Method for detecting the effectiveness of carbon dioxide preparations

Abstract

To provide a marker for detecting the effectiveness of a carbon dioxide preparation in ameliorating skin scales or horny layer moisture content in an individual, and a method for detecting the effectiveness of a carbon dioxide preparation in a subject using the marker.SOLUTION: A method for detecting the effectiveness of a carbon dioxide preparation in ameliorating a subject's skin scales is provided, including the step in which on-skin lipids taken from the subject are measured for the expression level of at least one selected from the group consisting of following genes: PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4 and SNORD94, and expression products of these genes.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a marker for detecting the effectiveness of a carbon dioxide preparation for improving scale or stratum corneum moisture content in an individual, and a method for detecting the effectiveness of a carbon dioxide preparation in a subject using the marker.

Background Art

[0002] The skin is an organ present in the outermost layer of the living body and is affected by various external environments such as temperature, humidity, ultraviolet rays, and bacteria. An important function of the skin is a physical barrier for protecting the body from these external environments. The barrier of the skin is the stratum corneum (horny layer) located in the outermost layer of the epidermis. The stratum corneum serves as a permeability barrier that delays transdermal evaporative water loss in a dry external environment. The stratum corneum is a multilayered tissue composed of flat anucleate keratinocytes, which are composed of keratinocytes embedded in a highly ordered lipid lamella composed of ceramides, free fatty acids, and cholesterol. The localization of these highly hydrophobic lipids within the extracellular domain of the stratum corneum suppresses the outward movement of water. Furthermore, natural moisturizing factors are present in the stratum corneum. Abnormalities of the stratum corneum associated with epidermal differentiation and lipid composition changes lead to a breakdown of the skin barrier function, which leads to the invasion of environmental allergens, immune responses, and inflammation in atopic dermatitis. On the skin surface with rough skin due to a breakdown of the skin barrier function, the texture becomes disrupted, and furthermore, scales are generated, resulting in a skin feel lacking smoothness. In scales, the stratum corneum peels off on the skin surface and turns up like a flap. Such a state is also called dry skin and can be regarded as a decrease in the water retention ability (moisturizing ability) of the stratum corneum.

[0003] Methods for evaluating skin condition include obtaining objective numerical values ​​for stratum corneum water content (Capacitance and Conductance values) and transepidermal water loss (TEWL values). Other methods include obtaining numerical data on skin surface shape using a dedicated camera-equipped probe (e.g., Surface Evaluation of Living Skin; SELS parameters), creating a replica of the skin surface using impression material and quantifying wrinkles and surface roughness using optical equipment, and visually evaluating the degree of skin dryness and skin roughness based on predetermined indicators by expert panelists.

[0004] Furthermore, methods have been reported for evaluating skin condition based on indicators of nucleic acid and protein expression in biological samples, in addition to quantifying phenotypes (phenotypes) that appear in findings and subjective perceptions using instruments or visual inspection (Patent Document 1 and Non-Patent Document 1). This is expected to lead to highly accurate and efficient evaluation of skin condition and provision of moisturizing technologies based on molecular mechanisms.

[0005] Traditionally, topical skin preparations such as lotions, ointments, and creams have been applied to moisturize the skin and improve rough skin. In recent years, carbon dioxide preparations containing carbon dioxide or carbon dioxide generators have been recognized for their moisture retention and scaling-improving effects, and various developments have been made to utilize these properties (for example, Patent Document 2). However, the skin is considered an organ where individual differences are easily observed, and the effects of topical skin preparations on the skin may also vary in effectiveness from person to person. The maximum effect of topical skin preparations is not necessarily achieved uniformly for all individuals. Therefore, if the effectiveness of topical skin preparations can be easily evaluated in advance, and the appropriate preparation can be selected and used for each individual, it will contribute to the early normalization of skin or improvement of rough skin.

[0006] In recent years, technologies have been developed to investigate the current and future physiological state of the human body by analyzing nucleic acids such as DNA and RNA in biological samples. Nucleic acids derived from living organisms can be extracted from bodily fluids such as blood, secretions, and tissues. More recently, it has been reported that RNA contained in skin surface lipids (SSL) can be used as a sample for biological analysis (Patent Document 3). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2015-227865 [Patent Document 2] Japanese Patent Publication No. 2014-129306 [Patent Document 3] International Public Gazette No. 2018 / 008319 [Non-patent literature]

[0008] [Non-Patent Document 1] Delattre et al. Exp Dermatol. 21:205-210, 2012. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention relates to a marker for detecting the effectiveness of a carbon dioxide formulation for improving scaling or stratum corneum moisture content in individuals, and a method for detecting the effectiveness of a carbon dioxide formulation in subjects using the marker. [Means for solving the problem]

[0010] The inventors collected silicic acid (SSL) from the skin of individuals who showed improvement in scaling and increased stratum corneum moisture content after applying a carbon dioxide-containing formulation, and those who did not, and comprehensively analyzed the RNA expression status contained in the SSL as sequencing information. As a result, they found that the subject's sensitivity to the carbon dioxide formulation was reflected in the expression level of a specific gene in the skin before application of the carbon dioxide formulation, and that the effectiveness of the carbon dioxide formulation in improving scaling or stratum corneum moisture content in the subject could be detected using the expression level of that gene as an indicator.

[0011] In other words, the present invention relates to the following 1) to 5). 1) A method for detecting the effectiveness of a carbon dioxide preparation for improving scaling in a subject, comprising the step of measuring the expression level of at least one selected from the group consisting of the following genes: PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94, and the expression products of said genes, in skin surface lipids collected from the subject. 2) A method for detecting the effectiveness of a carbon dioxide preparation in improving stratum corneum moisture content in a subject, comprising the step of measuring the expression level of at least one selected from the group consisting of the following genes: HERC3, BSG, and KHNYN, and the expression products of said genes, in skin surface lipids collected from the subject. 3) A marker for detecting the effectiveness of carbon dioxide formulations for improving scales, comprising at least one selected from the group consisting of the following genes: PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94, and the expression products of said genes. 4) A marker for detecting the effectiveness of carbon dioxide formulations in improving stratum corneum moisture content, comprising at least one selected from the group consisting of the following genes: HERC3, BSG, and KHNYN, and the expression products of said genes. 5) A kit for detecting the effectiveness of a carbon dioxide preparation for improving scale in a subject used in method 1), comprising an oligonucleotide that specifically hybridizes with the nucleic acid that is the marker, or an antibody that recognizes the protein that is the marker, or a kit for detecting the effectiveness of a carbon dioxide preparation for improving stratum corneum moisture content in a subject used in method 2). [Effects of the Invention]

[0012] According to the present invention, the effectiveness of a carbon dioxide preparation in improving scaling or stratum corneum moisture content in an individual can be easily detected without actually using the carbon dioxide preparation. Therefore, individuals can determine in advance whether a carbon dioxide preparation is effective for their own scaling or stratum corneum moisture content. [Brief explanation of the drawing]

[0013] [Figure 1] Graphs showing (a) the change in SEsc value, an indicator of scale formation, and (b) the change in Conductance value, an indicator of stratum corneum moisture content, for the non-responder group and the responder group. In the graph, "Placebo" represents the side where the placebo formulation was applied, and "Active" represents the side where the carbon dioxide formulation was applied. [Figure 2] A graph showing the relationship between RNA expression levels of target genes in SSL and SEsc values, which are a scale index. [Figure 3] A graph showing the relationship between RNA expression levels of target genes in SSL and Conductance values, which are an indicator of stratum corneum moisture content. [Modes for carrying out the invention]

[0014] All patent, non-patent, and other publications cited herein are incorporated herein by reference in their entirety.

[0015] In the present invention, the terms "nucleic acid" or "polynucleotide" mean DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and "RNA" includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA.

[0016] In the present invention, the term "gene" encompasses double-stranded DNA including human genomic DNA, single-stranded DNA (sense strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the sense strand, and fragments thereof, and means a sequence of bases constituting DNA that contains some biological information. In addition, the "gene" in the present invention includes not only the "gene" represented by a specific base sequence, but also its homologs (i.e., homologs or orthologs), variants such as gene polymorphisms, and derivatives. Here, the names of the genes disclosed in this specification follow the Official Symbol described in NCBI ([www.ncbi.nlm.nih.gov / ]).

[0017] In the present invention, the "expression product" of a gene is a concept that includes the transcription product and translation product of the gene. The "transcription product" is RNA generated by transcription from a gene (DNA), and the "translation product" means a protein encoded by the gene that is translationally synthesized based on RNA.

[0018] In the present invention, "skin surface lipid (SSL)" refers to the lipid-soluble fraction present on the skin surface and is sometimes called sebum. Generally, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin and exists on the skin surface in the form of a thin layer covering the skin surface. SSL contains RNA expressed in skin cells (see Patent Document 3). In the present invention, "skin" is a general term for a region including the stratum corneum, epidermis, dermis, hair follicles, and tissues such as sweat glands, sebaceous glands, and other glands, unless otherwise specified. In the present invention, "skin" is preferably human skin.

