JP5491133B2 - Dipeptidyl peptidase IV inhibitor - Google Patents

Description

  The present invention relates to a dipeptidyl peptidase IV inhibitor containing crocetin or a pharmacologically acceptable salt thereof as an active ingredient.

  Dipeptidyl peptidase IV (hereinafter also referred to as DPP4) specifically binds to a peptide containing proline (or alanine) second from the N-terminus, and cleaves the C-terminal side of the proline (or alanine) to cleave the dipeptide. It is a serine protease that is produced. DPP4 is expressed on epithelial and endothelial cells of a very wide range of tissues (eg, intestine, liver, lung, kidney and placenta), and on T cells, and activates T cells and becomes endothelial cells of cancer cells. It is thought to play an important role in adhesion of HIV, invasion of HIV into cells, metabolism of neuropeptides, and the like.

  On the other hand, diseases that can be improved by inhibiting DPP4 include arthritis, autoimmune diseases such as rheumatoid arthritis, acquired immune deficiency syndrome, transplant organ / tissue rejection, breast tumor and prostate tumor metastasis to the lung, It is thought to have skin diseases such as psoriasis and lichen planus, decreased pancreatic function, postprandial hyperglycemia, and impaired glucose tolerance. For this reason, various compounds having a DPP4 inhibitory action have been proposed so far.

  For example, 2- {6- [3 (R) -amino-piperidin-1-yl] -3-ethyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethyl} -benzonitrile TFA Salts (see Patent Document 1), dipeptide compounds formed from amino acids and thiazolidine groups or pyrrolidine groups and salts thereof (see Patent Document 2), compounds having a dipeptidyl peptidase IV inhibitory activity and having a tetrahydroisoquinoline skeleton or the like A pharmaceutical composition (see Patent Document 3) containing a pharmacologically acceptable salt as an active ingredient has been proposed.

  On the other hand, crocetin, which is a kind of carotenoid and extracted from plants with edible experience such as gardenia fruit and saffron pistil, is known to be used as an eye strain improving agent (see Patent Document 4). However, there is no report that it is useful as a DPP4 inhibitor.

JP 2007-314551 A JP-T-2002-516318 Japanese Patent Laid-Open No. 10-182613 JP 2007-31426 A

  An object of the present invention is to provide a novel dipeptidyl peptidase IV inhibitor using an extract derived from a plant with edible experience.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that crocetin, which is a kind of carotenoid pigment, has a DPP4 inhibitory action, and has completed the present invention.
That is, the present invention comprises a dipeptidyl peptidase IV inhibitor characterized by containing crocetin represented by the following formula (1) or a pharmacologically acceptable salt thereof as an active ingredient.

The dipeptidyl peptidase IV inhibitor of the present invention has excellent dipeptidyl peptidase IV inhibitory activity.
The dipeptidyl peptidase IV inhibitor of the present invention can be used for the prevention and treatment of diseases improved by inhibiting dipeptidyl peptidase IV.

  Crocetin used in the present invention is a compound (molecular weight 328.40) represented by the following formula (2).

  This crocetin is usually obtained by hydrolyzing crocin (a digentiobiose ester of crocetin), which is a carotenoid yellow pigment. Crocin is contained in the fruits of Rubiaceae gardenia (Gardenia augusta MERILIL var. Grandiflora HORT., Gardenia jasminoides ELLIS), dried saffron stigmas, and the like. Gardenia fruit is preferably used as an industrial raw material for obtaining crocin.

  There is no restriction on the method of extracting crocin from the gardenia fruit, for example, a dried gardenia fruit is pulverized and extracted using water, alcohol (for example, methanol, ethanol, etc.) or a mixture thereof. The method is used. For example, when the water / alcohol mixed solution (1: 1) is used, the extraction condition is preferably room temperature (about 0 to 30 ° C.) to 50 ° C. for about 1 to 18 hours, and about 30 to 40 ° C. for about 2 to 4 hours. Is more preferable. In order to further increase the extraction rate of crocin from the pulverized dried fruit, the extraction operation is usually repeated a plurality of times. The extract containing crocin is concentrated by a method known per se, and is usually stored refrigerated as a concentrated solution.

