JP5686365B2 - Collagen production promoter, photoaging inhibitor, moisturizing function improving agent and dermatological composition - Google Patents

Description

  The present invention is based on gamma-glutamyltranspeptidase (GGT) inhibitory activity, activates the ability to produce collagen in dermal fibroblasts of skin, reduces UV damage, and horny water content For skin containing ingredients that have a remarkable effect of maintaining and enhancing skin tension, gloss and moisturizing effect, and preventing and improving skin wrinkles and sagging, and photoaging. The present invention relates to a pharmaceutical composition.

  The skin has a three-layer structure of the epidermis, dermis, and fat layer (subcutaneous tissue). The dermis is a thick layer made of fibrous tissue and elastic tissue, most of which is composed of an extracellular matrix such as collagen. It gives elasticity and strength, and keeps the skin fresh and supple. However, due to the effects of external factors such as UV irradiation and drying, or aging, the production of collagen, which is a major component of the extracellular matrix, decreases and the amount of matrix metalloprotease, a collagenolytic enzyme, increases. As a result, the decrease, decomposition, and denaturation of collagen in the dermis accelerates, the skin barrier function decreases, the moisturizing function and elasticity are lost, the skin tension and gloss are lost, wrinkles Appears skin aging symptoms such as sagging.

  Occurrence of wrinkles, sagging, etc. seen in the aging change of the skin is a major aging phenomenon in appearance and is a serious problem for many middle-aged and elderly people. Conventionally, as a method for improving such skin symptom, it has been the mainstream to apply cosmetics containing collagen, but since it temporarily supplements the moisture retention of the skin surface, it is sufficient. It was not effective. Therefore, a highly safe active ingredient that improves the decrease in the amount of collagen is desired.

  Under such circumstances, the development of cosmetics for increasing the amount of collagen in the skin has been promoted for the purpose of preventing and improving aging of the skin, including maintaining skin tension and gloss and improving the moisture retention function of the skin. For example, it is reported that an extract derived from a natural product has a type 1 collagen production promoting action (Patent Documents 1 and 2), and ascorbic acid is used as a component having an action of stimulating collagen synthesis constituting a dermal extracellular matrix. Have been reported (Patent Document 3). In addition, the present inventors have found that the amount of collagen produced by dermal fibroblasts increases in association with a decrease in the amount of glutathione in the fibroblasts, and this mechanism enhances collagen production. The compound group which has this is reported (patent document 4).

  On the other hand, γ-glutamyltranspeptidase (GGT) is known as an enzyme that degrades intracellular glutathione. GGT plays an important role as an enzyme that degrades glutathione, which is a pool of major cysteine (Cys) outside the cell, and supplies Cys necessary for glutathione biosynthesis to the cell. It is thought that there is a physiological effect that controls the amount of glutathione. The present inventors have reported on GGT inhibitor having high activity and high selectivity in Patent Document 5.

JP 2007-186471 A JP 2007-302607 A Japanese Patent Application Laid-Open No. 2004-075646 JP 2006-151860 A WO 2007/066705 A1 Publication

Cutrin et al., Kidney International, Vol. 57 (2000), pp. 526-533) Aberkane et al., Biochem. Biophy. Res. Commun. 285, 1162-1167 (2001)

  The present invention not only promotes collagen production by exerting GGT inhibitory activity, but also contains a compound that can prevent damage due to ultraviolet rays and prevent photoaging, and can prevent and improve aging such as wrinkles and sagging of the skin. Provided a useful collagen production promoter, photoaging inhibitor and moisturizing function improving agent, and contains the collagen production promoter, photoaging inhibitor or moisturizing function improving agent, and is caused by skin wrinkles / sagging or UV damage. Provided is a composition for a dermatological agent that is effective for photoaging, moisturizing function improvement, and the like, and for effective skin conditioning.

  The present inventors have made various studies with the main purpose of solving the above problems, and as a result, found that a decrease in glutathione content in fibroblasts is associated with an increase in collagen production. As a result of intensive studies on the functions of GGT inhibitors, GGT inhibitors (i) reduce the amount of glutathione in fibroblasts and increase collagen production, (ii) prevent skin damage due to ultraviolet rays, and reduce photoaging. The present invention has been completed by discovering that it has a new function such as prevention and improvement, and (iii) the ability to increase the amount of keratin moisture.

  That is, the collagen production promoter of the present invention comprises a compound that inhibits γ-glutamyl transpeptidase as an active ingredient. The photoaging inhibitor of the present invention comprises a compound that inhibits γ-glutamyl transpeptidase as an active ingredient. The moisturizing function improving agent of the present invention comprises a compound that inhibits γ-glutamyl transpeptidase as an active ingredient.

  In the collagen production promoter, photoaging inhibitor or moisturizing function improving agent of the present invention, the compound preferably contains a phosphonic acid diester derivative having a leaving group. The compound is represented by the following general formula (1) (wherein at least one of R1 and R2 represents a leaving group, and at least one of R1 and R2 is represented by the following general formula (2) to general formula (6)): (In the formula, R3 is any of an aryl group optionally having substituent (s) and a heterocyclic residue optionally having substituent (s); R4, R5, R6, R7, R8 and R9; Each is at least one substituent selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, and an electron withdrawing group, and R4 It is preferable that two adjacent substituents among the substituents of ~ R8 may be bonded to each other to form a ring. The compound preferably contains 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid.

