JP2016214085A - Hair growth inhibitor evaluation or selection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a material having hair growth inhibitory action, and an evaluation and/or selection method therefor.SOLUTION: A hair growth inhibitor evaluation and/or selection method uses FABP4/5 expression or activity as an index. A hair growth inhibitor comprises a material that reduces FABP4/5 expression or activity as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a method for evaluating and / or selecting a hair growth inhibitor.

  In recent years, there has been an increasing demand for removal of body hair and whiskers for reasons of aesthetic appearance. As a method for removing these hairs, a mechanical method using a tweezer, a shaver, a hair removal device, or the like, and a chemical method using a hair removal agent or a hair removal agent are conventionally known. However, these known hair removal methods may involve physical or chemical irritation to the skin. In addition, since the known hair removal method cannot continuously suppress the growth of hair that is regenerated after removal, the hair removal process must be performed again after a certain period of time. There is a demand for a hair growth inhibitor that can suppress hair growth continuously and effectively, thereby reducing the number of hair removal treatments and reducing labor for hair removal.

  For efficient research and development of hair growth inhibitors, it is useful to develop a high-throughput screening model for hair growth inhibitors in vitro using cultured cells and the like. Examples of conventional in vitro screening models for hair growth regulators include direct observation of hair growth in human isolated hair follicle cultures and the method disclosed in Patent Document 1.

  FABPs (Fatty acid-binding proteins) are a family of intracellular fatty acid binding proteins, and are 14-15 kDa proteins having high binding properties to fatty acids and the like (Non-patent Documents 1 and 2). FABP4 and FABP5 are isoforms of FABPs and have high amino acid sequence and three-dimensional homology with each other. These are co-expressed in adipocytes and macrophages, while FABP4 is expressed with relatively high specificity in adipose tissue and macrophages, while FABP5 is widely expressed in epithelial cells. For FABP4 and FABP5, many studies have been conducted on inflammation and metabolism, particularly related to metabolic syndrome. For example, it has been reported that the genetic mutation of FABP4 and FABP5 or knockout mice each suppress the onset of atherosclerosis and diabetes. However, the function of FABP4 and FABP5 in hair tissue, particularly with respect to hair growth, has not been clarified.

Japanese Patent No. 5654808

Nature Reviews Drug Discovery, 2008, 7: 489-503 Diabetologia, 2013, 56: 10-21

  The present invention relates to a method for evaluating and / or selecting a hair growth inhibitor.

  The present inventors show that, among the FABPs family, FABP4 and FABP5 show locally high expression in the hair root and hair growth is carried out by administering to the hair follicle a compound that inhibits the activity of FABP4 or FABP5. Has been found to be suppressed. Based on these findings, the present inventors can evaluate and / or select a hair growth inhibitor using the inhibitory action on the expression or activity of FABP4 or FABP5 as an index, and the expression or expression of FABP4 or FABP5. It has been found that a substance having an activity inhibiting action can be used for hair growth inhibition.

Accordingly, the present invention provides the following:
Applying the test substance to a tissue or cell capable of expressing the FABP4 gene or FABP5 gene, or the FABP4 protein or FABP5 protein;
Measuring the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein, or the activity of the FABP4 protein or FABP5 protein in the tissue or cells;
Based on the measurement results, the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein, or a test substance that decreases the activity of the FABP4 protein or FABP5 protein is evaluated and / or selected as a hair growth inhibitor. To do,
A method for evaluating and / or selecting a hair growth inhibitor is provided.

The present invention also provides the following:
Applying the test substance to the FABP4 protein or FABP5 protein;
Measuring the activity of the FABP4 protein or FABP5 protein;
Evaluating and / or selecting a test substance that decreases the activity of the FABP4 protein or FABP5 protein as a hair growth inhibitor based on the measurement results;
A method for evaluating and / or selecting a hair growth inhibitor is provided.

  The present invention also provides a hair growth inhibitor containing as an active ingredient a substance that decreases the expression of FABP4 gene or FABP5 gene, the expression of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein.

The present invention also provides a compound represented by the following formula (I) or formula (II):
The present invention provides a hair growth inhibitor containing as an active ingredient.

  The present invention provides a method by which hair growth inhibitory effects of various substances can be more easily and rapidly evaluated. Moreover, this invention provides the new hair growth inhibitor based on FABP4 or FABP5 inhibitory action.

