JP2012008045A - Drug evaluation method - Google Patents
Drug evaluation method Download PDFInfo
- Publication number
- JP2012008045A JP2012008045A JP2010145179A JP2010145179A JP2012008045A JP 2012008045 A JP2012008045 A JP 2012008045A JP 2010145179 A JP2010145179 A JP 2010145179A JP 2010145179 A JP2010145179 A JP 2010145179A JP 2012008045 A JP2012008045 A JP 2012008045A
- Authority
- JP
- Japan
- Prior art keywords
- hair
- evaluation
- white hair
- white
- model animal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
本発明は薬剤評価方法に関する。更に詳しくは本発明は、体毛の白毛割合が経時変化する白髪モデル動物に供試薬剤を適用し、その白髪モデル動物の白毛評価によって前記供試薬剤の白毛制御効果を評価する薬剤評価方法に関する。
〔定義〕
本発明において、「体毛」とはヒト及び動物における各種の体毛を包含する概念であり、体毛を単に「毛」ということもある。「毛髪」とは、いわゆる頭髪、即ち、頭部における顔面部以外の部分に生える体毛をいう。ヒトの体毛として、毛髪、口髭、顎鬚、眉毛、睫毛の他に、腕・脚・胸に生える毛を好ましく例示することができる。毛髪、口髭、顎鬚、眉毛、睫毛を更に好ましく例示することができ、毛髪、口髭、顎鬚を特に好ましく例示することができる。
The present invention relates to a drug evaluation method. More specifically, the present invention relates to a drug evaluation in which a reagent is applied to a white hair model animal in which the white hair ratio of body hair changes over time, and the white hair control effect of the reagent is evaluated by evaluating the white hair of the white hair model animal. Regarding the method.
[Definition]
In the present invention, the “body hair” is a concept including various body hairs in humans and animals, and the body hair may be simply referred to as “hair”. “Hair” refers to so-called hair, that is, body hair that grows on the head other than the face. As human body hair, in addition to hair, mustache, beard, eyebrows, and eyelashes, hair that grows on arms, legs, and chests can be preferably exemplified. Hair, mustache, beard, eyebrows and eyelashes can be more preferably exemplified, and hair, mustache and beard can be particularly preferably exemplified.
また、「白毛」とはメラニン色素を有しない体毛をいい、「白髪」とはメラニン色素を有しない毛髪をいう。これに対して、例えば「黒毛」とはメラニン色素を有し黒色を呈する体毛をいい、「黒髪」とはメラニン色素を有し黒色を呈する毛髪をいう。なお、薬剤評価用のモデル動物については、一般的な称呼に従い「白髪モデル動物」と呼ぶが、本発明において実質的には「白毛モデル動物」の意味である。 Further, “white hair” refers to body hair that does not have a melanin pigment, and “white hair” refers to hair that does not have a melanin pigment. On the other hand, for example, “black hair” refers to body hair that has a melanin pigment and exhibits black color, and “black hair” refers to hair that has a melanin pigment and exhibits black color. The model animal for drug evaluation is referred to as a “white hair model animal” in accordance with a general designation, but in the present invention, it is substantially a meaning of “white hair model animal”.
メラニン色素を有し黒色、褐色、赤色、栗色等の色を呈する体毛を持つヒトの加齢に伴う白毛の発生は、美容上、外見上の大きな関心事であり、有効な白毛予防・治療剤が求められている。反面、中途半端に白毛化した状態（例えば毛髪における中途半端な白髪化による「ごましお頭」の状態や、口髭、顎鬚等における同様な状態）を嫌う心理から、白毛が多くなって来た時点で真っ白な美しい体毛への移行を促進する白毛促進剤が求められる場合もある。 The occurrence of white hair with aging in humans with melanin pigments and black, brown, red, chestnut-colored hair is a major cosmetic and cosmetic concern, and effective white hair prevention / There is a need for therapeutic agents. On the other hand, the amount of white hair has increased due to the psychology of disabling white hair (for example, “gummy head” due to half-hair whitening in hair, and similar conditions in mustache, beard, etc.). In some cases, a white hair promoter that promotes the transition to pure white and beautiful hair may be required.
そのため、従来より種々の化合物、組成物、抽出物等を用いた白毛制御剤（白毛予防・治療剤又は白毛促進剤）が提案されているが、白毛制御剤の開発に関連する技術として、白毛制御剤をスクリーニングするための評価方法も種々に提案されている。 For this reason, white hair control agents (white hair prevention / treatment agents or white hair promoters) using various compounds, compositions, extracts and the like have been proposed, but are related to the development of white hair control agents. As a technique, various evaluation methods for screening a white hair control agent have been proposed.
