HK1113739B - Prevention or improvement of wrinkles by using adam activation inhibitor - Google Patents

Description

Preventing or improving wrinkles with ADAM activity inhibiting substance

Technical Field

The present invention relates to the prevention or amelioration of wrinkles using a substance that inhibits ADAM activity. The present invention also relates to a method for evaluating an anti-wrinkle effect by using ADAM activity inhibition as an index.

Background

Wrinkles increase as a phenomenon of skin aging increases with age. Recently, from the viewpoint of beauty and the like, there is a remarkably increased interest in preventing and improving wrinkles particularly in women. Wrinkles can be roughly classified into large wrinkles, fine wrinkles, and wrinkled wrinkles, according to wrinkle generation sites, wrinkle generation mechanisms, and the like. Large wrinkles are deep wrinkles occurring at the back of the neck, etc., mainly due to photoaging. Fine wrinkles are relatively shallow wrinkles that occur around the eyes or mouth. Wrinkle wrinkles are wrinkled wrinkles that occur in non-exposed body parts such as the abdomen of the elderly.

To date, studies have been made on large wrinkles occurring mainly due to photoaging, and wrinkles have been considered as a symptom due to photoaging. As for photoaging, studies on the mechanism of occurrence and the like have been made, and animal models or human evaluation systems have been established. Most efforts to suppress wrinkles formed due to photoaging have so far attempted to prevent or alleviate the adverse effects of ultraviolet rays on the skin. For example, in order to prevent adverse effects of ultraviolet rays on the skin, cosmetics (sun screen cosmetics and sun protection cosmetics) containing various substances capable of absorbing, dispersing or blocking ultraviolet rays, such as titanium oxide, zinc oxide, p-methoxycinnamate, and p-aminobenzoate have been developed and used. Also, it has been found that free radicals generated in the skin due to ultraviolet radiation adversely affect fiber components such as collagen and elastin which constitute the skin, and cosmetics containing antioxidants have been proposed (see references, for example, PCT Japanese laid-open No. 2001-508809). Further, it has been considered that one of the main factors of skin photoaging is a phenomenon in which MMPs decomposing dermal connective tissues such as collagen and elastin are induced by ultraviolet radiation and thus the dermal connective tissues are destroyed by the MMPs. To prevent photoaging of the skin, for example, a composition for inhibiting photoaging of the skin, which comprises a UVA blocking agent, a UVB blocking agent, and an MMP inhibitor such as a retinoid, has been proposed (see references, for example, PCT japanese laid-open No. 2001-520677).

Recently, middle aged and elderly women have an increased interest in beauty and in fine wrinkles occurring around the eyes or mouth due to a decrease in water retention capacity of the horny layer of the epidermis with age or a decrease in sebum occurring due to a decrease in secretion of epidermal lipids with age. It is considered that fine wrinkles occur due to dryness of the skin. It is also considered that fine wrinkles are significantly different from large wrinkles occurring due to photoaging in the mechanism of occurrence of fine wrinkles and the morphological, histological, and biochemical changes of fine wrinkles. For example, it has been confirmed that the water content and the degree of small wrinkles with respect to the human horny layer are related to each other (see references, for example, Fragrance journal of Imokawa et al; 1992(11) 29-42). Further, it has been reported that epidermal wrinkles occur in the case where barrier function is continuously disrupted with unsaturated fatty acids (see references, for example, the Perfun Transactions of JinMasaki, 2001, Vol. 25, No.1, 34-38). However, it has not been sufficiently clarified what biochemical changes have occurred at the protein level or gene level in the skin of fine wrinkles, and prevention and improvement of fine wrinkles must rely on a method of protecting the skin from dryness by using moisturizers such as glycerin, sorbitol or plant fluid extracts or cosmetics containing collagen and the like. However, fine wrinkles cannot be sufficiently prevented and improved merely by protecting the skin from dryness by using a moisturizing agent or the like. Therefore, there is a strong demand for substances more effective in preventing and improving fine wrinkles.

In view of the above, the present invention has an object to clarify biochemical changes involved in the formation of fine wrinkles in the skin, specify a substance capable of inhibiting the biochemical changes, and more effectively prevent or improve wrinkles by using the substance. It is another object of the present invention to provide a method for enabling the anti-wrinkle effect of a test substance to be more effectively and easily evaluated by using the inhibition of the aforementioned biochemical changes as an index.

Disclosure of Invention

The inventors have found that repeated stripping of the tape from hairless mice results in a consequent disruption of the continuous skin barrier, with the same epidermal and dermal changes occurring in the skin of the mice as fine wrinkles occur in human skin. The inventors thus succeeded in establishing a mouse model of fine wrinkles. The present invention is based on the finding that in the mouse model of fine wrinkles described above, epidermal gene expression of a protein belonging to the ADAM (disintegrin and metalloprotease) family having disintegrin and metalloprotease domains (hereinafter referred to as ADAM), such as ADAM-9, ADAM-10 or ADAM-17, is increased, and epidermal gene expression of HB-EGF (heparin-binding epidermal growth factor-like growth factor) and amphiregulin released and activated from the cell membrane by the action of these ADAMs is increased. The present invention is also based on the discovery that the use of these ADAM inhibitors can inhibit epidermal and dermal thickening and wrinkle formation.