Expression of FABPs in human hair roots. n = 6, error bar = SD. Effect of compound of formula (I) on FABP4 / 5-fluorescent substrate binding. Each bar represents the fluorescence intensity of 1,8-ANS (n = 3, error bar = SD). **: p <0.01 (vs 0 μM compound of formula (I) / FABP4 or 5+, Dunnett's test). Effect of compound of formula (II) on FABP4 / 5-fluorescent substrate binding. Each bar represents the fluorescence intensity of 1,8-ANS (n = 3, error bar = SD). *: P <0.05, **: p <0.01 (vs 0 μM Formula (II) compound / FABP4 or 5+, Dunnett's test). The hair growth inhibitory effect in the cultured human hair follicle by the compound of formula (I). n = 16, error bar = SD. **: p <0.01 (vs Vehicle, Dunnett's test). The hair growth inhibitory effect in the cultured human hair follicle by the compound of formula (II). n = 16, error bar = SD. **: p <0.01 (vs Vehicle, Dunnett's test). Cytotoxicity evaluation of the compound of formula (I) and the compound of formula (II). Left: compound of formula (I), right: compound of formula (II). In both cases, n = 6 and error bar = SD.

  In the present specification, “hair growth inhibition” means an action of suppressing the growth of hair or hair follicles or an action of reducing hair diameter.

  In the present specification, examples of the “hair” whose growth is suppressed include head hair, beard, eyelashes, eyebrows, and other body hairs (for example, hair on limbs and trunk), preferably beards and limbs. Or body hair of the trunk.

  As used herein, “non-therapeutic” refers to a concept that does not include medical practice, that is, does not include methods for surgery, treatment, or diagnosis of humans, more specifically, a physician, or a medical professional or physician. It is a concept that does not include a method in which a person who receives an instruction performs surgery, treatment, or diagnosis on a human.

  FABP4 and FABP5 are proteins belonging to the FABPs family. In this specification, “FABP4 / 5” refers to the FABP4 gene, FABP5 gene, FABP4 protein, or FABP5 protein. Moreover, in this specification, "FABP4 / 5 protein" means FABP4 protein or FABP5 protein. In the present specification, “expression of FABP4 / 5” means expression of FABP4 gene or FABP5 gene, or expression of FABP4 protein or FABP5 protein. In the present specification, “activity of FABP4 / 5” refers to the activity of FABP4 protein or FABP5 protein.

  As used herein, “FABP4 gene” refers to GenBank Accession No. It refers to the human FABP4 gene represented by NM_001442.2, its homologue, paralogue or orthologue. Preferably, the “FABP4 gene” is a polynucleotide encoding a protein consisting of the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with the sequence and having a high binding property to fatty acids and the like. Refers to nucleotide. Further, in this specification, “FABP5 gene” refers to GenBank Accession No. This refers to the human FABP5 gene represented by NM_001444.2, its homologue, paralogue or orthologue. Preferably, the “FABP5 gene” is a polynucleotide encoding a protein consisting of the nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence having at least 90% identity with the sequence and having a high binding property to fatty acids and the like. Refers to nucleotide.

  As used herein, “FABP4 protein” refers to a polypeptide encoded by the above-mentioned “FABP4 gene”. More specifically, “FABP4 protein” refers to GenBank Accession No. It refers to the human FABP4 protein represented by NP_001433.1, its homologue, paralogue or orthologue. Preferably, the “FABP4 protein” refers to a protein having an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having at least 90% identity with the sequence and having a high binding property to fatty acids and the like. In the present specification, “FABP5 protein” refers to a polypeptide encoded by the “FABP5 gene”. More specifically, “FABP5 protein” refers to GenBank Accession No. It refers to the human FABP5 protein represented by NP_001435.1, its homologue, paralogue or orthologue. Preferably, the “FABP5 protein” refers to a protein having an amino acid sequence represented by SEQ ID NO: 4 or an amino acid sequence having at least 90% identity with the sequence and having a high binding property to fatty acids and the like.

  In the present specification, “at least 90% identity” with respect to amino acid sequences and nucleotide sequences means 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more. .

  Herein, nucleotide and amino acid sequence identity is calculated by the Lippman-Pearson method (Lipman-Pearson method; Science, 1985, 227: 1435-1441). Specifically, using the homology analysis (Search homology) program of genetic information software Genetyx-Win (Ver. 5.1.1; software development), analysis is performed with Unit size to compare (ktup) as 2. Is calculated by

  In one aspect, the present invention provides a method for evaluating the hair growth inhibitory action of various substances using the FABP4 / 5 inhibitory action as an index, or further selecting a hair growth inhibitor based on the evaluation. The method for evaluating and / or selecting the hair growth inhibitor of the present invention can be performed in vitro or ex vivo.

Thus, one embodiment of the present invention is the following:
Applying a test substance to a tissue or cell capable of expressing FABP4 / 5;
Measuring the expression level or activity of FABP4 / 5 in the tissue or cells;
Evaluation and / or selection of a test substance that reduces the expression level or activity of FABP4 / 5 as a hair growth inhibitor based on the measurement results,
A method for evaluating and / or selecting a hair growth inhibitor.