供試薬剤の白毛制御効果を評価する場合において、通常に考えられる直接的な指標として「白毛率」を挙げることができる。白毛率とは、検体である一定量の体毛中の毛を１本ずつ顕微鏡等で観察して、検体の全体毛中の白毛の比率（％）を算出するものである。例えばマウスの場合、体毛のメジュラー中にメラニンが見られるか否かで黒毛か白毛かを判定すれば、信頼性の高い結果が得られる。 In evaluating the white hair control effect of the reagent, a “white hair ratio” can be mentioned as a direct index that is usually considered. The white hair ratio is to calculate the ratio (%) of white hair in the whole hair of the specimen by observing hair in a certain amount of body hair as a specimen one by one with a microscope or the like. For example, in the case of a mouse, a highly reliable result can be obtained by determining whether black hair or white hair is based on whether or not melanin is found in the body hairs.
しかし、白毛率の算出には手間と時間がかかり、評価の迅速性に欠けるという欠点がある。例えば、１検体の白毛率を算出するために１時間程度も要する。又、この指標は、体毛の白毛化を結果として追認するものであって、評価時点以後の白毛化の進行に対する予測性を持たない。更にこの指標は、検体中の体毛１本ずつのミクロな観察結果の積上げに基づくものであり、検体中の全ての体毛をマスとして捉えた指標ではないから、通常の肉眼的な全体観察による白毛化体毛の外観的印象とは必ずしも一致しない。 However, calculation of the white hair rate takes time and effort, and there is a drawback that evaluation is not quick. For example, it takes about one hour to calculate the white hair rate of one specimen. Moreover, this index confirms the whitening of the body hair as a result, and has no predictability with respect to the progress of whitening after the evaluation time point. Furthermore, this index is based on the accumulation of microscopic observation results for each body hair in the sample, and is not an index that captures all body hair in the sample as a mass. It does not necessarily coincide with the appearance of hair on the hair.
白毛制御剤のその他の評価方法として、例えば以下の特許文献１〜特許文献３に開示されたものが挙げられる。これらの薬剤評価方法には、ヒト等の体毛の成長に関連する部分の組織あるいは細胞を採取して評価に利用する方法、ヒト等の体毛を集めて直接に観察する方法、白毛の発生に関して特定の表現形質を持つマウス等のモデル動物を利用する方法等が含まれる。 Examples of other evaluation methods for the white hair control agent include those disclosed in the following Patent Documents 1 to 3. These drug evaluation methods include methods for collecting and using tissue and cells of parts related to growth of human hair such as humans, collecting human hair such as humans and directly observing them, and generation of white hair A method using a model animal such as a mouse having a specific phenotype is included.
しかし、特許文献１〜特許文献３に開示された薬剤評価方法も、必ずしも十分に満足できるものではなかった。そこで本発明は、有効な白毛制御剤をスクリーニングできる新たな薬剤評価方法を提供することを、解決すべき技術的課題とする。 However, the drug evaluation methods disclosed in Patent Documents 1 to 3 are not always satisfactory. Therefore, an object of the present invention is to provide a new drug evaluation method capable of screening an effective white hair control agent.
（第１発明）
上記課題を解決するための本願第１発明の構成は、体毛の白毛割合が経時変化する白髪モデル動物に供試薬剤を適用し、前記白髪モデル動物の白毛評価によって前記供試薬剤の白毛制御効果を評価する方法であって、
前記白毛評価を以下（ａ）〜（ｃ）のいずれか１以上の評価指標に基づいて行う、薬剤評価方法である。
(First invention)
The configuration of the first invention of the present application for solving the above problem is that a reagent agent is applied to a white hair model animal in which the white hair ratio of body hair changes over time, and the white color of the reagent agent is determined by evaluating the white hair of the white hair model animal. A method for evaluating the hair control effect,
It is a medicine evaluation method in which the white hair evaluation is performed based on any one or more of the evaluation indices (a) to (c) below.
（ａ）白髪モデル動物の体毛の測色値。 (A) Colorimetric values of body hair of gray hair model animals.
（ｂ）白髪モデル動物における体毛発生部分の皮膚組織の観察結果。 (B) The observation result of the skin tissue of the hair generation part in the gray hair model animal.
