JP2012062294A - Agent for shortening hair growth telogen - Google Patents

Abstract

The present invention relates to a method for determining an endogenous factor that regulates the rest period of a hair growth cycle, screening for a substance that inhibits the activity or expression of the endogenous factor, and a hair growth rest period shortening agent or a hair loss preventing agent.
FGF18 present in hair follicles is a regulatory factor that regulates the rest period of the hair growth cycle, initiates the shedding period, and further defines the hair cycle domain. A method of screening a FGF18 activity inhibitor or an FGF18 gene expression inhibitor as a candidate for a hair growth rest period shortening agent, a hair loss preventing agent, and a hair leveling agent. In addition, a hair growth resting period shortening agent, a hair loss preventing agent, and a hair level adjusting agent containing an FGF18 activity inhibitory substance and / or an expression inhibitory substance such as a partial peptide of FGF18 and an anti-FGF18 antibody as active ingredients.
[Selection figure] None

Description

  The object of the present invention is to prevent or treat hair loss by shortening the rest period in the hair growth cycle and delaying the onset of the drop-out period, and a substance that regulates that the hair stops growing. The present invention relates to an inhibitor and a screening method thereof.

Hair loss and hair loss are said to have a large genetic component, but since they are accelerated with aging, many people suffer from not only men but also women. Alopecia is caused by androgenetic alopecia, which occurs frequently in aging men, diffuse alopecia, which often causes problems in women, alopecia areata, which is common in both men and women, and by the administration of cytotoxic drugs such as anticancer agents There are alopecia.
In order to solve these problems, many substances have been discovered and proposed as substances having a hair-growth effect and a hair-growth effect, and many substances have already been commercialized. Drugs recommended by the "Male Male Alopecia Clinical Practice Guidelines (2010)" as hair growth agents with confirmed hair effects are minoxidil (trade name Rogain, Reup) that promotes hair growth by vasodilation and male hormone action on the scalp It is only an internal medicine (trade name Propecia) that suppresses
In the case of androgenetic alopecia (AGA), one of the main causes is testosterone, a male hormone, is dihydrotestosterone (DHT) due to the action of 5α reductase. Although it has been described that DHT binds to the androgen receptor (AR) on papillary cells, thereby starting the hair follicle thinning process, the details of its molecular mechanism remain unclear. According to one theory, DHT hair follicle dwarfing involves cell apoptosis via TGF-β1, a cell growth factor-related molecule. If the follicular cells that should be maintained for 2 to 6 years according to the normal hair growth cycle are killed by apoptosis, the hair will gradually shift to the resting state. Therefore, even if taking oral medicine that suppresses the reaction to convert testosterone into DHT is effective in preventing male pattern baldness in men who tend to have excessive male hormones, side effects of suppressing DHT Some people are worried about this, and it is not effective for alopecia other than typical AGA caused by excessive male hormones.
By the way, in the case of diffuse alopecia other than male pattern alopecia as described above, alopecia areata that is frequent in both men and women, and alopecia caused by the administration of drugs such as anticancer agents, the onset of alopecia The mechanisms involved are also diversified, and the molecules involved in them kill the growth factor-related molecules such as TGF-β1 in male pattern alopecia, immune cell-related cytokines and chemokines in alopecia areata, and the hair follicle cells themselves. In addition to anti-cancer drugs, etc., there are various molecules, and various cells that produce and receive these molecules are involved.
Therefore, detailed study on the hair growth cycle is indispensable for fundamental hair loss prevention.

The hair growth cycle (FIG. 1) is defined by periodic changes in the hair follicle itself, the organ that produces the hair. That is, the hair follicle repeats three phases in this order: a growth period, a regression period, and a rest period. During the growth phase, the hair follicle tip reaches the subcutaneous fat layer deeper than the skin, and the hair follicles become thick and thick, forming a long hair shaft, and extending it long outside the body. This is the appearance of hair growth. In the regression phase, as the follicle constituent cells undergo apoptosis, the hair follicle retreats, and the tip of the hair follicle moves to the inside of the dermis closer to the skin surface than the subcutaneous fat layer. In the next resting phase, the follicle's constituent cells are thought to be quiescent in a small state with little proliferation or apoptosis. In these regression and rest periods, the appearance of hair growth stops, but the hair does not come off. Hair loss is thought to occur by an independent control in an independent phase called the hair loss phase, but the hair loss phase is not completely unrelated to the above three phases, but is in the late resting phase or the next growth It is said that it often happens in the middle of the period. However, pathological hair loss and the like may occur earlier than this, and in that case, hair follicles that do not have any hair appearance exist for a long period of time. Until now, little is known about the molecular mechanism that controls this hair loss phase. For these reasons, when explaining the hair growth cycle, the three phases of the growth phase, regression phase, and resting phase are always subject to explanation, but the hair loss phase is often omitted. The duration of each phase of the hair growth cycle varies depending on the species of animal, and it varies depending on the individual species, sex, body part, and hair type within the same species. Via each phase. In the case of human hair, there are individual differences and age differences, but in general, the growth period is 4 to 8 years for women, 3 to 6 years for men, the regression period is 1 to 2 weeks, and the rest period is about 3 months. Has been. In the case of mice, which are model animals frequently used for hair research, the growth phase is 18 to 19 days, the regression phase is 2 days, and the resting phase is about 3 to 5 weeks in the hair follicle existing on the back skin .
General alopecia means a state where there is no hair in appearance, or a state where the hair is very thin and short, and when the cause is viewed from the hair growth cycle, hair loss is insufficient. It can be said that hair loss has occurred earlier or excessively than normal, or that the next hair growth does not start even after the hair formed in the previous cycle has fallen off. The causes can be divided into (1) shortening of the growth period, (2) extension of the rest period, and (3) early start of the hair loss period. Known factors that define each period in the hair growth cycle are the cell growth factor Wnt family proteins that maintain the conventional growth phase (Non-patent document 1) and the cell growth factor FGF5 that induces the regression phase (Non-patent document 2). However, there have been few detailed studies on factors related to maintenance and control of resting phase and factors related to control of hair loss phase.
Therefore, in order to individually cope with the cause of alopecia of each alopecia patient, it is necessary to specify the action of factors that act in each phase of the hair growth cycle. In addition, if the rest period that takes a relatively long time can be shortened in the entire growth cycle, new hair grows before complete hair loss occurs, and hair loss itself can be prevented. It also increases so-called thinning hair prevention. Furthermore, if a factor that controls the hair loss period that has not been elucidated can be elucidated, the hair loss time can be directly delayed, which is a great gospel for alopecia patients.

WO2008 / 102782 WO2008 / 102783 Japanese Unexamined Patent Publication No. H4-224522

Shimizu, H, et al., J. Invest. Dermatol. 122, 239-245 (2004) Hebert, J.M., et al., Cell78, 1017-1025 (1994) Kawano, M., et al., J. Invest. Dermatol. 124,877-885 (2005) Ohbayashi, N., et al., Genes & Dev. 16,870-879 (2002) Tarutani, M., et al. Proc Natl Acad Sci USA 94,7400-7405 (1997) Ornitz DM, Itoh N: Fibroblast growth factors. Genome Biol 2: REVIEWS3005,2001 du Cros DL: Fibroblast growth factor and epidermal growth factor in hair development. J Invest Dermatol 101: 106S-113S.1993 du Cros DL, Isaacs K, Moore GP: Distribution of acidic and basic fibroblast growth factors in ovine skin during follicle morphogenesis. J Cell Sci 105: 667-674,1993 Hebert JM, Rosenquist T, Gotz J, Martin GR: FGF5 as a regulator of the hair growth cycle: Evidence from targeted and spontaneous mutations. Cell 78: 1017-1025,1994 Danilenko DM, Ring BD, Yanagihara D, Benson W, Wiemann B, Starnes CO, Pierce GF: Keratinocyte growth factor is an important intrinsic mediator of hair follicle growth, development, and differentiation. American J Pathol 147: 145-154, 1995 Marchese C, Chedid M, Dirsch OR, et al: Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin. J Exp Med 182: 1369-1376,1995 Guo L, Degenstein L, Fuchs E ： Keratinocyte growth factor is required for hair development but not for wound healing.Genes Dev 10 ： 165-175,1996 Rosenquist TA, Martin GR: Fibroblast growth factor signaling in the hair growth cycle: Expression of the fibroblast growth factor receptor and ligand genes in the murine hair follicle.Developmental Dynamics 205: 379-386,1996 Petho-Schramm A, Muller HJ, Paus R: FGF5 and the murine hair cycle. Arch Dermatol Res 288: 264-266, 1996 Mitsui S, Ohuchi A, Hotta M, Tsuboi R, Ogawa H: Genes for a range of growth factors and cyclin-dependent kinase inhibitors are expressed by isolated human hair follicles. Br J Dermatol 137: 693-698, 1997 Ortega S, Ittmann M, Tsang SH, Ehrlich M, Basilico C: Neuronal defects and delayed wound healing in mice lacking fibroblast growth factor-2. Proc Natl Acad Sci USA 95: 5672-5677,1998 Suzuki S, Kato T, Takimoto H, et al: Localization of rat FGF-5 protein in skin macrophage-like cells and FGF-5S protein in hair follicle: Possible involvement of two Fgf-5 gene products in hair growth cycle regulation. Invest Dermatol 111: 963-972,1998 Suzuki S, Ota Y, Ozawa K, Imamura T: Dual-mode regulation of hair growth cycle by two Fgf-5 gene products.J Invest Dermatol 114: 456-463,2000 Nakatake Y, Hoshikawa M, Asaki T, Kassai Y, Itoh N: Identification of a novel fibroblast growth factor, FGF-22, preferentially expressed in the inner root sheath of the hair follicle. Biochem Biophys Acta 1517: 460-463, 2001 Stenn KS, Paus R ： Controls of hair follicle cycling.Physiol Rev 81 ： 449-494,2001 Beyer TA, Werner S, Dickson C, Grose R: Fibroblast growth factor 22 and its potential role during skin development and repair.Exp Cell Res 287: 228-236,2003 Kawano M, Suzuki S, Suzuki M, Oki J, Imamura T ： Bulge- and basal layer-specific expression of fibroblast growth factor 13 (FHF-2) in mouse skin.J Invest Dermatol 122 ： 1084-1090,2004 Rendl, M., Polak, L. & Fuchs, E. BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties.Genes & Dev. 22,543-557 (2008)

The present invention determines an endogenous factor that controls the rest period in the hair growth cycle for a long period of time, elucidates its mechanism of action, determines an endogenous factor that controls the hair loss period, and determines the activity of the endogenous factor or The object of the present invention is to provide a novel hair growth quiescent shortening agent and hair loss inhibitor by screening a substance that inhibits expression.
A screening method for this purpose is another object of the present invention.

