JP7068942B2 - Tripeptide-containing composition - Google Patents

Description

The present invention relates to a composition containing a specific tripeptide (Leu-Arg-Ala) and the like.

The incretins glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are hormones involved in the maintenance of glucose homeostasis. DPP-4 (Dipeptidyl Peptidase-4, EC3.4.14.5) is an enzyme that decomposes incretins. By inhibiting DPP-4, the degradation of incretin is suppressed, so that the blood concentrations of GLP-1 and GIP are increased and insulin secretion is promoted. Therefore, inhibiting DPP-4 improves blood glucose control (particularly suppresses blood glucose elevation) and is effective in preventing or treating diabetes (particularly type 2 diabetes). A DPP-4 inhibitor (for example, sitagliptin) has already been put on the market as a therapeutic agent for type 2 diabetes.

International Publication No. 2017/217535 Japanese Unexamined Patent Publication No. 2017-043618 Japanese Unexamined Patent Publication No. 2016-003225

The challenge is to provide new means of inhibiting DPP-4.

The present inventors have found that a tripeptide consisting of Leu-Arg-Ala has a DPP-4 inhibitory activity, and have further improved it to complete the present invention.

The present invention includes, for example, the subjects described in the following sections.
Item 1.
A composition containing a tripeptide consisting of Leu-Arg-Ala.
A composition for inhibiting DPP-4, improving glucose metabolism, suppressing an increase in blood glucose level, or for antidiabetes.
Item 2.
Item 2. The composition according to Item 1, which is an oral composition.
Item 3.
Item 2. The composition according to Item 1 or 2, which is a food composition or a pharmaceutical composition.
Item 4.
Item 6. The composition according to any one of Items 1 to 3, wherein the tripeptide consisting of 10 to 300 μg of Leu-Arg-Ala is used so as to be ingested per adult day.
Item A-1.
For DPP-4 inhibition and improvement of glucose metabolism, which comprises a step of administering to a subject a tripeptide consisting of Leu-Arg-Ala, or a composition containing the tripeptide and a pharmaceutically or food hygiene-acceptable carrier. , Suppression of blood glucose elevation, or diabetes prevention or treatment method.
Item B-1.
A tripeptide consisting of Leu-Arg-Ala for DPP-4 inhibition, improvement of glucose metabolism, suppression of blood glucose elevation, or use in the prevention or treatment of diabetes, or the tripeptide and pharmaceutically or food hygiene acceptable. A composition comprising a carrier to be added.
Item C-1.
Use of a tripeptide consisting of Leu-Arg-Ala in the production of pharmaceuticals or foods for inhibiting DPP-4, improving glucose metabolism, suppressing blood glucose elevation, or preventing or treating diabetes.

The DPP-4 activity can be efficiently inhibited, whereby an effect of improving glucose metabolism, an effect of suppressing an increase in blood glucose level, an antidiabetic effect (preferably an effect of preventing or treating diabetes) and the like can be obtained.

The results of examining the DPP-4 inhibitory activity of the LRA peptide using the kit are shown. The lower the fluorescence intensity, the stronger the DPP-4 inhibitory activity.

Hereinafter, each embodiment included in the present invention will be described in more detail. The present invention preferably includes, but is not limited to, a composition containing a specific tripeptide, a method for administering a specific tripeptide to a subject, the use of the specific tripeptide, and the like. Includes all disclosed herein.

The compositions included in the present invention contain a particular tripeptide (Leu-Arg-Ala). In the present specification, the composition may be referred to as "the composition of the present invention". Further, in the present specification, a tripeptide consisting of an amino acid sequence: Leu-Arg-Ala may be referred to as "LRA peptide".

The LRA peptide can be prepared by chemical synthesis using a known peptide synthesis method. Examples of the peptide synthesis method include peptide synthesis methods such as an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DDC method, an active ester method, a carboimidazole method, and an oxidation-reduction method. .. These peptide synthesis methods can be carried out by either a solid phase synthesis method or a liquid phase synthesis method.

