Retinoic acid derivative

Abstract

The present invention provides a retinoic acid derivative that has high stability and water solubility. The present invention relates to a carboxylic acid ester compound of trehalose or a derivative thereof and retinoic acid.

Description

Retinoic acid derivatives

[0001] The present invention relates to retinoic acid derivatives.

[0002] Retinol is a type of vitamin A and is classified as a fat-soluble vitamin. Retinol is known to be useful for maintaining healthy skin, reducing wrinkles, improving sagging skin, and improving moisturizing. However, retinol is highly unstable to light and heat and has poor water solubility, which limits its storage and application. Retinoic acid, the active form of retinol, exhibits slightly higher water solubility and thermal stability than retinol, but its water solubility and stability are still insufficient.

[0003] Patent Document 1 discloses water-soluble aliphatic derivatives of retinoic acid. Patent Document 2 discloses trehalose derivatives having long-chain aliphatic acyl groups.

[0004] Special Publication No. 9-503499 Publication No. 3-47193

[0005] An object of the present invention is to provide a retinoic acid derivative which is highly stable and water-soluble.

[0006] The present inventors have discovered that a carboxylic acid ester compound of trehalose or a derivative thereof with retinoic acid is highly stable and water-soluble, and have completed the present invention.

[0007] The present disclosure includes the following embodiments. <1> A carboxylic acid ester compound of trehalose or a derivative thereof and retinoic acid. <2> The compound according to item 1, in which retinoic acid is bonded to the carbon atom at position 4 of trehalose. <3> The compound according to item 1, in which retinoic acid is bonded to the carbon atom at position 6 of trehalose. <4> The compound according to item 1, in which retinoic acid is bonded to the carbon atom at position 3 of trehalose. <5> The compound according to item 1, in which retinoic acid is bonded to the carbon atom at position 2 of trehalose. <6> The compound according to any one of items 1 to 5, in which retinoic acid is all-trans or all-cis. <7> The compound according to any one of items 1 to 5, in which retinoic acid is 7-cis-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, or 13-cis-retinoic acid. <8> A composition comprising, relative to 100 parts by mass of the compound according to item 2, 30 to 300 parts by mass of the compound according to item 3, 20 to 50 parts by mass of the compound according to item 4, and 20 to 50 parts by mass of the compound according to item 5. <9> A hyaluronic acid or collagen production enhancer comprising the compound according to item 1. <10> A hyaluronic acid or collagen production enhancer comprising the composition according to item 8. <11> A cosmetic or quasi-drug comprising the hyaluronic acid or collagen production enhancer according to item 9 or 10. <12> A skin turnover enhancer comprising the compound according to item 1. <13> A skin turnover enhancer comprising the composition according to item 8. <14> A cosmetic or quasi-drug comprising the skin turnover enhancer according to item 12 or 13.

[0008] The compound of the present invention has high stability and water solubility.In addition, the compound of the present invention has the effect of enhancing the production of hyaluronic acid or collagen or promoting skin turnover, so the compound of the present invention can be incorporated into cosmetics or quasi-drugs as a hyaluronic acid or collagen production enhancer or a skin turnover promoter.

[0009] The thermal stability of 6-TR (retinoic acid trehalose in which retinoic acid is bonded to the carbon atom at position 6 of trehalose), RA (retinoic acid), and RE (retinol) is shown. The uptake of RA into NHEK cells is shown. The uptake of 6-TR into NHEK cells is shown. The uptake of RA and 6-TR into NHDF cells is shown. The thermal stability of 4-TR (retinoic acid trehalose in which retinoic acid is bonded to the carbon atom at position 4 of trehalose) and RE is shown.

[0010] <<Retinoic Acid Derivatives>> The retinoic acid derivatives of the present invention are characterized by being carboxylic acid ester compounds of trehalose or a derivative thereof with retinoic acid.

[0011] Retinoic acid is C ２０ H ２８ O ２ The retinoic acid derivative of the present invention is a compound in which the terminal hydroxyl group of retinol is oxidized and converted to a carboxyl group. The retinoic acid derivative of the present invention is a compound in which an ester bond is formed between the terminal carboxyl group of retinoic acid and the hydroxyl group of trehalose or a derivative thereof.

[0012] Retinoic acid has four carbon-carbon double bonds, each of which may be independently in the cis or trans configuration. The retinoic acid constituting the retinoic acid derivative of the present invention may be in either the all-trans configuration or the all-cis configuration, but the all-trans configuration is preferred. All-trans configuration: All cis:

[0013] Furthermore, the retinoic acid constituting the retinoic acid derivative of the present invention may contain a mixture of cis- and trans-form carbon-carbon double bonds, including 7-cis-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, and 13-cis-retinoic acid.

[0014] <Trehalose or Derivatives Thereof> Trehalose is a non-reducing disaccharide in which two glucose molecules are bonded together via an α,α-1,1 bond. In the present invention, either anhydrous or hydrated trehalose can be suitably used. Examples of trehalose derivatives include those in which sugars or substituents have been introduced into the hydrogen atoms of some of the hydroxyl groups. Examples of the substituents include carboxylic acid ester groups such as acetate ester groups and benzoic acid groups; sulfate ester groups; fatty acid ester groups such as laurate ester groups, myristate ester groups, palmitate ester groups, stearate ester groups, oleate ester groups, linoleate ester groups, and linolenate ester groups; and ether groups such as methyl ether groups, benzyl ether groups, trityl ether groups, methylsilyl ether groups, and dodecyl ether groups. Examples of trehalose derivatives in which sugars have been introduced into the hydrogen atoms of hydroxyl groups include glucosyltrehalose, maltosyltrehalose, maltotriosyltrehalose, and maltotetraosyltrehalose.

