Composition for preventing or ameliorating sign of skin aging, and external preparation for skin

Abstract

Provided is a composition for preventing or ameliorating a sign of skin aging, the composition containing a ubiquinol dicarboxylic acid ester derivative represented by general formula (1), and also containing, as an active ingredient, an IL-6 mRNA expression inhibitor, an IL-8 mRNA expression inhibitor, an MMP1 mRA expression inhibitor, a CDKN1A mRNA expression inhibitor, an ATP production enhancer, a COL1A1 mRNA expression upregulator, a COL3A1 mRNA expression upregulator, a HAS2 mRNA expression upregulator, an elastin mRNA expression upregulator, a TGF-βR2 mRNA expression upregulator, and / or a neprilysin mRNA expression inhibitor. In general formula (1), R1 and R2 each independently represent a hydrogen atom or a substituent, wherein the substituent is selected from among dicarboxylic acid hemiester residues, salts thereof, and dicarboxylic acid double-ester residues; at least one of R1 and R2 is any one of dicarboxylic acid hemiester residues, salts thereof, and dicarboxylic acid double-ester residues; and n represents an integer from 1 to 10.

Description

Compositions and topical skin preparations for the prevention or improvement of skin aging. Related applications

[0001] This application claims priority to Japanese Patent Application No. 2025-39921, filed on 13 March 2025, and incorporates all the provisions contained herein.

[0002] The present invention relates to a composition and topical skin preparation for preventing or improving skin aging, such as wrinkles and sagging, which can be prevented or improved by suppressing the progression of cellular senescence in dermal fibroblasts and by suppressing at least one of the decrease, degeneration, and degradation of dermal extracellular matrix (ECM) components associated with skin aging.

[0003] Human skin aging consists of intrinsic physiological aging associated with aging and extrinsic photoaging caused by ultraviolet radiation exposure. The dermis is 10 times thicker than the epidermis and is mostly composed of extracellular matrix (ECM) components such as collagen fibers, elastic fibers, and hyaluronic acid produced by dermal fibroblasts (see Non-Patent Literature 1).

[0004] Aging of dermal fibroblasts causes a decrease and degeneration of ECM components, significantly impairing skin elasticity and greatly influencing the formation of skin signs of aging such as wrinkles and sagging (see Non-Patent Literature 2). Therefore, to prevent or improve skin signs of aging such as wrinkles and sagging, it is important to suppress cellular aging of dermal fibroblasts and to prevent the decrease and degeneration of ECM components due to skin aging.

[0005] Ubiquinone-10 (Uq10, coenzyme Q-10, CoQ10) is 2-decaisoprenyl-3-methyl-5,6-dimethoxy-1,4-benzoquinone, and in humans, ubiquinone-10 (Uq10) with n=10 isoprene chains is the major ubiquinone.

[0006] The two-electron reduced form of Uq10, ubiquinol-10 (UqH), is endogenous and is biosynthesized via two routes: one catalyzed by the mitochondrial enzyme Coq2 from 4-hydroxybenzoic acid and oligoprenyl pyrophosphate (see Non-Patent Document 3), and another catalyzed by the Golgi membrane enzyme UBIAD1 (see Non-Patent Document 4).

[0007] UqH possesses excellent antioxidant activity. Uq10 and UqH form a redox system and function as an electron transport chain that controls oxidative phosphorylation in the mitochondrial respiratory chain, playing an important role in ATP synthesis.

[0008] In humans, it is known that Uq10 levels decrease with age, and Uq10 levels in the skin also decrease (see Non-Patent Document 5). In other words, it is thought that UqH biosynthesis decreases with age.

[0009] The wrinkle-improving effect of Uq10 was first reported by Hoppe et al., who reported that it improves wrinkles by suppressing the promotion of collagenase expression induced by UVA irradiation in human dermal fibroblasts, thereby inhibiting collagen degradation or denaturation (see Non-Patent Literature 6). Since then, many applications of Uq10 and UqH to dermal fibroblasts have been reported, but their effects have varied considerably (see Non-Patent Literature 6-11).

[0010] Zang et al. reported that Uq10 increases type IV collagen production in dermal fibroblasts, increases elastin gene expression, and suppresses the expression of matrix metalloproteinase 1 (MMP1), a collagen-degrading enzyme (see Non-Patent Literature 9).

[0011] Mine et al. reported that a one-week administration of a water-soluble Uq10 formulation increased the gene expression of type I collagen, type IV collagen, type VII collagen, elastin, and heat shock protein (HSP) 47 in dermal fibroblasts, increasing type I collagen protein, but did not affect the gene expression of collagen-degrading enzymes, and that administration of Uq10 itself did not show any effect (see Non-Patent Literature 10).

[0012] Nakajima et al. reported that administering reduced UqH to dermal fibroblasts for 72 hours suppressed the increase in neprilysin (which has elastin-degrading elastase activity) induced by UV irradiation (see Non-Patent Literature 11).

[0013] The variation in the effects of Uq10 and UqH is thought to be partly due to the low uptake of Uq10 and UqH by dermal fibroblasts, as shown in Example 2 described later.

[0014] Both oxidized ubiquinone (Uq10) and its two-electron reduced form, ubiquinol (UqH), are known to be extremely unstable in the presence of light (see Non-Patent Document 12).

[0015] Furthermore, ubiquinone irradiation with light induces two types of phototoxicity: direct phototoxicity caused by reactive oxygen species generated by type I photochemical reactions, and indirect phototoxicity mediated by the reaction of singlet oxygen generated by type II photochemical reactions with biological components (see Non-Patent Documents 13, 14, and 1). In other words, when Uq10 or UqH are applied as topical skin preparations, there is a risk not only of a decrease in dosage due to photodegradation, but also of the occurrence of undesirable side effects.

[0016] Furthermore, as mentioned above, various effects of Uq10 and UqH on the reduction and degeneration of ECM components in dermal fibroblasts have been reported, but these effects lack reproducibility. It is thought that one of the reasons for such variability in effects is the low uptake of Uq10 and UqH into dermal fibroblasts, as shown in Example 2 below, and it is desirable to realize the effectiveness by improving uptake into dermal fibroblasts.

[0017] Furthermore, the present inventors have already proposed that ubiquinol dicarboxylic acid ester derivatives having a specific chemical structure can be obtained to have the advantageous effect of not requiring or being difficult to shield from light, having high photostability, and not exhibiting phototoxicity (see Patent Document 1).

[0018] Japanese Patent Publication No. 2021-113162

[0019] Hiroshi Shimizu: New Dermatology, 2nd Edition. Nakayama Shoten, 2011, pp.13-17.Kligman, AM, JAMA, 210: 2377-2380, 1969.Forsgren M., et al., Biochem J, 2004; 519-526.Mugoni V., et al., Cell, 2013; 152: 504-518.Kalen A., et al., Lipids, 24(7): 579-84 (1989).Hoppe. U., et al., BioFactors, 9, 371-378 (1999).Inui. M., et al., BioFactors, 32. 237-243 (2008).Muta-Takada. K., et al., BioFactors, 35, 435-441, 2009. Zang, M., et al., Int J Cosmet Sci., 34, 273-279, 2012. Mine Y., et al., J. Clin. Biochem. Nutr. 71(1), 29-33, 2021. Nakajima, H., et al., PLOS OINE 0161580. 1-29, 2016. Imada I., et al., Chem Pharm Bull, 12,1042-1046, 1964. Onoue S et al., Eur J Pharm, 2012; 46: 492-499. Setoguchi S. et al., BioFactors,46,983-994, 2020. Kurata S., et al., Exp. Cell Res. 206, 63-71 (1993).

[0020] However, the proposal in Patent Document 1 above does not clearly demonstrate that ubiquinol dicarboxylic acid ester derivatives having a specific chemical structure are effective against at least one of the progression of cellular senescence in adult human dermal fibroblasts and the decrease, degeneration, and degradation of dermal extracellular matrix (ECM) components due to skin aging; in other words, that ubiquinol dicarboxylic acid ester derivatives are effective in preventing or improving skin signs of aging such as wrinkles and sagging.

[0021] The present invention has been made in view of the above circumstances, and aims to provide a composition for preventing or improving skin signs of aging, such as wrinkles and sagging, and a topical skin preparation containing the composition for preventing or improving skin signs of aging, by suppressing the progression of cellular senescence of dermal fibroblasts and suppressing at least one of the decrease, degeneration, and degradation of dermal extracellular matrix (ECM) components associated with skin aging.

[0022] In order to achieve the aforementioned objectives, the present inventors conducted extensive research and found that the ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) is highly uptaken by adult human dermal fibroblasts, suppresses cellular senescence by inhibiting the production of senescence-associated secretory phenotype (SASP) factors in adult human dermal fibroblasts, and is effective in suppressing at least one of the following: the decrease in the ability to produce ECM components and the promotion of the expression of degrading enzymes associated with skin aging, which leads to a decrease in the production capacity of ECM components and the reduction, denaturation, and degradation of ECM components. Specifically, we have discovered that IL-6 mRNA expression inhibitors, IL-8 mRNA expression inhibitors, MMP1 mRNA expression inhibitors, CDKN1A mRNA expression inhibitors, ATP production promoters, COL1A1 mRNA expression promoters, COL3A1 mRNA expression promoters, HAS2 mRNA expression promoters, elastin mRNA expression promoters, TGF-βR2 mRNA expression promoters, and / or neprilysin mRNA expression inhibitors containing ubiquinol dicarboxylic acid ester derivatives represented by the following general formula (1) are extremely effective in preventing or improving skin signs of aging such as wrinkles and sagging, by suppressing the progression of cellular senescence in dermal fibroblasts and inhibiting at least one of the decrease, degeneration, and degradation of dermal extracellular matrix (ECM) components associated with skin aging, and have thus come to the present invention.

[0023] However, in the general formula (1) above, R1 and R2 each represent a hydrogen atom or a substituent selected from dicarboxylic acid hemiester residues and their salts and dicarboxylic acid double ester residues, R １ and R ２At least one of these is a dicarboxylic acid hemiester residue and / or a dicarboxylic acid double ester residue. n represents an integer from 1 to 10.

[0024] The present invention is based on the aforementioned findings by the inventors, and the means for solving the aforementioned problems are as follows.

[0025] The present invention provides a composition for the prevention or improvement of skin aging, which contains as an active ingredient at least one agent selected from IL-6 mRNA expression inhibitor, IL-8 mRNA expression inhibitor, MMP1 mRNA expression inhibitor, CDKN1A mRNA expression inhibitor, ATP production promoter, COL1A1 mRNA expression promoter, COL3A1 mRNA expression promoter, HAS2 mRNA expression promoter, elastin mRNA expression promoter, TGF-βR2 mRNA expression promoter, and neprilysin mRNA expression inhibitor.

[0026] The IL-6 mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of suppressing the expression of IL-6 mRNA that generates IL-6 from the interleukin-6 (IL-6) gene.

[0027] The IL-8 mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of suppressing the expression of IL-8 mRNA that generates IL-8 from the interleukin-8 (IL-8) gene.

