JP7315161B2 - peptide - Google Patents

Description

The present invention relates to ghrelin secretagogues, pharmaceutical compositions for stimulating appetite, pharmaceutical compositions for stimulating growth hormone secretion, and methods for stimulating appetite and/or stimulating growth hormone secretion.

Ghrelin is a gastrointestinal hormone that has appetite stimulating and growth hormone secretion promoting effects. In Japan, which is facing a super-aging society, development of a ghrelin secretagogue that contributes to improvement of age-related anorexia and decrease in growth hormone secretion is desired.

Patent Document 1 describes a low-molecular-weight peptide that activates the ghrelin receptor. Patent Literature 2 describes a growth hormone secretagogue composition derived from soy protein.

Japanese Patent Application Publication No. 2005-082489 International publication WO2017/150548

An object of the present invention is to provide a novel ghrelin secretagogue, a pharmaceutical composition for promoting appetite, a pharmaceutical composition for promoting growth hormone secretion, and a method for promoting appetite and/or growth hormone secretion.

The present inventors have made intensive studies to solve the above problems. As a result, it was found that a 2- to 20-amino acid peptide having an amino acid sequence SX (X represents an amino acid residue selected from V, L, I, M, F, W, Y, S, T, N, K, R and H) has ghrelin secretagogue activity. The present invention has been completed through further studies based on such findings.

That is, the present invention includes the following aspects.

Item 1, a ghrelin secretagogue comprising, as an active ingredient, a peptide having a length of 2 to 20 amino acids and having an amino acid sequence SX (X represents an amino acid residue selected from V, L, I, M, F, W, Y, S, T, N, K, R and H).

Item 2, A pharmaceutical composition for stimulating appetite, comprising a peptide of 2 to 20 amino acids in length having an amino acid sequence SX (X is an amino acid residue selected from V, L, I, M, F, W, Y, S, T, N, K, R and H) as an active ingredient.

Item 3, A pharmaceutical composition for promoting the secretion of growth hormone, comprising as an active ingredient a peptide of 2 to 20 amino acids having an amino acid sequence SX (X represents an amino acid residue selected from V, L, I, M, F, W, Y, S, T, N, K, R and H).

Item 4. A method for promoting appetite and/or growth hormone secretion, comprising the step of administering an effective amount of a peptide having a length of 2 to 20 amino acids and having an amino acid sequence SX (X is an amino acid residue selected from V, L, I, M, F, W, Y, S, T, N, K, R and H) to a patient in need thereof or a preliminary group.

Item 5, Use of a peptide of 2 to 20 amino acids in length having an amino acid sequence SX (X represents an amino acid residue selected from V, L, I, M, F, W, Y, S, T, N, K, R and H) for promoting appetite and/or secretion of growth hormone.

The ghrelin secretagogue and the pharmaceutical composition of the present invention exhibit appetite stimulating action and growth hormone secretion promoting action based on the secretagogue action of ghrelin. Based on these actions, it can be suitably used for the prevention and treatment of anorexia, decreased muscle mass, and locomotive syndrome. The ghrelin secretagogue and pharmaceutical composition of the present invention have low side effects and are suitable for long-term administration. In addition, the ghrelin secretagogue and pharmaceutical composition of the present invention are effective when administered orally.

A method for evaluating ghrelin secretory activity is schematically shown. 2 shows the evaluation results of ghrelin secretory activity. (A) Evaluation results of dipeptides SV, SF and SW, (B) evaluation results of dose dependence of dipeptide SV, and (C) evaluation results of constituent amino acids of dipeptide SV are shown. The left diagram schematically shows the known ghrelin secretagogue pathway. (A) shows the measurement results of intracellular cAMP concentration when ghrelin secretion is stimulated by dipeptide SV. (B) shows the measurement results of intracellular calcium ion concentration when ghrelin secretion was stimulated by dipeptide SV. This figure shows the effect of stimulating the feeding by dipeptide SV administration. Fig. 2 shows the effect of increasing blood ghrelin concentration by administration of dipeptide SV.

