JP2005304491A - Method for prognosis of hair growth or epilation of the skin using gene group expression advancing in hair follicle in stage of growth as index, and method for screening hair restorer and/or depilatory - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for prognosis of hair growth state or epilation state of the skin by detecting a factor relating to the mechanism of hair growth, epilation in a hair cycle, and to provide a new unconventional and effective hair restorer or depilatory. <P>SOLUTION: The invention relates to the method for testing the skin to predict the state of hair growth or epilation. The method uses the gene which expression advances in the hair follicle in the stage of growth as an index. The invention provides the method for screening the hair restorer or depilatory by using the gene as an index. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention provides a method for predicting the hair growth / hair loss state of the skin, as well as a method for screening a hair restorer and a hair remover.

  Hair is very important in terms of aesthetic appearance. Thus, hair loss due to congenital or acquired factors is a serious concern for many people, especially men. Especially in the modern society, which is called an aging society and a stress society, the hair of the head is exposed to a risk of hair loss due to various acquired causes. Corresponding to this, various attempts have been made to provide a better hair-restoring agent that exhibits a hair-restoring effect including promotion of hair growth and hair thickening. On the other hand, especially in the case of women, treatment of unwanted hair such as body hair and wrinkles in limbs, wrinkles, etc. is performed on the aesthetic appearance. For example, mechanical hair removal using a shaver, etc. Hair removal methods such as a method of removing from hair root and a method of removing unwanted hair by chemical action using a hair removal agent have been developed and used.

  Thus, there are situations in which hair growth is required for the hair and hair removal is required for the heels and limbs. In any case, various attempts have been made including research on hair follicles, but no precise treatment or treatment method has yet been found.

  A hair follicle is one of the appendages of the skin and is characterized as an organ that constantly grows with a certain period and has the ability to repeat self-renewal. The hair follicle is formed of so-called hair exposed to the outside of the skin, and the root sheath, hair matrix cells, and dermal papilla cells that form the hair shaft buried in the skin dermis. Developmentally, a hair follicle primordium is formed by the skin-dermis interaction, and a hair follicle is formed by extending the skin downward (growth stage). As the final stage of development, hair shaft elongation continues for a certain period of time, but eventually stops, and apoptosis causes the root sheath and hair matrix cells to regress (regression phase). In this series of processes, the hair follicles extending deep into the dermis are shortened to near the skin, and the size of the hair papilla is also reduced. Eventually, after a resting phase, the hair matrix cells are activated again and head for the growth phase of a new cycle. This cycle repeats almost throughout life (hair cycle).

  A number of factors are considered to be involved and function in a complex manner in such hair growth and hair loss mechanisms. Although it is known that keratin-related proteins and apoptosis-related enzymes such as caspases are involved in the hair cycle, there are many other proteins and genes that are not yet known to be involved. Is in the expected situation.

M. Naganuma et al., Journal of Dermatological Science 25 (2001) 29-35

  The present invention finds a factor involved in hair growth of the hair cycle, a hair removal mechanism, etc., provides a method for predicting the hair growth / hair removal situation of the skin, and further, a completely new and effective hair restorer, hair removal that has never existed before Providing a drug is an issue.

  The present inventor attempted exhaustive analysis of factors involved in hair cycle hair growth and hair loss mechanisms. Specifically, the present inventor has used several mice whose hair cycle was synchronized by simultaneously performing wax hair removal, and the hair follicle (hereinafter referred to as “growth”) in which wax hair removal was performed and the hair cycle on the 13th day shifted to the growth phase. As a result of microarray analysis of RNA derived from skin having a large number of skin follicles (sometimes referred to as “term hair follicles”), hair follicles of control mice that have not been subjected to wax hair removal (hereinafter sometimes referred to as “resting hair follicles”) It was found that the expression of the following genes was specifically enhanced as compared with the results of microarray analysis of a large number of skin-derived RNAs. Therefore, it has been clarified that by examining the expression of the following gene in human skin, it is possible to determine that the skin is easy to grow hair or to remove hair.

(1) A gene / lymphoid enhancement factor binding factor that is known to be involved in hair follicle formation and hair shaft formation, and whose expression in the growing hair follicle is 10 times higher than that in the resting hair follicle ( LEF-1)
Neuroblastoma myc-related oncogene 1
・ Fork head box N1
・ Connexin 30
・ Connexin 26
・ Tenacin C
・ Syndecan 1
・ Clusterin ・ Small proline rich protein 1A

