Semaphorin 3A expression promoter

Abstract

To provide new techniques for promoting the expression of semaphorin 3A.SOLUTION: A semaphorin 3A expression promoter contains a compound represented by formula (1) in the figure as an active ingredient. In formula (1), R1 represents a C6-14 aromatic hydrocarbon group or a C1-12 aliphatic hydrocarbon group; R2 represents a hydrogen atom or an aliphatic hydrocarbon group; and X represents a single bond, an oxygen atom or a sulfur atom.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a semaphorin 3A expression promoter, and more particularly to a semaphorin 3A expression promoter, a prophylactic and / or therapeutic agent for atopic dermatitis, a prophylactic and / or therapeutic agent for xeroderma, a prophylactic and / or therapeutic agent for allergic rhinitis, and a prophylactic and / or therapeutic agent for osteoporosis. [Background technology]

[0002] In lesions of atopic dermatitis and xerosis, a large number of nerve fibers proliferate within the epidermis, causing itching and hypersensitivity. Semaphorins are a group of proteins known to be involved in various pathologies, including immune response, bone metabolism control, and cancer progression. In normal skin (epidermis), semaphorin 3A (Sema3A, nerve repulsive factor) predominates over NGF (neuronal growth factor). However, in lesions of atopic dermatitis or xerosis, semaphorin 3A expression is reduced and NGF expression is increased. As a result, NGF predominates, inducing the penetration of nerve fibers into the epidermis. The increased proliferation of intraepidermal nerve fibers is thought to be due in part to the decreased expression of semaphorin 3A produced by epidermal keratinocytes. It has been reported that application or subcutaneous injection of semaphorin 3A to lesions of atopic dermatitis improved itching and dermatitis (Non-Patent Documents 1 and 2).

[0003] Decreased expression of semaphorin 3A is also observed in the nasal mucosal epithelium of patients with allergic rhinitis, and it has been reported that intranasal administration of semaphorin 3A alleviates rhinitis (Non-Patent Document 3). Semaphorin 3A is also known to increase bone mass by simultaneously inhibiting and promoting osteoclast differentiation (Non-Patent Document 4).

[0004] Plant-derived glycosphingolipids and green tea extracts have been reported to promote the expression of such semaphorin 3A (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2017-197501 [Patent Document 2] Patent Publication No. 2021-130641 [Non-patent literature]

[0006] [Non-Patent Document 1] Negi et al, J Dermatol Sci, 2012, 66: 37-43. [Non-patent document 2] Yamaguchi et al,J.Invest.Dermatol.,128(12),2842-9,2008 [Non-patent document 3] Sawaki et al.,J PharmacolSci 117,34-44,2011 [Non-patent document 4] Hayashi et al., Experimental Medicine, Vol. 31, No. 4, 2013 Summary of the Invention [Problem to be solved by the invention]

[0007] On the other hand, benzimidazole derivatives such as perbendazole, fenbendazole, and albendazole are known to be effective in anthelmintics, but there have been no reports examining whether they have the effect of promoting semaphorin 3A expression or the effect of preventing and / or treating atopic dermatitis, xerosis, allergic rhinitis, or osteoporosis. An object of the present invention is to provide a new technique for promoting the expression of semaphorin 3A. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors have found that certain benzimidazole derivatives, such as perbendazole, fenbendazole, and albendazole, have an excellent effect of promoting semaphorin 3A expression, and have completed the present invention.

[0009] That is, the present invention provides the following: <1> ~ <7> This provides: <1> A semaphorin 3A expression promoter (hereinafter also referred to as the semaphorin 3A expression promoter of the present invention) contains a compound represented by the following formula (1) (hereinafter also referred to as compound (1)) as an active ingredient.

[0010] [ka]

[0011] [In formula (1), R 1 represents an aromatic hydrocarbon group having 6 to 14 carbon atoms or an aliphatic hydrocarbon group having 1 to 12 carbon atoms, R 2 represents a hydrogen atom or an aliphatic hydrocarbon group, X represents a single bond, an oxygen atom, or a sulfur atom.

