Methods for evaluating the effectiveness of topical skin preparations

Abstract

To provide a marker for evaluating the effectiveness of an external preparation for skin, and a method for evaluating the effectiveness of an external preparation for skin using the marker and a method for selecting an effective external preparation for skin using the marker.SOLUTION: A method for evaluating the skin scale ameliorating effect of an external preparation for skin is provided, including the step in which on-skin lipids taken from the skin treated with the external preparation for skin are measured for the expression level of at least one selected from the group consisting of following genes: ARAP2, DENR, SPSB3, TMEM50A, SPNS2, and BICD2, and expression products of these genes.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a marker for evaluating the effectiveness of a topical skin preparation, and a method for evaluating the effectiveness of a topical skin preparation using the marker and a method for selecting an effective topical skin preparation.

Background Art

[0002] An important function of the skin is a physical barrier for protecting the body from pathogens, chemicals, and ultraviolet rays. The barrier of the skin is the stratum corneum (horny layer) located in the outermost layer of the epidermis. The stratum corneum has a role 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 anuclear keratinocytes, and is 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 in 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 that leads to the invasion of environmental allergens, immune reactions, and inflammation in atopic dermatitis. On the rough skin surface due to the breakdown of the skin barrier function, the texture is disrupted, and furthermore, scales are generated, resulting in a non-smooth touch. In the scales, the stratum corneum has an appearance as if it is peeled off and turned up on the skin surface. 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 values ​​of the skin surface shape (Surface Evaluation of Living Skin; SELS parameters) using a dedicated camera-equipped probe, 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. However, the wide variety of topical skin preparations makes selection difficult. If the effectiveness of such preparations could be easily evaluated, appropriate preparations could be used according to the skin condition, contributing to the early normalization of skin or improvement of rough skin. However, there is currently no sufficient method for easily evaluating the effectiveness of topical skin preparations based on molecular mechanisms.

[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 2). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2015-227865 [Patent Document 2] 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 project] [Problems that the invention aims to solve]

[0009] The present invention relates to a marker for evaluating the effectiveness of topical skin formulations, a method for evaluating the effectiveness of topical skin formulations using the marker, and a method for selecting effective topical skin formulations. [Means for solving the problem]

[0010] The inventors applied a topical skin preparation containing carbon dioxide, known to have moisture retention and scaling-improving effects, and a placebo preparation without carbon dioxide to either the left or right half of the entire face. After this, SSL (Skin Cell Saturation) was collected from the skin, and the expression status of RNA contained in the SSL was comprehensively analyzed as sequencing information. As a result, the inventors found that the expression level of a specific gene changed significantly in response to the improvement of scaling or increase in stratum corneum moisture content due to the repeated application of the topical skin preparation containing carbon dioxide, and that the effectiveness of the topical skin preparation on scaling or stratum corneum moisture content can be evaluated using the expression level of this specific gene as an indicator.

[0011] In other words, the present invention relates to the following 1) to 7). 1) A method for evaluating the scaling-improving effect of a topical skin preparation, comprising the step of measuring the expression level of at least one selected from the group consisting of the following genes: ARAP2, DENR, SPSB3, TMEM50A, SPNS2, and BICD2, and the expression products of said genes, in skin surface lipids collected from skin to which the topical skin preparation has been applied. 2) A method for selecting a topical skin preparation having a scaling-improving effect, comprising the step of measuring the expression level of at least one selected from the group consisting of the following genes: ARAP2, DENR, SPSB3, TMEM50A, SPNS2, and BICD2, and the expression products of said genes, in skin surface lipids collected from skin to which the topical skin preparation has been applied. 3) A method for evaluating the effect of a topical skin preparation on increasing stratum corneum moisture content, comprising the step of measuring the expression level of at least one selected from the group consisting of the following genes: STT3B, OVCA2, TMED7, LOC100190986, TGM2, VMP1, and ARAP2, and the expression products of said genes, in skin surface lipids collected from skin to which the topical skin preparation has been applied. 4) A method for selecting a topical skin preparation having a stratum corneum water content increasing effect, comprising the step of measuring the expression level of at least one selected from the group consisting of the following genes: STT3B, OVCA2, TMED7, LOC100190986, TGM2, VMP1 and ARAP2, and the expression products of said genes, in skin surface lipids collected from skin to which the topical skin preparation has been applied. 5) A marker for evaluating the scaling-improving effect of a topical skin preparation, comprising at least one selected from the group consisting of the following genes: ARAP2, DENR, SPSB3, TMEM50A, SPNS2, and BICD2, and the expression products of said genes. 6) A marker for evaluating the effect of topical skin preparations on increasing stratum corneum moisture content, comprising at least one selected from the group consisting of the following genes: STT3B, OVCA2, TMED7, LOC100190986, TGM2, VMP1, and ARAP2, and the expression products of said genes. 7) A kit for evaluating the scaling-improving effect of a topical skin preparation or selecting a topical skin preparation having a scaling-improving effect, used in method 1) or 2), which contains 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 evaluating the stratum corneum moisture content-increasing effect of a topical skin preparation or selecting a topical skin preparation having a stratum corneum moisture content-increasing effect, used in method 3) or 4). [Effects of the Invention]