[0019] In this invention, "scaling" refers to a condition in which the stratum corneum peels off and lifts from the skin surface due to dryness of the epidermis or keratinization disorders. "Improvement of scaling" refers to the improvement of the symptoms or condition of scaling, prevention, suppression, or delay of its worsening, or the reversal, prevention, suppression, or delay of the progression of the symptoms or condition of scaling. The degree of scaling can be expressed as an indicator of the proportion of the area occupied by peeled and flaked stratum corneum on the skin surface. For example, it can be expressed as the Skin Evaluation of Scaliness value (SEsc value) calculated using the SELS (Surface Evaluation of the Living Skin) program from images acquired by a 2D skin surface texture analyzer (Visioscan VC98, Courage + Khazaka Electronic GmbH). In the present invention, the scaling improvement effect corresponds to a reduction in the area occupied by peeled and flaked stratum corneum on the skin surface, preferably a reduction in the SEsc value.

[0020] In this invention, "stratum corneum moisture content" refers to the amount of water contained in the stratum corneum. An increase in stratum corneum moisture content means an increase in the amount of water contained in the stratum corneum, and includes an increase in water content due to replenishing water in the stratum corneum and an increase in water content due to suppressing the evaporation of water from the stratum corneum. The stratum corneum moisture content can be expressed, for example, by the Capacitance value measured by a Corneometer (Courage+Khazaha) or the electrical conductivity (Conductance) value of the skin surface measured by a SKICON 200-EX (IBS Corporation). In the present invention, the effect of increasing the stratum corneum moisture content is preferably the effect of increasing the stratum corneum moisture content that corresponds to an increase in the Conductance value.

[0021] In the present invention, a "carbon dioxide preparation" is a preparation capable of supplying carbon dioxide to the stratum corneum of the skin. The means of supplying carbon dioxide are not particularly limited and include, for example, aerosol cosmetics containing carbon dioxide as a propellant for topical skin preparations (JP 2014-129306, JP 2017-125003, etc.), effervescent topical skin preparations containing a carbon dioxide generating agent that reacts with water to generate carbon dioxide, nonwoven fabric sheets (JP 2015-105451, etc.), bath additives (JP 9-2942, JP 2000-191429, etc.), and pads (JP 2006-249025, etc.).

[0022] When carbon dioxide is encapsulated as a propellant in a topical skin preparation, it is preferable that the total amount of propellant contains 90% or more carbon dioxide by mass, more preferably 95% or more by mass, even more preferably 98% or more by mass, and even more preferably 100% by mass.

[0023] When preparing an aerosol formulation containing carbon dioxide as a propellant, the mass ratio of the topical skin preparation (undiluted solution) to the propellant containing carbon dioxide is preferably 94:6 to 99.5:0.5, more preferably 95:5 to 99:1, and even more preferably 96.5:3.5 to 98.5:1.5. As for the form of spraying the aerosol formulation, from the viewpoint of allowing the carbon dioxide contained as a propellant to remain on the skin for an extended period, it is preferable to use a foam type in which the topical skin preparation is dispensed in a foamy manner.

[0024] The topical skin preparation used as a concentrate can contain various ingredients commonly used in topical skin preparations such as cosmetics, quasi-drugs, and pharmaceuticals. Specifically, it is preferable to include ingredients such as powders, oils, surfactants, water-soluble thickeners, and water, from the viewpoint of dispensing the topical skin preparation in a foamy form, ensuring good foam spread on the skin, providing a superior user experience, and prolonging the foam to enhance the effect of carbon dioxide.

[0025] Examples of powders include one or more powders selected from silicon dioxide, titanium dioxide, (meth)acrylic acid or its salts / (meth)acrylate crosspolymers, and silicone lastomers, all having an average particle size of 0.01 to 30 μm. (Meth)acrylic acid or its salt / (meth)acrylate crosspolymer is a crosslinked (meth)acrylic acid ester resin powder obtained by copolymerizing at least one monomer selected from (meth)acrylic acid or its salt with at least one monomer selected from (meth)acrylate alkyl esters. (Meth)acrylic acid is a general term for acrylic acid and methacrylic acid, and (meth)acrylate alkyl is a general term for alkyl acrylate and alkyl methacrylate. Specifically, examples include lauryl methacrylate / ethylene glycol dimethacrylate / sodium methacrylate copolymer. Silicone elastomers are a general term for silicones and their derivatives having a crosslinked structure obtained by polymerization of monomers. For example, silicones and their derivatives obtained by polymerization or copolymerization of monomers selected from dimethicone, vinyl dimethicone, phenyl vinyl dimethicone, lauryl dimethicone, and lauryl polydimethylsiloxyethyl dimethicone are preferred, (dimethicone / vinyl dimethicone) crosspolymer, (dimethicone / phenyl vinyl dimethicone) crosspolymer, (vinyl dimethicone / lauryl dimethicone) crosspolymer, (vinyl dimethicone / methicone silsesquioxane) crosspolymer, and (PEG-15 / lauryl dimethicone) crosspolymer are more preferred, (dimethicone / vinyl dimethicone) crosspolymer, (vinyl dimethicone / lauryl dimethicone) crosspolymer, and (PEG-15 / lauryl dimethicone) crosspolymer are even more preferred, and (dimethicone / vinyl dimethicone) crosspolymer is even more preferred. Examples of (dimethicone / vinyl dimethicone) crosspolymers include, as solids, Trefil E-506S (100% effective content by mass) (manufactured by Toray Dow Corning), as mixtures with liquid oil, KSG-15 (7% effective content by mass), a mixture with decamethylcyclopentasiloxane (KF-995), KSG-16 (25% effective content by mass), a mixture with low viscosity dimethylpolysiloxane (KF-96A-6cs) (both manufactured by Shin-Etsu Chemical Co., Ltd.), and Trefil E-508 (70% effective content by mass), a mixture with dimethicone, and as mixtures with water, commercially available products such as BY29-119 (63% effective content by mass) and BY29-129 (63% effective content by mass) can be used. As the (dimethicone / phenylvinyl dimethicone) crosspolymer, commercially available products such as KSG-18A (15% by mass of effective content) (manufactured by Shin-Etsu Chemical Co., Ltd.), which is a mixture with diphenylsiloxyphenyl trimethicone (KF-56A), can be used as a mixture with liquid oil. From the viewpoint of improving the uniform application of carbon dioxide-containing foam to the skin and the penetration of carbon dioxide into the skin, the powder content in the stock solution of the present invention is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, even more preferably 0.8% by mass or more, and preferably 2.5% by mass or less, more preferably 2.0% by mass or less, and even more preferably 1.5% by mass or less. The specific range of content is preferably 0.1 to 2.5% by mass, more preferably 0.5 to 2.0% by mass, and even more preferably 0.8 to 1.5% by mass.

[0026] The oils used are not limited to those commonly used in cosmetics, and examples include hydrocarbon oils, silicone oils, ester oils, ether oils, and fluorinated oils. More specifically, examples include linear or branched hydrocarbon oils such as light isoparaffins, liquid paraffins, liquid isoparaffins, squalane, and squalene; silicone oils such as dimethylpolysiloxane, cyclomethicone, dimethicone, trisiloxane methyltrimethicone, ethyltrisiloxane, dimethylcyclopolysiloxane, methylphenylpolysiloxane, methylhydrogenpolysiloxane, and higher alcohol-modified organopolysiloxanes; and monoesters such as isononyl isononanoate, isotridecyl isononanoate, and alkyl benzoate (C12-15), which is an ester of benzoic acid with an aliphatic alcohol having 12 to 15 carbon atoms. Examples include oils, diester oils such as neopentyl glycol dicaprate, triester oils such as caprylic / capric triglyceride and triglyceryl 2-ethylhexanoate; ether oils such as alkyl-1,3-dimethylbutyl ether, dicaprylyl ether, and dicaprylyl ether; and fluorine oils such as fluoropolyether and perfluoroalkyl ether silicone. From the viewpoint of improving foam spread, one or more selected from hydrocarbon oils, ester oils, and silicone oils are preferred, with hydrocarbon oils, monoester oils, triester oils, and silicone oils being more preferred, and monoester oils and silicone oils being preferred. Regarding the viscosity of the oil, from the viewpoint of improving the penetration of carbon dioxide into the skin, it should be 1 mPa·s or more, preferably 2 mPa·s or more, more preferably 3 mPa·s or more, even more preferably 4 mPa·s or more, 100 mPa·s or less, preferably 50 mPa·s or less, more preferably 30 mPa·s or less, and even more preferably 20 mPa·s or less. Furthermore, the oil should have a viscosity of 1 to 100 mPa·s, preferably 2 to 50 mPa·s, more preferably 3 to 30 mPa·s, and even more preferably 4 to 20 mPa·s. Here, viscosity is measured at 25°C using a BM viscometer (manufactured by Toki Sangyo Co., Ltd.) (rotor No. 1, 60 rpm, 1 minute).