[0028] The MMP1 mRA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of suppressing the expression of MMP1 mRNA that generates MMP1 from the matrix metalloproteinase 1 (MMP1) gene.

[0029] The CDKN1A mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of suppressing the expression of CDKN1A mRNA that generates CDKN1A from the CDKN1A gene.

[0030] The ATP production promoter contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has an ATP production promoting effect in adult human dermal fibroblasts.

[0031] The COL1A1 mRNA expression promoter contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of COL1A1 mRNA, which generates COL1A1 from the type I procollagen α1 (COL1A1) gene.

[0032] The COL3A1 mRNA expression promoter contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of COL3A1 mRNA, which generates COL3A1 from the type III procollagen α1 (COL3A1) gene.

[0033] The HAS2 mRNA expression promoter contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of HAS2 mRNA that generates HAS2 from the hyaluronic acid synthase (HAS2) gene.

[0034] The elastin mRNA expression promoter contains, as an active ingredient, a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and which has the effect of promoting the expression of elastin mRNA that produces elastin from the elastin gene.

[0035] The TGF-βR2 mRNA expression promoter contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of TGF-βR2 mRNA, which generates TGF-βR2 from the TGF-β receptor 2 (TGF-βR2) gene.

[0036] The neprilysin mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has an inhibitory effect on the expression of neprilysin mRNA that generates neprilysin from the neprilysin gene, as an active ingredient.

[0037] Preferably, the composition for preventing or improving skin aging signs contains, as active ingredients, all agents selected from an IL-6 mRNA expression inhibitor, an IL-8 mRNA expression inhibitor, an MMP1 mRNA expression inhibitor, a CDKN1A mRNA expression inhibitor, an ATP production promoter, a COL1A1 mRNA expression promoter, a COL3A1 mRNA expression promoter, a HAS2 mRNA expression promoter, an elastin mRNA expression promoter, a TGF-βR2 mRNA expression promoter, and a neprilysin mRNA expression inhibitor.

[0038] Preferably, the composition for preventing or improving skin aging signs further contains a vitamin C derivative. Preferably, in the composition for preventing or improving skin aging signs, the vitamin C derivative is at least one selected from the group consisting of metal salts of ascorbic acid phosphate and trisodium ascorbyl palmitate phosphate.

[0039] Preferably, in the general formula (1) of the composition for preventing or improving skin aging signs, the dicarboxylic acid hemiester residue is bonded via a linear alkylene group having 2 to 4 carbon atoms between the carbonyl groups of the dicarboxylic acid residue. Preferably, in the general formula (1) of the composition for preventing or improving skin aging signs, the dicarboxylic acid diester residue is bonded via a linear alkylene group having 2 to 4 carbon atoms between the carbonyl groups of the dicarboxylic acid residue. In the composition for preventing or improving skin aging signs, R in the above general formula (1) １ and R ２ are each a hydrogen atom, HOOCCH ２ CH ２ CO-, HOOCCH ２ CH ２ CH ２ CO-, HOH ２ C-(CH(OH)) ４ CH ２ NHCH ３ ·HOOCCH ２ CH ２ CO-, CH ３ CH ２ OOCCH ２ CH ２ CO-, and (CH ３ ) ２ CHOOOCCH ２ CH２ At least one selected from CO-, R １ and R ２ At least one of them is HOOCCH ２ CH ２ CO-, HOOCCH ２ CH ２ CH ２ CO-, HOH ２ C-(CH(OH)) ４ CH ２ NHCH ３ HOOCCH ２ CH ２ CO-, CH ３ CH ２ OOCCH ２ CH ２ CO-, and (CH ３ ) ２ CHOOCCCH ２ CH ２ Preferably, it is at least one selected from CO-.

[0040] The topical skin preparation of the present invention contains the composition for preventing or improving skin aging according to the present invention.

[0041] According to the present invention, a composition for preventing or improving skin aging, such as wrinkles and sagging, can be provided, which suppresses the progression of cellular senescence in dermal fibroblasts and suppresses at least one of the decrease, degeneration, and degradation of dermal extracellular matrix (ECM) components associated with skin aging, thereby preventing or improving skin aging symptoms such as wrinkles and sagging, and a topical skin preparation containing the composition for preventing or improving skin aging is provided.

[0042] Furthermore, according to the present invention, by including the ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), the ubiquinol dicarboxylic acid ester derivative itself and its hydrolysis products can be efficiently taken up into adult human dermal fibroblasts.

[0043] Furthermore, according to the present invention, the ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) can suppress the gene expression of p21, which controls the gene expression of senescence-associated secretory phenotype (SASP) factors and SASP (IL-6, IL-8, etc.) expression in adult human dermal fibroblasts, and can suppress the protein production of IL-6 and IL-8, thereby providing a composition for preventing or improving skin signs of aging, such as wrinkles and sagging associated with skin aging.

[0044] Furthermore, according to the present invention, the ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) promotes ATP production in adult human dermal fibroblasts. That is, the ubiquinol dicarboxylic acid hemiester derivative can suppress the progression of cellular senescence in adult human dermal fibroblasts, thereby providing a composition for preventing or improving skin signs such as wrinkles and sagging associated with skin aging.

[0045] Furthermore, according to the present invention, the ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) genetically suppresses the denaturation or degradation of the ECM by suppressing the expression of the MMP1 gene, which is a degrading enzyme for type I collagen and type III collagen. The ubiquinol dicarboxylic acid ester derivative increases the gene expression of type I collagen, type III collagen, elastin, and hyaluronic acid synthase among the ECM components in adult human dermal fibroblasts, and can increase procollagen protein 1α1 (pro COL1A1) and hyaluronic acid, thereby providing a composition for preventing or improving skin signs such as wrinkles and sagging associated with skin aging.

[0046] Furthermore, according to the present invention, the ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) did not alter the gene expression of collagen IV. The ubiquinol dicarboxylic acid ester derivative increases the expression of TGF-β receptor 2 (TGF-βR2) in the TGF-β / Smad signaling pathway involved in the production of ECM components in adult human dermal fibroblasts, and promotes the phosphorylation and activation of Smad3 downstream of the signal. As a result, it is possible to promote the production of ECM components that use the TGF-β / Smad signaling pathway as the production pathway, thereby providing a composition for preventing or improving skin aging, such as wrinkles and sagging associated with skin aging.

[0047] Furthermore, according to the present invention, the ubiquinol dicarboxylic acid ester derivative represented by the general formula (1) can, when used in combination with a specific vitamin C derivative, further promote collagen production of the ubiquinol dicarboxylic acid ester derivative alone, promote extracellular secretion of collagen, and further increase the utilization capacity of ECM components, thereby providing a composition for preventing or improving skin signs such as wrinkles and sagging associated with skin aging.

[0048] Figure 1A shows the results of the uptake evaluation into adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 2, and shows the intracellular concentration of Uq10. Figure 1B shows the results of the uptake evaluation into adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 2, and shows the intracellular concentration of UqH. Figure 1C shows the results of the uptake evaluation into adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 2, and shows the intracellular concentrations of UqH-mono-SUC of compounds 1 to 3. Figure 1D shows the results of the uptake evaluation into adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 2, and shows the intracellular concentration of UqH-mono-GLUT of compound 5. Figure 1E shows the results of the uptake evaluation into adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative in Example 2, and shows the intracellular concentration of UqH-1,4-bis-GLUT of compound 5. Figure 1F shows the results of the uptake evaluation into adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative in Example 2, and shows the intracellular concentration of UqH-mono-GLUT of compound 6. Figure 1G shows the results of the uptake evaluation into adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative in Example 2, and shows the total intracellular concentration. In Figures 1A to 1F, significant differences were determined by One-way ANOVA and Dunnett multiple comparison tests, and are indicated as *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 vs C0ntrol. Figure 2A shows the results of the uptake evaluation of ubiquinol dicarboxylic acid ester derivatives administered to adult human dermal fibroblasts in Example 2, and shows the concentration of Uq10 in the culture medium. Figure 2B shows the results of the uptake evaluation of ubiquinol dicarboxylic acid ester derivatives administered to adult human dermal fibroblasts in Example 2, and shows the concentration of UqH in the culture medium. Figure 2C shows the results of the uptake evaluation of ubiquinol dicarboxylic acid ester derivatives administered to adult human dermal fibroblasts in Example 2, and shows the concentrations of UqH-mono-SUC in the culture medium of compounds 1 to 3.Figure 2D shows the results of the uptake evaluation of adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative in Example 2, and shows the UqH-mono-GLUT concentration of compound 5 in the culture medium. Figure 2E shows the results of the uptake evaluation of adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative in Example 2, and shows the UqH-1,4-bis-GLUT concentration of compound 5 in the culture medium. Figure 2F shows the results of the uptake evaluation of adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative in Example 2, and shows the total concentration in the culture medium. Figure 3A shows the relative expression of IL-6 mRNA in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative in Example 3. Figure 3B shows the expression of IL-6 protein in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative in Example 3. Figure 3C shows the relative expression of IL-8 mRNA in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 3. Figure 3D shows the expression of IL-8 protein in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 3. Figure 3E shows the relative expression of CDKN1A in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 3. Figure 3F shows ATP production in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 3. In Figures 3A to 3F, significant differences were determined by One-way ANOVA and Dunnett multiple comparison tests, and are indicated as *: p < 0.05, **: p < 0.01 vs Control. Figure 4A shows the COL1A1 mRNA expression level in adult human dermal fibroblasts after administration of compound 1 of Example 4. Figure 4B shows the elastin mRNA expression level in adult human dermal fibroblasts after administration of compound 1 of Example 4. Figure 4C shows the HAS2 mRNA expression level in adult human dermal fibroblasts after administration of compound 1 of Example 4. Figure 4D shows the MMP1 mRNA expression level in adult human dermal fibroblasts after administration of compound 1 of Example 4.In Figures 4A to 4D, statistical significance was determined by One-way ANOVA and Dunnett multiple comparison tests, and is indicated as *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 vs Control. Figure 5A shows the COL1A1 mRNA expression level in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 5. Figure 5B shows the COL3A1 mRNA expression level in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 5. Figure 5C shows the elastin mRNA expression level in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 5. Figure 5D shows the HAS2 mRNA expression level in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 5. Figure 5E shows the MMP1 mRNA expression level in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 5. Figure 5F shows the neprilysin (NEP1) mRNA expression level in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 5. Figure 6A shows the COL1A1 mRNA expression level in adult human dermal fibroblasts after administration of compound 5 of Example 6. Figure 6B shows the COL3A1 mRNA expression level in adult human dermal fibroblasts after administration of compound 5 of Example 6. Figure 6C shows the elastin mRNA expression level in adult human dermal fibroblasts after administration of compound 5 of Example 6. Figure 6D shows the HAS2 mRNA expression level in adult human dermal fibroblasts after administration of compound 5 of Example 6. In Figures 6A to 6D, statistical significance was determined by One-way ANOVA and Dunnett multiple comparison tests, and is indicated as *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs. Control. Figure 7A shows immunohistochemical staining (nuclear DAPI staining (blue), COL1A1 staining (green), Merge) of intracellular type I collagen expression in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid ester derivative of Example 7.Figure 7B shows the concentration of proCOL1A1 protein production in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 7. Figure 7C shows the concentration of hyaluronic acid production in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 7. In Figures 7B and 7C, statistical significance was determined by One-way ANOVA and Dunnett multiple comparison tests, and is indicated as *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs Control. Figure 8A shows the expression of TGF-β receptor 2 (TGF-βR2) in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 8. Figure 8B shows the phosphorylation of Smad3 in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 8. Figure 8C shows the TGF-βR2 immunostaining (Merge of nuclear DAPI staining (blue) and TGF-βR2 staining (green)) in adult human dermal fibroblasts after administration of the ubiquinol dicarboxylic acid derivative of Example 8. In Figures 8A and 8B, significant differences were determined by One-way ANOVA and Dunnett multiple comparison tests, and are indicated as *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs. Control. Figure 9A shows the immunostaining of intracellular type I collagen expression in adult human dermal fibroblasts after combined use of the ubiquinol dicarboxylic acid derivative and vitamin C derivative of Example 9. Figure 9B shows the pro COL1A1 concentration in adult human dermal fibroblasts after combined use of the ubiquinol dicarboxylic acid derivative and vitamin C derivative of Example 9. Figure 9C shows the concentration of proCOL1A1 in the culture supernatant of adult human dermal fibroblasts after the combined use of the ubiquinol dicarboxylic acid derivative and vitamin C derivative in Example 9. Figure 9D shows the total amount of proCOL1A1 in adult human dermal fibroblasts and in the supernatant after the combined use of the ubiquinol dicarboxylic acid derivative and vitamin C derivative in Example 9.In Figures 9B to 9D, AA represents ascorbic acid in combination, APS represents sodium ascorbate phosphate in combination, APM represents magnesium ascorbate phosphate in combination, AG represents ascorbic acid 2-glucoside in combination, and APPS represents sodium ascorbyl palmitate phosphate in combination. The concentration of the ubiquinol dicarboxylic acid derivative was 100 μM, the concentrations of AA, APS, APM, and AG were 100 μM, and the concentration of APPS was 10 μM. The proCOL1A1 concentration was measured after 48 hours of incubation. In Figures 9B to 9D, statistical significance was determined by One-way ANOVA and Dunnet multiple comparison tests, and is shown as follows: *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 vs Control (-), ##: p < 0.01, ###: p < 0.001, #####: p < 0.0001 vs Compound 1 (-), ψψ: p < 0.01, ψψψ: p < 0.001, ψψψψ: p < 0.0001 vs Compound 5 (-).