ADAMs such as ADAM-9, ADAM-10 or ADAM-17 are enzymes present on the surface of skin cells and play a role in releasing growth factors such as HB-EGF, amphiregulin, TNF- α and TGF- α from cell membranes in the skin and activating the above growth factors. It is considered that an important mechanism of formation of fine wrinkles is that ADAM is activated in the skin or expression thereof is increased, so that ADAM promotes release and activation of growth factors such as HB-EGF and causes thickening of epidermis and dermis to occur. Therefore, it is considered that by inhibiting the activity of ADAM in the skin, the activity improvement of HB-EGF and the like can be inhibited, epidermal and dermal thickening can be inhibited, and the formation of wrinkles, particularly fine wrinkles, can be prevented or improved. Fig. 1 is a schematic view illustrating a mechanism for preventing or improving wrinkles by inhibiting the activity of ADAM in the skin.

In one aspect of the present invention, the present invention provides a composition containing an effective wrinkle-preventing or-improving amount of a substance capable of inhibiting the activity of ADAM present in skin.

In another aspect of the present invention, the present invention provides a method for preventing or improving wrinkles, which comprises the step of inhibiting the activity of ADAM present in skin. Wrinkles can be prevented or improved by applying a substance capable of inhibiting the activity of ADAM to the skin. The method for preventing or improving wrinkles according to the present invention can be preferably used as a cosmetic method.

In a further aspect of the present invention, the present invention relates to the use of a substance capable of inhibiting ADAM activity for preventing or improving wrinkles. The substance capable of inhibiting the activity of ADAM can be used in cosmetic treatment for preventing or improving wrinkles.

In a further aspect of the invention, the invention relates to the use of a substance capable of inhibiting the activity of ADAM for producing a composition for preventing or improving wrinkles. There is no limitation on the kind of the composition. However, the composition should preferably be an article for external application to the skin, such as cosmetics, pharmaceuticals and quasi drugs. The composition should more preferably be a cosmetic product.

Examples of ADAM present in skin include ADAM-9, ADAM-10, ADAM-12, ADAM-15, ADAM-17, and ADAM-19. In particular, ADAM-9, ADAM-10 and ADAM-17 have been found to be deeply involved in the release and activation of cell growth factors.

The term "inhibiting activity of ADAM" as used herein encompasses inhibiting the enzymatic activity of ADAM and any effect for decreasing the activity of ADAM in skin, such as the effect of inhibiting gene expression and protein formation.

In another aspect of the present invention, the present invention provides a method for evaluating an anti-wrinkle effect, comprising the steps of:

1) contacting the test substance with skin, skin tissue or cells of a human or animal;

2) detecting the enzymatic activity or gene expression level of ADAM in said skin, skin tissue or cell;

3) evaluation of test substance by Using ADAM enzymatic Activity or Gene expression level as an index

The anti-wrinkle effect of (1).

An ADAM can be, for example, ADAM-9, ADAM-10, or ADAM-17.

For example, the anti-wrinkle effects of various test substances can be evaluated rapidly and effectively when using skin cells, particularly epidermal keratinocytes.

The term "anti-wrinkle effect" as used herein refers to any effect of preventing wrinkle formation or improving an already formed wrinkle.

When a substance capable of inhibiting ADAM activity is applied to the skin, the activity of ADAM present in the skin, such as ADAM-9, ADAM-10 or ADAM-17, can be inhibited, the release and activation of growth factors such as HB-EGF in epidermis and dermis due to skin barrier disruption caused by various main causes such as sebum reduction and face washing can be inhibited, and the thickening of epidermis and dermis can be inhibited. So that the formation of wrinkles, particularly fine wrinkles, can be significantly effectively prevented or improved.

In addition, with the method for evaluating an anti-wrinkle effect according to the present invention in which ADAM activity inhibition is used as an index, an anti-wrinkle substance having a great effect can be specified efficiently and easily.

Brief Description of Drawings

Fig. 1 is a schematic view showing a mechanism of preventing or improving wrinkles by inhibiting ADAM activity in epidermis;

FIG. 2A is a replicate image showing mouse skin being stripped by tape;

FIG. 2B is a photomicrograph showing a skin tissue section of mouse skin, which has been stained with HE (hematoxylin-eosin),

FIG. 3 is a graph showing changes in gene expression of ADAM-9, ADAM-17, HB-EGF and amphiregulin in a mouse model of fine wrinkles,

figure 4A is a reproduction image showing skin to which different drugs have been applied in a mouse model of fine wrinkles,

FIG. 4B is a photomicrograph showing a section of mouse skin tissue, which has been stained with HE (hematoxylin-eosin),

FIG. 5 is a graph showing the anti-wrinkle effect of ADAM activity-inhibiting substances, wherein the anti-wrinkle effect is obtained from visual judgment,

FIG. 6 is a graph showing the anti-wrinkle effect of ADAM activity-inhibiting substances, wherein the anti-wrinkle effect is expressed in terms of wrinkle area (%).

Best Mode for Carrying Out The Invention

ADAM is a multifunctional protein having two characteristics, i.e., adhesion and extracellular protein decomposition, and there are at least 30 families of ADAMs. Now, the number of kinds of ADAM families is increasing. ADAM is expressed in a variety of animals and in a variety of tissues. Examples of ADAM present in skin include ADAM-9, ADAM-10, ADAM-12, ADAM-15, ADAM-17, and ADAM-19. In particular, ADAM-9, ADAM-10 and ADAM-17 have been found to be deeply involved in the release and activation of cell growth factors.

The substance capable of inhibiting ADAM activity that can be used in the present invention can be selected from a wide variety of substances capable of reducing ADAM activity in skin. For example, the substance capable of inhibiting the activity of ADAM may be a substance capable of inhibiting the enzymatic activity of ADAM, or a substance capable of inhibiting ADAM gene expression or protein formation. In addition, the substance capable of inhibiting the activity of ADAM may be a natural substance such as a substance of animal origin or a substance of plant origin. Alternatively, the substance capable of inhibiting the activity of ADAM may be a synthetic substance.