In the above-mentioned peptide synthesis method, it is preferable to protect the amino group, the carboxy group, and / or the side chain functional group (for example, the guanidino group of arginine (Arg)) with a protecting group. As the protecting group, a known protecting group can be used without particular limitation, for example, a benzyloxycarbonyl group (Cbz), a tert-butoxycarbonyl group (Boc), a fluorenylmethoxycarbonyl group (Fmoc), a benzyl group. (Bz), p-toluenesulfonyl group (p-Ts) and the like can be mentioned.

Further, after synthesizing the LRA peptide by the above-mentioned peptide synthesis method, if necessary, a product purified by a known method can be used as the LRA peptide.

Since the LRA peptide has a DPP-4 inhibitory action, the composition of the present invention containing the LRA peptide can be used as a DPP-4 inhibitory composition, a glucose metabolism improving composition, a blood glucose level elevation suppressing composition, and the like. It can be used as an anti-diabetic (particularly for diabetes prevention or treatment) composition or the like. As described in detail below, the composition of the present invention can contain, for example, a pharmacologically or food hygiene-acceptable carrier or the like. Further, as an ingestion form of the composition of the present invention, for example, it can be preferably used as an oral composition. In addition, the composition of the present invention can be preferably used, for example, as a pharmaceutical composition or a food composition.

In the present specification, the food composition is not only a normal fresh or processed food composition, but also a food additive composition such as a salt substitute, a sweetener, an additive to a beverage, etc., for commercial use or household use. Includes compositions that are widely consumed as food and drink, such as food material compositions such as premixed foodstuffs. In particular, when it is used as a food composition, it can be preferably used as a food composition whose action and effect on blood glucose are clearly indicated on the product label. Specifically, for example, food compositions whose actions and effects such as improvement of glucose metabolism, suppression of blood sugar level rise, and prevention of diabetes are clearly indicated on the product label, people with high blood sugar, people who are concerned about blood sugar, etc. are targeted. Examples include food compositions that clearly indicate that this is the case on the product label.

In addition, in this specification, a pharmaceutical composition includes a pharmaceutical product and a quasi-drug. When the composition of the present invention is used as a pharmaceutical composition, the composition of the present invention may contain other components, if necessary, in addition to the LRA peptide. The other components are not particularly limited and may be appropriately selected depending on the intended purpose. For example, pharmaceutically acceptable bases, carriers and / or additives (for example, solvents, dispersants, emulsifiers, etc.) Buffering agents, stabilizers, excipients, binders, disintegrants, lubricants, etc.) and the like. The blending amount of the other components can be appropriately set according to the purpose.

When the composition of the present invention is used as a pharmaceutical composition, its administration form is not particularly limited, and examples thereof include oral administration and transvascular administration (particularly intravenous injection). Of these, oral administration is preferable.

When the composition of the present invention is used as a pharmaceutical composition, its dosage form is not particularly limited, and for example, tablets such as orally disintegrating tablets, chewable tablets, effervescent tablets, dispersion tablets, dissolving tablets; troches; powders; Suspensions; emulsions; elixirs; limonades; syrups; lotions; granules; capsules such as hard capsules and soft capsules; creams; ointments; suppositories; paps, tapes, microneedles , Transdermal / mucosal administration agents such as iontophoresis and electroporation; aerosol agents and the like. These forms can be prepared by a conventional method in combination with the LRA peptide and, if necessary, the other components described above.

When the composition of the present invention is used as a pharmaceutical composition, the blending amount of the LRA peptide in the pharmaceutical composition is not particularly limited as long as it exhibits the above-mentioned action of the LRA peptide, and may be appropriately set. can.

Further, when the composition of the present invention is used as a pharmaceutical composition, the dose, administration interval, administration target and the like are not particularly limited and can be appropriately set. For example, the dose can be appropriately set according to the age, gender, body weight, health condition of the subject, and other conditions of the subject. Further, for example, the administration interval may be once or a plurality of times (preferably 2 to 3 times) per day, or may be once or a plurality of times every several days to several weeks. In addition, the administration target may be humans and non-human mammals such as pets or livestock (for example, dogs, cats, cows, horses, sheep, llamas, rats, mice, etc.).

In particular, when the administration target is a human, the LRA peptide administration (intake) amount is preferably, for example, about 10 to 300 μg, more preferably about 15 to 200 μg, still more preferably about 20 to 100 μg, and even more preferably about 30 to 30 μg per adult day. About 80 μg is even more preferable, about 35 to 70 μg is still more preferable, and about 40 to 60 μg is particularly preferable.