  Moreover, the composition for dermatological agents containing the said collagen production promoter, a photoaging inhibitor, or a moisturizing function improving agent is also contained in this invention. Moreover, it is preferable that the composition for skin preparations is for improving wrinkles or skin elasticity.

  The collagen production promoter of the present invention comprises a GGT inhibitor that reduces the amount of glutathione in fibroblasts as an active ingredient, and has a function of significantly promoting collagen production in skin fibroblasts. is there. As a result, the collagen production promoter of the present invention has the effect of significantly promoting collagen production in skin fibroblasts, preventing and improving skin aging, specifically maintaining skin firmness and gloss. Effective in preventing and improving wrinkles and sagging by improvement, or maintaining and improving the moisture retention function of the skin.

  Moreover, the photoaging inhibitor of the present invention can also reduce damage to skin cells due to ultraviolet rays.

  Moreover, the moisturizing function improving agent of the present invention can improve the skin moisturizing function by increasing the amount of keratinous moisture in the skin.

  And the composition for dermatological agents containing the collagen production promoter by GGT inhibitory activity of this invention has the effect | action which improves collagen production in a skin fibroblast markedly, and also has skin elasticity and wrinkles. It can be improved. Moreover, the composition for dermatological agents containing the photoaging inhibitor by GGT inhibitory activity of this invention reduces the damage by ultraviolet rays, and exhibits an effective effect | action in prevention / improvement of photoaging. Moreover, the composition for dermatological agents containing the moisturizing function improving agent by GGT inhibitory activity of this invention exhibits the effect | action effective in the moisturizing function improvement and skin conditioning of a skin by raising the moisture content of a stratum corneum. . Therefore, for skin aging prevention / improvement or skin conditioning, specifically for skin firmness, wrinkle / sag prevention / improvement by maintaining and improving gloss, or photoaging prevention / improvement, or skin It can be effectively used for maintaining and improving the moisture retention function.

It is drawing which shows the measurement result of the amount of intracellular glutathione of the human skin origin fibroblast obtained in the Example. It is drawing which shows the result of the collagen staining image of the human skin origin fibroblast obtained in the Example. It is a figure which shows the change rate of the amount of stratum corneum water by the lotion continuous use obtained in the Example. It is a figure which shows a time-dependent change of the skin viscoelasticity of the lotion continuous use 3 months obtained in the Example. It is a figure which shows the wrinkle score 3 months after the lotion continuous use obtained in the Example. It is a figure which shows the influence of the GGT inhibitor on the survival rate of the human normal skin origin fibroblast under UV irradiation obtained in the Example. Is a diagram showing the effect of GGT inhibitor on increase of H ₂ O ₂ production level of human normal skin-derived fibroblasts in the UV irradiation under obtained in Example. Is a diagram showing the effect of GGT inhibitor on intracellular H ₂ O ₂ production amount of human skin fibroblasts enhanced by UV exposure obtained in Example.

  Conventionally, GGT has been known to be an enzyme that degrades extracellular glutathione. Therefore, generally speaking, inhibition of GGT activity prevents glutathione degradation, so that the amount of intracellular glutathione is also increased or maintained. For example, Non-Patent Document 1 is cited as an academic paper that supports this prediction. In this document, when acivicin, a GGT inhibitor, is administered to the rat kidney, the amount of glutathione increases compared to the control (Fig. 6). It has also been shown that GGT activity is reduced by acivicin administration (Fig. 1). That is, Non-Patent Document 1 shows that the addition of a GGT inhibitor suppresses the decomposition of glutathione, and as a result, the amount of glutathione increases. Non-Patent Document 2 describes data indicating that even when acivicin is administered to a V79GGT cell line highly expressing human GGT, no significant difference in intracellular glutathione level is observed (Table 2). . GGT is inhibited in a concentration-dependent manner by acivicin (Table 1). On the other hand, since the amount of glutathione in a cell is related to various factors such as biosynthesis, metabolism, and transport in addition to GGT, it is difficult to simply derive the relationship between GGT activity and the amount of glutathione. Furthermore, since acivicin, which has been frequently used as a GGT inhibitor, widely inhibits enzymes other than GGT and disturbs the biosynthetic system of cells in various ways, the amount of glutathione and GGT in conventional studies using acivicin The relationship remains unclear.

  Therefore, the present inventors have independently studied using a GGT-selective inhibitor instead of acivicin. As a result, the first time in the world that a selective GGT inhibitor reduces the amount of glutathione in fibroblasts. discovered. Such knowledge is not disclosed or suggested at all in, for example, Non-Patent Documents 1 and 2 and also in Patent Documents 4 and 5. Based on this novel finding, the present inventors have reduced the amount of intracellular glutathione by the present GGT inhibitor, resulting in promoting collagen production, reducing damage caused by ultraviolet rays, and increasing the amount of keratinous water. The present inventors have found a novel function of a GGT inhibitor such as being capable of being made, and thus completed the present invention. Therefore, it can be said that the present invention is completely new and original, which has been uniquely found by the present inventors.

  In addition, as shown in Examples described later, the present inventors have found that a GGT inhibitor can not only promote collagen production but also reduce damage to skin fibroblasts caused by ultraviolet irradiation. There is photoaging as skin aging. Photoaging is aging caused by exposure to ultraviolet rays, and one of the causes is oxidation of in vivo components by reactive oxygen species. Photoaging causes a decrease in the amount of collagen in the skin, which reduces the elasticity of the skin and causes wrinkles. It is known that an increase in collagen resolution is greatly involved in the decrease in collagen. As shown in the examples described later, the GGT inhibitor significantly reduced the amount of intracellular reactive oxygen species produced by exposure to ultraviolet rays, and thus it was revealed that it has a preventive effect or an improvement effect on photoaging. . In addition, the photoaging inhibitor as used in the field of this invention intends the composition which prevents and / or improves photoaging.

