A inhibitor of VEGF-A signaling-related protein gene expression and a screening method thereof

Abstract

To find new drugs that are effective in improving facial conditions from a novel perspective, such as suppressing the expression of VEGF-A signaling-related protein genes. [Solution] This disclosure provides an inhibitor of VEGF-A signaling-related protein gene expression containing 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof as an active ingredient. Furthermore, this disclosure provides a screening method for VEGF-A signaling-related protein gene expression inhibitors, comprising contacting candidate compounds with skin cells and selecting an inhibitor of VEGF-A signaling-related protein gene expression based on the degree of suppression of VEGF-A signaling-related protein gene expression in the skin cells.

Description

[Technical Field]

[0001] The present invention relates to an inhibitor of VEGF-A signaling-related protein gene expression and a screening method for the same. [Background technology]

[0002] The face is the most important part of the body that influences a person's impression, and swelling (edema) and redness can have a significant impact on others.

[0003] Swollen skin, such as facial swelling upon waking or swollen legs due to fatigue, is not only unhealthy but also a major enemy of beauty. Furthermore, if this swelling is left untreated, it can compress surrounding blood vessels, worsening the swelling and potentially leading to pathological edema.

[0004] The cutaneous vascular system is located in the dermis and consists of blood vessels and lymphatic vessels. To maintain homeostasis, tissue fluid that has moved outside the blood vessels must return to the veins. Skin veins efficiently deliver blood flow to the central nervous system. However, veins themselves have little capacity to take in tissue fluid. From this, it has become clear that tissues that take in tissue fluid, namely lymphatic vessels, are an essential structure in the skin as well.

[0005] Lymphatic vessels play a vital role in maintaining a stable microenvironment around cells by collecting waste products present in the skin and fluids and proteins that constantly leak from blood vessels. When fluids and other substances that leak out of blood vessels are not smoothly collected into the veins or lymphatic fluid and accumulate excessively between cells, this condition is generally called "edema" (swelling).

[0006] While methods for reducing facial swelling, such as massage, are known (Patent Document 1), there are very few ways to quantitatively measure swelling. As a result, it has been difficult to accurately determine how effective such methods are in reducing swelling.

[0007] Rosacea is a symptom that affects the impression one makes on others and is a source of distress for the person experiencing it. Rosacea is a condition in which the redness of the face persists and is mainly caused by skin inflammation or dilation of capillaries beneath the skin. Skin inflammation is caused by conditions such as atopic dermatitis (AD), and the dilation of capillaries beneath the skin causes the skin to turn red.

[0008] Various markers are used to clinically assess the overall severity of atopic dermatitis (AD). However, few markers are known to efficiently indicate the severity of local lesions. Vascular endothelial growth factor (VEGF), a potent activator of vascular permeability, is known to increase in AD lesions, and among these, the VEGF content in the stratum corneum (scVEGF) is known to be usable as a marker to assess the severity of AD lesions (Non-Patent Literature 1). [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2000-119157 [Patent Document 2] Patent No. 5593323 [Patent Document 3] Patent application No. 2023-133570 [Non-patent literature]

[0010] [Non-Patent Document 1] Amarbayasgalan T., et al., Int Arch Allergy Immunol. 2012;157(3):251-8. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] In the method described in Non-Patent Document 1, the VEGF protein in the stratum corneum being measured has extremely low expression levels and stability, and is below the detection limit in many samples, making it difficult to determine the expression level of VEGF protein and whether it is affecting edema.

[0012] In order to improve not only facial redness but also other facial conditions (such as edema), there has been a need for the development of a new method to accurately estimate these conditions. The inventors of this invention have found that the expression of VEGF-A signaling-related protein genes can serve as an indicator of improvement in facial conditions (such as edema) as well as facial redness.

[0013] Therefore, we set a new challenge: to find new drugs that are effective in improving facial conditions from a novel perspective, such as suppressing the expression of VEGF-A signaling-related protein genes. [Means for solving the problem]

[0014] As a result of diligent research by the present inventors, we have newly discovered that 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives act as an inhibitor of the expression of VEGF-A signaling-related protein genes, and have completed the present invention. Incidentally, 1-(2-hydroxyethyl)-2-imidazolidinone is known as a heparanase inhibitor and a laminin 511 expression promoter (Patent Documents 2-3), but it is not known to act as an inhibitor of the expression of VEGF-A signaling-related protein genes. Furthermore, the present invention has developed a screening method that provides a new inhibitor of VEGF-A signaling-related protein gene expression in the skin by using the expression of VEGF-A signaling-related protein genes as an indicator. In other words, the present invention includes the following:

[0015] [1] An inhibitor of VEGF-A signaling-related protein gene expression, containing 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof as an active ingredient. [2] The inhibitor according to item 1, wherein the 1-(2-hydroxyethyl)-2-imidazolidinone or its derivative contains a cyclic carboxamide derivative represented by the following general formula (1) or a salt thereof as an active ingredient. The inhibitor according to item 1. [Chemical formula] (However, in the general formula (1), n represents an integer of 1 to 3, and R , , represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group, X represents a group represented by -CH2- or -N(R 2 )-, and R 2 represents a hydrocarbon group having 1 to 6 carbon atoms which may be substituted with a hydrogen atom or a hydroxyl group.) [3] The inhibitor according to item 1 or 2, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes described in Table 1 below. [Table 1] [4] The inhibitor according to item 1 or 2, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes described in Table 2 below. [Table 2] [5] The inhibitor according to any one of items 1 to 4, which inhibits VEGF-A signaling in the epidermis. [6] The inhibitor according to any one of items 1 to 4, which is a skin external preparation. [7] A cosmetic characterized by containing the inhibitor according to any one of items 1 to 5.

[0016] [8] A method for screening an inhibitor of the expression of a VEGF-A signaling-related protein gene, comprising: contacting a candidate compound with skin cells, and selecting an inhibitor of the expression of the VEGF-A signaling-related protein gene using the degree of inhibition of the expression level of the VEGF-A signaling-related protein gene in the skin cells as an index. [9] The method according to item 8, wherein one or more VEGF-A signaling-related protein genes are selected from the genes listed in Table 3 below. [Table 3]

[10] The method according to item 8, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 4 below. [Table 4] [Effects of the Invention]

[0017] This disclosure provides an inhibitor of the expression of novel VEGF-A signaling-related protein genes. For example, by applying it, it is possible to improve facial conditions such as redness or facial edema (e.g., physiological edema). [Brief explanation of the drawing]

[0018]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 5

Figure 6

[0019] The following describes in detail one embodiment of the present invention, but the scope of the present invention is not limited to the embodiment described herein, and various modifications can be made without departing from the spirit of the invention. Furthermore, if multiple upper and lower limits are given for a particular parameter, any combination of these upper and lower limits can be used to create a suitable numerical range.

[0020] <Inhibitor of expression of VEGF-A signaling-related protein gene> In one embodiment, the present invention provides an inhibitor of VEGF-A signaling-related protein gene expression containing 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof as an active ingredient.

[0021] In this specification, "suppressant of VEGF-A signaling-related protein gene expression" means a drug that, when applied to a target requiring such suppression, can suppress the expression of VEGF-A signaling-related protein genes. The inventors of this application have for the first time discovered that 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives act as a drug that can suppress the expression of "VEGF-A signaling-related protein genes".