[0012] The marker for evaluating the scaling-improving effect or stratum corneum moisture-increasing effect of the topical skin preparation of the present invention provides an index for evaluating such effects of the topical skin preparation. By using the marker, it becomes possible to easily evaluate the scaling-improving effect or stratum corneum moisture-increasing effect of the topical skin preparation. Therefore, it becomes possible to accurately grasp the effectiveness of the topical skin preparation and provide a topical skin preparation suitable for the skin condition. [Brief explanation of the drawing]

[0013] [Figure 1] A graph showing the relationship between the logarithmic transformation of the RNA expression variation ratio of target genes in SSL and the ΔSEsc value, which is the change in the SEsc value, a scale index. [Figure 2] A graph showing the relationship between the logarithmic transformed value of the RNA expression fluctuation ratio of target genes in SSL and the ΔConductance value, which is the change in Conductance value, 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" includes double-stranded DNA containing human genomic DNA, single-stranded DNA (sense strand) containing cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the sense strand, and fragments thereof, and means those containing some biological information in the sequence information of the bases constituting the DNA. 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., homolog or ortholog), 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 encompassing 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 the protein encoded by the gene, which is synthesized by translation 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 2). 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 improvement of the scaling condition, prevention, suppression or delay of its worsening, or reversal, prevention, suppression or delay of the progression of the scaling condition. 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 by the Skin Evaluation of Scaliness value (SEsc value), which is a parameter that indicates the proportion of pixels with high brightness derived from peeled and flaked stratum corneum to the total number of pixels in the skin surface image, 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" is a scaling improvement effect that corresponds to a decrease in the area occupied by peeled and flaked stratum corneum on the skin surface, preferably a decrease in the SEsc value.

[0020] In this invention, "increase in stratum corneum moisture content" means an increase in the amount of water contained in the stratum corneum. An increase in stratum corneum moisture content 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). In the present invention, the "effect of increasing stratum corneum moisture content" is preferably the effect of increasing stratum corneum moisture content that corresponds to an increase in the Conductance value.

[0021] In the present invention, "topical skin preparation" refers to a preparation applied transdermally to the skin, which contains ingredients that penetrate into the skin and have a biological effect that improves skin function. Preferred topical skin preparations include those containing carbon dioxide as an active ingredient that improves skin function. The formulation form of topical skin preparations is not particularly limited and may include, for example, ointments, emulsions, creams, lotions, gels, aerosols, etc.

[0022] In this invention, "evaluation" can be replaced with terms such as detection, inspection, measurement, or determination. It should be noted that "evaluation," "detection," "inspection," "measurement," or "determination" are performed for non-therapeutic purposes such as cosmetic purposes, and do not include diagnoses made by a physician for therapeutic purposes.

[0023] (1. Marker) As shown in the examples described later, the inventors applied a carbon dioxide-containing topical skin preparation and a carbon dioxide-free placebo preparation to either the left or right half of a subject's entire face repeatedly. Then, they examined the change in the SEsc value, which is a scale index (the value obtained by subtracting the SEsc value before application from the SEsc value after application: ΔSEsc value), and the change in the Conductance value, which is an index of stratum corneum moisture content (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.

[0024] Furthermore, SSL was collected from the skin at the application site before and after repeated application, and the expression status of RNA contained in SSL was comprehensively analyzed as sequencing information. Genes that showed a significant change in expression before and after application at the carbon dioxide-containing formulation application site were extracted, and genes that did not show a significant change in expression before and after application at the placebo formulation application site were extracted. Then, 185 genes common to these two groups were identified as genes that did not show a significant change in expression with the placebo formulation application but showed a significant change in expression with the carbon dioxide-containing formulation application.

