TOPICAL COMPOSITION

Abstract

A composition for external use is provided, comprising a phosphate ester of uridine or a physiologically acceptable salt thereof as an active ingredient for moisturizing, anti-wrinkling, anti-sagging, wound healing, prevention of hair loss, hair growth, or prevention/treatment of atopic dermatitis.

Claims

1. A method for a purpose selected from the following (1) to (7), comprising applying a phosphate ester of uridine or a physiologically acceptable salt thereof as an active ingredient to skin: (1) increasing skin moisture retention, (2) reducing wrinkles, (3) reducing skin-sag, (4) treating a wound, (5) preventing hair loss, (6) promoting hair growth, or (7) preventing/treating atopic dermatitis.

2. The method according to claim 1, comprising applying the phosphate ester of uridine or a physiologically acceptable salt thereof as an independent active ingredient to skin.

3. The method according to claim 1, comprising applying no effective amount of any other active ingredient for increasing skin moisture retention, reducing wrinkles, reducing skin-sag, treating a wound, preventing hair loss, promoting hair growth, or preventing/treating atopic dermatitis.

4. The method according to claim 1, wherein the phosphate ester of uridine or a physiologically acceptable salt thereof is selected from uridine monophosphate, uridine diphosphate, uridine triphosphate, and a physiologically acceptable salt thereof.

5. The method according to claim 1, wherein the phosphate ester of uridine or a physiologically acceptable salt thereof is selected from uridine monophosphate and a physiologically acceptable salt thereof.

6. The method according to claim 1, wherein the phosphate ester of uridine or a physiologically acceptable salt thereof is selected from uridine 5′-monophosphate and a physiologically acceptable salt thereof.

7. (canceled)

8. The method according to claim 1, which promotes cell growth or promotes hyaluronic acid production.

9. The method according to claim 1, wherein the phosphate ester of uridine or a physiologically acceptable salt thereof is incorporated in a cosmetic, a medical drug for external use, or a quasi-medical drug for external use.

10. A method for promoting cell growth or promoting hyaluronic acid production, comprising applying a phosphate ester of uridine or a physiologically acceptable salt thereof as an active ingredient externally to skin.

11. The method according to claim 10, comprising applying the phosphate ester of uridine or a physiologically acceptable salt thereof as an independent active ingredient externally to skin.

12. The method according to claim 10, comprising applying no effective amount of any other active ingredient for promoting cell growth or promoting hyaluronic acid production.

13. The method according to claim 10, wherein the phosphate ester of uridine or a physiologically acceptable salt thereof is selected from uridine monophosphate, uridine diphosphate, uridine triphosphate, and a physiologically acceptable salt thereof.

14. The method according to claim 10, wherein the phosphate ester of uridine or a physiologically acceptable salt thereof is selected from uridine monophosphate and a physiologically acceptable salt thereof.

15. The method according to claim 10, wherein the phosphate ester of uridine or a physiologically acceptable salt thereof is selected from uridine 5′-monophosphate and a physiologically acceptable salt thereof.

16. (canceled)

17. The method according to claim 10, which is employed to increase skin moisture retention, reduce wrinkles, reduce skin-sag, treat a wound, prevent hair loss, promote hair growth, or prevent/treat atopic dermatitis.

18. The method according to claim 17, comprising applying no effective amount of any other active ingredient for increasing skin moisture retention, reducing wrinkles, reducing skin-sag, treating a wound, preventing hair loss, promoting hair growth, or preventing/treating atopic dermatitis.

19. The method according to claim 10, wherein the phosphate ester of uridine or a physiologically acceptable salt thereof is incorporated in a cosmetic, a drug for external use, or a quasi-drug for external use.

20. The method according to claim 1, wherein the method is for increasing skin moisture retention.

21. The method according to claim 1, wherein the method is for reducing wrinkles.

22. The method according to claim 1, wherein the method is for reducing skin-sag.

23. The method according to claim 1, wherein the method is for treating a wound.

24. The method according to claim 1, wherein the method is for preventing hair loss.

25. The method according to claim 1, wherein the method is for promoting hair growth.

26. The method according to claim 1, wherein the method is for preventing/treating atopic dermatitis.

27. The method according to claim 10, wherein the method is for promoting cell growth.

28. The method according to claim 10, wherein the method is for promoting hyaluronic acid production.

Description

BRIEF DESCRIPTION OF DRAWING

[0028] FIG. 1 depicts a graph showing the amount of hyaluronic acid produced when human epidermal keratinocytes were cultured in media containing various concentrations of UMP2Na or uridine in Test Example 1.