(2) A gene whose function in the hair follicle is unknown, but the function of the expressed protein is known, and its expression in the growing hair follicle is 10 times higher than that in the resting hair follicle Secretin / cytoplasmic poly A binding protein 1
Cytoplasmic poly A binding protein 2
・ Eukaryotic translation initiation factor 5
・ High glycine / tyrosine protein type I E5
・ Poliovirus receptor related 1
・ Ephrin receptor (EphA4)
-Potassium membrane voltage-gated channel-Isk-related subfamily member-Huntingtin interacting protein 1-related-Neuronal protein-Formin family protein FHOS2
・ Carbonic anhydrase 6
・ Cathepsin E
・ Gamma glutamyl transpeptidase ・ Serine protease inhibitor 1 (alpha-1, antitrypsin 1, serpin 1 alpha)
・ Glutathione peroxidase ・ Beta A4 crystallin ・ Plextrin homology domain ・ Family A ・ Member 2
・ Nucleoporin-like protein 2
-Bispecific phosphatase 2
・ Transmembrane 4 Superfamily member 4
・ Stimulated by retinoic acid gene 6
-Huntingtin interacting protein 1 related-Mitochondrial outer membrane 40 homolog (yeast) translocase-Aquaporin 1
・ Golgi autoantigen Golgin subfamily a4
・ Tubulin beta 5
・ Contains baculovirus IAP repeat 5
・ Ubiquitin conjugating enzyme E2S
・ Cytokeratin 12

(3) A gene involved in the hair follicle is unknown, the protein to be expressed is unknown, and the expression in the growing hair follicle is 10 times or more higher than that in the resting hair follicle: 1810008K03Rik (or 34) Cha gene (cation transport regulator gene) with% homology);
1110032D16Rik (shows high homology with KAP4-7);
4733401H21Rik (shows high homology with KAP6-1)

(4) A gene / basic keratin complex gene that is known to be involved in hair follicle formation and hair shaft formation, and whose expression in the growing hair follicle is 5 times or more higher than that in the resting hair follicle. 6a
・ Vitamin D receptor ・ P cadherin ・ Stratifin ・ Fin ・ Protoon cogene ・ Transforming growth factor alpha ・ Patched homolog 2
-Antigen identified by monoclonal antibody Ki67-Basonuclin

(5) Gene / run-related transcription factor 1 (Runx1, AML1), which is an unknown gene involved in hair follicles, and whose expression in growing hair follicles is 5 times or more higher than that in resting hair follicles
Cytoplasmic / activated T cell nuclear factor 3 (NFAT3)
・ Tight junction protein 2 (ZO-2)
・ Claudin 1
・ Ninjurin 1
・ Poliovirus receptor related 2
・ Presenilin 1
・ Tabbie-like protein 4
・ Naked Cuticle 2 Homolog (Drosophila)
Mitogen-activated protein kinase 13
・ Cyclin-dependent kinase 4
・ Harvey Rat Sarcoma Virus Oncogene ・ MAD Homolog (Drosophila) 6
・ RAB15
・ Ephrin receptor (EphB1)
・ Protein tyrosine phosphatase ・ Non-receptor type 11
Secretory carrier membrane protein 2
・ Transgelin 2
・ Kinesin Family Member 11
・ Microtubule-related protein ・ Cytidine 5′-triphosphate synthase ・ Lecithin-retinol acyltransferase ・ Protein dephosphorylation enzyme 1
・ Fused toes differential display and activated by p53 (differential display and activated by)
・ Binding mediator ・ Regulatory protein ・ Doricol phosphatase (beta D) mannosyltransferase 2
・ Repin 3
・ Adenylyl cyclase-related CAP protein homolog 1
・ BMP-2 induction kinase ・ Gasdermin ・ Cyclin E
・ Uridine phosphorylase ・ S phase kinase activator ・ Kinetocentric autoantigen A
・ Striatine casein Kappa
・ Solvent transport versus family 4 (anion exchanger) member 1
・ 4 1/2 LIM domain 2
Angiotensin converting enzyme (peptidyl peptidase A) 2
・ Ets homologous factor

In addition to the above genes, the following genes were also significantly increased in expression in the growing hair follicle as compared to the resting hair follicle.
(6) Gene / acid keratin complex 1 gene 3 which is a keratin-related gene whose expression in the growing hair follicle is 10 times higher than that in the resting hair follicle
・ Acid keratin complex 1 gene 1
・ Basic keratin complex gene 6g
Acid keratin complex 1 gene 17
Acid keratin complex 1 gene 19
・ Basic keratin complex gene 6a
・ Keratin-related protein 3-3
・ Keratin-related protein 9-1
・ Keratin related protein 14
・ Keratin-related protein 5-4
・ Keratin-related protein 12-4

  While many genes that have already been reported to cause abnormalities in hair follicle formation due to deficiency are up-regulated, genes that have no report related to hair follicles are related to hair shaft formation. It is a very surprising fact that the expression of is significantly enhanced in this way. It can be inferred that these genes are essential for hair shaft formation as well as those already reported.

