JP2008280291A - Dipeptidyl peptidase iv inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DPP4 inhibitor that may improve insulin-secreting ability and is highly safe and daily ingestible. <P>SOLUTION: The dipeptidyl peptidase IV inhibitor comprises hydrolyzable tannin, where the hydrolyzable tannin bears a valoneoyl group or the hydrolyzable tannin is rugosin A of a specific structure or eugeniflorin D2 of a specific structure. As the DPP4 inhibitor not only exhibits high inhibiting activity equal to that of well-known inhibitors but also is excellently safe, it promotes protection and proliferation of β-cells by incretin and therefore exhibits remarkable effects for prevention and treatments, particularly as a preventive agent, of diseases such as diabetes in mammals to which the DPP4 inhibitors are applicable. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to dipeptidyl peptidase IV inhibitors. More specifically, the present invention relates to a novel dipeptidyl peptidase IV inhibitor that can be used in the prevention and treatment of target diseases such as diabetes.

  Dipeptidyl peptidase IV (hereinafter abbreviated as DPP4) is a post-proline / alanine cleaved serine protease found in various organs and tissues. DPP4 has a variety of peptide hormones including glucagon-like polypeptide (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), glucagon-like polypeptide (GLP-2), and gastrin releasing polypeptide (GRP). It is thought to act on and regulate its activity.

  Diseases that can be improved by inhibiting DPP4 (target diseases for DPP4 inhibitors) include hyperglycemia, insulin resistance, obesity, lipid disorders, dyslipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, Since it is considered that there are high LDL levels, atherosclerosis, vascular restenosis, irritable bowel syndrome, inflammatory diseases, etc., the DPP4 inhibitor to be developed is expected to have a significant clinical effect.

  Among them, particularly for diabetes caused by a decrease in insulin secretion ability and insulin resistance, its effects have been confirmed.

  Diabetes is a blood vessel that is gradually damaged by hyperglycemia, resulting in complications such as diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, and the risk of developing arteriosclerosis. To raise. In recent years, rapidly increasing diabetes has been classified as type II, and so-called “insulin secretion ability” and “insulin sensitivity” are all impaired by stress, obesity, and lack of exercise. “Lifestyle related diseases”.

  In addition to developing insulin resistance as a characteristic of type II diabetes, there is no relative deficiency or absolute deficiency that prevents insulin secretion to compensate for it. Therefore, insulin secretagogues and insulin resistance improvers have been developed as oral diabetes drugs, mainly in patients with type II diabetes.

  Insulin secretory drugs include sulfonylurea drugs and meglitinide drugs because of their immediate effects, but both act on β-cells, which are insulin-secreting cells, and secrete insulin in a blood glucose-independent manner. On the other hand, there are biguanide drugs and thiazolidine derivatives as drugs for improving insulin sensitivity (resistance). The detailed mechanism of action of both is unknown, but the hypoglycemic effect of biguanides is mainly brought about by suppression of glucose release from the liver and suppression of hepatic gluconeogenesis. The hypoglycemic effect of thiazolidine derivatives acts on muscle and adipose tissue, and promotes glucose uptake. As other drugs, there is an α-glucosidase inhibitor that delays postprandial hyperglycemia so as to match postprandial insulin hypersecretion in patients with mild diabetes. This is an inhibitor of carbohydrases (mainly α-amylase and α-glucosidase).

  The above drugs are effective because they have improved blood parameters such as postprandial hyperglycemia, glycated hemoglobin level, fasting blood glucose level, etc., which are indicators of diabetes (for example, Non-patent Documents 1 and 2). , 3,4,5).

  However, oral diabetes drugs that have been developed so far are aimed at how to suppress hyperglycemia so that they do not progress to the complications caused by hyperglycemia. It is thought that it does not contribute at all to the improvement of “insulin secretion ability” which is one of the causes. Insulin secretagogues are thought to cause exhaustion of secretory cells and apoptosis due to stimulation of secretory cells over a long period of time, and accelerate the decrease in secretory capacity. In addition, there is a problem of hypoglycemia derived from showing a blood glucose-independent drop. In addition, there are side effects such as gastrointestinal tract, liver, kidney, and digestive disorders that are common to all, and its use requires caution.

