Dipeptidyl peptidase-IV inhibitor and food composition containing the same

Abstract

To provide a DPP-4 inhibitor containing a chemical compound which has an excellent DPP-4 inhibitory effect, and food and medical compositions containing the same.SOLUTION: A dipeptidyl peptidase IV inhibitor contains at least one tripeptide chosen among Val-Pro-Ile, Ile-Pro-Gln, Ile-Pro-Leu, Ile-Pro-Tyr and Ile-Pro-Lys as an effective ingredient. An appetite suppressant food composition contains the dipeptidyl peptidase IV inhibitor. A medical composition for prophylaxis or therapy of diabetes contains the dipeptidyl peptidase IV inhibitor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a dipeptidyl peptidase-IV inhibitor and a food composition containing the same, which can be used to suppress appetite and prevent and treat diabetes. [Background technology]

[0002] Glucagon-like peptide 1 (hereinafter sometimes referred to as "GLP-1"), an incretin, is a peptide released after a meal and acts on pancreatic β cells in a blood sugar (glucose)-dependent manner, causing insulin secretion and thereby lowering blood glucose levels. GLP-1 is known to be degraded by dipeptidyl peptidase-IV (hereinafter sometimes referred to as DPP-4). In other words, by inhibiting DPP-4, the degradation of GLP-1 is suppressed and the blood concentration increases. As a result, insulin secretion from pancreatic β cells is promoted, and blood glucose levels can be lowered.

[0003] Therefore, compounds that inhibit DPP-4 are expected to be therapeutic agents for diabetes, and research is being conducted. For example, it has been disclosed that catechins contained in tea act as DPP-4 inhibitors (Patent Document 1). It has also been reported that oligopeptides consisting of 3 to 7 amino acids, which are hydrolysates of milk protein, act as DPP-4 inhibitors (Patent Document 2), and further, it has been disclosed that dipeptides (Patent Document 3), tripeptides (Patent Documents 4 to 6, Non-Patent Documents 1 to 2), tetrapeptides (Patent Documents 7 to 8), and pentapeptides (Patent Document 9) have DPP-4 inhibitory effects. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-118939 A [Patent Document 2] Special Publication No. 2008-525430 [Patent Document 3] JP 2013-184962 A [Patent Document 4] International Publication No. 2013 / 125622 [Patent Document 5] International Publication No. 2013 / 133031 [Patent Document 6] JP 2016-136966 A [Patent Document 7] International Publication No. 2013 / 133032 [Patent Document 8] JP 2016-222601 A [Patent Document 9] JP 2015-84694 A [Non-patent literature]

[0005] [Non-Patent Document 1] "The Journal of Antibiotics" (Japan) 1984, Vol. 37, p422-425 [Non-Patent Document 2] "Food Chemistry" (Netherlands) 2019, Vol. 279, p70-79 Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have investigated the inhibitory effect of diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-leu) on human dipeptidyl peptidase-IV (hDPP-4) described in Non-Patent Document 1. However, the inhibitory effect of these oligopeptides on human DPP-4 was not sufficient. An object of the present invention is to provide DPP-4 inhibitors, including compounds having excellent inhibitory activity against human DPP-4. [Means for solving the problem]

[0007] The present inventors have conducted extensive research into DPP-4 inhibitors, including compounds that have excellent inhibitory activity against human DPP-4, and have surprisingly found that a tripeptide having a specific amino acid sequence has excellent inhibitory activity against human DPP-4. The present invention is based on these findings. Thus, the present invention provides [1] A dipeptidyl peptidase-IV inhibitor comprising, as an active ingredient, at least one tripeptide selected from the group consisting of Val-Pro-Ile, Ile-Pro-Gln, Ile-Pro-Leu, Ile-Pro-Tyr, and Ile-Pro-Lys; [2] The dipeptidyl peptidase-IV inhibitor according to [1], which contains Val-Pro-Ile as an active ingredient. [3] A food composition for appetite suppression, comprising the dipeptidyl peptidase-IV inhibitor according to [1] or [2], and [4] A pharmaceutical composition for preventing or treating diabetes, comprising the dipeptidyl peptidase-IV inhibitor according to [1] or [2]. Regarding. Effect of the Invention

