Stem cell activator

Abstract

Provided are: a novel stem cell activator; a topical skin preparation containing the stem cell activator; and a skin cosmetic containing the stem cell activator.　The stem cell activator according to the present invention contains, as an active ingredient, at least one compound selected from compounds represented by the following formula (1) (including optical isomers), salts of these compounds, and hydrates thereof. In the formula, R1 and R2 represent a hydrogen atom or a hydrocarbon group. R3 represents a hydrogen atom or an organic group. n represents an integer of 1 or more. The hydrocarbon group may be substituted.

Description

Stem cell activator

[0001] The present invention relates to a stem cell activating agent.

[0002] The basement membrane (or epidermal basement membrane) separating the epidermis from the dermis is a scaffold for cell adhesion, controlling cell adhesion, survival, differentiation, and migration, and playing an important role in constructing skin. In recent years, it has been revealed that the basement membrane is damaged by ultraviolet radiation and aging (e.g., rupture or multiplication of the basement membrane structure), resulting in a decrease in stem cells present on the basement membrane. When stem cells decrease, the skin turnover rate slows and the entire epidermis becomes thinner. This in turn leads to a decline in skin functions, such as a decrease in barrier function and a decrease in moisture content.

[0003] Patent Document 1 discloses that D-alanine or D-hydroxyproline promotes the production of laminin 332, and that the promotion of laminin 332 production can improve the function of the basement membrane.

[0004] More specifically, the following points are disclosed: 1. Laminin 332 is present in the basement membrane that separates the epidermis and dermis and plays an important role in the structure and function of the skin; 2. When laminin 332 is digested due to ultraviolet radiation or aging, the function of the basement membrane declines; 3. When human epidermal keratinocytes are cultured in a medium containing D-alanine or D-hydroxyproline, the amount of laminin 332 produced increases; 4. Promotion of laminin 332 production enhances the formation of the basement membrane, thereby inhibiting and / or improving skin aging.

[0005] International Publication No. 2011 / 040082

[0006] However, it is not known that the compound represented by the following formula (1) exhibits a stem cell activation effect by promoting the production of laminin 332 and type V collagen, nor is it known that the compound represented by the following formula (1) exhibits a stem cell activation effect by promoting the production of HBEGF and FGF2.

[0007] Therefore, an object of the present invention is to provide a novel stem cell activating agent. Another object of the present invention is to provide an external skin preparation having a stem cell activating effect. A further object of the present invention is to provide a cosmetic having a stem cell activating effect.

[0008] As a result of intensive research to solve the above problems, the present inventors have found that application of a compound represented by the following formula (1) to fibroblasts significantly increases the production of laminin 332 and type V collagen, which have cell adhesion properties, and that laminin 332 and type V collagen adhere cells to each other, thereby repairing damage to the basement membrane and activating stem cells. Furthermore, they have found that application of a compound represented by the following formula (1) to fibroblasts significantly increases the production of HBEGF and FGF2, and that the produced HBEGF and FGF2 bind to stem cells and promote stem cell self-renewal. The present invention was completed based on these findings.

[0009] That is, the present invention provides a stem cell activating agent containing, as an active ingredient, at least one compound selected from a compound represented by the following formula (1) (including optically active forms), a salt of the compound, and a hydrate thereof: [R １ , R ２ are the same or different and represent a hydrogen atom or a hydrocarbon group. ３ represents a hydrogen atom or an organic group. n represents an integer of 1 or more. The hydrocarbon group may have a substituent. The substituent may be a halogen atom, —COOR ａ , -CONR ａ ２ , -COR ａ , -CN, -NO ２ , -NHCOR ａ , -OR ａ , -SR ａ , -OCOR ａ , -SO ３ R ａ , and -SO ２ NR ａ ２ is a group selected from the group consisting of ａ represents a hydrogen atom or an aliphatic hydrocarbon group.

[0010] The present invention also provides the stem cell activating agent, which exhibits a stem cell activating effect by promoting the production of laminin and / or type V collagen.

[0011] The present invention also provides the stem cell activating agent, which is an agent for preventing or ameliorating a decrease in stem cells associated with a decrease in the production of laminin and / or type V collagen.

[0012] The present invention also provides the stem cell activating agent, which exhibits a stem cell activating effect by promoting the production of FGF2 and / or HBEGF.

[0013] The present invention also provides the stem cell activating agent, which is an agent for preventing or ameliorating a decrease in stem cells associated with a decrease in the production amount of FGF2 and / or HBEGF.

[0014] The present invention also provides an external skin preparation containing the stem cell activating agent.

[0015] The present invention also provides a cosmetic comprising the stem cell activating agent.

[0016] When TGF-β, a cytokine essential for tissue repair, binds to the TGF-β receptor, the transcription factors Smad2 and Smad3 (hereinafter sometimes referred to as "Smad2 / 3"), which are intracellular TGF-β signaling molecules, are phosphorylated. The phosphorylated and activated Smad2 / 3 translocates into the nucleus, where it induces the expression of laminin 332 and type V collagen, which have cell adhesion properties. The compound represented by formula (1) cooperates with TGF-β to enhance the phosphorylation of Smad2 / 3 and inhibit the dephosphorylation of phosphorylated Smad2 / 3, thereby increasing the expression levels of laminin 332 and type V collagen. The expressed laminin 332 and type V collagen then act to adhere cells to the basement membrane, repairing damage to the basement membrane. This improves the survival and differentiation potential of stem cells on the basement membrane and normalizes skin turnover.

[0017] Furthermore, the compound represented by formula (1) promotes the expression of FGF2, a growth factor for dermal stem cells, and HBEGF, a growth factor for epidermal stem cells. When dermal stem cells or epidermal stem cells receive these growth factors via receptors, self-renewal is promoted, thereby improving the regenerative ability of the skin.

[0018] The stem cell activating agent of the present invention contains a compound represented by formula (1) having the above-mentioned properties, and therefore can be suitably used as an agent for preventing or ameliorating a decrease in stem cells associated with a decrease in the production of laminin and / or type V collagen, or as an agent for preventing or ameliorating a decrease in stem cells associated with a decrease in the production of HBEGF and / or FGF2, or as an agent for preventing or ameliorating a decrease in stem cells associated with damage to the basement membrane.

[0019]

[0033] FIG. 1 shows the results of laminin expression level evaluation 1 for Nahlsgen.

[0034] FIG. 2 shows the results of laminin expression level evaluation 2 for Nahlsgen.

[0035] FIG. 3 shows the results of type V collagen expression level evaluation for Nahlsgen.

[0036] FIG. 4 shows the results of Smad2 / 3 phosphorylation evaluation 1 for Nahlsgen.

[0037] FIG. 5 shows the results of Smad2 / 3 phosphorylation evaluation 2 for Nahlsgen.

[0038] FIG. 6 shows the results of expression level evaluation of skin stem cell markers CD271 and CD105 for Nahlsgen.

[0039] FIG. 7 shows the results of expression level evaluation of skin stem cell growth factors FGF2 and HBEGF for Nahlsgen.

[0039] FIG. 8 shows the results of laminin expression level evaluation 1 for DL-AP4.

[0039] FIG. 9 shows the results of type V collagen expression level evaluation for DL-AP4.

[0039] FIG. 10 shows the results of Smad2 / 3 phosphorylation evaluation 1 for DL-AP4.

