (-COOH) or may have modifications such as an amide group. The amino acid residues of a peptide may be unmodified or may have modifications such as phosphate groups or sugar chains.

[0012] The peptide of the present invention may also be a salt (acid addition salt or base salt). Examples of acid addition salts include inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and perchloric acid, and salts of organic acids such as citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. Examples of base salts include alkali metal salts such as sodium, potassium, and lithium, and alkaline earth metal salts such as calcium and magnesium.

[0013] The peptide of the present invention may be a solvate. Examples of the solvate include solvates with water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, dimethoxyethane and other solvents.

[0014] The method for obtaining the above peptide is not limited. For example, it can be prepared by chemical synthesis using a known peptide synthesis method. Examples of the peptide synthesis method include peptide synthesis methods based on various chemical mechanisms such as the azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DDC method, active ester method, carbodiimidazole method, oxidation-reduction method and the like. These peptide synthesis methods can be carried out by either solid-phase synthesis method or liquid-phase synthesis method.

[0015] In the above peptide synthesis method, it is preferable to protect the amino group, carboxy group, and / or side chain functional group (for example, the guanidino group of arginine (Arg), etc.) with a protecting group. The protecting group is not particularly limited and known protecting groups can be used. Examples include fluorenylmethoxycarbonyl group (Fmoc), tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), benzyl group (Bz), p-toluenesulfonyl group (p-Ts) and the like. Depending on the protecting group, it is also called the Fmoc method, Boc method, etc.

[0016] In addition, after synthesizing a peptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 3 by the above peptide synthesis method, if necessary, a purified product obtained by a known method can be used. Examples of the purification method include methods using ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography, high performance liquid chromatography (HPLC) and the like.

[0017] The peptide of the present invention has an action of promoting the secretion of GLP-1 (Glucagon-like peptide-1). GLP-1 is a hormone that promotes insulin secretion and is a type of incretin.

[0018] GLP-1 is known to have various actions such as promoting insulin secretion, suppressing glucagon secretion, suppressing appetite (food intake suppression), delaying the discharge of gastric contents, etc. In addition, an action of preventing and / or improving the progression of dementia is also known. Therefore, the peptide of the present invention that promotes insulin secretion also has actions based on promoting insulin secretion such as appetite suppression action (food intake suppression action) and suppression of blood glucose level increase. Based on the appetite suppression action (food intake suppression action), the peptide of the present invention can be used for the prevention and treatment of overeating, obesity, etc. Therefore, the present invention provides a GLP-1 secretion promoter containing the above peptide. In one aspect, the present invention is an appetite suppressant containing the above peptide. In another aspect, the present invention is a blood glucose level increase suppressant containing the above peptide. In another aspect, the present invention is an obesity improvement agent containing the above peptide.

[0019] The promotion of the secretion amount of GLP-1 by the peptide of the present invention can be evaluated by a method known to those skilled in the art. For example, by adding a test substance to GLP-1 secreting cells such as STC-1 cells and quantifying GLP-1 in the medium recovered after culturing for a predetermined time, the secretion amount of GLP-1 can be measured. Quantification of GLP-1 can be performed by an immunochemical method such as ELISA (Enzyme-Linked ImmunoSorbent Assay).

[0020] The peptide of the present invention can be used as a component of a pharmaceutical composition, a food composition, etc. described later.

[0021] [Pharmaceutical Composition] The pharmaceutical composition of the present invention contains the above-mentioned peptide. Preferably, the above-mentioned peptide is included as an active ingredient. The pharmaceutical composition encompasses pharmaceuticals and quasi-drugs. The pharmaceutical composition of the present invention is suitably used for appetite suppression, suppression of blood glucose elevation, and / or improvement of obesity, and is suitably used for the treatment of obesity, bulimia, etc.

[0022] The composition of the present invention may contain, in addition to the peptide described above, other components as needed. These other components are not particularly limited and can be appropriately selected depending on the purpose. Examples include pharmaceutically acceptable bases, carriers, and / or additives (e.g., solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, etc.). The amount of these other components can be appropriately set depending on the purpose. When the composition of the present invention is used as a pharmaceutical composition, the mode of administration is not particularly limited, and examples include oral administration, intravascular administration (especially intravenous injection), etc. Among these, oral administration is preferred.

[0023] When the composition of the present invention is used as a pharmaceutical composition, the dosage form is not particularly limited and includes, for example, tablets such as orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, and dissolving tablets; lozenges; powders; suspensions; emulsions; elixirs; limonades; syrups; lotions; granules; capsules such as hard capsules and soft capsules; creams; ointments; suppositories; poultices, tapes, microneedles, transdermal / mucosal administration agents such as iontophoresis and electroporation; and aerosols. These forms can be prepared by conventional methods by combining the peptide described above with other components as needed.

[0024] When the composition of the present invention is used as a pharmaceutical composition, the amount of the peptide in the pharmaceutical composition is not particularly limited as long as it is an amount in which the above-mentioned effects of the peptide are exerted, and can be set as appropriate.