The peptide, which is the active ingredient in the present invention, has an amino acid sequence SX (X is an amino acid residue selected from V (valine), L (leucine), I (isoleucine), M (methionine), F (phenylalanine), W (tryptophan), Y (tyrosine), S (serine), T (threonine), N (asparagine), K (lysine), R (arginine), and H (histidine) and has a length of 2 to 20 amino acid residues). is a peptide of

The peptide preferably has 2 to 20 amino acid residues, more preferably 2 to 10 amino acid residues, even more preferably 2 to 5 amino acid residues, particularly preferably 2, 3, 4 or 5 amino acid residues.

When the peptide has an amino acid residue length of 3 or more, it has arbitrary amino acid residues on the N-terminal side and/or C-terminal side of the amino acid sequence NX. Amino acid residues may be natural or non-natural amino acids. Natural amino acids include the proteinogenic amino acid residues of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, as well as other amino acid residues such as selenocysteine, N-formylmethionine, pyrrolysine, pyroglutamine, and the like.

In one of the preferred embodiments of the invention said peptide is a dipeptide SV.

Amino acids constituting peptides may be L-form amino acids, D-form amino acids, or DL-form amino acids (if the D-form and L-form amino acids are mixed, both the racemic form and the amino acid in excess of one of the enantiomers are included). Peptides consisting of only L-amino acids or only D-amino acids, particularly peptides consisting of only L-amino acids, are preferred.

Moreover, when the peptide used in the present invention contains two or more asymmetric carbon atoms, it may be in the form of each enantiomer or diastereomer, or a mixture thereof at any ratio. Enantiomers or diastereomers can be separated by any known method, such as a method using an ordinary column, a method of using an optically active column, a method of introducing an optically active group and optically resolving it in the form of a derivative, followed by removing the optically active group, or a method of optically resolving by forming a salt with an optically active acid or base.

Peptides can have modifications. The amino terminus (N-terminus) of the peptide may be a free amino group (NH ₂ —) or modified such as an acetyl group (CH ₃ CO—). The carboxy terminus (C terminus) of the peptide may be a free carboxyl group (--COOH) or modified such as an amide group. The amino acid residues of the peptide may be unmodified or may have modifications such as phosphate groups and sugar chains.

The peptides of the present invention may be salts (acid addition salts or base salts). 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. Base salts include alkali metal salts such as sodium, potassium and lithium, alkaline earth metal salts such as calcium and magnesium, and the like.

The peptides of the invention may be solvates. Solvates include solvates of water (when hydrated), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethylsulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, dimethoxyethane, and the like.

Also, the peptide of the present invention can be obtained, for example, by a peptide synthesis method. That is, a raw material having a reactive carboxyl group and a raw material having a reactive amino group can be condensed by a conventional method in peptide synthesis, such as a method using an active ester such as HBTU or a method using a coupling agent such as carbodiimide, by a liquid phase method or a solid phase method, which are methods commonly used in peptide synthesis. When the resulting condensate has a protecting group, it can also be produced by removing the protecting group.

Functional groups that should not participate in the reaction in this reaction step are protected by protective groups. Examples of amino-protecting groups include benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of carboxyl group-protecting agents include groups capable of forming alkyl esters, benzyl esters, etc. In the case of the solid-phase method, the C-terminal carboxyl group is bound to a carrier such as chlorotrityl resin, chloromethyl resin, oxymethyl resin, p-alkoxybenzyl alcohol resin, and the like. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.

After completion of the condensation reaction, the protective group is removed, and in the case of the solid-phase method, the bond between the C-terminus of the peptide and the resin is further cleaved. Furthermore, the peptides of the invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reversed phase liquid chromatography, affinity chromatography and the like. Synthesis of the synthesized peptide is analyzed by a protein sequencer, GC-MS, etc., which reads the amino acid sequence from the C-terminus by the Edman degradation method.

The peptide of the present invention can also be synthesized by an enzymatic method (see WO2003/010307).

The peptide of the present invention has the effect of promoting the secretion of ghrelin. Ghrelin is a biologically derived peptide having the following structure. Specifically, it consists of 28 amino acid residues, and the third serine residue is modified with n-octanoic acid. Ghrelin exhibits appetite stimulating action and growth hormone secretion promoting action.