  In a first aspect, the present invention provides a skin inspection method for predicting the hair growth / hair loss state of the skin. This method is a gene whose expression in the growing hair follicle in the skin is 5 or 10 times higher than that in the resting hair follicle, in particular, secretin in the skin, cytoplasmic poly A binding protein 1, eukaryotic translation. Initiation factor 5, ephrin receptor (EphA4), carbonic anhydrase 6, cathepsin E, gamma glutamyl transpeptidase, serine protease inhibitor 1 (alpha-1 antitrypsin 1, serpin 1 alpha), beta A4 crystallin, nucleoporin-like protein 2. High glycine / tyrosine protein type I E5, stimulated by retinoic acid gene 6, aquaporin 1, cytokeratin 12, lymphoid enhancing factor binding factor (LEF-1) ), Neuroblastoma myc-related oncogene 1, fork Headbox N1, connexin 30, connexin 26, tenascin C, syndecan 1, clusterin, peptidylarginine deiminase IV, small proline rich protein 1A, acidic keratin complex 1 gene 3, acidic keratin complex 1 gene 1, basic keratin complex Body gene 6g, acid keratin complex 1 gene 17, acid keratin complex 1 gene 19, runt-related transcription factor 1 (Runx1, AML1), cytoplasmic / activated T cell nuclear factor 3 (NFAT3), ephrin receptor ( EphB1), cytidine 5′-triphosphate synthase, differential display and activated by p53 (differential display and activated by p53), BMP-2 induced kinase, vitamin D receptor, P cadherin, stratifine, fin・ Protoon Cozy , When the gene encoding one or more proteins selected from the group consisting of transforming growth factor alpha, patched homolog 2, monoclonal antibody Ki67, and basonucrin is enhanced, and / or Or, it is judged that the skin is likely to form a sensible hair shaft, and if the expression of the gene is reduced, it is judged that the skin is likely to be depilated and / or easily thinned. .

  Preferably, the protein is secretin, cytoplasmic poly A binding protein 1, eukaryotic translation initiation factor 5, ephrin receptor (EphA4), carbonic anhydrase 6, cathepsin E, gamma glutamyl transpeptidase, serine protease inhibitor 1 (alpha-1 antitrypsin 1, serpin 1 alpha), beta A4 crystallin, nucleoporin-like protein 2, high glycine / tyrosine protein type I E5, stimulated by retinoic acid gene 6 (stimulated by retinoic acid gene 6), aquaporin 1, cytokeratin 12, runt-related transcription factor 1 (Runx1, AML1), cytoplasmic / activated T cell nuclear factor 3 (NFAT3), ephrin receptor (EphB1), cytidine 5'-3 Phosphate synthase, diffalene Turbocharger is Le display and activation dead by p53 (differential display and activated by p53) and / or BMP-2-induced kinase.

  Preferably, the enhanced expression of the gene in the skin is determined by measuring the amount of the protein in the skin. More preferably, this measurement is performed by ELISA or RIA using an antibody specific for the protein.

  The enhancement of the expression of the gene in the skin may be determined by measuring the amount of mRNA encoding the protein extracted from the skin. Preferably, the mRNA is measured by a polymerase chain reaction method.

  The present invention also provides a method of screening for a hair growth promoting factor and / or a hair loss inhibiting factor and / or a hair follicle thickening promoting factor. In this method, the candidate drug is evaluated for the ability to enhance the expression of the gene and / or the activity of the protein product of the gene, and the candidate drug having the enhancing ability is evaluated as a hair growth promoting factor and / or a hair loss inhibiting factor and / or The hair follicle thickening promoting factor is selected. Preferably, the candidate drug having the enhancement ability is further applied to a hair cycle synchronization model animal, and a candidate drug having an effect of promoting hair growth and / or suppressing hair loss and / or promoting thickening of hair follicles is selected.

  In another aspect, the present invention provides a method for screening a hair growth inhibitory factor and / or a hair loss promoting factor and / or a hair follicle thickening inhibitory factor. In this method, the candidate drug is evaluated for the ability to inhibit the expression of the gene and / or the activity of the protein product of the gene, and the candidate drug having the inhibitory ability is evaluated as a hair growth inhibitor and / or a hair loss promoting factor and / or It is selected as a factor for inhibiting hair follicle thickening. Preferably, the candidate drug having the inhibitory ability is further applied to a hair cycle synchronization model animal, and a candidate drug having an effect of suppressing hair growth and / or promoting hair loss and / or suppressing hair follicle thickening is selected.

  In a further aspect, the present invention provides one or more of acetazolamide, sulfanilamide, and methazolamide, which are inhibitors of carbonic anhydrase 6, whose expression in anagen hair follicles is increased 10-fold or more as compared to telogen hair follicles. Provided is a pharmaceutical or external composition for skin containing a hair suppressing factor and / or a hair loss promoting factor and / or a hair follicle thickening suppressing factor. Such a composition can be used as a hair removal agent by exerting an effect of suppressing hair growth, promoting hair loss and / or suppressing thickening of hair follicles.

  In a second aspect, the present invention is a skin test method for predicting the hair growth / hair loss state of the skin, which is a polynucleotide (181008K03Rik) comprising the base sequence shown in SEQ ID NO: 1 in the skin or homologous thereto. Hybridization under high stringency conditions to a high Cha (cation transport regulator) gene, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 (1110032D16Rik), or a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 (4733401H21Rik) When the expression of a possible polynucleotide is increased, it is judged that the skin is likely to grow hair and / or to form a sensible hair shaft, and when the expression of the gene is reduced, hair is easily removed And / or judge that the skin is prone to thinning A method characterized in that.

  Preferably, the enhanced expression of the polynucleotide in the skin is determined by measuring the amount of mRNA complementary to the polynucleotide extracted from the skin. Preferably, the mRNA is measured by a polymerase chain reaction method.

