Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
  • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/34—Alcohols
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
  • A61K8/34—Alcohols
  • A61K8/345—Alcohols containing more than one hydroxy group
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/60—Sugars; Derivatives thereof
  • A61K8/602—Glycosides, e.g. rutin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/67—Vitamins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/67—Vitamins
  • A61K8/678—Tocopherol, i.e. vitamin E
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/49—Solubiliser, Solubilising system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/74—Biological properties of particular ingredients

Definitions

• the present invention relates to an aqueous composition and an external preparation for skin.
• This application claims priority based on Japanese Patent Application No. 2022-206769, filed on December 23, 2022, the contents of which are incorporated herein by reference.
• Patent Document 1 describes an antiallergic agent containing tocopheryl glycoside as an active ingredient, and lists tocopheryl glucoside as an example of the tocopheryl glycoside.
• Patent Document 2 discloses a delayed-type hypersensitivity preventive and therapeutic agent, an immunoglobulin E antibody production suppressant, and an anti-inflammatory agent, each of which contains tocopheryl glucoside as an active ingredient.
• an object of the present invention is to provide an aqueous composition of vitamin E glycoside and an external skin preparation containing said aqueous composition.
• aqueous composition comprising a vitamin E glycoside represented by the following general formula (1), 1,2-hexanediol, and water:
• R 1 , R 2 and R 3 each independently represent a hydrogen atom or a methyl group, Rx represents a monosaccharide, and Ry represents a group represented by the following formula (Ry-1) or (Ry-2)]
• a skin topical preparation comprising the aqueous composition according to any one of [1] to [4].
• the present invention provides an aqueous composition of vitamin E glycosides and a skin preparation for external use that contains the aqueous composition.
• the present invention provides an aqueous composition comprising a vitamin E glycoside represented by the following general formula (1), 1,2-hexanediol, and water.
• R 1 , R 2 and R 3 each independently represent a hydrogen atom or a methyl group, Rx represents a monosaccharide, and Ry represents a group represented by the following formula (Ry-1) or (Ry-2)]
• vitamin E glycoside a compound in which a monosaccharide is bound to vitamin E is referred to as vitamin E glycoside.
• Vitamin E is tocopherol and tocotrienol, and may be tocopherol alone, tocotrienol alone, or a mixture of both compounds.
• vitamin E glycoside is a compound in which a monosaccharide is bound to tocopherol or tocotrienol. More specifically, it is a compound in which tocopherol or tocotrienol is bound to the carbon atom at the anomeric position (asymmetric carbon atom) of a monosaccharide.
• vitamin E glycoside is a compound in which tocopherol is bound to the carbon atom at the anomeric position of a monosaccharide (tocopheryl glycoside).
• the vitamin E glycoside is a compound (tocotrienyl glycoside) in which tocotrienol is bound to the anomeric carbon atom of a monosaccharide.
• the vitamin E glycoside may be a mixture of a compound in which tocopherol is bound to the anomeric carbon atom of a monosaccharide and a compound in which tocotrienol is bound to the anomeric carbon atom of a monosaccharide.
• Tocotrienols may be chemically unstable.
• tocopherols are easily available as chemically synthesized products and are more chemically stable. For this reason, when vitamin E glycosides are used in skin external preparations and the like, it is preferable that the main component is tocopheryl glycoside or that the vitamin E glycosides are composed of tocopheryl glycosides.
• Tocopherol is a compound represented by the following general formula (2).
• Tocotrienol is a compound represented by the following general formula (3).
• R 1 , R 2 and R 3 each independently represent a hydrogen atom or a methyl group.
• the tocopherol may be any of ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -tocopherol, and ⁇ -tocopherol.
• the tocotrienol may be any of ⁇ -tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol, and ⁇ -tocotrienol.