[0015] The terminal carboxyl group of retinoic acid can be bonded to any carbon atom of trehalose via an ester structure.Isomers of retinoic acid derivatives include retinoic acid derivatives in which retinoic acid is bonded to the carbon atom at position 4 of trehalose, retinoic acid derivatives in which retinoic acid is bonded to the carbon atom at position 6 of trehalose, retinoic acid derivatives in which retinoic acid is bonded to the carbon atom at position 3 of trehalose, and retinoic acid derivatives in which retinoic acid is bonded to the carbon atom at position 2 of trehalose.These isomers may be used alone or in combination of any two or more thereof.

[0016] An example of a composition combining multiple isomers is a composition containing, per 100 parts by mass of a retinoic acid derivative in which retinoic acid is bonded to the carbon atom at position 4 of trehalose, 30 to 300 parts by mass of a retinoic acid derivative in which retinoic acid is bonded to the carbon atom at position 6 of trehalose, 20 to 50 parts by mass of a retinoic acid derivative in which retinoic acid is bonded to the carbon atom at position 3 of trehalose, and 20 to 50 parts by mass of a retinoic acid derivative in which retinoic acid is bonded to the carbon atom at position 2 of trehalose.

[0017] The amount of the retinoic acid derivative in which retinoic acid is bonded to the carbon atom at position 6 of trehalose is preferably 33 to 290 parts by mass, the amount of the retinoic acid derivative in which retinoic acid is bonded to the carbon atom at position 3 of trehalose is preferably 22 to 45 parts by mass, and the amount of the retinoic acid derivative in which retinoic acid is bonded to the carbon atom at position 2 of trehalose is preferably 22 to 45 parts by mass, per 100 parts by mass of the retinoic acid derivative in which retinoic acid is bonded to the carbon atom at position 4 of trehalose.

[0018] <Method for Synthesizing Retinoic Acid Derivatives> Retinoic acid derivatives can be produced by an ester-forming reaction between the hydroxyl group of trehalose or a derivative thereof and the carboxyl group of retinoic acid. Examples of ester-forming reactions include (i) a method in which an orthoacid hemiester is formed as an intermediate at the carboxyl group, followed by reaction with trehalose or a derivative thereof; (ii) a method in which a halogenating agent is reacted with the carboxyl group to synthesize an acyl halide, followed by reaction with trehalose or a derivative thereof; and (iii) a method in which an acid anhydride is formed between the carboxyl groups of retinoic acid, followed by reaction with trehalose or a derivative thereof. Among these, method (i) is preferred, and p-toluenesulfonic acid chloride (TsCl), N-methylimidazole (NMI), or the like can be used to promote the reaction.

[0019] In order to promote the ester-forming reaction, known catalysts such as TBTU (1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate, hereinafter simply referred to as TBTU), COMU ((1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinocarbenium hexafluorophosphate), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DCC (N,N'-dicyclohexylcarbodiimide), PyBOP (benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate), and CDI (N,N'-carbonyldiimidazole) may be used.

[0020] The reaction product can be purified by known methods such as ODS (octadecylsilane) chromatography, silica gel chromatography, gel filtration chromatography, and ion exchange chromatography.

[0021] <Physical Properties of Retinoic Acid Derivatives> The retinoic acid derivatives of the present invention have improved water solubility compared to retinoic acid. The retinoic acid derivatives preferably have a water solubility of 3 g / L or more, more preferably 5 g / L or more, and even more preferably 7 g / L or more at 30°C. The upper limit of the solubility in water at 30°C is not particularly limited, but is preferably 10 g / L or less.

[0022] The retinoic acid derivative of the present invention has improved stability compared to retinoic acid. The retinoic acid derivative is dissolved in N,N-dimethylformamide (DMF) to a concentration of 3.49 mM, and after standing at 50°C for 200 hours, the residual rate is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 93% or more, and particularly preferably 95% or more. The residual rate of the retinoic acid derivative can be evaluated by subjecting the solution after standing to HPLC analysis.

[0023] The retinoic acid derivative of the present invention preferably has a lower log P value than retinoic acid. The log P value is the logarithm of the 1-octanol / water partition coefficient of a compound, and means the ratio of the equilibrium concentrations of the solute in each solvent at partition equilibrium when the compound is dissolved as a solute in a two-phase solvent system of 1-octanol and water, and is generally expressed in the form of a logarithm to the base 10, "log P." The log P value is an index of hydrophobicity; the larger this value, the more hydrophobic the compound is, and the smaller this value, the more hydrophilic the compound is. The log P value is preferably 1 to 5, and more preferably 1.8 to 3. Within these ranges, skin permeability tends to be excellent.