  The present inventors have also confirmed that the GGT inhibitor has a function of increasing the amount of water in the stratum corneum of the skin. Thereby, providing the composition which improves and maintains the moisture retention function of the skin using a GGT inhibitor, and has a skin conditioning effect | action is also contained in this invention. In addition, the moisturizing function improving agent as used in the field of this invention intends the composition which improves the moisturizing function of skin (skin) and / or prevents that a moisturizing function falls (maintaining a moisturizing function).

  Hereinafter, an example of a specific embodiment of the present invention will be described.

  As the GGT inhibitor used in the present invention, a phosphonic acid diester derivative group (Patent Document 5) characterized by high enzyme selectivity and irreversible inhibitory activity can also be used. This group of phosphonic acid diester derivatives can be preferably used because of its particularly low biotoxicity.

  Specifically, for example, the following phosphonic acid diester derivative groups can be suitably used.

  1. General formula (1)

A phosphonic acid diester derivative represented by the formula (wherein at least one of R1 and R2 represents a leaving group):

2. General formula (1)
(In the formula, at least one of R1 and R2 is represented by the general formula (2) to the general formula (6).

(In the formula, R3 is any of an aryl group optionally having substituent (s) and a heterocyclic residue optionally having substituent (s); R4, R5, R6, R7, R8 and R9; Each is at least one substituent selected from the group consisting of a hydrogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, and an electron withdrawing group, and R4 To R8, two adjacent substituents may be bonded to each other to form a ring. A phosphonic acid diester derivative represented by

3. General formula (1)
(Wherein R1 is OR10, R2 is OR11, and R10 and R11 exclude a hydrogen atom).

  4). The phosphonic acid diester derivative shown in 3 above, wherein R10 is any one of an alkyl group which may have a substituent and an aryl group which may have a substituent, and R11 has a substituent. A phosphonic acid diester derivative which is an aryl group which may have.

  5. The phosphonic acid diester derivative shown in 4 above, wherein the substituent of the alkyl group which may have a substituent is a phenyl group which may have a substituent, a heterocyclic residue having nitrogen, an alkyl A phosphonic acid diester derivative which is at least one group selected from the group consisting of sulfanyl group, arylsulfanyl group, hydroxy group, carbamoyl group, amino group, guanidino group, alkoxy group, amide group, carboxy group and carboxy group equivalent .

  6). The phosphonic acid diester derivative shown in 4 above, wherein the alkyl group which may have the substituent of R10 is represented by the general formula (7)

(Wherein R12 represents any of an alkyl group which may have a substituent, an aryl group which may have a substituent, and a hydrogen atom, and R13 represents a hydrogen atom and a general formula (8)

(Wherein n1 is an integer of 0 to 4, n2 is any of 0 and 1, n3 is an integer of 0 to 4, X1 is either an amide group or an alkenyl group, X2 is a carboxy group, and Any one of the equivalents of a carboxy group represents any one of R14 represents either a hydrogen atom or a lower alkyl group. A phosphonic acid diester derivative.

7). A phosphonic acid diester derivative represented by the above 5 or 6, equivalent of carboxyl group and carboxy _{groups, -COOR, -CONR 2, -COR,} -CN, -NO 2, -NHCOR, -OR, -SR, A phosphonic acid diester derivative, which is at least one group selected from the group consisting of —OCOR, —SO ₃ R, and —SO ₂ NR ₂ , wherein R is any one of a hydrogen atom and an alkyl group.

  8). The phosphonic acid diester derivative shown in 6 above, wherein the substituent of the alkyl group which may have the substituent of R12 is a phenyl group which may have a substituent, or a heterocyclic residue having nitrogen A phosphonic acid diester derivative which is at least one group selected from the group consisting of an alkylsulfanyl group, an arylsulfanyl group, a hydroxy group, a carboxy group, a carbamoyl group, an amino group, a guanidino group, an alkoxy group, and an amide group.

  9. The phosphonic acid diester derivative shown in 4 above, wherein the substituent of the aryl group which may have the substituent may be an alkyl group which may be substituted with either a carboxy group or an equivalent of a carboxy group, A phosphonic acid diester derivative which is at least one group selected from the group consisting of an electron withdrawing group, a carboxy group, and an equivalent of a carboxy group.

  10. The phosphonic acid diester derivative shown in 4 above, wherein the aryl group which may have a substituent is a phenyl group which may have a substituent.

  11. The phosphonic acid diester derivative shown in the above 10, wherein the phenyl group which may have the substituent is represented by the general formula (9)

Wherein Y1 represents one group selected from the group consisting of —R ′, —OR ′, and an electron withdrawing group, and Y2 is substituted with either a carboxy group or an equivalent of a carboxy group And represents one group selected from the group consisting of an alkyl group optionally having a double bond, a hydrogen atom, a carboxy group, and an equivalent of a carboxy group, and two adjacent substituents Y1 And Y2 may be bonded to each other to form a ring, and R ′ represents either a hydrogen atom or an alkyl group optionally having a double bond.)