[0039] FIG. 11 shows the results of Smad2 / 3 phosphorylation evaluation 2 for DL-AP4. 1 shows the results of evaluating the expression levels of skin stem cell markers CD271 and CD105 for DL-AP4. 2 shows the results of evaluating the expression levels of skin stem cell growth factors FGF2 and HBEGF for DL-AP4. 3 shows the results of evaluation 1 of laminin expression for DL-AP4OMe. 4 shows the results of evaluation of type V collagen expression for DL-AP4OMe. 5 shows the results of evaluation 1 of Smad2 / 3 phosphorylation for DL-AP4OMe. 6 shows the results of evaluation 2 of Smad2 / 3 phosphorylation for DL-AP4OMe. 7 shows the results of evaluation 1 of Smad2 / 3 phosphorylation for DL-AP4OMe. 8 shows the results of evaluation 2 of Smad2 / 3 phosphorylation for DL-AP4OMe. 9 shows the results of evaluation 1 of expression levels of skin stem cell markers CD271 and CD105 for DL-AP4OMe. 10 shows the results of evaluation 1 of expression levels of skin stem cell growth factors FGF2 and HBEGF for DL-AP4OMe. 11 shows the results of evaluation 1 of expression levels of laminin, procollagen, and αSMA for Nahlsgen and others. FIG. 1 is a diagram showing the evaluation results of the expression levels of laminin, procollagen, and αSMA for Nahlsgen, etc. FIG. 2 is a diagram showing the evaluation results of the expression level of type V collagen for DL-AP3, etc. FIG. 3 is a diagram showing the evaluation results of the expression level of laminin α3 chain for DL-AP3, etc. FIG. 4 is a diagram showing the evaluation results of the expression level of skin stem cell marker CD271 for DL-AP3, etc.This figure shows the results of evaluating the expression level of skin stem cell marker CD105 for DL-AP3 etc. This figure shows the results of evaluating the expression level of skin stem cell growth factor FGF2 for DL-AP3 etc. This figure shows the results of evaluating the expression level of skin stem cell growth factor HBEGF for DL-AP3 etc. Nahlsgen H. ２ FIG. 10 shows the results of Smad3 phosphorylation evaluation 3 for O etc.

[0020] [Stem Cell Activating Agent] The stem cell activating agent of the present invention is a composition that has the effect of suppressing the decrease in stem cells (particularly stem cells present in the skin, such as epidermal stem cells and dermal stem cells) and improving their differentiation ability, and contains, as an active ingredient, at least one compound selected from a compound represented by the following formula (1), a salt of said compound, a hydrate of said compound, and a hydrate of a salt of said compound (hereinafter, sometimes referred to as "active ingredient (1)"):

[0021] The active ingredient (1) is a compound having a region with high electron density within the molecule. The active ingredient (1) acts cooperatively with TGF-β to promote the production of at least one selected from laminin, type V collagen, HBEGF, and FGF2, thereby activating stem cells. The active ingredient (1) with higher electron density tends to exhibit a more excellent stem cell activation effect.

[0022] The compound represented by the following formula (1) has at least one asymmetric atom. The carbon atom marked with * in the following formula (1) is the asymmetric atom. In the compound represented by the following formula (1), R １ , R ２ are different groups, the phosphorus atom in the formula is also an asymmetric atom, and therefore the compound represented by the following formula (1) has at least two optical isomers (four optical isomers when the phosphorus atom is also an asymmetric atom).

[0023] The stem cell activating agent of the present invention can use a mixture of optical isomers (i.e., a racemate or a diastereomeric mixture) as the compound represented by the following formula (1). It can also use an optically active substance (i.e., one enantiomer or one diastereomer) obtained by optical resolution of a racemate or a diastereomeric mixture. For optical resolution of a racemate or a diastereomeric mixture, well-known and commonly used methods can be used, such as physicochemical optical resolution methods and separation / resolution methods using chiral columns.

[0024] Therefore, examples of the stem cell activating agent of the present invention include those containing, as an active ingredient, at least one selected from the group consisting of a racemate, a diastereomeric mixture, an optically active form, a salt of these compounds, and a hydrate thereof, of a compound represented by the following formula (1):

[0025] [R １ , R ２ are the same or different and represent a hydrogen atom or a hydrocarbon group. ３ represents a hydrogen atom or an organic group. n represents an integer of 1 or more. The hydrocarbon group may have a substituent. The substituent may be a halogen atom, —COOR ａ , -CONR ａ ２ , -COR ａ , -CN, -NO ２ , -NHCOR ａ , -OR ａ , -SR ａ , -OCOR ａ , -SO ３ R ａ , and -SO ２ NR ａ ２ is a group selected from the group consisting of ａ represents a hydrogen atom or an aliphatic hydrocarbon group.

[0026] Examples of the organic group include a hydrocarbon group (R), an RO group, an RCO group, and an ROCO group, where R represents a hydrocarbon group.

[0027] The hydrocarbon group in the organic group may have a substituent. Examples of the substituent include a halogen atom, —COOR ａ , -CONR ａ ２ , -COR ａ , -CN, -NO ２ , -NHCOR ａ , -OR ａ , -SR ａ , -OCOR ａ , -SO ３ R ａ , -SO ２ NR ａ ２ The above-mentioned R ａ represents a hydrogen atom or an aliphatic hydrocarbon group.

[0028] The organic group may also be a group represented by the following formula (r3): １ , R ２ , and n are the same as above. In addition, the bond marked with a wavy line in the following formula is bonded to the carbon atom marked with an * in formula (1).

[0029] The hydrocarbon group includes an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, and a group in which these groups are bonded via a single bond.

[0030] The aliphatic hydrocarbon group includes C １－２０ (i.e., aliphatic hydrocarbon groups having 1 to 20 carbon atoms) are preferred, and examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, pentyl, hexyl, decyl, and dodecyl groups. １－２０ (Preferably C １－１０ , particularly preferably C １－３ ) alkyl group; C such as vinyl group, allyl group, 1-butenyl group ２－２０ (Preferably C ２－１０ , particularly preferably C ２－３ ) an alkenyl group such as an ethynyl group, a propynyl group, etc. ２－２０ (Preferably C ２－１０ , particularly preferably C ２－３ ) alkynyl groups and the like.

[0031] The alicyclic hydrocarbon group includes C ３－２０ Alicyclic hydrocarbon groups are preferred, and examples thereof include C groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl groups. ３－２０ (Preferably C ３－１５ , particularly preferably C ５－８ cyclopentenyl group, cyclohexenyl group, etc. ３－２０ (Preferably C ３－１５ , particularly preferably C ５－８ a cycloalkenyl group of a perhydronaphthalen-1-yl group, a norbornyl group, an adamantyl group, a tricyclo[5.2.1.0 ２，６ ] decan-8-yl group, tetracyclo[4.4.0.1 ２，５ .1 ７，１０ ] Bridged cyclic hydrocarbon groups such as dodecan-3-yl group and the like are also included.

[0032] The aromatic hydrocarbon group includes C ６－１４ (Especially C ６－１０ ) Aromatic hydrocarbon groups are preferred, such as phenyl and naphthyl groups.

[0033] An aromatic or non-aromatic heterocycle may be condensed with the alicyclic ring contained in the alicyclic hydrocarbon group or the aromatic ring contained in the aromatic hydrocarbon group.