Another embodiment of the present invention includes the following:
Applying the test substance to the FABP4 / 5 protein;
Measuring the activity of the FABP4 / 5 protein;
Evaluating and / or selecting a test substance that reduces the activity of the FABP4 / 5 protein as a hair growth inhibitor based on the measurement results;
A method for evaluating and / or selecting a hair growth inhibitor.

  The test substance used in the method of the present invention is not particularly limited as long as it is a substance desired to be used as a hair growth inhibitor. The test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, etc., and may be a compound, a composition or a mixture. Good.

  In one embodiment of the method of the present invention, as an example of a tissue or cell capable of expressing FABP4 / 5 to which a test substance is applied, a FABP4 gene or FABP5 gene, or a FABP4 protein or FABP5 protein is expressed. Examples include tissues or cells isolated from mammals, or cultures thereof. More detailed examples include skin epithelial tissue or cells collected from mammals, small intestinal tissue or cells such as duodenum and jejunum, adipose tissue or cells, thymic epithelial tissue or cells, and cultures thereof; hair follicles Skin culture; isolated hair follicle culture; organ culture hair follicle; small intestine primary culture cell; small intestine culture cells such as Caco-2 cells, IEC-6 cells, IEC-18 cells, STC-1 cells, GLUTag cells; 3T3 -L1 cells and the like.

  Alternatively, examples of tissues or cells capable of expressing FABP4 / 5, which are used in the method of the present invention, include mammals genetically modified to express FABP4 gene or FABP5 gene, or FABP4 protein or FABP5 protein. Or a culture thereof. The genetically modified mammalian tissue or cell, and the culture thereof, for example, can be obtained by introducing a gene encoding FABP4 protein or FABP5 protein into any tissue or cell of a mammal. It can be produced by transforming to express FABP4 / 5 or to enhance the expression of FABP4 / 5. Examples of a method for introducing a gene into a cell include, but are not limited to, vector introduction by electroporation or lipofection.

  Examples of mammals derived from tissues or cells capable of expressing FABP4 / 5 used in the method of the present invention include, but are not limited to, humans, mice, rats, hamsters, rabbits, pigs, monkeys and the like. Not.

  Application of a test substance to a tissue or cell capable of expressing FABP4 / 5 is, for example, adding a test substance in advance to a predetermined concentration in a medium and then seeding the tissue or cell in the medium. Alternatively, it can be carried out by adding a test substance at a predetermined concentration to a medium in which the tissue or cells are seeded. Alternatively, in the case of an isolated hair follicle culture or organ culture hair follicle, the test substance may be directly administered to the hair follicle or its surrounding tissue. The tissue or cells after application of the test substance are preferably cultured, for example, at room temperature (about 25 ° C.) to 37 ° C., usually for about 3 to 48 hours, preferably about 6 to 24 hours.

  The density | concentration at the time of seed | inoculation of the said tissue or cell with respect to the said culture medium will not be specifically limited if a cell is a density | concentration which can proliferate. Moreover, the addition concentration of the test substance is preferably 0.00001 to 10% (w / v), particularly preferably 0.0001 to 5% (w / v).

  As the medium for culturing the tissue or cells, a conventional medium can be used, and examples thereof include 10% FBS-containing Dulbecco's Modified Eagle's Medium. It is preferable to add growth additives such as serum, growth factors, insulin, and antibacterial agents to these media during cell passage and proliferation.

  Next, tissues or cells to which the test substance is applied are collected, and the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein is measured. The gene expression level can be measured according to a known method. For example, when detecting at the mRNA level, for example, after extracting total RNA from cells, the real-time RT-PCR method, RNase protection assay method, DNA array method, Northern blot analysis method, etc. are used. It can be carried out by detecting and quantifying mRNA transcribed from.

  The measurement of the expression level of FABP4 / 5 protein can be performed by a conventional immunoassay, for example, RIA method, EIA method, ELISA, bioassay method, Western blot, and the like. Western blots are desirable because they are cheap, simple and convenient. The activity of the FABP4 / 5 protein can be measured by measuring the amount of binding substrate bound to the FABP4 / 5 protein. For example, 1-anilinophthalene-8-sulfonic acid (1,8-ANS) may be used as the binding substrate, and the binding amount may be measured from the fluorescence intensity.

  Next, in the method of the present invention, a hair growth inhibitor is evaluated and / or selected based on the expression level or activity of FABP4 / 5 measured above. More specifically, a test substance that decreases the expression level or activity of FABP4 / 5 is evaluated and / or selected as a hair growth inhibitor.