[0025] Correlation analysis was performed between the expression changes of the 185 genes before and after application at the application site of the carbon dioxide-containing formulation and the ΔSEsc value at the same site. As a result, nine genes (Table 1) were found to show a significant positive or negative correlation with the ΔSEsc value at the application site of the carbon dioxide-containing formulation.

[0026] [Table 1]

[0027] The expression levels of the nine genes or their expression products shown in Table 1 reflect the scaling-improving effect of topical skin preparations. Therefore, genes or their expression products selected from the gene group shown in Table 1 can be used as evaluation markers, and the scaling-improving effect of topical skin preparations can be evaluated based on their expression levels. Of the nine genes shown in Table 1, the six genes marked with an asterisk (*) and shown in bold—ARAP2, DENR, SPSB3, TMEM50A, SPNS2, and BICD2—are genes that have not been previously reported to be related to scaling. Genes selected from this group, or their expression products, are novel markers for evaluating the scaling-improving effect of topical skin preparations. In the present invention, at least one selected from the group consisting of the six genes or their expression products is used as a marker, and the scaling-improving effect of a topical skin preparation is evaluated based on its expression level, or a topical skin preparation having a scaling-improving effect is selected. Furthermore, in addition to at least one selected from the group consisting of the six genes or their expression products, at least one selected from the group consisting of the three genes LCE3E, LCE3D, and CDSN or their expression products can be used as a marker, and the scaling-improving effect of topical skin preparations can be evaluated based on their expression levels, or topical skin preparations having a scaling-improving effect can be selected.

[0028] Of the nine genes shown in Table 1, seven genes—LCE3E, DENR, SPSB3, LCE3D, CDSN, SPNS2, and BICD2—showed a negative correlation between the ΔSEsc value at the application site of the carbon dioxide-containing formulation and the logarithmic value of the ratio of the expression level after formulation application to the expression level before formulation application (log2(Fold Change)). In other words, since a negative ΔSEsc value is defined as an improvement in scaling, LCE3E, DENR, SPSB3, LCE3D, CDSN, SPNS2, and BICD2 are positive markers that show a positive correlation with the degree of improvement in scaling by topical skin formulations. On the other hand, among the nine genes shown in Table 1, two genes, ARAP2 and TMEM50A, showed a positive correlation between log2 (Fold Change) at the application site of the carbon dioxide-containing preparation and the ΔSEsc value at the application site of the carbon dioxide-containing preparation. In other words, ARAP2 and TMEM50A are negative markers that show a negative correlation with the degree of improvement of scaling by topical skin preparations.

[0029] In the present invention, the six genes ARAP2, DENR, SPSB3, TMEM50A, SPNS2, and BICD2 can each serve as markers to evaluate the scaling-improving effect of topical skin preparations individually. Of these, preferably two or more, more preferably three or more, and even more preferably all six, are used in combination. Furthermore, it is preferable to use a combination of the six genes mentioned above and at least one selected from LCE3E, LCE3D, and CDSN. In the present invention, it is even more preferable to select all nine types shown in Table 1.

[0030] Furthermore, a correlation analysis was performed between the changes in the expression of the 185 genes before and after application at the application site of the carbon dioxide-containing formulation and the ΔConductance value at the same site. As a result, seven genes (Table 2) were found to show a significant positive or negative correlation with the ΔConductance value at the application site of the carbon dioxide-containing formulation.

[0031] [Table 2]

[0032] The expression levels of the seven genes or their expression products shown in Table 2 reflect the effect of topical skin preparations on increasing stratum corneum moisture content. Therefore, by selecting a gene or its expression product from the gene group shown in Table 2, the effect of topical skin preparations on increasing stratum corneum moisture content can be evaluated based on their expression levels. All seven genes shown in Table 2, namely STT3B, OVCA2, TMED7, LOC100190986, TGM2, VMP1, and ARAP2, are genes whose relationship with stratum corneum water content has not been reported to date. Therefore, genes selected from this group of genes, or their expression products, are novel markers for evaluating the effect of topical skin preparations on increasing stratum corneum water content. In the present invention, at least one selected from the group consisting of the seven genes or their expression products is used as a marker, and the effect of a topical skin preparation on increasing stratum corneum moisture content is evaluated based on its expression level, or a topical skin preparation having the effect of increasing stratum corneum moisture content is selected.