• Tocopherol and tocotrienol each have an asymmetric carbon atom at the 2nd position of the chroman ring, so there are d- and l-stereoisomers, as well as the dl-stereoisomer. Tocopherol and tocotrienol may each be any of these stereoisomers. Tocopherol and tocotrienol each have their own unique physiological activities, but because their chemical properties are similar, a mixture of the two is sometimes treated as vitamin E, and products sold as tocopherol may contain small amounts of tocotrienol. Products sold as tocotrienol may also contain small amounts of tocopherol.
• Rx represents a monosaccharide. More specifically, Rx is a monovalent group obtained by removing the hydroxyl group bonded to the carbon atom at the anomeric position from a monosaccharide.
• monosaccharides include glucose, mannose, galactose, fucose, rhamnose, and arabinose.
• the monosaccharide may be in the D-form, the L-form, or a mixture of these.
• the monosaccharide may be in the ⁇ -form or the ⁇ -form.
• Glucose is preferred as the monosaccharide.
• a compound in which vitamin E and glucose are bonded together is called vitamin E glucoside.
• a compound in which tocopherol and glucose are bonded together is called tocopheryl glucoside.
• a compound in which tocotrienol and glucose are bonded together is called tocotrienyl glucoside.
• vitamin E glucoside As a specific example of vitamin E glucoside, the compound in which ⁇ -tocopherol and ⁇ -D-glucose are bonded at the anomeric position is shown in formula (4) below. The compound in which ⁇ -tocotrienol and ⁇ -D-glucose are bonded at the anomeric position is shown in formula (5) below.
• Vitamin E glycosides can be synthesized by reacting tocopherol or tocotrienol with a glycoside derivative by a known reaction. For example, as in the method described in JP 2008-133275 A, tocopherol or tocotrienol is reacted with peracetylated sugar in the presence of a suitable acid catalyst. A derivative is then obtained in which tocopherol or tocotrienol is bonded to the anomeric position of the peracetylated sugar. The acetyl group is then removed from this derivative by a deprotection reaction, thereby synthesizing vitamin E glycosides. When the material used as vitamin E is a mixture of tocopherol and tocotrienol, a mixture of compounds having a structure derived from tocopherol and tocotrienol is obtained as vitamin E glycosides.
• 1,2-Hexanediol may be of a grade that can be used as a cosmetic ingredient, such as that available from Kankosha Co., Ltd. under the trade name KMO-6.
• the water used may be of a grade suitable for use in cosmetics, such as purified water.
• aqueous composition refers to a composition in which a solute is dissolved in a solvent mainly composed of water.
• vitamin E glycoside is dissolved as a solute in a mixed solvent containing at least water and 1,2-hexanediol.
• the proportion of water in the mixed solvent is preferably 60% by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, and particularly preferably 90% by mass or more, based on the total mass of the mixed solvent.
• the water content in the aqueous composition is preferably 80% by mass or more, more preferably 90% by mass or more, and even more preferably 95% by mass or more, based on the total mass of the aqueous composition.
• the water content in the aqueous composition is preferably in the range of 80 to 99.4% by mass, more preferably 90 to 99% by mass, and even more preferably 95 to 98% by mass, based on the total mass of the aqueous composition.
• the content of vitamin E glycoside in the aqueous composition of this embodiment is preferably 0.01 to 2% by mass relative to the total mass of the aqueous composition.
• the content of vitamin E glycoside is more preferably 0.1 to 1.0% by mass, and even more preferably 0.2 to 1.0% by mass.
• the content of 1,2-hexanediol in the aqueous composition of the present embodiment is preferably 0.5 to 20 mass % relative to the total mass of the aqueous composition, more preferably 1 to 10 mass %, and even more preferably 2 to 5 mass %.
• an organic solvent other than 1,2-hexanediol may be contained in the solvent.
• the organic solvent can be appropriately selected depending on the intended use of the aqueous composition.
• the organic solvent is a water-soluble alcohol such as ethanol
• the vitamin E glycoside dissolves sufficiently, and as a result, the physiological activity of the vitamin E glycoside can be fully exerted.
• the amount of 1,2-hexanediol in the aqueous composition is 20% by mass or less, the risk of problems such as irritation to the application site when applied as an external skin preparation is further reduced. Therefore, the content of 1,2-hexanediol in the aqueous composition is preferably 20% by mass or less.