12 A phosphonic acid diester derivative represented by the above 9 or 11, an electron withdrawing group, a halogen _{atom, -COOR ', - CONR' 2} , -COR ', - OCOR', - CF 3, -CN, -SR ', At least one group selected from the group consisting of —S (O) R ′, —SO ₂ R ′, —SO ₂ NR ′ ₂ , —PO (OR ′) ₂ , and —NO ₂ , wherein R ′ is A phosphonic acid diester derivative having the same meaning as described above.

13. A phosphonic acid diester derivative represented by the above 9 or 11, equivalent of carboxyl group and carboxy _{group, -COOR ", - CONR" 2} , -COR ", - CN, -NO 2, -NHCOR", - OR _{", -SR", - OCOR "} , - SO 3 R", - " is at least one group selected from ₂ made from a group, R" SO 2 NR and is not a hydrogen atom and a double bond A phosphonic acid diester derivative which is any of the possible alkyl groups.

  14 General formula (10)

(Wherein R12, R14, X2, Y1 and n3 represent the same meaning as described above).

  15. Formula (11)

(Wherein R12, R14, X2, Y1, n1 and n3 represent the same meaning as described above).

  16. Formula (12)

(In the formula, R15 represents a lower alkyl group, and W represents the general formula (13) to the general formula (16).

R16 represents either a hydrogen atom or a lower alkyl group, and Y1 and Y2 represent the same meaning as described above. A phosphonic acid diester derivative represented by

  17. Formula (17)

(Wherein R12 and Y1 represent the same meaning as described above).

  18. General formula (18)

(Wherein Y1 and Y2 represent the same meaning as described above).

  19. General formula (19)

(Wherein, M represents an alkali metal, and R17 represents an alkoxycarbonyl group including an optionally substituted aromatic hydrocarbon group), a metal of 2-substituted amino-4-phosphonobutanoic acid salt.

  20. General formula (20)

(Wherein R18 represents an aromatic hydrocarbon group which may have a substituent, and R17 represents the same meaning as described above), 2-substituted amino-4-phosphonobutanoic acid ester.

  In addition, the detailed manufacturing method and various properties of the phosphonic acid diester derivatives shown in the above 1 to 20 are disclosed in Patent Document 5. In the present invention, it is added that the description in Patent Document 5 can be incorporated as necessary, and the contents described in Patent Document 5 can be appropriately used in the present invention. That is, the disclosed content of Patent Document 5 is also a part of the present invention, and correction based on the described content of Patent Document 5 is also possible.

  In particular, in the general formula (12), the phosphonic acid diester derivative in which W is the general formula (13) or (15) is actually synthesized in the Example of Patent Document 5, and GGT inhibitory activity is also confirmed. This is preferable.

  In addition, it has been confirmed by the present inventors that the phosphonic acid diester derivative exhibits GGT activity when the leaving group is eliminated from the phosphate group. That is, GGT activity improves as the leaving group is easily removed. However, since the ease of leaving of the leaving group is related to the stability of the compound, it is not preferable to use a compound having a leaving group that is easily removed. Examples of the compound having an excellent balance between the detachability of the leaving group and the stability include the phosphonic acid diester derivatives of 1 to 20 above. In the general formula (12), R15 represents the general formula (13 ) Or (15) is more preferred, and 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid is particularly preferred.

  The phosphonic acid diester derivative may contain not only a simple substance and a mixture but also an optical isomer. For example, in the case of 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid used in the examples, since there are two chiral atoms, there are a total of four types of optical isomerism. It is a mixture of bodies.

  Collagen production promoter, photoaging inhibitor or moisturizing function improving agent by GGT inhibitory activity of the present invention is used for preventing or improving wrinkles and sagging by improving skin firmness and gloss, or for UV damage (photoaging). It can be applied to the skin as a composition for dermatological use for prevention / improvement or maintenance / improvement of the skin moisturizing function. It can also be used as a poultice, cosmetics, bath preparation, and sometimes as a subcutaneous injection. It can be arbitrarily selected according to the purpose. In particular, it can be widely used as a cosmetic, and can be in the form of creams, ointments, emulsions, solutions, gels, powders, granules, etc., and packs, lotions, powders, sticks, etc. . The preparation forms are also aqueous, solubilized, emulsified, powder, oil-liquid, gel, ointment, aerosol, water-oil two-layer, water-oil-powder three-layer, etc. can do. That is, if it is basic cosmetics, it can be widely used in the above-mentioned various dosage forms in the form of face wash, lotion, milky lotion, cream, gel, essence (beauty serum), pack, mask and the like. In addition, makeup cosmetics can be widely used in the form of foundations and toiletry products such as body soaps and soaps. Furthermore, as a pharmaceutical etc., it can utilize widely in forms, such as various ointments and creams. And the formulation form of the collagen production promoter by GGT inhibitory activity of this invention is not limited to these dosage forms and forms.

  The blending amount of the GGT inhibitor is not particularly limited, and varies depending on the type, properties, quality, and expected effect of the dosage form to be blended, but is, for example, 0.00001 to 50.0% by weight, more preferably 0. 0.0005 to 10.0% by weight, more preferably 0.0005 to 1.0% by weight. In particular, when penetration is promoted by, for example, converting a GGT inhibitor into a liposome, the effect can be expected even with a smaller amount.

  In addition to the above-described collagen production promoter component, the above-mentioned dermatological agent generally includes components that are usually used in dermatological agents such as cosmetics and pharmaceuticals, such as whitening agents, moisturizers, antioxidants, oily components, ultraviolet absorbers. , Surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients / vitamins, and the like can be appropriately blended as necessary.