[0027] One or more types of oils can be used, and from the viewpoint of improving the penetration of carbon dioxide into the skin and the feeling of a film on the skin after application, the content of the oils is 1% by mass or more in the undiluted solution, preferably 2% by mass or more, more preferably 4% by mass or more, preferably 25% by mass or less, preferably 15% by mass or less, and more preferably 12% by mass or less. Furthermore, the content of the oils in the undiluted solution is 1 to 25% by mass, preferably 2 to 15% by mass, and even more preferably 4 to 12% by mass.

[0028] Examples of surfactants include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants. However, in the present invention, nonionic surfactants are preferably used from the viewpoint of the amount of carbon dioxide dissolved in the stock solution, the promotion of solubility, and foaming properties during application. The nonionic surfactant used in the present invention is a nonionic surfactant with an HLB of 3 to 20. From the viewpoint of improving the feeling of foam penetration into the skin and the storage stability of the composition, the HLB of the nonionic surfactant is preferably 7 or higher, more preferably 9 or higher, and even more preferably 11 or higher. From the viewpoint of improving the feeling of a film on the skin after application, it is preferably 18 or lower, more preferably 16 or lower, and even more preferably 15 or lower. Furthermore, the HLB of the nonionic surfactant is preferably 7 to 18, more preferably 9 to 16, and even more preferably 11 to 15. Here, HLB (Hydrophilic-Lypophilic Balance) refers to the molecular weight of the hydrophilic group portion of the total molecular weight of a surfactant, and is determined by Griffin's formula. The HLB of a mixed surfactant composed of two or more nonionic surfactants is determined by averaging the HLB values ​​of each nonionic surfactant based on their blending ratio.

[0029] Specific examples of nonionic surfactants include sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, and polyoxyethylene hydrogenated castor oil. Sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene hydrogenated castor oil are preferred, sorbitan fatty acid esters and polyoxyethylene hydrogenated castor oil are more preferred, and polyoxyethylene hydrogenated castor oil is even more preferred. From the viewpoint of improving the penetration of carbon dioxide into the skin and the stability of the foam, polyoxyethylene hydrogenated castor oil preferably has an average number of ethylene oxide additions of 20 to 90, more preferably 30 to 80, and even more preferably 50 to 70.

[0030] Nonionic surfactants can be used individually or in combination of two or more types. From the viewpoint of improving foam spread, carbon dioxide penetration into the skin, and the feeling of a film on the skin after application, their content in the undiluted solution is preferably 0.05% by mass or more, more preferably 0.1% by mass or more, more preferably 0.3% by mass or more, and preferably 6% by mass or less, more preferably 2% by mass or less, and more preferably 1% by mass or less. Furthermore, the content of nonionic surfactants in the undiluted solution is preferably 0.05 to 6% by mass, more preferably 0.1 to 2% by mass, and more preferably 0.3 to 1% by mass.

[0031] Suitable water-soluble thickeners include those commonly used in cosmetics, such as carrageenan, dextrin, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, carboxyvinyl polymer, acrylic acid / alkyl methacrylate copolymer, xanthan gum, carboxymethyl chitin, and chitosan. These have the effect of increasing the viscosity of the undiluted solution, suppressing the rapid generation of foam, and improving stability. Furthermore, from the viewpoint of improving the penetration of carbon dioxide into the skin and the feeling of a film on the skin after application, carboxyvinyl polymer and acrylic acid / alkyl methacrylate copolymer are preferred, and acrylic acid / alkyl methacrylate copolymer is more preferred. Here, the acrylic acid-alkyl methacrylate copolymer is a copolymer of C10-30 alkyl acrylic acid and acrylic acid, methacrylic acid, or their lower alkyl esters, crosslinked with sucrose allyl ether or pentaerythritol allyl ether. Commercial products such as Pemlen TR-1, Pemlen TR-2, Carbopol ETD2020, Carbopol 1342, and Carbopol 1382 (all from Lubrizol Advanced Materials) can be used.

[0032] The water-soluble thickener can be one or more selected from the above, and its content is 0.1% by mass or more in the stock solution, preferably 0.15% by mass or more, more preferably 0.2% by mass or more, 1% by mass or less, preferably 0.8% by mass or less, and more preferably 0.5% by mass or less. Furthermore, the content of the water-soluble thickener is 0.1 to 1% by mass in the stock solution, preferably 0.15 to 0.8% by mass, and more preferably 0.2 to 0.5% by mass.

[0033] Water acts as a solvent, and its concentration in the stock solution is preferably 55% by mass or more, more preferably 65% ​​by mass or more, even more preferably 75% by mass or more, preferably 95% by mass or less, more preferably 93% by mass or less, and even more preferably 90% by mass or less. Furthermore, the water content in the stock solution is preferably 55 to 95% by mass, more preferably 65 to 93% by mass, and even more preferably 75 to 90% by mass.

[0034] From the viewpoint of improving stability, foam discharge, and carbon dioxide penetration into the skin, the viscosity of the stock solution at 25°C is preferably 500 mPa·s or more, more preferably 1000 mPa·s or more, even more preferably 1500 mPa·s or more, preferably 20000 mPa·s or less, more preferably 10000 mPa·s or less, and even more preferably 7000 mPa·s or less. The viscosity of the stock solution at 25°C is preferably 500 to 20000 mPa·s, more preferably 1000 to 10000 mPa·s, and even more preferably 1500 to 7000 mPa·s. Here, viscosity was measured at 25°C using a BM viscometer (manufactured by Toki Sangyo Co., Ltd.) with rotor No. 3, 12 rpm, and 1 minute. If the viscosity exceeded 10,000 mPa·s, the value was measured with rotor No. 3, 6 rpm, and 1 minute.

[0035] Carbon dioxide generating agents used in effervescent topical skin preparations typically consist of a carbon dioxide generating substance and an acidic substance, and these two substances react upon contact with water to generate carbon dioxide. Here, the acidic substance may be either an inorganic acid or an organic acid, and one or more of these may be used. Examples of organic acids include one or more selected from succinic acid, fumaric acid, malic acid, adipic acid, tartaric acid, benzoic acid, citric acid, pyrrolidone carboxylic acid, and salicylic acid. Specifically, examples of inorganic acids include one or more selected from phosphoric acid, boric acid, metasilicic acid, and anhydrous silicic acid. Among these, organic acids are preferred from the viewpoint of ensuring sufficient carbon dioxide generation, more preferably one or more selected from citric acid, malic acid, fumaric acid, succinic acid, and tartaric acid, and even more preferably one or more selected from citric acid, malic acid, and fumaric acid.

[0036] Examples of carbon dioxide generating substances include carbonates, specifically one or more selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, calcium carbonate, magnesium carbonate, and sodium sesquicarbonate. Among these, from the viewpoint of ensuring sufficient carbon dioxide generation, it is preferable that the carbonate be one or more selected from sodium carbonate and sodium bicarbonate.

[0037] Such a carbon dioxide generating agent may contain an acidic substance and a carbon dioxide generating substance in the same agent, or it may contain them separately in multiple agents.

[0038] The dosage form of the effervescent topical skin preparation may be solid, liquid, or gel. If solid, it may be granular, fine granule, or powder. The topical skin preparation may be in the form of a cosmetic, quasi-drug, or pharmaceutical, but it is preferable to use it as a cosmetic or quasi-drug. Specifically, various forms can be given, such as lotion, emulsion, beauty gel, pack, hair growth product, facial cleanser, cleansing product, shampoo, conditioner, etc., and the effervescent topical skin preparation may also be in the form of being supported on a sheet-like material. When using, it is preferable to mix it with a water-containing substance on the palm of the hand, in a container, or on a sheet-like material and use it by effervescence.

[0039] The sheet material is not particularly limited as long as it can support a powdered carbon dioxide generating agent (acidic substance and carbon dioxide generating substance) and can generate a sufficient amount of carbon dioxide within the sheet simply by pouring water or hot water on the sheet before or after application to the skin, but nonwoven fabric sheets are preferred. Specifically, examples include (A) powders selected from organic and inorganic acids, (B) carbonate powders, (C) substances having a melting point of 50°C or higher and 110°C or lower, and being solid at 25°C (for example, polymer compounds such as polyethers such as polyethylene glycol and cellulose derivatives such as hydroxyethylcellulose; higher fatty acids such as myristic acid and palmitic acid; higher alcohols such as cetyl alcohol, hexadecyl alcohol, cetearyl alcohol, and stearyl alcohol; sugars such as glucose and maltose), and (D) nonwoven fabric sheets containing fibers.

[0040] Examples of bath additives include a foaming tablet-type bath additive (Japanese Patent Publication No. 9-2942) containing (a) a solid excipient containing 10% by mass or more of a surfactant and 2 times the mass or more of a carbonate, (b) a carbonate or bicarbonate, and (c) an organic acid, wherein the content of component (a) is 3 to 20% by mass of the bath additive, and a foaming cosmetic (Japanese Patent Publication No. 2000-191429) containing (A) an acid that is solid at room temperature or an inorganic substance that exhibits acidity when dissolved in water, (B) a carbonate, and (C) a cooling agent or warming agent.