Among the 22 types of fibroblast growth factor (FGF) (Non-patent Document 7), FGF1, FGF2, FGF5, FGF7, FGF10, FGF13, and FGF22 are expressed in mouse and human skin tissues and hair follicle cells. It is reported that hair growth and skin regeneration are controlled (see Non-Patent Documents 8 to 23 and Patent Document 3). From the content of these documents, it is suggested that various FGFs play an important role in the proliferation and differentiation of skin cells, but the action of promoting and resting hair follicle growth, the associated hair growth stage and Much remains unknown about how individual factors of the FGF group are involved in the resting phase control action and hair loss promoting action.
In the above background, the inventors previously found that FGF18 is expressed at a high level in skin containing hair follicles, and reported that the expression level is particularly high in the resting phase of the hair growth cycle (Non-Patent Document). 4). After FGF18 is forced to induce hair follicle growth by hair removal in mouse resting skin hair follicles, FGF18 is allowed to remain in the dorsal skin, thereby significantly inhibiting hair growth. A patent application has been filed for a hair growth inhibitor comprising FGF18 and its activating substance and a hair growth agent comprising FGF18 inhibitor (Patent Documents 1 and 2). As described above, the effect of the FGF18 gene product introduced from the outside into hair follicle cells on hair growth has been elucidated, but what mechanism is the FGF18 gene endogenous to hair follicle cells in hair follicle cells? It was not well understood about the gene function, which is expressed in and how it is involved in the hair growth cycle.
Although there is a gene knockout technique as a method to directly elucidate the function of the endogenous gene, FGF18 is an indispensable factor for individual formation and life support such as chondrocyte and osteoblast proliferation and differentiation and lung formation. It is known that when normal systemic knockout is performed, the functional formation of an individual does not occur normally, and it dies in the embryonic period or immediately after birth (Non-patent Document 5).
Therefore, the present inventors recalled that the follicle cells knock out keratinocytes in a hair follicle-specific manner by knocking out the FGF18 gene that is selectively expressed at a high level in the hair follicle. Since it is a single type, keratin 5 was obtained with reference to a technique (Non-patent Document 6) in which a gene knockout method specific to keratin 5 positive cells was applied to the Pig-a gene involved in phosphatidylinositol glycan anchor synthesis. A mouse in which the Fgf18 gene was knocked out in a positive cell specific manner was obtained. Here, like keratin 14, keratin 5 is expressed in many cells such as epidermis, hair follicle, sebaceous gland in skin appendages such as skin and hair follicle, but Fgf18 is expressed at a high level. Since it is only a hair follicle in this, the obtained conditional knockout mouse can be substantially expressed as a “hair follicle-specific Fgf18 gene-deficient mouse”.
Hair follicle-specific FGF18-deficient mice grow healthy, have fertility, and have no detectable visual impairment, but surprisingly the state of progression of the hair growth cycle is wild type It was confirmed that it was significantly different. That is, the rest period of the hair growth cycle lasting 3 to 5 weeks or more in normal wild-type mice is completed in about 1 week in mice lacking hair follicle-specific FGF18, and the period required for one cycle is up to about 3 weeks. It was shortened. Moreover, the club hair in the hair follicle was prevented from falling off, and the start time of the hair removal period was delayed. As a result, the number of hair shafts per hair follicle increased more than twice. In addition, the progression of the hair growth cycle is extremely unaffected by the “hair cycle domain” structure on the body surface, and the hair growth phase repeats in a form of multiple stripes as aging progresses. Appears in For this reason, if hair follicle-specific FGF18-deficient mice are used, it is extremely difficult to perform reproducibility on the skin surface of the same individual in wild-type mice. It was found that a substance capable of promoting and suppressing hair and hair loss can be evaluated and screened, and a patent application was filed on the same date as this application.

From these results, the endogenous FGF18 in the hair follicle has a function of prolonging the hair growth cycle itself by extending the rest period of the hair growth cycle and dropping off the club hair in the hair follicle. It can be said that FGF18 is a diastolic extension controlling factor and a hair loss promoting factor.
Therefore, by administering to the hair follicle a substance that suppresses the expression of the endogenous FGF18 gene in the hair follicle or a substance that suppresses the activity of the FGF18 protein, the rest period of the hair growth cycle is shortened, and the speed of the entire hair growth cycle Can be accelerated, and hair loss and hair loss can be prevented. In other words, by searching for a substance that suppresses the expression of endogenous FGF18 gene in the hair follicle or a substance that suppresses the activity of the FGF18 protein, it was found that it is a resting period shortener of hair growth cycle and can provide a hair loss inhibitor. .
Obtaining the above knowledge, the present invention has been completed.

That is, the present invention includes the following.
[1] A hair growth rest period shortening agent, hair loss preventing agent or hair leveling regulator comprising an FGF18 activity inhibitor and / or an expression inhibitor as an active ingredient.
[2] The hair growth resting period shortening agent, hair loss preventing agent, or fur level adjusting agent according to [1], wherein the FGF18 activity inhibitor is a partial peptide of FGF18.
[3] The hair growth resting period shortening agent, hair loss preventing agent, or fur level adjusting agent according to [1], wherein the FGF18 activity inhibitor is an anti-FGF18 antibody.
[4] The hair growth resting period shortening agent, hair loss preventing agent, or fur level adjusting agent according to [1], wherein the FGF18 activity inhibitor is bitter gourd extract.
[5] The hair growth pause according to [1], wherein the FGF18 activity inhibitor is an expression vector incorporating a cDNA encoding a partial peptide of FGF18 having an activity of inhibiting FGF18 activity Period shortening agent, hair loss prevention agent or hair leveling agent.
[6] The hair growth quiescent shortening agent and hair loss prevention agent according to [1], wherein the FGF18 expression inhibitor is an expression vector incorporating an siRNA having an activity of inhibiting FGF18 expression. Or hair conditioner.
[7] The hair growth resting period shortening agent or hair loss prevention according to any one of [1] to [6], further comprising another hair growth promoter, hair growth promoter, or hair loss inhibitor Agent or hair conditioner.
[8] A method in which the FGF18 activity-inhibiting substance according to any one of [1] to [4] is screened, and the test substance is a candidate for a hair growth quiescence shortening agent, a hair loss prevention agent, or a hair level adjustment agent. A method comprising the following steps (a) to (c):
(A) a step of forcibly expressing on the cell surface by genetic manipulation using at least one FGF receptor gene selected from FGFR1c, FGFR2c, FGFR3c and FGFR4, and culturing the cells;
(B) a step of bringing a test substance into contact with a cell line having FGF receptor on the cell surface obtained in step (a) together with FGF18;
(C) In the step (b), a step of selecting a test substance observing the activity of inhibiting the cell growth promoting activity of FGF18 [9] The above [8], wherein the FGF receptor described in [8] is FGFR3c A screening method according to 1.
[10] The screening method according to [8], wherein the FGF receptor according to [8] is FGFR4.
[11] Any of [8] to [10] above, wherein the cell for forcibly expressing the FGF receptor according to [8] on the cell surface is a mouse IL-3-dependent Ba / F3 cell line A screening method according to 1.
[12] A method for screening the FGF18 expression-suppressing substance according to the above [1] to be a candidate for a hair growth quiescent period shortening agent, a hair loss preventing agent or a fur level adjusting agent, comprising: A method comprising the step of (d);
(A) preparing a cultured animal cell or experimental animal that expresses the FGF18 gene to an observable level;
(B) contacting the test substance with the animal cultured cells of (a) or contacting or administering to the experimental animal;
(C) (b) a step of monitoring the expression of the FGF18 gene in cultured animal cells or experimental animals after the step;
(D) A step of selecting a test substance having a function of inhibiting the expression of the FGF18 gene.
[13] In the step (c), the expression of the FGF18 gene is monitored by extracting mRNA from the experimental animal or animal cultured cells after the step (b) and analyzing the mRNA amount of the expressed FGF18. In the step (d), a test substance having a function of inhibiting the expression of the FGF18 gene is selected by selecting a system in which a low level of FGF18 mRNA is observed compared to the case where the test substance is not allowed to act. The method according to [12] above, wherein:

  In the present invention, a substance that suppresses the expression of the endogenous FGF18 gene in the hair follicle or a substance that suppresses the activity of the FGF18 protein is searched, and these substances are administered to the hair follicle to shorten the rest period of the hair growth cycle. And the speed of the entire hair growth cycle can be increased. In addition, hair loss can be prevented by delaying the hair loss period. That is, new hair grows before complete hair loss occurs, and hair loss itself can be prevented, so that the total amount of hair increases and so is thin hair prevention.