[0012] <2> A preventive and / or therapeutic agent for atopic dermatitis (hereinafter also referred to as the preventive and / or therapeutic agent for atopic dermatitis of the present invention) containing compound (1) as an active ingredient. <3> A preventive and / or therapeutic agent for xeroderma, which contains compound (1) as an active ingredient (hereinafter also referred to as the preventive and / or therapeutic agent for xeroderma of the present invention). <4> A preventive and / or therapeutic agent for allergic rhinitis (hereinafter also referred to as the preventive and / or therapeutic agent for allergic rhinitis of the present invention) containing compound (1) as an active ingredient.

[0013] <5> A preventive and / or therapeutic agent for osteoporosis (hereinafter also referred to as the preventive and / or therapeutic agent for osteoporosis of the present invention) containing compound (1) as an active ingredient. <6> An NGF expression inhibitor (hereinafter also referred to as the NGF expression inhibitor of the present invention) containing compound (1) as an active ingredient.

[0014] <7> The compound represented by formula (1) is one or more selected from perbendazole, fenbendazole, and albendazole. <1> ~ <6> The agent according to any one of the above. [Effects of the Invention]

[0015] The semaphorin 3A expression promoter of the present invention has an excellent effect of promoting semaphorin 3A expression, and the NGF expression inhibitor of the present invention has an excellent effect of inhibiting NGF expression. The agent for preventing and / or treating atopic dermatitis of the present invention has excellent effects for preventing and / or treating atopic dermatitis. The agent for preventing and / or treating xeroderma of the present invention has excellent effects for preventing and / or treating xeroderma. The agent for preventing and / or treating allergic rhinitis of the present invention has excellent effects for preventing and / or treating allergic rhinitis. The agent for preventing and / or treating osteoporosis of the present invention has excellent effects for preventing and / or treating osteoporosis. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows an increase in the amount of secreted Sema3A protein due to the addition of perbendazole. [Figure 2] FIG. 1 shows the effect of perbendazole in promoting Sema3A mRNA expression. [Figure 3] FIG. 1 shows the inhibitory effect of perbendazole on NGF mRNA expression. [Figure 4] A diagram showing the low toxicity of perbendazole. [Figure 5-1] FIG. 1 shows the effect of fenbendazole in promoting Sema3A mRNA expression. [Figure 5-2] FIG. 1 shows the effect of albendazole in promoting Sema3A mRNA expression. [Figure 6-1] FIG. 1 shows the inhibitory effect of fenbendazole on NGF mRNA expression. [Figure 6-2] FIG. 1 shows the inhibitory effect of albendazole on NGF mRNA expression. [Figure 7-1] FIG. 10 is a graph showing the effect of promoting semaphorin 3A expression when perbendazole is added to the stratum corneum surface of a three-dimensional epidermal model. [Figure 7-2] FIG. 10 is a graph showing the effect of promoting semaphorin 3A expression when perbendazole is added to a three-dimensional epidermal model culture medium. [Figure 8-1] FIG. 1 shows the low toxicity of perbendazole when added to the stratum corneum surface of a three-dimensional epidermal model. [Figure 8-2] FIG. 1 shows the low toxicity of perbendazole when added to a three-dimensional epidermal model culture medium. [Figure 9] Graph showing the relationship between the semaphorin 3A expression-promoting effect of perbendazole and transcription factors (c-JUN, c-FOS). [Figure 10] FIG. 1 shows the promoting effect of perbendazole on semaphorin 3A expression in the presence of an AP-1 inhibitor. DETAILED DESCRIPTION OF THE INVENTION

[0017] The semaphorin 3A expression promoter, NGF expression inhibitor, preventive and / or therapeutic agent for atopic dermatitis, xeroderma, allergic rhinitis, and osteoporosis of the present invention contain, as an active ingredient, a compound represented by the following formula (1): First, the compound (1) will be described in detail.