[0024] <<Hyaluronic acid or collagen production enhancer>> The hyaluronic acid or collagen production enhancer of the present invention is characterized by comprising the retinoic acid derivative.Retinoic acid derivatives are excellent in water solubility and stability, while having hyaluronic acid production ability and collagen production ability at the same level or higher than retinol and retinoic acid.It is presumed that retinoic acid derivatives bind to and activate retinoic acid receptors (RAR), which are nuclear receptors of cells, and that the activated retinoic acid receptors promote the expression of hyaluronic acid synthase genes and collagen genes, thereby enhancing the production of hyaluronic acid and / or collagen, but the present invention is not limited to this mechanism.It is expected that increasing the amount of hyaluronic acid and / or collagen in the skin will improve the firmness and elasticity of the skin.

[0025] Examples of hyaluronic acid synthase genes whose expression is promoted by hyaluronic acid production enhancers include HAS2 and HAS3.

[0026] The collagen production enhancer preferably inhibits collagen degradation. Examples of mechanisms for inhibiting collagen degradation include suppressing the expression of collagen-degrading enzyme genes. Examples of collagen-degrading enzyme genes whose expression is inhibited include genes encoding matrix metalloproteinases such as MMP1 and MMP3.

[0027] <<Skin Turnover Promoter>> The skin turnover promoter of the present invention is characterized by containing the retinoic acid derivative. Retinoic acid derivatives have excellent water solubility and stability, while also having the ability to promote skin turnover to the same extent as or greater than retinol and retinoic acid. It is presumed that retinoic acid derivatives promote skin turnover by promoting the proliferation of skin basal cells, promoting the differentiation of epidermal cells, inhibiting the binding between epidermal cells by suppressing the expression of intercellular adhesion molecules, and promoting the synthesis of extracellular matrix, but the present invention is not limited to this mechanism.

[0028] The skin turnover promoter preferably enhances the expression of a growth factor, such as HB-EGF (heparin-binding epidermal growth factor-like factor), FGF (fibroblast growth factor), IGF (epidermal growth factor), or EGF (epidermal growth factor).

[0029] <<Skin Barrier Function Improver>> The skin barrier function improver of the present invention is characterized by containing the retinoic acid derivative. Mechanisms for improving skin barrier function include promoting the formation of a cornified envelope by keratinocytes and moisturizing. For example, the formation of a cornified envelope is promoted by enhancing the expression of the IVL (involucrin) gene and the FLG (filaggrin) gene. Moisturizing is achieved by enhancing the expression of the AQP3 gene, the AQP9 gene, the PPARγ gene, and the like. It is expected that the improvement of skin barrier function will lead to the prevention, treatment, or amelioration of diseases and symptoms such as rough skin, dry skin, atopic dermatitis, and psoriasis.

[0030] <<Cosmetics or Quasi-drugs>> The cosmetics or quasi-drugs of the present invention are characterized by containing a hyaluronic acid or collagen production enhancer, or a skin turnover promoter. Retinoic acid derivatives are excellent in water solubility and stability, while being non-irritating to the skin and exhibiting cytotoxicity at levels comparable to or lower than those of retinol and retinoic acid, making them suitable for use as ingredients in cosmetics or quasi-drugs.

[0031] The formulation of the cosmetic product is not particularly limited, and examples thereof include aqueous compositions consisting of only an aqueous phase, oil-in-water emulsion compositions of O / W type (oil in water type), and water-in-oil emulsion compositions of W / O type (water in oil type).Among these, aqueous and oil-in-water emulsions are preferred.In the case of emulsions, it is preferred that the hyaluronic acid or collagen production enhancer, or the skin turnover promoter, is contained in the aqueous phase.

[0032] Examples of cosmetic forms include skin care cosmetics such as lotions, milky lotions, beauty serums, packs, and gel creams; hair care cosmetics such as shampoos, conditioners, and hair styling products; body care cosmetics such as packs, facial cleansers, body soaps, and hand creams; and makeup cosmetics such as foundations, lipsticks, lip balms, and lip glosses.

[0033] The route of administration of quasi-drugs may be either oral or parenteral. Oral dosage forms include liquids, tablets, powders, pills, fine granules, granules, suspensions, sugar-coated tablets, capsules, emulsions, syrups, extracts, microcapsules, and lozenges. Parenteral administration methods include transdermal, topical, nasal, sublingual, and transmucosal administration.

[0034] In addition to the hyaluronic acid or collagen production enhancer or skin turnover promoter, the cosmetics and quasi-drugs may contain carriers, preservatives, antioxidants, moisturizers, texture improvers, dispersants, emulsifiers, emulsion stabilizers, water-retaining agents, stabilizers, thickeners, ultraviolet absorbers, solvents, disintegrants, binders, colorants, and the like that are generally acceptable for cosmetics and quasi-drugs.

[0035] The concentration of the hyaluronic acid or collagen production enhancer, or the skin turnover promoter in the cosmetic or quasi-drug is not particularly limited, but is preferably 0.0005 to 1.0% by mass, more preferably 0.01 to 0.5% by mass, and even more preferably 0.01 to 0.2% by mass.

[0036] There are no particular limitations on the recipients of cosmetics or quasi-drugs, and they can be administered to both men and women. When cosmetics or quasi-drugs are applied to the skin, the application site is not particularly limited, and examples include the face, neck, chest, back, arms, elbows, hands, armpits, abdomen, vulva, buttocks, anal area, legs, fingers, toes, and soles.

[0037] The present invention will be described below with reference to examples, but is not limited to the following examples. Hereinafter, "parts" and "%" mean "parts by mass" and "% by mass", respectively, unless otherwise specified.