  In addition, functional materials for preventing diabetes include guava tea, mulberry leaves, indigestible dextrin and the like. Although the main effect is to suppress postprandial blood glucose elevation by inhibiting saccharide-degrading enzymes and sugar transporters, there are no reports that are particularly effective in preventing diabetes.

  On the other hand, new oral antidiabetic drugs are being developed in recent years compared to conventional oral antidiabetic drugs (for example, Patent Documents 1 and 2). Insulin secretion after feeding is induced by 50 to 70% by GLP-1 or GIP's gastrointestinal hormone "Incretin". Since incretin promotes glucose-dependent insulin secretion, the probability of hypoglycemia occurring at high concentrations is considered to be very low. Incretin is a multifunctional hormone that suppresses glucagon secretion, suppresses gastric excretion, protects and proliferates pancreatic β cells, and suppresses feeding, in addition to stimulating insulin secretion. have. However, since incretin has a half-life of several minutes and most of the secreted incretin is degraded, incretin functions are considered to be responsible for insulin secretion even in a normal part. .

Thus, “DPP4 inhibitors” are attracting attention for the purpose of improving the stability of incretins. For example, the pharmacological effects of DPP4 inhibitors confirmed in humans and model animals include a decrease in fasting and postprandial blood glucose levels, a decrease in glycated hemoglobin levels, and suppression of glucagon secretion (eg, Non-Patent Documents 6, 7, 8). , 9) In addition, in mice with onset of diabetes, knowledge showing protection and proliferation of β cells such as enlargement of β cell tissue, increase of insulin secretion amount and improvement of response to glucose has been obtained (for example, non-patent literature) Ten). Moreover, there is a report of a peptide derived from cheese having DPP4 inhibitory activity (for example, Patent Document 3). However, since all DPP4 inhibitors have significantly lower inhibitory activity than the known inhibitor diprotin A, a large amount of DPP4 inhibitor must be administered to achieve the desired effect. There may be a problem.
JP 2006-273873 A JP 2005-526811 A JP 2007-39424 A Kosaka, J. et al .; Pharmacology and Clinical 7; 669 (1997) Chiasson JL et al (1998); Diabetes Care 21; 1720-1725 Chiasson JL et al (2002); Lancet 359; 2072-2077 Hoffmann J, Spengler M (1994); Diabetes Care. 17 (6); 561-566 Lebovitz HE et al (2001); J. Clin. Endocrinol. Metab. 86 (1); 280-288. Aschner P, Kipnes MS et al (2006); Diabetes Care Dec; 29 (12): 2632-2637 Herman GA, Bergman A et al (2006); J Clin Endocrinol Metab. Nov; 91 (11): 4612-4619. Ristic S, Byiers S et al (2005); Diabetes Obes Metab. Nov; 7 (6): 692-698. Mari A, Sallas WM et al J (2005); Clin Endocrinol Metab. Aug; 90 (8): 4888-4894. Mu J, Woods J et al (2006) Diabetes Jun; 55 (6): 1695-1704

  Accordingly, an object of the present invention is to provide a DPP4 inhibitor that has the potential to improve insulin secretion ability and is highly safe and can be taken daily.

  From the viewpoint of safety and daily intake, the present inventors screened the inhibitory activity of DPP4 using an extract from a food material as a subject. As a representative example, there is an active substance in rose red petal (Rosa gallica). As a result, the present invention was completed by isolating, purifying and identifying active substances by various chromatography.

That is, the gist of the present invention is as follows.
(1) a dipeptidyl peptidase IV inhibitor containing hydrolyzable tannin,
(2) The dipeptidyl peptidase IV inhibitor according to (1) above, wherein the hydrolyzable tannin has a barone oil group,
(3) Hydrolyzable tannin is represented by the formula (1):

Lugosin A represented by the formula (2):

It is related with the dipeptidyl peptidase IV inhibitor of the said (2) description which is eugeniflorin D2 represented by these, or these pharmacologically acceptable salt, hydrate, or hemihydrate.