[0008] The DPP-4 inhibitor of the present invention exhibits excellent inhibitory activity against human dipeptidyl peptidase-IV and can prolong the action of glucagon-like peptide 1. The food of the present invention also has an appetite suppressing effect. The pharmaceutical composition of the present invention can be used for the prevention and treatment of diabetes. The tripeptide used in the present invention has a long half-life and can maintain its effectiveness for a long period of time. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows a chart of a mixture of tripeptides of VPX (X is any amino acid) separated by HPLC. [Diagram 2] FIG. 1 shows a chart of a mixture of tripeptides of IPX (X is any amino acid) separated by HPLC. [Diagram 3]1 is a graph showing the stability of Val-Pro-Ile (Example 1), Ile-Pro-Ile (Reference Example 1), and VPX-14, VPX-15, IPX-3, and IPX-13 (Reference Example 3) against human DPP-4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The dipeptidyl peptidase-IV inhibitor of the present invention contains at least one tripeptide selected from the group consisting of Val-Pro-Ile (hereinafter sometimes referred to as VPI), Ile-Pro-Gln (hereinafter sometimes referred to as IPQ), Ile-Pro-Leu (hereinafter sometimes referred to as IPL), Ile-Pro-Tyr (hereinafter sometimes referred to as IPY), and Ile-Pro-Lys (hereinafter sometimes referred to as IPK) as an active ingredient. The tripeptide is preferably Val-Pro-Ile.

[0011] The Val(V) is represented by the following formula (1): [ka] It means valine, which is represented by the formula: The Pro(P) is represented by the following formula (2): [ka] It means proline represented by the formula: The Ile(I) is represented by the formula (3): [ka] It means isoleucine represented by the formula: The Gln(Q) has the formula (4): [ka] It means glutamine represented by the formula: The Leu(L) formula (5): [ka] It means leucine represented by the formula: The Tyr(Y) is represented by the formula (6): [ka] It means tyrosine represented by the formula: The Lys(K) is represented by the formula (7): [ka] It means lysine represented by the formula:

[0012] The amino acids used in the present invention may be either D-type or L-type amino acids, but are preferably L-type amino acids. In the tripeptide used in the present invention, the carboxyl groups and amino groups of two amino acids are bonded by peptide bonds. The tripeptide has excellent stability. That is, it has excellent resistance to degradation by dipeptidyl peptidase-IV.

[0013] (Dipeptidyl peptidase-IV) Dipeptidyl peptidase-IV (DPP-4) degrades the incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (hereinafter sometimes referred to as GIP). It exists on cell membranes and in blood as a soluble protein. Human DPP-4 is a single-pass transmembrane protein consisting of 766 amino acids, with an intracellular domain of 6 amino acid residues and a transmembrane domain of 24 amino acid residues. The tripeptide used in the present invention can inhibit the action of DPP-4.

[0014] (Glucagon-like peptide 1) Glucagon-like peptide 1 (GLP-1) is generated by cleavage of preproglucagon to form GLP-1(1-37), which consists of 37 amino acids. GLP-1(7-37) and GLP-1(7-36)amide are then further cleaved at the N-terminal amino acids, and these have strong biological activities. The actions of GLP-1 include glucose-induced stimulation of insulin biosynthesis and secretion, inhibition of glucagon secretion, modulation of gene expression, trophic effects on β-cells, inhibition of food intake, and slowing of gastric emptying. On the other hand, for example, active GLP-1(7-37) loses its physiological activity when the dipeptide at the amino terminal is removed by the action of DPP-IV and it is converted to inactive GLP-1(9-37). As described above, the tripeptide of the present invention inhibits the action of DPP-4, which degrades GLP-1. The half-life of GLP-1 is reported to be 2 to 5 minutes, but the tripeptide of the present invention extends the half-life. Thus, the tripeptide of the present invention exhibits effects such as glucose-induced stimulation of insulin biosynthesis and secretion, inhibition of glucagon secretion, regulation of gene expression, nutritional effect on B cells, inhibition of food intake, and slowing of gastric emptying by inhibiting the degradation of GLP-1.

