Dermal papilla cell activation composition

Abstract

Provided is a dermal papilla cell activation composition for effectively activating dermal papilla cells. The dermal papilla cell activation composition contains oxytocin and / or an oxytocin receptor agonist. The dermal papilla cell activation composition may be a topical skin formulation, a topical scalp formulation, a composition for hair growth or hair restoration, a culture medium, or an additive for a culture medium.

Description

Composition for activating dermal papilla cells

[0001] The present invention relates to a composition for activating hair follicle papilla cells.

[0002] Non-Patent Document 1 describes that dermal papilla cells contained in hair follicles in the body play an important role in controlling the hair cycle.

[0003] On the other hand, Patent Documents 1 to 3 and Non-Patent Document 1 describe a technique for producing a cell aggregate having hair regeneration ability by co-culturing epithelial cells and mesenchymal cells (e.g., dermal papilla cells).

[0004] International Publication No. 2017 / 073625 International Publication No. 2020 / 225934 International Publication No. 2023 / 058429

[0005] Jiyu Hyun et al., "Morus alba Root Extract Induces the Anagen Phase in the Human Hair Follicle Dermal Papilla Cells", Pharmaceutics, 2021, 13, 1155Tatsuto Kageyama et al., In vitro hair follicle growth model for drug testing, Scientific Reports (2023) 13: 4847

[0006] However, it has not been easy to activate dermal papilla cells, nor has it been easy to promote hair growth or hair regeneration in vivo through activation of dermal papilla cells.

[0007] The present invention has been made in view of the above problems, and one of its objects is to provide a composition for activating hair follicle papilla cells that effectively activates hair follicle papilla cells.

[0008] [1] A composition for activating hair follicle dermal papilla cells according to one embodiment of the present invention for solving the above-mentioned problems contains oxytocin and / or an oxytocin receptor agonist. According to the present invention, a composition for activating hair follicle dermal papilla cells that effectively activates hair follicle dermal papilla cells is provided.

[0009] [2] The composition of [1] may contain oxytocin. [3] The composition of [1] or [2] may contain an oxytocin receptor agonist.

[0010] [4] Any of the compositions [1] to [3] above may be a topical preparation for skin. [5] Any of the compositions [1] to [4] above may be a topical preparation for scalp. [6] Any of the compositions [1] to [5] above may be a composition for hair growth or hair regeneration.

[0011] [7] Any of the compositions described in [1] to [3] above may be a culture medium. [8] Any of the compositions described in [1] to [3] above may be a culture medium additive.

[0012] According to the present invention, there is provided a composition for activating dermal papilla cells that effectively activates dermal papilla cells.

[0013] FIG. 1 is an explanatory diagram showing the results of evaluating the expression levels of hair growth-related genes in hair papilla cells cultured in Example 1. FIG. 2 is an explanatory diagram showing a micrograph of a cell aggregate formed in co-culture 1 of Reference Example. FIG. 3 is an explanatory diagram showing the results of evaluating the length of hair shaft-like structures formed in a cell aggregate in co-culture 1 of Reference Example. FIG. 4 is an explanatory diagram showing the results of evaluating the length of hair shaft-like structures formed in a cell aggregate in co-culture 2 of Reference Example. FIG. 5 is an explanatory diagram showing the results of evaluating the length of hair shaft-like structures formed in a cell aggregate in Example 2. FIG. 6 is an explanatory diagram showing the results of evaluating the expression levels of growth factor genes in a cell aggregate in Example 2. FIG. 7 is an explanatory diagram showing the results of evaluating the expression levels of hair growth-related genes and growth factor genes in hair papilla cells cultured in Example 3. FIG. 8 is an explanatory diagram showing the results of evaluating the length of hair shaft-like structures formed in a cell aggregate in Example 4. FIG. 9 is an explanatory diagram showing the results of evaluating the expression levels of hair growth-related genes in hair papilla cells cultured in Example 5. FIG. 10 is an explanatory diagram showing the results of evaluating the length of hair shaft-like structures formed in a cell aggregate in Example 6.

[0014] An embodiment of the present invention will be described below, although the present invention is not limited to this embodiment.

[0015] The composition for activating hair follicle dermal papilla cells according to this embodiment (hereinafter referred to as "the composition") contains oxytocin and / or an oxytocin receptor agonist. That is, the composition may be a composition containing oxytocin, a composition containing an oxytocin receptor agonist, or a composition containing oxytocin and an oxytocin receptor agonist.

[0016] The present composition activates dermal papilla cells. Therefore, the present composition is used to activate dermal papilla cells. In this regard, the inventors of the present invention have conducted extensive research into technical means for activating dermal papilla cells and unexpectedly found that oxytocin and an oxytocin receptor agonist each activate dermal papilla cells, leading to the completion of the present invention.

[0017] Oxytocin contained in this composition is a compound composed of nine amino acids and has the chemical formula C ４３ H ６６ N １２ O １２ S ２ It has a molecular weight of 1007.19 and a CAS (Chemical Abstracts Service) registration number of 50-56-6. The oxytocin contained in the composition may be chemically modified for the purpose of stabilization or the like, as long as the effects of the present invention are not impaired. Oxytocin is thought to activate dermal papilla cells via an oxytocin receptor expressed by the dermal papilla cells.

[0018] The oxytocin receptor agonist contained in the present composition is an agonist that acts on the oxytocin receptor, and is thought to activate dermal papilla cells via the oxytocin receptor expressed by the dermal papilla cells.

[0019] The oxytocin receptor agonist contained in the present composition is not particularly limited as long as it activates hair follicle papilla cells, and may be, for example, one or more selected from the group consisting of LIT-001 (CAS Registration No.: 2245072-21-1), WAY267464 (CAS Registration No.: 1432043-31-6), TC OT 39 (CAS Registration No.: 479232-57-0), and carbetocin (CAS Registration No.: 37025-55-1).

[0020] The composition contains oxytocin and / or an oxytocin receptor agonist as an active ingredient that activates dermal papilla cells. The activation of dermal papilla cells by the composition is confirmed, for example, by an increase in the expression levels of hair growth-related genes and / or growth factor genes in the dermal papilla cells.