Therefore, the peptide of the present invention that promotes the secretion of ghrelin also has actions based on the promotion of ghrelin secretion, such as an appetite-stimulating action (feeding promoting action) and a growth hormone secretion-promoting action. Based on their appetite-stimulating action (orexia-promoting action), the peptides of the present invention can be used, for example, for the treatment of anorexia in the elderly. It can be used for prevention of muscle loss, prevention or treatment of locomotive syndrome (locomotive syndrome), etc., based on its growth hormone secretion promoting action.

The promotion of ghrelin secretion by the peptide of the present invention can be evaluated by a method known to those skilled in the art. For example, the amount of ghrelin secreted can be measured by adding a test substance to ghrelin-secreting cells MGN3-1 described in the literature: Iwakura H et.al. Endocrinology. Ghrelin can be quantified by an immunochemical technique such as ELISA (Enzyme-Linked ImmunoSorbent Assay).

The peptide of the present invention can be provided as a pharmaceutical composition or food (food composition).

The administration route of the peptide of the present invention or a product containing the same is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted, and can be administered orally or parenterally. Among them, oral administration is preferable from the viewpoint of high effects.

The dosage of the present peptide varies depending on the administration method, the condition and age of the recipient, etc., but is usually 0.01 mg/kg to 500 mg/kg, preferably 0.05 mg/kg to 100 mg/kg, more preferably 0.1 to 30 mg/kg per day for adults. The peptide (active ingredient) of the present invention can be administered in the form of a pharmaceutical composition prepared by mixing with pharmaceutical carriers. As a pharmaceutical carrier, a substance commonly used in the pharmaceutical field and which does not react with the peptide of the present invention is used.

The peptide of the present invention can be used as a medicine or food per se, or can be used alone or together with an appropriate non-toxic carrier for oral intake, diluent or excipient, tablet (uncoated tablet, sugar-coated tablet, effervescent tablet, film-coated tablet, chewable tablet, etc.), capsule, troche, powder, fine granule, granule, liquid formulation, suspension, emulsion, paste, cream, injection (including cases where it is mixed with infusion such as amino acid infusion, electrolyte infusion), or enteric tablet, capsule, It can be made into food or pharmaceutical formulations such as sustained release formulations such as granules. The peptide content in food can be selected as appropriate, but is generally in the range of 0.01 to 100% by weight.

Specifically, examples of substances such as pharmaceutical carriers or oral ingestion carriers, diluents or excipients that can be added to pharmaceuticals or foods include lactose, glucose, mannite, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, carboxymethylcellulose sodium, hydroxypropyl starch, carboxymethylcellulose calcium, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, and light anhydrous. Silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, refined lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water, and the like.

Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like. These formulations are prepared according to a conventional method. Liquid formulations may be dissolved or suspended in water or other suitable solvents at the time of use. Moreover, tablets and granules may be coated by a known method. Injections are prepared by dissolving the peptide of the present invention in water, but if necessary, they may be dissolved in physiological saline or glucose solution, and buffers and preservatives may be added.

These formulations may contain the peptides of the invention in proportions of 0.01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other therapeutically valuable ingredients.

In order to produce a solid preparation for oral administration, the active ingredient and excipients such as lactose, starch, crystalline cellulose, calcium lactate, silicic anhydride, etc. are mixed to form a powder, or if necessary, a binder such as sucrose, hydroxypropylcellulose, polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose, carboxymethylcellulose calcium, etc. are added and wet or dry granulated to obtain granules. Tablets can be produced by compressing these powders and granules as they are or after adding a lubricant such as magnesium stearate and talc. These granules or tablets can be coated with an enteric base such as hydroxypropyl methylcellulose phthalate, methacrylic acid-methyl methacrylate polymer to form an enteric preparation, or ethylcellulose, carnauba wax, hydrogenated oil or the like to form a sustained release preparation. In addition, in order to manufacture capsules, powders or granules are filled into hard capsules, or active ingredients are coated with a gelatin film as they are or after being dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. to form soft capsules.