[0015] As described above, the tripeptide promotes the action of GLP-1 by dipeptidyl peptidase-IV inhibitory action. Therefore, it has the same action as GLP-1. That is, the dipeptidyl peptidase-IV inhibitor of the present invention can be used as a blood glucose level increase inhibitor, a hyperglycemia improving agent, a vascular endothelial dysfunction inhibitor, a vascular endothelial disorder inhibitor, a vascular disorder inhibitor, a vascular endothelial cell protector, and an appetite suppressant.

[0016] The appetite suppressant food composition of the present invention refers to one which contains the dipeptidyl peptidase-IV inhibitor of the present invention and a food or beverage. In addition, the food composition of the present invention can be used as a food composition for suppressing an increase in blood glucose level, improving hyperglycemia, suppressing a decrease in vascular endothelial function, suppressing vascular endothelial damage, suppressing vascular damage, protecting vascular endothelial cells, and suppressing appetite.

[0017] Specific examples of foods include fresh prepared foods such as salads; cooked foods such as steaks, pizzas, and hamburgers; stir-fried foods such as stir-fried vegetables; vegetables such as tomatoes, bell peppers, celery, bitter melon, carrots, potatoes, and asparagus, and prepared foods made from these vegetables; confectioneries such as cookies, bread, biscuits, dry bread, cakes, rice crackers, yokan, puddings, jellies, ice cream, chewing gum, crackers, chips, chocolate, and candies; noodles such as udon, pasta, and soba; fish paste products such as kamaboko, ham, and fish sausages; dairy products such as cheese, cream, and butter; condiments such as miso, soy sauce, dressing, ketchup, mayonnaise, soup base, noodle soup, curry powder, mirin, roux, and seasoning spices; soy foods such as tofu; konjac; and supplements.

[0018] Examples of beverages include coffee beverages; cocoa beverages; vegetable juices obtained from the above-mentioned vegetables; fruit juice beverages such as grapefruit juice, orange juice, grape juice, and lemon juice; tea beverages such as green tea, black tea, sencha, and oolong tea; alcoholic beverages such as beer, wine (red wine, white wine, sparkling wine, etc.), sake, plum wine, sparkling wine, whiskey, brandy, shochu, rum, gin, and liqueurs; dairy beverages; soy milk beverages; liquid diets; and sports drinks.

[0019] The food or beverage includes feed for animals, such as primates, including humans, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, and mice.

[0020] If desired, these foods or beverages may contain food additives and food ingredients, such as antioxidants, flavorings, acidulants, colorants, emulsifiers, preservatives, seasonings, sweeteners, spices, pH adjusters, stabilizers, antioxidants, vegetable oils, animal oils, sugars and sugar alcohols, vitamins, organic acids, fruit juice extracts, vegetable extracts, grains, beans, vegetables, meats, seafood, etc., either alone or in combination of two or more. The amounts of these food ingredients and food additives to be added may be appropriately determined within the range that does not impair the object of the present invention.

[0021] These foods or beverages can be subjected to common sterilization treatments, such as heat-pressure sterilization using retort and autoclave, batch sterilization, plate sterilization, electric heating sterilization, microwave heating sterilization, and steam sterilization such as injection and infusion.

[0022] Foods and beverages include functional foods (beverages) and health foods (beverages). In this specification, "health foods (beverages)" refers to foods or beverages that have some effect on health or that can be expected to have an effect, and "functional foods (beverages)" refers to foods or beverages that, among the above-mentioned "health foods (beverages), have been designed and processed to fully express a bioregulatory function (i.e., an immunostimulatory function). Functional foods and health foods can be in the form of granules, solids, liquids, capsules, gels, or tablets.

[0023] The pharmaceutical composition for preventing or treating diabetes of the present invention comprises a dipeptidyl peptidase-IV inhibitor of the present invention.