Improving either or both of the decrease in insulin secretion and insulin resistance, which are the causes of diabetes, can be said to be a true treatment for type II diabetes. In addition, as diabetes is called a “lifestyle-related disease”, it is possible that anyone can have it. Therefore, it is thought that prevention as well as treatment is important in the future society.
On the other hand, since the DPP4 inhibitor of the present invention exhibits a high inhibitory activity comparable to that of known inhibitors and is excellent in safety, the use of the DPP4 inhibitor of the present invention allows incretins to be used. It promotes the β-cell protection and proliferative action, and has a remarkable effect as a preventive agent in the prevention and treatment of DPP4 inhibitor target diseases such as diabetes in mammals.

  The present invention relates to a DPP4 inhibitor containing hydrolyzable tannin.

  The hydrolyzable tannin is an ester-bonded polyalcohol and phenol carboxylic acid, and is hydrolyzed by an acid or alkali and an enzyme. Examples of the hydrolyzable tannin include formula (1):

Lugosin A represented by the formula (2):

Eugeniflorin D2 represented by the formula, 1,4,6-tri-O-galloyl-β-D-glucose (1,2,4,6-tetra-O-galloyl-β-D-glucose), 1,2,4,6-tetra-O-galloyl-β-D-glucose (1,2,4,6-tetra-O-galloyl-β-D-glucose), 2,3,4,6-tetra -O-galloyl-β-D-glucose (2,3,4,6-tetra-O-galloyl-β-D-glucose), 1,2,3,6-tetra-O-galloyl-β-D- Glucose (1,2,3,6-tetra-O-galloyl-β-D-glucose), 1,2,3,4,6-penta-O-galloyl-β-D-glucose (1,2,3 , 4,6-penta-O-galloyl-β-D-glucose), stenophylanin A (Stenophyllanin A), stenophylanin B (Stenophyllanin B), and the like. Among them, hydrolyzable tannin having a valoneoyl group is preferable, and rugosin A and eugeniflorin D2 are more preferable because of their remarkable inhibitory activity. The barone oil group may be one or more and is not particularly limited.
In addition, a pharmacologically acceptable salt, hydrate, hemihydrate or the like of the above compound is also included in the hydrolyzable tannin.

  The hydrolyzable tannin can be obtained by chemical synthesis or isolated and purified from a plant containing hydrolyzable tannins, but is isolated and purified from the plant in terms of economy. It is preferable.

  The plant body includes rose red petal, periwinkle (scientific name Euphorbia prostrate Ait), periwinkle (scientific name Euphorbia supina), tachibana adek (scientific name Eugenia uniflora L), paprika, cats claw, walnuts, gray gum (scientific name) Eucalyptus cypellocarpa) and the like. Among these, from the viewpoint of easy extraction, it is preferable to use Tachibana adek for the isolation of Lugosin A, in addition to rose red petals, for the isolation of Arethonyxia, Konishixou, Eugeniflorin D2. Other hydrolyzable tannins are also contained in the plant body. Examples of the method for extracting hydrolyzable tannin from the plant include, for example, Chen L, Chen R et al (1992): Zhongguo Zhong Yao Za Zhi. Apr; 17 (4) 225-226, 255-256, Yamasaki T , Sato M et al (2002): J Nat Toxins. Aug; 11 (3) 165-171, Lee MH, Chiou JF et al (2000): Cancer Lett. Jun 30; 154 (2) 131-136, etc. Any known method may be used.

  For example, extraction from a plant can be performed at an arbitrary temperature and time by a conventional method such as a continuous method, a reflux method, a batch method, an immersion method, or an ultrasonic treatment. Specifically, the plant body is crushed with a mixer or the like, immersed in the extraction solvent at room temperature for 1 to 3 days, refluxed at the boiling temperature of the extraction solvent for 1 to 5 hours, or in the extraction solvent for a short time. Extraction is performed by sonication. Thereafter, the crushing residue is removed from the extract, and the extract is concentrated by reduced pressure or ultrafiltration. Further, if necessary, the solvent may be completely removed and dried or lyophilized. Moreover, you may perform processes, such as concentration, without removing a crushing residue.