[0022] VEGF-A protein is one of the vascular endothelial growth factors (VEGF). VEGF is known as a major angiogenic growth factor that regulates neovascularization. The biological effects of VEGF are mediated through specific VEGF receptors on the surface of endothelial cells. There are VEGF-A, VEGF-B, VEGF-C, and VEGF-D proteins, which mediate various reactions in the body by interacting with VEGF receptors such as VEGFR1, VEGFR2, and VEGFR3. Among these, VEGF-A / VEGFR2 signaling is known to significantly mediate cellular responses involved in angiogenesis.

[0023] The inhibitor of VEGF-A signaling-related protein gene expression provided by the present invention can, by suppressing the expression of VEGF-A signaling-related protein gene, treat or alleviate symptoms, diseases, or conditions in which overexpression of VEGF-A signaling-related protein gene is one of the causes. For example, since the expression of VEGF-A signaling-related protein gene affects facial edema (swelling) and redness, these symptoms can be treated or alleviated by applying the inhibitor of VEGF-A signaling-related protein gene expression provided by the present invention.

[0024] In one embodiment, the inhibitor of the present invention can alleviate edema, particularly physiological edema, by suppressing the expression of VEGF-A signaling-related protein genes. In this specification, "physiological edema" refers to a condition in which the face is swollen in a healthy state and is distinguished from a condition in which edema occurs in the face due to disease or injury.

[0025] In this specification, "VEGF-A signaling-related protein genes" refers to a group of genes that express proteins related to VEGF-A / VEGFR2 signaling, for example, genes that express human VEGF-A / VEGFR2 signaling-related proteins (154 types of proteins) as described at https: / / www.wikipathways.org / pathways / WP3888.html (accessed July 31, 2024). Among these, in the invention of this embodiment, it is preferable that the genes are selected from one or more genes that express the proteins listed in Table 5 below. [Table 5]

[0026] The specific amino acid sequences of VEGF-A signaling-related proteins or the sequences of the genes encoding them listed in this specification can be obtained, for example, by searching on UniProt (https: / / www.uniprot.org / ) by entering the abbreviation of the gene to be searched or the ID of the Uniprot Primary accession. The specific gene sequences that can be used in the present invention are not limited to the amino acid sequences or nucleic acid sequences of the genes encoding them that can be searched and obtained using the IDs listed in the Uniprot Primary accession in Table 5. For example, sequences that are 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identical to the amino acid sequences or nucleic acid sequences of the genes encoding them that can be obtained using the Primary accession in Table 5, as well as their splicing variants. Furthermore, the origin of the gene may be from any animal, but it is preferably from humans or other mammals (e.g., non-human primates), and most preferably from humans.

[0027] In this specification, "suppression of gene expression" means that the expression level of the target gene is reduced to a predetermined expression level, or that the expression level of the corresponding gene in the control is reduced.

[0028] "Genetic expression level" refers to a level that can be determined by detecting and measuring transcripts transcribed from any given gene, or translation products translated using those transcripts as a template. A transcript refers to, for example, an RNA chain transcribed using the DNA of a gene as a template, i.e., an RNA chain synthesized by RNA polymerase, and an RNA chain modified within the cell after transcription. An example of an RNA chain included in a transcript is messenger RNA (mRNA). These RNA chains also include those processed within the cell after transcription. A translation product refers to, for example, a polypeptide chain translated using a transcript transcribed by a gene as a template, i.e., a polypeptide chain synthesized by ribosomes, and a protein formed by the folding of that polypeptide chain. Translation products also include polypeptide chains or protein fragments.

[0029] Gene expression levels can be measured using known methods. For example, when measuring gene transcripts to determine gene expression levels, appropriate probes can be designed based on the sequence of the target gene, and then measured using methods such as quantitative PCR (qPCR), in situ hybridization, Northern blotting, DNA microarrays, and next-generation sequencing.

[0030] Furthermore, for example, when measuring the translation product of a gene to measure the gene expression level, the measurement may be performed using Western blotting, flow cytometry (FACS), or ELISA, which utilize antibodies to detect the protein translated by the target gene. Alternatively, it can be measured using omics methods (e.g., proteomics) that comprehensively analyze proteins and other components. Omics methods are preferred.

[0031] In one embodiment, gene expression levels can be compared by normalizing them by the expression level of any housekeeping gene. Examples of housekeeping genes that can be used for comparison include GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-actin, β2-microglobulin, and HPRT 1 (hypoxanthine phosphoribosyltransferase 1). The expression levels of housekeeping genes can be measured using the same method as described above for the genes used in the present invention, and then compared between different samples by normalizing them by the expression level of the said housekeeping gene.

[0032] In one embodiment of the present invention, applicable 1-(2-hydroxyethyl)-2-imidazolidinone (hereinafter sometimes referred to as "HEI") or its derivatives are not particularly limited as long as they have an inhibitory effect on the expression of VEGF-A signaling-related protein genes, and can be appropriately selected depending on the purpose, but cyclic carboxamide derivatives represented by the following general formula (1) or salts thereof are preferred.

[0033] [ka] (However, in the general formula (1) above, n represents an integer from 1 to 3, and R 1 represents a hydrocarbon group having 1 to 6 carbon atoms, which may be substituted with a hydrogen atom or a hydroxyl group, and X is -CH2- or -N(R 2 ) represents a group represented by R 2 (This represents a hydrocarbon group having 1 to 6 carbon atoms, which may be substituted with a hydrogen atom or a hydroxyl group.)

[0034] In the general formula (1) above, n is an integer between 1 and 3, but is preferably an integer between 1 and 2, and more preferably an integer of 1.

[0035] In the above general formula (1), if R1 is a hydrocarbon group having 1 to 6 carbon atoms that may be substituted with a hydroxyl group, the number of carbon atoms in the hydrocarbon group is preferably 1 to 4, more preferably 1 to 3, and even more preferably 2.

[0036] In the general formula (1) above, X is -N(R 2 If the group is represented by )-, then R 2 This refers to a hydrocarbon group having 1 to 6 carbon atoms, which may be substituted with a hydrogen atom or a hydroxyl group, but a hydrogen atom is preferred.

[0037] Among the cyclic carboxamide derivatives represented by the general formula (1) above, the compound represented by the following structural formula (1) is 1-(2-hydroxyethyl)-2-imidazolidinone.

[0038] [ka]

[0039] Derivatives of 1-(2-hydroxyethyl)-2-imidazolidinone are compounds other than the compound represented by structural formula (1) in the cyclic carboxamide derivatives represented by the general formula (1). There are no particular restrictions on the derivatives of 1-(2-hydroxyethyl)-2-imidazolidinone as long as they have an inhibitory effect on the expression of VEGF-A signaling-related protein genes, and they can be appropriately selected depending on the purpose, but 2-imidazolidinone, which is the compound represented by the following structural formula (2), or 1-(2-hydroxyethyl)-2-pyrrolidone, which is the compound represented by the following structural formula (3), are preferred.

[0040] [ka]

[0041] TIFF2026054100000010.tif43150

[0042] The salt of the cyclic carboxamide derivative represented by the general formula (1) is not particularly limited and may be an inorganic salt or an organic salt.

[0043] There are no particular restrictions on the inorganic salts used, and they can be appropriately selected depending on the purpose. Examples include hydrochloride salts, sulfate salts, phosphate salts, hydrobromide salts, sodium salts, potassium salts, magnesium salts, calcium salts, and ammonium salts.