  Preferably, the expression level or activity of FABP4 / 5 in a tissue or cell (test group) capable of expressing FABP4 / 5 to which the test substance is applied is compared with the expression level or activity of FABP4 / 5 in the control group. Based on the results, a test substance that can be used as a hair growth inhibitor is selected.

  Examples of the control group include tissues or cells capable of expressing the same FABP4 / 5 as in the test group, to which the test substance is not applied. The expression level or activity of FABP4 / 5 in the control group can be measured by the same procedure as described above.

  When the expression level or activity in the test group decreases compared to the control group, the test substance is evaluated as having a hair growth inhibitory action, and the test substance is selected as a hair growth inhibitor. For example, when the expression level or activity in the test group is statistically significantly lower than the expression level or activity in the control group, the test substance is evaluated as having a hair growth inhibitory effect. As another example, when the expression level or activity in the control group is 100%, the expression level or activity in the test group is 90% or less, preferably 80% or less, more preferably 60% or less. The test substance is evaluated as having a hair growth inhibitory action. A test substance evaluated to have a hair growth inhibitory action is selected as a hair growth inhibitor.

  In another embodiment of the method of the present invention, the test substance is applied to the FABP4 / 5 protein extracellularly. For example, the FABP4 / 5 protein is prepared by isolating the FABP4 protein or FABP5 protein from the tissue or cells capable of expressing the above-mentioned FABP4 / 5, or by chemical synthesis. A test substance and a binding substrate are applied to the obtained FABP4 / 5 protein, and then its activity is measured. The activity can be measured by measuring the amount of binding substrate bound to the FABP4 / 5 protein by the procedure described above.

  Next, a test substance that decreases the activity of the FABP4 / 5 protein measured above is evaluated and / or selected as a hair growth inhibitor. Preferably, the activity in the FABP4 / 5 protein (test group) to which the test substance is applied is compared with the activity in the control group, and a test substance that can be used as a hair growth inhibitor is selected based on the comparison result. The control group includes a group to which the test substance is not applied. The activity of the FABP4 / 5 protein in the control group can be measured by the same procedure as in the test group.

  When the activity in the test group decreases compared to the control group, the test substance is evaluated as having a hair growth inhibitory action, and the test substance is selected as a hair growth inhibitor. For example, when the activity in the test group is statistically significantly lower than the activity in the control group, the test substance is evaluated as having a hair growth inhibitory effect. As another example, when the activity in the control group is 100%, the test substance has hair growth when the activity in the test group is 90% or less, preferably 80% or less, more preferably 60% or less. Evaluated as having an inhibitory effect. A test substance evaluated to have a hair growth inhibitory action is selected as a hair growth inhibitor.

  In the above-described method of the present invention, the substance selected in the above procedure may be subjected to further screening, if necessary. For example, after administering a test substance evaluated or selected as having a hair growth inhibitory effect by the above method to a skin culture containing hair follicles, an isolated hair follicle culture, an organ culture hair follicle or the like, the hair or hair follicle By measuring the growth of the hair and comparing it with that of the control as necessary, a substance having a stronger hair growth inhibitory action can be further selected. Examples of the control include skin cultures containing hair follicles, isolated hair follicle cultures, organ culture hair follicles, etc., to which no test substance is administered. For example, when the length or hair diameter of the hair or hair follicle in the test substance administration group is statistically significantly lower than the length or hair diameter of the hair or hair follicle in the control group, or in the control group When the hair or hair follicle length or hair diameter is 90% or less, preferably 80% or less, more preferably 60% or less, the test substance is selected as a hair growth inhibitor.

  As described above, the hair growth inhibitor can be identified using the FABP4 / 5 inhibitory action as an index. Therefore, in one aspect, the present invention provides a hair growth inhibitor containing, as an active ingredient, a substance that reduces the expression level or activity of FABP4 / 5.

  In one embodiment, the hair growth inhibitor of the present invention contains a compound represented by the following formula (I) or formula (II) as an active ingredient. These compounds have the effect of reducing the expression level or activity of FABP4 / 5, and work as hair growth inhibitors when applied to hair follicles.

  In another embodiment, the hair growth inhibitor of the present invention contains a known FABP4 / 5 inhibitor, for example, a compound exemplified below (see Bioorganic & Medicinal Chemistry Letters, 2010, 20: 3675-3679) as an active ingredient. contains. These compounds act as hair growth inhibitors when applied to hair follicles.

  The hair growth inhibitor of the present invention can be used for hair growth inhibition, or for hair removal or hair removal. The use may be a therapeutic use but may also be a non-therapeutic use. Examples of non-therapeutic use include hair removal for cosmetic or aesthetic purposes, application of hair growth inhibitor of the present invention for hair removal or hair growth inhibition, and esthetician, hairdresser, barber, trimmer, etc. Use of the hair growth inhibitor of the present invention. In a preferred embodiment, the hair growth inhibitor of the present invention is topically administered to the skin.