[0038] [Production Example 1] <Synthesis of retinoic acid trehalose> 1,000 mg of retinoic acid, 4,200 mg of anhydrous trehalose, and 1,284 mg of TBTU (a condensing agent) were added to 120 g of pyridine, and the mixture was reacted at 27°C for 72 hours with stirring. After completion of the reaction, the product was extracted, washed, filtered, and then concentrated under reduced pressure. The resulting reaction product was then purified by medium-pressure preparative chromatography under the following conditions. [Medium-pressure preparative chromatography conditions] Normal-phase chromatography Column: DAISOGEL IR-60-63 / 210 (Osaka Soda Co., Ltd.) Mobile phase: acetonitrile and deionized water Reverse-phase chromatography Column: FS-1830FT (Organo Corporation) Mobile phase: methanol and deionized water Finally, 184.4 mg of retinoic acid trehalose was obtained.

[0039] <Confirmation of retinoic acid trehalose> The obtained retinoic acid trehalose was measured for 1H-NMR, 13C-NMR, DEPT135 spectrum, H-H COSY spectrum, and HMQC spectrum. As a result, it was confirmed that retinoic acid trehalose in which retinoic acid was bonded to the carbon atom at position 6 of trehalose was mainly obtained in Production Example 1. Hereinafter, evaluations were carried out using the retinoic acid trehalose bonded to the carbon atom at position 6 obtained in Production Example 1 (hereinafter sometimes abbreviated as 6-TR).

[0040] <Thermal Stability> 6-TR obtained in Production Example 1, retinoic acid (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter sometimes abbreviated as RA), and retinol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter sometimes abbreviated as RE) were each dissolved in N,N-dimethylformamide (DMF) to a concentration of 3.49 mM. Thereafter, each solution was allowed to stand at 50°C for 0 to 312 hours. After standing, the solutions were subjected to HPLC analysis under the following conditions, and the remaining percentage of each component relative to the amount at 0 hour was calculated. The results are shown in Figure 1.

[0041] [HPLC conditions] RA Column: YMC-Triart C18 (YMC, 250 x 4.6 mm I.D.) x 2 Solvent: 80% acetonitrile + 20% 1% ammonium acetate Flow rate: 1 mL / min Temperature: 40°C Detection: 354 nm

[0042] RE Column: Develosil RPAQUEOUS-AR-5 (Nomura Chemical Co., Ltd., 250 x 4.6 mm I.D.) x 2 Solvent: 100% acetonitrile Flow rate: 1 mL / min Temperature: 40°C Detection: 354 nm

[0043] 6-TR Column: Develosil RPAQUEOUS-AR-5 (Nomura Chemical Co., Ltd., 250 x 4.6 mm I.D.) x 2 Solvent: 80% acetonitrile Flow rate: 1 mL / min Temperature: 40°C Detection: 354 nm

[0044] The residual rates after 144 hours were 97.7% for 6-TR, 74.5% for RA, and 4.3% for RE. The residual rates after 312 hours were 93.6% for 6-TR, 71.2% for RA, and 0.9% for RE. 6-TR had significantly higher thermal stability than RA and RE.

[0045] <Solubility Measurement> Deionized water or PBS was added to a solution so that the concentration of 6-TR or RA was equivalent to 1%. The resulting solution was subjected to ultrasonic treatment for 10 minutes and then stirred at 30°C and 1,000 rpm for 1 hour using a block bath shaker (manufactured by AS ONE Corporation). The solution was then centrifuged at 15,000 rpm for 5 minutes. The supernatant was recovered from the centrifuged solution, and the solubility was measured by HPLC under the following conditions. The solubility (%) was calculated as the average of three experiments performed for each condition. The results are shown in Table 1. 6-TR had significantly higher solubility in deionized water and PBS than RA.

[0046] [HPLC conditions] Column: Develosil RPAQUEOUS-AR-5 (Nomura Chemical Co., Ltd., 250 x 4.6 mm I.D.) x 2 Solvent: 80% acetonitrile Flow rate: 1 mL / min Temperature: 40°C Detection: 354 nm

[0047]

[0048] <Hyaluronic Acid Production> Normal human epidermal keratinocytes (NHEK cells, manufactured by Lifeline Cell Technologies) were seeded onto a 96-well plate at 8,000 cells / well, and 100 μl of NHEK cell medium (EpiLife Medium supplemented with human keratinocyte growth supplement, manufactured by Thermo Fisher Scientific) was added. After 48 hours of culture, the culture supernatant was removed and replaced with medium containing 6-TR, RA, or RE at 2.5 μM, 5 μM, 10 μM, 20 μM, or 40 μM, respectively. After 24 hours of culture, the cell proliferation rate and hyaluronic acid production rate were measured for each sample. Cell proliferation rate was measured using the alamarBlue assay (alamarBlue Cell Viability Reagent, manufactured by Thermo Fisher Scientific). The amount of hyaluronic acid produced was measured by diluting the culture supernatant 40-fold with phosphate buffer containing 5% polyoxyethylene (20) sorbitan monolaurate and using a Hyaluronan ELISA assay (Hyaluronan DuoSet Kit, manufactured by R&D Systems). The results for cell proliferation rate are shown in Table 2, and the results for hyaluronic acid production are shown in Table 3. 6-TR, RA, or RE were each dissolved in ethanol before addition. For comparison, Tables 2 and 3 show the results when a medium containing only ethanol was used.