[0033] Of the seven genes shown in Table 2, three genes—STT3B, TMED7, and LOC100190986—showed a positive correlation between log2 (Fold Change) at the application site of the carbon dioxide-containing preparation and the ΔConductance value at the application site of the carbon dioxide-containing preparation. In other words, since a positive ΔConductance value is defined as an increase in stratum corneum moisture content, STT3B, TMED7, and LOC100190986 are positive markers that show a positive correlation with the increase in stratum corneum moisture content caused by topical skin preparations. On the other hand, of the seven genes shown in Table 2, four genes—OVCA2, TGM2, VMP1, and ARAP2—showed a negative correlation between log2 (Fold Change) at the application site of the carbon dioxide-containing formulation and the ΔConductance value at the application site of the carbon dioxide-containing formulation. In other words, OVCA2, TGM2, VMP1, and ARAP2 are negative markers that show a negative correlation with the increase in stratum corneum moisture content due to topical skin formulations.

[0034] In the present invention, seven genes, STT3B, OVCA2, TMED7, LOC100190986, TGM2, VMP1, and ARAP2, can each serve individually as markers for evaluating the effect of topical skin preparations on increasing stratum corneum moisture content. Of these, preferably two or more, more preferably three or more, and even more preferably all seven, are used in combination. In the present invention, it is even more preferable to select all seven genes shown in Table 2.

[0035] In one embodiment, the marker of the present invention is a nucleic acid marker such as the DNA of a gene shown in Table 1 or Table 2, 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.

[0036] The genes that can serve as markers for evaluating the scaling-improving effect or stratum corneum moisture-increasing effect of the aforementioned topical skin preparations (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 evaluating the scaling-improving effect or stratum corneum moisture-increasing effect of the topical skin preparations. 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.

[0037] 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, more preferably nucleic acids, and even more preferably mRNA, prepared from the SSL of a subject. 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] (2. Evaluation Method) In another embodiment, the present invention provides a method for evaluating the scaling-improving effect or stratum corneum moisture-increasing effect of a topical skin preparation using the marker of the present invention described in 1. above. The method for evaluating the scaling-improving effect or stratum corneum moisture-increasing effect of a topical skin preparation according to the present invention (hereinafter also referred to as "the method of the present invention") involves measuring the expression level of the marker of the present invention in SSL collected from skin to which the topical skin preparation has been applied, and further evaluating the scaling-improving effect or stratum corneum moisture-increasing effect of the topical skin preparation based on that expression level. In one embodiment, the method of the present invention evaluates the degree of the scaling improvement effect or the stratum corneum moisture content increase effect of a topical skin preparation using the expression level of the marker of the present invention as an indicator. In another embodiment, a topical skin preparation having a scaling-improving effect or a stratum corneum moisture-increasing effect is selected based on the expression level of the marker of the present invention.

[0042] Methods for applying topical skin preparations to the skin include applying the preparation to the skin, taking into consideration the actual environment, and spraying the preparation by dispensing it using an aerosol spray container, a trigger spray container (direct pressure or stored pressure type), etc.

[0043] Topical skin preparations can be applied to the skin by spraying, applying, or other methods, according to any application plan, and may be applied once to several times a day. The application period can be determined as appropriate, for example, from 1 day to 30 days, preferably from 3 days to 20 days, more preferably from 5 days to 10 days, and can be applied repeatedly and continuously.

[0044] (2.1 Analysis of marker expression) The skin used in the method of the present invention is the same as the skin from which SSL containing the marker of the present invention described above is collected. In one embodiment, the method of the present invention may further include a step of collecting SSL from the skin. The procedure for collecting SSL and the procedure for extracting markers from SSL are as described above.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Preferably, the marker used in the method of the present invention is SSL-derived RNA. In this case, the expression level of the RNA contained in the SSL is measured. Preferably, the expression level of the SSL-derived RNA is measured by converting the RNA extracted from the SSL into cDNA by reverse transcription, and then quantifying the cDNA or its amplification product using the method described above.

[0049] For reverse transcription of RNA, primers targeting a specific RNA to be analyzed may be used, but for more comprehensive nucleic acid preservation and analysis, it is preferable to use random primers. For this reverse transcription, a general reverse transcriptase or reverse transcription reagent kit can be used. Preferably, a reverse transcriptase or reverse transcription reagent kit with high accuracy and efficiency is used, examples of which include M-MLV Reverse Transcriptase and its variants, or commercially available reverse transcriptase or reverse transcription reagent kits, such as the PrimeScript® Reverse Transcriptase series (Takara Bio Inc.), the SuperScript® Reverse Transcriptase series (Thermo Scientific Inc.), SuperScript® III Reverse Transcriptase, and the SuperScript® VILO cDNA Synthesis kit (both from Thermo Scientific Inc.) are also suitable. 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.