[0034] The group in which an aliphatic hydrocarbon group and an alicyclic hydrocarbon group are bonded includes a cycloalkyl-substituted alkyl group such as a cyclopentylmethyl group, a cyclohexylmethyl group, or a 2-cyclohexylethyl group (for example, C ３－２０ Cycloalkyl-substituted C １－４ The group in which an aliphatic hydrocarbon group and an aromatic hydrocarbon group are bonded includes an aralkyl group (for example, C ７－１８ aralkyl group), alkyl-substituted aryl group (e.g., about 1 to 4 C １－４ alkyl-substituted phenyl or naphthyl groups).

[0035] The R ａThe aliphatic hydrocarbon group in may have a substituent. Examples of the substituent include a halogen atom, an oxo group, a hydroxyl group, a substituted oxy group (e.g., C １－４ Alkoxy group, C ６－１０ Aryloxy group, C ７－１６ Aralkyloxy group, C １－４ acyloxy group, etc.), carboxyl group, substituted oxycarbonyl group (e.g., C １－４ Alkoxycarbonyl group, C ６－１０ Aryloxycarbonyl group, C ７－１６ aralkyloxycarbonyl group, etc.), substituted or unsubstituted carbamoyl group (e.g., carbamoyl, methylcarbamoyl, etc. １－４ C such as alkyl-substituted carbamoyl and phenylcarbamoyl groups ６－１０ aryl-substituted carbamoyl group), cyano group, nitro group, substituted or unsubstituted amino group (e.g., mono- or di-C such as methylamino group, dimethylamino group, ethylamino group, diethylamino group, etc. １－４ alkylamino groups; 5- to 8-membered cyclic amino groups such as 1-pyrrolidinyl, piperidino, and morpholino groups; C groups such as acetylamino, propionylamino, and benzoylamino groups; １－１０ Examples include acylamino groups (sulfonylamino groups such as benzenesulfonylamino and p-toluenesulfonylamino), sulfo groups, heterocyclic groups, etc. The hydroxyl and carboxyl groups may be protected with protective groups commonly used in the field of organic synthesis.

[0036] n represents an integer of 1 or more, for example, a number selected from the integers of 1 to 10, preferably an integer of 2 to 8, particularly preferably an integer of 2 to 6, and most preferably 2, 4, and 6.

[0037] R １ As the substituent, a hydrogen atom, an optionally substituted aliphatic hydrocarbon group, or an optionally substituted aromatic hydrocarbon group is preferred, and a hydrogen atom, an optionally substituted alkyl group having 1 to 4 carbon atoms, or a phenyl group substituted with an alkyl group having 1 to 4 carbon atoms is particularly preferred. Examples of the substituent include a halogen atom, —COOH, —SO ３ H, and -SO ２ NHRａ (R ａ is the same as above. Among them, a group selected from the group consisting of a hydrogen atom or an alkyl group having 1 to 3 carbon atoms is preferred.

[0038] That is, the compound represented by formula (1) is １ is a hydrogen atom, an aliphatic hydrocarbon group which may have a substituent, or an aromatic hydrocarbon group which may have a substituent, and １ Particularly preferred are compounds in which R is a hydrogen atom, an optionally substituted alkyl group having 1 to 4 carbon atoms, or a phenyl group substituted with an alkyl group having 1 to 4 carbon atoms.

[0039] R １ Furthermore, in terms of increasing the electron density and thereby further improving the cooperativity with TGF-β, a hydrogen atom or an aliphatic hydrocarbon group which may have a substituent is preferred, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms which may have a substituent is particularly preferred, a hydrogen atom or an alkyl group having 1 to 4 carbon atoms is most preferred, and a hydrogen atom is particularly preferred.

[0040] That is, the compound represented by formula (1) has excellent synergistic properties with TGF-β, and is particularly effective in increasing the concentration of phosphorylated Smad2 / 3 and activating stem cells. １ is a hydrogen atom or an aliphatic hydrocarbon group which may have a substituent (preferably a hydrogen atom or an aliphatic hydrocarbon group, particularly preferably a hydrogen atom or an alkyl group having 1 to 4 carbon atoms), and １ Particularly preferred are compounds in which is a hydrogen atom.

[0041] R ２ As the alkyl group, a hydrogen atom or an aliphatic hydrocarbon group which may have a substituent is preferred, a hydrogen atom or an aliphatic hydrocarbon group which may have a substituent is particularly preferred, and a hydrogen atom or an alkyl group (for example, an alkyl group having 1 to 4 carbon atoms) is most preferred, in that the electron density is increased and thereby the cooperativity with TGF-β is further improved. A hydrogen atom is particularly preferred.

[0042] R ３As the alkyl group, a hydrogen atom or an aliphatic hydrocarbon group is preferred, and a hydrogen atom or an alkyl group (e.g., an alkyl group having 1 to 4 carbon atoms) is particularly preferred, since the electron density is increased and thereby the cooperativity with TGF-β is further improved.

[0043] As the compound represented by formula (1), compounds represented by the following formulas (1-1) to (1-5) (including optical isomers) are preferred. １ , R ２ , R ３ is the same as above.

[0044] As the compound represented by formula (1), the compounds represented by the above formulas (1-1), (1-3) and (1-5) (including optical isomers) are particularly preferred in terms of excellent stability.

[0045] The compound represented by formula (1) is preferably a compound represented by the following formula (including optical isomers): ２ ', R ３ ' may be the same or different and each represents a hydrogen atom or an alkyl group (e.g., C １－４ Alkyl group, preferably C １－２ R represents an alkyl group. ２ A hydrogen atom is particularly preferred as '.

[0046] As the compound represented by formula (1), the compounds represented by the above formulas (1-1-1), (1-2-1), (1-3-1), (1-4-1), and (1-5-1) (including optical isomers) are most preferred, in that they have excellent cooperativity with TGF-β, increase the concentration of phosphorylated Smad2 / 3, and are particularly effective in activating stem cells.

[0047] As the compound represented by formula (1), the compounds represented by the above formulas (1-1-1), (1-3-1), and (1-5-1) (including optical isomers) are particularly preferred in terms of excellent cooperativity with TGF-β, particularly excellent effects of increasing the concentration of phosphorylated Smad2 / 3 and activating stem cells, and excellent stability.

[0048] Examples of the compound represented by formula (1) that can be used include Nalsgen (registered trademark) or GGsTop [generic name: DL-2-amino-4-[(RSp)-(3-carboxymethylphenoxy)(methoxy)phosphoryl]butanoic acid], DL-2-amino-3-phosphonopropionic acid, DL-2-amino-4-phosphonobutyric acid, D(-)-2-amino-4-phosphonobutyric acid, L(+)-2-amino-4-phosphonobutyric acid, DL-2-amino-4-(methoxyphosphonyl)butyric acid, DL-2-amino-5-phosphonopentanoic acid, and DL-2-amino-6-phosphonohexanoic acid.

[0049] Examples of salts of the compound represented by formula (1) include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts, calcium salts and barium salts; salts with ammonia; salts with nitrogen-containing organic bases such as trimethylamine, triethylamine, tributylamine, pyridine, quinoline, piperidine, imidazole, picoline, dimethylaminopyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, cyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, N,N′-dibenzylethylenediamine and N-methyl-D-glucamine; salts with basic amino acids such as lysine, arginine and ornithine; transition metal salts; salts with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and boric acid; and salts with organic acids such as oxalic acid, acetic acid and p-toluenesulfonic acid.