[0049] (Composition for the Prevention or Improvement of Skin Aging) The composition for the prevention or improvement of skin aging of the present invention contains as an active ingredient at least one agent selected from IL-6 mRNA expression inhibitor, IL-8 mRNA expression inhibitor, MMP1 mRNA expression inhibitor, CDKN1A mRNA expression inhibitor, ATP production promoter, COL1A1 mRNA expression promoter, COL3A1 mRNA expression promoter, HAS2 mRNA expression promoter, elastin mRNA expression promoter, TGF-βR2 mRNA expression promoter, and neprilysin mRNA expression inhibitor, and preferably contains a vitamin C derivative, and further contains other components as necessary.

[0050] The present invention's composition for preventing or improving skin aging preferably contains all of the following as active ingredients: IL-6 mRNA expression inhibitor, IL-8 mRNA expression inhibitor, MMP1 mRNA expression inhibitor, CDKN1A mRNA expression inhibitor, ATP production promoter, COL1A1 mRNA expression promoter, COL3A1 mRNA expression promoter, HAS2 mRNA expression promoter, elastin mRNA expression promoter, TGF-βR2 mRNA expression promoter, and neprilysin mRNA expression inhibitor. This is preferable from the viewpoint of preventing or improving skin aging.

[0051] In the present invention, "composition for preventing and / or improving skin aging" is synonymous with "composition for preventing and improving skin aging" and "at least one of the composition for preventing skin aging and the composition for improving skin aging." In the present invention, "skin aging" refers to changes in the skin such as wrinkles and sagging that appear due to aging or the effects of ultraviolet rays, and also includes a decrease in skin firmness and a roughening of skin texture.

[0052] In this invention, by suppressing the spread of cellular senescence in dermal fibroblasts, which play a central role in human skin aging (consisting of physiological aging associated with aging and photoaging due to ultraviolet exposure), and by suppressing at least one of the following—decrease in the production capacity of dermal extracellular matrix (ECM) components, promotion of the expression of degrading enzymes, and reduction, degeneration, and degradation of ECM components—it is possible to prevent or improve skin signs such as wrinkles and sagging associated with human skin aging.

[0053] In particular, the ubiquinol dicarboxylic acid ester derivatives represented by the following general formula (1), contained in IL-6 mRNA expression inhibitors, IL-8 mRNA expression inhibitors, MMP1 mRNA expression inhibitors, CDKN1A mRNA expression inhibitors, ATP production promoters, COL1A1 mRNA expression promoters, COL3A1 mRNA expression promoters, HAS2 mRNA expression promoters, elastin mRNA expression promoters, TGF-βR2 mRNA expression promoters, and neprilysin mRNA expression inhibitors, are efficiently taken up by adult human dermal fibroblasts. Therefore, they can suppress the production of senescence-associated secretory (SASP) factors in dermal fibroblasts, thereby suppressing the spread of cellular senescence in dermal fibroblasts, which play a central role in skin aging, and promoting the production and suppressing the degradation of ECM components in dermal fibroblasts.

[0054] Furthermore, skin signs such as wrinkles and sagging associated with human skin aging significantly affect perceived age and appearance, and particularly contribute to a decline in the quality of life (QOL) of women. Therefore, preventing or improving skin signs can lead to an improvement in QOL.

[0055] <IL-6 mRNA Expression Inhibitor> The IL-6 mRNA expression inhibitor according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1), and has the effect of suppressing the expression of IL-6 mRNA that generates IL-6 from the interleukin-6 (IL-6) gene.

[0056] The interleukin-6 (IL-6) gene is the gene that codes for IL-6, a cytokine that regulates inflammatory responses. Skin aging is a major factor in cellular senescence, and the decrease in extracellular matrix production due to cellular senescence of dermal fibroblasts and the promotion of degeneration (degradation) by collagenase (protease) lead to a decrease in ECM components, causing skin signs of aging such as wrinkles and sagging. In cellular senescence, senescence-associated secretory (SASP) factors are secreted, affecting surrounding cells and tissues and promoting cellular senescence of dermal fibroblasts. Typical SASP factors include inflammatory cytokines such as IL-6 and IL-8. By suppressing IL-6 mRNA expression, it is possible to suppress the production of IL-6 and inhibit cellular senescence of dermal fibroblasts, thereby preventing or improving skin signs of aging such as wrinkles and sagging.

[0057] <Ubiquinol dicarboxylic acid ester derivatives represented by general formula (1)> However, in the above general formula (1), R １ and R ２ Each represents a hydrogen atom, or a substituent selected from dicarboxylic acid hemiester residues and their salts, and dicarboxylic acid double ester residues, R １ and R ２ At least one of these is a dicarboxylic acid hemiester residue and / or a dicarboxylic acid double ester residue. n represents an integer from 1 to 10.

[0058] The ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) is efficiently taken up by adult human dermal fibroblasts and can suppress the production of SASP factors and promote ATP production in adult human dermal fibroblasts. The ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) can be used as an active ingredient to suppress the spread of cellular senescence in dermal fibroblasts.

[0059] The ubiquinol dicarboxylic acid hemiester derivative represented by the above general formula (1) can promote the production and suppress the degradation of ECM components (collagen, hyaluronic acid, elastin, etc.) in adult human dermal fibroblasts, and thus can help prevent or improve signs of aging associated with skin aging (wrinkles, sagging, etc.).

[0060] The ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) may be included in a formulation alone or in a mixture, or it may be incorporated into the formulation as a salt or counterion mixture thereof.

[0061] In the general formula (1) above, the dicarboxylic acid hemiester residue is selected from dicarboxylic acid hemiester residues and their alkali metal salts and organic base salts. The carbonyl groups of the dicarboxylic acid residue are linked by a linear alkylene group having 2 to 4 carbon atoms. The alkylene group is preferably an ethylene group or a propylene group. The alkali metal salt is preferably a sodium salt or a potassium salt. The organic base salt is preferably a meglumine salt, a L-lysine salt, or a tromethamine salt.

[0062] The counterions of the counterion mixture of ubiquinol carboxylic acid hemiester having a dicarboxylic acid hemiester residue are preferably meglumine, tromethamine, and L-lysine. The molar ratio of the ubiquinol dicarboxylic acid hemiester to the counterion mixture is preferably 1:2 to 1:10.

[0063] In the general formula (1) above, the dicarboxylic acid double ester residues are bonded between the carbonyl groups of the dicarboxylic acid by a linear alkylene group having 2 to 4 carbon atoms. The alkylene group is preferably an ethylene group or a propylene group. The ester is preferably, for example, an ethyl ester or an isopropyl ester.

[0064] R in the above general formula (1) １ and R ２ are at least one selected from a hydrogen atom, HOOCCH ２ CH ２ CO-, HOOCCH ２ CH ２ CH ２ CO-, HOH ２ C-(CH(OH)) ４ CH ２ NHCH ３ ·HOOCCH ２ CH ２ CO-, CH ３ CH ２ OOCCH ２ CH ２ CO-, and (CH ３ ) ２ CHOOOCCH ２ CH ２ CO-, and it is preferable that at least one of R １ and R ２ is at least one selected from HOOCCH ２ CH ２ CO-, HOOCCH ２ CH ２ CH ２ CO-, HOH ２ C-(CH(OH)) ４ CH ２ NHCH ３ ·HOOCCH ２ CH ２ CO-, CH ３ CH ２ OOCCH ２ CH ２ CO-, and (CH ３ ) ２ CHOOOCCH ２ CH ２ CO-.

[0065] <Method for Producing Ubiquinol Dicarboxylic Acid Ester Derivative> Various methods for producing the ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) are conceivable, and a typical method is as shown in the following reaction formula.

[0066] However, in the above reaction equation, R １ and R ２ This has the same meaning as the general formula (1) above, where n is an integer from 1 to 10.

[0067] The ubiquinone represented by the above general formula (2) is reduced with a reducing agent to obtain ubiquinol represented by the above general formula (3), and this ubiquinol is reacted with a dicarboxylic acid anhydride in the presence of a catalyst to obtain a dicarboxylic acid hemiester. The ubiquinone represented by the above general formula (2), zinc powder, acetic acid, and acid anhydride are reacted by a conventional method to obtain ubiquinol dicarboxylic acid hemiester. This ubiquinol dicarboxylic acid hemiester is esterified with an alcohol by a conventional method to obtain ubiquinol dicarboxylic acid alcohol ester (double ester).

[0068] Examples of reducing agents used here include sodium borohydride, sodium hydrosulfite, tri-n-butylphosphine, zinc chloride, and stannous chloride.