  As the plant part used for extraction, any of seeds, leaves, roots, stems and the like can be used.

  As a solvent used for extraction, water and / or an organic solvent are preferable. Examples of the organic solvent include methanol, ethanol, acetone, ethyl acetate and the like. These may be used alone or in combination of two or more, or may be a mixed solvent of these and water. In particular, extraction with ethanol is preferable from the viewpoint that an extract having a DPP4 inhibitory action can be obtained efficiently.

In order to purify a fraction having DPP4 inhibitory action from the extract thus obtained, fractionation is performed.
In order to obtain the fraction, a method that can efficiently remove a desired fraction having a small DPP4 inhibitory action from the extract may be used. For example, liquid-liquid extraction, partition chromatography, adsorption chromatography, molecular Exclusion chromatography method, ion exchange chromatography method, etc. can be mentioned. Above all, in order to obtain a purified product, a method of fractionating the above extract by column chromatography packed with adsorption distribution type packing carrier, gel filtration carrier, etc. It is preferable to use it.

  For example, in the case of isolating and purifying rugosin A from rose red petal, after pulverizing the plant body, ethanol extraction is performed as an extract having a strong DPP4 inhibitory activity, and layer partitioning is performed with ethyl acetate and water, which has inhibitory activity An ethyl acetate layer is obtained. The ethyl acetate layer can be isolated and purified by reversed phase high performance liquid chromatography using normal phase and reversed phase open column methods, C18 column, C30 column, phenyl column.

  The fraction isolated and purified as described above contains hydrolyzed tannin, but may be a solid product dried by drying the solvent used for extraction. In addition, as a DPP4 inhibitor of the present invention, as long as it has a desired inhibitory activity, an extract (mixture) from a plant body as described above may be used as it is, or the fractions are mixed together. A thing may be used.

  The hydrolyzed tannin obtained as described above has DPP4 inhibitory activity. Among them, lugosin A and eugeniflorin D2 show an inhibitory activity effect comparable to that of diprotin A, which is known to have a strong DPP4 inhibitory activity.

  Moreover, since the hydrolyzable tannin is contained in plants and the like that have been used for food and medicine for a long time, such as cat's claw and paprika, the DPP4 of the present invention containing these hydrolyzable tannins. Inhibitors are also excellent in terms of safety.

  The DPP4 inhibitor of the present invention is a pharmaceutically acceptable solid composition such as a tablet, pill, capsule or fine granule, and a liquid composition such as an emulsion, solution or suspension. Ingestion is possible. In addition, parenteral ingestion can be administered as long as it is pharmaceutically acceptable.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

Example 1
Preparation of DPP4 Inhibitor Eleven types of hydrolyzed tannins used for screening are shown in Table 1.

These hydrolyzed tannins were obtained as follows.
Lugosin A (T11) is obtained by pulverizing rose red petal, followed by ethanol extraction as an extract having strong DPP4 inhibitory activity, and layer partitioning with ethyl acetate and water to obtain an ethyl acetate layer having inhibitory activity. The ethyl acetate layer was isolated and purified by reversed-phase high-performance liquid chromatography using normal-phase and reversed-phase open column methods, C18 columns, C30 columns, and phenyl columns.

  Terimagrandin I (T1), pedunclazine (T2), stenophylanin A, and B (T9, T10) were layered with ethyl acetate and water from an ethanol extract of walnut seeds, and the ethyl acetate layer was separated by HPLC. Separated and purified. The columns used for HPLC were “Diaion HP-20” (manufactured by Mitsubishi Kasei Co., Ltd.), open column method, C18 column, C30 column, and phenyl column in this order.

  1,4,6-tri-O-galloyl-β-D-glucose (T3), 1,2,4,6-tetra-O-galloyl-β-D-glucose (T4), 2,3,4, 6-tetra-O-galloyl-β-D-glucose (T5), 1,2,3,6-tetra-O-galloyl-β-D-glucose (T6), 1,2,3,4,6- Penta-O-galloyl-β-D-glucose (T7) and eugeniflorin D2 (T8) are divided into layers from ethanol extract of gray gum leaves with ethyl acetate and water, and the aqueous layer is divided into layers with 1-butanol. went. The obtained ethyl acetate layer and 1-butanol layer were isolated and purified by HPLC using “Diaion HP-20” open column method, C18 column, C30 column, and phenyl column.