[0044] There are no particular restrictions on the organic salts used, and they can be appropriately selected depending on the purpose. Examples include acetates, lactates, maleates, fumarates, tartrates, citrates, methanesulfonates, p-toluenesulfonates, triethanolamine salts, diethanolamine salts, and amino acid salts.

[0045] In an expression inhibitor of VEGF-A signaling-related protein genes according to one embodiment, 1-(2-hydroxyethyl)-2-imidazolidinone or its derivative may be a cyclic carboxamide derivative represented by general formula (1) or a salt thereof, used alone or in combination of two or more.

[0046] 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives may be synthesized by known methods or used as a commercially available product.

[0047] The content of 1-(2-hydroxyethyl)-2-imidazolidinone or its derivative in the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment is not particularly limited as long as it is sufficient to effectively exert the inhibitory effect on the expression of VEGF-A signaling-related protein genes, and can be appropriately selected according to the purpose. However, it is preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, even more preferably 0.01% by mass or more, even more preferably 0.1% by mass or more, and particularly preferably 1% by mass or more, relative to the total mass of the VEGF-A signaling-related protein gene expression inhibitor. The upper limit of the content of 1-(2-hydroxyethyl)-2-imidazolidinone or its derivative in the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment is not particularly limited as long as it is sufficient to effectively exert the inhibitory effect on the expression of the VEGF-A signaling-related protein gene, and can be appropriately selected according to the purpose. However, it is preferably 5% by mass or less, more preferably 4% by mass or less, even more preferably 3% by mass or less, even more preferably 2% by mass or less, and particularly preferably 1.5% by mass or less, relative to the total mass of the VEGF-A signaling-related protein gene expression inhibitor. In the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment, the lower and upper limits of the content of 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives can be appropriately combined, preferably 0.0001% to 5% by mass, more preferably 0.001% to 4% by mass, even more preferably 0.01% to 3% by mass, even more preferably 0.1% to 2% by mass, and particularly preferably 1% to 1.5% by mass, relative to the total mass of the VEGF-A signaling-related protein gene expression inhibitor. In addition, if the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment contains two or more 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives, the content of 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives refers to the total content of the two or more 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives.

[0048] <Other ingredients> An inhibitor of VEGF-A signaling-related protein gene expression according to one embodiment may contain other components other than 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives, as long as they do not inhibit the inhibitory effect on VEGF-A signaling-related protein gene expression. Examples of other components include pharmaceutically acceptable carriers or auxiliary agents such as diluents, binders, disintegrants, thickeners, dispersants, reabsorption enhancers, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonic agents, stabilizers, and pH adjusters. These may be used individually or in combination of two or more.

[0049] The content of other components in the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment is not particularly limited as long as it does not impair the inhibitory effect on the expression of the VEGF-A signaling-related protein gene, and can be appropriately selected according to the purpose. In addition, the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment may consist only of 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives.

[0050] [Method of administration and dosage] The method of use and dosage of the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment are not particularly limited as long as they can suppress the expression of the VEGF-A signaling-related protein gene, and can be appropriately selected according to the purpose. Examples of administration methods include topical administration, oral administration, and parenteral administration (e.g., intravenous administration, intraperitoneal administration, etc.), but topical administration is preferred, and application to the skin is more preferred.

[0051] The VEGF-A signaling-related protein gene expression inhibitor according to one embodiment can preferably inhibit the expression of the VEGF-A signaling-related protein gene by applying it to the skin. Thereby, for example, the swelling of the skin is alleviated. There are no particular restrictions on the usage method and dosage when applying to the skin, and it can be appropriately selected according to the dosage form, age, weight, swelling condition, etc. of the application target. As the usage method, several times a day is preferable, and 1 to 5 times is more preferable. At this time, it may be directly rubbed into the skin, or it may be impregnated into a base material such as gauze and then applied to the skin. As the dosage, 0.001 mL to 1 mL per 1 cm 2 of skin is preferable.

[0052] [Dosage form] There are no particular restrictions on the dosage form of the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment, and it can be appropriately selected according to the purpose. When used by topical administration as a skin external preparation, for example, solution systems, solubilized systems, emulsified systems, powder dispersion systems, water-oil two-layer systems, water-oil-powder three-layer systems, ointments, creams, emulsions, lotions, gels, aerosol dosage forms, etc. can be mentioned. Also, it may be a patch impregnated with these dosage forms in a base material. When used by oral administration, for example, solid preparations such as tablets, coated tablets, sugar-coated tablets, granules, powders, capsule preparations (e.g., hard, soft gelatin capsules, etc.); liquid preparations such as oral solutions, syrups (solutions, suspensions), etc. can be mentioned. When used by parenteral administration, for example, injection solutions, etc. can be mentioned.

[0053] [Manufacturing method] There are no particular restrictions on the manufacturing method of the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment, and it can be manufactured by appropriately selecting a method from known methods according to the dosage form.

[0054] (Cosmetics) The cosmetics according to one embodiment contain the VEGF-A signaling-related protein gene expression inhibitor according to one embodiment, and further contain other components as required.

[0055] <Inhibitor of VEGF-A signaling-related protein gene expression> The inhibitor of VEGF-A signaling-related protein gene expression contained in the cosmetic according to one embodiment is as described in the item of (Inhibitor of VEGF-A signaling-related protein gene expression).

[0056] The content of the inhibitor of VEGF-A signaling-related protein gene expression in the cosmetic according to one embodiment is not particularly limited as long as it can exhibit an inhibitory effect on the expression of the VEGF-A signaling-related protein gene, and can be appropriately selected according to the purpose.

[0057] <Other components> The cosmetic according to one embodiment may contain other components other than the inhibitor of VEGF-A signaling-related protein gene expression. The other components are not particularly limited, and can be appropriately selected from the components generally formulated in cosmetics according to the form of the cosmetic, etc. For example, glycols, glycerins, sugar alcohols, saccharides, oils, polymers, surfactants, powders, colorants, solvents, silicones, fragrances, drugs or medicinal components other than the inhibitor of VEGF-A signaling-related protein gene expression, pH adjusters, sequestering agents, etc. can be mentioned. These may be used alone or in combination of two or more.

[0058] <<Glycols>> Examples of glycols include propylene glycol, dipropylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, etc.

[0059] <<Glycerins>> Examples of glycerins include glycerin, diglycerin, polyglycerin, etc.

[0060] <<Sugar alcohols>> Examples of sugar alcohols include sorbitol, mannitol, maltitol, xylitol, and erythritol.

[0061] <<Sugars>> Examples of sugars include fructose, glucose, galactose, maltose, lactose, and trehalose.

[0062] <<Oil content>> Examples of oils include fats and oils, waxes, hydrocarbon oils, higher fatty acids, higher alcohols, ester oils, silicone oils, and oil-soluble agents. These may be used individually or in combination of two or more.

[0063] -Oils and fats- Examples of oils and fats include vegetable oils such as avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, sesame oil, peach kernel oil, wheat germ oil, sasanqua oil, castor oil, linseed oil, safflower oil, cottonseed oil, elm oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, cinnamon oil, Japanese tuni oil, jojoba oil, wheat germ oil, cocoa butter, coconut oil, hydrogenated coconut oil, palm oil, palm kernel oil, and Japanese wax kernel oil; and animal fats such as egg yolk oil, horse fat, beef fat, sheep fat, hydrogenated beef fat, pork fat, beef bone fat, and beef tallow.