  In addition, edetate disodium, edetate trisodium, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid and other chelating agents, caffeine, tannin, tranexamic acid and its derivatives, licorice extract, grabrizine, various crude drugs , Drugs such as tocopherol acetate, glycyrrhizic acid and derivatives thereof or salts thereof, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid and other whitening agents, glucose, fructose, mannose, sucrose, Sugars such as trehalose can also be appropriately blended.

  Further, for example, vegetable oils such as olive oil, avocado oil, palm oil, coconut oil, and hardened castor oil, and animal oils such as beef tallow and lard can be used as the fats and oils. Examples of waxes include beeswax, carnauba wax, whale wax, and lanolin. Examples of the mineral oil include liquid paraffin, petrolatum, paraffin, squalene, squalane and the like. For example, as fatty acids, in addition to natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, and oleic acid, synthetic fatty acids such as isononanoic acid and caproic acid can be used.

  Further, as alcohols, natural alcohols such as ethanol and isopropanol, synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol, ethylene glycol, polyethylene glycol, 1,3-butylene glycol, glycerin, batyl alcohol, Polyhydric alcohols such as pentaerythritol, sorbitol, mannitol, glucose and sucrose can also be used.

  In addition, esters such as isopropyl myristate, metal soaps such as aluminum stearate, gums and water-soluble polymer compounds such as gum arabic, carrageenan, gelatin, alginic acid and its salt, hyaluronic acid and its salt, chondroitin sulfate, carboxy You may mix | blend methylcellulose, carboxyethylcellulose, etc.

  Surfactants (anionic surfactants, cationic surfactants, amphoteric surfactants, nonionic surfactants), vitamins, amino acids, moisturizers, animals or plants, herbal extracts or extracts, microbial culture metabolites, α-Hydroxy acids, inorganic pigments, UV absorbers, astringents, bactericides / disinfectants, hair preparations, fragrances, pigments / colorants, other hormones, sequestering agents, pH adjusters, chelating agents, antiseptics Antifungal agent, refreshing agent, stabilizer, emulsifier, animal / plant protein and its degradation product, animal / plant polysaccharide and its degradation product, animal / plant glycoprotein and its degradation product, blood flow promoter, Anti-inflammatory agents / anti-allergic agents, cell activators, keratolytic agents, wound treatment agents, foaming agents, thickeners, oral preparations, deodorants / deodorants, bitterings, seasonings, enzymes, etc. are also available .

  The present invention also provides a compound that inhibits γ-glutamyltranspeptidase as an active ingredient in a collagen production promoter that promotes collagen production in skin fibroblasts by reducing the amount of glutathione in skin fibroblasts. Collagen production promoters or photoaging inhibitors can be included.

  In the embodiment of the present invention described above, the collagen production promoter can be read as a method of promoting collagen production using a GGT inhibitor, or the use of a GGT inhibitor for promoting collagen production. The photoaging inhibitor is a method of preventing and / or improving photoaging using a GGT inhibitor (a method of reducing UV damage), or the use of a GGT inhibitor for preventing and / or improving photoaging. (Use of GGT inhibitor to reduce UV damage) can also be read. The moisturizing function improving agent is a method for maintaining and / or improving the skin moisturizing function using a GGT inhibitor (a method for preventing the moisturizing function from being lowered), or for maintaining and / or improving the moisturizing function. It can also be read as “use of GGT inhibitor” (use of GGT inhibitor for preventing decrease in moisturizing function).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to these.

<1. Effect on intracellular glutathione content>
The following experiment was conducted to increase the amount of glutathione in the human normal skin-derived fibroblasts of the GGT inhibitor 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid (GGT5a). The effect of was confirmed.

Human normal skin-derived fibroblasts (CCD-1059SK, Dainippon Pharmaceutical Co., Ltd.) were subcultured 3-6 times in a DMEM medium containing 10% FBS (fetal bovine serum). Next, the cells were seeded on a TC petri dish (manufactured by Greiner) having a diameter of 35 mm so that the number of cells was 4.5 × 10 ⁵ , added with GGT5a, and cultured for 0 to 10 hours. Thereafter, the cells were collected using 500 μl of 25 mM Tris-HCl (pH 7.4) solution, and centrifuged at 100,000 × g for 30 minutes. To the supernatant (480 μl), 500 μl of 0.56 M Borate Buffer (pH 10.4) solution and 73 μl of o-phthalaldehyde solution were added, and the amount of intracellular glutathione was measured by high performance liquid chromatography (HPLC). In HPLC measurement, ODS Crestpak C18S (JASCO Corporation) was used as the column. Elution was performed by gradient elution using mobile phase A (30 mM sodium acetate) and mobile phase B (92.3% methanol / 7% acetonitrile). The observation was performed on the o-phthalaldehyde derivative at an excitation wavelength of 230 nm and an absorption wavelength of 444 nm.

As a result, it was found that the GGT inhibitor 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid (GGT5a) decreases the amount of intracellular glutathione (FIG. 1).
<2. Evaluation of collagen production ability>
In order to examine the collagen production ability improving action of the GGT inhibitor GGT5a, the following experiment was conducted.