[0024] The pharmaceutical composition of the present invention can be used for diseases or symptoms caused by dipeptidyl peptidase-IV other than diabetes, such as hyperglycemia, diabetic complications, vascular endothelial disorder, or vascular disorder. Furthermore, diseases induced by hyperglycemia or diabetes include diabetic microangiopathy (e.g., retinopathy, nephropathy, or neuropathy), and macrovascular complications (e.g., ischemic heart disease such as angina pectoris or myocardial infarction, cerebral infarction, arteriosclerosis obliterans, or gangrene). Therefore, the pharmaceutical composition of the present invention can be used as a pharmaceutical composition for preventing or treating these diseases.

[0025] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and can include oral and parenteral agents, with oral agents being preferred.The oral agents can include, for example, solid or powdered preparations such as fine granules, granules, tablets, capsules, and pills, as well as liquid preparations such as suspensions, emulsions, syrups, and extracts.The parenteral agents can include, for example, injections.

[0026] The pharmaceutical composition of the present invention may consist of the tripeptide or may contain the tripeptide. When the pharmaceutical composition of the present invention contains the tripeptide, it may contain other additives.

[0027] When the pharmaceutical composition of the present invention is an oral preparation, other additives can include excipients, binders, disintegrants, emulsifiers, lubricants, flow enhancers, diluents, preservatives, colorants, flavorings, taste enhancers, stabilizers, moisturizers, antiseptics, antioxidants, or suspending agents. Specific examples of such additives include gelatin, sodium alginate, starch, corn starch, white sugar, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soybean lecithin, sucrose, fatty acid esters, talc, magnesium stearate, polyethylene glycol, magnesium silicate, anhydrous silicic acid, and synthetic aluminum silicate.

[0028] When the pharmaceutical composition of the present invention is a parenteral preparation, other additives may include water-soluble solvents such as physiological saline or Ringer's solution, water-insoluble solvents such as vegetable oil or fatty acid ester, isotonic agents such as glucose or sodium chloride, solubilizing agents, stabilizers, preservatives, suspending agents, or emulsifiers.

[0029] The pharmaceutical composition of the present invention may contain the tripeptide in an amount of 90% by weight or more, 50% by weight or more, 10% by weight or more, or 1% by weight or more.

[0030] The dosage or intake of the pharmaceutical composition of the present invention can be appropriately adjusted depending on the formulation, and the age, sex, body weight, and severity of symptoms of the disease of the subject, but it is preferable that the pharmaceutical composition is administered or taken in an amount that can prevent the onset of diabetes, etc., or alleviate or treat the onset of diabetes, etc. Specifically, the amount can be 0.01 to 1000 mg / kg body weight / day, preferably 0.1 to 750 mg / kg body weight / day, more preferably 1 to 500 mg / kg body weight / day, even more preferably 5 to 400 mg / kg body weight / day, even more preferably 10 to 300 mg / kg body weight / day, even more preferably 15 to 200 mg / kg body weight / day, or most preferably 20 to 150 mg / kg body weight / day, calculated as the amount of tripeptide added.

[0031] Of course, the above administration method is an example, and other administration methods may be used. It is desirable that the administration method, dosage, administration period, administration interval, etc. of the pharmaceutical composition to humans be determined through a controlled clinical trial.

[0032] The pharmaceutical composition of the present invention can be administered to humans, but may also be administered to animals other than humans, including pets such as dogs, cats, rabbits, hamsters, guinea pigs, and squirrels; livestock such as cows and pigs; laboratory animals such as mice and rats; and animals kept in zoos, etc.

[0033] The pharmaceutical composition includes medicines and quasi-drugs. Medicines include, for example, herbal medicine preparations and herbal medicine preparations. Quasi-drugs include, for example, nutritional drinks and health medicines containing herbal medicines. EXAMPLES

[0034] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

[0035] Example 1 In this example, Val-Pro-Ile was synthesized and its inhibitory effect on DPP-4 was examined. The tripeptide Val-Pro-Ile was synthesized and purified by GenScript, Inc. The purity was 95% or higher. The human DPP-4 inhibition test was carried out as follows. A mixture of a serially diluted solution of the Val-Pro-Ile tripeptide (20 μL), a substrate solution (Gly-Pro-pNA; 2 mM; 2 μL), and Tris-HCl buffer (pH 8.0; 20 μL) was dispensed in 100 μL portions into a 96-well plate. 40 μL of human DPP-4 (hDPP-4: final concentration 0.025 units / mL) was added and incubated at 37°C for 60 minutes. The absorbance at 405 nm was measured using a microreader. The peptide concentration at which the absorbance was half was determined as the IC 50 The results are shown in Table 1.