Examples of the substance capable of inhibiting ADAM activity as described above include TAPI-1(N- (R) - (2- (hydroxyaminocarbonyl) methyl) -4-methylpentanoyl-L-Na 1-L-alanine 2-aminoethylamide; Immunex, Seattle, WA) and 4-methoxyphenyl hydroxamic acid. However, the substance capable of inhibiting the activity of ADAM is not limited to the above-mentioned examples.

The substance capable of inhibiting the activity of ADAM should preferably be used in a skin preparation for external use. However, the use of a substance capable of inhibiting the activity of ADAM is not limited to the above-described application.

The skin external preparation may contain only one substance capable of inhibiting the activity of ADAM. Alternatively, in the skin external preparation, at least two substances capable of inhibiting the activity of ADAM may be contained in combination and mixed. In addition, the mixing ratio of these substances capable of inhibiting ADAM activity in the skin external preparation may vary depending on the mode of use, the form of the product, and the like, and is not limited to a specific value. However, the mixing ratio of the substance capable of inhibiting ADAM activity, for example, should preferably fall within the range of 0.001 to 10 mass%, more preferably within the range of 0.005 to 5 mass%, and most preferably within the range of 0.01 to 1 mass% with respect to the total amount of the skin external preparation.

The term "skin preparation for external use" as used herein includes cosmetics, pharmaceuticals, quasi drugs, and the like. Also, the skin preparation for external use can take a wide variety of preparation forms such as an aqueous solution type, a solubilized type, an emulsion type, an oil type, a gel type, a cream type, an ointment type, an aerosol type, a water-oil bilayer type and an oil-water-powder trilayer type. Further, the skin external preparation may take the form of a support on a sheet-like base material.

In addition, the skin preparation for external use may have various product forms and may be used in various applications. For example, the skin external preparations can be used as external preparations for the skin of the face, body and head in the form of products of lotions, milky lotions, skin creams and masks.

The skin external preparation may contain, as necessary, other optional components generally used in skin external preparations such as cosmetics and pharmaceuticals, in addition to the substance capable of inhibiting the activity of ADAM. In addition, the skin preparation for external use can be produced according to a conventional method depending on the form of the intended preparation. For example, the above-mentioned substance capable of inhibiting ADAM activity and at least one component selected from the following components may be mixed together, and thus, a preparation for external application to the skin can be prepared.

In particular, the skin preparation for external use may contain at least one ultraviolet absorber. Examples of the ultraviolet absorber include benzoic acid type ultraviolet absorbers such as p-aminobenzoic acid (hereinafter abbreviated as PABA), PABA monoglyceride, N-dipropoxypPABA ethyl ester, N-diethoxypPABA ethyl ester, N-dimethylpPABA butyl ester and N, N-dimethylpPABA methyl ester; anthranilic acid type ultraviolet absorbers, such as N-acetyl anthranilic acid high menthol esters; salicylic acid type ultraviolet absorbent such as amyl salicylate, menthol salicylate, and homomenthyl salicylateAlcohol esters, octyl salicylate, phenyl salicylate, benzyl salicylate, and p-isopropyl phenyl salicylate; cinnamic acid type ultraviolet absorbent such as octyl cinnamate, ethyl 4-isopropyl cinnamate, methyl 2, 5-diisopropyl cinnamate, ethyl 2, 4-diisopropyl cinnamate, methyl 2, 4-diisopropyl cinnamate, propyl p-methoxy cinnamate, isopropyl p-methoxy cinnamate, isoamyl p-methoxycinnamate, octyl p-methoxycinnamate (2-ethylhexyl p-methoxycinnamate), 2-ethoxyethyl p-methoxycinnamate, cyclohexyl p-methoxycinnamate, ethyl α -cyano- β -phenylcinnamate, 2-ethylhexyl α -cyano- β -phenylcinnamate, glyceryl mono-2-ethylhexanoyl-di-p-methoxycinnamate and trimethoxycinnamate, methyl bis (trimethylsiloxane) silylisoprene; 3- (4' -methylbenzylidene) -d, l-camphor; 3-benzylidene-d, l-camphor; urocanic acid; ethyl urocanate; 2-phenyl-5-methylbenzeneAzole; 2, 2' -hydroxy-5-methylphenylbenzotriazole; 2- (2 '-hydroxy-5' -tert-octylphenyl) benzotriazole; 2- (2 '-hydroxy-5' -methylphenyl) benzotriazole; dibenzylazine (dibenzazine); di-methoxybenzoylmethane; 4-methoxy-4' -tert-butyl dibenzoylmethane; 5- (3, 3-dimethyl-2-norbornene (norbomylidene)) -3-pentan-2-one; and dimorpholinopyridazinone.

Examples of the ultraviolet dispersant that can be used include particles such as titanium oxide, particulate titanium oxide, zinc oxide, particulate zinc oxide, iron oxide, particulate iron oxide, cerium oxide.

Generally, the ultraviolet dispersing agent is used in the form of needle-shaped particles, spindle-shaped particles, spherical particles, and granular particles. In addition, the ultraviolet dispersing agent should preferably be fine particles having a particle diameter of at most 0.1. mu.m.

It is also preferable to use an ultraviolet dispersing agent which has been subjected to hydrophobization processing such as siloxane processing using methylhydrogenpolysiloxane or a silane coupling agent; processing the metal soap; fluorine processing using perfluoroalkyl phosphoric acid diethanolamine salt, perfluoroalkyl silane, or the like; or dextrin fatty acid ester processing.

Examples of liquid fats and oils that may be used include avocado oil, camellia oil, turtle fat, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg butter oil, sesame oil, persic oil, wheat germ oil, sasanqua oil, castor bean oil, linseed oil, safflower oil, cottonseed oil, perilla oil, soybean oil, peanut oil, tea seed oil, coconut oil, rice bran oil, china tung oil, japanese tung oil, jojoba oil, germ oil, and triglycerin.