  The present invention further relates to a method for screening for a hair growth promoting factor and / or a hair loss inhibiting factor and / or a hair follicle thickening promoting factor, wherein the candidate drug is a polymorph comprising the base sequence shown in SEQ ID NO: 1 in the skin. A polynucleotide capable of hybridizing under high stringency conditions to a nucleotide (181008K03Rik), a polynucleotide (1110032D16Rik) comprising the nucleotide sequence shown in SEQ ID NO: 2 and / or a polynucleotide (473733401H21Rik) comprising the nucleotide sequence shown in SEQ ID NO: 3 A method characterized by evaluating the ability to enhance the expression of hair and selecting a candidate drug having the enhancement ability as a hair growth promoting factor and / or a hair loss inhibiting factor and / or a hair follicle thickening promoting factor To do. Preferably, a candidate drug having the above-mentioned promoting ability is further applied to a hair cycle synchronization model animal, and a candidate drug having an effect of promoting hair growth and / or suppressing hair loss and / or promoting thickening of hair follicles is selected.

  The present invention further relates to a method for screening a hair growth inhibitory factor and / or a hair loss promoting factor and / or a hair follicle thickening inhibitory factor, wherein the candidate drug is a polymorph comprising the base sequence shown in SEQ ID NO: 1 in the skin. A polynucleotide capable of hybridizing under high stringency conditions to a nucleotide (181008K03Rik), a polynucleotide (1110032D16Rik) comprising the nucleotide sequence shown in SEQ ID NO: 2 and / or a polynucleotide (473733401H21Rik) comprising the nucleotide sequence shown in SEQ ID NO: 3 A method is provided for evaluating the ability to inhibit the expression of hair and selecting a candidate drug having the inhibitory ability as a hair growth inhibitory factor and / or a hair loss promoting factor and / or a hair follicle thickening inhibitory factor. To do. Preferably, the candidate drug having the inhibitory ability is further applied to a hair cycle synchronization model animal, and a candidate drug having an effect of suppressing hair growth and / or promoting hair loss and / or suppressing hair follicle thickening is selected.

  Hybridization can be performed according to a well-known method or a method analogous thereto, for example, the method described in J. Sambrook et al., Molecular Cloning 2nd, Cold Spring Harbor Lab. Press, 1989, and high stringency hybridization conditions include, for example, The conditions include that the sodium concentration is about 10 to 40 mM, preferably about 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C.

  According to the present invention, it is possible to provide a skin inspection method for predicting the hair growth / hair removal state of the skin, a new hair growth agent, and a hair removal agent.

  As described above, there is no report regarding the follicle formation and hair shaft formation in the hair cycle with respect to the above genes. As a result of microarray analysis of RNA from the skin as a control containing many resting hair follicles before wax hair removal and the skin containing many growing hair follicles on the 13th day after wax hair removal, It was found that the expression of each of the above genes was specifically enhanced in skin containing a large amount. Therefore, it can be presumed that the ease of hair growth and the ease of hair removal can be predicted by using these genes as indices. Furthermore, it is possible to efficiently find a drug having a hair-growth effect and a drug having a hair removal effect using these genes as indices. The hair-growth effect here means hair growth promotion, for example, increasing the number of hairs, suppressing hair loss, thickening hair, and the like. In addition, the hair removal effect referred to here means suppression of hair growth on body hair other than head hair, eyebrows, eyelashes, for example, eyelids, armpit hair, limbs, etc., for example, reduction in the number of hairs that grow, promotion of hair loss, Means hair thinning and length reduction.