[0050] When measuring the expression level of nucleic acid markers using PCR, if necessary, the RNA derived from SSL is reverse transcribed into cDNA, and then the SSL-derived DNA 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 (e.g., 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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 with a fluorescent substance, etc., 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).

[0056] 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.

[0057] 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.

[0058] 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 their 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, and may preferably have 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% 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.

[0059] 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.

[0060] (2.2 Evaluation method based on marker expression levels) In one embodiment of the method of the present invention, the scaling-improving effect or the stratum corneum moisture-increasing effect of the topical skin preparation is evaluated based on the expression level of the marker of the present invention (the marker of the present invention contained in SSL collected from the skin) derived from skin to which the topical skin preparation has been applied.

[0061] As described above, at least one marker selected from the group consisting of the nine genes or their expression products shown in Table 1 is a marker whose expression level at the application site of the carbon dioxide-containing formulation fluctuates in accordance with the scaling-improving effect of the carbon dioxide-containing formulation. Therefore, the present invention allows for the evaluation of the scaling-improving effect of topical skin formulations using the expression levels of the markers shown in Table 1 as indicators. In a preferred example of this embodiment, the scaling-improving effect of the topical skin preparation evaluated by the method of the present invention corresponds to a reduction in the proportion of the area occupied by peeled and flaked stratum corneum on the skin surface, for example, a reduction in the SEsc value of the topical skin preparation.

[0062] In this embodiment, the scaling-improving effect of the topical skin preparation can be evaluated by measuring the expression level of the marker of the present invention in SSL collected from skin after application of the topical skin preparation, and comparing the measured expression level of the marker with a preset reference value. In one example, the reference value may be the expression level of the marker of the present invention in a control area where the topical skin preparation is not applied, or in the area where the topical skin preparation is applied before application of the topical skin preparation. The expression level may also be a statistical value (e.g., the average value) of the expression level of the marker of the present invention measured from the skin of a population that has not applied the topical skin preparation. When using multiple types of markers as markers in the present invention, it is preferable to determine a reference value for each of the markers. Groups may be formed based on gender, race, or age. The setting of reference values ​​and the specific methods for evaluating the scaling-improving effect of topical skin preparations based on said reference values ​​can be carried out appropriately in accordance with the common technical knowledge of those skilled in the art.

[0063] In this embodiment, when the marker of the present invention is a positive marker (LCE3E, DENR, SPSB3, LCE3D, CDSN, SPNS2, and BICD2), if the expression level of the positive marker is higher than the reference value, the topical skin preparation is evaluated as having a scaling-improving effect. Furthermore, the higher the expression level is above the reference value, the higher the scaling-improving effect of the topical skin preparation is evaluated as. On the other hand, if the expression level of the positive marker is equal to or lower than the reference value, the topical skin preparation is evaluated as not having an effect on improving scaling. Furthermore, by comparing the expression levels of the positive marker of the present invention when two or more topical skin preparations are applied, it is possible to evaluate the degree of scaling improvement effect among the topical skin preparations. Topical skin preparations with higher expression levels of the positive marker of the present invention are evaluated as having a higher scaling improvement effect. Alternatively, by applying two or more topical skin preparations to a specific subject and comparing the expression levels of the positive marker of the present invention when each is applied, the degree of scaling improvement effect between the topical skin preparations in the subject can be evaluated. A topical skin preparation that shows a higher expression level of the positive marker of the present invention is evaluated as having a higher scaling improvement effect in the subject.

[0064] In this embodiment, when the markers of the present invention are negative markers (ARAP2 and TMEM50A), if the expression level of the negative markers is lower than the reference value, the topical skin preparation is evaluated as having a scaling-improving effect. Furthermore, the lower the expression level is compared to the reference value, the higher the scaling-improving effect of the topical skin preparation is evaluated as. On the other hand, if the expression level of negative markers is equal to or higher than the reference value, the topical skin preparation is evaluated as not having an effect on improving scaling. Furthermore, by comparing the expression levels of the negative marker of the present invention when two or more topical skin preparations are applied, it is possible to evaluate the degree of scaling improvement effect among the topical skin preparations. Topical skin preparations with lower expression levels of the negative marker of the present invention are evaluated as having a higher scaling improvement effect. Alternatively, by applying two or more topical skin preparations to a specific subject and comparing the expression levels of the negative markers of the present invention when each is applied, the degree of scaling improvement effect between the topical skin preparations in the subject can be evaluated. A topical skin preparation with a lower expression level of the negative markers of the present invention is evaluated as having a higher scaling improvement effect in the subject. The marker of the present invention is useful for the relative evaluation of the scaling-improving effects of multiple topical skin preparations.