Examples of solid fats and oils that may be used include cocoa butter, coconut oil, horse fat, hardened coconut oil, palm oil, beef tallow, mutton tallow, hardened beef tallow, palm kernel oil, lard, beef bone fat, Japanese wax kernel oil, hardened oil, beef tallow, Japanese wax, and hardened castor bean oil.

Examples of waxes that may be used include bees wax, candelilla wax, cotton wax, palm wax, insect wax, whale wax, peony wax, rice bran wax, lanolin, kapok wax, lanolin acetate, liquid lanolin, reed millet wax, lanolin fatty acid isopropyl ester, hexyl laurate, reduced lanolin, jojoba wax, hard lanolin, shellac wax, POE lanolin alcohol ether, POE lanolin alcohol acetate, POE cholesterol ether, lanolin fatty acid polyethylene glycol, and POE hydrogenated lanolin alcohol ether.

Examples of hydrocarbon oils which may be used include liquid paraffin, ozocerite, squalane, pristane, paraffin, ozocerite, squalene, petrolatum, microcrystalline wax, polyethylene wax, and Fischer-Tropsch wax.

Examples of higher fatty acids that may be used include lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, undecylenic acid, methylbenzoic acid, linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

Examples of higher alcohols that may be used include straight chain alcohols (e.g., lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, cetostearyl alcohol, and cetostearyl alcohol) and branched chain alcohols (e.g., monostearoyl glycerol ether (batyl alcohol), 2-decyltetradecylanonol alcohol, cholesterol, phytosterols, hexyldodecanol, octyldodecanol).

Examples of synthetic ester oils that may be used include isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, decyl dimethyl caprylate, cetyl acetate, myristyl acetate, lanolin acetate, isocetyl stearate, isocetyl isostearate, cholesterol 12-hydroxystearate, ethylene glycol di-2-ethylhexanoate, dipentaerythritol fatty acid, N-alkylethylene glycol mono-isostearate, neopentyl glycol di-decanoate, di-isostearyl malate, glycerol di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentylenethyl tetra-2-ethylhexanoate, pentylenethylene tetra-2-ethylhexanoate, isopropyl palmitate, N-butyl stearate, hexyl laurate, myristyl myristate, hexyl myristate, isopropyl palmitate, hexyl stearate, hexyl laurate, decyl myristate, hexyl laurate, decyl oleate, decyl laurate, glyceryl tri-2-ethylhexanoate, glyceryl trioctanoate, glyceryl tri-isopalmitate, trimethylolpropane tri-isostearate, cetyl-2-ethyl hexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, glyceryl tri-2-heptylundecanoate, methyl ricinoleate, oleyl oleate, acetyl glycerol, 2-heptylundecanoate palmitate, diisobutyl adipate, 2-octyldodecyl N-lauroyl-L-glutamate, di-2-heptylundecyladipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, 2-ethylhexyl succinate, glyceryl tri-isopalmitate, glyceryl tri-isostearate, glyceryl palmitoleate, 2-heptylundecanoate, glyceryl palmitoleate, triethyl citrate, polyoxyethylene and polyoxypropylene random polymer methyl ester.

Examples of silicone oils that can be used include chain polysiloxanes (such as dimethylpolysiloxane, methylphenylpolysiloxane, and diphenylpolysiloxane), cyclic polysiloxanes (such as octylmethylcyclotetrasiloxane, decamethyl-cyclopentasiloxane, and dodecamethylcyclohexasiloxane), silicone resins that have formed three-dimensional network structures, silicone rubbers, and various modified polysiloxanes (such as amino-modified polysiloxanes, polyether-modified polysiloxanes, alkyl-modified polysiloxanes, and fluorine-modified polysiloxanes).