[0029] FIG. 2 depicts a graph showing the amount of hyaluronic acid produced when human dermal fibroblasts were cultured in media containing various concentrations of UMP2Na or uridine in Test Example 1.

[0030] FIG. 3 depicts a graph showing the number of cells when human dermal fibroblasts are cultured in media containing various concentrations of UMP2Na or uridine in Test Example 2.

[0031] FIG. 4 depicts a graph showing the number of cells when human dermal papilla cells are cultured in media containing various concentrations of UMP2Na in Test Example 3.

[0032] FIG. 5 depicts a graph showing the amount of hyaluronic acid produced when human dermal fibroblasts were cultured in media containing various concentrations of AMP2Na in Test Example 4.

DESCRIPTION OF EMBODIMENT

[0033] The composition of the present invention comprises a phosphate ester of uridine or a physiologically acceptable salt thereof as an active ingredient for moisturizing, anti-wrinkling, anti-sagging, wound healing, prevention of hair loss, hair growth, prevention/treatment of atopic dermatitis, promotion of cell growth, or promotion of hyaluronic acid production.

[0034] In the present invention, an active ingredient means a substance that is independently capable of exerting a desired physiological action of the skin and is contained in a composition in the expectation that the composition containing the active ingredient exerts the desired physiological action. Examples of the desired physiological action include moisturizing, anti-wrinkling, anti-sagging, wound healing, prevention of hair loss, promotion of hair growth, prevention/treatment of atopic dermatitis, promotion of cell growth (for example, hyaluronic acid-producing cell (for example, keratinocyte, fibroblast), dermal papilla cell), and promotion of hyaluronic acid production. In the present invention, the active ingredient can independently exert an desired physiological action, and when used in combination with other ingredient(s), the desired physiological action may be enhanced additively or synergistically.

[0035] In the present invention, the wording “as an independent active ingredient” means that even without interaction with other active ingredient(s), a phosphate ester of uridine or a physiologically acceptable salt thereof may exert moisturizing effect, anti-wrinkling effect, anti-sagging effect, wound healing effect, hair loss prevention effect, hair growth promotion effect, atopic dermatitis prevention/treatment effect, promotion effect of cell growth (for example, hyaluronic acid-producing cell (for example, keratinocyte, fibroblast), dermal papilla cell), or promotion effect of hyaluronic acid production. The wording “as an independent active ingredient” does not preclude that the composition of the present invention comprises other active ingredient(s).

[0036] As used herein, “effective amount” means an amount that experts an effect of moisturizing, anti-wrinkling, anti-sagging, wound healing, prevention of hair loss, hair growth, prevention/treatment of atopic dermatitis, promotion of cell growth, or promotion of hyaluronic acid production.

[0037] As used herein, “other active ingredient(s) for moisturizing, anti-wrinkling, anti-sagging, wound healing, prevention of hair loss, hair growth, or prevention/treatment of atopic dermatitis” means ingredient(s) other than a phosphate ester of uridine or a physiologically acceptable salt thereof, known as an active ingredient for moisturizing, anti-wrinkling, anti-sagging, wound healing, prevention of hair loss, hair growth, or prevention/treatment of atopic dermatitis.

[0038] As used herein, “other active ingredient(s) for promoting cell growth or promoting hyaluronic acid production” means an ingredient other than a phosphate ester of uridine or a physiologically acceptable salt thereof, known as an active ingredient for promoting cell growth or promoting hyaluronic acid production.

[0039] As used herein, examples of a phosphate ester of uridine include uridine monophosphate (uridine 5′-monophosphate, uridine 3′-monophosphate, uridine 2′-monophosphate), uridine diphosphate, uridine triphosphate, uridine cyclic phosphate. Preferable examples of a phosphate ester of uridine include uridine monophosphate, more preferably uridine 5′-monophosphate.

[0040] Examples of a physiologically acceptable salt of a phosphate ester of uridine include alkali metal salts such as sodium salts, potassium salts and the like; alkaline earth metal such as calcium salts, magnesium salts, barium salts and the like; basic amino acid salts such as arginine, lysine and the like; ammonium salts such as ammonium salts, tricyclohexylammonium salts and the like; alkanolamine salts such as monoethanolamine salts, diethanolamine salts, triethanolamine salts, monoisopropanolamine salts, diisopropanolamine salts, triisopropanolamine salts and the like. Preferable salts includes alkali metal salts such as sodium salts. Examples of the alkali metal salts include uridine monophosphate monosodium salt, and uridine monophosphate disodium salt (hereinafter referred to as UMP2Na).