[0021] The hair growth-related gene is not particularly limited as long as it is a gene related to hair growth in a living organism, but for example, the composition may increase the expression level in dermal papilla cells of one or more genes selected from the group consisting of alkaline phosphatase gene, versican gene, LEF1 gene, WNT5A gene, BMP4 gene, NOG gene, SOX2 gene, and WIF1 gene. The expression level of the hair growth-related gene in dermal papilla cells is quantified by extracting RNA from the dermal papilla cells, performing reverse transcription, and then carrying out RT-PCR.

[0022] Specifically, the composition may increase the expression level of a hair growth-related gene (e.g., one or more selected from the group consisting of alkaline phosphatase gene, versican gene, LEF1 gene, WNT5A gene, BMP4 gene, NOG gene, SOX2 gene, and WIF1 gene) in hair papilla cells by 1.1 times or more, preferably 1.2 times or more, more preferably 1.3 times or more, even more preferably 1.4 times or more, and particularly preferably 1.5 times or more, compared to the expression level when a composition having the same composition but not containing oxytocin and an oxytocin receptor agonist is used.

[0023] The present composition is used, for example, to activate dermal papilla cells in a living body. That is, the present composition may be an external preparation for skin. The present composition as an external preparation for skin is used to activate dermal papilla cells contained in the skin of a living body (for example, dermal papilla cells contained in hair follicles of the skin).

[0024] The method for applying the present composition to the skin is not particularly limited as long as it allows the oxytocin and / or oxytocin receptor agonist contained in the present composition to reach the hair papilla cells contained in the skin. For example, coating, spraying, or injection onto the skin is preferably used.

[0025] The living body to which the composition is applied is not particularly limited as long as it is an animal having skin containing hair papilla cells, and may be a human or a non-human animal. Preferably, the non-human animal is a mammal. The mammal may be, for example, a primate (e.g., monkey), a rodent (e.g., mouse, rat, hamster, guinea pig, or rabbit), a carnivore (e.g., dog, cat), or an ungulate (e.g., pig, cow, horse, goat, or sheep).

[0026] The skin of a living body to which the present composition is applied is not particularly limited as long as it is skin containing hair papilla cells (e.g., skin containing hair follicles), but may be, for example, the scalp. That is, the present composition may be an external preparation for the scalp.

[0027] The present composition as an external scalp preparation is used to activate dermal papilla cells (e.g., dermal papilla cells contained in hair follicles on the scalp) contained in the scalp of a living organism (preferably a human). The present composition as an external scalp preparation is not particularly limited as long as it is used to allow oxytocin and / or an oxytocin receptor agonist contained in the composition to reach dermal papilla cells contained in the scalp, and may be, for example, a hair growth composition, a hair regrowth composition, a hair shampoo, a hair rinse, a hair conditioner, a hair treatment, or a hair tonic.

[0028] The present composition is preferably a composition for hair growth or hair regeneration. The present composition as a composition for hair growth or hair regeneration is used to activate dermal papilla cells (for example, dermal papilla cells contained in hair follicles of the skin) contained in the skin of a living body (for example, the scalp) and promote hair growth or hair regeneration in the skin.

[0029] Specifically, the present composition as a hair growth composition is used for hair growth (e.g., promoting hair growth, inhibiting hair loss, or inhibiting reduction, maintaining, and increasing hair diameter) by activating hair papilla cells contained in the hair follicles in the skin where hair grows (e.g., the scalp where hair grows).

[0030] Furthermore, as a hair growth composition, the present composition is used for hair growth (promoting the formation and growth of new hair) by activating dermal papilla cells contained in skin that contains dermal papilla cells but does not grow hair (for example, scalp that contains dermal papilla cells or hair follicles but does not grow hair).

[0031] The use of the composition is not particularly limited as long as the effects of the present invention can be obtained, and may be, for example, medical use or research use. That is, the composition may be used, for example, for the treatment or prevention of a disease related to hair growth or hair loss. In this case, the composition is applied, for example, to a living organism having a disease related to hair growth or hair loss. The living organism having a disease related to hair growth or hair loss may be, for example, a human or non-human animal having the disease, or a human or non-human animal that may suffer from the disease. The composition may also be used, for example, for research related to a disease related to hair growth or hair loss.

[0032] The disease related to hair growth or hair loss is not particularly limited, and may be, for example, one or more selected from the group consisting of androgenetic alopecia (AGA), female androgenetic alopecia (FAGA), postpartum alopecia, diffuse alopecia, seborrheic alopecia, pityriasis nobilis, traction alopecia, metabolic alopecia, pressure alopecia, alopecia areata, neurogenic alopecia, trichotillomania, alopecia universalis, and symptomatic alopecia.

[0033] Furthermore, the present composition is used, for example, to activate dermal papilla cells in vitro. That is, the present composition may be used, for example, to activate cultured dermal papilla cells.

[0034] Specifically, the composition may be a culture medium. The composition as a culture medium contains components necessary for the survival of cultured dermal papilla cells (e.g., minerals and buffers for maintaining appropriate osmotic pressure and pH). The composition as a culture medium may further contain components necessary for the metabolism of the cultured dermal papilla cells (e.g., sugars and / or amino acids), and may further contain components necessary for the proliferation of the cultured dermal papilla cells (e.g., growth factors (e.g., bFGF) and / or hormones (e.g., insulin)).

[0035] The present composition may also be an additive for a culture medium. The present composition as a culture medium additive is used in preparing a culture medium for activating cultured hair papilla cells. That is, the present composition as a culture medium additive is used by adding it to physiological saline (including that containing a buffer) for culturing hair papilla cells or to a basal medium (for example, a medium used for culturing hair papilla cells, such as DMEM or F12 medium).

[0036] The form of the composition is not particularly limited as long as the effects of the present invention are obtained, and an appropriate form may be appropriately adopted depending on the application. That is, the composition may be, for example, a fluid composition (e.g., a liquid or paste-like composition) or a non-fluid composition (e.g., a solid or gel-like composition). Specifically, the composition may be, for example, a liquid, a lotion, a suspension, an emulsion, an injection, a patch, an ointment, a cataplasm, a liniment, a spray, a lyophilized product, or a powder.