  The hair growth inhibitor of the present invention can also be used for production of a composition for inhibiting hair growth, or for removing or removing hair. Examples of the composition include pharmaceuticals, quasi-drugs, cosmetics, and other external compositions for hair growth inhibition or hair removal or hair removal. The said composition contains the hair growth inhibitor of the said this invention as an active ingredient for hair growth suppression or hair removal or hair removal. Furthermore, the composition is a pharmaceutically or cosmetically acceptable carrier or additive, or other active ingredient as necessary, as long as the hair growth inhibitory action of the hair growth inhibitor of the present invention is not lost. , Pharmacological ingredients, cosmetic ingredients and the like.

  Although the composition containing the hair growth inhibitor of the present invention can be prepared in any form, it can preferably be a skin external preparation. Examples of the external preparation for skin may include ointments, creams, lotions, gels, foams, patches, tapes, sprays and the like for hair growth inhibition or hair removal or hair removal. Alternatively, the external preparation for skin may be a moisturizing and whitening skin care product, an antiperspirant, a deodorant agent, a body soap, etc. having a hair growth inhibiting function.

  Alternatively, the hair growth inhibitor of the present invention can be added as a hair growth inhibiting component to any composition such as pharmaceuticals, quasi drugs, cosmetics, and other external compositions. For example, the hair growth inhibitor of the present invention can be added as a hair growth inhibitory component to a moisturizing or whitening skin care product, an antiperspirant, a deodorant agent, a body soap, and the like, thereby imparting a hair growth inhibitory effect.

  The content of the hair growth inhibitor of the present invention in the composition is preferably 0.0005% by mass or more, more preferably 0.001% by mass or more, and further preferably 0.005% by mass or more, Preferably it is 10 mass% or less, More preferably, it is 5 mass% or less, More preferably, it is 1 mass% or less.

  In yet another aspect, the present invention provides a method for inhibiting hair growth in a subject. The present invention also provides a method for removing the hair from a subject. The method includes administering an effective amount of the hair growth inhibitor of the present invention to the subject. The method may be a therapeutic method or a non-therapeutic method. Further, the method may be an in vitro method or an in vivo method.

  Examples of the target in the above method include animals for which hair growth suppression is desired and animals for which hair removal or hair removal is desired. The animal preferably includes humans and non-human mammals including dogs and cats, more preferably humans. Alternatively, examples of the subject include tissues, organs, and cells having animal-derived hair growth ability. Tissues, organs and cells having hair growth ability include skin cultures containing hair follicles, isolated hair follicle cultures, and organ culture hair follicles.

  In a preferred embodiment of the above method, the hair growth inhibitor of the present invention is locally administered (for example, applied) to the hair follicle of the subject for which hair growth inhibition is desired or the skin containing the hair follicle.

  An effective amount of administration of the hair growth inhibitor of the present invention in the above method may be an amount that can achieve inhibition of hair growth of the subject. Preferably, the effective amount is an amount that can reduce the hair follicle or hair elongation or hair diameter of the administration group to 90% or less, preferably 80% or less, more preferably 60% or less of the non-administration group. possible. The effective amount of administration can vary according to the subject's species, weight, sex, age, condition or other factors. The dose, route and interval of administration can be determined appropriately by those skilled in the art. A preferred route of administration is topical administration to the skin. In the case of an adult (60 kg), the standard dose is preferably 0.001 mg or more, more preferably 0.005 mg or more, and on the other hand, preferably 500 mg or less, more preferably 100 mg or less.

  In one embodiment, the hair growth suppression method, hair removal method or hair removal method of the present invention may be a non-therapeutic method. Examples of non-therapeutic methods include a method of administering the hair growth inhibitor of the present invention to a human or non-human mammal for which hair removal, hair removal or hair growth suppression is desired for cosmetic or aesthetic purposes, and an esthetician A method of applying the hair growth inhibitor of the present invention to the above-mentioned object by a hairdresser, a barber, a trimmer or the like.

  As exemplary embodiments of the present invention, the following compositions, production methods, uses or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

<1> Evaluation and / or selection method of hair growth inhibitor,
Applying the test substance to a tissue or cell capable of expressing the FABP4 gene or FABP5 gene, or the FABP4 protein or FABP5 protein;
Measuring the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein, or the activity of the FABP4 protein or FABP5 protein in the tissue or cells;
Based on the measurement results, the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein, or a test substance that decreases the activity of the FABP4 protein or FABP5 protein is evaluated and / or selected as a hair growth inhibitor. To do,
Including the method.