• the aqueous composition of this embodiment can be produced by dissolving vitamin E glycoside in a mixed solvent of water and 1,2-hexanediol.
• an aqueous composition can be obtained by placing a mixed solvent of water and 1,2-hexanediol in a suitable container, adding vitamin E glycoside, and stirring to dissolve the vitamin E glycoside.
• the aqueous composition of this embodiment is usually a colorless and transparent liquid.
• TRPV4 Transient receptor potential vanilloid 4
• aqueous composition containing tocopheryl glucoside as an example of the aqueous composition of this embodiment, and found that the aqueous composition has an excellent expression promoting effect.
• TRPV4 is a protein discovered as an osmosensitive receptor activated by low osmolarity. It has been reported that TRPV4 functions as a water loss sensor and inhibits water loss and enhances skin barrier function by being activated by 4 ⁇ -phorbol ester (4 ⁇ -PDD).
• Tight junctions (TJs) are deeply involved in the skin barrier function. TJs are intercellular adhesion structures formed in a belt shape around epithelial cells.
• TJs bring adjacent epithelial cells into close contact with each other and block the gaps, thereby preventing substances from entering or exiting through the gaps between cells.
• TJs are developed in the skin, trachea, intestinal tract, blood vessels, etc., and perform a barrier function to prevent the penetration of liquids inside the tissue and to prevent foreign substances from entering from the outside.
• Claudins (CLDN) and occludin (OCL) are known as membrane proteins localized in TJs.
• Claudin is known to have 24 members, and among them, claudin-1 (CLDN1) is responsible for strand formation in TJs and plays an important role in the formation of an intercellular barrier in the skin epidermis.
• the aqueous composition of the present embodiment has the effect of promoting the expression of the TRPV4 gene, the CLDN1 gene, and the OCL gene, and increasing the amount of TRPV4, OLDN1, and OCL.
• the aqueous composition of the present embodiment has the effect of suppressing the amount of transepidermal water loss. Therefore, the aqueous composition of the present embodiment can be used as an excellent skin roughness improving agent and/or skin moisturizing agent.
• the present invention provides an external preparation for skin.
• the external preparation for skin of this embodiment contains the aqueous composition.
• the aqueous composition of the present embodiment When used as a skin external preparation, it may contain other components in addition to the aqueous composition.
• other components include pharma- ceutically acceptable carriers.
• pharmaceutical acceptable carrier refers to a carrier that does not inhibit the physiological activity of an active ingredient and does not exhibit substantial toxicity to a subject to which it is administered.
• not substantially toxic means that the component is not toxic to a subject at a dose normally used.
• the pharma- ceutically acceptable carrier is not particularly limited, and examples thereof include excipients, binders, disintegrants, lubricants, stabilizers, diluents, solvents for injections, moisturizers, texture enhancers, surfactants, polymer/thickener/gelling agents, solvents, propellants, antioxidants, reducing agents, oxidizing agents, chelating agents, acids, alkalis, powders, inorganic salts, water, metal-containing compounds, unsaturated monomers, polyhydric alcohols, polymer additives, wetting agents, thickeners, tackifiers, oil-based raw materials, liquid matrices, fat-soluble substances, polymer carboxylates, and the like.
• the pharma- ceutical acceptable carrier may be used alone or in combination of two or more kinds.
• ingredients include preservatives, antibacterial agents, UV absorbers, skin whitening agents, vitamins and their derivatives, anti-inflammatory agents, anti-inflammatory agents, hair growth agents, blood circulation promoters, stimulants, hormones, anti-wrinkle agents, anti-aging agents, tightening agents, cooling agents, warming agents, wound healing promoters, irritation relief agents, analgesics, cell activators, plant, animal and microbial extracts, seed oils, antipruritics, keratin exfoliating and dissolving agents, antiperspirants, cooling agents, astringents, enzymes, nucleic acids, fragrances, dyes, colorants, dyes, pigments, anti-inflammatory and analgesic agents, antifungal agents, antihistamines, hypnotics and sedatives, tranquilizers, antihypertensive agents, antihypertensive diuretics, antibiotics, anesthetics, antibacterial substances, antiepileptic drugs, coronary vasodilators, herbal medicines, antipruri