[0037] The contents of oxytocin and oxytocin receptor agonist in the present composition are not particularly limited as long as they fall within the range in which the effects of the present invention can be obtained, and are determined appropriately depending on the application and form. That is, for example, when the present composition is a fluid hair-growth or hair-restoring composition, the contents of oxytocin and / or oxytocin receptor agonist that will achieve the desired effects can be determined by using multiple types of the composition with different contents of oxytocin and / or oxytocin receptor agonist and evaluating the effect of activating hair papilla cells and / or the hair-growth or hair-restoring effect.

[0038] Furthermore, for example, when the composition is a fluid culture medium, the content of oxytocin and / or oxytocin receptor agonist that will achieve the desired effect can be determined by culturing hair papilla cells using multiple types of culture media that differ in the content of oxytocin and / or oxytocin receptor agonist and evaluating the effect of activating the cultured hair papilla cells.

[0039] As described above, the content of oxytocin in the composition is not particularly limited as long as it is within a range in which the effects of the present invention can be obtained, and may be determined appropriately depending on the application and form. However, the content of oxytocin in the composition (e.g., the composition having fluidity) may be, for example, 0.01 μM or more, preferably 0.05 μM or more, more preferably 0.1 μM or more, even more preferably 0.5 μM or more, even more preferably 1 μM or more, even more preferably 3 μM or more, even more preferably 5 μM or more, even more preferably 7 μM or more, and particularly preferably 10 μM or more.

[0040] The upper limit of the oxytocin content in the composition is not particularly limited, and may be, for example, 1000 μM or less, 500 μM or less, or 200 μM or less. The oxytocin content in the composition may be specified by any combination of any one of the above-mentioned lower limit values ​​and any one of the above-mentioned upper limit values.

[0041] Furthermore, as described above, the content of the oxytocin receptor agonist in the composition is not particularly limited as long as it is within a range in which the effects of the present invention can be obtained, and may be determined appropriately depending on the application and form. However, the content of the oxytocin receptor agonist in the composition (e.g., the composition having fluidity) may be, for example, 0.01 μM or more, preferably 0.05 μM or more, more preferably 0.1 μM or more, even more preferably 0.5 μM or more, even more preferably 1 μM or more, even more preferably 5 μM or more, even more preferably 10 μM or more, even more preferably 15 μM or more, and particularly preferably 20 μM or more.

[0042] The upper limit of the content of the oxytocin receptor agonist in the composition is not particularly limited, and may be, for example, 1000 μM or less, 500 μM or less, or 200 μM or less. The content of the oxytocin receptor agonist in the composition may be specified by any combination of any one of the above-mentioned lower limit values ​​and any one of the above-mentioned upper limit values.

[0043] Furthermore, when the composition contains oxytocin and an oxytocin receptor agonist, the combination of the oxytocin content and the oxytocin receptor agonist content in the composition (e.g., the composition having flowability) may be specified by any combination of the oxytocin content specified as described above and the oxytocin receptor agonist content specified as described above.

[0044] The present composition may contain ingredients other than oxytocin and an oxytocin receptor agonist depending on the intended use and form. That is, the present composition may contain ingredients acceptable for use in, for example, pharmaceuticals, quasi-drugs, or scalp or hair cosmetics. Specifically, the present composition may contain one or more selected from the group consisting of excipients, stabilizers, dispersants, diluents, surfactants, emulsifiers, pH adjusters, preservatives, colorants, moisturizers, thickeners, antioxidants, preservatives, cooling agents, and fragrances.

[0045] Next, a specific example according to this embodiment will be described.

[0046] [Cell Culture] Using a two-dimensional culture system of dermal papilla cells, the effect of oxytocin on the expression of hair growth-related genes in the dermal papilla cells was evaluated. Specifically, commercially available human dermal papilla cells (PromoCell, passage number 4) were first suspended in a medium prepared by adding oxytocin (product code: 4084-v, CAS Registry Number: 50-56-6, Peptide Institute, Inc.) to a commercially available dermal papilla cell growth medium (Rohto Pharmaceutical Co., Ltd.) at concentrations of 0.1 μM, 1 μM, or 10 μM (hereinafter referred to as "oxytocin-added medium" in Example 1), or in the dermal papilla cell growth medium without added oxytocin (oxytocin concentration: 0 μM) (hereinafter referred to as "supplement-free medium" in Example 1), to prepare a cell suspension in which the dermal papilla cells were dispersed.

[0047] Next, the cell density at the start of culture was 2 × 10 ４ The cell suspension was seeded into the flat-bottom wells of a 24-well plate at an amount of 100 cells / well. The dermal papilla cells adhered to the bottom of the wells were then two-dimensionally cultured for 6 days. The medium was changed every 3 days.

[0048] [Expression Amounts of Hair Growth-Related Genes] RNA was extracted from the dermal papilla cells on day 6 of culture, and after reverse transcription, RT-PCR was carried out to quantify the expression amounts of the hair growth-related genes, alkaline phosphatase (ALP) gene, versican (VCAN) gene, LEF1 gene, WNT5A gene, BMP4 gene, and NOG gene.

[0049] The primers used for RT-PCR had the following nucleotide sequences: ALP forward primer: 5'-ATTGACCACGGGCACCAT-3'. ALP reverse primer: 5'-CTCCACCGCCTCATGCA-3'. VCAN forward primer: 5'-GGCACAAATTCCAAGGGCAG-3'. VCAN reverse primer: 5'-TCATGGCCCACACGATTAACA-3'. LEF1 forward primer: 5'-CTTCCTTGGTGAACGAGTCTG-3'. LEF1 reverse primer: 5'-TCTGGATGCTTTCCGTCAT-3'. WNT5A forward primer: 5'-TCCACCTTCCTCTTCACACTGA-3'. WNT5A reverse primer: 5'-CGTGGCCAGCATCACATC-3'. BMP4 forward primer: 5'-GCCCGCAGCCTAGCAA-3'. BMP4 reverse primer: 5'-CGGTAAAGATCCCGCATGTAG-3'. NOG forward primer: 5'-CTGGTGGACCTCATCGAACA-3'. NOG reverse primer: 5'-CGTCTCGTTCAGATCCTTTTCCT-3'. GAPDH forward primer used as a control: 5'-TGGAAGGACTCATGACCACAG-3'. GAPDH reverse primer: 5'-GGATGATGTTCTGGAGAGCCC-3'.