[0025] Furthermore, when the composition of the present invention is used as a pharmaceutical composition, the dosage, administration interval, target population, etc., are not particularly limited and can be set as appropriate. For example, the dosage can be set as appropriate according to the target population's age, sex, weight, health condition, and other conditions. Also, for example, the administration interval may be once or multiple times a day, preferably two to three times, or once or multiple times every few days to several weeks. In addition, the target population may be humans, as well as non-human mammals such as pets or livestock, such as dogs, cats, cattle, horses, sheep, llamas, rats, mice, etc.

[0026] In particular, when the target of administration is a human, the peptide dosage is preferably about 10 to 300 μg per day for an adult, more preferably about 15 to 200 μg, even more preferably about 20 to 100 μg, even more preferably about 30 to 80 μg, even more preferably about 35 to 70 μg, and particularly preferably about 40 to 60 μg.

[0027] Furthermore, while there are no particular restrictions on the duration of administration (intake), it is preferably 3 to 12 weeks or longer (3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or longer), and more preferably 9 to 12 weeks or longer. In particular, when the daily peptide dose is approximately 30 to 80 μg, it is preferably 5 to 12 weeks or longer, and when it is 10 μg or more but less than 30 μg, or more than 80 μg but 300 μg or less, it is preferably 9 to 12 weeks or longer.

[0028] When the target of administration is a non-human mammal, the administration method, dosage form, dose, administration interval, etc., can be appropriately determined by referring to the method used when the target of administration is a human.

[0029] [Food composition] The food composition of the present invention contains the above-mentioned peptide. Preferably, it contains the above-mentioned peptide as an active ingredient. The food composition of the present invention is suitably used for appetite suppression, suppression of blood glucose elevation, and / or improvement of obesity. Furthermore, based on its appetite-suppressing effect, it is suitably used for weight loss.

[0030] Food compositions include not only ordinary fresh or processed food compositions, but also a wide range of compositions consumed as food and beverages, such as food additive compositions including salt substitutes, sweeteners, and additives for beverages, as well as food ingredient compositions such as premixed food ingredients for commercial and household use. In one preferred embodiment of the present invention, the food composition is a food composition that clearly indicates the appetite-suppressing effect or other benefits of so-called health foods, functional foods, foods with functional claims, nutritional supplements, supplements, foods for specified health uses, etc.

[0031] In addition to the peptides mentioned above, the food composition may contain other components as needed. These other components are not particularly limited and can be appropriately selected depending on the purpose. Examples include food hygiene-compliant bases, carriers, additives, and other components and materials that can be used as food. In one preferred embodiment of the present invention, the peptide is not derived from any component, material, etc. that can be used as the above-mentioned food, but is added to the food.

[0032] The form of the food composition is not limited, and is not particularly limited, as long as it is in a form that can be taken orally. For example, in addition to forms such as hard capsules, soft capsules, supplements, chewable tablets, beverages, powdered beverages, granules, and films, when used as food or beverages, it can also be in the form of beverages such as tea-based beverages, sports drinks, beauty drinks, fruit juices, carbonated drinks, alcoholic beverages, soft drinks, jelly drinks, concentrated beverages that are diluted with water, hot water, carbonated water, etc., powders and granules that are dissolved or suspended in water or hot water before drinking, dried solids such as tablets, candy tablets, jellies, snacks, baked goods, fried goods, cakes, chocolates, gum, candy, gummies and other sweets, soups, noodles, rice dishes, bread, cereals and other foods. Preferred embodiments include various foods that use eggs as an ingredient. Specifically, these include bread, sandwiches, French toast, cakes, cookies, biscuits, ice cream, pudding, and other bread and confectionery products; Processed fish products containing eggs as a binder, such as hanpen, kamaboko, chikuwa, and surimi; Processed meat products such as ham and sausages; Other examples include mayonnaise, fried foods, boiled eggs, steamed egg custard, rolled omelet, egg tofu, custard cream, and custard pudding.

[0033] Furthermore, when the composition of the present invention is used as a food additive composition or a food ingredient composition, its form is not particularly limited, and examples include liquid, powder, flake, granular, and paste forms. More specifically, examples include seasonings (sweeteners, salt substitutes, soy sauce, vinegar, miso, sauces, ketchup, dressings, spices, herbs, etc.), flakes (rice seasoning, rice cooking additives, etc.), barbecue sauce, roux paste (curry roux paste, etc.), and food ingredient premixes.

[0034] In the food composition, the amount of the peptide is not particularly limited and can be set as appropriate.

[0035] [Appetite suppression method] The appetite suppression method of the present invention includes the step of administering the above-mentioned peptide to a target subject. The target population for administration is not particularly limited, as long as it is a population requiring appetite suppression, such as those with obesity, bulimia, or those at risk of developing these conditions. The target population includes humans and non-human mammals such as pets or livestock, with humans being preferred.

[0036] The preferred form of administration is the pharmaceutical composition or food composition described above. The method of administration can be appropriately selected depending on the form of the pharmaceutical or food composition. The dosage and administration interval can be selected as appropriate, in accordance with the descriptions in the "Pharmaceutical Composition" and "Food Composition" sections above.