• the topical skin preparation of this embodiment can be produced by mixing the aqueous composition and, optionally, a pharma- ceutically acceptable carrier and other ingredients and formulating the mixture according to standard methods (e.g., methods described in the Japanese Pharmacopoeia).
• the topical skin preparation of this embodiment may be a cosmetic.
• Types of cosmetic materials include, for example, hair cosmetics such as shampoo, conditioning shampoo, shampoo for dandruff, shampoo for hair color, shampoo with rinse, rinse, treatment, hair foam, hair mousse, hair spray, hair mist, hair gel, water grease, setting lotion, color lotion, hair tonic, hair liquid, hair blow, split end coat, permanent wave agent, straight perm agent, oxidative hair dye, hair bleach, hair color pre-treatment, hair color after-treatment, perm pre-treatment, perm after-treatment, hair manicure, and hair growth agent; facial cleanser, cleansing foam, washing powder, facial cleansing powder, cleanser, etc.
• hair cosmetics such as shampoo, conditioning shampoo, shampoo for dandruff, shampoo for hair color, shampoo with rinse, rinse, treatment, hair foam, hair mousse, hair spray, hair mist, hair gel, water grease, setting lotion, color lotion, hair tonic, hair liquid, hair blow, split end coat, permanent wave agent, straight perm agent, oxidative hair dye, hair bleach
• nourishing milk cleansing lotion, cleansing gel, cleansing mask, lotion, softening lotion, astringent lotion, cleansing lotion, multi-layered lotion, emulsion, emollient lotion, moisture lotion, milky lotion, nourishing lotion, nourishing milk, skin moisture, moisturizing emulsion, massage lotion, cleansing lotion, protective emulsion, sun protect, sun protector, UV care milk, sunscreen, makeup lotion, exfoliating smoother, elbow lotion, hand lotion, body lotion, cream, emollient cream, nourishing cream, nourishing cream, vanishing cream, moisture Basic cosmetics such as makeup cream, night cream, massage cream, cleansing cream, makeup cream, base cream, pre-makeup cream, sunscreen cream, suntan cream, hair removal cream, deodorant cream, shaving cream, keratin softening cream, gel, cleansing gel, moisture gel, soap, cosmetic soap, transparent soap, medicated soap, liquid soap, shaving soap, synthetic cosmetic soap, pack, mask, peel-off pack, powder pack, washing pack, oil pack, cleansing mask, essence, moisturizing essence, whitening essence
• the present invention provides a method for moisturizing skin, improving dry skin, or improving rough skin, comprising the step of applying the aqueous composition to the skin of a subject.
• the present invention provides a method for promoting the production of TRPV4, CLDN1, and OCL1, comprising applying the aqueous composition to the skin of a subject.
• the present invention provides the aqueous composition for moisturizing skin, improving dry skin, or improving rough skin.
• the present invention provides the aqueous composition for promoting the production of TRPV4, CLDN1, and OCL1.
• the present invention provides use of the aqueous composition for producing an external skin preparation for moisturizing skin, improving dry skin, or improving rough skin.
• the present invention provides use of the aqueous composition for producing an agent for promoting the production of TRPV4, CLDN1, and OCL1. In one embodiment, the present invention provides use of the aqueous composition for moisturizing skin, improving dry skin, or improving rough skin. In one embodiment, the present invention provides use of the aqueous composition for promoting the production of TRPV4, CLDN1, and OCL1.
• Example 1 (Confirmation of solubility of tocopheryl glucoside) The solubility of D- ⁇ -tocopheryl glucoside (hereinafter sometimes abbreviated as " ⁇ -TPG”) was investigated. As shown in Tables 1 and 2, water and 1,2-hexanediol (hereinafter sometimes abbreviated as “HD”) or ethanol (hereinafter sometimes abbreviated as “EtOH”) were mixed to prepare mixtures for each example. 9.9 g of each mixture was placed in each vial, and 0.01 g of solid ⁇ -TPG was added to each vial and stirred at room temperature. After stirring for 1 hour, the state of each vial was observed.
• HD 1,2-hexanediol
• EtOH ethanol
• Table 1 shows the results of dissolving 0.01 g of ⁇ -TPG in each mixture of water and HD (Examples 1 to 4).
• Table 2 shows the results of dissolving 0.01 g of ⁇ -TPG in each mixture of water and EtOH (Comparative Examples 1 and 2).