[0050] The salt of the compound represented by formula (1) can be produced by reacting the compound represented by formula (1) with, for example, a basic compound such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, calcium hydroxide, or barium hydroxide; ammonia; a nitrogen-containing organic base such as trimethylamine, triethylamine, tributylamine, pyridine, quinoline, piperidine, imidazole, picoline, dimethylaminopyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, cyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, N,N′-dibenzylethylenediamine, or N-methyl-D-glucamine; a basic amino acid such as lysine, arginine, ornithine; an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, or boric acid; or an organic acid such as oxalic acid, acetic acid, or p-toluenesulfonic acid.

[0051] The compound represented by formula (1) and the salt of said compound may be a hydrate (for example, a monohydrate to a pentahydrate, preferably a monohydrate).

[0052] The hydrate of the compound represented by formula (1) can be produced by subjecting the compound represented by formula (1) to a crystallization treatment using water and a water-soluble solvent.

[0053] The content of the compound selected from the compound represented by formula (1), a salt thereof, and a hydrate thereof in the stem cell activating agent of the present invention (the total content when two or more types are contained) is, for example, 0.001 to 500 μg / mL, preferably 0.005 to 300 μg / mL, more preferably 0.01 to 200 μg / mL, even more preferably 0.01 to 100 μg / mL, particularly preferably 0.01 to 10 μg / mL, most preferably 0.01 to 1.0 μg / mL, and especially preferably 0.01 to 0.5 μg / mL.

[0054] The mechanism of action of active ingredient (1) is thought to be as follows: 1. When TGF-β binds to the TGF-β receptor, the TGF-β receptor phosphorylates Smad2 / 3, a major transcription factor in the TGF-β signaling pathway. 2. The phosphorylated Smad2 / 3 binds to Smad4 to form a Smad complex. The Smad complex then translocates from the cytoplasm to the nucleus, induces gene expression in fibroblasts, and increases the production of fibrous proteins (specifically, laminin and type V collagen) and cytokines (specifically, HBEGF and FGF2) produced by fibroblasts. 3. Active ingredient (1) works cooperatively with TGF-β to promote phosphorylation of Smad2 / 3 and inhibit dephosphorylation of phosphorylated Smad2 / 3, thereby promoting gene expression of laminin, type V collagen, HBEGF, and FGF2 and increasing the production of laminin, type V collagen, HBEGF, and FGF2. This increases the concentrations of laminin, type V collagen, HBEGF, and FGF2 in the stem cell region. 4. Stem cells are activated by the increase in laminin and type V collagen concentrations. Specifically, laminin and type V collagen repair damage to the basement membrane, activating stem cells and improving their differentiation potential. 5. The increase in HBEGF and FGF2 concentrations promotes stem cell self-renewal, and the replicated stem cells differentiate into skin cells, regenerating the skin.

[0055] The active ingredient (1) acts together with TGF-β, a cytokine produced in vivo, to activate stem cells. The TGF-β that acts in cooperation with the active ingredient (1) may be only TGF-β produced in vivo, or may be TGF-β that is applied simultaneously or sequentially in combination with the active ingredient (1).

[0056] In order to enhance the stem cell activation effect of the active ingredient (1), it is preferable that the amount of TGF-β present is, for example, 2 ng / mL or more.

[0057] Laminin is a large protein composed of three types of chain proteins: α-chains, β-chains, and γ-chains. There are five types of α-chains, three types of β-chains, and three types of γ-chains. Therefore, there are many types of laminins in the laminin family (e.g., laminin-111, laminin-121, laminin-211, laminin-221, laminin-213, laminin-332, laminin-3A11, laminin-3A21, laminin-3A32, laminin-3B21, laminin-411, laminin-421, laminin-423, laminin-511, laminin-521, laminin-522, laminin-523, etc.) depending on the combination of α-chains, β-chains, and γ-chains.

[0058] The active ingredient (1) has the effect of significantly enhancing the production of laminins having β3 chains (for example, laminin 332) among the laminin family.

[0059] Active ingredient (1) also works in cooperation with TGF-β to promote the production of basic fibroblast growth factor (FGF2), a fibroblast growth factor. FGF2 binds to FGFR1, a receptor for dermal stem cells, transmitting a signal for cell proliferation and initiating self-renewal of dermal stem cells.

[0060] Active ingredient (1) also works cooperatively with TGF-β to promote the production of heparin-binding EGF-like growth factor (HBEGF), an epidermal growth factor. HBEGF binds to EGFR / ErbB1 and ErbB4, receptors for epidermal stem cells, transmitting a signal for cell proliferation and initiating self-renewal of epidermal stem cells.

[0061] The stem cell activating agent of the present invention is non-cytotoxic and has excellent safety. Therefore, the stem cell activating agent of the present invention can be used by adding it to, for example, external skin preparations and cosmetics.

[0062] When the stem cell activating agent of the present invention is applied to the skin, it has the effect of increasing laminin production (or laminin concentration) and HBEGF production (or HBEGF concentration) in the epidermal stem cell region, and the effect of increasing type V collagen production (or type V collagen concentration) and FGF2 production (or FGF2 concentration) in the dermal stem cell region.

[0063] Furthermore, an increase in the production of laminin or type V collagen (or the concentration of laminin or type V collagen) promotes the differentiation and maturation of stem cells, and promoting the differentiation and maturation of stem cells has the effect of preventing or improving skin diseases (e.g., atopic dermatitis, dry skin, decreased barrier function and cell turnover, age spots, wrinkles, sagging, decreased firmness and elasticity, etc.).

[0064] Furthermore, when the production amounts of HBEGF and FGF2 (or the concentrations of HBEGF and FGF2) increase, the self-renewal of stem cells is promoted, improving the regenerative ability of the skin.

[0065] Therefore, the stem cell activating agent of the present invention can be suitably used for preventing or ameliorating stem cell decline associated with reduced laminin and / or type V collagen production. That is, the stem cell activating agent of the present invention can be suitably used as an agent for preventing or ameliorating skin diseases associated with reduced laminin and / or type V collagen production. In addition, the stem cell activating agent of the present invention can also be suitably used as a laminin production promoter, a type V collagen production promoter, and an agent for preventing or ameliorating stem cell decline associated with basement membrane damage.

[0066] Furthermore, the stem cell activating agent of the present invention can be suitably used for preventing or ameliorating a decrease in stem cells associated with a decrease in the production of HBEGF and / or FGF2. That is, the stem cell activating agent of the present invention can be suitably used as an agent for preventing or ameliorating skin diseases associated with a decrease in the production of HBEGF and / or FGF2. In addition, the stem cell activating agent of the present invention can also be suitably used as an HBEGF production promoter and an FGF2 production promoter.

[0067] The stem cell activating agent of the present invention is also thought to exert an effect of enhancing immune function by working cooperatively with LPS (i.e., lipopolysaccharide) and IL-6 (i.e., cytokine) to enhance the phosphorylation and inhibit the dephosphorylation of NF-κB, a nuclear transcription factor of LPS signaling, and STAT3, a nuclear transcription factor of IL-6 signaling. In other words, the stem cell activating agent of the present invention can be used as a phosphorylation promoter or dephosphorylation inhibitor of NF-κB, a nuclear transcription factor of LPS signaling, and STAT3, a nuclear transcription factor of IL-6 signaling.