[0064] -Waxes- Examples of waxes include Japanese wax, beeswax, candelilla wax, cotton wax, carnauba wax, bayberry wax, privet wax, whale wax, montan wax, rice bran wax, lanolin, kapok wax, lanolin acetate, liquid lanolin, sugarcane wax, isopropyl lanolin fatty acid, hexyl laurate, reduced lanolin, jojoba wax, hard lanolin, and shellac wax.

[0065] -Hydrogen oil- Examples of hydrocarbon oils include linear, branched, or volatile hydrocarbon oils. Specific examples of hydrocarbon oils include liquid paraffin, squalane, isoparaffin, α-olefin oligomers, polybutene, microcrystalline wax, and hydrogenated polydecene.

[0066] -Higher fatty acids- Examples of high-grade fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, 12-hydroxystearic acid, undecylenic acid, lanolinic acid, isostearic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid.

[0067] -High-grade alcohol- Examples of higher alcohols include alcohols with six or more carbon atoms. Specific examples of higher alcohols include linear or branched higher alcohols such as lauryl alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol, myristyl alcohol, oleyl alcohol, cetostearyl alcohol, monostearyl glycerin ether (batyl alcohol), 2-decyltetradesinol, 2-octyldodecanol, lanolin alcohol, cholesterol, phytosterol, hexyldodecanol, isostearyl alcohol, and octyldodecanol.

[0068] -Ester oil- Examples of ester oils include glyceryl triisooctanoate, glyceryl triisopalmitate, octyl isopalmitate, isopropyl isostearate, isostearyl isostearate, isocetyl isostearate, hexyl isostearate, myristyl isostearate, isocetyl octanoate, cetyl isooctanoate, isostearyl octanoate, isodecyl isononanoate, octyldodecyl dimethyloctanoate, oleyl myristate, isostearyl erucate, isocetyl stearate, octyl stearate, isostearyl palmitate, isocetyl palmitate, palm Examples include octyl mitinate, isostearyl myristate, isocetyl myristate, octyldodecyl myristate, decyl myristate, isopropyl palmitate, isopropyl myristate, trimethylolpropane triisostearate, neopentyl glycol dicaprate, dioctyl sebacate, octyldodecyl 12-stearoyloxystearate, dioctyldodecyl stearoyl glutamate, dioctyl adipate, diisostearyl malate, octyl methoxycinnamate, octyldodecyl lactate, isostearyl lactate, and octyl paradimethylaminobenzoate.

[0069] -Silicone oil- Examples of silicone oils include volatile cyclic silicones, volatile dimethylpolysiloxanes, and methylphenyl silicones.

[0070] -Oil-soluble drugs- Examples of fat-soluble drugs include vitamin A, retinol, retinyl acetate, retinyl palmitate, benzyl nicotinate, dl-α-tocopherol nicotinate, vitamin D2, and tocopherol, among other fat-soluble vitamins.

[0071] <<Polymer>> Examples of polymers include plant-derived polymers, animal-derived polymers, microbial-derived polymers, starch-derived polymers, cellulose-derived polymers, alginate-derived polymers, vinyl-derived polymers, acrylic polymers, and inorganic water-soluble polymers. These polymers can act as thickeners.

[0072] Examples of plant-derived polymers include gum arabic, tragacanth gum, galactan, guar gum, carob gum, karaya gum, gellan gum, carrageenan, pectin, and agar.

[0073] Examples of animal-derived polymers include collagen, casein, albumin, and gelatin.

[0074] Examples of microbial polymers include xanthan gum, dextran, succinoglucan, and pullulan.

[0075] Examples of starch-based polymers include plant starches such as corn, wheat, potatoes, and rice; carboxymethyl starch; and methylhydroxypropyl starch.

[0076] Examples of cellulose-based polymers include methylcellulose, nitrocellulose, ethylcellulose, methylhydroxypropylcellulose, hydroxyethylcellulose, sodium cellulose sulfate, hydroxypropylcellulose, sodium carboxymethylcellulose, and crystalline cellulose.

[0077] Examples of alginate-based polymers include sodium alginate and propylene glycol alginate.

[0078] Examples of vinyl polymers include polyvinyl alcohol, polyvinyl acetate, polyvinyl methyl ether, polyvinylpyrrolidone, vinylpyrrolidone-vinyl acetate copolymers, and carboxyvinyl polymers.

[0079] Examples of acrylic polymers include sodium polyacrylate, polyethyl acrylate, polyacrylate alkanolamine, alkyl methacrylate and dimethylaminoethyl methacrylate copolymer, poly2-acrylamido-2-methylpropanesulfonic acid, and polymethacryloyloxytrimethylammonium.

[0080] Examples of inorganic water-soluble polymers include polyethyleneimine, cationic polymers, bentonite, aluminum magnesium silicate, laponite, hectorite, and anhydrous silicic acid.

[0081] <<Surfactants>> Examples of surfactants include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants.

[0082] Examples of anionic surfactants include soap bases, fatty acid soaps such as sodium laurate and sodium palmitate; higher alkyl sulfate salts such as sodium lauryl sulfate and potassium lauryl sulfate; alkyl ether sulfate salts such as polyoxyethylene (hereinafter sometimes abbreviated as "POE") lauryl sulfate triethanolamine and POE lauryl sulfate sodium; N-acyl sarcosinates such as sodium lauroyl sarcosinate; higher fatty acid amide sulfonic acids such as sodium N-myristoyl-N-methyl taurate and sodium coconut oil fatty acid methyl taulide; phosphate salts such as POE stearyl ether phosphate; monolauroyl monoethanolamide POE sulfosuccinate sodium, lauryl polypropylene Examples include sulfosuccinates such as sodium glycol sulfosuccinate; alkylbenzene sulfonates such as sodium linear dodecylbenzenesulfonate and linear dodecylbenzenesulfonate triethanolamine; N-acyl glutamates such as disodium N-stearoyl glutamate and monosodium N-stearoyl glutamate; higher fatty acid ester sulfates such as hydrogenated coconut oil fatty acid glycerin sulfate sodium; sulfurized oils such as belladonna oil; POE alkyl ether carboxylic acids; POE alkyl allyl ether carboxylic acid salts; higher fatty acid ester sulfonates; secondary alcohol sulfates; higher fatty acid alkylolamide sulfates; sodium lauroyl monoethanolamide succinate; and sodium caseinate.

[0083] Examples of cationic surfactants include alkyltrimethylammonium salts such as stearyltrimethylammonium chloride and lauryltrimethylammonium chloride; dialkyldimethylammonium salts such as distearyldimethylammonium chloride; alkylpyridinium salts such as cetylpyridinium chloride; alkylquaternary ammonium salts; alkyldimethylbenzylammonium salts; alkylisoquinolinium salts; dialkylmolionium salts; POE alkylamines; alkylamine salts; polyamine fatty acid derivatives; amyl alcohol fatty acid derivatives; and benzalkonium chloride.