Liquid preparations for oral administration may be prepared by dissolving the active ingredient and a sweetening agent such as sucrose, sorbitol, or glycerin in water to prepare a clear syrup, further adding essential oils, ethanol, or the like to prepare an elixir, or adding gum arabic, tragacanth, polysorbate 80, sodium carboxymethylcellulose, or the like to prepare an emulsion or suspension. Flavoring agents, coloring agents, preservatives and the like may be added to these liquid preparations as desired.

Specific forms of foods that can be prepared by adding or blending the peptide according to the present invention include, for example, beverages (coffee, cocoa, juice, soft drinks, mineral drinks, tea drinks, green tea, black tea, oolong tea, milk drinks, lactic acid beverages, yogurt drinks, carbonated drinks, other non-alcoholic drinks, alcoholic beverages, etc.), confectionery (hard candy, gum, gummy, jelly, pudding, mousse, cake, candy, cookie, cracker, biscuit, chocolate, ice cream, popsicle, sherbet, shaved ice, etc.), sprinkles, dressings, seasonings, processed soybean foods (tofu, miso, soy sauce, yuba, soybean flour, natto, etc.), processed meat foods (hamburgers, meatloaf, meatballs, meatballs, etc.), processed fish foods (kamaboko, chikuwa, etc.), retort foods, jelly-like foods (jelly, agar, jelly-like drinks, etc.), and the like. Foods that can be prepared by adding or blending the peptide of the present invention include so-called health foods, functional foods, foods with functional claims, dietary supplements, supplements, foods for specified health uses, foods for sick people/combined foods for sick people (Ministry of Health, Labor and Welfare, a type of food for special dietary uses), or foods for the elderly (a type of food for special dietary uses, Ministry of Health, Labor and Welfare), uncoated tablets, film-coated tablets, sugar-coated tablets, granules, powders, tablets, capsules (both hard capsules and soft capsules), chewable types, syrup types, and drinks. It can also be a type. Foods to which the peptide according to the present invention is added and/or blended can be prepared by a method known per se.

The peptide according to the present invention can be used as a lead compound for active ingredients of pharmaceuticals for stimulating appetite and/or stimulating secretion of growth hormone. By adding necessary chemical modifications to the peptide according to the present invention, it is possible to obtain an active pharmaceutical ingredient for stimulating appetite and/or promoting secretion of growth hormone, which ultimately satisfies stability, safety, and the like.

The peptide according to the present invention can also be used as a control in screening active ingredients of pharmaceuticals for stimulating appetite and/or stimulating growth hormone secretion. Specifically, the activity of promoting ghrelin secretion of a test substance to be screened is evaluated, and if the activity of promoting ghrelin secretion is higher than that of the peptide according to the present invention, the test substance can be selected as an active ingredient of a drug for promoting appetite and/or growth hormone secretion.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples. However, the following examples are not intended to limit the scope of the invention.

<Method>
(Evaluation of ghrelin secretory activity)
Ghrelin-secreting cells MGN3-1 were seeded in a 96-well plate at 1 x ¹⁰⁵ cells/well and cultured in the medium for 24 hours. After culturing, the cells were washed with DPBS, and 100 μL of test substance (Buffer: 50 μM sodium octanoate/DMEM) was added. Buffer to which no test substance was added was used as a control. After further culturing for 4 hours, the medium was collected and centrifuged to obtain a supernatant. 10 μL of 1N HCl was added to the supernatant and stored at -80°C.

The concentration of ghrelin (acylated ghrelin) in the sample was quantitatively evaluated by an ELISA method (Bertin Pharma, Ghrelin (Acylated) EIA Kit A05117).

The evaluation method is schematically shown in FIG.

(test substance (peptide))
Dipeptides SV, SL, SI, SM, SF, SW, SY, SS, ST, SN, SK, SR and SH synthesized by a conventional method were used as test substances.

(Statistical analysis)
The data obtained by the test was expressed as the sum of the mean (Mean) of the number of trials n and the standard error of the mean (SEM). A t-test was used for comparison between two groups. For comparisons between three or more groups, data were analyzed by one-way ANOVA followed by Tukey-Kramer test for multiple comparisons. When p<0.05 ("*" in the figure), it was determined that there was a significant difference.