[0018] [ka]

[0019] [In formula (1), R 1 represents an aromatic hydrocarbon group having 6 to 14 carbon atoms or an aliphatic hydrocarbon group having 1 to 12 carbon atoms, R 2 represents a hydrogen atom or an aliphatic hydrocarbon group, X represents a single bond, an oxygen atom, or a sulfur atom.

[0020] The compound (1) is preferably a compound represented by the following formula (1B).

[0021] [ka]

[0022] [In formula (1B), R 1 , R 2 and X have the same meanings as defined above.

[0023] In formulas (1) and (1B), X represents a single bond, an oxygen atom, or a sulfur atom. From the viewpoints of the semaphorin 3A expression-promoting effect, the NGF expression-suppressing effect, the atopic dermatitis preventive and / or therapeutic effect, the xeroderma preventive and / or therapeutic effect, the allergic rhinitis preventive and / or therapeutic effect, and the osteoporosis preventive and / or therapeutic effect, as well as low toxicity, X is preferably a single bond or a sulfur atom, and particularly preferably a single bond.

[0024] R 1 The aromatic hydrocarbon group represented by the formula (I) has 6 to 14 carbon atoms, preferably 6 to 12, and more preferably 6 to 8. Specific examples of the aromatic hydrocarbon group include aryl groups such as phenyl, tolyl, xylyl, and naphthyl. Among these, a phenyl group is preferred from the viewpoints of the semaphorin 3A expression-promoting effect, NGF expression-suppressing effect, atopic dermatitis preventive and / or therapeutic effect, xeroderma preventive and / or therapeutic effect, allergic rhinitis preventive and / or therapeutic effect, and osteoporosis preventive and / or therapeutic effect, as well as low toxicity.

[0025] R 1 The number of carbon atoms in the aliphatic hydrocarbon group represented by the formula (I) is 1 to 12, but from the viewpoints of the semaphorin 3A expression-promoting effect, NGF expression-suppressing effect, atopic dermatitis preventive and / or therapeutic effect, xeroderma preventive and / or therapeutic effect, allergic rhinitis preventive and / or therapeutic effect, and osteoporosis preventive and / or therapeutic effect, and low toxicity, it is preferably 1 to 10, more preferably 2 to 6, and particularly preferably 3 or 4. Also, R 2represents a hydrogen atom or an aliphatic hydrocarbon group, preferably an aliphatic hydrocarbon group. 2 The number of carbon atoms in the aliphatic hydrocarbon group represented by the formula (I) is preferably 1 to 10, more preferably 1 to 6, even more preferably 1 to 4, and particularly preferably 1 to 2, from the viewpoints of the semaphorin 3A expression-promoting effect, the NGF expression-suppressing effect, the atopic dermatitis preventive and / or therapeutic effect, the xeroderma preventive and / or therapeutic effect, the allergic rhinitis preventive and / or therapeutic effect, and the osteoporosis preventive and / or therapeutic effect, as well as low toxicity. R 1 , R 2 The aliphatic hydrocarbon group represented by the formula (I) may be linear or branched. Furthermore, the aliphatic hydrocarbon group is preferably an alkyl group. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, and a dodecyl group. R 1 Among the alkyl groups represented by the formula (I), an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group are preferred from the viewpoints of the semaphorin 3A expression promoting effect, the NGF expression suppressing effect, the atopic dermatitis preventive and / or therapeutic effect, the xeroderma preventive and / or therapeutic effect, the allergic rhinitis preventive and / or therapeutic effect, and the osteoporosis preventive and / or therapeutic effect, as well as low toxicity. 2 Among the alkyl groups represented by the formula (I), a methyl group and an ethyl group are preferred from the viewpoints of the semaphorin 3A expression-promoting effect, the NGF expression-suppressing effect, the atopic dermatitis preventive and / or therapeutic effect, the xeroderma preventive and / or therapeutic effect, the allergic rhinitis preventive and / or therapeutic effect, and the osteoporosis preventive and / or therapeutic effect, as well as low toxicity.

