JP6822648B2 - Tryptase inhibitor composition - Google Patents

Description

The present invention relates to a tryptase inhibitor composition, and more particularly to a tryptase inhibitor composition having a tryptase inhibitory action affecting skin wrinkle formation and capable of preventing skin aging.

Tranexamic acid or a salt thereof, especially tranexamic acid, is known to have anti-plasmin action, anti-allergic action, and anti-inflammatory action, and its efficacy / effect is considered to be related to systemic hyperfibrinolytic bleeding tendency (leukemia, regeneration). Defect anemia, purpura, etc. and abnormal bleeding during and after surgery); Abnormal bleeding that is thought to be associated with increased local fibrinolysis (pulmonary bleeding, epistaxis, genital bleeding, renal bleeding, abnormalities during and after prostate surgery) Bleeding); Symptoms of eczema and its similarities, urticaria, drug eczema / toxic eczema, erythema, swelling, pruritus, etc .; Pain and oral mucosal aphthae (see Non-Patent Document 1). It is also known that tranexamic acid or a salt thereof can be a therapeutic agent for pigmentation in addition to the above-mentioned actions, indications and effects (see Patent Document 1), but further studies on its usefulness are being conducted.

Trypsin is an intracellular trypsin-type protease (Non-Patent Document 2) isolated and purified from human lungs by Smith et al. In 1984, and trypsin M (Non-Patent Document 3), T4 isolated from mast cells. Tryptase TL1, TL2, TL3 (Non-Patent Document 4) isolated from lymphocytes, tryptase Clara (Non-Patent Document 5) isolated from Clara cells and the like are known. It is known that tryptase is increased and activated in human skin by irradiation with ultraviolet rays, and it decomposes type I collagen (Non-Patent Document 6), decomposes type IV collagen, and damages the basement membrane (non-patent). Patent Document 7), causing neutrophil migration by enhancing the permeability of peripheral blood vessels, promoting the decomposition of elastin by neutrophil-derived elastase (Non-Patent Document 8), and causing delayed keratinization in wound healing. (Non-Patent Document 9) is known.

As described above, tryptase causes reduction / degeneration of extracellular matrix components (ECM) such as collagen and elastin in the dermis due to ultraviolet rays, and skin aging (wrinkles, loss of texture, loss of elasticity, etc.) due to basement membrane damage and epidermal thickening. ) Is believed to be involved. In addition, an action of promoting degranulation of mast cells has been reported (Non-Patent Document 10), and it is considered to be involved in the generation of inflammation caused by ultraviolet rays.

Therefore, it is considered that inhibiting the tryptase activity is effective in preventing and improving wrinkles, sagging, rough skin, itch, inflammation and the like of the skin.

Compounds having tryptase inhibitory activity include 6'-amidino-2'-naphthyl4-guanidinobenzoate or a pharmaceutically acceptable salt thereof (Patent Document 2), catechins and / or glycosides thereof, flavones and flavones. / Or its glycosides, flavonols and / or its glycosides, flavanones and / or their glycosides, tannins (Patent Document 3), Paeoni lactiflar Pallas (Patent Document 4) and the like are known. However, the effect is not yet sufficient, and a compound having a stronger tryptase inhibitory activity is desired.

Japanese Unexamined Patent Publication No. 4-243825 Japanese Unexamined Patent Publication No. 2003-246730 JP-A-2007-186457 Japanese Unexamined Patent Publication No. 2013-129617

Japan Pharmaceutical Information Center Medical Japan Pharmaceutical 2002 (25th edition, Jiho Co., Ltd., p. 1369) T. J. Smith et al., Journal of Biological Chemistry Vol.259, P.11046-11051 (1984) L. B. Schwart et al. Journal of Biological Chemistry Vol.256, P.11939-11943 (1981) H. Kido et al. Journal of Biological Chemistry Vol.265, P.21979-21985 (1991) H. Kido et al. Journal of Biological Chemistry Vol.267, P.13573-13579 (1992) Fajard et al. The Journal of Immunology Vol.171, P1493-1499 (2003) Jouko Lohi, et al. Journal of Cellular Biochemistry Vol.50, P337-349 (1992) Shaoheng He et al. European Journal of Pharmacology Vol.328, P.89-97 (1997) M. Huttunen et al. Experimental Dermatology Vol.9, P258-265 (2000) Shaohen He, Journal of Pharmacology and Experimental Therapeutics Vol.286, P.289-297 (1998)

An object of the present invention is to provide a tryptase inhibitor composition having excellent safety and a tryptase inhibitory action, and a wrinkle improving composition containing the tryptase inhibitor composition.

As a result of investigating the tryptase inhibitory action of various substances in order to solve these problems, the present inventors have found that tranexamic acid has an excellent tryptase inhibitory action, and completed the present invention.

That is, the present invention relates to a tryptase inhibitor composition, which comprises tranexamic acid or a salt thereof. Furthermore, the present invention relates to wrinkle-improving compositions containing the tryptase inhibitor composition, and more particularly to those in which these compositions are oral compositions.