[0069] Ubiquinol dicarboxylic acid hemiesters are obtained by reacting ubiquinol with a dicarboxylic acid anhydride and N,N-dimethylpyridine as catalysts in pyridine anhydride from which dissolved oxygen has been removed with argon gas. Ubiquinol dicarboxylic acid hemiesters are also produced by heating ubiquinone, zinc, and dicarboxylic acid anhydride. Reaction in acetic acid and sodium acetate yields preferred results.

[0070] Ubiquinol dicarboxylic acid double esters are produced by esterifying synthesized ubiquinol dicarboxylic acid hemiesters using conventional methods.

[0071] <IL-8 mRNA Expression Inhibitor> The IL-8 mRNA expression inhibitor according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of suppressing the expression of IL-8 mRNA that generates IL-8 from the interleukin-8 (IL-8) gene.

[0072] The interleukin-8 (IL-8) gene, also known as the CXCL8 gene, encodes the human IL-8 protein. IL-8 is a type of chemokine secreted by various cells in response to inflammatory stimuli. IL-8 mRNA expression inhibitors can suppress IL-8 production, thereby inhibiting cellular senescence in dermal fibroblasts and preventing or improving skin aging symptoms such as wrinkles and sagging.

[0073] <MMP1 mRA Expression Inhibitor> The MMP1 mRA expression inhibitor according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of suppressing the expression of MMP1 mRNA that generates MMP1 from the matrix metalloproteinase 1 (MMP1) gene.

[0074] The MMP1 gene encodes MMP1, an enzyme that breaks down dermal extracellular matrix (ECM) components (collagen, hyaluronic acid, elastin, etc.). MMP1 mRA expression inhibitors can suppress the breakdown of ECM components by inhibiting the expression of the MMP1 gene, thereby preventing or improving skin aging signs such as wrinkles and sagging.

[0075] <CDKN1A mRNA Expression Inhibitor> The CDKN1A mRNA expression inhibitor according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of suppressing the expression of CDKN1A mRNA that generates CDKN1A from the CDKN1A gene.

[0076] CDKN1A encodes a potent cyclin-dependent kinase inhibitor that binds to and inhibits the activity of cyclin-cyclin-dependent kinase 2 or cyclin-cyclin-dependent kinase 4 complexes, thereby functioning as a regulator of G1 phase cell cycle progression. CDKN1A is one of the major regulators of aging and is an aging marker that regulates SASP factor expression. CDKN1A mRNA expression inhibitors can suppress CDKN1A production, thereby inhibiting cellular senescence in dermal fibroblasts and preventing or improving skin aging symptoms such as wrinkles and sagging.

[0077] <ATP Production Promoter> The ATP production promoter according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has an ATP production promoting effect in adult human dermal fibroblasts.

[0078] ATP production promoters can suppress the progression of cellular senescence in adult human dermal fibroblasts by promoting ATP production, thereby preventing or improving skin aging symptoms such as wrinkles and sagging.

[0079] <COL1A1 mRNA Expression Promoter> The COL1A1 mRNA expression promoter of the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of COL1A1 mRNA that generates COL1A1 from the type I procollagen α1 (COL1A1) gene.

[0080] The COL1A1 gene encodes the pro-α1 chain of type I collagen, which has a triple helix consisting of two α1 chains and one α2 chain. Type I collagen is the fibrillogenic collagen found in most connective tissues and is abundant in bone, cornea, dermis, and tendons. By promoting the production of type I collagen, an ECM component, in human dermal fibroblasts using a COL1A1 mRNA expression promoter, it is possible to prevent or improve skin aging signs such as wrinkles and sagging associated with human skin aging.

[0081] <COL3A1 mRNA Expression Promoter> The COL3A1 mRNA expression promoter according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of COL3A1 mRNA that generates COL3A1 from the type III procollagen α1 (COL3A1) gene.

[0082] By promoting the production of type III collagen, an ECM component, in human dermal fibroblasts using a COL3A1 mRNA expression promoter, it is possible to prevent or improve skin aging signs such as wrinkles and sagging associated with human skin aging.

[0083] <HAS2 mRNA Expression Promoter> The HAS2 mRNA expression promoter according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of HAS2 mRNA that generates HAS2 from the hyaluronic acid synthase (HAS2) gene.

[0084] Hyaluronic acid is a non-sulfated glycosaminoglycan composed of a disaccharide repeating structure of α-glucuronic acid (GlcA) and N-acetyl-α-glucosamine (GlcNAc). Hyaluronic acid is a major component of the extracellular matrix (ECM) in human dermal fibroblasts. By promoting the production of hyaluronic acid, an ECM component in human dermal fibroblasts, using HAS2 mRNA expression promoters, it is possible to prevent or improve skin aging signs such as wrinkles and sagging associated with skin aging.

[0085] <Elastin mRNA Expression Promoter> The elastin mRNA expression promoter according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of elastin mRNA that produces elastin from the elastin gene.

[0086] Elastin is a protein that is a component of elastic fibers and is produced by dermal fibroblasts. Elastic fibers are fibers that support collagen fibers. By promoting the production of elastin, an ECM component, in human dermal fibroblasts using elastin mRNA expression promoters, it is possible to prevent or improve skin signs associated with human skin aging, such as wrinkles and sagging.

[0087] <TGF-βR2 mRNA Expression Promoter> The TGF-βR2 mRNA expression promoter according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of promoting the expression of TGF-βR2 mRNA that generates TGF-βR2 from the TGF-β receptor 2 (TGF-βR2) gene.

[0088] TGF-β receptor 2 is a receptor that binds to transforming growth factor (TGF-β). TGF-β is a type of cytokine that regulates cell proliferation, differentiation, and apoptosis (programmed cell death), and promotes the transformation of fibroblasts. TGF-βR2 mRNA expression promoters increase TGF-βR2 in the TGF-β / Smad signaling pathway and activate Smad3, thereby promoting the production of ECM components in human dermal fibroblasts, and can prevent or improve skin signs associated with human skin aging, such as wrinkles and sagging.

[0089] <Neprilysin (NEP1) mRNA Expression Inhibitor> The neprilysin (NEP1) mRNA expression inhibitor according to the present invention contains a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), and has the effect of suppressing the expression of neprilysin mRNA that generates neprilysin from the neprilysin gene.

[0090] Neprilysin is a membrane-bound elastase (protein-degrading enzyme) present in dermal fibroblasts of the skin that breaks down elastin fibers, which are the cause of wrinkles. It has been reported that neprilysin expression is enhanced by interleukin-1α (IL-1α) due to ultraviolet rays such as UVB, which reduces skin elasticity (https: / / prtimes.jp / main / html / rd / p / 000000031.000043001.html, press release from Toujours Co., Ltd., accessed February 26, 2026). By suppressing the production of neprilysin, which breaks down elastin fibers, neprilysin mRNA expression inhibitors can prevent or improve skin signs associated with human skin aging, such as wrinkles and sagging.

[0091] <Vitamin C Derivatives> It is preferable that each agent in the composition for preventing or improving skin aging of the present invention further contains a vitamin C derivative. The ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1), when used in combination with a specific vitamin C derivative, can further promote collagen production by the ubiquinol dicarboxylic acid hemiester derivative alone in adult human dermal fibroblasts, promote the secretion of collagen outside the fibroblasts, and enhance its utilization as an ECM component. The vitamin C derivative is preferably at least one selected from, for example, metal salts of ascorbic acid phosphate (sodium, magnesium) and trisodium ascorbyl palmitate phosphate.

[0092] The present invention's composition for preventing or improving skin aging suppresses the progression of cellular senescence in dermal fibroblasts and inhibits at least one of the decrease, degeneration, and degradation of dermal extracellular matrix (ECM) components associated with skin aging. Therefore, it can prevent or improve skin signs such as wrinkles and sagging associated with skin aging, and is therefore suitably used in the topical skin preparations described below.

[0093] (Topical Skin Preparation) The topical skin preparation of the present invention contains the composition for preventing or improving skin aging of the present invention, and further contains other components as necessary. The content of the composition for preventing or improving skin aging of the present invention is preferably 0.01% to 1% by mass, and more preferably 0.1% to 0.5% by mass, based on the total amount of the topical skin preparation. Within this range, the composition for preventing or improving skin aging of the present invention can be stably incorporated, and an excellent effect of preventing or improving skin signs of aging can be exhibited.

[0094] Other ingredients include various additives commonly used in the formulation of topical skin preparations or topical pharmaceuticals. Examples of additives include water (purified water, hot spring water, deep sea water, etc.), alcohol, oils, surfactants, metal soaps, gelling agents, powders, alcohols, water-soluble polymers, film-forming agents, resins, UV protection agents, inclusion compounds, antibacterial agents, fragrances, deodorants, salts, pH adjusters, cooling agents, animal or microbial extracts, plant extracts, blood circulation promoters, astringents, anti-seborrheic agents, whitening agents, anti-inflammatory agents, free radical scavengers, cell activators, moisturizers, chelating agents, keratolytic agents, enzymes, hormones, vitamins, and the like. The preparation of topical skin preparations can be carried out according to conventional methods, and the amounts of the additives can also be determined according to conventional methods within a range that does not impair the effects of the present invention.

[0095] The form of the topical skin preparation of the present invention is not particularly limited, and examples include forms belonging to skin cosmetics such as lotions, creams, toners, serums, packs, facial cleansers, and makeup cosmetics; forms related to hair cosmetics such as shampoos, hair treatments, hair styling agents, hair tonics, and hair growth agents; and forms of topical pharmaceuticals such as dispersions, ointments, aerosols, patches, and poultices.

[0096] The following describes embodiments of the present invention, but the present invention is not limited in any way to these embodiments.

[0097] To demonstrate that the ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) (hereinafter sometimes referred to as "the compound of the present invention") is efficiently taken up by adult human dermal fibroblasts, examples are given in which it was evaluated by comparison with oxidized ubiquinone 10 (Uq10) and reduced ubiquinol (UqH).

[0098] To demonstrate that the compound of the present invention has the effect of suppressing cellular senescence in adult human dermal fibroblasts, examples are given in which the inhibitory effect on the production of senescence-related secretory phenomena (SASP) factors and the effect on ATP production were evaluated by comparison with Uq10 or UqH.

[0099] To demonstrate that the compound of the present invention is effective in gene expression related to the extracellular matrix (ECM) in adult human dermal fibroblasts, examples are given in which the compound was evaluated by comparison with Uq10 and / or UqH.

[0100] Examples illustrating that the compound of the present invention promotes the production of ECM components in adult human dermal fibroblasts are provided.

[0101] Examples illustrating the TGF-β / Smad signaling pathway involved in the production of ECM components in adult human dermal fibroblasts are provided.

[0102] Examples are provided to demonstrate that the compound of the present invention, when used in combination with a vitamin C derivative, further increases the production of ECM components in adult human dermal fibroblasts, and promotes the extracellular migration of ECM components, thereby increasing their utilization.

[0103] (Example 1) Compounds 1 to 9 of the present invention (ubiquinol dicarboxylic acid ester derivatives represented by the above general formula (1)) shown in Table 1 were produced by the methods shown in Production Methods A to E below. Next, the mass spectra (ionization method; FD method and FAB method) and 1H-NMR spectra of the obtained compounds are shown in Table 2.