[0026] R 1 The aromatic hydrocarbon group represented by R may be a substituted or unsubstituted aromatic hydrocarbon group. 1 , R 2 The aliphatic hydrocarbon group represented by may be a substituted aliphatic hydrocarbon group or an unsubstituted aliphatic hydrocarbon group. R 1 , R2 Examples of the substituent in include halogen atoms such as a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0027] As compound (1), from the viewpoints of the effect of promoting semaphorin 3A expression, the effect of suppressing NGF expression, the effect of preventing and / or treating atopic dermatitis, the effect of preventing and / or treating xeroderma, the effect of preventing and / or treating allergic rhinitis, and the effect of preventing and / or treating osteoporosis, as well as low toxicity, one or more compounds selected from perbendazole, fenbendazole, and albendazole are preferred, and perbendazole is particularly preferred. Compound (1) may be a commercially available product or may be synthesized according to a conventional method.

[0028] As shown in the Examples below, compound (1) has excellent effects of promoting semaphorin 3A expression, suppressing NGF expression, and preventing and / or treating atopic dermatitis, xeroderma, allergic rhinitis, and osteoporosis, and also has low cytotoxicity. Therefore, compound (1) can promote the expression of semaphorin 3A in cells and suppress the expression of NGF in cells, and is useful as a semaphorin 3A expression promoter, an NGF expression inhibitor, a preventive and / or therapeutic agent for atopic dermatitis, a preventive and / or therapeutic agent for xerosis, a preventive and / or therapeutic agent for allergic rhinitis, and a preventive and / or therapeutic agent for osteoporosis. Herein, "promoting semaphorin 3A expression" refers to promoting semaphorin 3A expression in cells, and "suppressing NGF expression" refers to suppressing NGF expression in cells. The cells are preferably skin cells, and include one or more types selected from epidermal keratinocytes, fibroblasts, and melanocytes. Furthermore, in this specification, "prevention and / or treatment of atopic dermatitis" refers to preventing and / or treating atopic dermatitis, and specifically refers to suppressing inflammation and itching caused by atopic dermatitis. Furthermore, in this specification, "prevention and / or treatment of xeroderma" refers to preventing and / or treating xeroderma, and specifically refers to suppressing inflammation and itching associated with xeroderma. In addition, in this specification, "prevention and / or treatment of allergic rhinitis" means preventing and / or treating allergic rhinitis, and specifically means alleviating nasal allergic symptoms such as runny nose, nasal congestion, and sneezing. In addition, in this specification, the term "prevention and / or treatment of osteoporosis" refers to the prevention and / or treatment of osteoporosis.

[0029] Furthermore, compound (1) can serve as a semaphorin 3A expression promoter, an NGF expression inhibitor, a preventive and / or therapeutic agent for atopic dermatitis, a preventive and / or therapeutic agent for xeroderma, a preventive and / or therapeutic agent for allergic rhinitis, or a preventive and / or therapeutic agent for osteoporosis, and can be used for promoting semaphorin 3A expression, suppressing NGF expression, preventing and / or treating atopic dermatitis, a preventive and / or therapeutic agent for xeroderma, a preventive and / or therapeutic agent for allergic rhinitis, or a preventive and / or therapeutic agent for osteoporosis. Furthermore, compound (1) can be used to produce a semaphorin 3A expression promoter, an NGF expression inhibitor, a preventive and / or therapeutic agent for atopic dermatitis, a preventive and / or therapeutic agent for xeroderma, a preventive and / or therapeutic agent for allergic rhinitis, or a preventive and / or therapeutic agent for osteoporosis. Here, the above-mentioned "use" may be administration or ingestion to a human or a non-human animal, and may be therapeutic or non-therapeutic use. Note that "non-therapeutic" is a concept that does not include medical procedures, i.e., a concept that does not include methods of surgery, therapy, or diagnosis on humans, more specifically, a concept that does not include methods of surgery, therapy, or diagnosis on humans by a physician or a person under the direction of a physician.