[0065] Furthermore, at least one marker selected from the group consisting of the seven genes or their expression products shown in Table 2 is a marker whose expression level at the application site of the carbon dioxide-containing preparation fluctuates in accordance with the stratum corneum moisture content-increasing effect of the carbon dioxide-containing preparation. Therefore, the present invention allows for the evaluation of the effect of topical skin formulations on increasing stratum corneum moisture content, using the expression levels of the markers of the present invention shown in Table 2 as indicators. In a preferred example of this embodiment, the effect of the topical skin preparation on increasing stratum corneum moisture content, as evaluated by the method of the present invention, is the effect of increasing stratum corneum moisture content that corresponds to an increase in the Conductance value of the topical skin preparation.

[0066] In this embodiment, the effect of the topical skin preparation on increasing stratum corneum moisture content can be evaluated by measuring the expression level of the marker of the present invention in SSL collected from skin after application of the topical skin preparation, and comparing the measured expression level of the marker with a preset reference value. The reference values ​​are as described above.

[0067] In this embodiment, when the marker of the present invention is a positive marker (STT3B, TMED7, and LOC100190986), if the expression level of the positive marker is higher than the reference value, the topical skin preparation is evaluated to have an effect of increasing stratum corneum moisture content. Furthermore, the higher the expression level is above the reference value, the greater the effect of the topical skin preparation in increasing stratum corneum moisture content is evaluated to be. On the other hand, if the expression level of the positive marker is equal to or lower than the reference value, the topical skin preparation is evaluated as not having an effect on increasing stratum corneum moisture content. Furthermore, by comparing the expression levels of the positive marker of the present invention when two or more topical skin preparations are applied, it is possible to evaluate the degree of the stratum corneum moisture content increase effect between the topical skin preparations. Topical skin preparations with higher expression levels of the positive marker of the present invention are evaluated as having a greater stratum corneum moisture content increase effect. Alternatively, by applying two or more topical skin preparations to a specific subject and comparing the expression levels of the positive marker of the present invention when each is applied, the degree of the effect of each topical skin preparation on increasing stratum corneum moisture content in the subject can be evaluated. A topical skin preparation that shows a higher expression level of the positive marker of the present invention is evaluated as having a greater effect on increasing stratum corneum moisture content in the subject.

[0068] In this embodiment, when the marker of the present invention is a negative marker (OVCA2, TGM2, VMP1, and ARAP2), if the expression level of the negative marker is lower than the reference value, the topical skin formulation is evaluated to have an effect of increasing stratum corneum moisture content. Furthermore, the lower the expression level is compared to the reference value, the higher the effect of the topical skin formulation in increasing stratum corneum moisture content is evaluated to be. On the other hand, if the expression level of the negative marker is the same as or higher than the reference value, the topical skin preparation is evaluated as not having an effect on increasing stratum corneum moisture content. Furthermore, by comparing the expression levels of the negative marker of the present invention when two or more topical skin preparations are applied, it is possible to evaluate the degree of the stratum corneum moisture content increase effect between the topical skin preparations. Topical skin preparations with lower expression levels of the negative marker of the present invention are evaluated as having a higher stratum corneum moisture content increase effect. Alternatively, by applying two or more topical skin preparations to a specific subject and comparing the expression levels of the negative markers of the present invention when each is applied, the degree of the effect of each topical skin preparation on increasing stratum corneum moisture content in the subject can be evaluated. A topical skin preparation with a lower expression level of the negative markers of the present invention is evaluated as having a higher effect on increasing stratum corneum moisture content in the subject. The marker of the present invention is useful for the relative evaluation of the stratum corneum moisture content-increasing effect of multiple topical skin formulations.

[0069] 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 relative to 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 relative to 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 markers are used as target markers, the scaling improvement effect or stratum corneum moisture content increase effect of the topical skin preparation can be evaluated by comparing the expression level of each target marker with the reference value and checking whether the expression level 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 markers differs from the reference value.