(1) Preparation of sample Human normal skin-derived fibroblasts (CCD-1059SK, Dainippon Pharmaceutical Co., Ltd.) were subcultured 3 to 6 times in DMEM medium containing 10% FBS (fetal bovine serum). Next, it is prepared on a culture slide (Culture slide: manufactured by Falcon) so that the number of cells becomes 1 × 10 ⁶ , cultured for 24 hours in DMEM medium containing 10% FBS, and further fixed on the slide. In order to match the cell cycle, the cells were cultured in DMEM medium alone for 24 hours. Then, it replaced | exchanged for the EMEM culture medium containing 10% FBS, GGT5a was added simultaneously, and it culture | cultivated for 24 hours, and prepared each sample group shown below. Moreover, the group which does not add a test compound as control, and the vitamin C (VC) addition group as positive control were prepared.

<Sample group>
1) Control group 2) GGT5a 10 μM addition group 3) VC 114 μM addition group (positive control)
(2) Measurement of collagen production amount With respect to the sample groups 1) to 3) prepared in (1), the collagen production amount was analyzed immunohistochemically by the following procedure.

The culture slide was washed 3 times with PBS solution for 5 minutes, 4% paraformaldehyde solution was added, and the mixture was allowed to stand at 4 ° C. overnight to fix the sample. After washing 3 times with PBS solution containing 0.1% Triton-X for 5 minutes, endogenous peroxidase was blocked with 3% H ₂ O ₂ solution for 5 minutes. Subsequently, non-specific reaction was blocked for 5 minutes using 10% normal goat serum. Then, the reaction of the primary antibody was performed for 60 minutes using an anti-rat type 1 collagen antibody (Anti-rat type I collagen antibody (LSL) 200-fold diluted solution). After washing with PBS solution for 5 minutes 3 times, the reaction of the secondary antibody was performed for 30 minutes using biotinylated goat anti-rabbit immunoglobulin antibody (Biotinylated Goat anti-rabbit immunogloblins antibody (DAKO) 400 times dilution). It was. After washing 3 times with PBS solution for 5 minutes, a reaction with an enzyme solution (horseradish peroxidase-labelled streptavidine-biotine complex (DAKO) 400-fold dilution) was performed for 30 minutes. After washing with PBS solution for 3 minutes for 5 minutes, a DAB (3,3-diaminobenzidin tetra-hydrocheloride) solution was reacted for 5 minutes to perform peroxidase color reaction. After washing with PBS solution for 3 minutes for 5 minutes, it was sealed with a water-soluble mounting medium to prepare a specimen. The staining intensity at the positive reaction (expression of type 1 collagen) location in the obtained specimen was quantified using NIH image software, and the amount of type 1 collagen produced was analyzed by image analysis.

  The analysis results of the obtained collagen production are shown in Table 1 and FIG.

  The values in the table were expressed as the ratio of the staining intensity of each sample when the control was 100 (value measured and averaged for 20 cells per slide).

As a result, as shown in Table 1 and FIG. 2, it was confirmed that the GGT inhibitor GGT5a exhibits a marked collagen production ability improving effect at a very low concentration as compared with VC used as a positive control.
<3. Evaluation of horny water content>
In order to verify the cosmetic effect of the GGT inhibitor GGT5a on the skin, it was evaluated whether or not the amount of keratin moisture changes due to the use of the lotion containing the GGT inhibitor.
(1) Test period / Subjects • Mid-December to late February (8 weeks continuous use, 3 evaluation measurements)
・ 1 healthy woman in 20s, 3 healthy women in 30s, 8 healthy women in 40s (12 in total)
(2) Test outline Skin lotion formulated with GGT5a (0.0005%) was used twice a day (morning and evening; placebo lotion, GGT5a blended lotion half face each) for 8 weeks, before and after skin condition Was measured and compared to evaluate the effect of the GGT inhibitor on the amount of keratinous moisture in the skin. In addition, after the test subject washed the face, the left and right cheeks were placed in a constant environment after being kept acclimatized in a constant temperature and humidity room (room temperature 20 ° C., relative humidity 50%) for about 20 minutes. Went to the subject. The formula for lotion is shown below. In addition, placebo lotion is the same prescription except having not mix | blended the GGT inhibitor.
(Prescription) (wt%)
Carboxymethyldextran sodium salt 0.05
Glycerin 2
BG (butylene glycol) 2
1,2-hexanediol 0.5
Pentylene glycol 1
Caprylyl glycol 0.1
Phenoxyethanol 0.2
Hyaluronic acid aqueous solution 2
Tuberose polysaccharide aqueous solution 1
Rose water 3
Citric acid 0.01
Sodium citrate 0.04
GGT inhibitor 0.0005
Purified water Total amount of 100 Using the skin surface moisture measuring device Corneometer CM825 (Courage + Khazaka electronic GmbH), measure each test site set on the left and right cheeks 10 times each, The rate of change was measured. The average value of 6 intermediate points excluding 2 points above and below the measured value was taken as the amount of horny moisture at that site. The graph shows the change in the amount of horny water after 4 weeks / 8 weeks of continuous use, where the average of subjects before continuous use is 100.

The results are shown in FIG. As shown in the figure, in the placebo lotion, the amount of stratum corneum decreased with time, whereas when the lotion containing GGT inhibitor was used continuously, a significant increase in the amount of stratum corneum was observed.
<4. Evaluation of skin elasticity improvement>
In order to verify the beauty effect on the skin of the GGT inhibitor GGT5a, a continuous evaluation test was performed on 10 female subjects. Ten subjects used a lotion containing a GGT inhibitor, and the skin was measured with a measuring device every month before use and after 1 month to 3 months, and evaluation was performed based on the results. . In addition, in order to evaluate the relative usefulness of the GGT inhibitor, a comparison was made with a cosmetic raw material “Matrixyl” that has already been recognized to improve wrinkles. Matricixyl is a known cosmetic ingredient that is a 3% blended sample and has been shown to improve wrinkles due to increased collagen synthesis. In addition, the prescription of the lotion used was the same prescription as above except that the concentration of the GGT inhibitor was 10 times 0.005% by weight. Moreover, the placebo lotion has the same composition except that no GGT inhibitor is blended. Matrixil formulated lotion is also a lotion having the same composition except that 3% of matrixil is blended in place of the GGT inhibitor.