[0084] Examples of nonionic surfactants include lipophilic nonionic surfactants and hydrophilic nonionic surfactants. Examples of lipophilic nonionic surfactants include sorbitan fatty acid esters such as sorbitan monooleate, sorbitan monoisostearate, sorbitan monolaurate, sorbitan monopalmitate, and sorbitan trioleate; glycerin polyglycerin fatty acids such as monocottonseed oil fatty acid glycerin, monostearate glycerin, sesquioleate glycerin, and monostearate glycerin malate; propylene glycol fatty acid esters such as monostearate propylene glycol; glycerin alkyl ethers; and POE-methylpolysiloxane copolymers. Examples of hydrophilic nonionic surfactants include POE sorbitan fatty acid esters such as POE sorbitan monooleate and POE sorbitan monostearate; POE sorbitan fatty acid esters such as POE sorbitan monolaurate, POE sorbitan monooleate, and POE sorbitan monostearate; POE glycerin fatty acid esters such as POE glycerin monooleate and POE glycerin distearate; POE fatty acid esters such as POE monooleate, POE distearate, and POE monodioleate; POE alkyl ethers such as POE lauryl ether, POE oleyl ether, and POE cholestanol ester; POE Examples include POE alkylphenyl ethers such as octylphenyl ether and POE nonylphenyl ether; POE-POP alkyl ethers such as POE-polyoxypropylene (hereinafter sometimes abbreviated as "POP") monobutyl ether, POE-POP cetyl ether, and POE-POP glycerin ether; POE beeswax-lanolin derivatives such as POE sorbitan beeswax; alkanolamides such as coconut oil fatty acid diethanolamide and fatty acid isopropanolamide; POE propylene glycol fatty acid esters; POE fatty acid amides, POE alkylamines; sucrose fatty acid esters, alkylethoxydimethylamine oxide, and the like.

[0085] Examples of amphoteric surfactants include imidazoline-based amphoteric surfactants such as 2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt, and betaine-based surfactants such as amidebetaine and sulfobetaine.

[0086] <<Powder>> The powder may be an inorganic powder or an organic powder.

[0087] Examples of inorganic powders include mica, talc, kaolin, sericite, muscovite, phlogopite, synthetic mica, rose mica, biotite, thiamite, synthetic mica, anhydrous silicic acid (silica), aluminum silicate, barium silicate, calcium silicate, magnesium silicate, strontium silicate, aluminum oxide, barium sulfate, zeolite, titanium dioxide, zinc oxide, and boron nitride.

[0088] Examples of organic powders include polyamide resin powder (nylon powder), polyethylene powder, polymethyl methacrylate powder, polystyrene powder, calcium carbonate powder, magnesium carbonate powder, styrene-acrylic acid copolymer resin powder, and cellulose powder.

[0089] <<Colorants>> Examples of colorants include pigments and dyes.

[0090] -Pigment- Examples of pigments include inorganic pigments, organic pigments, pearl pigments, and metal powder pigments.

[0091] Examples of inorganic pigments include inorganic white pigments such as titanium dioxide and zinc oxide; inorganic red pigments such as iron oxide (red iron oxide); inorganic yellow pigments such as yellow iron oxide and yellow ochre; black pigments such as black iron oxide and carbon black; inorganic green pigments such as chromium oxide, chromium hydroxide, and cobalt titanate; and inorganic blue pigments such as ultramarine and Prussian blue.

[0092] Examples of organic pigments include Red No. 3, Red No. 104, Red No. 106, Red No. 201, Red No. 202, Red No. 204, Red No. 205, Red No. 220, Red No. 226, Red No. 227, Red No. 228, Red No. 230, Red No. 401, Red No. 405, Red No. 505, Orange No. 203, Orange No. 204, Orange No. 205, Yellow No. 4, Yellow No. 5, Yellow No. 202, Yellow No. 203, Yellow No. 205, Yellow No. 401, Blue No. 1, Blue No. 404, Green No. 3, and their zirconium lake, barium lake, or aluminum lake counterparts.

[0093] Examples of pearl pigments include titanium dioxide-coated mica, colored titanium dioxide-coated mica, bismuth oxychloride, and fish scale foil.

[0094] Examples of metal powder pigments include aluminum powder and copper powder.

[0095] -Dye- Examples of pigments include natural pigments such as chlorophyll and beta-carotene.

[0096] <<Solvent>> Examples of solvents include water and lower alcohols. Examples of lower alcohols include alcohols with 5 or fewer carbon atoms. Specific examples of lower alcohols include methanol, ethanol, propyl alcohol, isopropyl alcohol, 2-amino-2-methyl-1-propanol, and 2-amino-2-methyl-1,3-propanediol.

[0097] <<Silicones>> There are no particular restrictions on the type of silicone used; they can be appropriately selected depending on the purpose. Examples include linear silicones, cyclic silicones, and modified silicones.

[0098] Examples of chain-like silicones include dimethylpolysiloxane (dimethicone), methylphenylpolysiloxane (phenylmethicone), and methylhydrogenpolysiloxane.

[0099] Examples of cyclic silicones include octamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, and dodecamethylcyclohexasiloxane.

[0100] Examples of modified silicones include amino-modified silicone oil, polyether-modified silicone oil, carboxy-modified silicone oil, alkyl-modified silicone oil, ammonium salt-modified silicone oil, and fluorine-modified silicone oil.

[0101] These silicones may be used individually or in combination of two or more types.

[0102] <<Drug or medicinal ingredient>> As for the drugs, there are no particular restrictions as long as they are drugs or active ingredients other than VEGF-A signaling-related protein gene expression inhibitors. Examples include vitamins, UV absorbers, chelating agents, preservatives, plant extracts, moisturizers, anti-inflammatory agents, whitening agents, cooling agents, amino acids, antioxidants, bactericides, and various skin nutrients.

[0103] -Vitamins- Examples of vitamins include water-soluble vitamins such as vitamin B6 hydrochloride, pantothenyl ethyl ether, pyridoxine hydrochloride, nicotinamide, pantothenic acid, and biotin.

[0104] - UV absorber - Examples of UV absorbers include benzoic acid-based UV absorbers such as para-aminobenzoic acid; anthranilic acid-based UV absorbers such as methyl anthranilate; salicylic acid-based UV absorbers such as octyl salicylate; cinnamic acid-based UV absorbers such as isopropyl para-methoxycinnamate and octyl para-methoxycinnamate; UV absorbers such as urocanic acid and ethyl urocanate; benzophenone-based UV absorbers such as 2-hydroxy-4-methoxybenzophenone and dihydroxybenzophenone; benzotriazole-based UV absorbers; and 2-phenylbenzimidazole-5-sulfonic acid.

[0105] -Chelating agent- Examples of chelating agents include citramalic acid, agalic acid, glyceric acid, shikimic acid, hinokitiol, gallic acid, tannic acid, caffeic acid, ethylenediamine-N,N,N',N'-tetraacetic acid disodium dihydrate (EDTA-2Na-2H2O), ethylene glycol diaminetetraacetic acid, diethylenetriaminepentaacetic acid, phytic acid, polyphosphate, metaphosphate, their analogs, their alkali metal salts, and their carboxylic acid esters.

[0106] - Preservatives - Examples of preservatives include benzoic acid, salicylic acid, para-hydroxybenzoic acid esters (e.g., methylparaben, ethylparaben, butylparaben, etc.), sorbic acid, parachlormethacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichlorocarbanilide, photosensitizer, phenoxyethanol, and chlorphenesin.