  The hydrolysis of crocin may be performed according to a conventional method, and is usually performed using an acid, an alkali or a suitable hydrolase. Examples of the acid include hydrochloric acid, sulfuric acid, and phosphoric acid. Examples of the alkali include sodium hydroxide, potassium hydroxide, sodium carbonate and potassium carbonate. Examples of hydrolases include β-glucosidase.

  Industrially, crocin is usually hydrolyzed using alkali. For example, an excess amount of an aqueous sodium hydroxide solution is added to the concentrate containing crocin, and preferably at room temperature (about 0 to 30 ° C.) to 70 ° C. for about 1 to 24 hours, preferably about 40 to under stirring. React at 60 ° C. for about 3-5 hours.

  After completion of hydrolysis with an alkali, an appropriate amount of an aqueous solution of an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as citric acid is added to the reaction solution, and the liquidity is about pH 4.0 or less, preferably about pH 1. Crystals of crocetin are precipitated by adjusting to 0 to 3.0. Alternatively, the reaction solution may be added to an aqueous solution of an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as citric acid to precipitate crocetin crystals. Thereafter, the liquid mixture containing the crocetin crystals is subjected to solid-liquid separation to obtain a suspension or slurry containing the crocetin crystals.

  When crocin is hydrolyzed using an acid, crocetin crystals are usually precipitated simultaneously with the hydrolysis, so that the reaction solution is obtained as a suspension containing crocetin. After completion of the reaction, the resulting suspension is subjected to solid-liquid separation to obtain a suspension or slurry containing crocetin crystals.

  The suspension or slurry containing the crocetin crystals obtained as described above contains crocetin crystals together with acids, neutralized salts, and impurities derived from the raw materials. For the purpose of removing these, A cleaning process is performed. The treatment may be performed by a known method such as washing the suspension or slurry with a sufficient amount of water, alcohol or a mixture thereof. The washing process is usually repeated several times until the desired purity is obtained. After the washing treatment, the suspension or slurry containing crocetin crystals is dried at a temperature not exceeding about 50 ° C. using, for example, a vacuum dryer to obtain purified crocetin. The purified crocetin is preferably sealed and stored in a container substituted with an inert gas such as nitrogen gas.

  The content of crocetin used in the present invention is calculated based on the following formula from the color value of the sample containing crocetin.

  In addition, the said color value is measured based on the following [color value measuring method].

[Color value measurement method]
1) A sample is accurately weighed so that the absorbance to be measured is in the range of 0.3 to 0.7, and dissolved in dimethyl sulfoxide (DMSO) to make exactly 100 ml.
2) Weigh exactly 5 ml, and add Kolthoff buffer solution (50 mM Na ₂ B ₄ O ₇ .10H ₂ O-50 mM Na ₂ CO ₃ , pH 10.0) to make 50 ml.
3) Weigh exactly 5 ml of the solution, and add Kolthoff buffer (pH 10.0) to make 50 ml.
4) Weigh exactly 5 ml, add Kolthoff buffer (pH 10.0) to make 50 ml, and use this solution as the test solution.
5) Using a Kolthoff buffer solution (pH 10.0) as a control, measure the absorbance A at the maximum absorption portion near 420 nm with a liquid layer length of 1 cm, and obtain the color value by the following formula.

  In the present invention, pharmacologically acceptable salts of crocetin include, for example, salts of group 1 elements such as sodium and potassium, salts of group 2 elements such as magnesium and calcium, pyridine, dimethylamine, diethylamine, Examples thereof include pharmaceutically acceptable salts of organic amino compounds such as ethanolamine.