Hair growth cycle In the figure, “anagen” is the time when hair follicle growth and hair shaft formation proceed, “catagen” is the time when hair follicles retreat, and “telogen” is the rest period It represents the period when hair follicle activity ceases, indicating that “anagen” is divided into “propagating anagen” and “autonomous anagen”, and “telogen” is divided into “refractory telogen” and “competent telogen”. In addition, the “exogenous period” in which the hair shaft falls off in appearance is not directly connected to the regression phase or resting phase, but the resting phase following the growth phase in which the hair shaft is completed or the next growth phase. It is supposed to occur as an independent phase during the period. FGF18 gene knockout schematic diagram a: Structure near the third exon of the mouse FGF18 gene genome In the figure, the thick black line shows the third exon and the line part shows the intron structure. EcoRI indicates the position of the restriction enzyme EcoRI. b: Schematic diagram of targeting vector structure and genomic homologous recombination. In the figure, “KIprobe” is a DNA probe used for recombination determination, genotype determination, etc. by Southern blotting, and its homologous position is indicated by a bold line. Boxed PGK-Neo ^r indicates the cassettes of neomycin resistance gene, the black triangles show the loxP sequence, gray triangles shows the FRT sequence. c: A state in which the cassette of neomycin resistance gene surrounded by FRT sequence is removed from the sequence of b by Flp recombinase. The state where the 3rd exon of the Fgf18 gene enclosed by the loxP sequence was removed from the sequence of d: c by Cre recombinase is shown. e: An example of the result of Southern blot analysis. The analysis results of hetero, homo, and wild type are shown. Hair follicle-specific FGF18-deficient mice a and b are hair growth cycles that are rapidly repeated in hair follicle-specific FGF18-deficient mice (K5Cre ^tg ; Fgf18 ^{flox / flox} : homo-knockout mice) (1) a: hair follicles at young age Changes in the hair growth cycle of the dorsal skin over time for a representative individual of a specific FGF18-deficient mouse (homo knockout mouse: lower) and a control hetero knockout mouse (K5Cre ^tg ; Fgf18 ^{+ / flox} : upper) What we observed. Each photo was taken after the hair that had grown was cut short with a clipper, and the color of the skin reflected the phase of the hair growth cycle. b: An example of a hair growth pattern on the dorsal skin of a representative adult individual of a hair follicle-specific FGF18-deficient mouse (homo knockout mouse: left) and a control wild type mouse (C57BL / 6: right) It is a thing. c and d are hair growth cycles that are rapidly repeated in hair follicle-specific FGF18-deficient mice (K5Cre ^tg ; Fgf18 ^{flox / flox} : homo-knockout mice) (2) c: hair follicle-specific FGF18-deficient mice (homogenous knock-out mice) The change of the hair growth cycle (Y axis) in the dorsal skin is plotted against the age of the mouse (X axis). d: Changes in the hair growth cycle in the dorsal skin of aging hair follicle-specific FGF18-deficient mice (homo knockout mice) were observed approximately every 1 week. Reflects hair growth. Illustration of hair cycle domains The figure represents the hair cycle domains present in the dorsal skin of mice. It has been empirically known that hair cycle domains exist in the mouse dorsal skin as shown by the partitions separated by vertical and horizontal lines in the figure, and the control of the hair cycle is different for each domain. However, the numbers and positions of the vertical and horizontal lines in the drawing are examples, and there are actually various patterns. Hair follicle-specific FGF18-deficient mice a and b are incomplete club hair loss in aging hair follicle-specific FGF18-deficient mice (homo knockout mice) (1) a: left: hair with advanced aging This shows the state of hair extracted from the back with a finger in a sac-specific FGF18-deficient mouse (homo knockout mouse). Right: Club hair removed from heterozygous knockout mice of the same age as the left as a control. b: The state of the hair follicle around the growing hair follicle. In the figure, the bar represents 200 μm. c and d are incomplete club hair loss in aging hair follicle-specific FGF18-deficient mice (2) c: hair follicle state around resting hair follicles. In the figure, the bar represents 200 μm. d: Enlarged view of the framed part of c. e and f are incomplete club hair loss in aging hair follicle-specific FGF18-deficient mice (3) e: Skin section containing growing hair follicles Immunostaining photograph. Double staining for keratin 15 and PCNA antigen. Crab hair follicles that snuggle up to the bulge area of PCNA positive growing follicles are observed. f: Only the signal of keratin 15 in part of the visual field of e is represented. It can be seen that keratin 15 is an antigen characteristic of skin stem cells, and the bulge region of the growing hair follicle and the bag-like structure that wraps the club hair are both keratin 15 positive and form a continuous structure. g and h are incomplete loss of club hair in aging hair follicle-specific FGF18-deficient mice (4) g: aging hair follicle-specific FGF18-deficient mice (homo knockout mice) Fluorescence observation image of a circular slice around the growing hair follicle. It is stained with a fluorescent dye, thioflavin T, which dyes the hair shaft protein. In the figure, the bar represents 100 μm. h: Magnified observation image of the same sample as e. It is merged with the phase difference observation image. In the figure, the bar represents 100 μm. Arrows indicate hair shafts that grow on the growing follicles containing melanin, and club hairs otherwise. i and j are incomplete loss of club hair in aging follicle-specific FGF18-deficient mice (5) i: as a control, heterogeneous knockout mice dorsal skin around growing hair follicles Fluorescence observation image of a sliced piece. It is stained with a fluorescent dye, thioflavin T, which dyes the hair shaft protein. In the figure, the bar represents 200 μm. j: Magnified observation image of the same sample as g. It is merged with the phase difference observation image. In the figure, the bar represents 100 μm. Arrows indicate hair shafts that grow on the growing follicles containing melanin, and club hairs otherwise. It is a figure which shows that the effect which FGF18 promotes the proliferation of an FGF receptor (FGFR) expression cell is suppressed by the FGF18 partial peptide. The analysis results using FGFR3c expressing cells (R3c) and FGFR4 expressing cells (R4) are shown. A: Results when test substance was added at 1 μg / ml <Control> Column 1: No sample other than FGF18 added. <R3c> Column 2: (d4). Column 3: (d16). Column 4: (d18). Column 5: (d22). Column 6: (d37). Column 7: (d48). Column 8: (d77). Column 9: (d95). <R4> Column 10: (d22). Column 11: (d37). Column 12: (d48). Column 13: (d67). Column 14: (d77). Column 15: (d95). B: Results when test substance was added at 100 ng / ml <Control> Column 1: No sample other than FGF18 added. <R3c> Column 2: (d4). Column 3: (d12). Column 4: (d16). Column 5: (d18). Column 6: (d37). Column 7: (d48). Column 8: (d67). Column 9: (d95). <R4> Column 10: (d37). Column 11: (d67). Inhibition of R4 / BaF3 cell proliferation by Goya hot water extract: By culturing FGFR4 / BaF3 cells with Goya hot water extract in the presence of FGF18, by adding 0.083%, 0.83% and 8.3% Goya hot water extract The inhibitory effect with respect to the proliferation promotion effect | action of FGFR4 / BaF3 cell of FGF18 is shown. In vivo analysis of Goya hot water extract: Ethanol solution of Goya hot water extract (containing 49.5% ethanol and 1% glycerol) on the back skin of C3H / He mice in the rest period of the hair cycle for 11 days (Day 5) And excluding the 6th day), and the hair growth state of the mouse back on the 10th day, 14th day, 17th day, 21st day, and 24th day is shown by a hair growth score.

Hereinafter, the present invention will be described in detail.
1. Hair growth cycle within the hair follicle The hair growth cycle (FIG. 1) is defined by periodic changes in the hair follicle itself, the organ that produces the hair. In other words, the hair follicle repeats three phases in this order: growth phase (anagen), regression phase (catagen), and telogen phase. The growth period (anagen) is further divided into “propagating anagen” and “autonomous anagen”, and the telogen is divided into “refractory telogen” and “competent telogen”. During the growth period, the hair follicle tip reaches the subcutaneous fat layer deeper than the skin, and the hair follicles become larger and thicker, forming a long hair shaft, and extending it long outside the body. This is the appearance of hair growth. In the regression phase, as the follicle constituent cells undergo apoptosis, the hair follicle retreats, and the tip of the hair follicle moves to the inside of the dermis closer to the skin surface than the subcutaneous fat layer. In the next resting phase, the follicle's constituent cells are thought to be quiescent in a small state with little proliferation or apoptosis. In mice with colored hair, melanin pigments are synthesized in the early stages of growth, and the skin becomes blue. Therefore, the progress of the hair follicle growth cycle can be evaluated by looking at the blue color from the outside of the skin. Further, when the skin is incised during the growth period and viewed from the back side, hair follicles containing a large amount of melanin are arranged at a high density, so that the back side of the skin can be visually recognized as black. On the other hand, in the rest period, it can be visually recognized that the back side of the skin remains white. In general, in the mouse experimental system, the growth period is the period from the 1st to the 19th day after the hair loss, and the regression period is the period from the 20th to the 21st day. In addition, it is known that after hair removal, a rest period starts on the 21st to 22nd days, and the rest period lasts for 3 to 5 weeks or more. Prior to the present invention, it was reported that BMP (bone morphogenetic protein) signals, which are a group of cell growth factors, are involved as regulators of “refractory telogen”, which is the first short period of telogen. (Non-patent document 24). However, the control factor of "competent telogen", which accounts for a large percentage of the rest period and follows "refractory telogen", was not known at all. For the first time in the present invention, it was found that the regulatory factor of “competent telogen” is Fgf18 factor. Therefore, it has been found for the first time in the present invention that the regulator that substantially extends the resting phase is Fgf18 factor. In addition, there is a possibility that the Fgf18 factor also functions as a control factor of “refractory telogen”, and future verification is awaited.
In these regressive and resting phases, the appearance of hair growth stops, but hair does not escape, and hair loss occurs by an independent control called an exogenous phase (exogen). Though thought, the molecular mechanism that controls this hair loss phase has been largely unknown. In the present invention, it has been found for the first time that one of the factors controlling the start of the hair loss period is Fgf18.

2. Production of hair follicle-specific FGF18 gene knockout mouse The “hair follicle-specific FGF18 gene knockout mouse” of the present invention is prepared as follows.
Exon 3 in the mouse Fgf18 gene is knocked out specifically for keratin 5 positive cells using the Cre-loxP method. By deleting exon 3, a part of the secretion signal of Fgf18 protein and its downstream are not expressed, and the biological activity of Fgf18 protein is lost. A method for specifically deleting FGF18 gene in keratin 5 positive cells was performed with reference to Non-Patent Document 6.
The nucleotide sequence of the genomic region containing exon 3 of the mouse-derived Fgf18 gene can be obtained from public databases such as GenBank (Entrez Gene; Gene ID: 14172, updated on 21-Jul-2010; Official Symbol: Fgf18 provided by MGI; Official Full Name: fibroblast growth factor 18 provided by MGI; Primary source: MGI: 1277980), neomycin resistance gene cassette sandwiched between two FRT sequences is inserted upstream, and this neomycin cassette and exon 3 are two loxP A targeting vector sandwiched between sequences was prepared (FIG. 1). Using this targeting vector, Fgf18 gene exon 3 allele was targeted to mouse ES cells. This cell was injected into a blastocyst of a C57BL / 6 mouse and transplanted into the uterus of an ICR mouse, and the resulting offspring of a chimera was mated with a C57BL / 6 mouse. Individuals with the introduced target gene were selected from among them (F1). F1 individuals were crossed with Flpe transgenic mouse individuals (distributed from RIKEN BRC: RIKEN RBRC01834; Non-Patent Document 7) to obtain individuals lacking the neomycin resistance gene cassette sandwiched between FRT sequences (F2). Next, F2 mice were crossed with K5Cre transgenic mice (distributed from KARD, University KARD: CARD ID323; Non-Patent Document 6) to remove Fgf18 gene exon 3 specifically for keratin 5 positive cells. The obtained K5Cre ^tg ; Fgf18 ^{+ / flox} mice were crossed to obtain K5Cre ^tg ; Fgf18 ^{flox / flox} mice.
By the way, keratin 5, like keratin 14, is expressed in many cells such as epidermis, hair follicle, sebaceous gland in skin appendages such as skin and hair follicle. However, only hair follicles express Fgf18 at a high level. Therefore, the conditional knockout mouse obtained as described above can be expressed as a mouse in which the Fgf18 gene is knocked out in a hair follicle-specific manner.