[0107] -Plant extract- Examples of plant extracts include Houttuynia cordata extract, Phellodendron amurense extract, Licorice extract, Peony extract, Paeonia suffruticosa extract, Luffa gourd extract, Saxifraga stolonifera extract, Eucalyptus extract, Clove extract, Horse chestnut extract, Cornflower extract, Seaweed extract, and Thyme extract. These plant extracts may also contain caffeine, tannins, etc.

[0108] - Humectant - Examples of the humectant include polyethylene glycol (hereinafter sometimes abbreviated as "PEG"), sodium chondroitin sulfate, sodium hyaluronate, sodium lactate, glucosamine, cyclodextrin, and the like. Further, the glycols, the glycerins, the sugar alcohols, and the saccharides can also be used as the humectant.

[0109] - Anti-inflammatory agent - Examples of the anti-inflammatory agent include azulene, glycyrrhizin, lysozyme chloride, pyridoxine hydrochloride, sulfur, and the like.

[0110] - Whitening agent - Examples of the whitening agent include arbutin, 4-methoxysalicylic acid, tranexamic acid, vitamin C, ethyl vitamin C, magnesium ascorbyl phosphate, glucosyl ascorbate, kojic acid, and the like.

[0111] - Cooling agent - Examples of the cooling agent include L-menthol, camphor, and the like.

[0112] - Antioxidant - Examples of the antioxidant include ascorbic acid, α-tocopherol, carotenoid, and the like.

[0113] <<pH adjuster (neutralizing agent)>> Examples of the pH adjuster include potassium hydroxide, sodium hydroxide, triethanolamine, sodium carbonate, lactic acid, citric acid, sodium citrate, glycolic acid, succinic acid, tartaric acid, malic acid, sodium hydrogen carbonate, ammonium hydrogen carbonate, and the like.

[0114] <<Metal ion sequestering agent>> Examples of the metal ion sequestering agent include disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, and the like.

[0115] In the cosmetic composition according to one embodiment, there are no particular restrictions on the content of other components, as long as they do not impair the effects of the cosmetic composition according to one embodiment, and they can be appropriately selected depending on the purpose.

[0116] [Application] There are no particular restrictions on the uses of the cosmetic composition according to one embodiment, and can be appropriately selected according to the purpose. Examples include lotions, emulsions, creams, essences, jellies, gels, ointments, packs, masks, and foundations.

[0117] < Screening method for inhibitor of expression of VEGF-A signaling-related protein gene > In one embodiment, the present invention provides a method for screening inhibitors of VEGF-A signaling-related protein gene expression, comprising contacting candidate compounds with skin cells and selecting an inhibitor of VEGF-A signaling-related protein gene expression using the degree of suppression of the expression level of the VEGF-A signaling-related protein gene in the skin cells as an indicator.

[0118] In one embodiment, the above method may involve selecting one or more genes from those listed in Table 6 below. [Table 6]

[0119] In one embodiment, it is preferable that the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 7 below. [Table 7]

[0120] The amino acid sequence of the above gene or the specific sequence of the gene encoding it can be obtained, for example, by searching on UniProt (https: / / www.uniprot.org / ) by entering the abbreviation of the gene to be searched or the ID of the Uniprot Primary accession. The specific sequences of the genes that can be used in the present invention are not limited to the amino acid sequences or nucleic acid sequences of the genes encoding them that can be searched and obtained using the IDs listed in the Uniprot Primary accession in Table 5. For example, sequences that are 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identical to the amino acid sequences or nucleic acid sequences of the genes encoding them that can be obtained using the Primary accession in Table 5, as well as splicing variants thereof. Furthermore, the origin of the gene may be any animal, but it is preferably from humans or non-human mammals (e.g., non-human primates), and most preferably from humans.

[0121] In one embodiment, the skin cells applicable to screening may be primary skin cells collected from a living organism or a skin cell line, preferably epidermal cells. In this specification, "epidermal cells" includes all cells at different stages of differentiation that constitute them, and mainly consist of cells also called keratinocytes. In this specification, keratinocytes are one of the cells that make up the epidermis, and in the epidermal tissue of a living organism, they divide in the deepest layer (basal layer) and migrate to the surface, differentiating into the spinous layer, granular layer, and then the stratum corneum, before eventually becoming dead skin cells that are shed. The keratinocytes used in the method of one embodiment of the present invention may be primary cultured cells collected from living tissue, cells that have been previously isolated and / or proliferated and are commercially available or distributed, cells that have been made into a cell line, or cells differentiated from pluripotent stem cells such as ES cells, iPS cells, or Muse cells. The method for culturing keratinocytes may follow known methods, and the culture medium and culture conditions used may be determined by referring to known methods.

[0122] By using the degree of suppression of the expression level of the VEGF-A signaling-related protein gene in the aforementioned skin cells as an indicator, an inhibitor of VEGF-A signaling-related protein gene expression can be selected. For example, as a control, if the expression level of the VEGF-A signaling-related protein gene in skin cells exposed to the candidate drug is suppressed compared with the expression level of skin cells not exposed to the candidate drug, the candidate drug can be selected as an inhibitor of VEGF-A signaling-related protein gene expression. The gene expression level can be measured by any method. For example, when measuring the gene transcript to measure the gene expression level, an appropriate probe can be designed by referring to the sequence of the target gene, and then measured using methods such as quantitative PCR (qPCR), in situ hybridization, Northern blotting, DNA microarray, or next-generation sequencing.

[0123] Furthermore, for example, when measuring the translation product of a gene to measure the gene expression level, the measurement may be performed using Western blotting, flow cytometry (FACS), or ELISA, which utilize antibodies to detect the protein translated by the target gene. Alternatively, it can be measured using omics methods (e.g., proteomics) that comprehensively analyze proteins and other components. Omics methods are preferred.

[0124] In one embodiment, gene expression levels can be compared by normalizing them by the expression level of any housekeeping gene. Examples of housekeeping genes that can be used for comparison include GAPDH (glyceraldehyde-3-phosphate dehydrogenase), β-actin, β2-microglobulin, and HPRT 1 (hypoxanthine phosphoribosyltransferase 1). The expression levels of housekeeping genes can also be measured using the same method as described above for the genes used in the present invention, and then compared between different samples by normalizing them by the expression level of the said housekeeping gene.

[0125] In this specification, "candidate substance" refers to a substance to be screened, and may, but is not limited to, small molecule compounds, peptides, proteins, nucleic acids, tissue extracts or cell culture supernatants of mammals (e.g., mice, rats, pigs, cattle, sheep, monkeys, humans, etc.), plant-derived compounds or extracts (e.g., herbal extracts, compounds derived from herbal medicines), and microbial-derived compounds, extracts, or culture products. [Examples]

[0126] The embodiments will be described in more detail below with reference to examples, comparative examples, and test examples, but the embodiments are not limited to these examples, comparative examples, and test examples. In the examples and comparative examples shown in Table 1 below, the amount of each ingredient is expressed as "mass %" relative to the total mass of the lotion.

[0127] In each of the tests described below, lotions and emulsions with the compositions and proportions shown in Table 1 were prepared by conventional methods.

[0128] [Table 8] [Table 9]

[0129] Thirty-nine healthy Japanese women aged 20 to 60 participated as panelists. They applied 0.3 mL of the lotion from Example 1 to half of their face twice a day, morning and evening. The left and right halves of the faces were randomly assigned; 19 panelists applied the lotion from Example 1 to the right side of their face's midline, and 20 panelists applied it to the left side of their face's midline. The half of the face to which the lotion from Example 1 was applied was designated "Q Half" (Figure 1).