[0041] Examples of pads include a pad (Japanese Patent Publication No. 2006-249025) that comprises a water-absorbing base layer, a gas-barrier protective layer provided to cover the base layer, and a carbon dioxide generating agent sealed between the protective layer and the base layer, and that can be attached to the skin at the periphery of the protective layer.

[0042] In the present invention, the term "detection" of the effectiveness of a carbon dioxide preparation may be rephrased as "inspection," "measurement," or "evaluation." The terms "detection," "inspection," "measurement," "determination," or "evaluation" of the effectiveness of a carbon dioxide preparation used herein do not include a diagnosis by a physician.

[0043] (1. Marker) As shown in the examples described later, the inventors used a carbon dioxide formulation containing carbon dioxide and a placebo formulation without carbon dioxide as test products. After transdermally applying them to either the left or right half of each subject's face, the inventors examined the change in SEsc value (a scaling index) (the value obtained by subtracting the SEsc value before application from the SEsc value after application: ΔSEsc value) and the change in Conductance value (a stratum corneum moisture content index) (the value obtained by subtracting the Conductance value before application from the Conductance value after application: ΔConductance value) for the skin at the application site. At this time, using the change in SEsc value and the change in Conductance value as indicators, subjects who showed a relative improvement in scaling or an increase in stratum corneum moisture content with the carbon dioxide formulation compared to those with the placebo formulation were defined as the Responder group, while subjects who did not show improvement in scaling or an increase in stratum corneum moisture content were defined as the non-Responder group. Furthermore, before applying the test product, SSL was collected from the skin at the application site, and the RNA expression status contained in the SSL was comprehensively analyzed as sequencing information. As a result, the expression patterns of 175 genes shown in Table 1 were significantly different between the Responder group and the non-Responder group before the application of the carbon dioxide formulation.

[0044] [Table 1-1]

[0045] [Table 1-2]

[0046] Furthermore, when the discriminant formula (a predictive model that distinguishes between the responder group and the non-responder group) using the 175 genes shown in Table 1 was used to calculate the estimated error rate, it was 15%, meaning the accuracy rate was high at 85%, demonstrating that it is possible to predict the effectiveness of carbon dioxide preparations on the scales or stratum corneum moisture content of subjects. The genes shown in Table 1 are thought to reflect the subject's sensitivity to carbon dioxide preparations in their expression. Therefore, even without actually using carbon dioxide preparations, it is possible to detect the effectiveness of carbon dioxide preparations on the scales or stratum corneum moisture content of subjects based on their expression levels, using genes selected from the gene group shown in Table 1 or their expression products as evaluation markers. Furthermore, based on the detection results, subjects (Responders) whose scales or stratum corneum moisture content improve with the use of carbon dioxide preparations can be selected.

[0047] Of the 175 genes, the 104 genes shown in Table 1A are positive markers with high expression levels in the responder group. On the other hand, the 71 genes shown in Table 1B are negative markers with low expression levels in the responder group.

[0048] Furthermore, as shown in the examples described later, Pearson correlation analysis was performed on the expression levels of the 175 RNAs listed in Table 1 before carbon dioxide preparation application and the ΔSEsc and ΔConductance values ​​at the carbon dioxide preparation application site. As a result, eight genes—PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94—were found to show a significant positive or negative correlation with the ΔSEsc value at the carbon dioxide preparation application site. These eight genes have not been previously reported to be related to scales. Furthermore, it was found that three genes, HERC3, BSG, and KHNYN, showed a significant positive or negative correlation with the ΔConductance value at the application site of the carbon dioxide formulation. These three genes have not been previously reported to be related to stratum corneum moisture content. Furthermore, using a discriminant formula (a predictive model to determine whether a subject is in the responder group or not) that utilizes eight genes—PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94—and three genes—a total of 11 genes, the estimated error rate was 15%, meaning the accuracy rate was a high 85%, demonstrating that it is possible to predict the effectiveness of carbon dioxide preparations on the scales or stratum corneum moisture content of subjects. Therefore, a gene selected from these 11 gene groups, or its expression product, can be used as an evaluation marker, and the effectiveness of the carbon dioxide preparation on the subject's scales or stratum corneum moisture content can be detected based on its expression level. Furthermore, based on the detection results, subjects (Responders) whose scales or stratum corneum moisture content improves with the use of the carbon dioxide preparation can be selected.

[0049] Furthermore, by selecting a gene or its expression product from eight gene groups—PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94—as an evaluation marker, the effectiveness of carbon dioxide formulations, particularly against scaling, can be detected with greater accuracy based on their expression levels. Based on these detection results, responders who are particularly effective in improving scaling can be selected from among those responding to carbon dioxide formulations. Furthermore, by using genes selected from three gene groups—HERC3, BSG, and KHNYN—or their expression products as evaluation markers, the effectiveness of carbon dioxide formulations in subjects, particularly in terms of stratum corneum moisture content, can be detected with greater accuracy based on their expression levels. Based on these detection results, responders who are particularly effective in improving stratum corneum moisture content can be selected from among the responders to carbon dioxide formulations.

[0050] Table 2 shows the 164 genes remaining after excluding eight genes (PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94) and three genes (HERC3, BSG, and KHNYN) from the genes shown in Table 1.

[0051] [Table 2-1]

[0052] [Table 2-2]

[0053] In this invention, at least one of 11 genes or their expression products is selected as a marker, consisting of eight genes (PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94) and three genes (HERC3, BSG, and KHNYN). The effectiveness of a carbon dioxide preparation on scales or stratum corneum moisture content is detected based on its expression level. Based on the detection results, subjects (Responders) whose scales or stratum corneum moisture content improve with the use of the carbon dioxide preparation can be selected. Furthermore, it is possible to detect the effectiveness of the carbon dioxide preparation on the subject's scales or stratum corneum moisture content based on the expression levels of at least one marker selected from the group consisting of the 11 genes or their expression products, along with at least one marker selected from the group consisting of the 164 genes or their expression products shown in Table 2. Based on the detection results, it is also possible to select a responder to the carbon dioxide preparation.

[0054] In the present invention, eight genes—PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94—and three genes—HERC3, BSG, and KHNYN, for a total of eleven genes, can each individually serve as a marker for evaluating the effectiveness of a carbon dioxide formulation on the scales or stratum corneum moisture content of a subject. However, from the viewpoint of improving accuracy, preferably two or more of these, more preferably three or more, even more preferably five or more, and even more preferably all eleven genes are used in combination.

[0055] Furthermore, in this invention, at least one selected from the group consisting of eight genes or expression products of these genes—PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94—is used as a marker, and the effectiveness of the carbon dioxide formulation on subjects, particularly its effectiveness against scaling, is detected with higher accuracy based on its expression level. Based on the detection results, responders to the carbon dioxide formulation who are particularly effective in improving scaling are selected. Furthermore, at least one selected from the group consisting of the eight genes or their expression products, along with three genes (HERC3, BSG, and KHNYN) and at least one selected from the group consisting of the 164 genes or their expression products shown in Table 2, can be used as markers to detect the effectiveness of carbon dioxide formulations in subjects, particularly in terms of scaling improvement, based on their expression levels. Based on these detection results, it is also possible to select responders to carbon dioxide formulations who are particularly effective in improving scaling.

[0056] In the present invention, eight genes, PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94, can each individually serve as markers for evaluating the effectiveness of carbon dioxide preparations on subjects, particularly their effectiveness against scaling. However, from the viewpoint of improving accuracy, preferably two or more of these genes, more preferably three or more, and even more preferably all eight genes are used in combination.

[0057] Furthermore, in this invention, at least one selected from the group consisting of three genes, HERC3, BSG, and KHNYN, or their expression products, is used as a marker, and the effectiveness of the carbon dioxide formulation on subjects, particularly its effectiveness on stratum corneum moisture content, is detected with higher precision based on its expression level. Based on the detection results, responders to the carbon dioxide formulation that are particularly effective in improving stratum corneum moisture content are selected. Furthermore, at least one selected from the group consisting of the three genes or their expression products, along with eight genes (PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94) and at least one selected from the group consisting of the 164 genes or their expression products shown in Table 2, can be used as markers to detect the effectiveness of carbon dioxide formulations in subjects, particularly their effectiveness in improving stratum corneum moisture content, based on their expression levels. Based on these detection results, it is also possible to select responders to carbon dioxide formulations who are particularly effective in improving stratum corneum moisture content.

[0058] In the present invention, the three genes HERC3, BSG, and KHNYN can each serve individually as markers for evaluating the effectiveness of carbon dioxide formulations in subjects, particularly their effectiveness in terms of stratum corneum moisture content. However, from the viewpoint of improving accuracy, preferably two or more of these genes, and more preferably all three, are used in combination.

[0059] In one embodiment, the marker of the present invention is a nucleic acid marker such as the DNA of the gene shown in Table 1, or its transcript, RNA. In another embodiment, the marker of the present invention is a protein marker which is the translation product of the gene. Preferably, the marker of the present invention is a nucleic acid marker.