<Test example>
Test Example 1: Evaluation of ghrelin secretory activity (1)
The effect of the sample dipeptide on ghrelin secretion was evaluated using ghrelin-secreting cell MGN3-1.

The results are shown in Table 1 (n=1). Values are ratios to control ghrelin concentrations.

It was found that the tested dipeptides have the activity of increasing ghrelin secretion and promoting ghrelin secretion.

Test Example 2: Evaluation of ghrelin secretory activity (2)
(A) Dipeptides SV, SF and SW were evaluated for their effects on ghrelin secretion with n=8 trials. The test was conducted in the same procedure as in Test Example 1.

The results are shown in Figure 2A. In the figure, the horizontal axis "Ghrelin (% of control)" indicates the ratio (percentage) to the measured value of ghrelin secretion when control (only buffer without test substance added) was added to MGN3-1. As with the results obtained in Test Example 1, it was revealed that the dipeptides SV, SF and SW have the activity of promoting ghrelin secretion.

(B) The test was performed in the same manner as in Test Example 1, with dipeptide SV concentrations of 100 μM, 300 μM and 1000 μM in the sample (n=4).

The results are shown in Figure 2B. It was revealed that the dipeptide SV has a dose (concentration)-dependent activity of promoting ghrelin secretion.

(C) Amino acids S, V, and a mixture of S and V (S+V) were tested in the same manner as in Test Example 1 (n=4).

The results are shown in Figure 2C. The dipeptide SV has the activity of promoting ghrelin secretion, but the constituent amino acids S and V alone and the mixture of S and V did not have the activity of promoting ghrelin secretion.

Test Example 3 Mechanism of Action The mechanism of action leading to promotion of ghrelin secretion was verified. The effects of peptide SV on intracellular cAMP concentration and intracellular calcium ion concentration ([Ca ²⁺ ]) were evaluated.

For measurement of intracellular cAMP concentration, ghrelin-secreting cells MGN3-1 were seeded in a 96-well plate at 1 x ¹⁰⁵ cells/well and cultured in the medium for 24 hours. After culturing, the cells were washed with DPBS, and 45 μL of test substance (Buffer: Krebs-Ringer-HEPES buffer containing 0.5 mM IBMX) was added. After culturing for another 30 minutes, intracellular cAMP was quantified using HitHunter cAMP Assay for small molecules (DiscoveRx).

The intracellular calcium ion concentration was measured according to the method described in literature: Kagebayashi T et.al. Mol. Nutr. Food Res. 2012 Sep;56(9):1456-63.

The results are shown in FIG. Noradrenaline (NA) is known to promote ghrelin secretion through an increase in intracellular cAMP concentration and intracellular calcium ion concentration. Trimeric G proteins of the Gs type are known to be involved. In contrast, the dipeptide SV promotes ghrelin secretion without changes in intracellular cAMP concentration and intracellular calcium ion concentration, and thus is thought to be through a pathway different from the known pathways.

Test Example 4: In vivo feeding-promoting effect and ghrelin-secreting effect Dipeptide SV was administered to mice (ddY mice, male, 34-42 g), and effects on food intake and blood ghrelin concentration were evaluated. Specifically, dipeptide SV dissolved in physiological saline was orally administered (po) to mice at a dose of 0.3 mg/kg or 1.0 mg/kg. Mice receiving saline alone served as controls. Food intake was calculated by feeding pre-weighed solid feed (CE-2, CLEA Japan) after SV administration and measuring the feed weight over time (0, 20, 60, 120 and 240 minutes after administration). Regarding blood ghrelin concentration (Plasma acetylated ghrelin), food was removed after SV administration, and serum ghrelin concentration was measured 2 hours (120 minutes) later.

The results are shown in Figures 4A and 4B. Oral administration of the dipeptide SV was found to increase food intake and blood ghrelin levels in mice.

Claims (2)

A ghrelin secretagogue containing dipeptide SV and/or SF as an active ingredient. A pharmaceutical composition or food composition for promoting appetite or growth hormone secretion, comprising dipeptide SV and/or SF as an active ingredient.

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