<2> Preferably, the tissue or cell capable of expressing the FABP4 gene or FABP5 gene, or the FABP4 protein or FABP5 protein is selected from the group consisting of the following (1) to (4), Method:
(1) Skin epithelial tissue or cells collected from mammals, small intestine tissue or cells, adipose tissue or cells, thymic epithelial tissue or cells, or cultures thereof;
(2) a skin culture containing hair follicles, an isolated hair follicle culture, or an organ culture hair follicle;
(3) Small intestine primary cultured cells, Caco-2 cells, IEC-6 cells, IEC-18 cells, STC-1 cells, GLUTag cells, or 3T3-L1 cells; or (4) FABP4 gene or FABP5 gene, or FABP4 protein Or a mammalian tissue or cell genetically modified to express the FABP5 protein, or a culture thereof.

<3> Preferably, the method further comprises measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the control group, <1> or <2> The method according to <1> or <2>, wherein the gene or FABP5 gene, or tissue or cell capable of expressing the FABP4 protein or FABP5 protein is not applied with the test substance.

<4> Preferably, the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the expression level of FABP4 protein or FABP5 protein measured in the control group measured in <1> above Alternatively, the method according to <3>, wherein the test substance is selected as a hair growth inhibitor when the activity is statistically significantly decreased.

<5> Preferably, the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the expression level of FABP4 protein or FABP5 protein measured in the control group measured in <1> above Alternatively, the method according to <3>, wherein the test substance is selected as a hair growth inhibitor when the activity is 90% or less, preferably 80% or less, more preferably 60% or less when the activity is 100%.

<6> Evaluation method and / or selection method of hair growth inhibitor,
Applying the test substance to the FABP4 protein or FABP5 protein;
Measuring the activity of the FABP4 protein or FABP5 protein;
Evaluating and / or selecting a test substance that decreases the activity of the FABP4 protein or FABP5 protein as a hair growth inhibitor based on the measurement results;
Including the method.

<7> Preferably, the method further comprises measuring the activity of the FABP4 protein or FABP5 protein in the control group, and the control group is the FABP4 protein or FABP5 protein to which the test substance is not applied. Method.

<8> Preferably, when the activity of the FABP4 protein or FABP5 protein measured in <6> above is statistically significantly lower than the activity measured in the control group, the test substance is <7> The method according to <7>, which is selected as a hair growth inhibitor.

<9> Preferably, the activity of the FABP4 protein or FABP5 protein measured in <6> above is 90% or less, preferably 80% or less, more preferably 100% when the activity measured in the control group is 100%. <7> The method according to <7>, wherein the test substance is selected as an agent for suppressing hair growth when is 60% or less.

<10> A hair growth inhibitor comprising, as an active ingredient, a substance that decreases the expression of FABP4 gene or FABP5 gene, the expression of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein.

<11> A hair growth inhibitor containing a compound represented by formula (I) or formula (II) described in the present specification as an active ingredient.

<12> A composition for hair growth inhibition, hair removal or hair removal, comprising the hair growth inhibitor according to <10> or <11> above.

<13> Use of the hair growth inhibitor according to the above <10> or <11> for the production of a composition for hair growth inhibition, hair removal or hair removal.

<14> Use of the hair growth inhibitor as described in <10> or <11> above for hair growth inhibition, or for hair removal or hair removal.

<15> A method for suppressing hair growth of a subject, comprising administering the hair growth inhibitor of the above <10> or <11> to the subject in an effective amount.

<16> A method for removing hair from a subject, comprising administering to the subject an effective amount of the hair growth inhibitor according to <10> or <11> above.

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Example 1 Expression analysis of FABPs in human hair roots Total RNA was extracted from human hair roots, and gene expression was analyzed by DNA array (Affymetrix, Human Genome U133 Plus 2.0 Array). As a result, as shown in FIG. 1, the expression of FABP4 and FABP5 was remarkable in the human hair root, and the expression of other FABPs was hardly observed. From this result, it was suggested that FABPs related to hair growth are FABP4 and FABP5.

Example 2 Screening of hair growth inhibitors based on FABP4 / 5 activity (principle)
Hair growth inhibitors were screened using competitive binding of fluorescent substrates and test substances to FABP4 or FABP5 based on the report of Liu et al. (J Neurochem, 2008, 106: 2015-2029). The principle is shown in Schema 1 below. 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) (A) is a substance having binding ability to FABP4 protein and FABP5 protein (Kd = 1.2 μM and 11.7 μM, respectively). 1,8-ANS is a substance that emits fluorescence only in a hydrophobic environment, and does not emit fluorescence in a hydrophilic environment such as in a buffer solution (B). However, when FABP4 protein or FABP5 protein is present, 1,8-ANS binds to FABP4 protein or FABP5 protein, and as a result, the periphery of the 1,8-ANS molecule becomes a hydrophobic environment and becomes fluorescent. (C). On the other hand, when a substance having a smaller binding constant to the FABP4 protein or FABP5 protein further coexists, the substance binds to the FABP4 protein or FABP5 protein preferentially over 1,8-ANS, and as a result, is in a hydrophobic environment. Since 1,8-ANS decreases, fluorescence decreases (D). Therefore, when fluorescence is attenuated when a test substance is added simultaneously with 1,8-ANS, the test substance is more likely to bind to FABP4 protein or FABP5 protein than 1,8-ANS, that is, FABP4 protein or FABP5. It is judged to be a substance that binds to protein and suppresses its activity.