[0130] Next, the panelists were asked to apply 0.3 mL of Comparative Example 1 lotion to the half of their face opposite to the side to which they had applied the lotion from Example 1, twice a day, in the morning and evening. The opposite half of the face to the side to which the panelist had applied the lotion from Example 1 means, for example, that if the panelist applied the lotion from Example 1 to the left side of the midline of their face, they applied the lotion from Comparative Example 1 to the right side of the midline of their face. The half of the face to which the lotion from Comparative Example 1 was applied was designated as "P half of the face" (Figure 1).

[0131] After applying the lotions of Example 1 and Comparative Example 1, participants were asked to apply 0.3 mL of the emulsion with the composition shown in Table 9 to both the Q and P halves of their face twice a day, in the morning and evening.

[0132] The lotions and emulsions of Example 1 and Comparative Example 1 were applied for 6 weeks. The compositions of the lotions of Example 1 and Comparative Example 1 were not disclosed to the panelists.

[0133] (Test 1) <Visual assessment of swelling> For the half of the face to which the lotion of Example 1 was applied (half Q) and the half of the face to which the lotion of Comparative Example 1 was applied (half P), photographs of the left and right sides of the face were taken with a digital camera from a position 45 degrees diagonally from the front, with the midline of the face considered as the front. Using the left and right facial photographs as a pair, four expert evaluators performed a visual judgment on "which side of the face was more swollen." Figure 3A is a schematic diagram illustrating the pair of left and right facial photographs. Note that the expert evaluators were not disclosed which half of the face each lotion of Example 1 and Comparative Example 1 was applied to when performing the visual judgment.

[0134] The evaluation was conducted before the first application of the lotion in Example 1 and Comparative Example 1 (hereinafter referred to as "Week 0"), and 3 weeks and 6 weeks after the start of application of the lotion in Example 1 and Comparative Example 1. In the evaluation at each week, four expert evaluators each performed five evaluations (Figure 2). The evaluation was conducted based on the following evaluation criteria. The scores of each expert evaluator were allocated to the Q half of the face and the P half of the face, respectively, and for each week's evaluation, the average value a of the scores from the five evaluations by each expert evaluator was calculated for one panelist. Next, the average value b of the average value a for the 39 panelists at Week 0, 3 weeks, and 6 weeks was calculated. Next, the average value c of the average value b for the four expert evaluators was calculated. The standard error of the average value b was also calculated.

[0135] -Judgment criteria- Rating -1 point: Q: Half of my face looks swollen. Rating: 1 point: P Half of the face looks swollen.

[0136] The results are shown in Figure 3. In Figure 3, "*" indicates a significant difference based on a paired t-test (two-sided t-test) comparing the pre-treatment (week 0) and 6 weeks after the start of treatment, with a p-value of 0.013. Before treatment (week 0), there was no difference between the P and Q halves of the face. On the other hand, 6 weeks after the start of treatment, the P half of the face appeared significantly more swollen; in other words, the Q half of the face did not appear as swollen.

[0137] (Exam 2) <Quantitative evaluation of edema> Before the first application of the lotion in Example 1 and Comparative Example 1 (week 0), and 3 weeks and 6 weeks after the start of application of the lotion in Example 1 and Comparative Example 1, a 3D image acquisition and analysis device (VECTRAHandy H2 (VEC-H2, Canfield) was used. Using a Scientific camera, data representing the three-dimensional shape of the panelists' faces (three-dimensional facial image data) was captured. Three-dimensional facial image data was obtained when the panelists' faces were in a horizontal position (specifically, when the midline of the face was held perpendicular to the direction of gravity) and when they were in a vertical position (specifically, when the midline of the face was held parallel to the direction of gravity). Next, using a computer, the amount of gravity-induced swelling (VCswelling) and gravity-induced shrinkage (VCshrinking) were calculated for each half of the face to which the lotion of Example 1 was applied (Q half of the face) and the amount of gravity-induced shrinkage (P half of the face) to which the lotion of Comparative Example 1 was applied, based on the change in three-dimensional shape between the three-dimensional facial image when the panelists' faces were in a horizontal position and the three-dimensional facial image when the face was held vertically. Next, the amount of facial swelling (Sc) was calculated based on the amount of gravity-induced swelling (VCswelling) and gravity-induced shrinkage (VCshrinking).

[0138] -Calculation of gravitational bulge (VCswelling)- Figure 4A is a diagram illustrating gravity bulge according to one embodiment of the present invention. Three-dimensional facial images are obtained when the subject's face is in a horizontal position and when the subject's face is in a vertical position. When the three-dimensional shape in the horizontal position is compared with the three-dimensional shape in the vertical position, the volume is larger in the area within the dashed line in Figure 4A when the face is in the vertical position. In this way, the difference in volume between the three-dimensional shape in the horizontal position and the three-dimensional shape in the vertical position of the subject is compared with the three-dimensional shape in the vertical position, and this difference in volume is defined as the amount of gravity bulge (VCswelling).

[0139] -Calculation of gravity shrinkage (VCshrinking)- Figure 4B is a diagram illustrating gravity-induced indentation according to one embodiment of the present invention. Three-dimensional facial images are obtained when the subject's face is in a horizontal position and when the subject's face is in a vertical position. When the three-dimensional shape in the horizontal position and the three-dimensional shape in the vertical position are compared, the volume is smaller in the vertical position in the area within the dashed line in Figure 4B. In this way, the difference in volume between the two positions (i.e., horizontal and vertical) of the area where the volume is smaller in the vertical position is defined as the amount of gravity-induced indentation (VCshrinking).

[0140] -Calculation of facial swelling (Sc)- Facial swelling (Sc) was calculated using the following formula (1). Three-dimensional facial images were obtained for each panelist three times at 0 weeks, 3 weeks, and 6 weeks, and the average value of these measurements was taken as the facial swelling (Sc) of that panelist.

[0141]

number

[0142] Next, the improvement in facial swelling (Sc) was calculated using the following formulas (3) and (4).

[0143] Difference in facial swelling (Sc) between left and right sides = [Q: Swelling amount on one half of the face (Sc)] - [P: Swelling amount on the other half of the face (Sc)] ... Equation (3) Improvement in facial swelling (Sc) = [Difference in facial swelling (Sc) between the left and right sides at week 0] - [Difference in facial swelling (Sc) between the left and right sides at week n] ... Equation (4) (However, in equation (4), n = 3 or 6)

[0144] Figure 5 shows a graph illustrating the change in the amount of facial swelling (Sc) from week 0 to week 6 of the study. In Figure 5, "*" indicates p<0.05. From these results, the p-value between week 0 and week 6 was less than 0.05, indicating that the Q half of the face, to which the lotion of Example 1 was applied, showed a significant improvement in the amount of swelling (Sc) compared to the P half of the face, to which the lotion of Comparative Example 1 was applied.

[0145] The results of Test Example 1 and Test Example 2 showed that edema-reducing agents containing 1-(2-hydroxyethyl)-2-imidazolidinone or its derivatives as active ingredients have excellent edema-reducing effects.