[0070] (2.3 Selection of topical skin formulations based on marker expression levels) In the method of the present invention, as described above, the expression level of the marker of the present invention is used as an indicator to evaluate the scaling improvement effect or the stratum corneum moisture content increase effect of the topical skin preparation. As an example, when using the expression level of the marker of the present invention, which is a positive marker, as an indicator, the expression level of the positive marker is measured for SSL collected from skin after application of the topical skin preparation. If the measured expression level of the marker is higher than the reference value, the topical skin preparation is evaluated as having a scaling-improving effect or an effect of increasing stratum corneum moisture content, and the topical skin preparation can be selected as a preparation having a scaling-improving effect or an effect of increasing stratum corneum moisture content. Here, an arbitrary numerical value may be defined for the difference between the measured expression level of the positive marker and the reference value, and the topical skin preparation may be selected as a preparation having a scaling-improving effect or an effect of increasing stratum corneum moisture content only if the measured expression level of the marker is higher than or equal to this defined value. Alternatively, if the expression level of a positive marker at the application site of a topical skin preparation after application is higher than the expression level of a positive marker at the application site of another topical skin preparation after application, then the topical skin preparation is evaluated as having a scaling-improving effect or a stratum corneum moisture-increasing effect, and its effect is considered superior. In such cases, the topical skin preparation can be selected as a preparation with a superior scaling-improving effect or stratum corneum moisture-increasing effect. Alternatively, by applying two or more topical skin preparations to a specific subject and comparing the expression levels of the positive marker of the present invention when each is applied, the topical skin preparation that exhibits a higher expression level of the positive marker of the present invention can be selected as the more suitable topical skin preparation that has a greater effect on improving scaling or increasing stratum corneum moisture content in the subject. As another example, if the expression level of the marker of the present invention, which is a negative marker, is used as an indicator, the expression level of the negative marker is measured for SSL collected from skin after application of the topical skin preparation. If the measured expression level of the marker is lower than the reference value, the topical skin preparation is evaluated as having a scaling-improving effect or an effect of increasing stratum corneum moisture content, and the topical skin preparation can be selected as a preparation having a scaling-improving effect or an effect of increasing stratum corneum moisture content. Here, an arbitrary numerical value may be defined for the difference between the measured expression level of the negative marker and the reference value, and the topical skin preparation may be selected as a preparation having a scaling-improving effect or an effect of increasing stratum corneum moisture content only if the measured expression level of the marker is lower than or equal to this defined value. Alternatively, if the expression level of negative markers at the application site of a topical skin preparation after application is lower than the expression level of negative markers at the application site of another topical skin preparation after application, then the topical skin preparation is evaluated as having a scaling-improving effect or a stratum corneum moisture-increasing effect, and its effect is superior. In such cases, the topical skin preparation can be selected as a preparation with a superior scaling-improving effect or stratum corneum moisture-increasing effect. Alternatively, by applying two or more topical skin preparations to a specific subject and comparing the expression levels of the negative markers of the present invention when each is applied, the topical skin preparation with a lower expression level of the negative markers of the present invention can be selected as the more suitable preparation, indicating a higher effect in improving scaling or increasing stratum corneum moisture content in the subject.

[0071] (3. Evaluation Kit) In a further embodiment, the present invention provides a kit for evaluating the scaling-improving effect or the stratum corneum moisture-increasing effect of a topical skin preparation according to the method of the present invention described in Section 2 above, or for selecting a topical skin preparation having a scaling-improving effect or a stratum corneum moisture-increasing effect. 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 indices 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 for evaluating the scaling-improving effect or stratum corneum moisture-increasing effect of topical skin preparations, or for selecting topical skin preparations that have a scaling-improving effect or a stratum corneum moisture-increasing effect. Furthermore, the kit of the present invention may further include a biological sample collection device (for example, the SSL absorbent material or SSL adhesive material), a reagent for extracting the marker of the present invention from the biological sample (for example, a nucleic acid purification reagent), a preservative for the sample collection device after biological sample collection, a storage container, and the like. [Examples]

[0072] Example 1: Evaluation of the effect of RNA extracted from SSL on improving scales or increasing stratum corneum moisture content. 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-containing formulation). The following human trials were conducted using both of these formulations as test samples.

[0073] [Table 3]

[0074] 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 subjects after observing the skin surface of both cheeks. Twenty subjects were selected based on the presence of scaling on both cheeks. The selected subjects applied the test products, placebo cream and carbon dioxide-containing cream, 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, skin surface images were taken, and sebum (SSL) samples were collected before use and one week after use. On the day of measurement one week after use, the test product was applied two hours before measurement.

[0075] 3) Measurement of physical properties and skin surface Image acquisition 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.

[0076] 4) SSL collection, RNA extraction, pretreatment, sequencing Before using the test product and one week after use, sebum (SSL) was collected from each half of the face (left and right) using an oil-absorbing film (5.0 cm x 8.0 cm, Hakugen Earth Co., Ltd.) before washing the face as described above. After cutting the oil-absorbing film containing the collected SSL into 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.