[0068] The stem cell activating agent of the present invention can be used as a preparation in the form of, for example, a paste, gel, liquid, emulsion, cream, aerosol, or mist.

[0069] The stem cell activating agent of the present invention can be used as a topical agent (ointment, cream, lotion, gel, mists, etc.), a patch (adhesive plaster, tape, etc.), a spray (aerosol, mists), etc.

[0070] [Topical Skin Preparations, Cosmetics] The topical skin preparation of the present invention contains the stem cell activating agent (or the active ingredient (1)). The topical skin preparation is, for example, a topical skin preparation having a stem cell activating effect, and is preferably a topical skin preparation for preventing or ameliorating a skin disease accompanied by a decrease in the production of laminin and / or type V collagen, a topical skin preparation for preventing or ameliorating a skin disease accompanied by a decrease in the production of HBEGF and / or FGF2, a topical skin preparation for preventing or ameliorating a decrease in stem cells accompanied by damage to the basement membrane, or a topical skin preparation for preventing or ameliorating a skin disease accompanied by damage to the basement membrane (e.g., dryness, decreased barrier function, decreased turnover, age spots, wrinkles, sagging, decreased firmness, decreased elasticity, etc.).

[0071] The content of the stem cell activating agent in the topical skin preparation is such that the concentration of the active ingredient (1) (when two or more kinds are contained, the total concentration) is in the range of, for example, 0.5 to 70 μM (preferably 10 to 60 μM, particularly preferably 30 to 60 μM, and most preferably 40 to 60 μM).

[0072] The cosmetic of the present invention contains the stem cell activating agent (or the active ingredient (1)). The cosmetic is, for example, a cosmetic having a stem cell activating effect, and is preferably a cosmetic for preventing or ameliorating a skin disease accompanied by a decrease in the production of at least one selected from laminin, type V collagen, HBEGF, and FGF2, or a cosmetic for preventing or ameliorating a decrease in stem cells accompanied by damage to the basement membrane, or a cosmetic for preventing or ameliorating a skin disease accompanied by damage to the basement membrane (for example, dryness, decreased barrier function, decreased turnover, age spots, wrinkles, sagging, decreased firmness, decreased elasticity, etc.).

[0073] The content of the stem cell activating agent in the cosmetic is such that the concentration of the active ingredient (1) (when two or more types are contained, the total concentration) is in the range of, for example, 0.5 to 70 μM (preferably 10 to 60 μM, particularly preferably 30 to 60 μM, and most preferably 40 to 60 μM).

[0074] In addition to the stem cell activating agent, the topical skin preparations and cosmetics can contain, as needed, ingredients commonly used in dermatological drugs and cosmetics (e.g., moisturizers, antioxidants, oils, UV protection agents, surfactants, thickeners, alcohols, powdered ingredients, coloring materials, aqueous ingredients, various skin nutrients, etc.).

[0075] The topical skin preparations include, for example, moisturizers, agents for treating keratosis, etc. The dosage forms of the topical skin preparations include topical preparations (creams, ointments, gels, lotions, liquids, tinctures), patches (cataplasms, plasters, tapes, patches), and aerosol preparations.

[0076] Examples of the cosmetics include basic cosmetics such as lotion, emulsion, serum, face cream, eye cream, and lip cream; base makeup cosmetics such as foundation, face powder, and concealer; and makeup cosmetics such as blush, lipstick, mascara, and eye shadow.

[0077] There is no particular limit to the number of times that topical skin preparations or cosmetics can be used, and this can be adjusted as appropriate depending on the symptoms and purpose.

[0078] The above-described configurations and combinations of the present invention are merely examples, and the configurations may be added, omitted, substituted, or modified as appropriate without departing from the spirit of the present invention. Furthermore, the present invention is not limited to the embodiments, but is limited only by the claims.

[0079] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0080] Example 1 The compound represented by the following formula (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., sometimes referred to as "Nahlsgen" in this specification) was used as a stem cell activating agent.

[0081]

[0082] Example 2 A compound represented by the following formula (DL-2-amino-4-phosphonobutyric acid, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.; sometimes referred to herein as "DL-AP4") was used as a stem cell activating agent. DL-AP4 does not absorb moisture at room temperature and is non-deliquescent. As a result, it has excellent storage stability and is easy to handle. On the other hand, Nahlsgen in Example 1 does not crystallize at room temperature and is oily, and when lyophilized, an amorphous powder solid is obtained, but it is deliquescent and therefore easily decomposes at room temperature.

[0083]

[0084] Example 3 The compound represented by the following formula (DL-2-amino-4-(methoxyphosphonyl)butyric acid, sometimes referred to herein as "DL-AP4OMe") was used as a stem cell activating agent.

[0085]

[0086] Example 4 The compound represented by the following formula (DL-2-amino-3-phosphonopropionic acid (sometimes referred to herein as "DL-AP3")) was used as a stem cell activating agent.

[0087]

[0088] Example 5 The compound represented by the following formula (DL-2-amino-5-phosphonopentanoic acid (sometimes referred to herein as "DL-AP5")) was used as a stem cell activating agent.

[0089]

[0090] Example 6 The compound represented by the following formula (DL-2-amino-6-phosphonohexanoic acid (sometimes referred to herein as "DL-AP6")) was used as a stem cell activating agent.

[0091]

[0092] Example 7 A salt of D-2-amino-4-phosphonobutyric acid and D-arginine (sometimes referred to herein as "D-AP4·D-Arg") was used as a stem cell activating agent.

[0093] Example 8 A salt of L-2-amino-4-phosphonobutyric acid and L-arginine (sometimes referred to herein as "L-AP4·L-Arg") was used as a stem cell activating agent.

[0094] Example 9 A salt of D-2-amino-4-phosphonobutyric acid and L-arginine (sometimes referred to herein as "D-AP4·L-Arg") was used as a stem cell activating agent.

[0095] Example 10 A salt of L-2-amino-4-phosphonobutyric acid and D-arginine (sometimes referred to herein as "L-AP4·D-Arg") was used as a stem cell activating agent.

[0096] Example 11 A salt of DL-2-amino-4-phosphonobutyric acid and DL-arginine (sometimes referred to herein as "DL-AP4.DL-Arg") was used as a stem cell activating agent.

[0097] Example 12 L-2-amino-4-phosphonobutyric acid (sometimes referred to herein as "L-AP4") was used as a stem cell activating agent.

[0098] Example 13 Nahlsgen monohydrate (referred to herein as "Nahlsgen.H") ２O) was used as a stem cell activating agent.

[0099] (Evaluation) The stem cell activating agents of the examples were evaluated by the following method.

[0100] (Laminin Expression Level Evaluation 1) A stem cell activator was added to mouse fetal fibroblasts in a culture medium to a final concentration of 20 μg / mL, followed by incubation at 37°C for 8 hours. TGF-β was then added to a final concentration of 0 ng / mL, 2 ng / mL, or 10 ng / mL, and the cells were further incubated at 37°C for 16 hours. Proteins were then extracted from the culture medium, and the laminin β3 chain and γ1-actin (control) were detected for the obtained proteins by Western blotting. The same experiment was conducted as above, except that the stem cell activator was not used. This was used as a blank test.