• ⁇ -TPG did not dissolve in water. However, it was completely soluble in EtOH or HD. Using high concentrations of EtOH or HD is not preferable for aqueous compositions. Therefore, the solubility of ⁇ -TPG was examined when these compounds were reduced, that is, in aqueous compositions containing low concentrations of EtOH or HD. In mixtures containing 5% or 10% by mass of EtOH, ⁇ -TPG did not dissolve (Table 2). In contrast, when a mixture was prepared using HD, ⁇ -TPG dissolved even in mixtures with a low HD concentration of about 0.5% by mass, and a clear aqueous composition was obtained (Table 1).
• Example 2 (Confirmation of activity of aqueous composition containing ⁇ -TPG and HD: Expression of TRPV4 gene)
• Normal human epidermal keratinocytes (NHEK, manufactured by Kurashiki Boseki Co., Ltd.) were seeded on a plastic petri dish at a seeding density of 10,000 cells/ cm2 and cultured in serum-free growth medium for normal human epidermal keratinocytes (manufactured by Kurashiki Boseki Co., Ltd.) for 24 hours. 8.94 g of water, 1.0 g of HD, and 0.06 g of ⁇ -TPG were mixed and stirred to obtain an aqueous composition.
• the aqueous composition was added to the culture medium in which the above-mentioned NHEK cells were cultured for 24 hours so that the final concentration of ⁇ -TPG was 10 ⁇ M. After the addition of ⁇ -TPG, the culture solution was further cultured for 48 hours. Then, the cells were irradiated with 60 mJ/ cm2 UVB using a UVB irradiation device manufactured by UVP. Then, fresh medium for growing normal human epidermal keratinocytes was added to the culture solution, and the cells were further cultured for 24 hours. In this manner, the cells for evaluation were obtained.
• the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was quantified as an internal standard gene.
• GAPDH is a housekeeping gene whose expression does not change with the addition of compounds.
• the expression level of the GAPDH gene in NHEK cells was used as the standard to standardize the expression level of the TRPV4 gene.
• Primer GAPDH (ID: HA067812) manufactured by Takara Bio Inc. was used as a primer for amplifying the GAPDH gene.
• Primer TRPV4 (ID: HA186338) manufactured by Takara Bio Inc. was used as a primer for amplifying the TRPV4 gene.
• ⁇ -TPG D- ⁇ -tocopheryl glucoside
• TPA tocopherol acetate
• ⁇ -TP ⁇ -tocopherol
• ⁇ -TP Sigma-Aldrich
• ⁇ -TP ⁇ -tocopherol
• the expression level of the TRPV4 gene was calculated as a relative value when the expression level of the TRPV4 gene in the control cells was set to 1.
• the control cells were prepared by culturing NHEK cells without adding the test compound to the cell culture medium and without irradiating with UVB. HD was added to the cell culture medium of the control cells so that the HD concentration was the same as that in the cell culture medium when the above-mentioned ⁇ -TPG was added and cultured.
• the culture conditions for the NHEK cells were the same as when the above-mentioned ⁇ -TPG was added, except that ⁇ -TPG was not added and UVB irradiation was not performed.
• Comparative cells were prepared by culturing NHEK cells without adding a test compound to the cell culture medium and irradiating UVB.
• HD was added to the cell culture medium of the comparative cells so that the HD concentration in the cell culture medium was the same as that in the cell culture medium when the ⁇ -TPG was added.
• the culture conditions of the comparative cells were the same as those in the case where the ⁇ -TPG was added, except that the ⁇ -TPG was not added.
• the expression level of the TRPV4 gene in the comparative cells was determined by the same method as above.
• aqueous composition containing tocopheryl glucoside but not HD was also prepared, but tocopheryl glucoside was not sufficiently dissolved in the aqueous composition, and therefore, the preparation of cells for evaluation and the measurement of the expression level of the TRPV4 gene were not performed.
• tocopheryl glucoside has the effect of significantly promoting the expression of the TRPV4 gene in cells irradiated with UVB.
• the effect of tocopheryl glucoside in promoting the expression of the TRPV4 gene was significantly higher than the effect of ⁇ -TP, ⁇ -TP, and TPA in promoting the expression of the TRPV4 gene.
• Example 3 (Confirmation of activity of aqueous compositions of ⁇ -TPG and HD: Effect of promoting expression of TJ-related genes)
• Normal human epidermal keratinocytes (NHEK, manufactured by Kurabo Industries, Ltd.) were subjected to the same procedure as in Experimental Example 2 to obtain cells for evaluation.
• Total RNA was extracted from the thus obtained cells for evaluation, and cDNA was synthesized.
• quantitative real-time PCR was performed using the above cDNA as a template to quantify the expression levels of the claudin 1 (CLDN1) gene and the occludin (OCL) gene in the NHEK cells.