[0049]

[0050]

[0051] In samples containing 6-TR, RA, or RE at concentrations of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, the ethanol concentrations in the medium were 0.17%, 0.33%, 0.66%, 1.33%, and 2.65%, respectively. Due to the influence of ethanol, a decrease in hyaluronic acid production was confirmed under conditions of ethanol concentrations of 1.33% and 2.65%. When comparing conditions under the same concentration, 6-TR had a greater effect on enhancing hyaluronic acid production than RA and RE, and the cell proliferation rate was comparable or higher.

[0052] "Medium" in Tables 2 and 3 indicates the results of the control to which only medium was added. * in Tables 2 and 3 indicates that the p-value in the Dunnett test is less than 0.05, and there is a significant difference between the control group (Medium) and the comparison group with a confidence probability of 95% or higher. ** in Tables 2 and 3 indicates that the p-value in the Dunnett test is less than 0.01, and there is a significant difference between the control group (Medium) and the comparison group with a confidence probability of 99% or higher. SD in Tables 2 and 3 indicates standard deviation. The same applies to Table 3 and subsequent tables.

[0053] <Toxicity to NHEK Cells> Normal human epidermal keratinocytes (NHEK cells) were seeded onto a 96-well plate at 6,000 cells / well with 100 μl of NHEK cell medium. After 24 hours of culture, the culture supernatant was removed and replaced with medium containing 2.5 μM, 5 μM, 10 μM, 20 μM, or 40 μM of 6-TR, RA, or RE, respectively. After 24 hours of culture, each sample was evaluated for cytotoxicity. The cytotoxicity of the culture supernatant was measured using an LDH assay (Cytotoxicity LDH Assay Kit-WST, Dojindo Laboratories). The results are shown in Table 4. The cytotoxicity (%) shown in Table 4 is the percentage of damaged cells measured by LDH assay, calculated as a ratio of the control group (medium) to 0% and the total cell lysis group to 100%. 6-TR, RA, and RE were each dissolved in ethanol before addition. For comparison, Table 4 also shows the results when medium containing only ethanol was used.

[0054]

[0055] For samples containing 6-TR, RA, or RE at concentrations of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, the ethanol concentrations in the medium were 0.17%, 0.33%, 0.66%, 1.33%, and 2.65%, respectively. RA was cytotoxic at concentrations above 20 μM, RE at concentrations above 10 μM, and 6-TR at concentrations above 20 μM. Thus, the cytotoxicity of 6-TR against NHEK cells was lower than that of RE and comparable to that of RA.

[0056] <Toxic Effect on NHDF Cells> Normal human dermal fibroblasts (NHDF cells, manufactured by Lifeline Cell Technologies) were seeded onto a 96-well plate at 6,000 cells / well with 100 μl of NHDF cell medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (manufactured by HyClone), manufactured by Shimadzu Diagnostics). After 24 hours of culture, the culture supernatant was removed and replaced with medium containing 10 μM, 20 μM, 40 μM, 80 μM, or 160 μM of 6-TR, RA, or RE, respectively. Then, after 24 hours of culture, each sample was evaluated for cell proliferation rate and cytotoxicity. Cell proliferation rate was measured using the alamarBlue assay (alamarBlue Cell Viability Reagent, manufactured by Thermo Fisher Scientific). Cytotoxicity of the culture supernatant was measured by LDH assay (Cytotoxicity LDH Assay Kit-WST, Dojindo Laboratories). The results of cell proliferation rate are shown in Table 5, and the results of cytotoxicity are shown in Table 6. 6-TR, RA, or RE was dissolved in ethanol before addition. For comparison, Tables 5 and 6 show the results using a medium containing only ethanol. For samples containing 6-TR, RA, or RE at concentrations of 10 μM, 20 μM, 40 μM, 80 μM, and 160 μM, the ethanol concentrations in the medium were 0.65%, 1.30%, 2.60%, 5.20%, and 10.40%, respectively.

[0057]

[0058]

[0059] Due to the effect of ethanol, cell proliferation was inhibited at ethanol concentrations of 5.20% and 10.40%. On the other hand, 6-TR was confirmed to promote cell proliferation at low concentrations (10 μM and 20 μM). Furthermore, RA was cytotoxic at concentrations of 80 μM or higher, RE at concentrations of 40 μM or higher, and 6-TR at concentrations of 160 μM or higher. In other words, the cytotoxicity of 6-TR against NHDF cells was lower than that of RA and RE.

[0060] <Uptake into NHEK cells> Experiments were performed with reference to the method for quantifying retinoid uptake into vascular endothelial cells (HUVEC) over time described in the following literature. Literature: Guntner, A. S.; Doppler, C.; Wechselberger, C.; Bernhard, D.; Buchberger, W. Cells 2020, 9, 2048. First, normal human epidermal keratinocytes (NHEK cells) were cultured at a concentration of 3.0 × 10 ５ cells / 3 ml or 4.5 x 10 ５ The cells were seeded onto a 60 mm dish to a concentration of 100 cells / 3 ml. NHEK cell medium was used. After 24 hours of culture, the culture supernatant was removed and replaced with medium containing RA or 6-TR at a final concentration of 5 μM. After culturing for the specified time, the cells were recovered. The resulting cell pellet was suspended in 300 μl of acetonitrile and subjected to ultrasonic disruption. The suspension was centrifuged, and the supernatant was recovered and dried under vacuum. The cells were then resuspended in 100 μl of 80% acetonitrile and subjected to HPLC analysis under the following conditions. The results for each culture time after the addition of RA are shown in Figure 2, and the results for each culture time after the addition of 6-TR are shown in Figure 3.