[0060] The genes that can serve as markers for detecting the effectiveness of carbon dioxide preparations on the scales or stratum corneum moisture content of the aforementioned subjects (hereinafter also referred to as "target genes") include genes that have substantially identical base sequences to the base sequences of the DNA constituting the said gene, insofar as they can serve as biomarkers for detecting the effectiveness of carbon dioxide preparations on the scales or stratum corneum moisture content of the subjects. Here, substantially identical base sequences mean, for example, that when searching using the homology calculation algorithm NCBI BLAST with the conditions expected value = 10; gap allowed; filtering = ON; match score = 1; mismatch score = -3, the base sequences of the DNA constituting the said gene have 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more identity.

[0061] The markers of the present invention can be prepared from SSL (Surface Extract) collected from the skin according to conventional methods. For example, commercially available kits can be used to prepare nucleic acids or proteins from SSL. Preferably, the markers of the present invention are nucleic acids or proteins prepared from the SSL of a subject, more preferably nucleic acids, even more preferably RNA, and even more preferably mRNA. The skin from which SSL is collected can be any animal from which SSL can be collected, but human skin is preferred, and healthy human skin is even more preferred. The parts of the skin from which SSL is collected can be any part of the body, such as the head, face, neck, trunk, hands and feet, and areas with high sebum secretion, such as facial skin, are preferred.

[0062] Any means used for the collection or removal of SSL from the skin can be employed for collecting SSL from the skin. Preferably, SSL absorbent materials, SSL adhesive materials, or instruments for scraping off SSL from the skin, as described later, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has an affinity for SSL, and examples include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include methods of absorbing SSL onto a sheet material such as oil-blotting paper or oil-blotting film, methods of adhering SSL to a glass plate or tape, and methods of scraping off and collecting SSL with a spatula or scraper. To improve the adsorption of SSL, an SSL absorbent material containing a highly lipid-soluble solvent beforehand may be used. On the other hand, since the adsorption of SSL is inhibited if the SSL absorbent material contains a highly water-soluble solvent or water, it is preferable that the content of highly water-soluble solvents or water is low. It is preferable to use the SSL absorbent material in a dry state.

[0063] The collected SSL may be stored for a certain period of time. In order to minimize the degradation of the contained RNA, it is preferable to store the collected SSL under low temperature conditions as quickly as possible after collection. The storage temperature conditions for the RNA-containing SSL in this invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C, even more preferably -20±20°C to -40±20°C, even more preferably -20±10°C to -40±10°C, even more preferably -20±10°C, and even more preferably -20±5°C. The storage period for the RNA-containing SSL under these low temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours to 12 months, more preferably 6 months or less, for example, 1 day to 6 months, and even more preferably 3 months or less, for example, 3 days to 3 months.

[0064] For the extraction of nucleic acids or proteins from SSL, methods commonly used for the extraction or purification of nucleic acids or proteins from biological samples can be used. Examples of nucleic acid extraction or purification methods include the phenol / chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or methods using columns such as TRIzol®, RNeasy®, or QIAzol®, methods using special magnetic particles coated with silica, methods using Solid Phase Reversible Immobilization magnetic particles, and extraction using commercially available RNA extraction reagents such as ISOGEN. For protein extraction or purification, commercially available protein extraction reagents such as QIAzol Lysis Reagent (Qiagen) can be used.

[0065] (2. Evaluation Method) In another embodiment, the present invention provides a method for evaluating the effectiveness of a carbon dioxide formulation on the scales or stratum corneum moisture content of a subject using the marker of the present invention described in 1. above. In the method for evaluating the effectiveness of a carbon dioxide preparation on the scales or stratum corneum moisture content of a subject according to the present invention (hereinafter also referred to as "the method of the present invention"), the expression level of the marker of the present invention is measured for SSL collected from the subject, and the effectiveness of the carbon dioxide preparation on the scales or stratum corneum moisture content of the subject is further evaluated based on the expression level.

[0066] The subjects in this invention include, for example, human or non-human mammals who exhibit a decrease in scale and / or stratum corneum moisture content and who desire or need improvement in scale and / or an increase in stratum corneum moisture content. The subjects are preferably human.

[0067] In one embodiment, the method of the present invention may further include a step of collecting SSL from a subject. The procedure for collecting SSL and the procedure for extracting markers from SSL are as described above.

[0068] (2.1 Analysis of marker expression) The expression level of the marker of the present invention can be measured according to methods for quantifying nucleic acids or proteins commonly used in the art. The expression level of the marker to be measured may be an expression level based on the absolute amount of the marker in SSL, or a relative expression level to other standards or to the expression level of all nucleic acids or all proteins.

[0069] For example, the expression level of nucleic acid markers can be measured according to the gene expression analysis procedures commonly used in the field. Examples of gene expression analysis methods include PCR, multiplex PCR, real-time PCR, hybridization (DNA chip, DNA microarray, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.), sequencing, chromatography, and other methods for quantifying nucleic acids or their amplified products. If the nucleic acid is RNA, it is preferable to convert the RNA to cDNA by reverse transcription before quantifying it using the aforementioned methods.

[0070] The expression level of the protein marker can be measured using protein quantification methods commonly used in the field, such as immunoassays (e.g., Western blotting, ELISA, immunostaining, etc.), fluorescence methods, electrophoresis, protein chips, chromatography, mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS), 1-hybrid methods (PNAS 100, 12271-12276 (2003)), 2-hybrid methods (Biol. Reprod. 58, 302-311 (1998)), etc. Alternatively, the expression level of the marker of the present invention may be measured by measuring molecules that interact with nucleic acids or proteins that are the markers of the present invention. Examples of molecules that interact with the markers of the present invention include DNA, RNA, proteins, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, and their phosphorylated, alkylated, and glycosidic compounds, etc., and complexes of any of the above.

[0071] Preferably, the marker used in the method of the present invention is mRNA derived from SSL. In this case, the expression level of mRNA contained in SSL is measured. Preferably, the mRNA extracted from SSL is converted to cDNA by reverse transcription, and then the expression level of the SSL-derived mRNA is measured by quantifying the cDNA or its amplification product using the method described above.

[0072] For mRNA reverse transcription, primers targeting a specific RNA to be analyzed may be used, but for more comprehensive nucleic acid preservation and analysis, random primers are preferable. General reverse transcriptases or reverse transcription reagent kits can be used for this reverse transcription. Preferably, highly accurate and efficient reverse transcriptases or reverse transcription reagent kits are used, such as M-MLV Reverse Transcriptase and its variants, or commercially available reverse transcriptases or reverse transcription reagent kits, for example, PrimeScript® Reverse Transcriptase series (Takara Bio Inc.), SuperScript® Reverse Transcriptase series (Thermo Scientific Inc.), SuperScript® III Reverse Transcriptase, SuperScript® VILO cDNA Synthesis kit (both from Thermo Scientific Inc.). In the extension reaction during reverse transcription, it is preferable to adjust the temperature to preferably 42°C ± 1°C, more preferably 42°C ± 0.5°C, and even more preferably 42°C ± 0.25°C, while adjusting the reaction time to preferably 60 minutes or more, more preferably 80 to 120 minutes.

[0073] When measuring the expression level of nucleic acid markers using PCR, if necessary, mRNA derived from SSL is reverse transcribed into cDNA, and then the mRNA derived from SSL is amplified using a primer pair. In PCR, only one specific DNA to be analyzed may be amplified using a primer pair that targets that specific DNA, or multiple specific DNAs may be amplified simultaneously using multiple primer pairs. Preferably, the PCR is multiplex PCR. Multiplex PCR is a method of simultaneously amplifying multiple gene regions by using multiple primer pairs simultaneously in the PCR reaction system. Multiplex PCR can be performed using commercially available kits (for example, the Ion AmpliSeqTranscriptome Human Gene Expression Kit; Life Technologies Japan Co., Ltd., etc.). The temperature for the annealing and extension reactions in the PCR is difficult to generalize as it depends on the primers used, but when using the multiplex PCR kit described above, it is preferably 62°C ± 1°C, more preferably 62°C ± 0.5°C, and even more preferably 62°C ± 0.25°C. Therefore, in the PCR, the annealing and extension reactions are preferably performed in one step. The duration of the annealing and extension reaction steps can be adjusted depending on the size of the DNA to be amplified, but is preferably 14 to 18 minutes. The conditions for the denaturation reaction in the PCR can be adjusted depending on the DNA to be amplified, but is preferably 95 to 99°C for 10 to 60 seconds. Reverse transcription and PCR at the above temperatures and times can be performed using a thermal cycler commonly used for PCR.

[0074] The purification of the reaction product obtained by the PCR is preferably carried out by size separation of the reaction product. Size separation allows the target PCR reaction product to be separated from primers and other impurities contained in the PCR reaction solution. DNA size separation can be carried out, for example, by a size separation column, a size separation chip, or magnetic beads suitable for size separation. Preferred examples of magnetic beads suitable for size separation include Solid Phase Reversible Immobilization (SPRI) magnetic beads such as Ampure XP.