[0037] [Methods to suppress blood sugar level increases] The present invention's method for suppressing blood glucose elevation includes the step of administering the above-mentioned peptide to a target. The target population for administration is not particularly limited, as long as it is a population that requires suppression of blood glucose elevation in individuals with obesity, bulimia, hypertension, or those at risk of developing these conditions. The target population includes humans and non-human mammals such as pets or livestock, with humans being preferred.

[0038] The preferred form of administration is the pharmaceutical composition or food composition described above. The method of administration can be appropriately selected depending on the form of the pharmaceutical or food composition. The dosage and administration interval can be selected as appropriate, in accordance with the descriptions in the "Pharmaceutical Composition" and "Food Composition" sections above.

[0039] [Methods to improve obesity] The present invention provides a method for improving obesity, which includes the step of administering the above-mentioned peptide to a target subject. The target population for administration is not particularly limited, as long as it is a person who requires improvement of obesity or pre-obesity. The target population includes humans and non-human mammals such as pets or livestock, and is preferably humans.

[0040] The preferred form of administration is the pharmaceutical composition or food composition described above. The method of administration can be appropriately selected depending on the form of the pharmaceutical or food composition. The dosage and administration interval can be selected as appropriate, in accordance with the descriptions in the "Pharmaceutical Composition" and "Food Composition" sections above. [Examples]

[0041] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0042] Manufacturing example The following three types of peptides of the present invention were manufactured by an external company. The peptides were synthesized by the Fmoc method and purified by HPLC. (1) A peptide consisting of the amino acid sequence VLVNA (SEQ ID NO: 1) (2) Peptide consisting of amino acid sequence EKLTEWT (SEQ ID NO: 2) (3) Peptide consisting of amino acid sequence VLLPDEVSG (SEQ ID NO: 3)

[0043] Examples (1) Preparation of peptide lysis medium The three peptides obtained in the above manufacturing example were dissolved in HEPES Buffer of the following composition to prepare a sample solution with a final concentration of 1 mM. A control sample solution without peptides was also prepared as a control. HEPES Buffer The following components were mixed in ultrapure water (MilliQ water). After adjusting the pH to 7.4, the mixture was made up to 100 mL with ultrapure water (MilliQ water). Then, it was filtered and sterilized using a 0.22 μm filter (Merck, Millipore brand) to obtain 10 × HEPES Buffer. This was diluted 10-fold for use in the experiment. 140 mM NaCl 818.6 mg 4.5 mM KCl 33.55 mg 20 mM HEPES 2 mL 1.2 mM CaCl2·2H2O 17.64 mg 1.2 mM MgCl2·6H2O 24.4 mg 10 mM D-glucose 180.156 mg 0.1% BSA 100mg

[0044] (2) Preparation of cells Add DMEM medium (Merck, Sigma brand, product number D5796) to each well of a 96-well plate and infuse 3.3 × 10⁶ small cell carcinoma-derived STC-1 cells. 4 Seeds were sown to a cell / well ratio and incubated for 24 hours at 37°C in a 5% CO2 environment.

[0045] (3) Evaluation of GLP-1 secretion activity The cells described above were washed with 100 μL of PBS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 100 μL of the peptide-containing sample solution was added, and the cells were incubated at 37°C in a 5% CO2 environment for 10 minutes. Each sample solution and control sample were tested with n=4. Next, the entire supernatant was collected in a test tube and centrifuged at 800 × g for 5 minutes at 4°C to separate the supernatant from the precipitate. The supernatant was then centrifuged again in the same manner, and the resulting supernatant was used as the evaluation sample. The obtained evaluation sample was stored at -80°C.

[0046] As an indicator of GLP-1 secretion, the GLP-1 concentration in the evaluation samples was measured by ELISA using the Multi Species GLP-1 Total ELISA, EZGLP1T-36K (Merck, Millipore brand) according to the protocol provided with the kit. The results are shown in Figure 1. GLP-1 secretion levels are shown as relative amounts to the control, using mean ± SEM. In all samples, a statistically significant difference (p<0.01) was observed compared to the control using the Dunnett method.

Claims

1. Amino acid sequence VLVNA (SEQ ID NO: 1), Amino acid sequence EKLTEWT (SEQ ID NO: 2) or A peptide consisting of the amino acid sequence VLLPDEVSG (SEQ ID NO: 3).

2. A GLP-1 secretion promoter comprising the peptide described in claim 1.

3. A pharmaceutical composition comprising the peptide described in claim 1.

4. A pharmaceutical composition according to claim 3 for the purpose of suppressing appetite, suppressing the rise in blood sugar levels, and / or improving obesity.

5. A food composition comprising the peptide described in claim 1.

6. The food composition according to claim 5, wherein the peptide is added.

7. A food composition according to claim 5 for the purpose of suppressing appetite, suppressing the rise in blood sugar levels, and / or improving obesity.

8. A method for suppressing appetite, comprising the step of administering the peptide described in claim 1.

9. A method for suppressing an increase in blood glucose levels, comprising the step of administering the peptide described in claim 1.

10. A method for improving obesity, comprising the step of administering the peptide described in claim 1.

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