  The dipeptidyl peptidase IV inhibitor of the present invention is prepared by mixing the above crocetin or a pharmacologically acceptable salt thereof as it is, or appropriately adding a pharmaceutical additive, a food additive, a food material, etc. For example, drinks, etc.), powders, granules, tablets, microcapsules, soft capsules, hard capsules, oil and fat compositions, O / W emulsions, W / O emulsions or solubilized liquids, and the like. .

  Examples of pharmaceutical additives, food additives, and food materials used in the preparation of the above preparations include excipients (lactose, dextrin, corn starch, crystalline cellulose, etc.), lubricants (magnesium stearate, sucrose fatty acid ester, glycerin). Fatty acid esters, etc.), disintegrating agents (carboxymethylcellulose calcium, anhydrous calcium hydrogen phosphate, calcium carbonate, etc.), binders (starch glue solution, hydroxypropyl cellulose solution, gum arabic solution, etc.), solubilizers (gum arabic, polysorbate 80) Etc.), sweeteners (sugar, fructose glucose liquid sugar, honey, aspartame, etc.), coloring agents (β-carotene, edible tar dye, riboflavin, etc.), preservatives (sorbic acid, methyl parahydroxybenzoate, sodium sulfite, etc.), Thickener (alginic acid Thorium, sodium carboxymethylcellulose, sodium polyacrylate, etc.), antioxidants (BHT, BHA, ascorbic acid, tocopherol, etc.), flavors (mint, strawberry flavor, etc.), acidulants (citric acid, lactic acid, DL-malic acid, etc.) ), Seasoning (DL-alanine, sodium 5'-inosinate, sodium L-glutamate, etc.), emulsifier (glycerin fatty acid ester, sucrose fatty acid ester, etc.), pH adjuster (citric acid, trisodium citrate, etc.), Vitamins, minerals, amino acids and the like can be mentioned.

  In the case of the above preparation, the content of crocetin or a pharmacologically acceptable salt thereof is usually about 0.0001 to 50% by mass, preferably about 0.0001 to 50% by mass in terms of 100% by mass of crocetin in 100% by mass of the formulation, preferably About 0.001-20 mass%, More preferably, it is about 0.01-10 mass%.

  Furthermore, the dipeptidyl peptidase IV inhibitor of the present invention can take the form of food and drink. Examples of the food and drink include soft drinks, drops, candy, chewing gum, chocolate, gummy, yogurt, ice cream, pudding, jelly confectionery, and cookies.

  In the case of the above food and drink, the content of crocetin or a pharmacologically acceptable salt thereof is usually about 0.00003 to 10% by mass in terms of 100% by mass of crocetin in 100% by mass of the food and drink, Preferably it is about 0.01-5 mass%.

  When the above preparation and food and drink are taken orally, the daily dose of crocetin or a pharmacologically acceptable salt thereof is about 0.1 to 500 mg in terms of crocetin with a purity of 100% by mass. is there.

  Since the dipeptidyl peptidase IV inhibitor of the present invention is excellent in dipeptidyl peptidase IV inhibitory activity, autoimmune diseases such as arthritis and rheumatoid arthritis, acquired immune deficiency syndrome (AIDS), tumor metastasis, psoriasis and flattening It can be used for the prevention and treatment of skin diseases such as lichen, and for the purpose of promoting insulin secretion, improving pancreatic function, improving postprandial hyperglycemia, improving glucose tolerance, improving insulin resistance and the like.

  Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited thereto.