[0104] <Manufacturing Method A> Dissolve 10 g (1.16 mmol) of ubiquinone in 150 ml of ethyl acetate, add 2.8 mmol of sodium borohydride suspended in 15 ml of methanol, and stir at room temperature until the yellow solution becomes colorless. Add 100 ml of distilled water saturated with argon gas to the reaction mixture and wash the ethyl acetate layer. After liquid-liquid separation, dehydrate the isopropyl ether layer with anhydrous sodium sulfate and remove the solvent under reduced pressure to obtain ubiquinol. Add anhydrous dicarboxylic acid, 4-N,N-dimethylaminopyridine hydrochloride, and anhydrous pyridine 75 ml to the ubiquinol, replace the atmosphere with argon gas, and stir at room temperature for 24 hours. Remove the solvent under reduced pressure, suspend the residue in ethyl acetate, and after liquid-liquid separation of the ethyl acetate layer with distilled water, aqueous sodium bicarbonate solution, and 0.1 N aqueous hydrochloric acid solution, dehydrate the ethyl acetate layer with anhydrous sodium sulfate and concentrate under reduced pressure. The residue was separated and purified by silica gel flash chromatography (eluent: n-hexane:ethyl acetate) to obtain a mixture of ubiquinol-1(4)-mono-dicarboxylic acid hemiesters and ubiquinol-1,4-bis-dicarboxylic acid hemiester.

[0105] <Manufacturing Method B> 1.16 mmol of ubiquinone, 8.26 mmol of zinc, 20.0 mmol of anhydrous dicarboxylic acid, 7.31 mmol of sodium acetate, and 66.6 mmol of acetic acid are refluxed at 85°C for 3 hours. After cooling to room temperature, 200 ml of ethyl acetate and 100 ml of purified water are added to the resulting white solid to extract the ethyl acetate-soluble fraction (100 ml x 3 times). The extract is dehydrated with anhydrous sodium sulfate, the solvent is removed under reduced pressure, and the residue is separated and purified by silica gel flash chromatography (eluent: n-hexane:ethyl acetate) to obtain a mixture of ubiquinol-1(4)-mono-dicarboxylic acid hemiesters and ubiquinol-1,4-bis-dicarboxylic acid hemiester.

[0106] <Manufacturing Method C> The ubiquinol-1(4)-mono-dicarboxylic acid hemiester mixture separated in Manufacturing Methods A and B is separated and purified by preparative high-performance liquid chromatography (column) to obtain ubiquinol-1-mono-dicarboxylic acid hemiester and ubiquinol-4-mono-dicarboxylic acid hemiester.

[0107] <Manufacturing Method D> Dissolve 2 mmol of ubiquinol-1,4-bis-dicarboxylic acid hemiester in 2 mol of 1N aqueous sodium hydroxide solution or 2 mol of aqueous organic base (meglumine, L-lysine, or tromethamine), and freeze-dry. Recrystallize with methanol-acetonitrile to obtain ubiquinol-1,4-bis-dicarboxylic acid hemiester sodium salt, ubiquinol-1,4-bis-dicarboxylic acid hemiester alkali metal salt, or organic base salt.

[0108] <Manufacturing Method E> Dissolve ubiquinol-1,4-bis-dicarboxylic acid hemiester in a primary alcohol, add hydrochloric acid, and stir at room temperature. Remove the solvent under reduced pressure to obtain ubiquinol-1,4-bis-dicarboxylic acid alcohol ester.

[0109] Table 1 summarizes the chemical formulas of compounds 1 to 9 and their corresponding manufacturing methods. For compounds 1 to 6 and compounds 8 to 9, mass spectrometry (m / z, FD-MS or FAB-MS) and nuclear magnetic resonance spectroscopy ( １ Table 2 shows the H-NMR, δ (ppm, internal standard TMS) results.

[0110]

[0111]

[0112] (Example 2) <Evaluation of uptake by adult human dermal fibroblasts after administration of ubiquinol dicarboxylic acid derivatives> The uptake of ubiquinol dicarboxylic acid derivatives by adult human dermal fibroblasts was evaluated in comparison with oxidized ubiquinone 10 (Uq10) and reduced ubiquinol-10 (UqH).

[0113] Adult human fibroblasts were seeded in a 6-well plate (2 x 10⁻¹⁰ ５Cells / well cells were cultured for 48 hours in 10% FBS DMEM medium. Then, the medium was replaced with 0.1% FBS DMEM medium containing 0.1% polyoxyethylene (40) hydrogenated castor oil (manufactured by Nikko Chemicals Co., Ltd.), 0.3% ethanol (manufactured by Fujifilm Wako Pure Chemical Industries Ltd.), and 0.05% glycerin (manufactured by Fujifilm Wako Pure Chemical Industries Ltd.), to which compounds 1, 2, 3, 5, 6, Uq10, and UqH were added, and the cells were cultured for another 48 hours. After harvesting the medium and cells and performing extraction, compounds 1, 2, 3, 5, 6, Uq10, and UqH were measured by LC / MS / MS under the following measurement conditions.

[0114] Uq10, UqH, compound 1, compound 2, compound 3, compound 5, and compound 6 were administered at a concentration of 100 μM, and the intracellular and culture medium drug levels after 48 hours are shown in Figures 1A to 1G and 2A to 2F, respectively. The uptake levels of Uq10 (see Figure 1A), UqH (see Figure 1B), compound 1, compound 2, compound 3, compound 5, and compound 6 themselves (see Figures 1C, 1D, 1E, and 1F) are shown (for the compound 1 administration group, only monoesters were detected in the cells after 48 hours, so the sum of monoesters is shown; for the compound 5 administration group, the sum of monoesters and bisesters are shown). Total uptake was calculated as the sum of the intracellular Uq10, UqH, and compound levels that increased after administration (see Figure 1G).

[0115] Figures 2A to 2F show the drug concentrations in the culture medium. When total uptake is shown as a Uq10 dose ratio, the doses of Uq10, UqH, compound 1, compound 2, compound 3, compound 5, and compound 6 result in 1.0 times, 1.1 times, 45.6 times, 31.4 times, 51.6 times, 58.9 times, and 28.4 times, respectively. When the sum of intracellular Uq10, UqH, and monoester is shown as a Uq10 dose ratio, the doses of Uq10, UqH, compound 1, compound 2, and compound 6 result in 1.0 times, 1.1 times, 45.6 times, 31.4 times, 51.6 times, 58.9 times, and 28.4 times, respectively. 3. Administration of compound 5 and compound 6 resulted in increases of 1.0, 1.1, 45.6, 31.4, 51.6, 7.9, and 28.4 times, respectively. When the sum of intracellular Uq10 and UqH is expressed as the Uq10 administration ratio, the increases were 1.0, 1.0, 3.0, 2.7, 5.1, 0.4, and 0.4 times for administration of Uq10, UqH, compound 1, compound 2, compound 3, compound 5, and compound 6.

[0116] From these results, it is clear that compounds 1, 2, 3, 5, and 6 are better taken up by adult human dermal fibroblasts compared to Uq10 administration and UqH administration.

[0117] [LC / MS / MS Measurement Conditions] Mass spectrometer: LCMS-8050 LIQUID CHROMATOGRAPH MASS SPECTROMETER (manufactured by Shimadzu Corporation) was used. High-performance liquid chromatography (HPLC) system: Shimadzu HPLC System [System controller (CBM-20A), Pump (LD-20AD), Degasser (DGU-20As), UV detector (SPD-20A), Auto injector (SIL-20AC HT)] was used. Column: Shim-pack XR-C8 3.0 × 75 mm, particle size 2.2 μm (manufactured by Shimadzu Corporation) was used. The mobile phase consisted of a methanol solution of 10 mM ammonium acetate and 0.1% acetic acid, and an ethanol solution of 10 mM ammonium acetate and 0.1% acetic acid, used in gradient mode. The flow rate was 0.2 mL / min, the sample cooler was set to 4°C, and the column oven to 40°C. Ionization was performed using the Electrospray Ionization (ESI) method, measured in MRM mode.

[0118] (Example 3) <Effects of Ubiquinol Dicarboxylic Acid Ester Derivatives on the Generation of Senescence-Associated Secretory Phenomena (SASP) Factors and ATP Production in Adult Human Dermal Fibroblasts> Cellular senescence is the main factor in skin aging, and the decrease in the generation of extracellular matrix (ECM) components due to cellular senescence of dermal fibroblasts and the promotion of degeneration (degradation) by collagenase (protease) lead to a decrease in ECM components, causing skin signs of aging such as wrinkles and sagging. In cellular senescence, SASP factors are secreted, affecting surrounding cells and tissues and promoting aging. Typical SASP factors are inflammatory cytokines such as IL-6 and IL-8, and matrix metalloproteinase 1 (MMP1), which degrades collagen and elastin, is also a SASP factor, so suppressing cellular senescence of dermal fibroblasts is important for maintaining their function. CDKN1A is one of the major regulators of aging and is an aging marker that regulates SASP factor expression.

[0119] Therefore, the effect of the compound of the present invention (a ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1)) on SASP factor production in adult human dermal fibroblasts was evaluated by its effect on the expression of IL-6, IL-8, MMP1 mRNA and CDKN1A, and their protein expression. Furthermore, since mitochondrial function is known to decline in cellular senescence, the effect of the compound of the present invention on ATP production in adult human dermal fibroblasts was investigated.

[0120] Normal human dermal fibroblasts (adult) (Promocell, Heidelberg, Germany) were seeded in a 6-well plate (2 x 10⁻¹⁰ ５ Cells / Well) FBS 10% DMEM medium was incubated for 48 hours, then the medium was replaced with FBS 0.1% DMEM medium containing 0.1% polyoxyethylene (40) hydrogenated castor oil (Nikko Chemicals Co., Ltd.), 0.3% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.05% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.), to which each compound was added, and incubated for another 48 hours. The concentrations of Uq10, UqH, compound 1, and compound 5 were set to 30 μM and 100 μM, respectively.

[0121] After 48 hours, cell samples were washed with cold phosphate-buffered saline (PBS), lysed with lysis / binding buffer, and total RNA was extracted using an RNA extraction kit (High Pure RNA Isolation Kit, Roche, Basel, Switzerland). 250 ng of RNA was reverse transcribed into cDNA using the Rever Tra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan). 1 μL of cDNA template, a primer set for the target gene, and Light Cycle 480 SYBR Green Master (Roche) were mixed, and mRNA expression was measured using the Light Cycle 480 system (Roche).

[0122] Standard PCR conditions consisted of one cycle at 95°C (5 minutes, enzyme activation), 45 cycles at 95°C (10 seconds, thermal denaturation), 55°C (10 seconds, annealing), and 72°C (10 seconds, extension reaction), and one cycle at 50°C (10 seconds, cooling). mRNA expression levels were determined by dividing by the amount of Glucuronidase Beta (GUS) in each sample. The primer sets for the target gene are shown in Table 3.