3. Characteristics of hair follicle-specific Fgf18 gene knockout mice (3-1) Overall characteristics
The FGF18 gene is expressed in bone, myocardium, fetal lung, and other tissues in mammals such as mice, and is thought to play an important role in individual formation and life support. However, only hair follicles express Fgf18 at high levels in skin appendages such as the skin and hair follicles. In addition to the skin, keratin 5 gene is expressed at a high level in only a few tissues such as bone, tongue and bronchus. Therefore, in the conditional knockout mouse obtained as described above, It is only the hair follicle that the physiological function of the Fgf18 gene is substantially lost compared to the type of mouse.
The hair follicle-specific Fgf18 gene knockout mouse obtained in the present invention is very good in the health condition of the individual, and the characteristics specifically observed are as follows.
1) Since the rest period is shortened to 1/3 to 1/5 of the hair growth cycle, the entire hair growth cycle progresses quickly.
2) The hair growth cycle on the back skin proceeds in an orderly manner in a band shape. At this time, the forward speed near the neck is the fastest and the rearward tail is the slowest, but the speed of the forward speed advances in an orderly manner, and when the shaving is repeated, a plurality of horizontal stripes are formed. The number of horizontal stripes at that time is proportional to aging.
3) Although the number of hair follicle cells does not increase, the rate of hair removal from the hair follicle is slowed. As a result, the number of hair shafts in the hair follicle is large, and the total number of hair shafts on the skin surface is more than double that of the wild type. It becomes.

(3-2) Characteristic of fast progression of hair growth cycle The hair follicle-specific Fgf18 gene knockout mouse of the present invention undergoes hair growth at a much faster cycle than the wild type. In particular, of the hair growth cycle, the rest period is short (FIG. 3c), and it is shown that it ends in about one week and enters the next growth period. On the other hand, in the case of the wild type mouse, it is well known that the rest period starting from about 48 days of FIG. 3c usually lasts 3 to 5 weeks. As a result, the period required for one cycle was shortened to about 3 weeks, compared to about 6 to 8 weeks or more for wild-type mice.
That is, it can be seen that the inhibition of the expression of the endogenous Fgf gene in the hair follicle shortened the rest period of the hair growth cycle and at the same time increased the speed of the hair growth cycle itself.

(3-3) Inherent phenotype of hair growth cycle phase It has been conventionally known that the hair growth cycle on the back skin of mice tends to be the fastest in the front part near the neck and the slowest in the rear part of the tail. However, since the beginning of the hair growth cycle begins at a plurality of positions on the back skin, when the back skin of a wild type mouse is shaved, as shown in the right of FIG. . In order to explain this, it was common to assume the presence of a control system that determines the hair growth cycle divided into blocks called “hair cycle domains” in the back skin of mice (FIG. 4). It has been empirically known that hair cycle domains exist in the back skin of the mouse as shown by the partitions separated by the vertical and horizontal lines in FIG. 4, and the control of the hair cycle is different for each domain. However, the numbers and positions of the vertical and horizontal lines in FIG. 4 are merely examples, and there are actually various patterns.
On the other hand, the hair follicle-specific Fgf18 gene knockout mouse of the present invention does not show such a hair cycle domain, and it is highly possible that the factor that determines the control system that determines the hair growth cycle by being divided into blocks is lost, The tendency of the progression rate of the original hair growth cycle that the hair growth cycle near the neck is the fastest and the tail back is the slowest, and as a result, the hair follicles of each phase become zebra's It shows the appearance of orderly arrangement like stripes.
In other words, the expression of the endogenous FGF gene in the hair follicle was inhibited, and it was found that the boundary line between domains in the direction perpendicular to the mouse body axis was lost among the hair cycle domains. .

(3-4) Characteristics of a large number of hair shafts in the hair follicle In the hair follicle-specific FGF18 gene knockout mouse of the present invention, the number of hair follicles per unit area of the skin does not increase, but the progression of the hair growth cycle is accelerated Nevertheless, the start of the hair loss period is delayed, and hair loss as a product of the previous cycle is unlikely to occur. Within the hair follicle, there are many club hairs that are close to the growing hair shaft growing on the growing hair follicle and have not fallen off. That is, it can be seen that the inhibition of the expression of the endogenous FGF gene in the hair follicle delayed the start of the hair loss period and prevented the club hair from falling off.

4). Actions of FGF18 gene and gene product FGF18 protein in hair follicle cells
Only by comparing the phenotype of Fgf18 gene knockout mice with that of wild-type mice has it been possible to elucidate the regulatory action of the endogenous FGF18 gene in hair follicle cells or the expression product FGF18 in hair follicles. Specifically, FGF18 is presumed to have the following three actions.
1) FGF18 has a function of maintaining the resting period of hair follicles. That is, endogenous FGF18 in the hair follicle is considered to be a regulatory factor that extends the rest period of the hair growth cycle.
2) FGF18 has the function of removing club hair from the skin. That is, endogenous FGF18 in the hair follicle is considered to be a regulatory factor that initiates the hair shedding phase that has not been elucidated so far.
3) FGF18 has a function of determining a boundary line between hair cycle domains sharing a direction orthogonal to an animal body axis (assumed to be the X axis) (tentatively assumed to be the Y axis). That is, it is considered to be one of the control factors that determine the hair cycle domain on the skin surface, that is, the control factor that controls the start time of the hair cycle.

5. FGF18 activity inhibitory substance 4. As shown in the above, since the endogenous FGF18 expressed in the hair follicle has the actions 1) to 3), the substance that inhibits or suppresses the action of the endogenous FGF18, that is, the “FGF18 activity inhibitor” The following effects can be expected.
1) Since it is considered to have an action of shortening the rest period of the hair growth cycle and, consequently, increasing the speed of the entire hair growth cycle, the effect as a rest period shortening agent or a hair growth cycle promoter of the hair growth cycle. Have
2) Since it can be said that it has the effect of delaying the hair dropout period, it has the effect of acting as a hair loss inhibitor, hair loss prevention agent or hair loss prevention agent.
3) With respect to the start time of the hair growth cycle, the variation in the direction orthogonal to the body axis is suppressed, and hair growth is promoted in an orderly growth cycle only in the body axis direction, so that there is an effect of aligning the hairs.
That is, the present invention relates to a hair growth cycle resting period shortening agent or hair growth cycle promoting agent (also referred to as “hair growth resting period shortening agent”), hair loss preventing agent, hair loss preventing agent or hair loss preventing agent (also referred to as “hair loss” It is referred to as “inhibitor” or “hair conditioner”.
The FGF18 activity inhibitor having the action of the “hair growth resting period shortening agent”, “hair loss prevention agent” or “hair conditioner” according to the present invention may be, for example, a partial fragment of FGF18.
FGF18 is synthesized as a 207-amino acid polypeptide in the cytoplasm of production cells in humans and mice, and when it is secreted outside the cell, the N-terminal signal peptide is cleaved, resulting in physiological activity as a secreted body consisting of 181 amino acids. Demonstrate. It reacts with at least four of the seven types of FGF receptor subclasses (FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b, and FGFR4), that is, FGFR1c, FGFR2c, FGFR3c, and FGFR4.
In the present invention, “FGF18” is not limited to human origin, and for example, FGF18 derived from other mammals can be used. Examples of other mammals include, but are not limited to, mice, rats, chickens, turkeys, cows, pigs, sheep and rabbits. For example, if a probe prepared based on the base sequence of human-derived FGF18 shown in SEQ ID NO: 1 is used according to a standard method, a gene encoding FGF18 can be isolated from mammals other than humans.
The nucleotide sequence and amino acid sequence of human-derived FGF18 after removal of the signal sequence are shown in SEQ ID NOs: 1 and 2, respectively. The nucleotide sequence and amino acid sequence of mouse-derived FGF18 after removal of the signal sequence are shown in SEQ ID NOs: 3 and 14, respectively. As can be seen by comparing the amino acid sequences shown in SEQ ID NOs: 2 and 14, FGF18 has a very high homology in mammals, and it can be understood that the functions of FGF18 in mammals are almost the same. .

By the way, in general, an antagonist binds to a receptor, but unlike a biological substance (ligand) that stimulates the receptor, it does not cause a biological reaction or causes a relatively weak biological reaction, and it naturally binds. It refers to a substance that inhibits the binding between a biological substance and a receptor and does not cause or attenuate a biological response. As described above, FGF18 reacts with at least FGFR1c, FGFR2c, FGFR3c, and FGFR4 among the seven types of FGF receptor subclasses. Therefore, in the present invention, “FGF18 antagonistic action” refers to FGFR1c, FGFR2c, FGFR3c, and FGFR4. A substance that does not cause or attenuates the response by FGF18 to cells expressing any of the receptors. FGF18 antagonists interfere with the binding of FGF18 to the receptor, thereby inhibiting the resting period extension action and the hair drop-out period initiation action in the hair growth cycle caused by FGF18, resulting in the effect of promoting the hair growth cycle. It will have the effect of preventing hair loss.
Taking the case of human FGF18 as an example of an FGF18 partial peptide having an FGF18 antagonistic action, the 32-151st region in the amino acid sequence shown in SEQ ID NO: 2 is a region called a core sequence that is highly common in the FGF family. Since the three-dimensional structure constructed only from this region has basic binding ability to the receptor but is not considered to be completely active, the amino acid sequence of the region corresponding to the region The partial peptide consisting of is a partial peptide considered to have an FGF18 antagonistic action. FGF18 partial peptide containing a core sequence corresponding to the region, which has sufficient binding ability to a receptor such as FGFR4 and does not have a sufficient length to cause an FGF18 response , Has an FGF18 antagonistic action.
As a result of actual experimental confirmation, as shown in FIG. 3, in SEQ ID NO: 14 , even when 16 methionines introduced from the N-terminus for translation initiation were deleted, they exert an antagonistic action, It can be said that a partial peptide having preferably 22 amino acids, more preferably 77 amino acids, and most preferably 95 amino acids has an even stronger FGF18 antagonistic action. That is, at least the N-terminal amino acid sequence does not lose the receptor binding ability even if it is deleted up to 95, and rather it can be said that the antagonistic action is strengthened, so the N-terminal amino acid sequence starts from the 32nd position. It is clear that all of the above core sequences are not essential sequences for FGF receptor binding, and even when the N-terminal amino acid is further removed, it can be said to be an FGF18 antagonist. In addition, it is clear from this that the partial peptide corresponding to the C-terminal side of the core sequence corresponding to the C-terminal side of the core sequence has an FGF18 antagonistic action when the 31st amino acid from the C-terminal is deleted. In addition, even when the C-terminal amino acid sequence is further largely deleted, it is sufficiently expected that the receptor binding ability is not completely lost, so 31 or more from the C-terminus, for example, 43, 57, Partial peptides lacking 67, 82, 94, 108, 113, 125, etc. amino acids are also considered to be strong FGF18 antagonistic partial peptides. And since this antagonistic activity is maintained even if this removes the N-terminal side and the C-terminal side at the same time, not only the peptide containing at least the 32-151st amino acid sequence, but preferably It can be said that a peptide comprising amino acid sequence 77 to 151, more preferably 95 to 151, also has an FGF18 antagonistic action.