Further, the preparation for external application to the skin may contain, for example, moisturizers such as polyethylene glycol, glycerin, 1, 3-butylene glycol, erythritol, sorbitol, xylitol and maltitol; thickeners such as cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methylhydroxypropyl cellulose, methyl cellulose, carboxymethyl cellulose, paradox, carrageenan, pectin, mannan, curdlan, chondroitin sulfate, starch, galactan, dermatan sulfate, glycogen, gum arabic, heparan sulfate, hyaluronic acid, sodium hyaluronate, tragacanth, keratan sulfate, chondroitin, xanthane gum, mucopolysaccharides sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, dextran, cutin sulfate, locust bean gum, succinoglucan, charonin acid, chitin, chitosan, carboxymethyl chitin and agar; lower alcohols such as ethanol; antioxidants such as butylated hydroxytoluene, vitamin E and phytin; antibacterial agents such as benzoic acid, salicylic acid, sorbic acid, alkyl p-hydroxybenzoate, and hexachlorophene; organic acids such as acylsarcosine (e.g., sodium lauroyl N-methyl-glycine (carposine)), glutathione, citric acid, malic acid, tartaric acid, and lactic acid; vitamins such as vitamin a and its derivatives, vitamin B such as vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctoate, vitamin B2 and its derivatives, vitamin B12, vitamin B15 and its derivatives, vitamin C such as ascorbic acid, ascorbyl sulfate, ascorbyl phosphate and ascorbyl dipalmitate, vitamin E such as α -tocopherol, β -tocopherol, γ -tocopherol and vitamin E acetate, vitamin D, vitamin H, pantothenic acid and pantetheine; various medicines such as nicotinic acid amide, benzyl nicotinate, gamma-oryzanol, allantoin, glycyrrhizic acid (salt), glycyrrhizic acid and its derivatives, hinokitiol, bisabolol, eucalyptol, thymol, inositol, saponins such as saikoside, daucosterol, luffa saponin and pimaponin, panthenyl ethyl ether, ethynylgestodene, tranexamic acid, arbutin, sarcin and placenta extract; plant extracts such as Rumex japonicus Houtt, Sophora flavescens Aiton, Nuphar japonicum DC, citrus, sage (Salvia officinalis), Achillea alpina L, Malva sylvestris L, Mauritiana macrophylla L, Swertiana japonica (Swie Herb), Thymus vulgaris L (Thymus vulgaris L), Angelica japonica root, bitter orange peel, Betula alba, Equisetum arvense L, Luffa cylindrica, Aesculus hippocastanum L (horse chestnut), Saxifraga stolonifera (Saxifraga longifolia), Gardenia jasminoides (Aranea), Lilium Montanium, Lilium (Artemisia japonica L), Aloe barbata (Lilium japonicum L), extract of Pacifolia japonica (Pacifia japonica L), extract of Pacifica (Pacifia japonica L), extract of Pacifia japonica (Pacifica japonica), extract of Pacifia japonica (Pacifia japonica), Pacifia japonica (Pacifia japonica L), extract of Pacifica (Pacifia japonica L), Pacifia japonica (Pacifica japonica L), extract of Pacifia japonica (Pacifica, Pacifia japonica L), Pacifia japonica (Pacifica extract of Pacifia japonica, Pacifia japonica L), Pacifolia (Pacifia japonica L), Pacifia japonica (Pacifolia) Iris extract, catechu palmatum extract, Ginkgo biloba L (Ginkgo biloba) extract, Thymus quinquecostata cell extract, Foeniculum vulgare Mill (fennel) extract, oolong tea extract, lilium aquaticum extract, rosehip extract, japanese savory (Isodon japonica (Burm) Hara) extract, scutellaria root extract, phellodendron amurense bark extract, wild sesame variety (Lamium album ml. var. barbatum) (sieb. et Zucc.) extract, licorice extract, Gardenia jasminoides Ellis fortunei (Lour.) kurz extract, black tea extract, chinese tamark twining extract, potentilla chinensis (potentilla scholaris) extract, rose extract, rosewood extract, rosemary extract, and rosemary extract; a colorant; nonionic surfactants such as sorbitan monolaurate, sorbitan monopalmitate, sorbitan sesquioleate, sorbitan trioleate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostearate, polyethylene glycol monooleate, polyoxyethylene alkyl ether, polyethylene glycol diethanolamide, lauroyl diethanolamide, fatty acid isopropanolamide, maltitol hydroxy fatty acid esters, alkylated polysaccharides, alkyl glycosides and sugar esters; cationic surfactants such as octadecyl trimethyl ammonium chloride, benzalkonium chloride, and dodecyl amine oxide; anionic surfactants such as sodium palmitate, sodium laurate, sodium laurylate, potassium lauryl sulfate, triethanolamine alkyl sulfates, turkey red oil, linear dodecylbenzene sulfates, polyoxyethylene cured castor oil maleate, and acyl methyl taurines; an amphoteric surfactant; a neutralizing agent; antioxidants such as gamma-tocopherol and butylated hydroxytoluene; and preservatives such as phenoxyethanol and parabens.

In the method according to the present invention, the above-mentioned substance capable of inhibiting ADAM activity can be applied to the skin in one of various forms, whereby the substance can be applied to the skin, and thus the object of the present invention can be achieved. The ADAM activity-inhibiting substance can be used alone. Alternatively, the above-mentioned substance capable of inhibiting the activity of ADAM may be used in admixture with any other component. Further, there is no limitation on the skin site to which the above-mentioned substance capable of inhibiting ADAM activity is applied. In particular, the above-mentioned substance capable of inhibiting ADAM activity can be applied to every part of the skin on the body surface, including the skin of the head. The method according to the invention should preferably be used as a cosmetic method, but is not limited to use as a cosmetic method.

The method for evaluating an anti-wrinkle effect according to the present invention includes the step of contacting a test substance with skin, skin tissue, or skin cells of a human or an animal.

The method for evaluating the anti-wrinkle effect according to the present invention can be performed by using one of various skins of humans and animals, and thus the object of the present invention can be achieved. For example, the method for evaluating the anti-wrinkle effect according to the present invention can be performed by using the skin of hairless mice, whose skin has been subjected to tape stripping for peeling off the stratum corneum of the skin. In particular, the skin of hairless mice can be stripped of tape, which can strip the stratum corneum of the skin and thus continuously disrupt the skin barrier. In this case, the skin of the mouse shows fine wrinkles, and the enzymatic activity or gene expression of ADAM such as ADAM-9, ADAM-10 or ADAM-17 is increased. It is considered that one of the causes of the formation of fine wrinkles is to promote the release and activation of growth factors such as HB-EGF by increasing the activity of ADAM, and thus cause the occurrence of thickening of epidermis and dermis. Therefore, it is considered that an anti-wrinkle substance can be efficiently specified by specifying a substance capable of reducing the expression of ADAM gene, which has been increased by the above-mentioned continuous skin barrier disruption, for example, with the method for evaluating an anti-wrinkle effect according to the present invention.

The skin cells used in the method for evaluating an anti-wrinkle effect according to the present invention are epithelial cells forming the epidermal area of the skin. Examples of epithelial cells include epidermal cells such as epidermal keratinocytes, sebaceous gland cells, mammary gland cells, small intestine epithelial cells, and other mucosal epithelial cells. Among the examples of epithelial cells listed above, epidermal keratinocytes, which are an example of epidermal cells, have an advantage that the cells can be easily cultured. In addition, this detection method is a technique related to the state of epidermis. Thus, epidermal keratinocytes are the preferred skin cells for the present method. In addition, the skin cell supply source should preferably be human. Still further, the skin cells used in the present method should preferably be cultured cells. The cultured cells can be prepared by a conventional method.