The administration (ingestion) period is not particularly limited, but is 3 to 12 weeks or longer (3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more). It is preferably 9 to 12 weeks or more, and more preferably 9 to 12 weeks or more. In particular, when the daily LRA peptide dose is about 30 to 80 μg, it is preferably 5 to 12 weeks or more, and when it is 10 μg or more and less than 30 μg or more than 80 μg and 300 μg or less, 9 It is preferably 12 weeks or more.

When the administration target is a non-human mammal, the administration form, dosage form, dose, administration interval and the like can be appropriately set with reference to the case where a human is the administration target.

When the composition of the present invention is used as a food composition, the composition of the present invention may contain other components, if necessary, in addition to the LRA peptide. The other ingredients are not particularly limited and can be appropriately selected according to the purpose. For example, bases, carriers, additives, and other ingredients / materials that can be used as foods, etc., which are acceptable in terms of food hygiene. Can be exemplified.

When the composition of the present invention is used as a food composition, the form of ingestion thereof is oral ingestion. In this case, the form of the composition of the present invention is not particularly limited, and it can be used for general foods, foods with health claims, and foods for special purposes. For example, foods for specified health use, foods with nutritional function, foods with functional claims, foods for the sick, foods for people with swallowing difficulties, health supplements, dietary supplements, hospital foods, nursing foods, processed foods, beverages, etc. may be mentioned. .. These can be prepared by a conventional method. The dosage form of the food composition includes, for example, hard capsules, soft capsules, supplements, chewable tablets, beverages, powdered beverages, granules, films, and the like, and when used as foods and drinks, for example, tea-based beverages. Dissolve or suspend in sports drinks, beauty drinks, fruit juice drinks, carbonated drinks, alcoholic drinks, soft drinks, jelly drinks, concentrated type drinks diluted with water or hot water, carbonated water, etc., water or hot water, etc. Drinkable powders and granules, dried solids such as tablets, tablet confectionery, jellies, snacks, baked confectionery, fried confectionery, cakes, chocolate, gum, candy, gummy and other confectionery, soups, noodles, rice and rice , Serials, etc. can also be made into food forms. Of these, in normal life, supplement types, chewable tablets, one-shot drink types and the like are preferable, and when ingested for the purpose of enhancing exercise effects, beverage forms such as sports drinks are also preferable. In particular, in the case of foods with health claims, health supplements, dietary supplements, etc., for example, granules, capsules, tablets and tablets (including chewable agents), beverages, etc., in order to facilitate continuous ingestion. (Beverage powder, drink agent, etc.), jelly agent, etc. are preferable.

Further, when the composition of the present invention is used as a food additive composition or a food material composition, its form is not particularly limited, and for example, it is in the form of liquid, powder, flakes, granules, or paste. Things can be mentioned. More specifically, seasonings (sweeteners, salt substitute compositions, soy sauce, vinegar, miso, sauces, ketchup, dressings, spices, herbs, etc.), flakes (sprinkle, rice cooking additives, etc.), roasted meat sauce, roux paste (Curry sauce paste, etc.), premixed foodstuffs, etc. can be mentioned.

When the composition of the present invention is used as a food composition, the blending amount of the LRA peptide in the food composition is not particularly limited as long as it exhibits the above-mentioned action of the LRA peptide, and may be appropriately set. can.

Further, when the composition of the present invention is used as a food composition, the intake amount, intake interval, intake target and the like are not particularly limited and can be appropriately set. For example, the intake amount can be appropriately set according to the age, sex, weight, health condition of the target, and other conditions of the target. For example, the intake interval may be once or multiple times (preferably 2 to 3 times) per day, or once or multiple times every few days to several weeks when the above-mentioned amount is ingested. In addition, the intake target may be humans and non-human mammals such as pets or livestock (for example, dogs, cats, cows, horses, sheep, llamas, rats, mice, etc.).

In particular, when the administration target is a human, the LRA peptide intake is preferably, for example, about 10 to 300 μg, more preferably about 15 to 200 μg, further preferably about 20 to 100 μg, and even more preferably about 30 to 80 μg per adult day. Even more preferably, about 35 to 70 μg is still more preferable, and about 40 to 60 μg is particularly preferable.