In addition, antibodies that bind to FGF18 or FGF18-reactive receptors, that is, anti-FGF18 antibodies, anti-FGFR3c antibodies, and anti-FGFR4 antibodies also inhibit the binding of FGF18 to its receptors, so It inhibits the action of maintaining the rest period of the growth cycle for a long period of time, the action of promoting the start of hair loss, or the action of defining the hair growth cycle start time for each hair cycle domain.
Therefore, in the present invention, a substance that inhibits the action of the FGF18 via the receptor, such as a substance that inhibits the binding of FGF18 and its receptor, including the FGF18 antagonist, is referred to as “FGF18 activity inhibitory substance”.
Such a substance having an inhibitory action on FGF18 activity may be a substance selected from a group of substances unrelated to FGF18. By applying this screening method, a substance having an FGF18 activity inhibitory action can be easily obtained. Also in the examples of the present application, the gourd extract having an FGF18 activity inhibitory action is selected by the screening method, and the goya extract inhibits the FGF18 hair follicle growth inhibitory action and has a hair follicle growth action. It was confirmed. Thus, the FGF18 activity inhibitor in the present invention inhibits the binding of FGF18 to the receptor, thereby maintaining the rest period of the hair growth cycle of the hair follicle by FGF18 for a long period of time, and hair loss. Inhibits the initiation promoting action of hair, and thus has a hair growth resting period shortening action and a hair loss preventing action, but is observed as a hair growth promoting action when observed for a short period of time.
When these FGF18 activity-inhibiting substances are peptides, proteins or glycoproteins, it is of course possible to administer them as preparations containing these peptides, but of course, expression vectors incorporating DNA encoding such peptides, etc. It can also be administered in the state of For example, a preparation containing an expression vector incorporating a DNA encoding FGF18 partial peptide as an active ingredient can also be used as a hair growth rest period shortening agent, hair loss preventing agent or hair leveling agent.
These FGF18 activity-inhibiting substances can be used alone or in combination as a hair growth resting phase shortening agent, a hair loss preventing agent or a hair leveling agent.

6). Screening method for FGF18 activity inhibitor
The method of screening for an FGF18 activity inhibitor and using the test substance as a candidate for a hair growth quiescence shortening agent, a hair loss prevention agent or a hair leveling agent is specifically the following steps (a) to (c): Is included.
(A) a step of forcibly expressing on the cell surface by genetic manipulation using at least one FGF receptor gene selected from FGFR1c, FGFR2c, FGFR3c and FGFR4, and culturing the cells;
(B) a step of bringing a test substance into contact with a cell line having FGF receptor on the cell surface obtained in step (a) together with FGF18;
(C) In the step (b), a step of selecting a test substance observing the activity of inhibiting the cell growth promoting activity of FGF18. Here, as the FGF receptor gene, FGF18 is bound and the receptor is attached to the cell surface. At least one of the receptor genes of FGFR1c, FGFR2c, FGFR3c, and FGFR4, which has been confirmed to have a cell proliferating action of FGF18 on cells having the above, is used. Preferably, FGFR3c or FGFR4 gene is used. In the step (b), when the test substance is brought into contact with the cell, typically, the test substance is directly administered to the cell culture solution. However, particularly when the test substance is a protein, the test substance is used. It is also possible to introduce a gene encoding in a FGF receptor-expressing cell.
The cell for forcibly expressing the FGF receptor on the cell surface may be any cell that can be cultured, but is preferably a mouse IL-3-dependent Ba / F3 cell line.
At the time of screening, cell proliferation ability of FGF receptor-expressing cells is decreased by 5% or more, preferably 10% or more, compared with the case of culturing for 48 hours or more, for example, about 72 hours and adding FGF18 alone. It is preferable to screen using the above as a guide.
The parent cells into which the FGF receptor gene has not been introduced can be used for the control test, and the test substance is used to replace FGF18 added in step (b) with IL-3 and perform the same operation. It is preferable to provide a step of confirming that the test substance does not inhibit the cell growth promoting activity of IL-3 for these parent cells.

7). FGF18 expression inhibitor and screening method thereof
Since FGF18 is an endogenous factor present in hair follicles, substances that inhibit the transcription and translation of the FGF18 gene in hair follicle cells, that is, substances that reduce the expression of FGF18 gene also reduce the FGF18 concentration in hair follicle cells. Inhibits the action of FGF18 to maintain the rest period of the hair growth cycle in the hair follicle for a long period of time, the action of promoting the start of hair shedding, or the action of defining the hair growth cycle start time for each hair cycle domain Therefore, the hair growth resting period shortening agent, the hair loss preventing agent or the hair leveling agent has a hair growth promoting action and a hair growth action.
Such a substance that inhibits the expression of the FGF18 gene can be designed by a known method as an siRNA against the FGF18 gene or an expression vector thereof, and its activity can be expressed by using cultured animal cells or experimental animals. It can be confirmed by monitoring whether or not it is inhibited.
Alternatively, such a substance that inhibits the expression of the FGF18 gene can be screened by monitoring whether the expression of the FGF18 gene is inhibited using cultured animal cells or experimental animals.

Specifically, first, animal cultured cells or experimental animals that express the FGF18 gene to an observable level are prepared, and the test substance is contacted with the animal cultured cells or experimental animals or administered to the experimental animals. The experimental animal means, for example, mice, rats, chickens, turkeys, cows, pigs, sheep and rabbits, excluding humans. Examples of test substances include, but are not limited to, plant extracts, peptides, proteins, non-peptide compounds, low molecular compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts, and the like. These substances may be novel substances or known substances. When the test substance is brought into contact with cells or animals, the test substance is typically added to a cell culture medium or administered to a laboratory animal, but particularly when the test substance is a protein. It is also possible to introduce a gene encoding a test substance into an FGF receptor-expressing cell.
Next, the expression of the FGF18 gene in the animal cultured cells or experimental animals is monitored. The expression of the FGF18 gene in cultured animal cells or experimental animals can be analyzed, for example, using an ordinary method such as ELISA using FGF18 antibody, or the amount of mRNA of the FGF18 gene in the cells or experimental animals can be quantitatively reversed. It can be monitored by a method such as analysis by a copy PCR method or a Northern blot method.
As a result of any of these analyses, the expression level of the FGF18 gene in cultured animal cells cultured in the absence of the test substance is compared with that in the cultured animal cells or experimental animals cultured in the presence of the test substance. If the expression level of the FGF18 gene is reduced, it can be determined that the test substance may have a function of promoting hair growth or hair growth. Specifically, if the amount of mRNA is reduced to 0.8 times or less, preferably 0.7 times or less, more preferably 0.5 times or less as compared with the case where the test substance is not allowed to act, the substance is surely an expression inhibitor of FGF18. It can be said. The expression level of FGF18 mRNA in cultured keratinocytes, cultured dermal cells, and cultured dermal papilla cells varies depending on the culture conditions and cell types. Screening should be performed based on the decrease in
The FGF18 expression-inhibiting substance screened through such steps can be used alone or in combination as a hair growth quiescent shortening agent, a hair loss prevention agent or a hair leveling agent.

8). Hair growth resting period shortening agent, hair loss preventing agent or hair leveling agent The substances described in the above-mentioned "FGF18 activity inhibitory substance" and / or "FGF18 expression inhibitory substance" are, for example, adapted for skin application. It is formulated in the form of a solution, cream, ointment, gel, lotion, shampoo or aerosol, and is provided as a hair growth resting period shortening agent, hair loss preventing agent or hair leveling agent. The hair growth resting period shortening agent, the hair loss preventing agent or the hair leveling agent is particularly effective when applied not only to humans but also to pet animals, domestic animals using hair, experimental animals and the like.
In particular, the hair growth resting period shortening agent, hair loss preventing agent or hair leveling agent comprises a pharmacologically acceptable carrier adapted for topical administration and an FGF18 activity inhibitor and / or expression suppressor. It is administered in the form of The hair growth resting phase shortening agent, hair loss preventing agent or hair level regulator containing the FGF18 activity inhibitor and / or expression inhibitor is usually about 0.01 to about 100 μg / day / cm ² in a pharmaceutically acceptable carrier, preferably Contains from about 0.1 to about 10 μg / day / cm ² of active compound. In other words, the concentration of FGF18 is usually about 0.01 to about 100 μg / day / cm ² , preferably about 0.1 to about 10 μg / day / cm ² of active compound in a pharmaceutically acceptable carrier.
Furthermore, the pharmacologically acceptable carrier adapted for topical administration is not particularly limited, but is an ointment such as hydrophilic petrolatum or polyethylene glycol ointment, a paste such as rubber such as xanthan gum, alcohol, aqueous or Solutions such as buffers, gels such as aluminum hydroxide or sodium alginate gels, albumins such as human or animal albumin, collagens such as human or animal collagen, alkylcelluloses, hydroxyalkylcelluloses and alkylhydroxyalkylcelluloses Cellulose, methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose and hydroxypropylcellulose, pluronic exemplified by Pluronic F-127 (P Mention may be made of polymers such as luronic ™ polyols; tetronics such as tetronic 1508 and alginates such as sodium alginate.
As the FGF18 activity-inhibiting substance according to the present invention, an FGF18 activity-inhibiting protein such as an FGF18 partial peptide, or an expression vector incorporating a peptide-encoding DNA can be used. Can be offered at. The expression vector is provided with a sequence such as a promoter for expressing an FGF18 partial peptide in animal cells, but is not particularly limited. Examples of expression vectors that can be used include, but are not limited to, plasmid vectors and virus vectors.

In addition, the hair growth resting period shortening agent, hair loss preventing agent or hair level regulator according to the present invention may contain an FGF18 expression inhibitory substance as an active ingredient. That is, the hair growth quiescent period shortening agent, hair loss preventing agent or hair level regulator according to the present invention can be provided as gene therapy using an siRNA expression vector for FGF18. The expression vector has a sequence such as a promoter for expressing siRNA in animal cells, but is not particularly limited. Examples of expression vectors that can be used include, but are not limited to, plasmid vectors and virus vectors. Moreover, the FGF18 expression inhibitor is not limited to the siRNA expression vector for FGF18.
Methods for introducing a hair growth resting period shortening agent, a hair loss prevention agent or a hair level regulator for gene therapy into cells include gene introduction methods using viral vectors, non-viral gene introduction methods (Nikkei Science, 1994) The April issue, pages 20-45, experimental medicine extra number, 12 (15) (1994), experimental medicine separate volume “Basic technology of gene therapy”, Yodosha (1996)) can be applied.
As a gene transfer method using a viral vector, for example, a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a pox virus, a poliovirus, a simbis virus, or other DNA virus or RNA virus is encoded with TR4 or mutant TR4. The method of introducing and incorporating DNA to be used. Of these, methods using retroviruses, adenoviruses, adeno-associated viruses, and vaccinia viruses are particularly preferred. Non-viral gene transfer methods include a method in which an expression plasmid is directly administered into muscle (DNA vaccine method), a liposome method, a lipofection method, a microinjection method, a calcium phosphate method, an electroporation method, and the like. The vaccine method and the liposome method are preferred.