[0077] 5) Data Analysis The expression levels (read counts) of SSL-derived RNA obtained for each subject before and after application of the test product 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.

[0078] 6) RNA expression analysis Based on the obtained RNA expression levels (normalized count values) derived from SSL before and after application of the test product for each subject, RNAs that met the Wald test result p<0.05 (differentially expressed genes) before and after application of the carbon dioxide-containing formulation were extracted. Subsequently, similarly, RNAs that did not meet the Wald test result p<0.05 (p>=0.05) (undifferentiatedly expressed genes) before and after application of the placebo formulation were extracted. 185 RNAs common to both of these extracted RNAs, i.e., RNAs whose expression did not change with placebo application but changed with carbon dioxide-containing formulation application, were identified. Of these, 97 showed increased expression and 88 showed decreased expression with carbon dioxide-containing formulation application.

[0079] 7) Correlation analysis For 185 RNAs identified by RNA expression analysis, the logarithmic value (Log2(Fold Change)) of the ratio of expression levels after application to expression levels before application on the side coated with the carbon dioxide-containing formulation was calculated. Furthermore, the ΔSEsc and ΔConductance values, which represent the changes in SEsc and Conductance values, were calculated by subtracting the SEsc and Conductance values ​​before application from the SEsc and Conductance values ​​after application on the side where the carbon dioxide-containing formulation was applied. Pearson correlation analysis was performed between Log2 (Fold Change) and ΔSEsc values, and only RNAs satisfying p<0.05 were extracted. The RNAs shown in Table 4 and Figure 1 were extracted. Furthermore, Pearson correlation analysis was performed between Log2 (Fold Change) and ΔConductance values, and only RNAs satisfying p<0.05 were extracted. The RNAs shown in Table 5 and Figure 2 were extracted.

[0080] [Table 4]

[0081] [Table 5]

[0082] As shown in Table 4 and Figure 1, seven genes—LCE3E, DENR, SPSB3, LCE3D, CDSN, SPNS2, and BICD2—were positive markers whose expression levels at the application site of the carbon dioxide-containing formulation showed a negative correlation with the ΔSEsc value at the application site of the carbon dioxide-containing formulation. On the other hand, the two genes ARAP2 and TMEM50A were negative markers, showing a positive correlation between their expression levels at the application site of the carbon dioxide-containing formulation and the ΔSEsc value at the application site. Furthermore, as shown in Table 5 and Figure 2, the three genes STT3B, TMED7, and LOC100190986 were positive markers, showing a positive correlation between their expression levels at the application site of the carbon dioxide-containing formulation and the ΔConductance value at the application site. On the other hand, four genes—OVCA2, TGM2, VMP1, and ARAP2—were negative markers, showing a negative correlation between their expression levels at the application site of the carbon dioxide-containing formulation and the ΔConductance value at the application site.

Claims

1. A method for evaluating the effect of a topical skin preparation on reducing the degree of scaling, wherein the method is: The expression level of ARAP2 or its expression product is measured as a marker in surface lipids collected from skin treated with a topical skin preparation. This includes comparing the expression level of the marker with a predetermined reference value, and evaluating that the topical skin preparation reduces the degree of scaling if the expression level of the marker is lower than the reference value. The reference value is the expression level of the marker contained in surface lipids of the skin collected from the skin before application of the topical skin preparation, in a method.

2. A method for selecting a topical skin preparation having the effect of reducing the degree of scaling, the method being: The expression level of ARAP2 or its expression product is measured as a marker in surface lipids collected from skin treated with a topical skin preparation. This includes comparing the expression level of the marker with a predetermined reference value, and if the expression level of the marker is lower than the reference value, selecting the topical skin preparation as one that has the effect of reducing the degree of scaling. The reference value is the expression level of the marker contained in surface lipids of the skin collected from the skin before application of the topical skin preparation, in a method.

3. The method according to claim 1 or 2, wherein the degree of scaling is the ratio of the area occupied by the peeled and flaked stratum corneum on the skin surface.

4. The method according to any one of claims 1 to 3, wherein the expression level of ARAP2 or its expression product is the expression level of mRNA.

5. A kit for use in the method according to any one of claims 1 to 4, comprising an oligonucleotide that specifically hybridizes with the nucleic acid of ARAP2, or an antibody that recognizes the protein of the expression product of ARAP2.

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