[Preparation of dipeptidyl peptidase IV inhibitor]
To 150 g of pulverized dried fruit of gardenia, 300 ml of a methanol / water mixture (1: 1) was added, and the mixture was stirred at room temperature for 3 hours, followed by suction filtration. The operation of adding 300 ml of methanol / water mixture (1: 1) to the residue and stirring for 30 minutes at room temperature followed by suction filtration was repeated twice to obtain a total of about 900 ml of extract as filtrate. This extract was concentrated at 60 ° C. under reduced pressure using a rotary evaporator to obtain about 50 g of a concentrate containing crocin.
The obtained concentrate and 17 g of 40% by mass aqueous sodium hydroxide solution were mixed and subjected to a hydrolysis reaction at 50 ° C. for 3.5 hours with stirring. After completion of the reaction, the reaction solution was acidified by adding it to 420 ml of a 4% by mass phosphoric acid aqueous solution, and then left at room temperature for about 3 hours. The deposited precipitate was collected by centrifugation (10,000 × g, 10 minutes), and further washed and centrifuged with 100 ml of water twice. The obtained pasty solid was vacuum-dried at 50 ° C. for 8 hours to obtain about 1.2 g of crocetin.
To 1 g of the obtained crocetin, 18 ml of dimethylformamide was added and dissolved at 80 ° C. Insoluble matter was filtered through quantitative filter paper (No. 5C, Advantech Toyo Co., Ltd.), and the filtrate was left at 10 ° C. for 3 days. Next, the mother liquor containing the produced crocetin crystals was added to a glass filter No. After filtration with 3 and washing with 20 ml of methanol, the crystals were vacuum dried at 50 ° C. to obtain about 0.16 g of a dipeptidyl peptidase IV inhibitor consisting of purified crocetin.

[Test example]
[DPP4 inhibitory activity measurement test]
Measurement of DPP4 inhibitory activity was carried out by the following method using DDPIV Drug Discovery kit (manufactured by BIOMOL) according to the protocol of the kit. The kit contains a DPP4 solution, a glycyl-proline-7-amino-4-methylcoumarin (H-Gly-Pro-AMC) solution, and isoleucylthiazolidine (P32 / 98).

  To a 96-well microplate, 25 μl of measurement buffer (50 mM Tris, pH 7.5) is added, followed by 15 μl of DPP4 solution (17.3 μU / μl) and various concentrations (10, 50, 100, 200, 400 μM) of test substance After adding 10 μl of the solution and mixing, it was allowed to stand at 37 ° C. for 10 minutes. Thereafter, 50 μl of glycyl-proline-7-amino-4-methylcoumarin (H-Gly-Pro-AMC) solution (0.5 mM) was added and mixed as a substrate, and the mixture was heated to 37 ° C. to initiate the reaction. Ten minutes after the start of the reaction, AMC released by DPP4 activity was measured for fluorescence intensity (excitation wavelength: 380 nm, fluorescence wavelength: 460 nm) using a microplate reader (INFINEITE 200; manufactured by TECAN). In addition, it replaced with the test substance solution and performed the same measurement using the measurement buffer, The result was used as control. Moreover, it replaced with the test substance solution and DPP4 solution, performed the same measurement using the measurement buffer solution, and made the result the blank. Based on the measurement results and the following formula, the inhibition rate of DPP4 activity was calculated for each final concentration (1, 5, 10, 20, 40 μM) of the added test substance.

Based on these inhibition rates, a graph is prepared by plotting the final concentration of the test substance on the horizontal axis and the DPP4 inhibition rate on the vertical axis. From this graph, the concentration of the test substance (IC ₅₀ value when inhibiting DPP4 activity by 50%) ) Was calculated. For comparison, a known isoleucyl thiazolidine as a compound showing excellent DPP4 inhibitory activity was tested in the same manner, and an IC ₅₀ value was calculated. The results are shown in Table 1.

  From the results of Table 1, it is clear that the dipeptidyl peptidase IV inhibitor of the present invention has dipeptidyl peptidase IV inhibitory activity comparable to that of isoleucyl thiazolidine by containing crocetin as an active ingredient.

Claims (1)

Below (1) crocetin the formula or dipeptidyl peptidase IV inhibitor characterized by containing a pharmacologically acceptable cormorants Ru salt thereof as an active ingredient, (however, autoimmune diseases, skin diseases, diabetes Except those used for the prevention or treatment of cancer or insulin resistance) .