[0061] [HPLC conditions] Column: YMC-Triart C18 (YMC, 250 x 4.6 mm I.D.) x 2 Solvent: 80% acetonitrile + 20% 1% ammonium acetate Flow rate: 1 mL / min Temperature: 40°C Detection: 355 nm

[0062] The amount of RA uptake in the cells increased over time up to 6 hours, and that of 6-TR up to 24 hours. Furthermore, in 6-TR-treated cells, no peak was observed at the RA elution position up to 24 hours. This confirmed that 6-TR was not degraded by RA in NHEK cells and was stable within the cells.

[0063] <Uptake into NHDF cells> Normal human dermal fibroblasts (NHDF cells) were cultured at 1.0 x 10 ６The cells were seeded onto a 60 mm dish at a concentration of 1000 cells / 3 ml. NHDF cell medium was used. After 24 hours of culture, the culture supernatant was removed and replaced with medium containing RA or 6-TR at a final concentration of 10 μM. After culturing for the specified time, the cells were recovered. The resulting cell pellet was suspended in 300 μl of acetonitrile and subjected to ultrasonic disruption. The suspension was centrifuged, and the supernatant was recovered and dried in vacuo. The cells were then resuspended in 100 μl of 80% acetonitrile and subjected to HPLC analysis under the following conditions. The results for each culture time after the addition of RA or 6-TR are shown in Figure 4.

[0064] The amount of RA uptake in the cells increased over time up to 6 hours, and that of 6-TR up to 24 hours. Furthermore, in 6-TR-treated cells, no peak was observed at the RA elution position up to 24 hours. This confirmed that 6-TR was not degraded into RA in NHEK cells.

[0065] <Permeability> For each of 6-TR, RA, and RE, the log P value was calculated based on each chemical formula using ALOGPS2.1 (URL: http: / / www.vcclab.org / lab / logps / ). The "log P value" is the logarithmic value of the 1-octanol / water partition coefficient of a compound, and refers to the ratio of the equilibrium concentrations of the solute in each solvent at partition equilibrium when the compound is dissolved as a solute in a two-phase solvent system of 1-octanol and water. It is generally expressed in the form of a logarithm to the base 10, "log P." In other words, the log P value is an index of hydrophobicity; the larger the value, the more hydrophobic the compound, and the smaller the value, the more hydrophilic the compound. Note that the log P value calculated based on the molecular structure will be referred to as the "clog P value" hereinafter.

[0066] The clog P value of 6-TR was 1.93, that of RA was 5.7, and that of RE was 6.4. From the viewpoint of skin permeability, a log P value of approximately 1.8 to 3 is ideal, and therefore, it was confirmed that 6-TR has high skin permeability.

[0067] [Production Example 2] <Synthesis of retinoic acid trehalose> Under a nitrogen atmosphere, 60 mL of DMF, 3.0 g of retinoic acid, 2.3 g of p-toluenesulfonic acid chloride (TsCl), and 2.5 g of N-methylimidazole (NMI) were added and stirred at room temperature for 0.5 hours. In a separate container, 3.5 g of anhydrous trehalose and N-ethyldiisopropylamine (DIEA) were dissolved in 90 mL of DMF. This solution was added, and the mixture was stirred at 27°C for an additional 72 hours. After completion of the reaction, the product was extracted, washed, filtered, and concentrated under reduced pressure. The resulting reaction product was then purified by medium-pressure preparative chromatography under the following conditions.

[0068] [Medium-pressure preparative chromatography conditions] Normal phase chromatography Column: DAISOGEL IR-60-63 / 210 Mobile phase: acetonitrile and deionized water

[0069] Reverse phase chromatography Column: FS-1830FT Mobile phase: methanol and deionized water

[0070] <Confirmation of retinoic acid trehalose> The substance obtained by purification was fractionated using reverse phase chromatography, and each of the obtained components was measured for 1H-NMR, 13C-NMR, DEPT135 spectrum, H-H COSY spectrum, and HMQC spectrum. As a result, it was found that a mixture of the following substances was obtained in Production Example 2. The compositional ratio of 4-TR, 6-TR, 3-TR, and 2-TR in this mixture was 100 parts by mass of 4-TR, 75 parts by mass of 6-TR, 25 parts by mass of 3-TR, and 25 parts by mass of 2-TR. 6-TR (all-trans-retinoic acid is bonded to the carbon atom at position 6 of trehalose) 13cis-6TR (13-cis-retinoic acid is bonded to the carbon atom at position 6 of trehalose) 4-TR (all-trans-retinoic acid is bonded to the carbon atom at position 4 of trehalose) 13cis-4TR (13-cis-retinoic acid is bonded to the carbon atom at position 4 of trehalose) 3-TR (all-trans-retinoic acid is bonded to the carbon atom at position 3 of trehalose) 13cis-3TR (13-cis-retinoic acid is bonded to the carbon atom at position 3 of trehalose) 2-TR (all-trans-retinoic acid is bonded to the carbon atom at position 2 of trehalose) 13cis-2TR (13-cis-retinoic acid is bonded to the carbon atom at position 2 of trehalose)

[0071] The mixture of retinoic acid trehalose isomers obtained in Production Example 2 (hereinafter sometimes abbreviated as MixTR) and 4-TR and 6-TR obtained by separating it were evaluated below.