[0050] [Results] Figure 1 shows the results of quantifying the expression levels of the VCAN gene, ALP gene, NOG gene, LEF1 gene, WNT5A gene, and BMP4 gene in dermal papilla cells. In Figure 1, the horizontal axis shows the concentration (µM) of oxytocin (OXT) added to the medium, and the vertical axis shows the relative gene expression level (arithmetic mean value, n = 3) of dermal papilla cells cultured in an oxytocin-added medium, when the gene expression level of dermal papilla cells cultured in an oxytocin-free medium (oxytocin concentration 0 (zero) µM) is set to "1."

[0051] As shown in Figure 1, the expression levels of hair growth-related genes in dermal papilla cells cultured in an oxytocin-added medium were higher than those in dermal papilla cells cultured in an oxytocin-free medium. Furthermore, it was confirmed that the expression levels of hair growth-related genes in dermal papilla cells tended to increase as the concentration of oxytocin added to the medium increased. In particular, the expression levels of the VCAN gene, ALP gene, and NOG gene, among others, the NOG gene, were significantly increased by oxytocin. Thus, it was confirmed that oxytocin activates dermal papilla cells, effectively increasing the expression levels of hair growth-related genes in the dermal papilla cells.

[0052] Furthermore, the number (proliferation) of cultured dermal papilla cells was also quantified (not shown), and the presence or absence of oxytocin in the medium did not significantly affect the change in cell number over time. In other words, it was confirmed that oxytocin activated the dermal papilla cells without inhibiting their proliferation. Furthermore, the results of immunofluorescence staining (not shown) confirmed that the cultured dermal papilla cells expressed oxytocin receptors. In other words, it was thought that oxytocin activated the dermal papilla cells via the oxytocin receptors expressed by the dermal papilla cells. Reference example

[0053] The inventors of the present invention have previously demonstrated that co-culturing epithelial cells and mesenchymal cells dispersed in a medium results in the formation of a cell aggregate containing the epithelial cells and mesenchymal cells, and that transplanting the cell aggregate into a living organism can regenerate new hair at the transplant site in the living organism (Patent Document 1: WO 2017 / 073625).

[0054] The inventors of the present invention have also developed a culture method for forming cell aggregates that grow hair in vitro by co-culturing epithelial cells and mesenchymal cells in a medium containing a low concentration of a specific extracellular matrix (Patent Document 2: WO 2020 / 225934, Patent Document 3: WO 2023 / 058429), and have further used this culture method to establish an in vitro model for evaluating hair growth-promoting substances (Non-Patent Document 2: Tatsuto Kageyama et al., In vitro hair follicle growth model for drug testing, Scientific Reports (2023) 13: 4847). This Reference Example describes the establishment of this in vitro model.

[0055] [Cell Preparation] Commercially available human fetal epidermal keratinocytes (ScienCell Research Laboratories) (hereinafter referred to as "F epithelial cells" in this Reference Example) and commercially available human adult hair follicle keratinocytes (ScienCell Research Laboratories) (hereinafter referred to as "A epithelial cells" in this Reference Example) were used as epithelial cells (both of which were passaged 2 or 3).

[0056] As mesenchymal cells, commercially available human fetal dermal fibroblasts (ScienCell Research Laboratories) (hereinafter referred to as "F mesenchymal cells" in this Reference Example) and commercially available human adult hair papilla cells (PromoCell) (hereinafter referred to as "A mesenchymal cells" in this Reference Example) that were also used in Example 1 above were used (both mesenchymal cells were passaged 2 or 3).

[0057] [Co-culture 1] First, epithelial cells (F epithelial cells or A epithelial cells) and mesenchymal cells (F mesenchymal cells or A mesenchymal cells) were suspended and mixed in a medium prepared by adding Matrigel (Matrigel (registered trademark), Basement Membrane Matrix, CORNING (registered trademark)) or type I collagen (Cellmatrix type IA, Nitta Gelatin Co., Ltd.) as an extracellular matrix to a concentration of 2 v / v% (hereinafter referred to as "matrix-added medium" in the Reference Examples). The epithelial cells and mesenchymal cells were each suspended in a concentration of 5 × 10 ３ A cell suspension was prepared in which the cells were dispersed at a density of 100 cells / 200 μL.

[0058] Next, 200 μL of the resulting cell suspension was added to each non-cell-adhesive round-bottom well of a 96-well plate to seed epithelial cells and mesenchymal cells. After seeding, the 96-well plate was immediately centrifuged (100 × g) for 2 minutes. After centrifugation, the 96-well plate was immediately transferred to a 4°C refrigerator and left for at least 30 minutes. The 96-well plate was then transferred to a 37°C incubator to initiate co-culture of epithelial cells and mesenchymal cells. Medium changes were performed every two days by removing 100 μL of the 200 μL medium in each well and replacing it with 100 μL of matrix-free DMEM / F12 medium (hereinafter referred to as "unsupplemented medium" in the Reference Examples).

[0059] The co-culture was performed under the following 12 conditions, which differed depending on the combination of epithelial cells, mesenchymal cells, and extracellular matrix added to the medium: (1) FF-M (F epithelial cells + F mesenchymal cells + Matrigel); (2) FA-M (F epithelial cells + A mesenchymal cells + Matrigel); (3) AF-M (A epithelial cells + F mesenchymal cells + Matrigel); (4) AA-M (A epithelial cells + A mesenchymal cells + Matrigel); (5) FF-C (F epithelial cells + F mesenchymal cells + collagen); (6) FA-C (F epithelial cells + A mesenchymal cells + collagen); (7) AF-C (A epithelial cells + F mesenchymal cells + collagen); (8) AA-C (A epithelial cells + A mesenchymal cells + collagen); (9) FF-No (F epithelial cells + F mesenchymal cells + no matrix); (10) FA-No (F epithelial cells + A mesenchymal cells + no matrix); (11) AF-No (A epithelial cells + F mesenchymal cells + no matrix); (12) AA-No (A epithelial cells + A mesenchymal cells + no matrix).