[0101] The results when Nahlsgen was used as a stem cell activator are shown in Figure 1. As can be seen from Figure 1, the expression level of laminin 332 increases when cells are stimulated with TGF-β, but when cells are stimulated with TGF-β and Nahlsgen is added, the expression of laminin 332 is significantly enhanced in a Nahlsgen concentration-dependent manner compared to when cells are stimulated with TGF-β alone.

[0102] The results when DL-AP4 was used as a stem cell activator are shown in Figure 8. As can be seen from Figure 8, the expression level of laminin 332 increases when cells are stimulated with TGF-β, but when cells are stimulated with TGF-β and DL-AP4 is added, the expression of laminin 332 is significantly enhanced in a DL-AP4 concentration-dependent manner compared to when cells are stimulated with TGF-β alone.

[0103] The results when DL-AP4OMe was used as a stem cell activator are shown in Figure 14. As can be seen from Figure 14, the expression level of laminin 332 increases when cells are stimulated with TGF-β, but when cells are stimulated with TGF-β and DL-AP4OMe is added, the expression of laminin 332 is significantly enhanced in a DL-AP4OMe concentration-dependent manner compared to when cells are stimulated with TGF-β alone.

[0104] (Laminin expression level evaluation 2) The same procedure as in laminin expression level evaluation 1 was performed, except that the timing of applying TGF-β was changed from 8 hours after application of the stem cell activation agent to 5 hours after application of the stem cell activation agent, and the incubation time after application of TGF-β until protein extraction was changed from 16 hours to 4 hours.

[0105] The results when Nahlsgen was used as a stem cell activator are shown in Figure 2. As can be seen from Figure 2, the expression level of laminin 332 increases when cells are stimulated with TGF-β, but when cells are stimulated with TGF-β and Nahlsgen is added, the expression of laminin 332 is significantly enhanced in a Nahlsgen concentration-dependent manner compared to when cells are stimulated with TGF-β alone.

[0106] (Evaluation of Type V Collagen Expression Levels) A stem cell activator was added to mouse fetal fibroblasts in a culture medium to a final concentration of 20 μg / mL, followed by incubation at 37°C for 5 hours. TGF-β was then added to a final concentration of 0 ng / mL, 0.05 ng / mL, or 0.1 ng / mL, and the cells were further incubated at 37°C for 4 hours. RNA was then extracted, and the resulting RNA was subjected to real-time PCR to detect Type V collagen. The same experiment was conducted as above, except that no stem cell activator was used. This was used as a blank test.

[0107] The results when Nahlsgen was used as a stem cell activator are shown in FIG.

[0108] The results when DL-AP4 was used as a stem cell activator are shown in FIG.

[0109] The results when DL-AP4OMe was used as a stem cell activator are shown in FIG.

[0110] The results when DL-AP3, DL-AP4, DL-AP5, and DL-AP6 were used as stem cell activators are shown in FIG.

[0111] Figures 3, 9, and 15 show that stimulation of cells with TGF-β increases the expression level of type V collagen, but when cells are stimulated with TGF-β and a stem cell activator is added, the expression of type V collagen is significantly enhanced in a concentration-dependent manner of the stem cell activator compared to stimulation with TGF-β alone.

[0112] As can be seen from Figure 22, stimulation of cells with TGF-β increases the expression level of type V collagen, but when cells are stimulated with TGF-β and a stem cell activator (particularly DL-AP3, DL-AP4, or DL-AP6) is added, the expression of type V collagen is significantly enhanced in a concentration-dependent manner of the stem cell activator compared to stimulation with TGF-β alone.

[0113] (Smad2 / 3 Phosphorylation Evaluation 1) Mouse fetal fibroblasts were cultured at 37°C for 8 hours after adding a stem cell activator to the culture medium to a final concentration of 20 μg / mL. TGF-β was then added to a final concentration of 0 ng / mL, 0.1 ng / mL, or 0.5 ng / mL, and the cells were further cultured at 37°C for 1 hour. Proteins were then extracted from the culture medium, and the resulting proteins were subjected to Western blotting to detect phosphorylated Smad2 and phosphorylated Smad3, as well as Smad2 and Smad3 as controls. The same experiment was conducted as above, except that the stem cell activator was not used. This was used as a blank test.

[0114] The results when Nahlsgen was used as a stem cell activator are shown in FIG.

[0115] The results when DL-AP4 was used as a stem cell activator are shown in FIG.

[0116] The results when DL-AP4OMe was used as a stem cell activator are shown in FIG.

[0117] Figures 4, 10, and 16 show that stimulation of cells with TGF-β increases phosphorylation of Smad2 / 3, but when cells are stimulated with TGF-β and a stem cell activator is added, phosphorylation of Smad2 / 3 is significantly enhanced in a stem cell activator concentration-dependent manner compared to stimulation with TGF-β alone.

[0118] (Evaluation of Smad2 / 3 Phosphorylation 2) Mouse embryonic fibroblasts were cultured at 37°C for 5 hours after adding a stem cell activator to the culture medium to a final concentration of 20 μg / mL. TGF-β was then added to a final concentration of 0 ng / mL, 2 ng / mL, or 10 ng / mL, and the cells were further cultured at 37°C for 4 hours. Proteins were then extracted from the culture medium, and the resulting proteins were subjected to Western blotting to detect phosphorylated Smad2 and phosphorylated Smad3, as well as Smad2 and Smad3 as controls. The same experiment was conducted as above, except that the stem cell activator was not used. This was used as a blank test.

[0119] The results when Nahlsgen was used as a stem cell activator are shown in FIG.

[0120] The results when DL-AP4 was used as a stem cell activator are shown in FIG.

[0121] The results when DL-AP4OMe was used as a stem cell activator are shown in FIG.

[0122] Figures 5, 11, and 17 show that stimulation of cells with TGF-β increases phosphorylation of Smad2 / 3, but that stimulation of cells with TGF-β and addition of a stem cell activator significantly enhances the duration of Smad2 / 3 phosphorylation compared to stimulation with TGF-β alone.

[0123] (Smad3 Phosphorylation Evaluation 3) Mouse fetal fibroblasts were cultured at 37°C for 8 hours after adding a stem cell activator to the culture medium to a final concentration of 20 μg / mL. TGF-β was then added to a final concentration of 0 ng / mL or 10 ng / mL, and the cells were further cultured at 37°C for 16 hours. Proteins were then extracted from the culture medium, and the resulting proteins were subjected to Western blotting to detect phosphorylated Smad3 and control Smad3. The same experiment was conducted as above, except that the stem cell activator was not used. This was used as a blank test.

[0124] Nahlsgen H as a stem cell activator ２The results when O, DL-AP4·DL-Arg, D-AP4·D-Arg, L-AP4·L-Arg, D-AP4·L-Arg, L-AP4·D-Arg, DL-AP3, and DL-AP4 were used are shown in Figure 28.

[0125] As can be seen from Figure 28, stimulation of cells with TGF-β increases Smad3 phosphorylation, but when cells are stimulated with TGF-β and a stem cell activator (particularly DL-AP4·DL-Arg, D-AP4·D-Arg, L-AP4·L-Arg, D-AP4·L-Arg, L-AP4·D-Arg, DL-AP4) is added, the duration of Smad3 phosphorylation is significantly enhanced compared to stimulation with TGF-β alone.