[0041] In the present invention, a phosphate ester of uridine or a physiologically acceptable salt thereof may be a single type of a phosphate ester of uridine or a salt thereof, or may be a mixture of a plurality of types of phosphate ester of uridine or a salt thereof.

[0042] The amount of a phosphate ester of uridine or a physiologically acceptable salt thereof contained in the composition of the present invention may vary depending on the type of a phosphate ester of uridine or a physiologically acceptable salt thereof, the use or form of the composition etc., but may be optionally selected from, for example, a range of usually 0.0001 to 20% by weight, preferably 0.0001 to 10% by weight, based on the total weight of the composition. Preferably from 0.001% by weight to 10% by weight, more preferably from 0.01% by weight to 10% by weight, more preferably from 0.01% by weight to 5% by weight, particularly preferably from 0.1% by weight to 10% by weight, particularly preferably from 0.5% by weight to 5% by weight, also preferably from 0.05% by weight to 3% by weight, and even more preferably from 0.1% by weight to 1% by weight are exemplified.

[0043] Further examples of the upper limit of the amount of a phosphate ester of uridine or a physiologically acceptable salt thereof contained in the composition of the present application include, relative to the total weight of the composition, preferably 10% by weight, 7% by weight, 5% by weight, 3% by weight, and 2% by weight, and particularly preferably 1% by weight.

[0044] Further examples of the lower limit of the amount of a phosphate ester of uridine or a physiologically acceptable salt thereof contained in the composition of the present application include, relative to the total weight of the composition, preferably 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, and 0.7% by weight, and particularly preferably 1% by weight.

[0045] The compositions of the present invention may be prepared in a variety of forms in combination with a pharmaceutically or cosmetically acceptable bases or carriers, in addition to a phosphate ester of uridine or a physiologically acceptable salt thereof. As a pharmaceutically or cosmetically acceptable base or carrier, conventionally known ones may be used. The composition of the present invention may comprise, if required, a wide variety of known ingredients used for externally-applied compositions suitable for the skin and/or mucosa, such as cosmetics and externally-applied medical/quasi-medical drugs. Examples of such ingredients include surfactants, colorants (dyes, pigments)), flavors, antiseptics, bactericides (antibacterials), thickeners, antioxidants, sequestering agents, cooling agents, deodorizers, humectants, UV absorbers, UV dispersants, vitamins, plant extracts, astringents, anti-inflammatory agents (antiphlogistic agents), whiteners, cell activators, vasodilators, blood circulation accelerators, skin function accelerators, and the like.

[0046] Among the above ingredients, specific examples of the surfactant include anionic surfactants such as higher fatty acid soaps, alkyl sulfates, polyoxyethylene alkyl ether sulfates, alkyl ether phosphates, N-acylamino acid salts, acyl N-methyl taurine salts, and the like; cationic surfactants such as alkyltrimethylammonium chlorides, dialkyldimethylammonium chlorides and the like; amphoteric surfactants such as alkyldimethylaminoacetate betaines, alkylamidedimethylaminoacetate betaines, 2-alkyl-N-carboxy-N-hydroxyimidazolinium betaines and the like; nonionic surfactants such as polyoxyethylene bases, polyhydric alcohol ester bases, ethylene oxide/propylene oxide block copolymers and the like. Any high molecular weight surfactants or natural surfactants can also be used without limitation.

[0047] Examples of antiseptic include ethyl p-hydroxybenzoate, salicylic acid, sorbic acid and the like. Specific examples of thickener include xanthane gum, carboxymethyl cellulose sodium, carboxyvinyl polymers and the like. Specific examples of sequestering agent include sodium salts of ethylenediamine tetra acetic acid, phosphoric acid, citric acid and the like.

[0048] The composition of the present invention may be used as an externally-applied preparation to be spread or sprayed to the skin. More specifically, the composition of the present invention may be widely used as a cosmetic or an externally-applied medical drug, an externally-applied quasi-medical drug (dermatological preparation). Preferable among the above are cosmetics given that cosmetics can be used on a daily basis to bring about promotion of hyaluronic acid production in the skin on a daily basis. Examples of such externally-applied preparation include a wide variety of hair care products such as hair restorers and hair growth preparations, as well as, shampoos, rinses, and hair lotions (including tonics and liquids) that have hair restoration and/or hair growth effect(s).