[0030] The semaphorin 3A expression promoter, NGF expression inhibitor, atopic dermatitis preventive and / or therapeutic agent, xeroderma preventive and / or therapeutic agent, allergic rhinitis preventive and / or therapeutic agent, and osteoporosis preventive and / or therapeutic agent of the present invention can be used as a pharmaceutical, quasi-drug, cosmetic, or food that is effective for promoting semaphorin 3A expression, suppressing NGF expression, preventing and / or treating atopic dermatitis, xeroderma preventive and / or treatment, allergic rhinitis preventive and / or treatment, and osteoporosis preventive and / or treatment, or as an ingredient to be incorporated into a pharmaceutical, quasi-drug, cosmetic, or food. The food products may be health functional foods (e.g., functional foods, foods for specified health uses, foods with nutritional functions, etc.) that are based on the concept of promoting semaphorin 3A expression, suppressing NGF expression, preventing and / or treating atopic dermatitis, preventing and / or treating xerosis, preventing and / or treating allergic rhinitis, and / or treating osteoporosis, and may be labeled as such if necessary.

[0031] The semaphorin 3A expression promoter, NGF expression inhibitor, atopic dermatitis preventive and / or therapeutic agent, xeroderma preventive and / or therapeutic agent, allergic rhinitis preventive and / or therapeutic agent, and osteoporosis preventive and / or therapeutic agent of the present invention may be administered orally, by injection, or topically on the skin, with topical application or injection being preferred, and topical application being more preferred. Examples of such forms in which the skin is used as the application site include topical skin preparations, bath additives, and injectable preparations. The term "skin" encompasses the skin of the face, body, hands, feet, and scalp.

[0032] When the semaphorin 3A expression promoter, NGF expression inhibitor, atopic dermatitis preventive and / or therapeutic agent, xeroderma preventive and / or therapeutic agent, allergic rhinitis preventive and / or therapeutic agent, and osteoporosis preventive and / or therapeutic agent of the present invention are orally administered, their dosage forms include solid preparations such as tablets, granules, fine granules, powders, pills, capsules, and dry syrups; semisolid preparations; and liquid preparations such as suspensions and syrups. These dosage forms may be used in the case of any of pharmaceuticals, quasi-drugs, and foods. Furthermore, when preparing a composition for oral administration, additives such as excipients, disintegrants, binders, and lubricants may be contained in addition to the active ingredients. Specific examples of the food include health functional foods and supplements in the dosage form of tablets, as well as various foods such as wafers, biscuits, and gum.

[0033] When the semaphorin 3A expression promoter, NGF expression inhibitor, atopic dermatitis preventive and / or therapeutic agent, xeroderma preventive and / or therapeutic agent, allergic rhinitis preventive and / or therapeutic agent, or osteoporosis preventive and / or therapeutic agent of the present invention is used transdermally (as an external skin preparation), its dosage form may be an ointment, cream, emulsion, gel, paste, lotion, spray, patch, etc. Furthermore, the external skin preparation may be in the form of, for example, a lotion, serum, pack, emulsion, cream, sunscreen, sun oil, etc. When the application site is the skin, various ingredients used as base materials or additives in ordinary external preparations or bath additives can be contained in addition to the above-mentioned active ingredients.