Hair cycle synchronized mouse The body hair of mice around 8 weeks of age is mainly in the resting period of the hair cycle. When this is removed with a hair removal wax, the hair rapidly regenerates from the rest period to the growth stage, and the hair is regenerated. At that time, the skin color changes from pink to gray blue. Hair growth starts around 9 days after the hair removal treatment, and hair shaft formation is completed around 13 days. The skin color of the mouse that has started to grow is changed from gray blue to pink again. Since there is not much individual difference in the hair cycle of mice, it is possible to synchronize the hair cycle among a plurality of mice by simultaneously performing such a dewaxing wax treatment on a plurality of mice (Japanese Patent Laid-Open No. 9-22214). . In the present invention, the skin containing many growing hair follicles was the skin of a mouse on the 13th day after wax hair removal treatment, and the skin containing many resting hair follicles was the skin immediately before the wax hair removal.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides a skin inspection method for predicting the hair growth / hair loss state of skin, preferably human skin. In this method, a gene whose expression in the growing hair follicle in the skin is 5 or 10 times higher than that in the resting hair follicle, that is, secretin, cytoplasmic poly A binding protein 1, eukaryotic translation initiation factor 5, ephrin receptor (EphA4), carbonic anhydrase 6, cathepsin E, gamma glutamyl transpeptidase, serine protease inhibitor 1 (alpha-1 antitrypsin 1, serpin 1 alpha), beta A4 crystallin, nucleoporin-like protein 2, High glycine / tyrosine protein type I E5, stimulated by retinoic acid gene 6, aquaporin 1, cytokeratin 12, lymphoid enhancing factor binding factor (LEF-1), Neuroblastoma myc-related oncogene 1, forkhead N1, Connexin 30, Connexin 26, Tenascin C, Syndecan 1, Clusterin, Peptidylarginine Deiminase IV, Small Proline Rich Protein 1A, Acid Keratin Complex 1 Gene 3, Acid Keratin Complex 1 Gene 1, Basic Keratin Complex Gene 6g, acid keratin complex 1 gene 17, acid keratin complex 1 gene 19, runt-related transcription factor 1 (Runx1, AML1), cytoplasmic / activated T cell nuclear factor 3 (NFAT3), ephrin receptor (EphB1) ), Cytidine 5′-triphosphate synthase, differential display and activated by p53, BMP-2 induced kinase, vitamin D receptor, P cadherin, stratifine, fin Proto-oncogene, trance When the expression of a gene selected from the group consisting of a gene encoding a protein selected from the group consisting of forming growth factor alpha, patched homolog 2 and monoclonal antibody Ki67 is increased compared to those in the control skin, hair growth occurs It is characterized by determining that the skin is easy to form an easy or sensible hair shaft. As an evaluation criterion, for example, the expression of the above gene in the skin is 10% or more, or 20% or more, or 30% or more, or 50% or more, or 70% or more, or 100% or more, higher than those in the control skin. If so, it may be determined that the skin is easy to grow hair or to form a sensible hair shaft. In addition, as an evaluation criterion, for example, the expression of the above-mentioned gene in the skin is 10% or more, or 20% or more, or 30% or more, or 50% or more, or 70% or more, or 100% compared to those in the control skin. If it has decreased more than this, it may be determined that “the skin is easy to remove hair or to be thinned easily”.

  The present invention also relates to a skin test method for predicting the hair growth / hair loss state of skin, preferably human skin, which is a polynucleotide (181008K03Rik) comprising the base sequence shown in SEQ ID NO: 1 in the skin or homologous thereto. High under high stringent conditions against a high Cha (cation transport regulator) gene, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 (1110032D16Rik), or a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 (4733401H21Rik) Provided is a method characterized in that when the expression of a polynucleotide capable of hybridization is enhanced as compared with the expression in normal skin, it is determined that the skin is susceptible to stain formation. As the evaluation criteria, for example, the expression of the above-mentioned polynucleotide in the skin is 10% or more, or 20% or more, or 30% or more, or 50% or more, or 70% or more, or 100% or more compared with those in the control skin. If it is enhanced, it may be determined that “the skin is easy to grow hair or to form a sensible hair shaft”. In addition, as an evaluation criterion, for example, the expression of the polynucleotide in the skin is 10% or more, or 20% or more, or 30% or more, or 50% or more, or 70% or more, or 100, compared with those in the control skin. If it has decreased by more than%, it may be determined that the skin is easy to remove hair or to be thinned easily.

  Hybridization can be performed according to a well-known method or a method analogous thereto, for example, the method described in J. Sambrook et al., Molecular Cloning 2nd, Cold Spring Harbor Lab. Press, 1989, and high stringency hybridization conditions include, for example, The conditions include that the sodium concentration is about 10 to 40 mM, preferably about 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C.

  The skin to be examined may be any part of the skin where hair growth or hair loss is a concern, such as the scalp, face, heels, arms, limbs. If you want to predict the ease of hair growth and the formation of a sensible hair shaft, you may use skin derived from the skin where hair follicles are not formed, such as palms and soles, as control skin. . Moreover, when it is desired to evaluate the ease of hair removal and the ease of thinning, it may be a portion where healthy hair is formed as a control, such as the temporal region and the back of the head.

  The increase or decrease of the gene in the skin is determined, for example, by measuring the amount of protein encoded by the gene in the skin. Preferably, this measurement uses an antibody specific for the protein, and is well known in the art, for example, an immunostaining method using a fluorescent substance, a dye, an enzyme, etc., a Western blot method, an immunoassay method such as an ELISA method. It can be carried out by various methods such as RIA method. It can also be determined by extracting RNA from the skin and measuring the amount of mRNA encoding the protein. Extraction of mRNA and measurement of the amount thereof are also well known in the art. For example, RNA is quantified by quantitative polymerase chain reaction (PCR).

  Expression of a polynucleotide capable of hybridizing to the gene in the skin under high stringency conditions can also be determined by extracting RNA from the skin and measuring the amount of mRNA corresponding to the polynucleotide. Extraction of mRNA and measurement of the amount thereof are also well known in the art. For example, RNA is quantified by quantitative polymerase chain reaction (PCR).

  As described above, as a result of microarray analysis of RNA derived from skin containing many vegetative hair follicles and skin containing many quiescent hair follicles, the present invention shows that the above genes are present in vegetative hair follicle skin compared to quiescent hair follicle skin. Based on the finding that the expression of is specifically enhanced.