For example, in the case where the skin cells are human epidermal keratinocytes, human epidermis can be prepared from an excess skin section obtained by restorative surgery, dermatology, surgical operation, or the like, by processing of removing adipose tissue and blood therein from the skin section and separating a dermal portion thereof by protease treatment, or the like. In addition, epidermal keratinocytes can be isolated from the human epidermis thus prepared and can be primary-cultured by a conventional method using a KGM medium and can be subcultured by a conventional method using a protease or the like. In this way, the desired cultured human epidermal keratinocytes can be obtained. Epidermal keratinocytes of the type described are commercially available. Commercially available epidermal keratinocytes may also be used in the present invention.

The detection of the enzymatic activity of ADAM can be carried out by, for example, measuring the release amount of ADAM substrate such as HB-EGF.

In addition, for detecting the expression level of a gene to be detected, for example, cDNA can be amplified and/or assayed by using RT-PCR, Northern blot technique, or the like. Alternatively, proteins can be detected and/or assayed by using ELISA, Western blot techniques, and the like.

The step of evaluating the anti-wrinkle effect of the test substance by using the enzymatic activity or gene expression level of ADAM as an index may include, for example, a technique in which skin, skin tissue, or skin cells to which the test substance is not added or to which a substance known not to affect the activity of ADAM is added are used as a control, and the enzymatic activity or gene expression level of ADAM when the test substance is added is compared with the enzymatic activity or gene expression level of ADAM in the control. For example, as a result of comparison with the enzyme activity or gene expression level of ADAM of a control, it is possible to specify that a substance that decreases the enzyme activity or gene expression level of ADAM is found as a substance having an anti-wrinkle effect (i.e., an anti-wrinkle substance or a wrinkle preventing or improving agent).

There is no limitation in the manner of using the method for evaluating an anti-wrinkle effect according to the present invention. For example, the method for evaluating an anti-wrinkle effect according to the present invention can be used for screening and evaluating an anti-wrinkle substance and the like.

The invention will be further illustrated by the following non-limiting examples.

Examples

Preparation of Fine wrinkle mouse model

It is believed that in the event that a number of major factors, such as decreased sebum and washing of the face, weaken or disrupt the skin barrier and increase water evaporation from the skin surface, the skin dries out and fine wrinkles form. Therefore, the adhesive tape was repeatedly peeled off from the skin of hairless mice, thereby peeling off the horny layer andthe skin barrier is broken. In this way a fine wrinkle mouse model can be prepared. In particular, the percutaneous water loss (TEWL) can be dropped to 4mg/cm with cellophane tape by adjusting the number of times the tape is peeled off (measured by using a water evaporation amount measuring device MEECO provided by Meeco corporation, USA)²H to 6mg/cm²In the range of/, the tape was peeled off from the skin on the left back side of hairless mice (HR-1, male, six weeks old, supplied by Hoshino Experimental Animals). Typically, the stripping of the tape is repeated approximately four times in the initial stage. The stripping of the tape is repeated from about seven to eight times in the final stage. The tape was peeled off at three stages per week and continued for four weeks. The right side of the back of hairless mice was left as the non-treated area.

FIG. 2A is a replicate image showing the skin of a mouse that has been subjected to stripping of the tape in the manner described above. Fig. 2B is a photomicrograph showing a skin tissue section of mouse skin, which has been stained with HE (hematoxylin-eosin). As shown in the reproduction (FIG. 2A), the skin trench deepened at a 24 hour period (24h) after the start of stripping the tape and at a 48 hour period (48h) after the start of stripping the tape. The direction of the skin channel was the same as the predetermined direction at the one-week (1W) stage after the start of the tape stripping. At the four week (4W) stage after the start of the tape stripping, a wrinkled appearance appeared. In addition, as shown in the photomicrograph of the skin tissue (fig. 2B), it was found that the epidermis began to thicken at the 24-hour period after the start of peeling the tape when compared with the non-treated region (NT). The skin remained thickened at the two week stage (2W) after the start of the tape stripping. Table 1 below shows morphological changes, histological changes, and the like of the fine wrinkle mouse model that has been prepared in the above-described manner, and shows morphological changes, histological changes, and the like of the photoaging mouse model that has been prepared with ultraviolet irradiation (10 weeks).

TABLE 1 comparison between Fine wrinkles mouse model and photoaging mouse model

The skin of mice that had been subjected to the peeling tape in the above manner showed the same changes as those of fine wrinkles of human skin. In addition, the skin of a mouse that has been subjected to a release tape in the above-described manner is significantly different from a photoaging mouse model that has been prepared with ultraviolet radiation in terms of wrinkle appearance, epidermal hypertrophy, and the like. It was thus shown that with the above method it is possible to prepare a fine wrinkle mouse model that is significantly different from the large wrinkle mouse model prepared by photoaging.

Study of Biochemical alterations in the Fine wrinkle mouse model

With respect to the fine wrinkle mouse model that has been prepared in the above-described manner, it is inferred that hypertrophy of the epidermis and dermis has occurred, and that cell growth factors in the epidermis and dermis have been activated by barrier disruption stimulation that has occurred by peeling off the adhesive tape. Therefore, the gene expression of the cell growth factors HB-EGF and amphiregulin present in the epidermis and the gene expression of the enzymes ADAM-9 and ADAM-17 which exert the action of releasing the above growth factors and activated growth factors from the cell membrane were investigated.