[0123] Normal human dermal fibroblasts (adult) (Promocell, Heidelberg, Germany) were placed in a 6-well plate (2.0 × 10⁶). ５ Cells / well seeds were seeded and cultured for 48 hours in a 10% FBS-containing medium. Then, the medium was replaced with a DMEM medium containing 0.1% FBS with 0.1% polyoxyethylene (40) hydrogenated castor oil (Nikko Chemicals Co., Ltd.), 0.3% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.05% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) to which each compound was added, and cultured for another 48 hours. The concentrations of Uq10, UqH, compound 1, and compound 5 were set to 30 μM and 100 μM, respectively. The expression levels of IL-6 and IL-8 proteins in the supernatant were measured using an ELISA kit (R&D Systems Inc., USA & Canada).

[0124] The primary antibody prepared with reagent diluent was placed in a 96-well plate and left overnight at room temperature. Thereafter, each well was washed three times with a washing solution, and then a reagent diluent was added. After standing at room temperature for 1 hour, the wells were washed three times again, and the sample supernatant was added. After further leaving the plate at room temperature for 2 hours, the plate was washed three times, the secondary antibody prepared with reagent diluent was added, and the plate was left at room temperature for 2 hours. Thereafter, the plate was washed three times again, avidin-HRP was added, and the plate was left for 20 minutes at room temperature protected from light. Finally, the plate was washed three times, the prepared HRP chromogenic substrate for ELISA was added, and the plate was left for 20 minutes at room temperature protected from light. After standing, a reaction stop solution was added to terminate the reaction, and the absorbance was measured at a wavelength of 450 nm.

[0125] Normal human dermal fibroblasts (adult) (Promocell, Heidelber, Germany) were seeded in a 96-well plate at (1.0×10 ４ cells / well), cultured in a medium containing 10% FBS for 48 hours, then the culture medium was replaced with a 0.1% FBS DMEM medium containing 0.1% polyoxyethylene (40) hydrogenated castor oil (manufactured by Nikko Chemicals Co., Ltd.), 0.3% ethanol (manufactured by Fujifilm Wako Pure Chemical Corporation), and 0.05% glycerin (manufactured by Fujifilm Wako Pure Chemical Corporation), added with each test compound, and cultured for 48 hours. The concentrations of Uq10, UqH, Compound 1 and Compound 5 were set to 10 μM, 30 μM and 100 μM. 100 μL of CellTiter-Glo 2.0 Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) was added to the cell culture plate, shaken for 2 minutes, and then allowed to stand for 10 minutes. Luminescence was measured with a Veritas Microplate Luminometer (Turner Biosystems), and the ATP amount was calculated as the luminescence amount relative to the control.

[0126] Compounds 1 and 5 dose-dependently suppressed the gene expression of IL-6 and IL-8, representative SASP factors (see Figures 3A and 3C), and also dose-dependently suppressed their protein expression (see Figures 3B and 3D). Compound 1 also significantly suppressed the expression of the p21 gene (CDKN1A), which halts the cell cycle in cellular senescence (see Figure 3E). Furthermore, as shown in Example 4 described later, compound 1 dose-dependently suppressed the gene expression of MMP1 (see Figure 4D).

[0127] The compounds of the present invention (ubiquinol dicarboxylic acid ester derivatives represented by the above general formula (1)) suppress the production of interleukins (IL-6, IL-8), which are SASP factors, and the expression of the matrix metalloproteinase 1 (MMP1) gene, as well as the expression of the aging marker CDKN1A, in adult human dermal fibroblasts, thus suppressing cellular senescence. Furthermore, compounds 1 and 5 were shown to significantly promote ATP production in adult human dermal fibroblasts independently of cell proliferation (see Figure 3F), thereby suppressing functional decline due to cellular senescence. Therefore, it is clear that compounds 1 and 5 are effective in suppressing the progression of cellular senescence in adult human dermal fibroblasts and in preventing or improving skin signs such as wrinkles and sagging associated with skin aging.

[0128] (Example 4) <Dose-dependent effect of ubiquinol dicarboxylic acid derivative on gene expression related to the extracellular matrix in adult human dermal fibroblasts> The effect of compound 1 on gene expression related to the generation and degeneration (degradation) of the extracellular matrix was investigated at doses of 10 μM, 30 μM, and 100 μM.

[0129] Normal human dermal fibroblasts (adult) (Promocell, Heidelberg, Germany) were seeded in a 6-well plate (2 x 10⁻¹⁰ ５Cells / Well) FBS 10% DMEM medium was incubated for 48 hours, then the medium was replaced with a DMEM medium containing compound 1 added to FBS 0.1% containing 0.1% polyoxyethylene (40) hydrogenated castor oil (manufactured by Nikko Chemicals Co., Ltd.), 0.3% ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.05% glycerin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and incubated for another 48 hours. The concentrations of compound 1 were 10 μM, 30 μM, and 100 μM.

[0130] After 48 hours, cell samples were washed with cold phosphate-buffered saline (PBS), lysed with lysis / binding buffer, and total RNA was extracted using an RNA extraction kit (High Pure RNA Isolation Kit, Roche, Basel, Switzerland). 250 ng of RNA was reverse transcribed into cDNA using the River Tra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan). 1 μL of cDNA template and a primer set for the target gene were mixed, and mRNA expression was measured using the Light Cycle 480 system (Roche).

[0131] Standard PCR conditions consisted of one cycle at 95°C (5 minutes, enzyme activation), 45 cycles at 95°C (10 seconds, thermal denaturation), 55°C (10 seconds, annealing), and 72°C (10 seconds, extension reaction), and one cycle at 50°C (10 seconds, cooling). mRNA expression levels were determined by dividing by the amount of Glucuronidase Beta (GUS) in each sample. The primer sets for the target gene are shown in Table 3.

[0132] Figures 4A to 4D show the gene expression levels of type I procollagen α1 (COL1A1), hyaluronic acid synthase (HAS2), elastin, and MMP1 after administration of compound 1. Administration of compound 1 significantly and dose-dependently increased the expression levels of COL1A1 mRNA above 10 μM (see Figure 4A), elastin mRNA above 30 μM (see Figure 4B), and hyaluronic acid synthase (HAS2) mRNA above 10 μM (see Figure 4C). MMP1 mRNA expression significantly and dose-dependently decreased above 10 μM (see Figure 4D).

[0133] Therefore, it was revealed at the genetic level that compound 1 dose-dependently promotes the production of ECM components (type I collagen, elastin, and hyaluronic acid) in adult human dermal fibroblasts, reduces MMP1 mRNA expression, and suppresses the denaturation (degradation) of ECM components.

[0134] - Primer Set -

[0135] (Example 5) <Comparison of the effects of ubiquinol dicarboxylic acid derivatives on gene expression of extracellular matrix (ECM) components in adult human dermal fibroblasts, Uq10, and UqH> The drug concentration of compound 1 was fixed at 100 μM, which showed a clear effect on mRNA expression of ECM components in adult human dermal fibroblasts. The effects of the ubiquinol dicarboxylic acid derivatives on the gene expression of type I, type III, and type IV procollagen 1α (COL1A1, COL3A1, COL4A1), elastin, hyaluronic acid synthase (HAS2), MMP1, and neprilysin (NEP1) were compared with the effects of Uq10 and UqH.

[0136] Compounds 1, 2, and 3 significantly increased the expression of COL1A1 mRNA (Figure 5A), COL3A1 mRNA (Figure 5B), Elastin mRNA (Figure 5C), and HAS2 mRNA (Figure 5D). Uq10 showed no effect on any gene expression. UqH significantly increased COL1A1 mRNA, but the effect was small. COL4A1 mRNA expression remained unchanged for all tested compounds: 1, 2, 3, Uq10, and UqH. Although Uq10 has been reported to increase the gene expression of type I, type IV, and type VII collagen (see Non-Patent Documents 9 and 10), no increase in type IV collagen gene expression was observed under the conditions of this study.

[0137] MMP1 mRNA expression was significantly reduced by administration of compound 1, compound 2, and compound 3, but no significant change was observed with UqH and Uq10 administration (see Figure 5E). Despite the lower sum of intracellular Uq10 and UqH levels with compound 5 administration compared to Uq10 and UqH administration (Example 2, Figure 1A, Figure 1B), an effect was observed. This is likely due to the higher intracellular amount of monoester compared to Uq10 and UqH administration, and the fact that the monoester of compound 5 has a semiquinone structure and is thought to have antioxidant properties, suggesting that the monoester contributes to the effect of compound 5 administration. Conflicting reports exist regarding Uq10's effect on MMP1 expression, with some reporting a reduction (see Non-Patent Literature 9) and others showing no effect (see Non-Patent Literature 10), but no effect was observed with Uq10 under the conditions of this study. Gene expression of the elastase neprilysin (NEP1) was significantly reduced by compound 1 and compound 5 (see Figure 5F). No significant decrease was observed with Uq10 administration or UqH administration. This was inconsistent with reports that UqH reduces neprilysin gene expression (see Non-Patent Document 11).

[0138] Therefore, it was revealed at the genetic level that compounds 1, 2, 3, and 5, which are ubiquinol dicarboxylic acid ester derivatives with superior cellular uptake compared to Uq10 and UqH, promote the production of ECM components (type I collagen, type III collagen, elastin, and hyaluronic acid) in adult human dermal fibroblasts, reduce MMP1, and suppress the denaturation (degradation) of ECM components.

[0139] (Example 6) <Effect of Compound 5 on the expression of extracellular matrix-related genes in adult human dermal fibroblasts> The effect of Compound 5 on the expression of extracellular matrix-related genes in adult human dermal fibroblasts was evaluated by comparison with Uq10. The evaluation was performed according to the same method as in Example 5.

[0140] Administration of compound 5 (100 μM) significantly increased the expression of COL1A1 mRNA (Figure 6A), COL3A1 mRNA (Figure 6B), Elastin mRNA (Figure 6C), and HAS2 mRNA (Figure 6D). It did not show a significant effect on the expression of COL4A1 mRNA or MMP1 mRNA.

[0141] Therefore, it was revealed at the genetic level that the administration of compound 5, which showed a higher total uptake of Uq10, UqH, and monoester into adult human dermal fibroblasts compared to Uq10 administration, promotes the production of ECM components (type I collagen, type III collagen, elastin, and hyaluronic acid).

[0142] (Example 7) <Effects of ubiquinol dicarboxylic acid derivatives on the production of extracellular matrix (ECM) components in adult human dermal fibroblasts> To demonstrate that ubiquinol dicarboxylic acid derivatives can promote the production of ECM components by promoting mRNA expression of ECM components in adult human dermal fibroblasts, the effects of ubiquinol dicarboxylic acid derivatives represented by general formula (1) on the production of type I procollagen a1 protein (pro COL1A1) and hyaluronic acid were evaluated in comparison with Uq10.