[0060] [Result 1] Figure 2 shows phase-contrast micrographs of cells in wells on day 10 of culture for AA-M, FF-M, FF-C, FA-M, AF-M, and AF-C, out of the 12 conditions mentioned above. Figure 3 also shows the results of measuring the length of the hair shaft-like structures formed in the cell aggregates using a fluorescence microscope (BZ-X810, Keyence Corporation) for eight conditions using matrix-supplemented medium (FF-M, FA-M, AF-M, AA-M, FF-C, FA-C, AF-C, and AA-C).

[0061] In Figure 3, the horizontal axis indicates the culture days on which the length of the hair shaft-like structure was measured (culture day 4 (D4), day 6 (D6), day 8 (D8), and day 10 (D10)), and the vertical axis indicates the measured length (mm) of the hair shaft-like structure. Here, the length of the hair shaft-like structure formed in the cell aggregate was measured as the linear distance from the base of the hair shaft-like structure (the connection part with the cell aggregate) to the tip of the hair shaft-like structure (the free end).

[0062] As shown in Figures 2 and 3, in the five conditions of AA-M, FF-M, FF-C, AF-M, and AF-C, cells spontaneously aggregated over the course of culture time after seeding, forming one cell aggregate in each well. In these cell aggregates, hair shaft-like structures were formed by the fourth day of culture, and the length of the hair shaft-like structures then increased until the tenth day of culture.

[0063] In particular, under the AA-M condition, the length of the hair shaft-like structures formed in the cell aggregates was statistically significantly greater than that of the cell aggregates cultured under the other seven conditions using matrix-supplemented medium on the 10th day of culture (arithmetic mean, n = 24).

[0064] Therefore, a co-culture system under AA-M conditions, which uses human adult hair follicle keratinocytes as epithelial cells, human adult dermal papilla cells as mesenchymal cells, and Matrigel as extracellular matrix, was considered to be the most preferable in vitro model that mimics hair growth from hair follicles in vivo, with the growth of hair shaft-like structures from cell aggregates as an indicator.

[0065] [Co-culture 2] Next, the usefulness of the above-described AA-M co-culture system as an in vitro model was confirmed using minoxidil (CAS registration number: 38304-91-5), a hair growth-promoting substance proven to promote hair growth in vivo. Specifically, co-culture was initiated by seeding human adult hair follicle keratinocytes and human adult dermal papilla cells suspended in matrix-supplemented medium into non-cell-adhesive round-bottom wells under the same AA-M conditions as in "Co-culture 1" described above. Medium changes were performed every two days by removing 100 μL of the 200 μL of medium in the wells and replacing it with 100 μL of unsupplemented medium.

[0066] Then, on day 4 of the culture, the medium in some of the wells was replaced with a medium prepared by adding minoxidil to a concentration of 10 μM (hereinafter referred to as "MNX-added medium" in the Reference Examples), and co-culture was continued for 6 days in the MNX-added medium until day 10. Meanwhile, for comparison, the medium in some of the other wells was replaced with a supplement-free medium on day 4 of the culture, and co-culture was continued until day 10. The medium was replaced once every two days.

[0067] [Result 2] Figure 4 shows the results of evaluating the relative lengths of the hair shaft-like structures on days 6, 8, and 10 of culture (D6, D8, and D10 on the horizontal axis, respectively) using a fluorescence microscope (BZ-X810, Keyence Corporation), where the length of the hair shaft-like structures on day 4 of culture (D4 on the horizontal axis) is set to "1" for cell aggregates cultured in a supplement-free medium from day 4 onwards (0 µM MNX in the figure) and cell aggregates cultured in an MNX-supplemented medium from day 4 onwards (10 µM MNX in the figure) (arithmetic mean, n = 24).

[0068] As shown in Figure 4, in the cell aggregates cultured in MNX-supplemented medium from day 4 onwards, the length of the hair shaft-like structures increased more rapidly than in the cell aggregates cultured in MNX-free medium.

[0069] Furthermore, on days 8 and 10 of culture, the relative length of hair shaft-like structures in cell aggregates cultured in MNX-supplemented medium was statistically significantly greater than that of cell aggregates cultured in MNX-free medium.

[0070] Thus, the above-described AA-M co-culture system showed a hair growth-promoting effect in response to minoxidil similar to that of in vivo hair follicles. In other words, the above-described AA-M co-culture system was confirmed to be useful as an in vitro model for evaluating whether a candidate substance has a hair growth-promoting effect outside the body by using the length of the hair shaft-like structures in the cell aggregates as an indicator.

[0071] [In vitro model] Using the in vitro model (AA-M co-culture system) established in the above-mentioned "Reference Example," the effect of oxytocin on the growth of hair shaft-like structures in cell aggregates was evaluated. That is, first, in the same manner as in the above-mentioned "Reference Example," human adult hair follicle keratinocytes and human adult dermal papilla cells were suspended and mixed in a medium prepared by adding Matrigel (Matrigel (registered trademark), Basement Membrane Matrix, CORNING (registered trademark)) at a concentration of 2 v / v% to commercially available DMEM / F12 medium (Advanced Dulbecco's Modified Eagle Medium / Ham's F-12, GIBCO (registered trademark)) (hereinafter referred to as "supplement-free medium" in Example 2) without adding oxytocin, and the hair follicle keratinocytes and dermal papilla cells were each suspended at a concentration of 1 x 10 ４ A cell suspension was prepared in which the cells were dispersed at a density of 100 cells / 200 μL.

[0072] Next, 200 μL of the resulting cell suspension was added to each non-cell-adhesive round-bottom well of a 96-well plate to seed epithelial cells and mesenchymal cells. After seeding, the 96-well plate was immediately centrifuged (100 × g) for 2 minutes. After centrifugation, the 96-well plate was immediately transferred to a 4°C refrigerator and left to stand for at least 30 minutes. The 96-well plate was then transferred to a 37°C incubator to initiate co-culture of epithelial cells and mesenchymal cells. Medium changes were performed every two days by removing 100 μL of the 200 μL medium in each well and replacing it with 100 μL of unsupplemented medium.

[0073] On day 4 of the culture, the medium in some of the wells was replaced with medium prepared by adding oxytocin to a concentration of 10 μM to a supplement-free medium (hereinafter referred to as "OXT-added medium" in Example 2), and co-culture was then carried out for 6 days in the OXT-added medium until day 10. Meanwhile, for comparison, the medium in some of the other wells was replaced with a supplement-free medium on day 4 of the culture, and co-culture was carried out until day 10. The medium was replaced once every two days.