The composition of the present invention is a highly safe tryptase inhibitor composition that can reliably inhibit the enzymatic activity of tryptase. Therefore, by using the composition, it is possible to improve various diseases and lesions based on the tryptase inhibitory action, and particularly to improve wrinkles due to the influence of aging and ultraviolet rays. In addition, the tryptase inhibitor composition of the present invention and the wrinkle improving composition containing the tryptase inhibitor composition can be used in the form of oral preparations, food and drink compositions, supplements, external preparations, cosmetics and the like. However, it is particularly effective when used as an oral administration composition such as an internal preparation, a food and drink composition, and a supplement.

FIG. 1 is a diagram showing test results of a tryptase inhibitory activity test using a tranexamic acid solution or a control administration solution. TA in the figure means tranexamic acid. FIG. 2 is a diagram showing the measurement results of the amount of water evaporation measured after the test substance or the control substance was administered to the test animal for 20 days. FIG. 3 is a diagram showing the measurement results of the skin water content measured after the test substance or the control substance was administered to the test animal for 20 days. FIG. 4 is a photograph showing the skin thickness observed after administration of the test substance or the control substance to the test animal for 20 days, and a diagram showing the measurement results. FIG. 5 is a photograph showing a mouse back condition observed after administration of a test substance or a control substance to a test animal for 20 days. FIG. 6 is a photograph of mast cells in the skin after staining observed after administration of the test substance or control substance to the test animal for 20 days. FIG. 7 is a diagram showing the measurement results of the amount of collagen I protein in the skin measured after the test substance or the control substance was administered to the test animal for 20 days. FIG. 8 is a diagram showing the measurement results of the amount of ceramide in the skin measured after the test substance or the control substance was administered to the test animal for 20 days.

The tranexamic acid used in the present invention is a known compound ((trans-4-aminomethylcyclohexanecarboxylic acid, CAS Registry Number: 1197-18-8)), and the production method thereof is not particularly limited, and a commercially available product is used. Alternatively, those produced based on a known method may be used. Examples of the salt of tranexamic acid include mineral acid salts such as hydrochloride, nitrate and sulfate, organic acid salts such as methanesulfonate, and sodium salts. Alkali metal salts such as potassium salt, calcium salt, and magnesium salt, alkaline earth metal salts, and the like can be used. As the tranexamic acid or a salt thereof used in the present invention, tranexamic acid is particularly preferable.

The tryptase inhibitor composition and the wrinkle improving composition of the present invention can be administered in oral or parenteral dosage forms. Examples of the form of the oral administration composition include tablets, capsules, powders, fine granules, pills, liquids, syrups, lozenges, jellies, etc., which can be administered as pharmaceuticals, foods, etc. Is. In addition, as a form of parenteral administration composition, ointment, ointment, liquid, lotion, cream, gel, milky lotion, aerosol, liniment, tape, pap, external powder, injection, drip. Agents, packs, bathing agents and the like can be mentioned, and these can be administered as pharmaceuticals, non-pharmaceutical products, cosmetics and the like. In the present invention, it is preferable to use an orally administered composition.

The formulation of the composition according to the present invention can be produced by a known production technique according to a desired dosage form, and if necessary, a known excipient, disintegrant, binder, lubricant, stable Agents, preservatives, colorants, surfactants, pH adjusters, flavoring agents, flavoring agents, sweeteners, preservatives, flavors, aqueous components, alcohols, sugars, water and the like can be added as additive components. .. In addition, components such as anti-aging agents, antioxidants, whitening agents, and various crude drugs can be blended as long as the effects of the composition according to the present invention are not diminished.

The daily amount of the tryptase inhibitor composition of the present invention to be used may be appropriately determined in consideration of the gender, age, symptoms, administration method, number of administrations, administration time, etc. of the user, and is used within a range that does not cause side effects. It can be done and is not particularly limited. For example, when used as an oral composition, the dose of tranexamic acid or a salt thereof is 1 to 5000 mg per day, preferably 10 to 3000 mg per day, more preferably 200 to 1000 mg per day, and particularly preferably. Is 420-750 mg per day. When used as a parenteral administration composition, tranexamic acid or a salt thereof is 0.01 to 20.0% by mass, preferably 0.6 to 5.0% by mass, more preferably 0 in the parenteral administration composition. .Mixed in a proportion of 6 to 3.0% by mass.

Hereinafter, the present invention will be described in more detail by way of examples. The present invention is not limited thereto.

(Example 1) Tryptase inhibitory activity test 1. Sample Preparation A sample tranexamic acid solution was prepared by dissolving tranexamic acid (manufactured by Daiichi Fine Chemical Workers) in distilled water so that the final concentration was 1,3,9,36 mM.

2. 2. Evaluation of tryptase inhibitory activity rh skin b1 Tryptase (manufactured by Promega Co., Ltd.) was used as the tryptase. A tryptase solution (final concentration 16.7 ng / mL) was prepared using distilled water and mixed with a sample tranexamic acid solution having each of the above concentrations to prepare an enzyme mixture. As a substrate, N- (p-Tosyl) -Gly-Pro-Lys4-nitroanilid acetate salt (manufactured by Sigma-Aldrich) was used, and 100 mM Tris-Hcl (pH 8.0) was used so that the final concentration was 1.5 mM. It was dissolved to prepare a substrate solution.