  Therefore, it is presumed that by suppressing the expression of the above gene in the skin and / or the activity of the above protein as the gene product, it is possible to suppress hair growth, promote hair loss and / or suppress hair follicle thickening. The

  Accordingly, the present invention relates to a hair removal medicament comprising a compound containing a compound or herbal medicine that inhibits the expression of the gene as a hair growth inhibitory factor, a hair loss promoting factor and / or a hair follicle thickening inhibitory factor or An external composition for skin is provided. The composition according to the present invention can be effectively used for depilation and thinning of the skin.

  In another aspect, by promoting the expression of the gene in the skin and / or the activity of the protein, which is the gene product, it is possible to promote hair growth, suppress hair loss and / or promote thickening of the hair follicle. It is inferred.

  Accordingly, the present invention further provides a hair-growth medicament or skin comprising a drug containing a compound or herbal medicine that promotes the expression of the gene as a hair growth promoting factor, a hair loss promoting factor and / or a hair follicle thickening inhibiting factor. An external composition is provided. The composition according to the present invention can be effectively used for hair growth and thickening of the skin.

  The pharmaceutical or external composition for skin of the present invention is applied in the form of, for example, an aqueous solution, oil solution, other solution, emulsion, cream, gel, suspension, microcapsule, powder, granule, capsule, solid preparation and the like. After preparing into these forms by a conventionally known method, a lotion preparation, emulsion, cream, ointment, plaster, haptic agent, aerosol, water-oil two-layer system, water-oil-powder three-layer As a system, injection, internal preparation (tablet, powder, granule, pill, syrup, troche, etc.), suppository, etc., it can be applied, affixed, sprayed, injected, drunk, inserted into the body. The said composition contains the said compound and the chemical | medical agent containing a crude drug, for example, based on the composition whole quantity, for example, 0.001 mM-1M, Preferably it contains about 0.01-100 mM, More preferably, about 0.1-10 mM. Will.

  Among these dosage forms, external preparations for skin such as lotion preparations, emulsions, creams, ointments, plasters, haptics, aerosols are suitable dosage forms for the purpose of the present invention. The topical skin preparations listed here include pharmaceuticals, quasi-drugs (ointments, etc.), cosmetics [basic cosmetics such as facial cleansers, emulsions, creams, gels, essences (beauty serums), packs and masks; foundations Makeup cosmetics such as lipsticks; oral cosmetics, aromatic cosmetics, hair cosmetics, body cosmetics, and the like]. In particular, the pharmaceutical or external composition for skin of the present invention is suitable for application as a hair restorer or hair remover.

  In the pharmaceutical or skin external composition of the present invention, conventionally known excipients and fragrances, fats and oils, surfactants, preservatives, sequestering agents, water-soluble polymers, depending on the desired dosage form. , Powder components such as thickeners and pigments, UV protection agents, humectants, antioxidants, pH adjusters, cleaning agents, desiccants, emulsifiers and the like are appropriately blended. Furthermore, it is possible to mix | blend this other medicinal component with the pharmaceutical or external composition for skin within the range which does not impair the effect by the mixing | blending.

Method for screening hair growth promoting factor and / or hair loss inhibiting factor and / or hair follicle thickening promoting factor The present invention further provides hair growth promoting factor and / or hair loss inhibiting factor and / or hair follicle promoting hair thickening. Also provided are methods of screening for factors. In this method, a candidate drug is evaluated for its ability to promote the expression of the gene and / or activate the activity of the protein as its gene product, and the candidate drug having the ability is evaluated as a hair growth promoting factor and / or hair loss. It is selected as an inhibitory factor and / or a factor promoting hair thickening of hair follicles.

  In a preferred embodiment, the screening method further comprises applying the candidate drug having the inhibitory ability to a blotting model animal to obtain a candidate drug having an effect of promoting hair growth and / or suppressing hair loss and / or promoting hair follicle thickening. Comprising selecting.

  The step of confirming the effect of promoting hair growth and / or suppressing hair loss and / or promoting hair thickening of hair follicles can be carried out using this candidate drug using a model animal, for example, a hair cycle synchronized mouse. In addition to mice, various animals such as rats and rabbits can be used. In a preferred embodiment, after preparing a solution of this drug, for example, an aqueous solution, it is repeatedly applied to the skin of a model animal, and the presence or absence of the above effect can be determined by evaluating hair growth and hair thickening. it can.

Method for screening hair growth inhibitory factor and / or hair loss promoting factor and / or hair follicle thickening inhibitory factor The present invention further relates to a hair growth inhibiting factor and / or hair loss promoting factor and / or hair follicle inhibiting hair thickening. Also provided are methods of screening for factors. In this method, the candidate drug is evaluated for the ability to inhibit the expression of the gene and / or the activity of the protein that is the gene product thereof, and the candidate drug having the inhibitory ability is evaluated as a hair growth inhibitor and / or a hair loss promoting factor and It is selected as a hair follicle thickening inhibitory factor.
In a preferred embodiment, the screening method further comprises applying the candidate drug having the inhibitory ability to a blotting model animal to obtain a candidate drug having an effect of suppressing hair growth and / or promoting hair loss and / or suppressing hair follicle thickening. Comprising selecting.