[0143] Normal human dermal fibroblasts (adult) (Promocell, Heidelberg, Germany) were placed in a 24-well plate (1.0 × 10⁶). ５Cells were seeded (cells / well) and cultured for 48 hours in a medium containing 10% FBS. Then, the medium was replaced with a DMEM medium containing 0.1% FBS with 0.1% polyoxyethylene (40) hydrogenated castor oil (Nikko Chemicals Co., Ltd.), 0.3% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.05% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) to which each compound was added, and cultured for another 48 hours. The doses of Uq10, UqH, compound 1, and compound 5 were 100 μM. The cells were washed with cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 minutes. Cells were permeabilized with PBS containing 0.1% Triton X-100, blocked with Blocking One Hist (Nacalai Tesque Co., Ltd.) at room temperature for 1 hour, and then reacted with Polychloroal Antibody to Collagen type I (1:200; ORIGENE, Rockville, MD 20850, USA) at room temperature for 1 hour. After washing with PBS, the cells were incubated with Goat Anti-Rabbit IgG FITC Conjugate (1:400; Tokyo Chemical Industry Co., Ltd.) at room temperature for 1 hour. Cell nuclei were visualized using PLUS Antifade Mounting Medium with DAPI (VectorLabs, Newark, CA, USA).

[0144] Nuclear DAPI staining, intracellular type I collagen (COL1A1) staining, and their binding (merge) diagrams are shown in Figure 7A. Compared to the control, intracellular COL1A1 levels were slightly higher with the administration of Uq10 and UqH, but with the administration of compound 1 and compound 5, COL1A1 staining was clearly stronger, and the intracellular COL1A1 concentration was clearly higher compared to Uq10 and UqH.

[0145] Normal human dermal fibroblasts (adult) (Promocell, Heidelberg, Germany) were placed in a 6-well plate (2.0 × 10⁶). ５Cells / well seeds were seeded and cultured for 48 hours in a 10% FBS-containing medium. Then, the culture medium was replaced with a 0.1% FBS-containing DMEM medium containing 0.1% polyoxyethylene (40) hydrogenated castor oil (Nikko Chemicals Co., Ltd.), 0.3% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.05% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.), to which each compound was added, and cultured for another 48 hours. The concentrations of compound 1 and compound 5 were set to 100 μM. The amount of proCOL1A1 protein and hyaluronic acid produced in the supernatant was measured using an ELISA kit (R&D Systems Inc., USA & Canada).

[0146] The primary antibody prepared with the reagent diluent was placed in a 96-well plate and left at room temperature overnight. Afterward, each well was washed three times with the washing solution, and the reagent diluent was added. After standing at room temperature for 1 hour, the wells were washed again three times, and the sample supernatant was added. The plate was then left at room temperature for another 2 hours, washed three times, and the secondary antibody prepared with the reagent diluent was added. The plate was then left at room temperature for 2 hours. Afterward, the plates were washed three more times, avidin-HRP was added, and the plates were left at room temperature, away from light, for 20 minutes. Finally, the plates were washed three more times, and the prepared ELISA HRP chromogenic substrate was added. The plates were left at room temperature, away from light, for 20 minutes. After standing, the reaction was stopped by adding the reaction stop solution, and the absorbance was measured at a wavelength of 450 nm.

[0147] The proCOL1A1 concentration was significantly increased by the administration of compound 1 and compound 5, but not by the administration of Uq10 (see Figure 7B). It was revealed that compound 1 and compound 5 promote the expression of CO1A1 mRNA and increase type I collagen protein. The hyaluronic acid concentration was significantly increased by the administration of compound 1, compound 5, and Uq10, but not by the administration of UqH (see Figure 7C). It was revealed that the increase in hyaluronic acid was due to the promotion of HAS2 mRNA expression by compound 1 and compound 5 (see Figures 4C and 5D) (see Figure 7C).

[0148] Therefore, it is clear that compounds 1 and 5 increase the production of ECM components by increasing the gene expression of ECM components produced by adult human dermal fibroblasts. Furthermore, the ability to suppress the gene expression of MMP1, an enzyme that degrades type I and type III collagen, which are the main components of ECM, strongly suggests the possibility of suppressing the denaturation (degradation) of ECM components.

[0149] Combining the above results, it is clear that the compound of the present invention (ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1)) improves the decrease and degeneration (degradation) of ECM components due to cellular senescence of dermal fibroblasts. Since the decrease and degeneration of ECM components are major factors in wrinkle formation, it is clear that the compound of the present invention is effective in preventing or improving skin signs such as wrinkles and sagging associated with skin aging.

[0150] (Example 8) The TGF-β signaling pathway, which is involved in the production of extracellular matrix (ECM) components in adult human dermal fibroblasts, is known to be involved in the production of ECM components. The involvement of the ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1) in the TGF-β signaling pathway was investigated.

[0151] Normal human dermal fibroblasts (adult) (Promocell, Heidelberg, Germany) were placed in a 6-well plate (2.0 × 10⁶). ５Cells were seeded (cells / well) and cultured for 48 hours in a medium containing 10% FBS. Then, the culture medium was replaced with a DMEM medium containing 0.1% FBS with 0.1% polyoxyethylene (40) hydrogenated castor oil (Nikko Chemicals Co., Ltd.), 0.3% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.05% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.) to which each compound was added, and cultured for another 48 hours. The dosage of each compound was 100 μM. The cells were washed with cold phosphate-buffered saline (PBS), lysed in RIPA buffer containing a cocktail of phosphatase inhibitors and protease inhibitors (Nacalai Tesque Co., Ltd., Kyoto, Japan), and recovered. The sample was centrifuged at 10,000 x g for 10 minutes at 4°C, and the supernatant was collected using a bicinchoninate protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) to determine the protein concentration. The sample (20 μg of total protein) was mixed with a reducing reagent solution (Nacalai Tesque Co., Ltd.) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 15% SDS-PAGE (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan).

[0152] Subsequently, the protein was transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). After blocking with Blocking One Buffer (manufactured by Nakalai Tesque Co., Ltd.), the membrane is treated with Anti-phospho-Smad3 (Ser423 / 425) rabbit antibody (1:1000; Sigma-Aldrich, St. Louis, MO, USA), Anti-SMAD3 rabbit antibody (1:1000; Sigma-Aldrich), Anti-TGF-β Receptor II (E5M6F) Rabbit antibody, and GAPDH mouse. After 1 hour of treatment with an antibody (1:10000; Sigma-Aldrich), followed by washing, treatment with either an anti-rabbit IgG, HRP-linked antibody or an anti-mouse IgG, HRP-linked antibody (1:10000; Cell Signaling Technology, Danvers, MA, USA) for 1 hour, the signal was visualized using Immunostar LD (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0153] Normal human dermal fibroblasts (adult) (Promocell, Heidelberg, Germany) were placed in a 24-well plate (1.0 × 10⁶). ５ Cells were seeded (cells / well) and cultured for 48 hours in a medium containing 10% FBS. Then, the culture medium was replaced with a 0.1% DMEM medium containing FBS with 0.1% polyoxyethylene (40) hydrogenated castor oil (Nikko Chemicals Co., Ltd.), 0.3% ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.05% glycerin (Fujifilm Wako Pure Chemical Industries, Ltd.), to which each compound was added, and cultured for another 48 hours. The dosage of each compound was 100 μM. The cells were washed with cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 minutes.

[0154] Cells were permeabilized with PBS containing 0.1% Triton X-100, blocked with Blocking One Hist (Nacalai Tesque Co., Ltd.) at room temperature for 1 hour, then reacted with TGF-β Receptor II (E5M6F) Rabbit mAb (1:200; Cell Signaling Technology Inc, Danvers, MA, USA) at room temperature for 1 hour, washed with PBS, and incubated with Goat Anti-Rabbit IgG FITC Conjugate (1:400, Tokyo Chemical Industry Co., Ltd.) at room temperature for 1 hour. Cell nuclei were visualized using PLUS Antifade Mounting Medium with DAPI (VectorLabs, Newark, CA, USA).

[0155] Compounds 1 and 5 did not affect TGF-β1 and TGF-β2 mRNA expression in adult human dermal fibroblasts, but increased TGF-β receptor 2 (TGF-βR2) mRNA expression and TGF-βR2 protein expression, while Uq10 and UqH did not increase either (see Figure 8A). In TGF-βR2 immunostaining of adult human dermal fibroblasts, compounds 1 and 5 clearly increased intracellular TGF-βR2 expression (see Figure 8C). Compounds 1 and 5 significantly increased Smad3 phosphorylation downstream of the TGF-β signaling pathway, while Uq10 and UqH had no effect (see Figure 8B).

[0156] Compounds 1 and 5 were found to regulate the gene expression of ECM components by increasing TGF-βR2 in the TGF-β / Smad signaling pathway and activating Smad3. Therefore, it was found that the compounds of the present invention (ubiquinol dicarboxylic acid ester derivatives represented by the above general formula (1)) promote the generation of ECM components using the TGF-β / Smad signaling pathway as at least one signaling pathway.

[0157] (Example 9) <Combined effect of vitamin C derivative on collagen production in adult human dermal fibroblasts by ubiquinol dicarboxylic acid ester derivative> It has been shown that vitamin C derivatives promote collagen production in human dermal fibroblasts and promote the extracellular release of collagen through post-translational modification (Non-patent Literature 15). Therefore, we investigated the effect of compound 1 on promoting type I collagen expression and the effect of vitamin C derivative on extracellular release in adult human dermal fibroblasts.

[0158] First, using the same method as in Example 7 above, the concentrations of Compound 1 and Compound 5 were set to 100 μM, and the vitamin C derivatives were set to 100 μM each: ascorbic acid (AA), sodium ascorbic acid phosphate (APS), magnesium ascorbic acid phosphate (APM), and ascorbic acid 2-glucoside (AG). Trisodium ascorbyl palmitate phosphate (APPS) was set to 10 μM.

[0159] Cell nuclei (DAPI) and intracellular type I collagen (COL1A1) were stained with compound 1, APS, and APS + compound 1 after culture. Compound 1 clearly promoted intracellular COL1A1 expression (see Figure 9A). On the other hand, intracellular COL1A1 staining in cells cultured with APS + compound 1 was clearly reduced compared to cells cultured with compound 1 alone, indicating that APS addition reduced intracellular COL1A1 (see Figure 9A). The amount of intracellular proCOL1A1 (Figure 9B), the amount of proCOL1A1 in the supernatant (Figure 9C), and the total amount of type I collagen in the cell and supernatant (Figure 9D) are shown. The amount of proCOL1A1 protein increased with compound 1 and compound 5, and increased even more significantly with compound 1 in combination with APS, APM, or APPS, revealing that the generated type I collagen was efficiently secreted extracellularly. There was no increasing effect with the combination of AA or AG.

[0160] Compounds 1 and 5, when used in combination with alkali metal salts of ascorbic acid phosphate and trisodium ascorbyl palmitate phosphate (APS, APM, APPS) in adult human fibroblasts, efficiently increase type I collagen production and cell secretion, improving the utilization of type I collagen in the dermis. Therefore, it has been shown that they are effective in preventing or improving skin signs of aging such as wrinkles and sagging.

[0161] The following are specific formulation examples of topical skin preparations of the present invention that contain the compound of the present invention (ubiquinol dicarboxylic acid ester derivative represented by the above general formula (1)). However, the present invention is not limited to the following. The amounts of each component listed in the formulation examples are shown in "mass%" and are all values ​​converted to pure content.