A substrate solution was added to 50 μL / well of the enzyme mixture at each concentration, and the mixture was incubated at 37 ° C. for 5 minutes, and then the absorbance was measured with an absorptiometer (measurement condition: 410 nm). 100 mM Tris-Hcl (pH 8.0) was added to the blank instead of the substrate solution, and measurement was performed.

3. 3. Tryptase activity The tryptase inhibition rate by tranexamic acid was calculated by the following formula.
Tryptase inhibition rate (%) = 100 × (1-sample absorbance / control absorbance)
The calculation result of the inhibition rate is shown in FIG.

From the results of the above test, it was confirmed that tranexamic acid exhibits a tryptase inhibitory effect in a concentration-dependent manner.

(Example 2) Anti-wrinkle test 1. Preparation of test substance The following experiment was conducted using tranexamic acid (manufactured by Daiichi Fine Chemicals) dissolved in distilled water so as to have a dose of 750 mg / kg as the test substance.

2. 2. Test animals Three-week-old NOA / Jcl mice (male and female) were purchased from Japan Claire Co., Ltd., and 5 animals per group were pre-reared for 1 week, and then the health condition was observed with the naked eye and good ones were used as test animals. used. The NOA / Jcl mouse is a mouse that expresses dry skin and is used as a model for verifying the effect of an external moisturizer.

3. 3. Administration of test substance The test group was a group in which 0.1 mL of the test substance was orally administered once a day for 20 days per 10 g of the body weight of the test animal mouse, and 0.1 mL of distilled water alone was orally administered once a day for 20 days. The group was used as a control group. Each of these groups was configured with n = 5.

4. Measurement After an oral administration period of 20 days, the amount of water evaporation (TEWL; g / m ² / h), the amount of skin water (Corneometer value), and the thickness of the epidermis (μm) were measured, and the condition of the back of the mouse was observed. Observation of cells, comparison of the amount of collagen I in the skin, and comparison of the amount of ceramide in the skin were carried out based on the following measurement methods.

The amount of water evaporation was measured using a Tewameter (registered trademark) TM300 (Courage + manufactured by Khazaka Electronic GmbH). The measurement result of the amount of water evaporation is shown in FIG.

The skin water content was measured using a Corneometer (registered trademark) CM825 (manufactured by Courage & Khazaka Electronic GmbH). The measurement result of the skin water content is shown in FIG.

The skin thickness was measured by staining skin sections with haematoxylin and eosin (H & E) staining. As for the thickness, the thickness of the epidermis at 5 places was randomly measured in one field of view, and this was performed for 5 fields of view, and the average value was taken as the skin thickness. A photograph showing the skin thickness and measurement results are shown in FIG.

The back state of the mouse was observed by visually observing the back of the test animal to confirm the presence or absence of wrinkles and the degree of wrinkles. A photograph showing the state of the back of the mouse is shown in FIG.

For observation of mast cells in the skin, skin sections were stained with toluidine blue, and the number of mast cells stained bluish purple was observed. A photograph of mast cells in the skin after staining is shown in FIG. In the figure, the cells indicated by arrows are mast cells.

The amount of collagen I protein in the skin and the amount of ceramide in the skin were measured using western blotting, and the analysis of the test group sample and the control group sample was performed using Multi-Gauge software (manufactured by FUJIFILM Corporation). I went. The measurement result of the amount of collagen I protein in the skin is shown in FIG. 7, and the measurement result of the amount of ceramide in the skin is shown in FIG.

5. Test Results The mice in the tranexamic acid-administered group showed lower TEWL measurements, higher skin water content measurements, and higher epidermal thickness than the control group mice. From this, it is recognized that the administration of tranexamic acid improved the water content in the skin.

In addition, administration of tranexamic acid decreased the number of mast cells in the skin and increased the amount of collagen I in the skin. Furthermore, it was confirmed from the observation of the back photographs of the mice that the formation of wrinkles was suppressed in the mice in the tranexamic acid-administered group as compared with the mice in the control group.

From the above results, tranexamic acid has a tryptase inhibitory action, and further, a water evaporation amount in the skin, a skin water amount, an epidermal thickness improving action, a skin mast cell reducing action, and a skin collagen I amount increasing action. It was clarified that it has an effect of improving wrinkles.

INDUSTRIAL APPLICABILITY The present invention provides a tryptase inhibitor composition, particularly an oral composition effective for improving wrinkles.

Claims (3)

A tryptase inhibitor composition comprising tranexamic acid or a salt thereof ( provided that the skin texture, luster, sagging, rough skin, inflammatory skin disease, deterioration of skin barrier function due to stress, acne skin Excludes tryptase inhibitory compositions for prevention or amelioration.) The tryptase inhibitor composition according to claim 1, wherein the tryptase inhibitor composition is for improving wrinkles. The tryptase inhibitor composition according to claim 1 or 2, wherein the tryptase inhibitor composition is an orally administered composition.