[0049] When the composition of the present invention is used for hair growth or preventing hair loss, the composition of the present invention is preferably formulated into a hair cosmetic exemplified above. The composition of the present invention may also be used as an agent for promoting growth of/preventing loss of hairs such as eyebrows, eyelashes and the like which are desired to increase/elongate, in addition to head hairs. The wording “hair growth” means that the existing hair, including downy hair, grows healthy, and includes that a sprout of new hair is promoted. The wording “prevention of hair loss” means preventing loss of the existing hairs.

[0050] When the composition of the present invention is used for hair growth or prevention of hair loss, the composition of the present invention may be used for use associated with these (for example, prevention or treatment of thinning hair, promotion of hair emergence, hair growth promotion, treatment of post-illness/postpartum hair loss, hair restoration and the like).

[0051] The composition of the present invention may be any form without limitation insofar as it is applicable to the skin or mucosa. Examples of the form include pastes, mousses, gels, liquids, emulsions, suspensions, creams, ointments, solids, sheets, aerosols, sprays, and liniments. Examples of cosmetic include lotions; emulsions such as emollient emulsions, milky lotions, nourishing emulsions, cleansing emulsions and the like; creams such as emollient creams, massage creams, cleansing creams, makeup creams; lip creams and like. Examples of hair care product such as hair nourishing agents and hair restoring agents include hair tonics, hair creams, hair lotions, aerosols (air sprays), mousses, shampoos, rinses, liquids and the like.

[0052] The composition of the present invention may be directly applied to or sprayed onto the skin or mucosa as a cosmetic or an externally-applied medical/quasi-medical drug. The composition can be applied to the skin or mucosa once to 5 or 6 times per day in an effective amount for a target effect according to the age of the user (human), the gender, the intended use, the condition of the affected part of the skin, etc. The period of use of the composition of the present invention is not limited, but it is preferably used continuously to keep the effect of cell growth promotion and/or hyaluronic acid production promotion effectively, and the composition may be used, for example, for one month (preferably 2 months or more).

[0053] By promoting cell growth and/or promoting hyaluronic acid production, the composition of the present invention may exhibit the effects of anti-aging, moisturizing, anti-wrinkling, anti-sagging, hair growth, prevention of hair loss, wound healing, prevention/treatment of atopic dermatitis, prevention/treatment of senile xerosis and the like. Thus, the composition of the present invention may be used as a cosmetic or an externally-applied medical/quasi-medical drug for the purpose of anti-aging, moisturizing, anti-wrinkling, anti-sagging, hair growth, prevention of hair loss, wound healing, prevention/treatment of atopic dermatitis, prevention/treatment of senile xerosis and the like.

[0054] One aspect of the present invention includes a composition which does not contain acetylglucosamine, glucuronic acid, a salt of glucuronic acid, or a derivative thereof which are proposed to be blended into a cosmetic and the like in combination with a phosphate ester of uridine by JP 2014-88329 A.

[0055] As used herein, the derivative of acetylglucosamine has the following chemical formula (1):

##STR00001##

(R.sup.1 is a hydrogen atom or an alkyl group having 2 to 18 carbon atoms. R.sup.2, R.sup.3, and R.sup.4 are a hydrogen atom or an acyl group having 2 to 18 carbon atoms, and all may be the same or any may be different. The configuration at position 1 may be either α or β. Provided that all of R.sup.1, R.sup.2, R.sup.3, and R.sup.4 must not be hydrogen atoms.)

[0056] Specifically, the derivative are octyl(2-acetamide-2-deoxy)β-D-glucopyranoside, 2-acetamide-1,3,4,6-tetra-O-acetyl-2-deoxy-β-D-glucopyranoside, 2-acetamide-2-deoxy-6-O-octanoyl-α-D-glucopyranose, octyl(2-acetamide-2-deoxy-6-O-octanoyl)β-D-glucopyranoside, butyl(2-acetamide-2-deoxy)β-D-glucopyranoside, pentyl(2-acetamide-2-deoxy)β-D-glucopyranoside, lauryl(2-acetamide-2-deoxy)β-D-glucopyranoside, 2-acetamide-2-deoxy-6-O-palmitoyl-α-D-glucopyranose, geranyl(2-acetamide-2-deoxy)β-D-glucopyranoside, ethyl(2-acetamide-3,4,6-tri-O-acetyl-2-deoxy)β-D-glucopyranoside, pentyl(2-acetamide-3,4,6-tri-O-acetyl-2-deoxy)β-D-glucopyranoside, octyl(2-acetamide-3,4,6-tri-O-acetyl-2-deoxy)β-D-glucopyranoside, and geranyl(2-acetamide-3,4,6-tri-O-acetyl-2-deoxy)β-D-glucopyranoside.