[0126] (Evaluation of skin stem cell expression levels) A stem cell activator was added to mouse fetal fibroblasts in a culture medium to a final concentration of 20 μg / mL, followed by incubation at 37°C for 5 hours. TGF-β was then added to a final concentration of 0 ng / mL or 0.1 ng / mL, and the cells were further incubated at 37°C for 4 hours. RNA was then extracted, and the stem cell markers CD271 and CD105 were detected using real-time PCR on the resulting RNA. The same experiment was conducted as above, except that the stem cell activator was not used. This was used as a blank test.

[0127] The results when Nahlsgen was used as a stem cell activator are shown in FIG.

[0128] The results when DL-AP4 was used as a stem cell activator are shown in FIG.

[0129] The results when DL-AP4OMe was used as a stem cell activator are shown in FIG.

[0130] The results when DL-AP3, DL-AP4, DL-AP5, and DL-AP6 were used as stem cell activators are shown in FIGS. 24 and 25.

[0131] 6, 12, and 18 show that stimulation of cells with TGF-β increases the expression of stem cell markers CD271 and CD105, but that stimulation of cells with TGF-β and addition of a stem cell activator significantly enhances the expression of stem cell markers CD271 and CD105 compared to stimulation with TGF-β alone. Furthermore, stimulation of cells with the stem cell activator alone also increased the expression of stem cell markers CD271 and CD105.

[0132] 24 and 25 show that stimulation of cells with TGF-β increases the expression levels of stem cell markers CD271 and CD105, but that when cells are stimulated with TGF-β and a stem cell activator (particularly DL-AP3, DL-AP4, or DL-AP6) is added, the expression of stem cell markers CD271 and CD105 is significantly enhanced compared to stimulation with TGF-β alone. Furthermore, the expression of stem cell markers CD271 and CD105 also increased when cells were stimulated with a stem cell activator alone. These results demonstrate that the stem cell activator of the present invention has an excellent effect of activating stem cells.

[0133] (Evaluation of FGF2 expression level) A stem cell activator was added to mouse fetal fibroblasts in a culture medium to a final concentration of 20 μg / mL, followed by incubation at 37°C for 5 hours. TGF-β was then added to a final concentration of 0 ng / mL, 0.05 ng / mL, or 0.1 ng / mL, and the cells were further incubated at 37°C for 4 hours. RNA was then extracted, and FGF2 was detected from the resulting RNA by real-time PCR. The same experiment was conducted as above, except that no stem cell activator was used. This was used as a blank test.

[0134] The results when Nahlsgen was used as a stem cell activator are shown in FIG.

[0135] The results when DL-AP4 was used as a stem cell activator are shown in FIG.

[0136] The results when DL-AP4OMe was used as a stem cell activator are shown in FIG.

[0137] The results when DL-AP3, DL-AP4, DL-AP5, and DL-AP6 were used as stem cell activators are shown in FIG.

[0138] Figures 7, 13, and 19 show that stimulation of cells with TGF-β increases the expression level of FGF2, but when cells are stimulated with TGF-β and a stem cell activator is added, FGF2 expression is significantly enhanced compared to stimulation with TGF-β alone.

[0139] As can be seen from Figure 26, stimulation of cells with TGF-β increases the amount of FGF2 expression, but when cells are stimulated with TGF-β and a stem cell activator (particularly DL-AP3, DL-AP4, or DL-AP6) is added, FGF2 expression is significantly enhanced compared to stimulation with TGF-β alone.

[0140] (Evaluation of HBEGF Expression Levels) A stem cell activator was added to mouse fetal fibroblasts in a culture medium to a final concentration of 20 μg / mL, followed by incubation at 37°C for 5 hours. TGF-β was then added to a final concentration of 0 ng / mL, 0.05 ng / mL, or 0.1 ng / mL, and the cells were further incubated at 37°C for 4 hours. RNA was then extracted, and HBEGF was detected using real-time PCR on the resulting RNA. The same experiment was conducted as above, except that no stem cell activator was used. This was used as a blank test.

[0141] The results when Nahlsgen was used as a stem cell activator are shown in FIG.

[0142] The results when DL-AP4 was used as a stem cell activator are shown in FIG.

[0143] The results when DL-AP4OMe was used as a stem cell activator are shown in FIG.

[0144] The results when DL-AP3, DL-AP4, DL-AP5, and DL-AP6 were used as stem cell activators are shown in FIG.

[0145] Figures 7, 13, and 19 show that stimulation of cells with TGF-β increases the expression level of HBEGF, but when cells are stimulated with TGF-β and a stem cell activator is added, HBEGF expression is significantly enhanced compared to stimulation with TGF-β alone.

[0146] As can be seen from Figure 27, when cells are stimulated with TGF-β, the expression level of HBEGF increases, but when cells are stimulated with TGF-β and a stem cell activator (particularly DL-AP3, DL-AP4, DL-AP6) is added, the expression of HBEGF is significantly enhanced compared to when cells are stimulated with TGF-β alone.

[0147] (Evaluation of expression levels of laminin, procollagen, and αSMA) A stem cell activator was added to mouse fetal fibroblasts in a culture medium to a final concentration of 20 μg / mL, followed by incubation at 37°C for 8 hours. TGF-β was then added to a final concentration of 10 ng / mL, and the cells were further incubated at 37°C for 16 hours. Proteins were then extracted from the culture medium, and the laminin β3 chain, procollagen, αSMA, and γ1-actin (control) were detected using Western blotting. The same experiment was conducted as above, except that the stem cell activator was not used. This was used as a blank test.

[0148] Figure 20 shows the results when Nahlsgen, DL-AP4·DL-Arg, D-AP4·D-Arg, L-AP4·L-Arg, D-AP4·L-Arg, L-AP4·L-Arg, DL-AP4, L-AP4, and DL-AP4·OMe were used as stem cell activators.

[0149] As stem cell activators, Nahlsgen, Nahlsgen H ２ The results when O, DL-AP4·DL-Arg, DL-AP3, DL-AP4, DL-AP5, DL-AP6, and DL-AP4·OMe were used are shown in Figure 21.

[0150] 20 and 21 show that stimulating cells with TGF-β and adding a stem cell activator significantly increases the expression of laminin 332, procollagen, and αSMA. αSMA (α smooth muscle actin) is a protein specific to smooth muscle cells. Increased expression of αSMA indicates that self-renewal of smooth muscle cells has been promoted.

[0151] (Evaluation of laminin α3 chain expression level) A stem cell activator was added to mouse fetal fibroblasts in a culture medium to a final concentration of 20 μg / mL, followed by incubation at 37°C for 5 hours. TGF-β was then added to a final concentration of 0 ng / mL, 0.05 ng / mL, or 0.1 ng / mL, and the cells were further incubated at 37°C for 4 hours. RNA was then extracted, and the laminin α3 chain was detected using real-time PCR on the resulting RNA. The same experiment was conducted as above, except that the stem cell activator was not used. This was used as a blank test.

[0152] The results when DL-AP3, DL-AP4, DL-AP5, and DL-AP6 were used as stem cell activators are shown in Figure 23. Figure 23 shows that stimulating cells with TGF-β and adding stem cell activators (particularly DL-AP3, DL-AP4, and DL-AP6) significantly increased the expression of laminin α3 chain.