(1) Test content Subjects: 10 general women in their 30s to 50s Test period: Mid-May to late August Number of measurements: 4 times (before continuous use, 1st month, 2nd month, 3rd month)
Equipment used: Cutometer (Courage + Khazaka electronic GmbH)
Measurement site: cheek (left / right)
In addition, all measurements were performed after acclimatizing the skin to room temperature for 20 minutes under constant temperature and humidity (20 ° C., RH 50%) after washing the face.

(2) Sample Placebo lotion 1. 2. Toner lotion containing Matrixyl (3%) Toner lotion containing GGT inhibitor (0.005%) The subjects used samples after washing their face twice a day in the morning and evening. The test subjects were divided into 2 groups (groups A and B) of 5 people, each applied to the face in the following manner, and then a special moisturizing cream was applied to the entire face.

Group A: Left half face: Matrix (3%) lotion
Right half face: lotion containing GGT inhibitor (0.005%) Group B ... left half face: placebo lotion
Right half face: GOT inhibitor (0.005%) formulated lotion Note that the moisturizing cream is a moisturizing cream prepared for this test, and is formulated to obtain the minimum moisturizing effect. No ingredients with improvement, whitening and anti-inflammatory effects are included.

FIG. 4 shows the results of examining the change in skin elasticity over time. Relative values are plotted on a graph when each numerical value before continuous use is 100, and the vertical axis represents the average change rate (unit:%) of the elasticity parameter R7 (Ur / Uf; unit: none). The higher the value is, the higher the elasticity of the skin is. As shown in the figure, placebo decreased from one month onward, while those using matrixyl and GGT inhibitor showed a continuous increase in the number from one month onward. Matrixil shows almost no change after 2 to 3 months, but the GGT inhibitor shows a large increase from 2 to 3 months.
<5. Evaluation of wrinkle improvement>
Using the facial image analyzer “VISIA evolution (TM) (Canfield Co., Ltd.)”, the above-mentioned site where the GGT inhibitor-containing lotion was used continuously (hereinafter referred to as GGT inhibitor (+) site), “Matrixyl formulation” Photographs of “parts where lotion was used continuously” (same as Matrixil (+) part) and “parts where placebo lotion was used” (same, placebo part) were taken every month. )) Was detected as wrinkles, and the value representing the ratio of the wrinkles in the analysis frame was evaluated as a “wrinkle score”.

  The results are shown in FIG. For each of the GGT inhibitor (+) site, matrixyl (+) site, and placebo site, the wrinkle score after 3 months of lotion was shown as a relative value when the state before the start of the test was taken as 100. The smaller the value of the wrinkle score, the less wrinkled the skin.

The wrinkle score after 3 months was significantly increased to 259.1% at the placebo site, compared with 86.6% at the Matrixyl (+) site and 80.9% at the GGT inhibitor (+) site. Decreased. As a result, it was confirmed that the collagen production promoting effect of the GGT inhibitor led to the improvement of wrinkles.
<6. Evaluation of UV damage reduction function>
In the presence of the GGT inhibitor GGT5a, the damage when the skin-derived fibroblasts were exposed to ultraviolet rays was evaluated. The specific experimental method is as follows.
(1) Culture of human normal skin-derived fibroblasts Human normal skin-derived fibroblasts consist of 10% fetal bovine serum (FBS), penicillin (penicillin, 50 units / ml, Meiji Seika) and streptomycin (50 mg / ml). ml, Meiji Seika) was adjusted to a Dulbecco's modified Eagle's medium (DMEM) medium (Nissui Pharmaceutical) so that the number of cells was 1.0 × 10 ⁵ cells / ml, and adjusted to 37 ° C. and 5% CO ₂ . The cells were cultured in an incubator and the medium was changed every two days. When the cells became confluent, the main culture was started, and the medium was replaced with a DMEM medium. At the same time, a GGT inhibitor was added to a concentration of 10 μM and cultured for 3 hours, followed by ultraviolet irradiation. Thereafter, the intracellular reactive oxygen species production was measured for 1 hour and then subjected to experiments. For measurement of cell viability, the cells were cultured for 24 hours and then subjected to experiments.
(2) UV irradiation to human normal skin-derived fibroblasts A UV lamp (TOSHIBA germicidal lamp GL15) was used for UV irradiation. Using a UV-340 UV LIGHT METER (Sato Corporation), the height of the UV lamp was adjusted so that the UV intensity was 20 μW. The petri dish was placed under a UV lamp, the lid was opened, and UV was irradiated for 1 minute.
(3) Measurement of cell viability (MTT method)
The methylthiazole tetrazoleum (MTT) method is a method in which living cells are stained with a dye and the relative cell mass is simply measured. The almost dye-free tetrazolium salt taken up into the cell is reduced by mitochondrial oxidoreductase to form an insoluble colored substance (formazan). Since the amount of formazan produced corresponds to the number of living cells, this dye was extracted with an organic solvent and the absorbance was measured.