[0075] The purified PCR reaction product may be subjected to further processing necessary for subsequent quantitative analysis. For example, the purified PCR reaction product may be prepared into a suitable buffer solution for DNA sequencing, the PCR primer region in the PCR-amplified DNA may be cleaved, or adapter sequences may be further added to the amplified DNA. For instance, the purified PCR reaction product can be prepared into a buffer solution, the amplified DNA can be subjected to removal of PCR primer sequences and adapter ligation, and the resulting reaction product can be amplified as needed to prepare a library for quantitative analysis. These operations can be performed, for example, using the 5×VILO RT Reaction Mix included with the SuperScript® VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.), the 5×Ion AmpliSeq HiFi Mix included with the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.), and the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel, according to the protocols included with each kit.

[0076] When measuring the expression level of nucleic acid markers using real-time PCR, if necessary, the RNA derived from SSL is reverse-transcribed into cDNA, and then PCR is performed using primers that have been pre-labeled with radioisotopes (RI), fluorescent substances, etc. The resulting labeled double-stranded DNA is detected and quantified.

[0077] When measuring the expression level of nucleic acid markers using Northern blot hybridization, for example, SSL-derived RNA is transferred onto a membrane according to a standard procedure, and then probe DNA labeled with an RI, fluorescent substance, etc., is hybridized to the RNA. By detecting the signal derived from the label from the double helix of the formed labeled probe DNA and RNA, the expression level of the nucleic acid marker can be measured.

[0078] When measuring the expression level of nucleic acid markers using DNA microarrays, for example, a microarray is used in which nucleic acids (cDNA or DNA) that specifically hybridize to the target nucleic acid marker are immobilized on a support. After labeling nucleic acids (cDNA or cRNA) prepared from SSL, they are bound to the microarray, and the expression level of the nucleic acid marker in SSL can be measured by detecting the label on the microarray. The nucleic acid immobilized on the microarray can be any nucleic acid that hybridizes specifically to the target nucleic acid marker (i.e., substantially only to the target nucleic acid marker) under stringent conditions, and may be a nucleic acid having the entire sequence of the nucleic acid marker of the present invention, or a nucleic acid consisting of a partial sequence. Examples of such "partial sequences" include nucleic acids consisting of at least 15 to 25 bases. Here, stringent conditions can be washing conditions of approximately "1×SSC, 0.1%SDS, 37°C", preferably conditions of approximately "0.5×SSC, 0.1%SDS, 42°C", and even more preferably conditions of approximately "0.1×SSC, 0.1%SDS, 65°C". Stringent hybridization conditions are described, for example, in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press (2001).

[0079] When measuring the expression levels of nucleic acid markers using sequencing, a next-generation sequencer (e.g., Ion S5 / XL system, Life Technologies Japan Co., Ltd.) can preferably be used. The expression level of DNA or RNA can be measured based on the number of reads (read count) generated by sequencing.

[0080] When measuring the expression levels of multiple nucleic acid markers by sequencing, the aforementioned read count can be used as expression level data. Alternatively, the RPM (Reads per million mapped reads) value of the read count, corrected for differences in the total number of reads between samples in the read count, the logarithm of the RPM value (Log2RPM value, or Log2(RPM+1) value), the count value corrected using DESeq2 (Love MI et al., Genome Biol, 2014) (Normalized count value), or its logarithm (Log2(Normalized count+1) value) can be used as expression level data. Alternatively, as expression level data, commonly used quantitative values ​​for RNA-seq, such as Fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), and transcripts per million (TPM), can be used.

[0081] Probes or primers used for measuring nucleic acid markers may, for example, be primers for specifically amplifying the nucleic acid marker of the present invention, or probes for specifically detecting the nucleic acid marker. Here, "specific" means that the nucleic acid can be recognized or detected in such a way that substantially only the marker of the present invention is detected in Northern blotting, or substantially only the marker of the present invention is amplified in PCR, etc., that a product or detectable substantially derived from the marker of the present invention is produced. These probes or primers can be designed based on the nucleotide sequence of the nucleic acid marker. Specific examples of probes or primers include oligonucleotides or complementary strands consisting of the entire or partial sequence of the nucleic acid marker of the present invention. The "complementary strand" is not limited to a perfectly complementary sequence, as long as it specifically recognizes the target marker; preferably it is a sequence with 80% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity. Sequence identity can be determined by algorithms such as NCBI BLAST described above. Examples of primers used for measuring the nucleic acid marker include those that can perform specific annealing and chain extension for the target nucleic acid marker, and which have a chain length of preferably 10 bases or more, more preferably 15 bases or more, even more preferably 20 bases or more, preferably 100 bases or less, more preferably 50 bases or less, and even more preferably 35 bases or less. Examples of probes used for measuring the nucleic acid marker include those capable of specific hybridization with the target nucleic acid marker, preferably having a chain length of 10 bases or more, more preferably 15 bases or more, preferably 100 bases or less, more preferably 50 bases or less, and even more preferably 25 bases or less. The probe or primer can be DNA or RNA, and may be synthetic or natural. Probes used for hybridization are usually labeled.

[0082] When measuring the expression level of a protein marker using an immunoassay, for example, an antibody against the protein marker can be brought into contact with a biological sample, and the protein marker bound to the antibody can be quantified. For example, in Western blotting, a primary antibody against the protein marker is used, followed by labeling the primary antibody with a secondary antibody labeled with an RI, fluorescent substance, enzyme, etc., and then the expression level of the protein marker can be measured by measuring the signal derived from the label. The antibody against the protein marker may be a polyclonal antibody or a monoclonal antibody. These antibodies can be manufactured according to known methods.

[0083] (2.2 Detection method based on marker expression level) In one embodiment of the present invention, the effectiveness of a carbon dioxide formulation on the scales or stratum corneum moisture content of a subject is detected based on a predictive model constructed using data on the expression levels of the markers of the present invention. For example, by applying a carbon dioxide formulation to any population with a sufficient sample size beforehand, and using the expression level of the target gene or its expression product of the present invention before application as an explanatory variable, and performing machine learning with the objective variable being whether the labeled carbon dioxide formulation is effective for the subject (Responder group) or not (non-Responder group) based on the change in SEsc value (a scaling index obtained by subtracting the SEsc value before application from the SEsc value after application of the carbon dioxide formulation: ΔSEsc value) and the change in Conductance value (a stratum corneum moisture content index obtained by subtracting the Conductance value before application from the Conductance value after application of the carbon dioxide formulation: ΔConductance value) obtained from the population before and after application, an optimal predictive model is constructed to detect the effectiveness of the carbon dioxide formulation on the scaling or stratum corneum moisture content of the subject from the expression level. Here, the labeling of the group as Responder or non-Responder is based on the ΔSEsc and ΔConductance values ​​before and after application of the carbon dioxide preparation. Preferably, a placebo preparation without carbon dioxide components is applied in parallel with the application of the carbon dioxide preparation, and the ΔSEsc and ΔConductance values ​​before and after application of the placebo preparation are calculated as reference values. The group is then labeled as Responder or non-Responder based on a comparison between the carbon dioxide preparation application and the placebo preparation application. Next, based on the constructed predictive model, the effectiveness of the carbon dioxide formulation on the scales or stratum corneum moisture content of a subject can be detected from the expression level of the target gene or its expression product in the subject to be tested. When constructing a predictive model, it is preferable to use a population where the attributes of the target subjects (gender, race, age, etc.) are matched. As for expression levels, it is preferable to use the read count value, which is expression level data, the RPM value obtained by correcting the difference in the total number of reads between samples from the read count value, the value obtained by converting the RPM value to a base-2 logarithm (Log2RPM value) or the base-2 logarithm obtained by adding an integer 1 (log2(RPM+1) value), or the count value corrected using DESeq2 (Love MI et al. Genome Biol. 2014) (Normalized count value) or the base-2 logarithm obtained by adding an integer 1 (log2(Normalized count+1) value) as an indicator. Alternatively, values ​​calculated by fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), transcripts per million (TPM), etc., which are common quantitative values ​​for RNA-seq, may also be used. Furthermore, signal values ​​obtained by microarray methods and their corrected values ​​may also be used. Furthermore, when analyzing only specific target genes using RT-PCR or similar methods, it is preferable to either convert the expression level of the target gene to a relative expression level based on the expression level of housekeeping genes for analysis, or to quantify the absolute copy number (absolute quantification) using a plasmid containing the region of the target gene for analysis. The copy number obtained by digital PCR may also be used. On the other hand, for the ΔSEsc value and ΔConductance value, for example, the natural logarithm value obtained by adding the integer 1 to the measured value (ln(measured value + 1) value) can be used.