[0072] <Thermal Stability> 4-TR obtained in Production Example 2 and retinol (RE) were each dissolved in N,N-dimethylformamide (DMF) to a concentration of 3.49 mM. Each solution was then allowed to stand at 50°C for 0 to 192 hours. HPLC analysis was performed on the solutions after standing, and the residual percentage of each component relative to the amount at 0 hours was calculated. The results are shown in Figure 5. The residual percentage after 192 hours was 95.1% for 4-TR and 6.2% for RE. 4-TR had significantly higher thermal stability than RE.

[0073] <Solubility Measurement> Deionized water was added to a 4-TR concentration equivalent to 1%. The resulting solution was subjected to ultrasonic treatment for 10 minutes and then stirred at 30°C and 1,000 rpm for 1 hour using a block bath shaker (manufactured by AS ONE Corporation). The solution was then centrifuged at 15,000 rpm for 5 minutes. The supernatant was recovered from the centrifuged solution, and the solubility was measured by HPLC under the following conditions. The solubility (%) of 4-TR was calculated as the average value of three experiments. The results are shown in Table 7. In Table 7, the solubility results of retinoic acid trehalose obtained in Production Example 1 (solubility in deionized water in Table 1) are shown as the solubility of 6-TR. All compounds exhibited much higher solubility in water than retinoic acid. [HPLC conditions] Column: Develosil RPAQUEOUS-AR-5 (Nomura Chemical Co., Ltd., 250 x 4.6 mm I.D.) x 2 Solvent: 80% acetonitrile Flow rate: 1 mL / min Temperature: 40°C Detection: 354 nm

[0074]

[0075] <Hyaluronic Acid Production> Normal human epidermal keratinocytes (NHEK cells) were seeded onto a 96-well plate at 5,000 cells / well with 100 μl of NHEK cell medium. After 48 hours of culture, the culture supernatant was removed and replaced with medium containing 4-TR, 6-TR, and MixTR at 31.25 nM, 62.5 nM, 125 nM, 250 nM, or 500 nM, 1,000 nM, respectively. After 48 hours of culture, the cell proliferation rate and hyaluronic acid production rate were measured for each sample. Cell proliferation rate was measured using the alamarBlue assay (alamarBlue Cell Viability Reagent, Thermo Fisher Scientific). The amount of hyaluronic acid produced was measured by diluting the culture supernatant 40-fold with phosphate buffer containing 5% polyoxyethylene (20) sorbitan monolaurate and using a Hyaluronan ELISA assay (Hyaluronan DuoSet Kit, manufactured by R&D Systems). The results for cell proliferation rate are shown in Table 8, and the results for hyaluronic acid production are shown in Table 9. 4-TR, 6-TR, and MixTR were each dissolved in ethanol before addition. To perform measurements under conditions where ethanol has minimal effect on cell proliferation, the test was conducted under lower concentration conditions than in the test for hyaluronic acid production using Production Example 1. Increased hyaluronic acid production was also confirmed in samples containing 4-TR, 6-TR, or MixTR, and it was also confirmed that hyaluronic acid production tends to increase in a concentration-dependent manner.

[0076]

[0077]

[0078] <Effect of MixTR on gene expression in NHEK cells> Normal human epidermal keratinocytes (NHEK cells) were cultured at 1.0 × 10 ５Cells were seeded at 1000 cells / well and cultured for 1 day. The culture supernatant was removed, and the medium was replaced with NHEK cell medium containing 1 μM each of RA, RE, and MixTR, and cultured for 24 hours. After culture, RNA was extracted from the cells using an RNeasy Mini kit (Qiagen), and cDNA was synthesized using SuperScript VILO Mastermix (Thermo Fisher Scientific). The expression levels of the following genes were measured by qPCR. qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). - HB-EGF (cell growth factor) - IVL (factor that contributes to the formation of the cornified envelope of the epidermis) - HAS3 (epidermal hyaluronic acid synthesis factor) - AQP3 (aquaporin 3, water / glycerol channel factor) Gene expression levels were corrected by the expression level of an internal standard gene (GAPDH), and then the gene expression levels in cells cultured in the presence of each sample were calculated as relative values, with the average of the control group (Medium) set to 1. The results are shown in Tables 10A, 10B, 11A, and 11B.

[0079]

[0080]

[0081]

[0082]

[0083] As shown in Tables 10A and 10B, MixTR increased the expression of the HB-EGF gene and the IVL gene to the same extent as RE. This result suggests that MixTR has the effect of promoting epidermal cell turnover. As shown in Tables 11A and 11B, MixTR increased the expression of the HAS3 gene and the AQP3 gene to the same extent as RE. This result suggests that MixTR has the effect of moisturizing the skin.