[0036] Example 2 In this example, Ile-Pro-Gln was synthesized and the inhibitory effect of DPP-4 was examined. The procedure of Example 1 was repeated, except that the tripeptide Ile-Pro-Gln was synthesized instead of the tripeptide Val-Pro-Ile. The results are shown in Table 1.

[0037] Example 3 In this example, Ile-Pro-Leu was synthesized and the inhibitory effect of DPP-4 was examined. The procedure of Example 1 was repeated, except that the tripeptide Ile-Pro-Leu was synthesized instead of the tripeptide Val-Pro-Ile. The purity was 94.8%. The results are shown in Table 1.

[0038] Example 4 In this example, Ile-Pro-Tyr was synthesized and the inhibitory effect of DPP-4 was examined. The procedure of Example 1 was repeated, except that the tripeptide Ile-Pro-Tyr was synthesized instead of the tripeptide Val-Pro-Ile. The results are shown in Table 1.

[0039] Example 5 In this example, Ile-Pro-Lys was synthesized and its inhibitory effect on DPP-4 was examined. The procedure of Example 1 was repeated, except that the tripeptide Ile-Pro-Lys was synthesized instead of the tripeptide Val-Pro-Ile. The results are shown in Table 1.

[0040] 《Reference example 1》 In this reference example, Ile-Pro-Ile was synthesized and the inhibitory effect of DPP-4 was examined. The procedure of Example 1 was repeated, except that the tripeptide Ile-Pro-Ile was synthesized instead of the tripeptide Val-Pro-Ile. The purity was 99%. The results are shown in Table 1.

[0041] 《Reference example 2》 In this example, the inhibitory effect of DPP-4 was examined using Val-Pro-Leu. The procedure of Example 1 was repeated, except that the tripeptide Val-Pro-Leu was purchased from BioVision. The results are shown in Table 1.

[0042] [Table 1]

[0043] 《Reference example 6》 In this example, a mixture of tripeptides of VPX (X is any amino acid) and a mixture of tripeptides of IPX (X is any amino acid) were synthesized, and each tripeptide was separated by HPLC. The synthesis of the mixture of VPX and the mixture of IPX was outsourced to GenScript. Figure 1 shows the HCLP chart for VPX, and Figure 2 shows the HCLP chart for IPX.

[0044] Stability testing The stability of each tripeptide was examined using Val-Pro-Ile obtained in Example 1, Ile-Pro-Ile obtained in Reference Example 1, and VPX-14, VPX-15, IPX-3, and IPX-13 obtained in Reference Example 3. 200 μL of each peptide was added to 50 μL of human DPP-4 (final concentration 0.0625 units / mL) and incubated at 37° C. for 1 hour or 2 hours. Samples at 0 hours, 1 hour, and 2 hours were analyzed by HPLC. The results are shown in FIG. 3. Val-Pro-Ile obtained in Example 1 exhibited excellent stability. [Industrial Applicability]

[0045] The dipeptidyl peptidase-IV inhibitor of the present invention can be used as a food composition or a pharmaceutical composition for the treatment of diseases caused by dipeptidyl peptidase-IV, including diabetes.

Claims

1. A dipeptidyl peptidase-IV inhibitor comprising a tripeptide of Ile-Pro-Tyr or Ile-Pro-Lys as an active ingredient.

2. 13. A food composition for suppressing appetite, comprising the dipeptidyl peptidase-IV inhibitor according to claim 1.

3. A pharmaceutical composition for preventing or treating diabetes, comprising the dipeptidyl peptidase-IV inhibitor according to claim 1.

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