[0034] The content of compound (1) in the semaphorin 3A expression promoter, NGF expression inhibitor, atopic dermatitis preventive and / or therapeutic agent, xeroderma preventive and / or therapeutic agent, allergic rhinitis preventive and / or therapeutic agent, and osteoporosis preventive and / or therapeutic agent of the present invention is preferably 0.001% by mass or more and 80% by mass or less, and when the semaphorin 3A expression promoter, NGF expression inhibitor, atopic dermatitis preventive and / or therapeutic agent, xeroderma preventive and / or therapeutic agent, allergic rhinitis preventive and / or therapeutic agent, and osteoporosis preventive and / or therapeutic agent of the present invention is a pharmaceutical product, the content is more preferably 0.01% by mass or more and 40% by mass or less. On the other hand, when the semaphorin 3A expression promoter, NGF expression inhibitor, a prophylactic and / or therapeutic agent for atopic dermatitis, xeroderma, allergic rhinitis, or osteoporosis of the present invention is a quasi-drug, cosmetic, or food, the content of compound (1) in the quasi-drug, cosmetic, or food is more preferably 0.001% by mass or more and 10% by mass or less.

[0035] The semaphorin 3A expression promoter, NGF expression inhibitor, preventive and / or therapeutic agent for atopic dermatitis, xeroderma, allergic rhinitis, and osteoporosis of the present invention is preferably ingested or applied in an amount of 0.001 to 5 mg per day as Compound (1) for an adult. [Example]

[0036] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0037] [Test example] (1) Materials and reagents T-5224 was a product of AdooQ Bioscience. c-Jun, c-Fos, and Control siRNA were purchased from Horizon Discovery. The benzimidazole anthelmintics perbendazole, fenbendazole, and albendazole were purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO) before use.

[0038] (2) Culture of NHEKs Normal human epidermal keratinocytes (NHEK) were cultured according to Lonza's protocol. NHEKs were cultured in 6-well tissue culture plates, and perbendazole (0.01 μM, 0.1 μM, 1 μM, 10 μM), fenbendazole (1 μM, 10 μM), or albendazole (1 μM, 10 μM) was added to the proliferating cells at 50–80% cell density. Forty-eight hours after addition of the anthelmintic drugs, the cell culture supernatant was collected and analyzed for Sema3A protein expression by ELISA. Total RNA was extracted and mRNA expression was analyzed by quantitative real-time PCR.

[0039] (3) siRNA knockdown analysis To prove that c-Jun and c-Fos, components of the transcription factor AP-1, are involved in the promotion of Sema3A expression by perbendazole, we used small interfering RNA (siRNA) to suppress the expression of c-Jun and c-Fos, then stimulated the cells with perbendazole and analyzed the expression level of Sema3A mRNA. NHEKs were transfected with siRNA against c-Jun and c-Fos or control siRNA (final concentration 40 nM) using Lipofectamine RNAi MAX (Invitrogen). Cells were cultured in antibiotic-free medium, and after 48 hours, the medium was changed to one containing 1 μM perbendazole to stimulate the cells. After an additional 24 hours, total RNA was extracted from the cells, and the mRNA expression of each transcription factor and Sema3A was analyzed by quantitative real-time PCR. The sequences of the siRNAs used (a mixture of four different siRNA sequences (SEQ ID NOs: 1 to 12)) are shown in Table 1.

[0040] [Table 1]

[0041] (4) Analysis using inhibitors To demonstrate that the transcription factor AP-1 is involved in the perbendazole-induced Sema3A expression promotion, we added the AP-1 inhibitor T-5224 (final concentration 10 μM) to NHEKs to inhibit AP-1 binding to DNA, then stimulated the cells with 1 μM perbendazole and analyzed the expression level of Sema3A.

[0042] (5) Analysis of mRNA expression of each gene The transcription level of each gene was analyzed by quantitative real-time PCR. Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and reverse-transcribed to cDNA using the PrimeScript RT reagent kit (Takara). Quantitative real-time PCR was performed using the Applied Biosystems 7900HT Fast Real-time PCR system (Applied Biosystems) with TB Green Premix Ex Taq (Takara) according to the manufacturer's instructions. The primers used are listed in Table 2 (SEQ ID NOS: 13-22). Ribosomal protein S18 (RPS18) was used as a standard gene. The amount of each mRNA was normalized to the amount of RPS18 and finally expressed as a ratio to the mRNA in the control (solvent-added) group.