9. Application to in vitro hair regeneration system
Using an FGF18 activity inhibitor and / or expression suppressor, an in vitro hair regeneration system for regenerated skin tissue can be constructed. Here, the skin tissue means a tissue composed of various skin cells obtained by culturing isolated skin stem cells. The various skin cells are not particularly limited, and mean skin epidermis epithelial cells, skin epithelial basal layer cells, various cells constituting hair follicles, dermis cells, fat cells and the like. The cells used when regenerating skin tissue may be any of heterogeneous cells, allogeneic allogeneic cells, and allogeneic autologous cells.
First, the method for controlling the differentiation of skin stem cells into various cells of the skin is not particularly limited in the present invention because there is no established technique. For example, by using growth factor receptors that show different expression at the stage of spontaneous differentiation and adding corresponding growth factors to the medium, cells with different differentiation directions can be selected selectively. Can be amplified. After selectively amplifying cells with different differentiation directions, skin tissue can be made.
There is no specific technique for producing an artificial skin tissue.Therefore, the present invention is not limited in the present invention. After formation, epithelial cells are layered and integrated, then epithelial cell surface is exposed to air to promote epidermis, instead of dermis layer formed by biodegradable components Various methods such as a method using a layered film can be employed. The present invention also applies to a skin tissue preparation method in so-called regenerative medicine, in which skin stem cells are separated from skin tissue collected from humans, and the skin tissue is prepared using the separated skin stem cells. At this time, the newly prepared skin tissue may be assumed to be returned to the same person as the collected human for treatment, or transplanted for treatment to another person different from the collected human. It may be assumed.
In such a skin tissue preparation method, the rest period of the hair follicle growth cycle is shortened in the regenerated skin tissue by adding an FGF18 activity inhibitor and / or an expression inhibitor to the medium at an appropriate time. As a result, the speed of the hair growth cycle itself can be increased, and hair loss in the skin tissue can be prevented. Furthermore, in the method for preparing skin tissue, the proliferation of skin cells can be promoted by adding an FGF18 activity inhibitor and / or expression suppressor to the medium at an appropriate time, and the volume of the entire skin tissue is increased. Can be achieved.

10. Goya extract The FGF18 activity inhibitory substance obtained by this screening method was a bitter gourd extract, and its hair growth promoting effect was also confirmed as shown in the Examples.
Goya, which is the raw material of Goya extract, is a plant belonging to the genus Cucurbitaceae, and its scientific name is Momordica charantia, which is also called bitter gourd or pickaxe.
In obtaining the extract from bitter gourd, all parts of the plant can be used, and each part can be used alone or in an appropriate mixture as an extraction raw material. Moreover, the thing of the dry state and the non-dry state can be used for the property of the plant body used as a raw material.

  In order to obtain an extract used in the present invention from bitter gourd, these raw materials may be chopped or pulverized as necessary, and extracted by a commonly used extraction method using an appropriate solvent. Although the solvent used for extraction is not specifically limited, For example, water, anhydrous, or a water-containing organic solvent can be illustrated. The anhydrous or hydrous organic solvent may be one or more selected from monohydric alcohols, polyhydric alcohols or derivatives thereof, ketones, esters, ethers, petroleum ethers, aliphatic hydrocarbons, halogen compounds, and aromatic hydrocarbons. Can be illustrated. Specific examples of the solvent include water, methanol, ethanol, butanol, acetone, and ethyl acetate. These can be used alone or in combination of two or more. Among these, it is particularly preferable to use water or a monohydric alcohol such as methanol or ethanol. In the extraction, the amount of the solvent used is not particularly limited, but is 0.1-1000 times by weight, preferably 1-100 times by weight, more preferably 2-50 times by weight with respect to bitter gourd as an extraction raw material.

  The extraction method using the solvent can be performed according to a conventional method. For example, with respect to the extraction temperature, extraction may be performed at a temperature of about room temperature, or may be performed at a temperature near the boiling point of the solvent used. As for the extraction operation, a method of extracting by immersing dried pulverized product or pulverized product of bitter gourd at room temperature for 1 to 30 days, a method of performing reflux extraction at a temperature about the boiling point of the extraction solvent, or the like can be used. .

As shown in the examples below, the substance comprising the extract of bitter gourd of the present invention has FGF18 inhibitory activity for inhibiting cell growth by FGF18 against Ba / F3 cells expressing the FGFR4 gene. In the examples, when cells in which FGFR4, which is one of the FGF receptors, is forcibly expressed on the cell surface by gene expression manipulation are used, goya extract against cell proliferation by FGF18 in the presence of FGF18. It was shown that the addition was inhibited in a concentration-dependent manner. This action of the bitter gourd extract did not show a growth inhibitory effect on cell growth in the absence of FGF18 at the same time, indicating that it was not a cytotoxic effect but an FGF18 activity inhibitory action .
That is, the substance comprising the bitter gourd extract of the present invention is an FGF18 activity inhibitory substance that inhibits cell growth by FGF18.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.
Unless otherwise specified, genetic cloning (Molecular Cloning-A laboratory Manual, Sambrook
and Russell, Inc.).

[Example 1] Fgf18 gene hair follicle-specific deficient (knockout) mouse production A BAC clone containing the mouse Fgf18 gene was obtained from a 129 mouse BAC clone library. Using this clone, a targeting vector for deletion of exon 3 was constructed by introducing the loxP-FRT [PGK-Neo ^r ] FRT sequence upstream of exon 3 of the Fgf18 gene and introducing the loxP sequence downstream (Fig. 2). In addition, the targeting vector preparation was outsourced to Phoenix Bio as “targeting vector for exon 3 deletion of mouse Fgf18 gene”.
This vector was introduced into ES cells derived from 129 mice, and cells in which the Fgf18 gene was recombined with this sequence (referred to as targeted) were selected from the cell population and cloned. One of these ES cell lines was used for the production of transgenic mice. This cell was injected into a blastocyst of a C57BL / 6 mouse (dark brown hair, Japan SLC), and the uterus of a female ICR mouse (white body hair, Japan SLC) made into a pseudopregnant state. Introduced in. Chimera mice were distinguished from body hair color for pups delivered by the mice. These operations were performed on 63 blastocysts, and 16 chimeric mouse pups were obtained. These mice were bred and mated with C57BL / 6 mice after sexual maturation, resulting in 35 pups. Analysis of these genomes showed that 15 individuals contained the targeted Fgf18 gene (referred to as F1).
Next, in order to remove the [PGK-Neo ^r ] cassette, F1 mice were mated with Flpe transgenic mice, and 67 offspring mice were obtained. When the Fgf18 gene allele was analyzed for these, 12 individuals had the target sequence (the introduced loxP-FRT [PGK-Neo ^r ] FRT-Exon3-loxP as loxP-FRT-Exon3-loxP). (F2).
The Flpe transgenic mouse used here was obtained from RIKEN BRC (RIKEN RBRC01834; Non-Patent Document 8).
Next, F2 mice were further mated with K5-Cre transgenic mice for the purpose of excluding Fgf18 gene exon 3 in a hair follicle-specific manner. The obtained pups were weaned when they reached 4 weeks of age, and a part of the tail including the skin was excised, and the genotype of the Fgf18 gene and the expression of Cre were confirmed. Of the 37 weaned individuals, 6 K5Cre ^tg ; Fgf18 ^{+ / flox} mice (1 female, 5 males) were obtained. 29 mice were obtained by crossing K5Cre ^tg ; Fgf18 ^{+ / flox} mice. Analysis of these genotypes showed that one individual was K5Cre ^tg ; Fgf18 ^{flox / flox} (hereinafter referred to as Fgf18 homo-deleted). Similarly, by crossing K5Cre ^tg ; Fgf18 ^{+ / flox} mouse colonies, a large number of homozygous Fgf18 deletions were obtained.
Hereinafter, this Fgf18 homo-deleted product is referred to as an Fgf18 gene hair follicle-specific deficient animal.
The K5Cre transgenic mouse used here was obtained from KARD University CARD (CARD ID323; Non-Patent Document 9).
The presence of DNA lacking the Fgf18 third exon in these mouse skins was confirmed by Southern blot. An example is shown.
In addition, the genotype of the offspring produced in the cross between K5Cre ^tg ; Fgf18 ^{+ / flox} mice was in accordance with the usual Mendel's law.

[Example 2] Shortening of rest period and rapidly repeated hair growth cycle in Fgf18 gene hair follicle-specific deficient (knockout) mice
The Fgf18 gene hair follicle-specific deficient mice survived, grew, were reproducible, and were in good health. It was found that a characteristic hair growth cycle wave was exhibited by trimming the body hair growing on the back skin. Fig. 3a shows a representative individual (homo knockout body) of a Fgf18 gene hair follicle-specific deficient mouse [K5Cre ^tg ; Fgf18 ^{flox / flox} ], and one of one pair of Fgf18 genes as its target. This is a photograph of a representative individual (hetero knockout body) [K5Cre ^tg ; Fgf18 ^{+ / flox} ], which is only destroyed in the control group, after 32 days of age. Prior to taking these pictures, the hair was cut with a clipper so that the color of the body surface can be seen. At 32 days of age, the dorsal skin is in the initial physiological growth period for all mice and shows a black color. At 37 days of age, the area near the neck turns pinkish, indicating that it has entered a rest period. At 40 days of age, the entire back skin is at rest. In the case of the homomono-out body, it can be seen that the skin has a bluish color at 47 days of age and has entered the next growth period (FIG. 3a). In contrast, the control hetero knockout has a much longer rest period (FIG. 3a).
The hair growth cycle in the dorsal skin of the Fgf18 gene hair follicle-specific deficient mouse (68 days old) progresses smoothly from the front position (position close to the neck) to the rear position (position close to the tail). It can be seen that in addition to hair follicle morphogenesis (cycle indicated by M in FIG. 3c), two cycles (1 and 2 in FIG. 3c) have already started and the third cycle (3 in FIG. 3c) has been entered (see FIG. 3c). Fig. 3b left). In contrast, in wild-type mice (89 days of age), the location and timing of the next growth phase varies from individual to individual, and even within one individual's back skin, the “hair growth domain” Different phases are shown (Fig. 3b right and Fig. 4). Therefore, in the Fgf18 gene hair follicle-specific deficient mice, the transition of each phase in the general hair growth cycle as a function of mouse age can be shown in the same manner as in Fig. 2a (Fig. 3C).
Here, the phase of the hair growth cycle in the dorsal skin close to the neck of the mouse (indicated by the dotted line in Fig. 3b) is determined by the color of the skin and the hair growth history of the previous week. Expressed as a period. As a result, it was found that in the Fgf18 gene hair follicle-specific deficient mice, the rest period lasted only about 1 week, and each growth cycle was about 3 weeks as a whole including the rest period. In contrast, in wild-type mice, the rest period usually lasts 3 to 5 weeks or longer.
FIG. 3d shows three representative mice of the Fgf18 gene hair follicle-specific deficient mice with advanced aging. These mice have been raised for one week after shaving their back skin, and photographed including the hair that grew during the week. As a characteristic phenotype, in the Fgf18 gene hair follicle-specific deficient mice that have advanced in this way, the hair follicles of the same growth cycle phase are arranged so as to form a stripe in appearance. Furthermore, as a feature, it was found that the number of stripes increased with age, depending on the age of the mouse. In the aging mice, the period required for one hair growth cycle to take one rotation was about 3 weeks in the anterior position (Fig. 3d), but about 4 weeks in the posterior position ( Fig. 3d). Therefore, it can be understood that in the Fgf18 gene hair follicle-specific deficient mice with advanced aging, the difference in the growth cycle length between the anterior position and the posterior position shortens the distance between stripes.