• the expression level of GAPDH was quantified as an internal standard gene.
• the expression levels of the CLDN1 gene and the OCL gene were standardized based on the expression level of the GAPDH gene in the NHEK cells.
• a primer for amplifying the GAPDH gene a primer GAPDH (ID: HA067812) manufactured by Takara Bio Inc. was used.
• a primer for amplifying the CLDN1 gene a primer CLDN1 (ID: HA103348) manufactured by Takara Bio Inc. was used.
• a primer OCL ID: HA036753 manufactured by Takara Bio Inc. was used.
• ⁇ -TPG ⁇ -TPG
• TPA ⁇ -TP
• ⁇ -TP ⁇ -TP
• the expression level of each gene was calculated as a relative value when the expression level of each gene in the control cells was set to 1.
• the control cells were prepared in the same manner as the control cells in Experimental Example 2.
• the expression levels of the CLDN1 gene and the OCL gene in the comparative cells were determined by the same method as described above.
• the comparative cells were prepared by the same procedure as the comparative cells in Experimental Example 2.
• An aqueous composition containing tocopheryl glucoside but not HD was also prepared, but tocopheryl glucoside was not sufficiently dissolved in the aqueous composition. Therefore, the preparation of cells for evaluation and the measurement of the expression levels of the CLDN1 gene and the OCL gene were not performed.
• Example 4 TJ formation ability evaluation test
• a skin model (model "EP1-200", manufactured by Kurabo Industries, Ltd.) was pre-cultured for 1 hour under conditions of 37°C and 5% CO2 .
• ⁇ -TPG dissolved at a concentration of 0.2% by mass in a 0.5% by mass HD aqueous solution was added to the surface of the skin model, and the model was further cultured for 24 hours.
• ⁇ -TPG was removed, and the model was washed three times with phosphate buffer, after which the transepidermal water loss (TEWL) was measured.
• TEWL transepidermal water loss
• a cyclone water loss meter manufactured by Nippon Ash Co., Ltd. was used to measure the TEWL.
• ⁇ -TPG dissolved in 0.5% HD by mass
• ⁇ -TP dissolved in 1% HD by mass at a concentration of 0.2% by mass was used, and the same procedure as above was carried out to measure the TEWL.
• each skin model was collected and embedded in paraffin, and tissue slice specimens were prepared using a sectioning device.
• the tissue slice specimen was deparaffinized and dehydrated to obtain a skin tissue sample.
• the skin tissue sample was subjected to immunostaining.
• Rabbit anti-TRPV4 antibody, rabbit anti-occludin antibody, or rabbit anti-claudin 1 antibody (all manufactured by Abcam) was used as the primary antibody.
• Each antibody was diluted with phosphate buffer containing 0.05% by mass Tween-20 at a dilution ratio of rabbit anti-TRPV4 antibody (1:100), rabbit anti-occludin antibody (1:100), or rabbit anti-claudin 1 antibody (1:100).
• horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (manufactured by Abcam) was used after dilution at 1:1000 with phosphate buffer containing 0.05% by mass Tween-20.
• the skin tissue sample was washed. Thereafter, a color reaction was carried out using a peroxidase color-developing substrate, 3,3'-diaminobenzidine tetrahydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). Each skin tissue sample was observed and photographed under an inverted microscope (Nikon TS1). Three independent images were obtained, and quantitative analysis of brightness values correlated with protein expression levels was performed using Image J software (NIH).

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Epidemiology (AREA)
• Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Birds (AREA)
• Dermatology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Emergency Medicine (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• Molecular Biology (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Cosmetics (AREA)
• Saccharide Compounds (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (1)

Family

ID=91588777

Family Applications (1)

Country Status (6)

Cited By (1)

Citations (8)

Family Cites Families (3)

• 2023
  • 2023-12-20 EP EP23907096.4A patent/EP4640207A1/en active Pending
  • 2023-12-20 CN CN202380075331.9A patent/CN120112272A/zh active Pending
  • 2023-12-20 WO PCT/JP2023/045728 patent/WO2024135736A1/ja not_active Ceased
  • 2023-12-20 KR KR1020257019265A patent/KR20250107248A/ko active Pending
  • 2023-12-20 JP JP2024523574A patent/JP7619529B2/ja active Active
  • 2023-12-21 TW TW112149964A patent/TWI874061B/zh active

Patent Citations (8)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events