(Test substance)
The FABP4 protein-specific activity inhibitor FABP4 Inhibitor (formula (I) compound) was purchased from Santa Cruz Biotechnology, Inc. In addition, triazolopyrimidinone derivatives (5-((3-chloro-2-methylphenoxy) methyl) -2-phenyl- [1, which are reported to broadly inhibit the activity of FABP3 protein, FABP4 protein and FABP5 protein are reported. 2,4] triazolo [1,5-α] pyrimidin-7 (4H) -one; compound of formula (II)) was synthesized by the method described in Reference Example 1 described later. Each test substance was dissolved in dimethylsulfoxide (DMSO) so as to have a concentration of 10 mM, and then diluted with ethanol to a predetermined concentration. The ethanol / DMSO concentration ratio was adjusted to be the same between the samples.

(Method)
Recombinant human FABP4 protein and recombinant human are cloned by cloning human FABP4 or FABP5 cDNA into pGEX-6P-2 vector (GE healthcare) and transforming it into E. coli strain BL21 (GE healthcare) for expression of recombinant protein. Each of the FABP5 proteins was obtained. To a 96-well plate, the assay buffer (10 mM potassium phosphate, 40 mM KCl, pH 8.0) containing the FABP4 protein or FABP5 protein obtained above was added to a final volume of 200 μL, and the above formula (I ) And a compound of formula (II), and 1,8-ANS (Life technologies), which is a fluorescent substrate, were added. Thereafter, the mixture was incubated at room temperature for 5 minutes, and fluorescence was measured with an EnVision 2104 Multilabel reader (PerkinElmer) (excitation wavelength: 355 nm, measurement wavelength: 480 nm).

(result)
A result is shown to FIGS. 2-3 and Tables 1-2. The compound of formula (I) significantly inhibited the binding between the fluorescent substrate and the FABP4 protein in a concentration-dependent and statistically significant manner, but did not inhibit the binding between the fluorescent substrate and the FABP5 protein (FIG. 2 and Table 1). . On the other hand, the compound of the formula (II) significantly inhibits the binding between the fluorescent substrate and the FABP4 protein in a concentration-dependent and statistically significant manner, and also binds the fluorescent substrate to the FABP5 protein when the added amount is 10 μM. It was statistically significantly inhibited (FIG. 3 and Table 2). Therefore, it was shown that the compound of formula (I) is an inhibitor of FABP4 protein, and the compound of formula (II) is an inhibitor of FABP4 and FABP5 proteins.

Example 3 Evaluation of Hair Growth Inhibitory Action of Test Substance Using Human Isolated Hair Follicle Culture A human scalp specimen was disinfected by immersing in a 0.1% hibiten solution for 1 minute, and then washed with PBS. Thereafter, hair follicles were isolated using a forceps and a scalpel in a William E medium (Life technologies) under a stereomicroscope. The isolated hair follicles contained 2 mM L-Glutamine (Life technologies), 10 μg / mL Insulin (Life technologies), 40 ng / mL Hydrocortisone (Sigma), 1% Antibiotics (Sigma) in William E medium. (24-well plate, 300 μL) was cultured under conditions of 37 ° C. and 5% CO ₂ . As a test substance, an ethanol / DMSO solution of the same compound of formula (I) or formula (II) as prepared in Example 2 was added to the medium at a predetermined concentration. Alternatively, only ethanol / DMSO solvent was added to the medium as a control. The cultured hair follicles were photographed with time under a microscope, and the length of the hair follicle was calculated by image analysis based on the scale photographed under the same conditions. Image analysis was performed using NewQube (version 4.0.3, Nexus), and the amount of increase from the initial value was defined as the amount of hair elongation.

  The results are shown in FIGS. The compounds of formula (I) and formula (II) suppressed human hair follicle elongation significantly and statistically in a concentration range of 3 to 10 μM. As shown in Example 2, since the compounds of formula (I) and formula (II) are FABP4 protein and / or FABP5 protein activity inhibitors, the expression or activity of FABP4 and / or FABP5 is determined from the results of this example. It became clear that the hair growth can be suppressed by suppressing the hair.