  The step of confirming the effect of suppressing hair growth and / or promoting hair loss and / or suppressing thickening of hair follicles can be carried out using this candidate drug using a model animal, for example, a hair cycle synchronized mouse. In addition to mice, various animals such as rats and rabbits can be used. In a preferred embodiment, after preparing a solution of this drug, for example, an aqueous solution, it is repeatedly applied to the skin of a model animal, and the presence or absence of the above-described effect can be determined by evaluating hair loss and hair thinning.

  Hereinafter, the present invention will be described more specifically with specific examples. In addition, this invention is not limited by this.

Preparation of hair cycle synchronized mice Three C57BL mice (8 weeks old) three back skins (2 cm × 4 cm) were simultaneously subjected to hair removal treatment with a hair removal wax (SURGI-WAX) to prepare mice with synchronized hair cycles.

RNA collection from the skin The entire skin of the back of the mouse was collected, cut into 1 cm squares, frozen with liquid nitrogen, crushed, and extracted with ISOGEN (Nippon Gene, manufacturer recommended protocol), RNeasy (Qiagen) Purified by The skin of mice containing many growth follicles was subjected to wax hair removal treatment to obtain mouse-derived skin on the 13th day. As a control, mouse skin immediately before the wax hair removal treatment was used.

Microarray Sample Reaction Agilent cDNA Microarray Sample Sample preparation and hybridization were in accordance with Agilent recommended protocols. Labeled cDNA was synthesized from 10 μg of total RNA using a cDNA labeling kit (Agilent). After purification with Qiaquick (Qiagen, manufacturer recommended protocol), the solvent was dried with Speed Vac. Next, the slide glass was washed after hybridization using a mouse cDNA microarray kit (Agilent, G410A). Data reading was performed with an Agilent scanner.

Analysis result As a result of comprehensive analysis of the expression of about 9,000 kinds of genes, it was found that the expression of the following genes was significantly enhanced in the skin containing many growing hair follicles compared to the control skin. It was. Since the expression of these genes is enhanced in the skin containing many growing hair follicles, it can be predicted that hair shafts will be formed in the future by using these genes as an index. The results are shown in the following table.

Effect of Carbonic Anhydrase 6 Inhibitor on Hair Follicle Formation Each of five C57BL mice synchronized with the hair cycle was coated with 100 mM, 10 mM acetoazolamide and a solvent (dimethyl sulfoxide). Apply from the 5th day after wax hair removal, 100 μl once a day on the back, and hair follicle formation by measuring the skin darkness of the back skin on the 6th day (11 days after wax removal) with a mexameter. The degree was evaluated.

  The result is shown in FIG. From this result, it became clear that hair growth can be suppressed by delaying the initiation of hair growth by inhibiting the activity of carbonic anhydrase 6 with acetazolamide. Therefore, it was suggested that by suppressing the expression of a gene whose expression is enhanced in the growing hair follicle, it is possible to suppress hair growth promotion, promote hair loss and / or make hair thin.

Effect of carbonic anhydrase 6 inhibitor on hair follicle formation.

Claims (18)