[0043] [Table 2]

[0044] (6) Sema3A protein expression analysis The expression level of Sema3A protein was analyzed by ELISA. The amount of Sema3A in the culture supernatant was measured using an ELISA kit for human Sema3A (Uscn Life Science) according to the manufacturer's instructions.

[0045] (7) Analysis of cell viability of NHEKs after the addition of perbendazole The cell viability of NHEKs was analyzed using Cell-Counting Kit-8 (Dojindo Laboratories). Cells were cultured in a 96-well culture plate, and perbendazole was added to cells in the proliferative phase at a cell density of 50-80%. 48 hours after drug addition, CCK-8 solution was added to each well according to the manufacturer's instructions, and the plate was placed in a CO2 incubator for 3 hours to allow for color reaction. Absorbance at 450 nm was then measured using an ARVOX4 multilabel plate reader (Perkin Elmer).

[0046] (8) Cultivation of a three-dimensional human epidermal model The three-dimensional cultured human epidermis model, LabCyte EPI-MODEL (J-TEC), was cultured in the assay medium provided with the kit according to the J-TEC manufacturer's instructions. The day before the experiment, the cells were pre-cultured overnight in a 24-well tissue culture plate. The following day, 50 μL of perbendazole (1 μM or 10 μM) diluted in assay medium was applied dropwise to the surface of the stratum corneum of the epidermal model to stimulate the cells, or the epidermal model was cultured in medium containing perbendazole (1 μM or 10 μM) to stimulate the cells from the basal layer side. After 24 hours of culture, the epidermis was harvested and disrupted with a biomasher. Total RNA was extracted using an RNeasy Mini Kit (Qiagen), and mRNA expression was analyzed by quantitative real-time PCR. Additionally, a solution of 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Dojindo Chemical Industries) diluted in assay medium (final concentration: 0.5 mg / mL) was added to the outside of the culture cup. After 3 hours of incubation in a CO2 incubator, the epidermis was harvested and immersed in 300 μL of isopropanol to extract the pigment. After standing overnight in the dark at room temperature, the extract was subjected to absorbance measurement at 450 nm using an ARVOX4 multilabel plate reader (Perkin Elmer).

[0047] (9) Statistical processing Using the statistical processing software Graph Pad Prism 9 (Graph Pad Software), significant differences were determined by Dunnett's test, Tukey's test, or Student's t-test.* P < 0.05 was considered statistically significant.

[0048] (Test results) Figure 1 shows the results of ELISA to quantify Sema3A protein secreted into the culture supernatant of NHEKs to which perbendazole had been added (compared to the group without perbendazole (0 μM)). *** P < 0.001, **** (P < 0.0001 indicates a significant difference). As shown in Figure 1, the addition of perbendazole to NHEKs increased the amount of Sema3A protein secreted into the culture supernatant.

[0049] Figure 2 shows the results of quantitative real-time PCR analysis of Sema3A mRNA expression in perbendazole-treated NHEKs (compared to the no-perbendazole (0 μM) group). *** P < 0.001, **** (P < 0.0001 indicates a significant difference). As shown in Figure 2, the addition of perbendazole to NHEKs increased the expression level of Sema3A mRNA.

[0050] Figure 3 shows the results of quantitative real-time PCR analysis of NGF mRNA expression in perbendazole-treated NHEKs (compared to the no-perbendazole (0 μM) group). **** (P < 0.0001 indicates a significant difference). As shown in Figure 3, NGF mRNA expression was reduced by the addition of perbendazole.

[0051] FIG. 4 shows the results of analyzing the cell viability of NHEKs to which perbendazole had been added using Cell-counting kit-8. As shown in Figure 4, perbendazole had low cytotoxicity.