[0057] The salts of glucuronic acid are potassium salt, sodium salt, and other physiologically acceptable salts. The derivatives of glucuronic acid are glucuronolactone, and glucuronoxylan.

[0058] Another aspect of the present invention includes a composition which does not contain a purine nucleic acid-related substance which are proposed to be blended into a cosmetic and the like in combination with a phosphate ester of uridine by WO 2003/084485 and WO 2005/034902.

[0059] In the present specification, purine nucleic acid-related substances are adenine, adenosine, adenosine phosphate [adenosine 2′-monophosphate, adenosine 3′-monophosphate, adenosine 5′-monophosphate, adenosine 5′-diphosphate, adenosine 5′-triphosphate, cyclic adenosine phosphate, adenylosuccinic acid, nicotinamide adenine monodinucleotide (NMN), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD)], metabolites thereof (hypoxanthine, inosine, inosinic acid), and salts thereof; guanine, guanosine, guanosine phosphate [guanosine 3′-monophosphate, guanosine 5′-monophosphate, guanosine 5′-diphosphate, and guanosine 5′-triphosphate, and the like], metabolites thereof [xanthylic acid, xanthin], and salts thereof. The above salts are alkali metal salts such as sodium salts, potassium salts; alkaline earth metal salts such as calcium salts, magnesium salts, barium salts; basic amino acid salts such as arginine, lysine; ammonium salts such as ammonium salts, tricyclohexylammonium salts; various kinds of alkanolamine salts such as monoethanolamine salts, diethanolamine salts, triethanolamine salts, monoisopropanolamine salts, diisopropanolamine salts, and triisopropanolamine salts.

[0060] The present invention provides a method for increasing skin moisture retention, a method for reducing wrinkle, a method for reducing skin-sag, a method for treating a wound, a method for preventing hair loss, a method for promoting hair growth, a method for preventing/treating atopic dermatitis, a method for promoting cell growth (for example hyaluronic acid-producing cell (for example keratinocyte, fibroblast), dermal papilla cell), and a method for promoting hyaluronic acid production. The method is carried out by applying a phosphate ester of uridine or a physiologically acceptable salt thereof to the skin.

[0061] According to the method of the invention, the application of the substance to the skin can be achieved by spreading, spraying, or sticking of the composition of the present invention to the skin.

[0062] In the method of the invention, there is no limitation on the frequencies with which a phosphate ester of uridine or a physiologically acceptable salt thereof is applied to the skin and also the application amounts thereof are not limited. For example, a phosphate ester of uridine or a physiologically acceptable salt thereof is applied to the skin in an appropriate amount from one to five or six times per day according to the age of the application target, the gender, the intended use, the condition of the affected part of the skin, etc. For example, when the method of the invention is carried out by using the composition of the present invention, a single dose can be suitably adjusted such that the amount of the composition applied to the skin is within the range of 0.5 to 10 mg/cm.sup.2. The period of use of the method of the present invention is not limited, but it is preferably to be used continuously for a effectively-sustaining promotion of cell growth and a effectively-sustaining promotion of hyaluronic acid production, for example, for one month (preferably for two months or more).

EXAMPLE

[0063] The present invention is explained in further detail with reference to Examples and Test Examples. However, the scope of the invention is not limited to these Examples.

[0064] In the following Test Examples, “w/v %” means a weight (g) contained in 100 mL.

[Example 1] Lotion (pH6.5)

[0065]

TABLE-US-00001 UMP2Na 3.0 (% by weight) Polyoxyethylene (E.O.60) hydrogenated castor oil 0.7 Ethanol 5.0 Glycerin 2.0 Antiseptic Suitable quantity Flavor Suitable quantity pH adjuster Suitable quantity Purified water Balance Total 100.0% by weight

[0066] A lotion is prepared according to the above formulation in a routine manner.

[Example 2] Emulsion (pH6.5)

[0067]

TABLE-US-00002 UMP2Na 1.5 (% by weight) Carboxyvinyl polymer 0.3 Decaglyceryl monomyristate 2.0 Squalane 5.0 Ethanol 1.0 Glycerin 6.0 Antiseptic Suitable quantity pH adjuster Suitable quantity Purified water Balance Total 100.0% by weight

[Example 3] Hair Restorer (pH7.0)

[0068]

TABLE-US-00003 UMP2Na 3 (% by weight) Polyoxyethylene polyoxypropylene decyl 0.5 tetradecyl ether Ethanol 30 1,3-Butylene glycol 2.5 Antiseptic Suitable quantity Flavor Suitable quantity pH Adjuster Suitable quantity Purified water Balance Total 100.0% by weight

[0069] A hair restorer is prepared according to the above formulation in a routine manner.