[Example 3] The hair growing on the incomplete physiological hair loss resting hair follicle in the Fgf18 gene hair follicle-specific deficient mouse is a hair that has been completed in the previous hair growth cycle, and is club hair (club hair). hair). In general, in a mouse, club hair growing on a resting hair follicle can be picked up gently with a fingertip with a glove attached and pulled out gently. In the Fgf18 gene hair follicle-specific deficient mouse (homo knockout mouse), it was found that the hair pulled out in this way was a bundle of a plurality of hairs (FIG. 5a, left).
Such bundles were rare in the subject hetero knockout mice (Figure 5a, right). When the length of the pulled hair was measured for 200 or more hairs in each group, it was almost the same in both the homo knockout mouse (6.40 +/− 0.53 mm) and the hetero knockout mouse (6.38 +/− 0.65 mm). Furthermore, there was no difference in the distribution of hair types (guard, awl, and zigzag hairs). These facts strongly suggested that the growth phase of the hair growth cycle is proceeding normally in the Fgf18 gene hair follicle-specific deficient mice.
Fgf18 gene hair follicle-specific deficient mice (homogeneous knockout mice) and subjects with advanced aging, showing all hair follicles in the 3.7 mm2 area of the skin showing the characteristic that the hair cycle is in the growing phase and the number of hair shafts present there The number of hair follicles on the dorsal skin and the number of hair shafts present therein were measured. (Table 1)

Table 1: Number of hair shafts held by hair follicle cells
From this result, although the number of hair follicles in the Fgf18 gene hair follicle-specific deficient mice is not different from the control group, since the number of hair shafts retained in each hair follicle is large, the number of hair shafts as a whole I can understand that it is more than twice.

Furthermore, in the aging Fgf18 gene hair follicle-specific deficient mice (homo knockout mice) and the subject hetero knockout mice, the state of the hair follicles on the dorsal skin was magnified and observed. Then, it became clear that there were many club hairs that did not fall off, close to the growing hair shaft growing on the growing hair follicle (FIG. 5c). Furthermore, when the skin was observed with full focus imaging, it was confirmed that the bag-like structure that wraps the club hair that had not fallen off was snuggled up in the vicinity of the bulge region of the growing hair shaft.
In addition, in an aging Fgf18 gene hair follicle-specific deficient mouse, a section obtained by cutting the hair follicle of the back skin was prepared, stained with thioflavin T (yellow) and DAPI (red), and observed. Then, in homo knockout mice, it was shown that each hair follicle coexists with growing hair shafts rich in melanin and, in many cases, 3 to 6 club hairs without melanin (FIG. 5f). . On the other hand, in the target hetero knockout mice, the number of club hairs contained in the growing hair follicles was 1 to 2 at most. The distribution is as shown in Table 1. From these observations, it was suggested that in the Fgf18 gene hair follicle-specific deficient mice (homo knockout mice), the mechanism for the normal loss of club hair (hair loss period) was incomplete. Even so, since the number of remaining hair shafts per hair follicle is less than the number of hair growth cycles considered to be complete (10 to 12 cycles), some hair shafts may be incomplete. The dropout was thought to have occurred. From the above, it has been shown for the first time that deletion of FGF18 activity inhibits hair loss of club hair.

[Example 4] Inhibition of FGF18 activity by a partial peptide of FGF18
As a partial peptide of FGF18, a partial polypeptide (d4˜) in which amino acids from the 4th to the 95th excluding methionine introduced for translation initiation were removed from the N-terminus of SEQ ID NO: 14 corresponding to the amino acid sequence of mouse FGF18. d95: represented by the number of amino acids removed from the N-terminus excluding methionine).
In addition, the amino acid sequence (base sequence) of each partial polypeptide corresponds to the following sequence numbers.
d4: SEQ ID NO: 15 (SEQ ID NO: 4)
d12: SEQ ID NO: 16 (SEQ ID NO: 5)
d16: SEQ ID NO: 17 (SEQ ID NO: 6)
d18: SEQ ID NO: 18 (SEQ ID NO: 7)
d22: SEQ ID NO: 19 (SEQ ID NO: 8)
d37: SEQ ID NO: 20 (SEQ ID NO: 9)
d48: SEQ ID NO: 21 (SEQ ID NO: 10)
d67: SEQ ID NO: 22 (SEQ ID NO: 11)
d77: SEQ ID NO: 23 (SEQ ID NO: 12)
d95: SEQ ID NO: 24 (SEQ ID NO: 13)

FGF18 stimulates four types of FGF receptors, FGFR1c, FGFR2c, FGFR3c, and FGFR4, but they have different strengths, and as a result of the summation of stimulation to these receptors, the resting phase of the hair growth cycle Is held for a long period of time, the start of hair loss is advanced, and the start of the hair growth cycle is considered to be controlled for each domain.
According to the teachings of the literature, one of the FGF receptors FGFR1c, FGFR2c, FGFR3c, and FGFR4 was introduced into the Ba / F3 cell line (obtained from Riken BRC), a mouse IL-3-dependent proB cell, by gene expression manipulation. Thus, cells in which FGFR is forcibly expressed on the cell surface were prepared (Ornitz, DM., Xu, J., Colvin, JS., McEwen, DG., MacArthur, CA., Coulier, F., Gao). , G. and Goldfarb, M., 1996. Receptor specificity of the fibroblast growth factor family. J. Biol. Chem. 271 (25): 15292-15297; Yoneda, A., Asada, M., Oda, Y., Suzuki, M. and Imamura, T., 2000. Engineering of an FGF-proteoglycan fusion protein with heparin-independent, mitogenic activity. Nat. Biotec. 18 (6): 641-644).

When using these cells and stimulating each receptor with 30 ng / ml FGF18, each FGF18 partial peptide coexists at a concentration of 1 μg / ml (Fig. 3A) or 100 ng / ml (Fig. 3B). Antagonist activity was examined to see if it inhibits DNA synthesis of cells stimulated with FGF18 (this is 100%). That is, the activity of suppressing the cell proliferation stimulating effect by FGF18 was analyzed. The result is shown in FIG. The correspondence between each column and the test sample is as follows.
(FIG. 6A) Column 1: No sample other than FGF18 added (control). Column 2: (d4). Column 3: (d16). Column 4: (d18). Column 5: (d22). Column 6: (d37). Column 7: (d48). Column 8: (d77). Column 9: (d95). Column 10: (d22). Column 11: (d37). Column 12: (d48). Column 13: (d67). Column 14: (d77). Column 15: (d95).
(FIG. 6B) Column 1: No sample other than FGF18 added (control). Column 2: (d4). Column 3: (d12). Column 4: (d16). Column 5: (d18). Column 6: (d37). Column 7: (d48). Column 8: (d67). Column 9: (d95). Column 10: (d37). Column 11: (d67).
FIG. 6A shows that when the test substance is tested at a concentration of 1 μg / ml, d16, d22, d37, d48, d77, d95, etc. strongly inhibit the activity of FGF18 on FGFR3c / BaF3 cells. It can also be seen that d22, d48, d77, d95, etc. strongly inhibit the activity of FGF18 on FGFR4 / BaF3 cells. Other partial peptides also show various inhibitory effects as shown in the figure.
FIG. 6B shows that when the test substance is tested at a concentration of 100 ng / ml, d16, d95, etc. strongly inhibit the activity of FGF18 on FGFR3c / BaF3 cells.
By using these FGF18 partial peptides, which are inhibitors of FGF18 activity, it is possible to cancel the hair growth cycle resting phase control action and hair loss initiation controlling action by FGF18 in hair growth control. Can shorten hair, prevent hair loss, and align the fur.
Similarly, the partial polypeptide (dc25 to dc125: represented by the number of amino acids removed from the C terminus) from which amino acids from the C terminus to the 25 th to 125 th amino acids of SEQ ID NO: 14 corresponding to the amino acid sequence of mouse FGF18 are removed. ) Was produced.
In addition, the amino acid sequence (base sequence) of each partial polypeptide corresponds to the following sequence numbers.
dc25: SEQ ID NO: 34 (SEQ ID NO: 25)
dc43: SEQ ID NO: 35 (SEQ ID NO: 26)
dc57: SEQ ID NO: 36 (SEQ ID NO: 27)
dc67: SEQ ID NO: 37 (SEQ ID NO: 28)
dc82: SEQ ID NO: 38 (SEQ ID NO: 29)
dc94: SEQ ID NO: 39 (SEQ ID NO: 30)
dc108: SEQ ID NO: 40 (SEQ ID NO: 31)
dc113: SEQ ID NO: 41 (SEQ ID NO: 32)
dc125: SEQ ID NO: 42 (SEQ ID NO: 33)
Of these FGF18 partial peptides, the partial peptides except dc25 have the same inhibitory action on FGF18 activity as the above partial peptide from which the N-terminus has been removed. Since it can be released, the hair growth resting period can be shortened, hair loss can be prevented, and the level of hair can be aligned.

[Example 5] Screening for FGF18 activity inhibitory substance using FGFR4-expressing cells The prepared FGFR4-expressing cells (R4 / Ba / F3 cells) were cultured together with various plant extracts in the presence of FGF18. A commercially available FGF18 protein was used as a positive control. Using Cell Counting Kit-8 (manufactured by Dojindo Laboratories Co., Ltd., sold by Wako Pure Chemical Industries, Ltd.), the number of cells after culturing for a certain period of time was changed to 450 nm with the amount of WST-8 formazan produced. Determined by measurement.
As a result, it was found that the bitter gourd extract acts as an inhibitor of FGF18 activity and inhibits the proliferation of FGFR4 / BaF3 cells by FGF18.

[Example 6] Cell growth inhibitory activity of FGF18 activity inhibitory substance of the present invention To 1.5 g of dried goya, 30 ml of distilled water was added and boiled for 15 minutes. The extract obtained as described above was filtered with a filter paper, and the filtrate was collected to obtain a hot water extract of bitter gourd. To 1.5 g of dried goya, 30 ml of 70% ethanol aqueous solution was added, followed by extraction by immersion for 7 days at room temperature. The extract thus obtained was filtered with a filter paper to obtain a 70% ethanol extract of bitter gourd.