The intake period is not particularly limited, but is preferably 3 to 12 weeks or longer (3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks or more). , 9-12 weeks or more, more preferably. In particular, when the daily intake of LRA peptide is about 30 to 80 μg, it is preferably 5 to 12 weeks or more, and when it is 10 μg or more and less than 30 μg or more than 80 μg and 300 μg or less, 9 It is preferably 12 weeks or longer.

The human to be ingested is not particularly limited, but a human having a desire or need for improving glucose metabolism, suppressing an increase in blood glucose level, or preventing diabetes is preferable.

When the ingestion target is a non-human mammal, the morphology, ingestion amount, ingestion interval, etc. can be appropriately set with reference to the case where the ingestion target is human.

In addition, in this specification, "includes" also includes "consisting essentially" and "consisting of" (The term "comprising" includes "consisting essentially of" and "consisting of."). The present invention also includes all combinations of the constituent elements described herein.

In addition, the various properties (property, structure, function, etc.) described for each embodiment of the present invention described above may be combined in any way in specifying the subject included in the present invention. That is, the present invention includes all subjects consisting of any combination of each of the combinable properties described herein.

Hereinafter, the present invention will be described in more detail, but the present invention is not limited to the following examples.

Production Example 1: Synthesis of Tripeptide by Fmoc Method A tripeptide consisting of Leu-Arg-Ala was synthesized in solid phase by the Fmoc method. After purifying the obtained LRA peptide by HPLC, the sequence was analyzed by a protein sequencer, and as a result, it was confirmed that it was a tripeptide consisting of Leu-Arg-Ala.

Production Example 2: Synthesis of Tripeptide by Boc Method We requested Peptide Institute, Ltd. to synthesize a tripeptide consisting of Leu-Arg-Ala by the Boc method. The delivered LRA peptide was confirmed to be a tripeptide composed of Leu-Arg-Ala by RP-HPLC, mass spectrometry and amino acid analysis.

Example 1: Measurement of DPP-4 inhibitory activity DPP-4 inhibitory activity measurement kit: The DPP-4 inhibitory activity of the LRA peptide was measured using the DPP4 Activity Fluorometric Assay Kit (BioVision). The kit is a kit for measuring the enzymatic activity of DPP-4 by a fluorescence method, and more specifically, a labeled substrate (H-Gly-Pro-) to which AMC (7-Amino-4-Methyl Coumarin) is bound. AMC: DPP4 substrate) is a kit that can examine DPP-4 activity by measuring the fluorescence intensity generated by cleavage by DPP-4. As can be seen from this, when the DPP-4 activity is inhibited, the fluorescence intensity decreases.

The LRA peptide or sitagliptin (DPP-4 inhibitor: positive control included in the kit) synthesized in Production Example 1 above is diluted with the DPP4 assay buffer included in the kit, and 25 μl each is dispensed into a 96-well plate, and an additional 50 μl is added. The enzyme mix was added and incubated at 37 ° C. for 10 minutes. Then, 25 μl of reaction mix (23 μl DPP4 assay buffer and 2 μl DPP4 substrate) was added to each well, and the fluorescence intensity was measured with a plate reader (37 ° C./excitation wavelength 360 nm, fluorescence wavelength 460 nm).
In the dilution operation, 25 μl of the reaction mix was added to dilute the LRA peptide to 5000 μM when the sample volume in the well reached 100 μl. In addition, sitagliptin was diluted to 0.3 μM when the sample volume in the well reached 100 μl by adding 25 μl of the same reaction mix.

In addition, the same operation was performed on a sample prepared to have an LRA peptide of 0 μM (that is, DPP4 assay buffer only) and a dipeptide (YY peptide) consisting of an amino acid sequence: Tyr-Tyr (synthesized in the same manner as the LRA peptide) of 5000 μM. The fluorescence intensity was measured.

The results are shown in FIG.

Claims (3)

A composition containing a tripeptide consisting of Leu-Arg-Ala.
A composition for inhibiting DPP-4, improving glucose metabolism, suppressing an increase in blood glucose level, or for antidiabetes. The composition according to claim 1, which is an oral composition. The composition according to claim 1 or 2, which is a food composition or a pharmaceutical composition.

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