Example 4 Cytotoxicity Evaluation of Test Substance Human kidney (293A) cells were purchased from ATCC and cultured in DMEM (Life technologies, High glucose, 10% inactivated FBS) at 37 ° C. and 5% CO ₂ conditions. The cultured cells were seeded in a 96-well plate, and the test substance was diluted to a predetermined concentration and added the next day. As the test substance, the same ethanol / DMSO solution of the compound of formula (I) or formula (II) as prepared in Example 2 was used. 48 hours after addition of the test substance, using alamarBlue (AbD Serotec), fluorescence (excitation wavelength: 544 nm, fluorescence wavelength: 590 nm) as an indicator of the number of viable cells was detected according to the attached instruction manual, and a control (vehicle; ethanol / DMSO solvent) The ratio (%) of the number of viable cells was calculated.

  The results are shown in FIG. The compounds of formula (I) and formula (II) did not show cytotoxicity in the concentration range up to 30 μM. Therefore, the compounds of formula (I) and formula (II) can effectively suppress hair growth in a concentration range that does not show cytotoxicity.

Reference Example 1 Synthesis of compound of formula (II)

  First, 1.61 g of 3-phenyl-1H-1,2,4-triazol-5-amine was suspended in 12 mL of acetic acid, and 1.50 mL of ethyl 4-chloroacetoacetate was added. This was stirred at 130 ° C. for 15 hours and cooled to room temperature. After filtration, washing with 3 mL of acetonitrile, drying and 5- (chloromethyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (compound 1 ) Was obtained as a white powder (263 mg, 10% yield).

The product obtained above was analyzed by ¹ H-NMR, and 5- (chloromethyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidine-7 (4H)- Confirmed that it was on.
¹ H-NMR (MeOD) δ: 8.28-8.24 (2H, m), 7.55 (3H, dd, J = 5.2, 2.1 Hz), 6.25 (1H, s), 4.69 (2H, s).

  Next, 246 mg of 3-chloro-2-methylphenol was dissolved in 3 mL of dimethylacetamide, and 85.2 mg of sodium hydride (60%) was added at room temperature. After 20 minutes, 270 mg of 5- (chloromethyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (Compound 1) was added and at 180 ° C. Heated for 40 hours. After cooling to room temperature, 5 mL of water was added, and the mixture was extracted 4 times with 50 mL of ethyl acetate. The obtained upper layers were combined, washed with 10 mL of saturated brine, and dried over anhydrous magnesium sulfate. After filtration, the solvent was distilled off to obtain the compound of formula (II) as a crude product. This was purified by silica gel column chromatography (chloroform-methanol). Subsequently, the product was purified by Diaion HP-20 (water-methanol) to obtain the compound of formula (II) as a white powder (10.3 mg, yield 2.7%).

The product obtained above was subjected to ¹ H-NMR analysis and ¹³ C-NMR analysis to confirm that it was a compound of formula (II).
¹ H-NMR (MeOD) δ: 8.23-8.22 (2H, m), 7.48 (3H, dd, J = 5.2, 2.0 Hz), 7.14 (1H, t, J = 8.3 Hz), 7.03 (1H, d, J = 8.3 Hz), 6.95 (1H, d, J = 8.3 Hz, 6.21 (1H, s), 5.11 (2H, s), 2.43 (3H, s).
¹³ C-NMR (MeOD) δ: 163.7, 162.3, 161.4, 159.0, 158.8, 136.4, 132.6, 131.4, 129.9, 128.6, 128 5, 126.4, 123.2, 111.6, 95.6, 70.9, 13.3.

Claims (4)

A method for evaluating and / or selecting a hair growth inhibitor, comprising:
Applying the test substance to a tissue or cell capable of expressing the FABP4 gene or FABP5 gene, or the FABP4 protein or FABP5 protein;
Measuring the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein, or the activity of the FABP4 protein or FABP5 protein in the tissue or cells;
Based on the measurement results, the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein, or a test substance that decreases the activity of the FABP4 protein or FABP5 protein is evaluated and / or selected as a hair growth inhibitor. To do,
Including the method. A method for evaluating and / or selecting a hair growth inhibitor, comprising:
Applying the test substance to the FABP4 protein or FABP5 protein;
Measuring the activity of the FABP4 protein or FABP5 protein;
Evaluating and / or selecting a test substance that decreases the activity of the FABP4 protein or FABP5 protein as a hair growth inhibitor based on the measurement results;
Including the method.   A hair growth inhibitor comprising, as an active ingredient, a substance that decreases the expression of FABP4 gene or FABP5 gene, the expression of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein. Compounds represented by the following formula (I) or formula (II):
A hair growth inhibitor, containing as an active ingredient.