Test Example 1: Evaluation of Hyaluronic Acid Production Promoting Effect of UMP2Na and Uridine on Cultured Human Epidermal Keratinocytes and Dermal Fibroblasts

[0070] Pre-cultured primary-cultured human epidermal keratinocytes (manufactured by Kurabo Industries, Ltd.) were cultured in a growth factor-containing EpiLife liquid medium (manufactured by ThermoFisher Scientific) in a 96-well microplate, and then the medium was replaced with the growth-factor-free medium containing an each concentration (0, 10.sup.−6, 10.sup.−5, 10.sup.−4, 10.sup.−3, 10.sup.−2 mol/L) of UMP2Na or uridine.

[0071] Pre-cultured primary-cultured dermal fibroblasts (manufactured by Kurabo Industries, Ltd.) were cultured in a serum-containing Medium106 liquid medium (manufactured by ThermoFisher Scientific) in a 96-well microplate, and then the medium was replaced with the serum-free medium containing an each concentration (0, 10.sup.−6, 10.sup.−5, 10.sup.−4, 10.sup.−3, 10.sup.−2 mol/L) of UMP2Na or uridine.

[0072] These were cultured for 48 and 72 hours under a condition of 37° C. and 5% CO.sub.2. After the culturing, the culture supernatant in each well was collected, and the amount of hyaluronic acid in the culture supernatant was detected using a hyaluronic acid quantification kit (manufactured by Cosmo Bio) and measured with a microplate reader.

[0073] FIG. 1 shows the results of epidermal keratinocytes.

[0074] FIG. 2 shows the results of dermal fibroblasts.

Test Example 2: Evaluation of Promoting Effect of UMP2Na and Uridine on Cultured Human Dermal Fibroblasts Growth

[0075] Pre-cultured primary-cultured human dermal fibroblasts (manufactured by Kurabo Industries, Ltd.) were cultured in a serum-containing Medium106 liquid medium (manufactured by ThermoFisher Scientific) in a 96-well microplate. After confirming the stable adhesion, the medium was replaced with the same medium containing an each concentration (0, 10.sup.−6, 10.sup.−5, 10.sup.−4, 10.sup.−3, 10.sup.−2 mol/L) of UMP2Na or uridine. This was cultured for 48, 72, and 96 hours under a condition of 37° C. and 5% CO.sub.2. After each culturing time, the number of cells in each well was detected using WST-1 reagent (manufactured by Takara Bio Inc.) and then measured with a microplate reader.

[0076] The results are shown in FIG. 3.

Test Example 3: Evaluation of Promoting Effect of UMP2Na on Cultured Human Dermal Papilla Cell Growth

[0077] Pre-cultured primary-cultured human dermal papilla cells (manufactured by Takara Bio Inc.) were cultured in a dermal papilla cell growth medium (manufactured by Takara Bio Inc.) in a 96-well microplate. After confirming the stable adhesion, the medium was replaced with the medium containing various concentrations of the drug.

[0078] This was cultured for 72, 96, and 120 hours under a condition of 37° C. and 5% CO.sub.2. After each culturing time, cell nuclei were stained, and the number of cells in each well was counted.

[0079] The results are shown in FIG. 4.

Test Example 4: Evaluation of Hyaluronic Acid Production Promoting Effect of Adenosine Monophosphate Disodium on Cultured Human Dermal Fibroblasts

[0080] Pre-cultured primary-cultured dermal fibroblasts (manufactured by Kurabo Industries, Ltd.) were cultured in a serum-containing Medium106 liquid medium (manufactured by ThermoFisher Scientific) in a 96-well microplate. After the culturing, the medium was replaced with the serum-free medium containing an each concentration (0, 10.sup.−8, 10.sup.−6, 10.sup.−4 w/v %) of adenosine monophosphate disodium (AMP2Na). This was cultured for 48 and 96 hours under a condition of 37° C. and 5% CO.sub.2. After the culturing, the culture supernatant in each well was collected, and the amount of hyaluronic acid in the culture supernatant was detected using a hyaluronic acid quantification kit (manufactured by Cosmo Bio) and measured with a microplate reader.

[0081] The results are shown in FIG. 5.