  A skin test method for predicting the hair growth / hair loss state of the skin, including secretin in the skin, cytoplasmic poly A binding protein 1, eukaryotic translation initiation factor 5, ephrin receptor (EphA4), carbonic ann Hydrase 6, cathepsin E, gamma glutamyl transpeptidase, serine protease inhibitor 1 (alpha-1 antitrypsin 1, serpin 1 alpha), beta A4 crystallin, nucleoporin-like protein 2, high glycine / tyrosine protein type I E5, Stimu Rated by retinoic acid gene 6, aquaporin 1, cytokeratin 12, lymphoid enhancing factor binding factor (LEF-1), neuroblastoma myc-related oncogene 1, fork Headbox N1, connexin 30, connex 26, tenascin C, syndecan 1, clusterin, peptidyl arginine deiminase IV, small proline rich protein 1A, acidic keratin complex 1 gene 3, acidic keratin complex 1 gene 1, basic keratin complex gene 6g, acidic keratin complex Body 1 gene 17, acidic keratin complex 1 gene 19, runt-related transcription factor 1 (Runx1, AML1), nuclear factor 3 of cytoplasmic / activated T cells (NFAT3), ephrin receptor (EphB1), cytidine 5′- Triphosphate synthase, differential display and activated by p53, BMP-2 induced kinase, vitamin D receptor, P cadherin, stratifin, fin proto-oncogene, transforming growth Factor alpha, When a gene encoding one or a plurality of proteins selected from the group consisting of the humid homolog 2, the antigen identified by the monoclonal antibody Ki67, and bathonucrin is enhanced, it forms a hair shaft that is likely to grow and / or is wise. A method characterized in that it is determined that the skin is easy to skin, and when the expression of the gene is reduced, it is determined that the skin is easy to remove hair and / or to be thinned.   The protein is secretin, cytoplasmic poly A binding protein 1, eukaryotic translation initiation factor 5, ephrin receptor (EphA4), carbonic anhydrase 6, cathepsin E, gamma glutamyl transpeptidase, serine protease inhibitor 1 (alpha-1) Antitrypsin 1, Serpin 1 alpha), beta A4 crystallin, nucleoporin-like protein 2, high glycine / tyrosine protein type I E5, stimulated by retinoic acid gene 6 , Aquaporin 1, cytokeratin 12, runt-related transcription factor 1 (Runx1, AML1), nuclear factor 3 of cytoplasmic / activated T cells (NFAT3), ephrin receptor (EphB1), cytidine 5′-triphosphate synthase, Differential It is Ray and activation dead by p53 (differential display and activated by p53) and / or BMP-2-induced kinase, method of claim 1.   The method according to claim 1 or 2, wherein the enhanced expression of the gene in the skin is determined by measuring the amount of the protein in the skin.   The method according to claim 3, wherein the measurement is performed by ELISA or RIA using an antibody specific for the protein.   The method according to any one of claims 1 to 4, wherein the enhanced expression of the gene in the skin is determined by measuring the amount of mRNA encoding the protein extracted from the skin.   The method according to claim 5, wherein the mRNA is measured by a polymerase chain reaction method.   A method for screening a hair growth promoting factor and / or a hair loss inhibiting factor and / or a hair follicle thickening promoting factor, wherein the candidate drug is a gene expression and / or a protein of the gene defined in claim 1 or 2 A method characterized by evaluating the ability to enhance the activity of a product and selecting a candidate drug having the enhancement ability as a hair growth promoting factor and / or a hair loss inhibiting factor and / or a hair follicle thickening promoting factor.   The method further comprises applying the candidate drug having the enhancement ability to a hair cycle synchronization model animal and selecting a candidate drug having an effect of promoting hair growth and / or suppressing hair loss and / or promoting hair follicle thickening. The method of claim 7.   A method for screening a hair growth inhibitory factor and / or a hair loss promoting factor and / or a hair follicle thickening inhibitory factor, wherein a candidate drug is a gene expression and / or a protein of the gene as defined in claim 1 or 2 A method characterized by evaluating the ability to inhibit the activity of a product, and selecting a candidate drug having the inhibitory ability as a hair growth inhibitory factor and / or a hair loss promoting factor and / or a hair follicle thickening inhibitory factor.   The method further comprises applying the candidate drug having the inhibitory ability to a hair cycle synchronization model animal and selecting a candidate drug having an effect of suppressing hair growth and / or promoting hair loss and / or suppressing hair follicle thickening. 10. The method of claim 9.   A skin test method for predicting the hair growth / hair loss state of a skin, comprising a polynucleotide comprising a base sequence represented by SEQ ID NO: 1 in the skin (181008K03Rik) and a polynucleotide comprising a base sequence represented by SEQ ID NO: 2 (1110032D16Rik ) And / or when the expression of the polynucleotide capable of hybridizing to the polynucleotide (4733401H21Rik) comprising the base sequence shown in SEQ ID NO: 3 under high stringency conditions is increased, hair growth is likely and / or sensible. A method characterized by determining that the skin is easy to form a hair shaft, and, when expression of the gene is reduced, determining that the skin is easy to remove hair and / or to be thinned.   12. The method of claim 11, wherein the enhanced expression of the polynucleotide in the skin is determined by measuring the amount of mRNA complementary to the polynucleotide extracted from the skin.   The method according to claim 12, wherein the mRNA is measured by a polymerase chain reaction method.   A method for screening a hair growth promoting factor and / or a hair loss inhibiting factor and / or a hair follicle thickening promoting factor, wherein the candidate drug is a polynucleotide comprising the base sequence represented by SEQ ID NO: 1 in the skin (181008K03Rik), Increases the expression of polynucleotides capable of hybridizing under high stringency conditions to the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 (1110032D16Rik) and / or the polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 (4733401H21Rik) A method comprising evaluating the ability and selecting a candidate drug having the enhancement ability as a hair growth promoting factor and / or a hair loss inhibiting factor and / or a hair follicle thickening promoting factor.   The method further comprises applying the candidate drug having the promoting ability to a hair cycle synchronization model animal, and selecting a candidate drug having an effect of promoting hair growth and / or suppressing hair loss and / or promoting hair follicle thickening. The method of claim 14.   A method for screening a hair growth inhibitory factor and / or a hair loss promoting factor and / or a hair follicle thickening inhibitory factor, wherein the candidate drug is a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 in the skin (181008K03Rik), Inhibits the expression of polynucleotides capable of hybridizing under high stringency conditions to the polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 (1110032D16Rik) and / or the polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 (4733401H21Rik) A method comprising evaluating the ability and selecting a candidate drug having the inhibition ability as a hair growth inhibitory factor and / or a hair loss promoting factor and / or a hair follicle thickening inhibitory factor.   The method further comprises applying the candidate drug having the inhibitory ability to a hair cycle synchronized model animal and selecting a candidate drug having an effect of suppressing hair growth and / or promoting hair loss and / or suppressing hair follicle thickening. The method of claim 16.   A hair removal composition comprising one or more of acetoazolamide, sulfanilamide, and metazolamide as a hair growth inhibitory factor and / or a hair loss promoting factor and / or a hair follicle thickening inhibitory factor.

Images