[0146] (Exam 3) <Extraction, pretreatment, and omics analysis of stratum corneum proteins> 1. Extraction and pretreatment of stratum corneum proteins Three layers of stratum corneum were collected from the same area of ​​the cheeks (P half and Q half) of the same subjects (subjects) as in Test 1, using D-squame tape (Promotool), before application of the lotion (week 0), and 3 and 6 weeks after the start of lotion application in Example 1 and Comparative Example 1. The third layer was used as the proteomics sample (Figure 1). The reagents and equipment used for protein extraction and pretreatment were mainly those included in EasyPep® 96 MS Sample Prep Kits (Thermo).

[0147] First, the stratum corneum tape was immersed in Lysis Buffer, then sonicated to extract the protein, and the supernatant was obtained by centrifugation. Next, Reduction Solution and Alkylation Solution were added to each sample and mixed, and incubated at 50°C for 10 minutes, followed by cooling at room temperature for 10 minutes. Then, Trypsin / Lys-C solution was added, and the mixture was incubated at 37°C for 3 hours to digest the peptide of the protein. Next, Digestion Stop solution was added and mixed to stop the digestion. Then, the peptide was purified using a Peptide Clean-Up Plate, and the resulting solution was dried using a centrifugal evaporator. Finally, the solution was redissolved in 0.1% formic acid solution and prepared as a sample for LC-MS / MS analysis.

[0148] 2. LC-MS / MS measurement The UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) and Orbitrap Fusion Lumos (Thermo Fisher Scientific) were used as the LC-MS / MS system, along with a nano HPLC capillary column (ODS, inner diameter 75 μm × 120 mm, particle size: 3.0 μm, manufactured by Nikkyo Technos Co., Ltd.). A 0.1% formic acid solution was used as mobile phase A, and a 0.1% acetonitrile solution as mobile phase B. The gradient was performed under the following conditions.

[0149] 0 min, 2% B; 90 min, 30% B; 95 min, 60% B; 100 min, 60% B; 105 min, 2% B; 110 min, 2 % B

[0150] Data-dependent acquisition was used for data acquisition. The separated peptides were electrosprayed with a Nanospray Flex NG source under a 1.8kV voltage, and the ion transfer tube was set to 275°C. The RF level of the ion funnel was 45, the resolution of the MS1 Orbitrap was 120,000 (at m / z 200), and the MS1 AGC target and maximum injection time were 4 × 10⁻¹⁶. 5 The time was set to 50ms. Precursor ions with charges from +2 to +7 were isolated for MS2 sequencing. The MS2 isolation window was 1.6 Da, and the AGC target was 5 × 10⁻¹⁴. 4 The dynamic exclusion time was set to 20 seconds, and the mass accuracy to ±10 ppm. The resolution and maximum injection time of MS2 were 15000 (at m / z 120) and 22 ms, respectively, and the signal intensity threshold was 2.5 × 10⁻¹⁶. 4 The settings were adjusted. Precursor ions were dissociated by high-energy collision-induced dissociation (HCD) with a normalized collision energy of 30%. Mass calibration was performed automatically for each injection using a lock mass system with peaks at m / z = 391.2843 and 445.12.

[0151] 3. LC-MS / MS Data Analysis Database searches and protein / peptide identification and quantification were performed using Proteome Discoverer (version 2.5) and the Sequest and Amanda databases. MS / MS spectra were searched using the Swiss-Prot and TrEMBL human databases. Protein N-terminal acetylation and methionine oxidation were selected as dynamic modifications. Carbamide methylation of cysteine ​​residues was set as a static modification. The minimum peptide length was set to 7 amino acids, the maximum mass to 5000 Da, and up to two missed cleavages were set for each peptide. Filtering was performed with an FDR (False Discovery Rate) of 0.01 for both peptides and proteins. Both unique peptides and razor peptides were selected for label-free quantification (LFQ) calculations. Other unspecified parameters were set to the default settings of Proteome Discoverer.

[0152] 4. Data Analysis The identified and quantified protein / peptide data were further processed and visualized in Microsoft Excel. Principal component analysis (PCA) using Proteome Discover was performed to verify the types and amounts of proteins obtained from the facial stratum corneum.

[0153] Data compiled in Excel was read using R (version 4.2.1), and the data from 37 panelists, excluding those who dropped out of the study midway or whose quantified protein values ​​were abnormally low, was analyzed using R. Considering the metabolic period of the stratum corneum, data from weeks 0 and 6 of the study were extracted, and missing values ​​were imputed using the multiple imputation method (100 repetitions) with the R package missRanger (version 2.4.0). Rather than simply calculating using the average value for each panelist, individual differences among panelists were taken into consideration, and a mixed linear model analysis was performed to show the difference in how changes occurred with 1-(2-hydroxyethyl)-2-imidazolidinone added and with placebo, using paired models. For the mixed linear model, cAIC was used to compare whether to consider placebo and 1-(2-hydroxyethyl)-2-imidazolidinone added at week 0 as different models or the same model, and the model that considered them the same was adopted. The interaction term from the analysis results of the mixed linear model was used to quantify the changes between week 0 and week 6 of the experiment as a correlation coefficient.

[0154] 5.Results Table 10 shows the results for proteins related to the VEGFA-VEGFA2 Signaling Pathway among all proteins that could be quantified and analyzed. When we examined the correlation coefficients, which quantify how these proteins changed between week 0 and week 6 of the experiment, we found that there were more proteins with negative correlation coefficients than with positive correlation coefficients, as shown in the histogram in Figure 6. From this, we found that among the proteins related to the VEGFA-VEGFA2 Signaling Pathway quantified in the stratum corneum, there were more proteins that changed in a suppressive direction than proteins that changed in a suppressive direction upon addition of 1-(2-hydroxyethyl)-2-imidazolidinone.

[0155] [Table 10-1] [Table 10-2]

Claims

1. An inhibitor of VEGF-A signaling-related protein gene expression, containing 1-(2-hydroxyethyl)-2-imidazolidinone or a derivative thereof as an active ingredient.

2. The 1-(2-hydroxyethyl)-2-imidazolidinone or its derivative contains a cyclic carboxamide derivative represented by the following general formula (1) or a salt thereof as an active ingredient. The inhibitor according to claim 1. 【Chemistry 1】 (However, in the general formula (1) above, n represents an integer from 1 to 3, and R １ represents a hydrocarbon group having 1 to 6 carbon atoms, which may be substituted with a hydrogen atom or a hydroxyl group, and X is -CH ２ - or -N(R ２ ) represents a group represented by -, R ２ (This represents a hydrocarbon group having 1 to 6 carbon atoms, which may be substituted with a hydrogen atom or a hydroxyl group.)

3. The inhibitor according to claim 1, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 1 below. Table 1

4. The inhibitor according to claim 1, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 2 below. Table 2

5. The inhibitor according to claim 1, which suppresses VEGF-A signaling in the epidermis.

6. The inhibitor according to claim 1, which is a topical skin preparation.

7. A cosmetic composition characterized by containing the inhibitor described in claim 1.

8. A screening method for inhibitors of VEGF-A signaling-related protein gene expression, A method comprising contacting candidate compounds with skin cells and selecting an inhibitor of VEGF-A signaling-related protein gene expression, using the degree of suppression of the expression level of the VEGF-A signaling-related protein gene in the skin cells as an indicator.

9. The method according to claim 8, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 3 below. Table 3

10. The method according to claim 8, wherein the VEGF-A signaling-related protein gene is selected from one or more of the genes listed in Table 4 below. Table 4

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