[0074] [Length of hair shaft-like structures] After the start of co-culture, hair follicle keratinocytes and dermal papilla cells spontaneously aggregated, and one cell aggregate was formed in each well by day 4 of culture. Furthermore, on day 4 of culture, the formation of a hair shaft-like structure was confirmed in each cell aggregate. Therefore, the length of the hair shaft-like structure formed in each cell aggregate was measured using a fluorescence microscope (BZ-X810, Keyence Corporation) on days 4, 6, 8, and 10 of culture, as in the above-mentioned "Reference Example."

[0075] [Expression Amount of Growth Factor Genes] RNA was extracted from the cell aggregates on day 10 of culture, and after reverse transcription, RT-PCR was carried out to quantify the expression amounts of the growth factor genes, VEGFA (vascular endothelial growth factor A) gene and PDGFB (platelet-derived growth factor subunit B) gene.

[0076] The primers used for RT-PCR had the following base sequences: VEGFA forward primer: 5'-ACTTCTGGGCTGTTCTCG-3'. VEGFA reverse primer: 5'-TCCTCTTCCTTCTCTTCTTC-3'. PDGFB forward primer: 5'-GAAGGAGCCTGGGTTCCC-3'. PDGFB reverse primer: 5'-TTTCTCACCTGGACAGGT-3'. GAPDH forward primer used as a control: 5'-TGGAAGGACTCATGACCACAG-3'. GAPDH reverse primer: 5'-GGATGATGTTCTGGAGAGCCC-3'.

[0077] [Results] Figure 5 shows the results of evaluating the relative lengths of the hair shaft-like structures on days 6, 8, and 10 of culture (D6, D8, and D10 on the horizontal axis, respectively) for cell aggregates cultured in a supplement-free medium from day 4 onwards ("0 μM OXT" in the figure) and cell aggregates cultured in an OXT-supplemented medium from day 4 onwards ("10 μM OXT" in the figure) (arithmetic mean values, n = 24), where the length of the hair shaft-like structures on day 4 of culture ("D4" on the horizontal axis) is set to "1."

[0078] As shown in Figure 5, in the cell aggregates cultured in OXT-supplemented medium from day 4 onwards, the length of the hair shaft-like structures increased more rapidly than in the cell aggregates cultured in OXT-free medium.

[0079] Furthermore, on days 8 and 10 of culture, the relative length of the hair shaft-like structures in the cell aggregates cultured in OXT-supplemented medium was statistically significantly greater than that of the cell aggregates cultured in OXT-free medium, confirming the hair growth-promoting effect of oxytocin.

[0080] Fig. 6 shows the results of quantifying the expression levels of the VEGFA gene and the PDGFB gene in the cell aggregates on day 10 of culture. In Fig. 6, the horizontal axis shows the concentration of oxytocin added to the medium ("0 µM", "10 µM"), and the vertical axis shows the relative gene expression level (arithmetic mean value, n = 3) in the cell aggregates cultured in the OXT-supplemented medium (10 µM) when the gene expression level in the cell aggregates cultured in the supplement-free medium (0 µM) is set to "1".

[0081] As shown in Figure 6, the expression levels of both the VEGFA gene and the PDGFB gene in the cell aggregates cultured in OXT-supplemented medium were statistically significantly higher than those in the cell aggregates cultured in OXT-free medium. In other words, it was confirmed that oxytocin effectively increases the expression levels of the VEGFA gene and the PDGFB gene in the cell aggregates of the in vitro model.

[0082] The hair cycle in the body is composed of the growth phase (anagen), regression phase (catagen), and resting phase (telogen), and it is known that VEGF expression in dermal papilla cells plays an important role in promoting hair growth, including the transition from telogen to growth phase (Non-Patent Document 1: Jiyu Hyun et al., "Morus alba Root Extract Induces the Anagen Phase in the Human Hair Follicle Dermal Papilla Cells", Pharmaceutics, 2021, 13, 1155). Therefore, the results shown in Figure 6 suggest that oxytocin enhanced the expression of growth factor genes in dermal papilla cells contained in the cell aggregates of the in vitro model, and it is thought that this enhanced expression of growth factor genes in dermal papilla cells may contribute to the hair growth promotion by oxytocin shown in Figure 5.

[0083] [Cell Culture] Using a two-dimensional culture system of dermal papilla cells, the effect of an oxytocin receptor agonist on the expression of hair growth-related genes in the dermal papilla cells was evaluated. Specifically, first, commercially available human dermal papilla cells (PromoCell, passage number 4) were cultured in a medium prepared by adding WAY-267464 (Product Name: WAY 267464 dihydrochloride, Catalog No.: 3933, CAS Registry Number: 1432043-31-6, Tocris Bioscience), an oxytocin receptor agonist, to a commercially available dermal papilla cell growth medium (PromoCell) at a concentration of 20 μM (referred to as "WAY-supplemented medium" in Example 3), or in a medium prepared by adding LIT-001 (Cat. No.: HY-124733A, CAS Registry Number: 1432043-31-6), another oxytocin receptor agonist, to the dermal papilla cell growth medium. The dermal papilla cells were suspended in a medium prepared by adding LIT-001 and WAY-267464 (2245072-21-1, MedChemExpress) at a concentration of 20 μM (referred to as "LIT-added medium" in Example 3), or in the dermal papilla cell proliferation medium to which LIT-001 and WAY-267464 had not been added (referred to as "supplement-free medium" in Example 3), to prepare a cell suspension in which the dermal papilla cells were dispersed.

[0084] Next, the cell density at the start of culture was 2 × 10 ４ The cell suspension was seeded into the flat-bottom wells of a 24-well plate at an amount of 100 cells / well. The dermal papilla cells adhered to the bottom of the wells were then two-dimensionally cultured for 3 days. The medium was not changed during this 3-day culture period.

[0085] [Expression levels of hair growth-related genes] RNA was extracted from the dermal papilla cells on day 3 of culture, and after reverse transcription, RT-PCR was carried out to quantify the expression levels of the hair growth-related genes VCAN gene, ALP gene, NOG gene, and LEF1 gene, and the expression level of the growth factor gene VEGFA gene. RT-PCR used primers having the same base sequences as those used in the above-mentioned "Example 1" and "Example 2."