[0084] For constructing a prediction model, publicly known algorithms such as those used in machine learning can be utilized. Examples of machine learning algorithms include Random Forest, Support Vector Machine (SVM linear), Support Vector Machine (SVM rbf), Neural Network, Generalized Linear Model, Regularized Linear Discriminant Analysis, Regularized Logistic Regression, and Lasso (Least Absolute Shrinkage and Selection Operator) Regression. By inputting validation data into the constructed prediction model and calculating predicted values, the model whose predicted values ​​best match the observed values, for example, the model with the highest accuracy, can be selected as the optimal prediction model. Additionally, the recall rate, precision, and their harmonic mean (F-score) can be calculated from the predicted and observed values, and the model with the highest F-score can be selected as the optimal prediction model. Furthermore, the root mean square error (RMSE) between the predicted and actual values ​​can be used as an accuracy evaluation metric for the prediction model, and the model with the smallest RMSE can be selected as the optimal prediction model.

[0085] In another embodiment of the method of the present invention, the effectiveness of the carbon dioxide formulation on the scales or stratum corneum moisture content of a subject can be evaluated by measuring the expression level of the marker of the present invention in SSL collected from a subject and comparing the measured expression level of the marker with a preset reference value. In one example, the reference value can be predetermined based on the relationship between the SEsc value, which is a scale index, the Conductance value, which is an index of stratum corneum moisture content, and the expression level of the target gene or its expression product of the present invention. For example, a carbon dioxide preparation is applied to a certain population, and the population is divided into a Responder group and a non-Responder group based on the change in the SEsc value before and after application (the value obtained by subtracting the SEsc value before application from the SEsc value after application of the carbon dioxide preparation: ΔSEsc value) and the change in the Conductance value (the value obtained by subtracting the Conductance value before application from the Conductance value after application of the carbon dioxide preparation: ΔConductance value). The grouping into Responder group and non-Responder group is performed based on the ΔSEsc and ΔConductance values ​​before and after application of the carbon dioxide preparation. Preferably, a placebo preparation without carbon dioxide components is applied in parallel with the application of the carbon dioxide preparation, and the ΔSEsc and ΔConductance values ​​before and after application of the placebo preparation are calculated as reference values. The grouping into Responder group and non-Responder group is then performed by comparing the results of carbon dioxide preparation application and placebo preparation application. Then, values ​​determined by referring to statistical values ​​such as the mean and standard deviation of the expression levels of target genes or their expression products in each group can be set as criteria for determining whether or not a person belongs to each group. When using multiple genes as target genes, it is preferable to determine a reference value for each gene or its expression product. Preferably, the study group consists of subjects whose attributes (gender, race, age, etc.) are consistent. The method for determining the reference value is not particularly restricted and can be determined according to known methods. For example, it can be obtained from an ROC (Receiver Operating Characteristic Curve) curve created using a discriminant formula (predictive model). In an ROC curve, the vertical axis plots the probability (sensitivity) that the carbon dioxide preparation is effective in the responder group, and the horizontal axis plots the value obtained by subtracting the probability (specificity) that the carbon dioxide preparation is not effective in the non-responder group from 1 (false positive rate). Regarding the "true positive (sensitivity)" and "false positive (1-specificity)" shown in the ROC curve, the value (Youden index) at which "true positive (sensitivity)" - "false positive (1-specificity)" is maximized can be used as the reference value.

[0086] In this embodiment, if the marker of the present invention is a positive marker, and the expression level of the measured marker derived from the subject is equal to or higher than that of the Responder group, the subject is evaluated as someone for whom the carbon dioxide formulation is effective in treating scales and / or stratum corneum moisture content; in other words, the carbon dioxide formulation is effective in treating scales and / or stratum corneum moisture content in the subject (shows a scaling improvement effect and a stratum corneum moisture content increase effect). Alternatively, if the expression level of the measured marker derived from the subject is higher than that of the non-Responder group, the subject is similarly evaluated as someone for whom the carbon dioxide formulation is effective in treating scales and / or stratum corneum moisture content. On the other hand, if the expression level of the measured markers derived from the subject is equivalent to or lower than that of the non-responder group, or lower than that of the responder group, the subject will not be evaluated as someone for whom the carbon dioxide preparation is effective in treating scaling and / or stratum corneum moisture content. In other words, the carbon dioxide preparation will be evaluated as ineffective for scaling and / or stratum corneum moisture content in the subject (no effect on scaling improvement, no effect on increasing stratum corneum moisture content).

[0087] In this embodiment, if the marker of the present invention is a negative marker, and the expression level of the measured marker derived from the subject is equal to or lower than that of the Responder group, the subject is evaluated as someone for whom the carbon dioxide formulation is effective in treating scale and / or stratum corneum moisture content; in other words, the carbon dioxide formulation is effective in treating the subject's scale and / or stratum corneum moisture content (shows a scaling improvement effect and a stratum corneum moisture content increase effect). Alternatively, if the expression level of the measured marker derived from the subject is lower than that of the non-Responder group, the subject is similarly evaluated as someone for whom the carbon dioxide formulation is effective in treating scale and / or stratum corneum moisture content. On the other hand, if the expression level of the measured markers derived from the subject is equivalent to or higher than that of the non-responder group, or higher than that of the responder group, the subject will not be evaluated as someone for whom the carbon dioxide preparation is effective in treating scaling and / or stratum corneum moisture content. In other words, the carbon dioxide preparation will be evaluated as ineffective for scaling and / or stratum corneum moisture content in the subject (no effect on scaling improvement, no effect on increasing stratum corneum moisture content).

[0088] According to the present invention, if a carbon dioxide preparation is evaluated as effective against a subject's scales or stratum corneum moisture content (shows an effect of improving scales and increasing stratum corneum moisture content), the subject can be selected as a suitable subject for application of the carbon dioxide preparation. Conversely, if a carbon dioxide preparation is evaluated as ineffective against a subject's scales or stratum corneum moisture content (shows no effect of improving scales and no effect of increasing stratum corneum moisture content), the subject can be excluded from application of the carbon dioxide preparation.

[0089] In one embodiment of the method of the present invention, if the expression level of the marker of the present invention is preferably 91% or less, more preferably 83% or less, and even more preferably 77% or less of the reference value, the expression level of the marker may be judged to be lower than the reference value. If the expression level of the marker of the present invention is preferably 110% or more, more preferably 120% or more, and even more preferably 130% or more of the reference value, the expression level of the marker may be judged to be higher than the reference value. Alternatively, the difference between the expression level of the marker derived from the subject and the reference value can be judged, for example, by whether the two are statistically significant or not. When multiple genes are used as target genes, the effectiveness of the carbon dioxide formulation on the subject's scales or stratum corneum moisture content can be evaluated based on whether the expression levels of a certain percentage, for example, 50% or more, preferably 70% or more, more preferably 90% or more, and even more preferably 100%, of the genes or their expression products meet the standard value.

[0090] (3. Evaluation Kit) In a further embodiment, the present invention provides a kit for evaluating the effectiveness of a carbon dioxide formulation on the scales or stratum corneum moisture content of a subject according to the method of the present invention described in Section 2 above. In one embodiment, the kit of the present invention comprises reagents or instruments for measuring the expression level of the markers of the present invention described above. For example, the kit of the present invention may comprise reagents for amplifying or quantifying the nucleic acid markers of the present invention (e.g., reverse transcriptase, PCR reagents, primers, probes, sequencing adapter sequences, etc.), or reagents for quantifying the protein markers of the present invention (e.g., reagents for immunological measurement, antibodies, etc.). Preferably, the kit of the present invention contains oligonucleotides that specifically hybridize with the nucleic acid markers of the present invention (e.g., primers or probes for PCR), or antibodies that recognize the protein markers of the present invention. Preferably, the kit of the present invention comprises indicators or guidance for evaluating the expression level of the markers of the present invention. For example, the kit of the present invention may comprise guidance explaining reference values ​​for the expression level of each marker in order to evaluate the effectiveness of the carbon dioxide formulation on the scales or stratum corneum moisture content of a subject. The kit of the present invention may further comprise a biological sample collection device (e.g., the SSL absorbent material or SSL adhesive material described above), reagents for extracting the markers of the present invention from a biological sample (e.g., nucleic acid purification reagents), a preservative or storage container for the sample collection device after biological sample collection, etc. [Examples]

[0091] Example 1 1) Test sample Of the formulation components shown in Table 3, components (1), (2), and (15) were stirred at 60°C, and then (10) was mixed and stirred. The remaining components were then added and stirred until homogenized, and cooled to 25°C to obtain the stock solution, which was designated as "placebo cream" (placebo formulation). The obtained stock solution was filled into a pressure-resistant container, sealed, and then filled with carbon dioxide to obtain "carbon dioxide-containing cream" (carbon dioxide formulation). The following human trials were conducted using both of these formulations as test samples.

[0092] [Table 3]

[0093] 2) Human trials This study was conducted in compliance with the ethical principles based on the Declaration of Helsinki. Fifty healthy men aged 25-50 were selected as potential participants for the study. The skin surface of both cheeks was observed, and 20 men who showed scaling on both cheeks were selected as subjects. The selected subjects applied a placebo cream (placebo formulation) and a carbon dioxide-containing cream (carbon dioxide formulation) to either the left or right half of their face twice a day (after washing their face in the morning and evening) for one consecutive week. Subjects were instructed beforehand to apply three pumps of placebo cream and a coin-sized amount of carbon dioxide-containing cream, ensuring that each application amount was 0.5g. Physical properties were measured and skin surface images were taken before use and one week after use. On the day of the measurement one week after use, the test product was applied two hours prior to the measurement.