[0153] In summary, the configuration of the present invention and its variations are described below. [1] Use of a composition comprising, as an active ingredient, at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof, as a stem cell activating agent. [2] Use of a composition comprising, as an active ingredient, at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof, as a stem cell activating agent that exhibits a stem cell activating effect by promoting the production of laminin and / or type V collagen. [3] Use of a composition comprising, as an active ingredient, at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof, as a prophylactic agent for preventing a decrease in stem cells associated with a decrease in laminin and / or type V collagen production, or as an ameliorative agent for ameliorating a decrease in stem cells associated with a decrease in laminin and / or type V collagen production. [4] Use of a composition comprising at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof as an active ingredient, as a stem cell activating agent that exhibits a stem cell activation effect by promoting the production of HBEGF and / or FGF2. [5] Use of a composition comprising at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof as an active ingredient, as a prophylactic agent for preventing a decrease in stem cells associated with a decrease in the production of HBEGF and / or FGF2, or as an ameliorator for ameliorating a decrease in stem cells associated with a decrease in the production of HBEGF and / or FGF2. [6] A composition for activating stem cells, comprising at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof as an active ingredient. [7] A composition for activating stem cells by promoting the production of laminin and / or type V collagen, comprising, as an active ingredient, at least one compound selected from a compound represented by formula (1) (including optically active forms), a salt of said compound, and a hydrate thereof.[8] A composition for activating stem cells by promoting the production of HBEGF and / or FGF2, comprising as an active ingredient at least one compound selected from a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof. [9] A composition for preventing or ameliorating a decrease in stem cells associated with a decrease in laminin and / or type V collagen production, comprising as an active ingredient at least one compound selected from a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof.

[10] A composition for preventing or ameliorating a decrease in stem cells associated with a decrease in HBEGF and / or FGF2 production, comprising as an active ingredient at least one compound selected from a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof.

[11] A method for using a composition comprising as an active ingredient at least one compound selected from a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof, for activating stem cells.

[12] A method for using a composition containing at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof as an active ingredient, to activate stem cells by promoting the production of laminin and / or type V collagen.

[13] A method for using a composition containing at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof as an active ingredient, to activate stem cells by promoting the production of HBEGF and / or FGF2.

[14] A method for using a composition containing at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof as an active ingredient, to prevent or ameliorate a decrease in stem cells associated with a decrease in the production of laminin and / or type V collagen.

[15] A method for preventing or ameliorating a decrease in stem cells associated with a decrease in the production of HBEGF and / or FGF2, comprising using a composition containing, as an active ingredient, at least one compound selected from the group consisting of a compound represented by formula (1) (including an optically active substance), a salt of the compound, and a hydrate thereof.

[16] A method for treating a disease accompanied by a reduction in stem cells, comprising the step of administering to a patient suffering from a disease accompanied by a reduction in stem cells a composition comprising, as an active ingredient, at least one compound selected from the compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof.

[17] A method for treating a disease accompanied by a reduction in stem cells due to a reduction in laminin and / or type V collagen production, comprising the step of administering to a patient suffering from a disease accompanied by a reduction in stem cells due to the suppression of laminin and / or type V collagen production a composition comprising, as an active ingredient, at least one compound selected from the compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof.

[18] A method for treating a disease accompanied by a reduction in stem cells due to the suppression of HBEGF and / or FGF2 production, comprising the step of administering to a patient suffering from a disease accompanied by a reduction in stem cells due to the suppression of HBEGF and / or FGF2 production a composition comprising, as an active ingredient, at least one compound selected from the compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof.

[19] A method for activating skin stem cells, the method comprising applying to skin with damaged basement membrane a composition containing, as an active ingredient, at least one compound selected from the compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof.

[20] A method for improving the production of laminin and / or type V collagen in skin, the method comprising applying to skin with damaged basement membrane a composition containing, as an active ingredient, at least one compound selected from the compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof.

[21] A method for improving the production of HBEGF and / or FGF2 in skin, the method comprising applying to skin with damaged basement membrane a composition containing, as an active ingredient, at least one compound selected from the compound represented by formula (1) (including an optically active form), a salt of the compound, and a hydrate thereof.

[22] A stem cell activating agent comprising, as an active ingredient, at least one compound selected from the group consisting of a compound represented by formula (1) (including optically active compounds), a salt of said compound, and a hydrate thereof.

[23] The stem cell activating agent according to

[22] , which exhibits a stem cell activating effect by promoting the production of laminin and / or type V collagen.

[24] The stem cell activating agent according to

[22] , which is an agent for preventing or ameliorating a decrease in stem cells associated with a decrease in the production of laminin and / or type V collagen.

[25] The stem cell activating agent according to

[22] , which exhibits a stem cell activating effect by promoting the production of HBEGF and / or FGF2.

[26] The stem cell activating agent according to

[22] , which is an agent for preventing or ameliorating a decrease in stem cells associated with a decrease in the production of HBEGF and / or FGF2.

[27] An external skin preparation comprising the stem cell activating agent according to any one of

[22] to

[26] .

[28] A cosmetic preparation comprising the stem cell activating agent according to any one of

[22] to

[26] .

[0154] The stem cell activating agent of the present invention works in cooperation with TGF-β to enhance phosphorylation of Smad2 / 3 and inhibit dephosphorylation of phosphorylated Smad2 / 3, thereby increasing the expression levels of laminin 332 and type V collagen and repairing basement membrane damage. Furthermore, the stem cell activating agent of the present invention works in cooperation with TGF-β to increase the expression levels of FGF2 and HBEGF, which are growth factors for stem cells, thereby promoting stem cell self-renewal. Because the stem cell activating agent of the present invention has the above-mentioned effects, it can be suitably used as an agent for preventing or ameliorating stem cell decline associated with decreased laminin and / or type V collagen production, or as an agent for preventing or ameliorating stem cell decline associated with decreased FGF2 and HBEGF production, or as an agent for preventing or ameliorating stem cell decline associated with basement membrane damage.

Claims

1. A stem cell activating agent comprising, as an active ingredient, at least one compound selected from a compound represented by the following formula (1) (including optically active forms), a salt of said compound, and a hydrate thereof: [R １ , R ２ are the same or different and represent a hydrogen atom or a hydrocarbon group. ３ represents a hydrogen atom or an organic group. n represents an integer of 1 or more. The hydrocarbon group may have a substituent. The substituent may be a halogen atom, —COOR ａ , -CONR ａ ２ , -COR ａ , -CN, -NO ２ , -NHCOR ａ , -OR ａ , -SR ａ , -OCOR ａ , -SO ３ R ａ , and -SO ２ NR ａ ２ is a group selected from the group consisting of ａ represents a hydrogen atom or an aliphatic hydrocarbon group.

2. The stem cell activating agent according to claim 1, which exhibits a stem cell activating effect by promoting the production of laminin and / or type V collagen.

3. The stem cell activating agent according to claim 1, which is an agent for preventing or ameliorating the decrease in stem cells associated with a decrease in the production of laminin and / or type V collagen.

4. The stem cell activating agent according to claim 1, which exhibits a stem cell activating effect by promoting the production of FGF2 and / or HBEGF.

5. The stem cell activating agent according to claim 1, which is an agent for preventing or ameliorating the decrease in stem cells associated with a decrease in the production of FGF2 and / or HBEGF.

6. An external preparation for skin containing the stem cell activating agent according to any one of claims 1 to 5.

7. A cosmetic comprising the stem cell activating agent according to any one of claims 1 to 5.

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