The cells were adjusted to a concentration of 1.0 × 10 ⁵ cells / ml, seeded 1.0 × 10 ³ cells in a plastic petri dish having a diameter of 35 mm, and each sample was added. After completion of the main culture, 200 μl of 0.5% MTT solution was added to 1 ml of the medium, and further incubated for 2 hours in a CO ₂ incubator. After completely removing the medium with an aspirator or a micropipette, 1 ml of 0.04N HCl / isopropanol was added, and reddish purple formazan was dissolved while pipetting. Thereafter, the absorbance was measured at a wavelength of 570 nm using an absorptiometer (UV mini 1240).
(4) Measurement of production amount of intracellular hydrogen peroxide (H ₂ O ₂ ) Reactive oxygen species (ROS) production amount was measured by labeling intracellular peroxides with DCFH-DA. In addition, the sample was subjected to protein quantification by the Bradford method, and the amount of ROS produced per protein amount was calculated. Two methods were used to measure the amount of reactive oxygen species produced: an absorbance method and a fluorescence microscope method. The measuring method is as follows.

(A) Absorbance method-Add 30 μl of 2.4 mM DCFH-DA solution 30 minutes before the end of the main culture.
・ After the main culture is complete, remove the medium and wash twice with 1 ml of PBS (on ice)
-Add 150 μl of Hanks' solution, scrape the cells with a rubber policeman, and put 150 μl cell solution into a 96-well plate (2 holes). Separately store the entire remaining amount for protein determination.
Fluorescence was measured at a wavelength of 485 nm and a fluorescence wavelength of 535 nm with a multi-label counter (Wallac 1420 ARVOsx).
-Ultrasonic {4 min, (30 s interval) x 3 times} is applied to the sample cell solution that has been stored frozen to destroy the cell membrane.
-Centrifugation (15000 rpm, 5 min, 4 ° C).
・ For protein quantification, the absorbance at 595 nm is measured with an absorptiometer (UV mini 1240).

(B) Fluorescence microscopy ・ Add 5 μl of 2.4 mM DCFH-DA solution 30 minutes before the end of the main culture.
-After the main culture is completed, the medium is removed and washed twice with 1 ml of PBS (on ice).
・ Remove the petri dish wall with pliers, add a small amount of H ₂ O, and cover it with a cover glass (24 mm × 24 mm).
・ Observe and photograph under a fluorescence microscope (OLYMPUS LSC101).

  The results are shown below. First, FIG. 6 shows the results of examining the effect of a GGT inhibitor on the survival rate of human normal skin-derived fibroblasts under ultraviolet irradiation. As shown in the figure, the survival rate of dermal fibroblasts was reduced by about 20% by irradiation with ultraviolet rays. Moreover, when a GGT inhibitor was added, the tendency which recovered the survival rate fallen by the ultraviolet-ray was recognized.

Next, FIG. 7 and FIG. 8 show the results of examining the effect of a GGT inhibitor on the increase in the amount of H ₂ O ₂ produced by normal human skin-derived fibroblasts under ultraviolet irradiation. As shown in the figure, the amount of H ₂ O ₂ produced by normal human skin-derived fibroblasts was higher than that of the non-irradiated group when irradiated with ultraviolet rays. On the other hand, the GGT inhibitor decreased the amount of H ₂ O ₂ increased by ultraviolet irradiation.

[Prescription example: serum]
A cosmetic liquid having the following formulation was produced by a conventional method.
(Composition) (wt%)
Sorbit 4.0
Glycerin 3.0
Butylene glycol 3.0
Polyethylene glycol 1500 5.0
POE (20) oleyl alcohol ether 0.5
Sucrose fatty acid ester 0.2
Methylcellulose 0.2
GGT inhibitor 0.005
Purified water Amount of 100 [Prescription example: Emulsion]
An emulsion having the following formulation was produced by a conventional method.
(Prescription) (wt%)
Glycerin 1.5
Sucrose fatty acid ester 1.5
Sorbitan monostearate 1.0
Squalane 7.5
Dipropylene glycol 5.0
GGT inhibitor 0.005
Purified water Total amount of 100 [Prescription example: Cream]
A cream having the following formulation was produced by a conventional method.
(Prescription) (wt%)
Glycerin 3.0
Butylene glycol 3.0
Squalane 19.0
Stearic acid 5.0
Glycerol monostearate 5.0
Sorbitan monostearate 12.0
Polyethylene sorbitan monostearate 38.0
Sodium edetate 0.03
GGT inhibitor 0.005
Total amount of purified water is 100

  ADVANTAGE OF THE INVENTION According to this invention, the collagen production promoter which improves a collagen production amount by exhibiting GGT inhibitory activity is provided. The GGT inhibitor also has an effect of reducing damage caused by ultraviolet rays and an effect of increasing the amount of stratum corneum. For this reason, according to the present invention, a dermatological agent suitable for preventing or improving aging such as wrinkles and sagging of the skin, preventing and improving photoaging due to ultraviolet rays, and maintaining and improving the moisture retention function of the skin is provided. It is.

Claims (5)

A collagen production promoter comprising 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid as an active ingredient. A photo-aging inhibitor comprising 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid as an active ingredient. A moisturizing function improving agent comprising 2-amino-4-{[3- (carboxymethyl) phenyl] (methyl) phosphono} butanoic acid as an active ingredient. A composition for dermatology, comprising the collagen production promoter according to claim 1, the photoaging inhibitor according to claim 2 , or the moisturizing function improving agent according to claim 3 . The composition for skin preparation according to claim 4 , which is used for improving wrinkles or skin elasticity.