[0086] [Results] Figure 7 shows the results of quantifying the expression levels of the VCAN gene, ALP gene, NOG gene, LEF1 gene, and VEGFA gene in dermal papilla cells. In Figure 7, the horizontal axis shows the type of medium used ("(-)": supplement-free medium, "WAY": WAY-added medium, "LIT": LIT-added medium), and the vertical axis shows the relative gene expression levels (arithmetic mean, n = 3) of dermal papilla cells cultured in WAY-added medium or LIT-added medium, when the gene expression level of dermal papilla cells cultured in supplement-free medium is set to "1."

[0087] As shown in Figure 7, the expression levels of the VCAN gene and the ALP gene in dermal papilla cells cultured in a WAY-supplemented medium were statistically significantly higher than those in dermal papilla cells cultured in a supplement-free medium.

[0088] Furthermore, the expression levels of the NOG gene, LEF1 gene, and VEGFA gene in dermal papilla cells cultured in LIT-supplemented medium were statistically significantly higher than those in dermal papilla cells cultured in LIT-free medium.

[0089] That is, it was confirmed that oxytocin receptor agonists activate dermal papilla cells and effectively increase the expression levels of hair growth-related genes and / or growth factor genes in the dermal papilla cells.

[0090] [In vitro model] Using the in vitro model (AA-M co-culture system) established in the above-mentioned "Reference Example," the effect of an oxytocin receptor agonist on the growth of hair shaft-like structures in cell aggregates was evaluated. That is, first, in the same manner as in the above-mentioned "Reference Example," human adult hair follicle keratinocytes and human adult dermal papilla cells were suspended and mixed in a medium prepared by adding Matrigel (Matrigel (registered trademark), Basement Membrane Matrix, CORNING (registered trademark)) at a concentration of 2 v / v% to commercially available DMEM / F12 medium (Advanced Dulbecco's Modified Eagle Medium / Ham's F-12, GIBCO (registered trademark)) (hereinafter referred to as "supplement-free medium" in Example 4) without adding WAY-267464 or LIT-001, and the hair follicle keratinocytes and dermal papilla cells were each suspended at a concentration of 1 x 10 ４ A cell suspension was prepared in which the cells were dispersed at a density of 100 cells / 200 μL.

[0091] Next, 200 μL of the resulting cell suspension was added to each non-cell-adhesive round-bottom well of a 96-well plate to seed epithelial cells and mesenchymal cells. After seeding, the 96-well plate was immediately centrifuged (100 × g) for 2 minutes. After centrifugation, the 96-well plate was immediately transferred to a 4°C refrigerator and left to stand for at least 30 minutes. The 96-well plate was then transferred to a 37°C incubator to initiate co-culture of epithelial cells and mesenchymal cells. Medium changes were performed every two days by removing 100 μL of the 200 μL medium in each well and replacing it with 100 μL of unsupplemented medium.

[0092] On day 4 of the culture, the medium in some of the wells was replaced with medium prepared by adding WAY-267464 to a concentration of 20 μM to a supplement-free medium (referred to as "WAY-added medium" in Example 4), or medium prepared by adding LIT-001 to a concentration of 20 μM to a supplement-free medium (referred to as "LIT-added medium" in Example 4), and co-culture was then carried out for 6 days in the WAY-added medium or LIT-added medium until day 10. The medium was replaced once every two days. Meanwhile, for comparison, the medium in some of the other wells was replaced with a supplement-free medium on day 4 of the culture, and co-culture was carried out until day 10. The medium was replaced once every two days.

[0093] [Length of hair shaft-like structures] After the start of co-culture, hair follicle keratinocytes and dermal papilla cells spontaneously aggregated, and one cell aggregate was formed in each well by day 4 of culture. Furthermore, on day 4 of culture, the formation of a hair shaft-like structure was confirmed in each cell aggregate. Therefore, the length of the hair shaft-like structure formed in each cell aggregate was measured using a fluorescence microscope (BZ-X810, Keyence Corporation) on days 4, 6, 8, and 10 of culture, as in the above-mentioned "Reference Example."

[0094] [Results] Figure 8 shows the results of evaluating the relative lengths of the hair shaft-like structures on days 6, 8, and 10 of culture (D6, D8, and D10 on the horizontal axis, respectively) for cell aggregates cultured in a supplement-free medium from day 4 onwards ("(-)" in the figure), cell aggregates cultured in a WAY-supplemented medium from day 4 onwards ("WAY" in the figure), and cell aggregates cultured in a LIT-supplemented medium from day 4 onwards ("LIT" in the figure), where the length of the hair shaft-like structures on day 4 of culture ("D4" on the horizontal axis) is set to "1" (arithmetic mean, n = 24).

[0095] As shown in Figure 8, the length of the hair shaft-like structures increased more rapidly in the cell aggregates cultured in WAY-supplemented medium and in the cell aggregates cultured in LIT-supplemented medium from day 4 onwards compared to the cell aggregates cultured in non-supplemented medium from day 4 onwards.

[0096] Furthermore, on days 6, 8, and 10 of culture, the relative lengths of the hair shaft-like structures in the cell aggregates cultured in the WAY-supplemented medium and in the LIT-supplemented medium were statistically significantly greater than those of the cell aggregates cultured in the supplement-free medium. In other words, the hair growth-promoting effect of the oxytocin receptor agonist was confirmed.

[0097] [Cell Culture] Using a two-dimensional culture system of dermal papilla cells, the effect of a combination of oxytocin and an oxytocin receptor agonist on the expression of hair growth-related genes in the dermal papilla cells was evaluated. Specifically, commercially available human dermal papilla cells (PromoCell, passage 4) were first suspended in a medium prepared by adding oxytocin at a concentration of 10 μM to a commercially available dermal papilla cell growth medium (PromoCell) and further adding LIT-001 at a concentration of 20 μM (referred to as "OXT+LIT-added medium" in Example 3), or in a medium prepared by adding oxytocin at a concentration of 10 μM to the dermal papilla cell growth medium without adding LIT-001 (referred to as "OXT-added medium" in Example 3), to prepare a cell suspension in which the dermal papilla cells were dispersed.