[0094] 3) Measurement of physical properties and selection of non-responder and responder groups Before using the test product and one week after use, the entire face was washed, and then acclimatized for at least 15 minutes in a variable environment chamber (temperature 20°C ± 2°C, humidity 40 ± 5%). Subsequently, the Conductance, an indicator of stratum corneum moisture content, was measured on the left and right cheeks using SKICON 200-EX (IBS), and magnified images of the skin surface were acquired using Visioscan VC98 (Courage + Khazaka Electronics GmbH). Furthermore, the SEsc value, an indicator of scale, was calculated from the acquired magnified skin surface images using the SELS (Surface Evaluation of the Living Skin) program. The changes in Conductance and SEsc values ​​obtained were calculated before and after application of placebo cream (placebo formulation) and carbon dioxide-containing cream (carbon dioxide formulation), respectively. At this time, subjects who showed greater improvement in at least one of the changes in Conductance or SEsc after application of carbon dioxide-containing cream compared to placebo cream were defined as the Responder group (12 subjects), and subjects who showed no improvement in either were defined as the non-Responder group (8 subjects). Figure 1 shows (a) the change in SEsc value, an index of scale formation, and (b) the change in Conductance value, an index of stratum corneum moisture content, for the non-responder group and the responder group.

[0095] 4) SSL collection, RNA extraction, pretreatment, sequencing Before using the test sample and before washing the face for the aforementioned physical property measurements, sebum (SSL) was collected from both sides of the face using an oil-absorbing film (5.0 cm × 8.0 cm, Hakugen Earth). After cutting the oil-absorbing film containing the collected SSL to an appropriate size, RNA was transferred to the aqueous layer using QIAzol Lysis Reagent (Qiagen) according to the provided protocol. RNA was extracted from the aqueous layer using the RNA extraction spin column of the RNeasy Mini Kit (QIAGEN) according to the provided protocol. The extracted RNA was reverse transcribed using the SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) at 42°C for 90 minutes to synthesize cDNA. Random primers included in the kit were used as primers for the reverse transcription reaction. From the obtained cDNA, a library containing DNA derived from the 20802 gene was prepared by multiplex PCR. Multiplex PCR was performed using the Ion AmpliSeqTranscriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.) under the following conditions: [99°C, 2 min → (99°C, 15 sec → 62°C, 16 min) × 20 cycles → 4°C, Hold]. The obtained PCR products were purified using Ampure XP (Beckman Coulter, Inc.), followed by buffer reconstitution, primer sequence digestion, adapter ligation and purification, and amplification to prepare the library. The prepared library was loaded onto an Ion 540 Chip and sequenced using the Ion S5 / XL system (Life Technologies Japan Co., Ltd.). The genes from which each read sequence originated were determined by gene mapping using the human genome reference sequence, hg19 AmpliSeq Transcriptome ERCC v1.

[0096] 5) Data Analysis The expression levels (read counts) of SSL-derived RNA obtained for each subject before application of the test sample were corrected using the DESeq2 method. However, only genes for which expression levels without missing values ​​were obtained for more than 90% of the subjects were used for the following analysis. The normalized count values ​​corrected using the DESeq2 method were used for the analysis.

[0097] 6) RNA expression analysis Based on the obtained RNA expression levels (Normalized count values) derived from SSL in each subject, 175 genes were extracted from Tables 1A and 1B as RNAs (differentially expressed genes) that satisfied p<0.05 in the Student's t test results in the Responder group and non-Responder group before application of the test material.

[0098] 7) Correlation analysis Pearson correlation analysis was performed on 175 genes identified by RNA expression analysis, comparing their expression levels with the changes in Conductance and SEsc values ​​(ΔConductance and ΔSEsc values) at the carbon dioxide application site. As a result, RNAs with a high correlation to ΔConductance or ΔSEsc values, satisfying p<0.05, were extracted. Table 4 shows the genes correlated with the extracted ΔConductance, and Table 5 shows the genes correlated with the ΔSEsc value. Figures 2 and 3 show scatter plots illustrating the correlation between the expression levels of each correlated gene and the ΔConductance or ΔSEsc value.

[0099] [Table 4]

[0100] [Table 5]

[0101] As shown in Table 4 and Figure 2, eight genes—PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94—were found to show a significant positive or negative correlation with the ΔSEsc value at the carbon dioxide formulation application site. Furthermore, as shown in Table 5 and Figure 3, three genes, HERC3, BSG, and KHNYN, were found to show a significant positive or negative correlation with the ΔConductance value of the carbon dioxide formulation application site.

[0102] 8) Building a predictive model Expression levels of 175 genes, in which expression differences were observed between the Responder and non-Responder groups before application of the test sample, were converted from RPM values ​​following a negative binomial distribution to base-2 logarithms (Log2(RPM+1) values) by adding an integer 1 to approximate a normal distribution. The resulting Log2(RPM+1) values ​​of expression levels were used as explanatory variables, and a predictive model was constructed to determine whether a group belonged to the Responder or non-Responder group as the dependent variable. The random forest algorithm was specified as a method in the "caret" package of the R language, and the optimal value of the number of variables used to construct a decision tree in one iteration (mtry value) was tuned. Using the mtry value determined by tuning, the random forest algorithm was executed, and the estimated error rate (OOB error rate) was calculated.

[0103] The results are shown in Table 6. As shown in Table 6, the estimated error rate was 15% (correct rate 85%), confirming that the effectiveness of the carbon dioxide formulation can be predicted before application based on the expression level data of the 175 genes mentioned above.

[0104] [Table 6]

[0105] Furthermore, using the 11 genes mentioned above that showed a correlation with the change in physical properties among the 175 genes, a predictive model was constructed to distinguish between the responder group and the non-responder group using the same method, and the estimated error rate (OOB error rate) was calculated.

[0106] The results are shown in Table 7. As shown in Table 7, the estimated error rate was 15% (correct rate 85%), confirming that the effectiveness of the carbon dioxide formulation can be predicted before application based on the expression level data of the 11 genes mentioned above.

[0107] [Table 7]

Claims

1. A method for detecting the effectiveness of a carbon dioxide preparation for improving scaling in a subject, wherein the expression level of at least one selected from the group consisting of the following genes: PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4 and SNORD94, and the expression products of said genes, is measured in surface lipids of the skin taken from the subject, wherein detecting the effectiveness of the carbon dioxide preparation allows for determining in advance whether the carbon dioxide preparation has a scaling-improving effect on the subject's own scaling without actually using the carbon dioxide preparation.

2. The method according to claim 1, comprising measuring the expression level in order to select subjects in whom the carbon dioxide preparation is detected to be effective as subjects whose scaling will improve with the use of the carbon dioxide preparation.

3. A method for detecting the effectiveness of a carbon dioxide preparation for improving the stratum corneum moisture content of a subject, wherein the expression level of at least one selected from the group consisting of the following genes: HERC3, BSG, and KHNYN, and the expression products of said genes, is measured in skin surface lipids collected from the subject, wherein detecting the effectiveness of the carbon dioxide preparation allows for determining in advance whether or not the carbon dioxide preparation has an effect on increasing the stratum corneum moisture content of the subject without actually using the carbon dioxide preparation.

4. The method according to claim 3, wherein a subject in whom the carbon dioxide preparation is detected to be effective is selected as a subject whose stratum corneum moisture content will improve by the use of the carbon dioxide preparation, by measuring the expression level.

5. The method according to any one of claims 1 to 4, wherein the expression level of the gene or its expression product is the expression level of mRNA.

6. A marker for detecting the effectiveness of a carbon dioxide preparation for improving scales in a subject, comprising at least one selected from the group consisting of the following genes: PSMA7, SNORA5A, VPS37C, C22orf28, RAPGEF2, NT5C2, HSPA4, and SNORD94, and the expression products of said genes, wherein detecting the effectiveness of the carbon dioxide preparation allows for determining in advance whether the carbon dioxide preparation has an effect on improving the scales of the subject without actually using the carbon dioxide preparation.

7. A marker for detecting the effectiveness of a carbon dioxide preparation for improving stratum corneum moisture content in a subject, comprising at least one selected from the group consisting of the following genes: HERC3, BSG, and KHNYN, and the expression products of said genes, wherein detecting the effectiveness of the carbon dioxide preparation allows for determining in advance whether or not the carbon dioxide preparation has an effect on increasing the stratum corneum moisture content of the subject without actually using the carbon dioxide preparation.

8. A kit for use in the method according to any one of claims 1 to 5, comprising an oligonucleotide that specifically hybridizes with a nucleic acid that is a marker according to claim 6 or 7, or an antibody that recognizes a protein that is a marker according to claim 6 or 7.

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