The expression level was measured by RT-PCR. Specifically, total RNA was extracted from dorsal skin samples sampled with time on the left side (peel tape treatment: T) and the right side (non-treatment: N) of the fine wrinkle mouse model that had been prepared in the above manner. In addition, after the cDNA has been prepared, RT-PCR is performed by using primers specific to the target gene to be tested. Further, as a reference, a housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) having the property that the expression amount in each cell is the same as a predetermined amount was determined in the same manner as described above.

The results as shown in fig. 3 were obtained. As shown in FIG. 3, with regard to ADAM-9 and ADAM-17, gene expression was increased at the 24-hour period after the tape had been peeled off from the skin and at the 48-hour period after the tape had been peeled off from the skin. In addition, with respect to HB-EGF and amphiregulin released and activated by ADAM belonging to the same HB-EGF family, gene expression was increased at a 24-hour period after a tape had been peeled off on the skin to a one-week period after the tape had been peeled off on the skin.

Research on anti-wrinkle effect of drug applied to fine wrinkle mouse model

The anti-wrinkle effects of various drugs applied on a fine wrinkle mouse model as shown in table 2 below were investigated.

TABLE 2

^*: n-hydroxy-2- [ [ (4-methoxyphenyl) sulfonyl group]3-picolyl]Amino group]-3-methylbutaneamide hydrochloride](J.Med.chem.1997, Vol.40, page 2525-2332)

In particular, the tape was peeled off in the above manner on hairless mice. After each tape stripping treatment, 100 μ l of each of the drugs listed in table 2 was applied to the skin of hairless mice. Fig. 4A is a duplicate image showing skin to which different drugs have been applied in a mouse model of fine wrinkles, wherein duplicate images have been obtained at one, two, and four week stages after application of each drug. Fig. 4B is a photomicrograph showing a mouse skin tissue section at a four-week stage after each drug, in which the skin tissue section has been stained with HE (hematoxylin-eosin). The following table 3 shows the results obtained according to the evaluation criteria described below.

O: improvements in or relating to

And (delta): no changes were found

X: deterioration of

TABLE 3 anti-wrinkle Effect of various drugs on the Fine wrinkle mouse model

TAPI-1, an ADAM activity inhibitor, inhibited the formation of fine wrinkles and significantly inhibited the thickening of epidermis and dermis in a fine wrinkle mouse model. CGS27023A (N-hydroxy-2- [ [ (4-methoxyphenyl) sulfonyl ] 3-picolyl ] amino) -3-methylbutaneamide hydrochloride) (j.med.chem.1997, volume 40, page 2525-2332) and glycerin as a humectant, which showed an effect of preventing wrinkle formation on a mouse model of photoaging as MMP inhibitors, were not able to inhibit wrinkle formation at all and epidermal and dermal thickening as a moisturizing agent for a mouse model of fine wrinkles. In addition, unsaturated fatty acid oleic acid, which has an effect of promoting skin roughening, promotes wrinkle formation and accelerates thickening of epidermis and dermis.

From the above results, it was revealed that the formation of fine wrinkles cannot be sufficiently prevented or improved only with the moisturizing agent. It has also been shown that when the activity of ADAM in the skin is inhibited, and thus the release and activation of growth factors are inhibited, thickening of the epidermis and dermis can be inhibited, and the formation of fine wrinkles can be effectively prevented or improved. Further, from the results that CGS27023A as an MMP inhibitor showing an effect of inhibiting wrinkle formation in a mouse model of photoaging could not inhibit wrinkle formation, it was suggested that the effect of inhibiting wrinkle formation by the substance capable of inhibiting ADAM activity of the present invention is obtained by a mechanism of action different from that of an MMP inhibitor.

Search for novel ADAM activity inhibitory substance and evaluation of anti-wrinkle effect

Novel compounds capable of inhibiting ADAM activity were sought, and anti-wrinkle effects of the novel compounds were evaluated using a fine wrinkle mouse model. Unless otherwise indicated, the mixing ratio of the compounds is expressed in mass%.

(1) Screening for ADAM Activity inhibitory substance

First, a compound having ADAM enzyme inhibitory activity was screened by using HB-EGF-AP/HT-1080 (human fibrosarcoma-derived cultured cell modified to achieve forced expression of fusion protein HT-800 in which thermostable Alkaline Phosphatase (AP) was added to the N-terminus of human HB-EGF). On the cell surface of the used cell line HB-EGF-AP/HT-1080, the HB-EGF full-length molecule was expressed in the form of fusion with alkaline phosphatase. In the case of phorbol ester-stimulated cells, ADAM enzyme on the cell membrane surface was activated to cleave HB-EGF molecule. Since alkaline phosphatase has bound to HB-EGF which has been cleaved into a released form, in the case of measuring the alkaline phosphatase activity in the culture supernatant, the ADAM enzyme inhibitory activity of the compound can be indirectly measured.

In particular, the number of cells has been adjusted to 2.0X 10⁵Individual cells/well of HB-EGF-AP/HT-1080 were seeded into 96 well microplate at a rate of 0.2 ml/well and cultured overnight at 37 ℃. After removing the medium and washing with PBS (-), the medium containing the test substance was added at a rate of 0.1 ml/well and incubated at 37 ℃ for 30 minutes. This is done for pretreatment. Thereafter, the supernatant was removed, and a medium containing the test substance and 60nM TPA (phorbol ester: 12-o-tetradecanoyl phorbol acetate; Sigma P8139) was added at a rate of 0.2 ml/well. Then incubated for 60 minutes and treated as such. After treatment, 0.1ml of culture supernatant per well was transferred to wells of a microplate for alkaline phosphatase activity detection. In addition, incubation was carried out at 65 ℃ for 10 minutes, and the endogenous alkaline phosphatase was thus inactivated. Further, 1mg/ml of an AP substrate (p-nitrophenylphosphate, Wako; 141-02341) was added to each well at a rate of 0.1 ml/well, and the absorbance in each well was immediately measured at a wavelength of 405 nm. After incubation for 2 hours at room temperature in the dark, the absorbance in each well was measured again at a wavelength of 405 nm. The absorbance of each well was determined as a value obtained by subtracting the absorbance measured immediately at the stage after the addition of the AP substrate from the absorbance measured at the stage after the two-hour incubation. The inhibition rate was calculated by the following formula:

inhibition rate (%) - (a0-AS)/(a0-a100) × 100

Where a0 represents the absorbance of the 0% inhibition control (TPA only medium), a100 represents the absorbance of the 100% inhibition control (TPA only medium), and AS represents the absorbance of the sample.