[0084] <Toxic Effect of MixTR on NHEK Cells> Normal human epidermal keratinocytes (NHEK cells) were seeded onto a 96-well plate at 8,000 cells / well with 100 μL of NHEK cell medium. After 24 hours of culture, the culture supernatant was removed and replaced with medium containing RA, RE, or MixTR at 2.5 μM, 5 μM, 10 μM, 20 μM, or 40 μM, respectively. After 24 hours of culture, each sample was evaluated for cell proliferation and cytotoxicity. Cell proliferation was measured using the alamarBlue assay (alamarBlue Cell Viability Reagent, Thermo Fisher Scientific). Cell proliferation was calculated as a ratio of the percentage of the sample containing only medium (Medium) to 100%. Cytotoxicity was measured for the culture supernatant using an LDH assay (Cytotoxicity LDH Assay Kit-WST, Dojindo Laboratories). The results for cell proliferation rate are shown in Table 12, and the results for cytotoxicity are shown in Table 13. RA, RE, or MixTR were each dissolved in ethanol before addition. For comparison, Tables 12 and 13 show the results using a medium containing only ethanol. For samples containing RA, RE, or MixTR at concentrations of 20 μM and 40 μM, the ethanol concentrations in the medium were 1.33% and 2.65%, respectively. Cytotoxicity was observed for RA at concentrations of 20 μM or higher, RE at concentrations of 10 μM or higher, and MixTR at concentrations of 20 μM or higher. In other words, the cytotoxicity of MixTR was lower than that of RE and comparable to that of RA.

[0085]

[0086]

[0087] <Effect of MixTR on gene expression in NHDF cells> Normal human dermal fibroblasts (NHDF cells) were cultured at 1.0 × 10 ５　The cells were seeded at 1000 cells / well and cultured for 1 day. The medium was replaced with NHDF cell medium containing 5 μM each of RA, RE, and MixTR, and cultured for 48 hours. After culture, RNA was extracted from the cells using an RNeasy Mini kit (Qiagen), and cDNA was synthesized using SuperScript VILO Mastermix (Thermo Fisher Scientific). Expression levels of the MMP1 (collagen-degrading enzyme) gene were measured by qPCR. qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Gene expression levels were corrected by the expression level of an internal standard gene (GAPDH), and the average of the control group (Medium) was set to 1. The gene expression levels in cells cultured in the presence of each sample were calculated as relative values. The results are shown in Table 14.

[0088]

[0089] As shown in Table 14, the expression level of the MMP1 gene was significantly reduced in the MixTR-administered group compared to the control group (Medium). This result suggests that MixTR is expected to have the effect of suppressing collagen degradation.

[0090] The present disclosure may include the following embodiments. <1> A carboxylic acid ester compound of trehalose or a derivative thereof and retinoic acid. <2> The compound according to item 1, in which retinoic acid is bonded to the carbon atom at position 4 of trehalose. <3> The compound according to item 1, in which retinoic acid is bonded to the carbon atom at position 6 of trehalose. <4> The compound according to item 1, in which retinoic acid is bonded to the carbon atom at position 3 of trehalose. <5> The compound according to item 1, in which retinoic acid is bonded to the carbon atom at position 2 of trehalose. <6> The compound according to any one of items 1 to 5, in which retinoic acid is all-trans or all-cis. <7> The compound according to any one of items 1 to 6, in which retinoic acid is 7-cis-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, or 13-cis-retinoic acid. <8> A composition comprising, relative to 100 parts by mass of the compound according to item 2, 30 to 300 parts by mass of the compound according to item 3, 20 to 50 parts by mass of the compound according to item 4, and 20 to 50 parts by mass of the compound according to item 5. <9> A hyaluronic acid or collagen production enhancer comprising the compound according to any one of items 1 to 7. <10> A hyaluronic acid or collagen production enhancer comprising the composition according to item 8. <11> A cosmetic or quasi-drug comprising the hyaluronic acid or collagen production enhancer according to item 9 or 10. <12> A skin turnover enhancer comprising the compound according to any one of items 1 to 7. <13> A skin turnover enhancer comprising the composition according to item 8. <14> A cosmetic or quasi-drug comprising the skin turnover enhancer according to item 12 or 13.

Claims

1. A carboxylic acid ester compound of trehalose or its derivatives with retinoic acid.

2. The compound of claim 1, wherein retinoic acid is bound to the carbon atom at position 4 of trehalose.

3. The compound of claim 1, wherein retinoic acid is bound to the carbon atom at position 6 of trehalose.

4. The compound of claim 1, wherein retinoic acid is bound to the carbon atom at position 3 of trehalose.

5. The compound of claim 1, wherein retinoic acid is bound to the carbon atom at position 2 of trehalose.

6. The compound according to any one of claims 1 to 5, wherein the retinoic acid is all-trans or all-cis.

7. The compound of any one of claims 1 to 5, wherein the retinoic acid is 7-cis-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, or 13-cis-retinoic acid.

8. A composition comprising, per 100 parts by mass of the compound according to claim 2, 30 to 300 parts by mass of the compound according to claim 3, 20 to 50 parts by mass of the compound according to claim 4, and 20 to 50 parts by mass of the compound according to claim 5.

9. A hyaluronic acid or collagen production enhancer comprising the compound according to any one of claims 1 to 7.

10. A hyaluronic acid or collagen production enhancer comprising the composition according to claim 8.

11. A cosmetic or quasi-drug containing the hyaluronic acid or collagen production enhancer according to claim 9 or 10.

12. A skin turnover promoter comprising the compound according to any one of claims 1 to 7.

13. A skin turnover promoter comprising the composition according to claim 8.

14. A cosmetic or quasi-drug comprising the skin turnover promoter according to claim 12 or 13.

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