[0052] Figure 5-1 shows the results of quantitative real-time PCR analysis of Sema3A mRNA expression in NHEKs treated with fenbendazole (compared to the group without fenbendazole (0 μM)). **** (P < 0.0001 indicates a significant difference). As shown in Figure 5-1, fenbendazole also promoted Sema3A mRNA expression when added to NHEKs. Figure 5-2 shows the results of quantitative real-time PCR analysis of Sema3A mRNA expression in albendazole-treated NHEKs (compared to the albendazole-free (0 μM) group). **** (P < 0.0001 indicates a significant difference). As shown in Figure 5-2, albendazole also promoted Sema3A mRNA expression when added to NHEKs.

[0053] Figure 6-1 shows the results of quantitative real-time PCR analysis of NGF mRNA expression in NHEKs treated with fenbendazole (compared to the group without fenbendazole (0 μM)). **** (P < 0.0001 indicates a significant difference). As shown in Figure 6-1, NGF mRNA expression was also suppressed in NHEKs treated with fenbendazole. Figure 6-2 shows the results of quantitative real-time PCR analysis of NGF mRNA expression in albendazole-treated NHEKs (compared to the albendazole-free (0 μM) group). **** (P < 0.0001 indicates a significant difference). As shown in Figure 6-2, NGF mRNA expression was also suppressed in NHEKs treated with albendazole.

[0054] Figure 7-1 shows the results of analyzing Sema3A mRNA expression by real-time PCR when perbendazole was added to the stratum corneum surface of a three-dimensional culture human epidermal model and cells were stimulated from the stratum corneum side (compared to the group without perbendazole (0 μM)). ** P < 0.01, **** (P < 0.0001 indicates a significant difference). As shown in Figure 7-1, application of perbendazole solution to the stratum corneum surface of the three-dimensional cultured human epidermis model increased Sema3A mRNA expression. Figure 7-2 shows the results of analyzing Sema3A mRNA expression by real-time PCR when perbendazole was added to the medium of a three-dimensional culture human epidermal model and cells were stimulated from the basal layer side (compared to the group without perbendazole (0 μM)). * P < 0.05, ** P < 0.01 indicates a significant difference). As shown in Figure 7-2, Sema3A mRNA expression also increased when cells were stimulated from the basal layer side with perbendazole solution diluted in the medium.

[0055] Figure 8-1 shows the results of an analysis of cell viability when perbendazole was added to the stratum corneum surface of a three-dimensional cultured human epidermal model, and Figure 8-2 shows the results of an MTT assay analysis of cell viability when perbendazole was added to the culture medium of a three-dimensional cultured human epidermal model and stimulation was performed from the basal layer side. As shown in Figures 8-1 and 8-2, when cells of the three-dimensional cultured human epidermal model were stimulated with perbendazole from the stratum corneum side (Figure 8-1) or the basal layer side (Figure 8-2), no significant decrease in cell viability was observed.

[0056] Figure 9 shows the results of analyzing Sema3A mRNA expression by real-time PCR when perbendazole was added after knockdown of the transcription factors c-Jun and c-Fos in NHEKs (compared to the siControl group without perbendazole (0 μM)). **** Significant difference (P < 0.0001). Compared to the siControl group with 1 μM perbendazole added, ††††Significant difference (P < 0.0001). As shown in Figure 9, the promoting effect of perbendazole on Sema3A expression was significantly suppressed by knockdown of c-Jun and c-Fos, which are components of the transcription factor AP-1.

[0057] Figure 10 shows the results of analyzing Sema3A mRNA expression by real-time PCR when NHEKs were stimulated with perbendazole in the presence of an inhibitor of the transcription factor AP-1 (compared to the group without perbendazole (0 μM)). **** Significant difference at P < 0.0001. ††††Significant difference at P < 0.0001 compared to the T-5224-free (0 μM) group with 1 μM perbendazole. As shown in FIG. 10, the increase in Sema3A expression caused by the addition of perbendazole was also suppressed when the cells were treated with the AP-1 inhibitor T-5224.

Claims

[Claim 1] A semaphorin 3A expression promoter having one or more active ingredients selected from perbendazole, fenbendazole and albendazole.

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