  In addition, each hydrolyzable tannin was identified using NMR, MS, etc., compared with spectral data of various substances, and further using a chemical method such as hydrolysis.

A 100 μM solution of 11 types of hydrolyzed tannins shown in Table 1 was prepared in methanol.
15 μl of 25 mM Tris-HCl buffer solution, 5 μl of 2 ng / μl of human-derived DPP4 solution diluted with buffer (R & D Systems), 5 μl of 100 μM tannins or methanol were added and left at room temperature for 5 minutes. The reaction was started by adding 25 μl of 40 μM glycylproline-4-methylcoumaryl-7-amide (Peptide Institute) prepared in 25 mM Tris-HCl buffer. For detection, 7-amino-4-methylcoumarin released by DPP4 was measured with a fluorescence detector (Fluoroscan Ascent: Thermoelectron) for 96 well plates. The excitation wavelength was 390 nm and the measurement wavelength was 460 nm.
The activity was obtained by subtracting the fluorescence value shown when containing only the substrate from the fluorescence value measured after the reaction for 5 minutes. For the calculation of the inhibition rate, the ratio of the activity value when each tannin was included and the activity value when the tannin was not included was calculated, and the value obtained by subtracting the value from 1 was multiplied by 100.
as a result. At 10 μM, Eugeniflorin D2 showed an inhibition rate of 50.9%, and Lugosin A showed an inhibition rate of 54.9%.
These results are shown in Table 2. The screening results are shown in FIG.

  In the present invention, the inhibition experiment is the same as the other inhibition experiments. However, since the product is fluorescent, the fluorescence value was obtained and evaluated by the inhibition rate.

(Example 2)
IC50 (50% inhibitory concentration) was determined for iugeniflorin D2 and rugosin A, which were found to be active in Example 1, and compared with diprotin A, which is known as a DPP4 inhibitor. The experimental method for IC50 measurement is almost the same as in Example 1, except that rugosin A has a final concentration of 40, 10, 1 μM, iugeniflorin D2 has a final concentration of 50, 10, 2.5 μM, and diprotin A has a final concentration of 10, 4, 1 μM. The inhibition rate was obtained and obtained by numerical calculation. As a result, the inhibitory effect was strong in the order of diprotin A> lugosin A> eugeniflorin D2, but it was found that lugosin A has an inhibitory effect comparable to diprotin A. As a result of calculating IC50, it was found to be diprotin A: 4.5 μM, rugosin A: 6.3 μM, and iugeniflorin D2: 13.3 μM. These results are shown in Table 2.

  The present invention can be suitably used as a prophylactic and / or therapeutic agent for a DPP4 inhibitor target disease such as diabetes containing the DPP4 inhibitor. In addition to diabetes, DPP4 inhibitor target diseases include hyperglycemia, insulin resistance, obesity, lipid disorders, dyslipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL level, high LDL level, Examples include atherosclerosis, vascular restenosis, irritable bowel syndrome, and inflammatory diseases.

  Moreover, the DPP4 inhibitor of this invention can be suitably mix | blended using a well-known technique with compositions, such as a foodstuff and a pharmaceutical.

FIG. 1 is a graph showing DPP4 inhibitory activity of eleven types of hydrolyzable tannin compounds used in Example 1. The percentages shown in each bar indicate the inhibition rate. FIG. 2 is a graph showing the inhibition rates at respective concentrations of diprotin A, rugosin A, and eugeniflorin D2. A straight line plot is shown by logarithm concentration display.

Claims (3)

  A dipeptidyl peptidase IV inhibitor containing hydrolyzable tannin.   The dipeptidyl peptidase IV inhibitor according to claim 1, wherein the hydrolyzable tannin has a barone oil group. Hydrolyzed tannin is represented by the formula (1):

Lugosin A represented by the formula (2):

The dipeptidyl peptidase IV inhibitor according to claim 2, which is Eugeniflorin D2 represented by the formula:

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