In the same manner as in Example 5, the activity of the FGF18 activity inhibitory substance of the present invention was measured using FGFR4 / BaF3 cells. Specifically, the measurement was performed as follows. RPMI1640 medium containing 50 μl of 10% FBS, 1% Antibiotic G-418 Sulfate (Promega Corporation, V7983) was added to each well of a 96-well cell culture plate. Next, after adding 10 μl of various concentrations of test solutions prepared by diluting each sample with water, 5 × 10 ⁴ FGFR4 / BaF3 in RPMI1640 medium containing 10% FBS and 1% Antibiotic G-418 Sulfate 50 μl of the cell suspension in which the cells were suspended was added and gently stirred. Furthermore, heparin / 10% FBS / 1% Antibiotic G-418 Sulfate / RPMI1640 medium, 10 μl (heparin final concentration 5 μg / ml), and FGF18 (Peprotech Inc., 100-28) solution 10 μl (FGF18 final concentration 3 ng / ml) ml), and cultured in a carbon dioxide incubator at 37 ° C. in the presence of 5% CO 2 for 72 hours. FGFR4 / BaF3 cells were proliferated after adding 10 μl of Cell Counting Kit-8 (manufactured by Dojindo Laboratories, Inc., sold by Wako Pure Chemical Industries, Ltd.) / PBS solution to each well after 72 hours of culture. The culture was performed for a long time, and the color development at 450 nm accompanying the production amount of WST-8 formazan was measured and determined.

In the measurement, 10 μl (FGF18 final concentration 3 ng / ml) of FGF18 (Peprotech Inc., 100-28) solution was used as a positive control, and water or ethanol solution ( The final concentration of the ethanol solution was adjusted to 1% or less) and measured using 10 μl. The cell growth inhibition rate (%) was calculated by the following formula (A).
[Formula (A)]
Cell growth inhibition rate (%) = 100 × {(absorbance when adding FGF18 and water or ethanol solution) − (absorbance when adding FGF18 and test solution)} / absorbance when adding FGF18 and water or ethanol solution−water or ethanol Absorbance when adding solution)

  The results for the Goya hot water extract are shown in FIG. In the presence of FGF18, when a Goya hot water extract with a final concentration of 8.3% was used as the test solution, the amount of color development decreased, and it was confirmed that the Goya hot water extract inhibits the growth of FGFR4 / BaF3 cells by FGF18. It was. On the other hand, the absorbance when FGF18 was not added and when the test solution was added was almost the same as the absorbance when FGF18 was not added and when the water or ethanol solution was added, and the cytotoxic effect of the test solution was hardly observed. .

[Example 7] In vivo analysis of FGF18 activity inhibitor of the present invention
In order to see the in vivo effects of FGF18 activity inhibitors, we tested C3H / He mice in the resting period of the hair cycle.
To 1.1 g of dried goya, 15 ml of distilled water was added and boiled for 20 minutes. After the extract returned to room temperature, it was filtered through filter paper, and the same amount of ethanol was added to the filtrate to prepare a 50% ethanol solution of Goya hot water extract. Further, the solution was passed through a 0.45 μm Millex HV sterilization filter (Millipore), and then glycerol was added to 1% to prepare a test solution. The Goya hot water extract (containing 49.5% ethanol and 1% glycerol) thus obtained was applied to the back skin of C3H / He mice in the rest period of the hair cycle. Seven week old C3H / He male mice were anesthetized and the back hair was gently shaved with a clipper. After shaving, 200 μl of Goya hot water extract (containing 49.5% ethanol and 1% glycerol) or water-ethanol-glycerol solution (containing 49.5% water, 49.5% ethanol, and 1% glycerol) each as a test solution Group 5 mice were applied to the dorsal skin (day 0). Similarly, it was applied every day for 11 days (excluding the 5th and 6th days), and the hair growth on the back of the mice on the 10th, 14th, 17th, 21st and 24th days was visually observed. The hair growth score was obtained. The hair growth score is 1) pigmentation: 1 point, 2) short hair level: 2 points, 3) normal hair level: 3 points, and the ratio of the area of each hair growth state to the total shaved area The percentage was calculated by the following formula. According to this calculation method, when the total shaved area is restored to the normal level, the score is 100 points.
Hair growth score = (Proportion of pigmentation area (%) × 1 + Proportion of short fur area (%) × 2 + Proportion of normal fur area (%) × 3) / 3
As a result of observing the hair growth on the back of the mice on the 10th, 14th, 17th, 21st, and 24th day after the first application, as shown in FIG. Compared to the group (water-ethanol-glycerol solution (containing 49.5% water, 49.5% ethanol, 1% glycerol) application group), the hair growth score was particularly high after the 21st day, and the hair growth was significantly promoted. .
As described above, it was demonstrated that the Goya hot water extract, which is an FGF18 activity inhibitory substance, has an action of inhibiting the original activity of FGF18 even in in vivo analysis on the surface of the back of the mouse. In this experiment, since only data up to 24th day was acquired, it was observed as a “hair growth promoting effect”, but if the observation was continued further, the effect was that the rest period of the hair growth cycle was shortened and It can be said that this is based on the effect of increasing the total amount of hair due to the fact that hair removal does not start.

[Example 8] Hair shampoo using Goya hot water extract Formulation and manufacturing method of hair shampoo using Goya hot water extract according to the present invention will be described.
A hair shampoo was produced according to the following formulation and production method.
(Prescription)
Component Weight%
1. Diluent obtained in Example 5 0.1
2. Sodium lauryl ether sulfate ethanol 20
3. Sodium lauryl sulfate 10
4.1,3 Butylene glycol 1
5. Perfume appropriate amount 6. Purified water remaining amount (100 in total)
(Manufacturing method)
The prescription ingredients were heated to 80 ° C., stirred and mixed, and then cooled by stirring to produce the hair shampoo of the present invention.

[Example 9] Hair Liquid Using Goya Hot Water Extract A hair liquid formulation and production method using the Goya hot water extract of the present invention will be described.
A hair liquid was produced according to the following formulation and production method.
(How to)
Component Weight%
1. Diluent obtained in Example 5 0.1
2. Ethanol 40
3. Glycerin 1
4. Perfume appropriate amount 5. Purified water remaining amount (100 in total)
(Manufacturing method)
After adding the formulation components and dissolving with stirring, purified water was added to produce a hair liquid.

Example 10
The formulation and manufacturing method of the hair cream using the bitter gourd hot water extract which is this invention are shown.
A hair cream was produced according to the following formulation and production method.
(Prescription)
Component Weight%
1. Diluent obtained in Example 5 0.1
2. Liquid paraffin 40
3. Vaseline 1
4. Cetostearyl alcohol 1
5. Methylpolysiloxane 1
6. Methyl paraoxybenzoate 0.2
7. Propylene glycol 5
8. Perfume proper amount 9. Purified water remaining amount (100 in total)
(Manufacturing method)
The formulation ingredients were mixed with stirring to produce the hair cream of the present invention.

  According to the present invention, it is possible to provide an effective hair growth rest period shortening agent, hair loss prevention agent or hair leveling preparation agent, and by blending these, shampoos, hair liquids and the like having hair growth promoting activity can be used for treating alopecia In addition, non-human animals and other experimental animals with accelerated hair growth cycles and pets with uniform fur can be provided.

Claims (13)

  A hair growth resting phase shortening agent, hair loss preventing agent or hair level regulator comprising an FGF18 activity inhibitor and / or an expression inhibitor as an active ingredient.   The FGF18 activity inhibitor is a partial peptide of FGF18, wherein the hair growth resting period shortening agent, hair loss preventing agent, or hair leveling agent is described.   2. The hair growth resting period shortening agent, hair loss preventing agent or hair level adjusting agent according to claim 1, wherein the FGF18 activity inhibitor is an anti-FGF18 antibody.   2. The hair growth resting period shortening agent, hair loss preventing agent or hair leveling regulator according to claim 1, wherein the FGF18 activity inhibitor is bitter gourd extract.   2. The hair growth quiescent shortening agent according to claim 1, wherein the FGF18 activity inhibitor is an expression vector incorporating a cDNA encoding a partial peptide of FGF18 having an activity of inhibiting the activity of FGF18. Hair loss prevention agent or hair leveling agent.   The hair growth resting period shortening agent, hair loss preventing agent or hair level regulator according to claim 1, wherein the FGF18 expression inhibitor is an expression vector incorporating siRNA having an activity of inhibiting FGF18 expression. .   The hair growth resting period shortening agent, hair loss preventing agent or hair level adjusting agent according to any one of claims 1 to 6, further comprising another hair growth promoting agent, hair growth promoting agent, or hair loss inhibitor. A method of screening the FGF18 activity inhibitor according to any one of claims 1 to 4 and using the test substance as a candidate for a hair growth quiescence shortening agent, a hair loss prevention agent or a hair leveling agent, comprising: A method comprising steps a) to (c).
(A) a step of forcibly expressing on the cell surface by genetic manipulation using at least one FGF receptor gene selected from FGFR1c, FGFR2c, FGFR3c and FGFR4, and culturing the cells;
(B) a step of bringing a test substance into contact with a cell line having FGF receptor on the cell surface obtained in step (a) together with FGF18;
(C) a step of selecting a test substance observing the activity of inhibiting the cell growth promoting activity of FGF18 in step (b)   The screening method according to claim 8, wherein the FGF receptor according to claim 8 is FGFR3c.   The screening method according to claim 8, wherein the FGF receptor according to claim 8 is FGFR4.   The screening method according to any one of claims 8 to 10, wherein the cell for forcibly expressing the FGF receptor according to claim 8 on the cell surface is a mouse IL-3-dependent Ba / F3 cell line. A method for screening the FGF18 expression-suppressing substance according to claim 1 to be a candidate for a hair growth quiescence shortening agent, a hair loss prevention agent or a hair leveling agent, comprising the following (a) to (d): A method comprising the steps.
(A) preparing a cultured animal cell or experimental animal that expresses the FGF18 gene to an observable level;
(B) contacting the test substance with the animal cultured cells of (a) or contacting or administering to the experimental animal;
(C) (b) a step of monitoring the expression of the FGF18 gene in cultured animal cells or experimental animals after the step;
(D) A step of selecting a test substance having a function of inhibiting the expression of the FGF18 gene. In the step (c), mRNA is extracted from the experimental animal or animal cultured cell after the step (b), and the expression of the FGF18 gene is monitored by analyzing the mRNA level of the expressed FGF18. ) In the step, selecting a test substance having a function of inhibiting the expression of the FGF18 gene by selecting a system in which a low level of FGF18 mRNA is observed compared to the case where the test substance is not allowed to act. 13. A method according to claim 12, characterized.