[0162] (Formulation Example 1) -Lotion- (Ingredients) Amount (mass%) (1) Glycerin 5.0 (2) 1,3-Butylene Glycol 6.5 (3) Polyoxyethylene (20 E.O.) Sorbitan Monolaurate 1.2 (4) Ethyl Alcohol 8.0 (5) Compound 1*1 0.05 (6) Lactic Acid 0.05 (7) Sodium Lactate 0.1 (8) 2-Ethylhexyl Paramethoxycinnamate 3.0 (9) Appropriate amount of preservative (10) Appropriate amount of fragrance (11) Purified water remaining amount 100.0% by mass *1: Ubiquinol 1,4-Bis-Hemisuccinate

[0163] <Manufacturing Method> A solution obtained by mixing and dissolving ingredients (3), (4), and (8) to (10) was mixed with a solution obtained by mixing and dissolving ingredients (1), (2), (5) to (7), and (11) to make a homogenized lotion.

[0164] (Formulation Example 2) -Lotion- (Ingredients) Amount (mass%) (1) Glycerin 5.0 (2) 1,3-Butylene Glycol 6.5 (3) Polyoxyethylene (20 E.O.) Sorbitan Monolaurate 1.2 (4) Ethyl Alcohol 8.0 (5) Compound 7*2 0.05 (6) Lactic Acid 0.05 (7) Sodium Lactate 0.1 (8) 2-Ethylhexyl Paramethoxycinnamate 3.0 (9) Appropriate amount of preservative (10) Appropriate amount of fragrance (11) Purified water remaining amount 100.0% by mass *2: Ubiquinol 1,4-Bis-Hemisuccinate Meglumine Salt

[0165] <Manufacturing Method> A solution obtained by mixing and dissolving ingredients (3), (4), and (8) to (10) was mixed with a solution obtained by mixing and dissolving ingredients (1), (2), (5) to (7), and (11) to make a homogenized lotion.

[0166] (Formulation Example 3) - Oil-in-Water Emulsion - (Ingredients) Amount (mass%) (1) Polyoxyethylene (10 E.O.) Sorbitan Monostearate 1.0 (2) Polyoxyethylene (60 E.O.) Sorbitol Tetraoleate 0.5 (3) Glyceryl Monostearate 1.0 (4) Stearic Acid 0.5 (5) Behenyl Alcohol 0.5 (6) Squalane 8.0 (7) Compound 5*3 0.1 (8) Preservative 0.1 (9) Carboxyvinyl Polymer 0.1 (10) Sodium Hydroxide 0.05 (11) Ethyl Alcohol 5.0 (12) Purified Water Remaining Amount (13) Appropriate Amount of Fragrance Total 100.0% by mass *3: Ubiquinol 1,4-Bis-Hemiglutarate

[0167] <Manufacturing Method> Components (8) to (9) were added to component (12) and swollen, then component (10) was added and mixed, and the mixture was heated to 70°C to prepare the aqueous phase. Components (1) to (6) were heated to 70°C and added to the aqueous phase to emulsify. This emulsion was cooled to room temperature, components (7), (11) and (13) were added and mixed uniformly to obtain an emulsion.

[0168] (Formulation Example 4) - Oil-in-Water Emulsion - (Ingredients) Amount (mass%) (1) Polyoxyethylene (10 E.O.) Sorbitan Monostearate 1.0 (2) Polyoxyethylene (60 E.O.) Sorbitol Tetraoleate 0.5 (3) Glyceryl Monostearate 1.0 (4) Stearic Acid 0.5 (5) Behenyl Alcohol 0.5 (6) Squalane 8.0 (7) Compound 8*4 0.1 (8) Preservative 0.1 (9) Carboxyvinyl Polymer 0.1 (10) Sodium Hydroxide 0.05 (11) Ethyl Alcohol 5.0 (12) Purified Water Remaining Amount (13) Appropriate Amount of Fragrance Total 100.0% by mass *4: Ubiquinol 1,4-Bis-Succinate Ethyl Ester

[0169] <Manufacturing Method> Components (8) to (9) were added to component (12) and swollen, then component (10) was added and mixed, and the mixture was heated to 70°C to prepare the aqueous phase. Components (1) to (6) were heated to 70°C and added to the aqueous phase to emulsify. This emulsion was cooled to room temperature, components (7), (11) and (13) were added and mixed uniformly to obtain an emulsion.

[0170] (Formulation Example 5) -Non-aqueous emulsion- (Ingredients) Amount (mass%) A. Oil phase liquid paraffin 20 Lanolin alcohol 5 Lanolin fatty acid glyceryl 10 Oil-soluble licorice extract 1 Stearyl glycyrrhetinate 2 Ethyl parahydroxybenzoate 2 B. Glycerin phase glycerin 25 Propylene glycol 10 Erythritol 20 Adenosine triphosphate 2 Pyridoxine hydrochloride 3 Compound 1*1 0.1 Appropriate amount of fragrance Total 100.0 mass% *1: Ubiquinol 1,4-bis-hemisuccinate -Manufacturing method- The oil phase of A and the glycerin phase of B were heated and melted at 70°C. The oil phase of A was added to the glycerin part of B and emulsified with an emulsifier. The emulsion was cooled using a heat exchanger to obtain an emulsion.

[0171] (Formulation Example 6) -Non-aqueous emulsion- (Ingredients) Amount (mass%) A. Oil phase liquid paraffin 20 Lanolin alcohol 5 Lanolin fatty acid glyceryl 10 Oil-soluble licorice extract 1 Stearyl glycyrrhetinate 2 Ethyl parahydroxybenzoate 2 B. Glycerin phase glycerin 25 Propylene glycol 10 Erythritol 20 Adenosine triphosphate 2 Pyridoxine hydrochloride 3 Compound 9*5 0.1 Appropriate amount of fragrance Total 100.0 mass% *5: Ubiquinol 1,4-bis-succinate isopropyl ester -Manufacturing method- The oil phase of A and the glycerin phase of B were heated and dissolved at 70°C. The oil phase of A was added to the glycerin phase of B and emulsified in an emulsifier. The emulsion was cooled using a heat exchanger to obtain an emulsion.

Claims

1. A composition for the prevention or improvement of skin aging, comprising as an active ingredient at least one agent selected from IL-6 mRNA expression inhibitors, IL-8 mRNA expression inhibitors, MMP1 mRNA expression inhibitors, CDKN1A mRNA expression inhibitors, ATP production promoters, COL1A1 mRNA expression promoters, COL3A1 mRNA expression promoters, HAS2 mRNA expression promoters, elastin mRNA expression promoters, TGF-βR2 mRNA expression promoters, and neprilysin mRNA expression inhibitors. However, the IL-6 mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has an inhibitory effect on the expression of IL-6 mRNA that generates IL-6 from the interleukin-6 (IL-6) gene; the IL-8 mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has an inhibitory effect on the expression of IL-8 mRNA that generates IL-8 from the interleukin-8 (IL-8) gene; the MMP1 mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has an inhibitory effect on the expression of MMP1 mRNA that generates MMP1 from the matrix metalloproteinase 1 (MMP1) gene; the CDKN1A mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has an inhibitory effect on the expression of CDKN1A mRNA that generates CDKN1A from the CDKN1A gene. The ATP production promoter comprises a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has an ATP production promoting effect in adult human dermal fibroblasts; the COL1A1 mRNA expression promoter comprises a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has an expression promoting effect on COL1A1 mRNA that generates COL1A1 from the type I procollagen α1 (COL1A1) gene; the COL3A1 mRNA expression promoter comprises a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has an expression promoting effect on COL3A1 mRNA that generates COL3A1 from the type III procollagen α1 (COL3A1) gene; and the HAS2The mRNA expression promoter contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has the effect of promoting the expression of HAS2 mRNA that produces HAS2 from the hyaluronic acid synthase (HAS2) gene; the elastin mRNA expression promoter contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has the effect of promoting the expression of elastin mRNA that produces elastin from the elastin gene; the TGF-βR2 mRNA expression promoter contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has the effect of promoting the expression of TGF-βR2 mRNA that produces TGF-βR2 from the TGF-β receptor 2 (TGF-βR2) gene; and the neprilysin mRNA expression inhibitor contains a ubiquinol dicarboxylic acid ester derivative represented by the following general formula (1) and has the effect of suppressing the expression of neprilysin mRNA that produces neprilysin from the neprilysin gene. However, in the above general formula (1), R １ and R ２ Each represents a hydrogen atom, or a substituent selected from dicarboxylic acid hemiester residues and their salts, and dicarboxylic acid double ester residues, R １ and R ２ At least one of these is a dicarboxylic acid hemiester residue and / or a dicarboxylic acid double ester residue. n represents an integer from 1 to 10.

2. The composition for preventing or improving skin aging according to claim 1, comprising as active ingredients all agents selected from IL-6 mRNA expression inhibitor, IL-8 mRNA expression inhibitor, MMP1 mRNA expression inhibitor, CDKN1A mRNA expression inhibitor, ATP production promoter, COL1A1 mRNA expression promoter, COL3A1 mRNA expression promoter, HAS2 mRNA expression promoter, elastin mRNA expression promoter, TGF-βR2 mRNA expression promoter, and neprilysin mRNA expression inhibitor.

3. The composition for preventing or improving skin aging according to claim 1, further containing a vitamin C derivative.

4. The composition for preventing or improving skin signs according to claim 3, wherein the vitamin C derivative is at least one selected from metal salts of ascorbic acid phosphate and trisodium ascorbyl palmitate phosphate.

5. The composition for preventing or improving skin aging according to claim 1, wherein the dicarboxylic acid hemiester residue in the general formula (1) is bonded between the carbonyl groups of the dicarboxylic acid residue by a linear alkylene group having 2 to 4 carbon atoms.

6. The composition for preventing or improving skin aging according to claim 1, wherein the dicarboxylic acid double ester residue in the general formula (1) is bonded between the carbonyl groups of the dicarboxylic acid residue by a linear alkylene group having 2 to 4 carbon atoms.

7. R in the aforementioned general formula (1) １ and R ２ are a hydrogen atom, HOOCCH ２ CH ２ CO-, HOOCCH ２ CH ２ CH ２ CO-, HOH ２ C-(CH(OH)) ４ CH ２ NHCH ３ ·HOOCCH ２ CH ２ CO-, CH ３ CH ２ OOCCH ２ CH ２ CO-, and (CH ３ ) ２ CHOOCCH ２ CH ２ CO-; the composition for preventing or improving skin aging signs according to claim 1, wherein at least one of R １ and R ２ is at least one selected from the group consisting of HOOCCH ２ CH ２ CO-, HOOCCH ２ CH ２ CH ２ CO-, HOH ２ C-(CH(OH)) ４ CH ２ NHCH ３ ·HOOCCH ２ CH ２ CO-, CH ３ CH ２ OOCCH ２ CH ２ CO-, and (CH ３ ) ２ CHOOCCH ２ CH ２ CO-.

8. A topical skin preparation containing the composition for preventing or improving skin aging according to any one of claims 1 to 7.

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