As a result, it was found that HB-EGF release was efficiently inhibited with 4-methoxyphenyl hydroxamic acid, and it was revealed that 4-methoxyphenyl hydroxamic acid could inhibit ADAM activity. The 4-methoxyphenyl hydroxamic acid can be represented by the following structural formula. In addition, the release inhibition rate of 4-methoxyphenyl hydroxamic acid is shown in table 4 below.

4-methoxyphenyl hydroxamic acid

TABLE 4

(2) Evaluation of anti-wrinkle Effect of ADAM Activity inhibitory substance

The anti-wrinkle effects of TAPI-1 and 4-methoxyphenyl hydroxamic acid as ADAM activity inhibitors were investigated by using a fine wrinkle mouse model.

In particular, the skin on the left back of hairless mice (HR-1, male, six weeks old, supplied by Hoshino Experimental animals) was taped off in three stages per week for four weeks so that the transcutaneous Water loss (TEWL) (measured by using the Water evaporation Capacity measuring device MEECO supplied by the American Meeco Corp.) could fall at 4mg/cm²H to 8mg/cm²In the range of/h. After each stage of the peel-tape treatment was completed, 100. mu.l of TAPI-1 or 4-methoxyphenyl hydroxamic acid was applied to the skin on the left side of the back of the mouse. At this time, 4-methoxyphenyl hydroxamic acid was dissolved in 50% aqueous ethanol solution so that the concentration of 4-methoxyphenyl hydroxamic acid may become equal to 1%, and 4-methoxyphenyl hydroxamic acid was used in the form of the resulting solution. In addition, TAPI-1 (supplied by Peptide Research Institute) was evaluated at a concentration of 1 mM. Further, as a negative pairIn the same manner as described above, a 50% aqueous ethanol solution (i.e., vehicle) was applied.

The state of wrinkle occurrence after four weeks had elapsed is indicated by a visually judged score. The state of occurrence of wrinkles was evaluated by the following scale.

0: without wrinkles

1: shallow wrinkles

2: obvious wrinkles

3: deep wrinkles

The state of occurrence of wrinkles is indicated by a score of every 0.5. Large scores indicate deep wrinkles. The mean and standard deviation of each group were calculated. The results shown in FIG. 5 were obtained.

As shown in fig. 5, the mean of the vehicle scores was equal to 1.17. In addition, the average value for the TAPI-1 score is equal to 0.43, while the average value for the 4-methoxyphenyl hydroxamic acid score is equal to 0.91. Each of TAPI-1 and 4-methoxyphenyl hydroxamic acid as ADAM activity inhibitory substances showed a significant wrinkle inhibitory effect in comparison with vehicle.

Thereafter, the replicas of the mouse back were analyzed by using a wrinkle analysis apparatus (supplied by Hamano Engineering) and the wrinkle area (%) was calculated. A low percent wrinkle area indicates that wrinkle formation has been inhibited. The results shown in FIG. 6 were obtained.

As can be seen in FIG. 6, the mean value of the area (%) of the TAPI-1 wrinkles and the mean value of the area (%) of the 4-methoxyphenyl hydroxamic acid wrinkles are significantly lower than the mean value (%) of the area of the vehicle wrinkles. Thus, each of TAPI-1 and 4-methoxyphenyl hydroxamic acid as ADAM activity inhibitory substances showed a high wrinkle inhibitory effect.

From the above results, it was revealed that each of TAPI-1 and 4-methoxyphenyl hydroxamic acid as ADAM activity inhibitory substances can inhibit the release of HB-EGF as an epidermal growth factor by inhibiting ADAM enzyme, and can prevent or improve wrinkles.

Claims (5)

1. Use of 4-methoxyphenyl hydroxamic acid for producing an agent for preventing or ameliorating wrinkles, wherein the 4-methoxyphenyl hydroxamic acid is capable of inhibiting the activity of disintegrin and metalloprotease present in the skin.
2. 2. The use according to claim 1, wherein the wrinkle preventive or improving agent is a cosmetic.
3. 3. A simple cosmetic method for preventing or improving wrinkles, comprising the step of applying to the skin a substance capable of inhibiting the activity of disintegrin and metalloprotease present in the skin, wherein the substance capable of inhibiting the activity of disintegrin and metalloprotease present in the skin is 4-methoxyphenyl hydroxamic acid.
4. 4. The simple cosmetic method for preventing or improving wrinkles as claimed in claim 3, wherein the disintegrin and metalloprotease are selected from the group consisting of disintegrin and metalloprotease-9, disintegrin and metalloprotease-10 and disintegrin and metalloprotease-17.
5. 5. Use of a substance capable of inhibiting the activity of disintegrin and metalloprotease for cosmetically preventing or ameliorating wrinkles for non-therapeutic purposes, wherein the substance capable of inhibiting the activity of disintegrin and metalloprotease is 4-methoxyphenyl hydroxamic acid.