[0098] Next, the cell density at the start of culture was 2 × 10 ４ The cell suspension was seeded into the flat-bottom wells of a 24-well plate at an amount of 100 cells / well. The dermal papilla cells adhered to the bottom of the wells were then two-dimensionally cultured for 3 days. The medium was not changed during this 3-day culture period.

[0099] [Expression levels of hair growth-related genes] RNA was extracted from the dermal papilla cells on day 3 of culture, and after reverse transcription, RT-PCR was carried out to quantify the expression levels of the hair growth-related genes, LEF1 gene, VCAN gene, and NOG gene. RT-PCR used primers having the same nucleotide sequences as those used in "Example 1" above.

[0100] [Results] Figure 9 shows the results of quantifying the expression levels of the LEF1 gene, VCAN gene, and NOG gene in dermal papilla cells. In Figure 9, the horizontal axis shows the type of medium used ("OXT": OXT-added medium, "OXT+LIT": OXT+LIT-added medium), and the vertical axis shows the relative gene expression levels (arithmetic mean, n=3) of dermal papilla cells cultured in OXT+LIT-added medium, when the gene expression level of dermal papilla cells cultured in OXT-added medium is set to "1."

[0101] 9, the expression levels of the LEF1 gene and NOG gene in dermal papilla cells cultured in an OXT+LIT-supplemented medium were statistically significantly higher than those in dermal papilla cells cultured in an OXT-supplemented medium. In other words, it was confirmed that the oxytocin receptor agonist further enhances the activation effect of oxytocin on dermal papilla cells.

[0102] [In vitro model] Using the in vitro model (AA-M co-culture system) established in the above-mentioned "Reference Example," the effect of a combination of oxytocin and an oxytocin receptor agonist on the growth of hair shaft-like structures in cell aggregates was evaluated. That is, first, in the same manner as in the above-mentioned "Reference Example," human adult hair follicle keratinocytes and human adult dermal papilla cells were suspended and mixed in a medium prepared by adding Matrigel (Matrigel (registered trademark), Basement Membrane Matrix, CORNING (registered trademark)) at a concentration of 2 v / v% to commercially available DMEM / F12 medium (Advanced Dulbecco's Modified Eagle Medium / Ham's F-12, GIBCO (registered trademark)) (hereinafter referred to as "supplement-free medium" in Example 6) without adding oxytocin or LIT-001, and the hair follicle keratinocytes and dermal papilla cells were each suspended at a concentration of 1 x 10 ４ A cell suspension was prepared in which the cells were dispersed at a density of 100 cells / 200 μL.

[0103] Next, 200 μL of the resulting cell suspension was added to each non-cell-adhesive round-bottom well of a 96-well plate to seed epithelial cells and mesenchymal cells. After seeding, the 96-well plate was immediately centrifuged (100 × g) for 2 minutes. After centrifugation, the 96-well plate was immediately transferred to a 4°C refrigerator and left to stand for at least 30 minutes. The 96-well plate was then transferred to a 37°C incubator to initiate co-culture of epithelial cells and mesenchymal cells. Medium changes were performed every two days by removing 100 μL of the 200 μL medium in each well and replacing it with 100 μL of unsupplemented medium.

[0104] On the fourth day of culture, the medium in some of the wells was replaced with a medium prepared by adding oxytocin to a concentration of 10 μM and further adding LIT-001 to a concentration of 20 μM (hereinafter referred to as "OXT+LIT-added medium" in Example 6), and then co-culture was continued for 6 days in the OXT+LIT-added medium until the 10th day. The medium was replaced once every two days.

[0105] On the other hand, for comparison, on day 4 of culture, the medium in some of the other wells was replaced with a medium prepared by adding oxytocin at a concentration of 10 μM without adding LIT-001 to the supplement-free medium (hereinafter referred to as "OXT-supplemented medium" in Example 6), and co-culture was then continued for 6 days in the OXT-supplemented medium until day 10. The medium was replaced once every two days.

[0106] [Length of hair shaft-like structures] After the start of co-culture, hair follicle keratinocytes and dermal papilla cells spontaneously aggregated, and one cell aggregate was formed in each well by day 4 of culture. Furthermore, on day 4 of culture, the formation of a hair shaft-like structure was confirmed in each cell aggregate. Therefore, the length of the hair shaft-like structure formed in each cell aggregate was measured using a fluorescence microscope (BZ-X810, Keyence Corporation) on days 4, 6, 8, and 10 of culture, as in the above-mentioned "Reference Example."

[0107] [Results] Figure 10 shows the results of evaluating the relative lengths of the hair shaft-like structures on days 6, 8, and 10 of culture (D6, D8, and D10 on the horizontal axis, respectively) for cell aggregates cultured in OXT-supplemented medium from day 4 onwards ("OXT" in the figure) and cell aggregates cultured in OXT+LIT-supplemented medium from day 4 onwards ("OXT+LIT" in the figure), where the length of the hair shaft-like structures on day 4 of culture ("D4" on the horizontal axis) is set to "1" (arithmetic mean, n = 18).

[0108] As shown in Figure 10, the length of the hair shaft-like structures increased more rapidly in the cell aggregates cultured in OXT+LIT-supplemented medium from day 4 onwards compared to the cell aggregates cultured in OXT-supplemented medium from day 4 onwards.

[0109] Furthermore, on days 8 and 10 of culture, the relative length of the hair shaft-like structures in the cell aggregates cultured in the OXT+LIT-supplemented medium was statistically significantly greater than that in the cell aggregates cultured in the OXT-supplemented medium. In other words, it was confirmed that the oxytocin receptor agonist enhances the hair growth-promoting effect of oxytocin.

Claims

1. A composition for activating hair follicle papilla cells, comprising oxytocin and / or an oxytocin receptor agonist.

2. The composition of claim 1, which contains oxytocin.

3. The composition of claim 1, comprising an oxytocin receptor agonist.

4. A composition according to any one of claims 1 to 3, which is an external preparation for the skin.

5. The composition according to claim 4, which is an external agent for the scalp.

6. A composition according to any one of claims 1 to 3, which is a hair growth or hair regrowth composition.

7. A composition according to any one of claims 1 to 3, which is a culture medium.

8. The composition according to any one of claims 1 to 3, which is an additive for culture media.

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