（ｃ）白髪モデル動物の体毛におけるメラニン量の測定値。 (C) The measured value of the amount of melanin in the body hair of a gray hair model animal.
この明細書において、「白髪モデル動物」にヒトは含まれない。又、「体毛の白毛割合が経時変化する」とは、長期にわたり徐々に体毛の白毛割合が増大する場合から、極めて短期間に一挙に白毛割合が増大する場合までを含む。又、（ｂ）でいう「体毛発生部分の皮膚組織」とは、少なくとも皮膚の体毛発生部分である毛胞（毛根）部分を含む皮膚組織を言う。 In this specification, “white hair model animals” do not include humans. Further, “the white hair ratio of the body hair changes with time” includes from the case where the white hair ratio of the body hair gradually increases over a long period of time to the case where the white hair ratio increases all at once in a very short time. In addition, the “skin tissue of the hair generation part” referred to in (b) refers to a skin tissue including at least a hair follicle (hair root) part that is a body hair generation part of the skin.
（第２発明）
上記課題を解決するための本願第２発明の構成は、前記第１発明に係る薬剤評価方法において、（ａ）の測色値が、L＊a＊b＊表色系の明度指数L＊の測色値である、薬剤評価方法である。
(Second invention)
The structure of the second invention of the present application for solving the above problem is that, in the medicine evaluation method according to the first invention, the colorimetric value of (a) is the lightness index L * of the L * a * b * color system. It is a drug evaluation method that is a colorimetric value.
（第３発明）
上記課題を解決するための本願第３発明の構成は、前記第１発明又は第２発明に係る薬剤評価方法において、（ｂ）の体毛発生部分の皮膚組織の観察結果が、毛胞部分の色素細胞生産組織における色素幹細胞数である、薬剤評価方法である。
(Third invention)
The structure of the third invention of the present application for solving the above-described problem is that, in the drug evaluation method according to the first invention or the second invention, the observation result of the skin tissue of the hair generation part of (b) is a pigment of the hair follicle part. It is a drug evaluation method which is the number of pigment stem cells in a cell producing tissue.
この第３発明において、色素幹細胞数は、単一の毛胞（毛根）部分の色素細胞生産組織における色素幹細胞数のカウント数を用いても良いが、統計的な信頼性の点から、好ましくは複数ケ所（例えば７０箇所程度）の毛胞部分についての色素幹細胞数の合計値を用い、あるいはその合計値を毛胞部分の箇所数で除した平均値を用いることが、より好ましい。 In this third invention, the number of pigment stem cells may be the count of the number of pigment stem cells in the pigment cell-producing tissue of a single hair follicle (follicle) part, but from the viewpoint of statistical reliability, It is more preferable to use the total value of the number of pigment stem cells for a plurality of follicular portions (for example, about 70 locations) or use an average value obtained by dividing the total value by the number of follicular portions.
（第４発明）
上記課題を解決するための本願第４発明の構成は、前記第３発明に係る薬剤評価方法において、色素幹細胞数を計数するに当たり、白髪モデル動物の皮膚組織を採取し、ｃ−ｋｉｔとｄｃｔの二重染色により免疫組織染色し、以下（１）〜（３）の全ての条件を満たす細胞を色素幹細胞と認定する、薬剤評価方法である。
(Fourth invention)
The structure of the fourth invention of the present application for solving the above-mentioned problem is that, in the drug evaluation method according to the third invention, when counting the number of pigment stem cells, the skin tissue of a white hair model animal is collected, and c-kit and dct This is a drug evaluation method in which immunohistochemical staining is performed by double staining, and cells satisfying all of the following conditions (1) to (3) are recognized as pigment stem cells.
（１）体毛バルジ領域に存在する細胞である。 (1) Cells present in the hair bulge region.
（２）ｄｃｔの発現状態によって色素細胞であると判定できる。 (2) It can be determined that it is a pigment cell by the expression state of dct.
（３）ｃ−ｋｉｔの発現状態によって未分化細胞であると判定できる。 (3) It can be determined that the cell is an undifferentiated cell depending on the expression state of c-kit.
この第４発明において、「ｃ−ｋｉｔとｄｃｔの二重染色」とは、実際には「ｃ−ｋｉｔの遺伝子産物（タンパク質）とｄｃｔの遺伝子産物（タンパク質）との二重染色」を意味する。「ｄｃｔの発現状態によって」とは、ｄｃｔの発現産物であるタンパク質が認められる状態を言う。「ｃ−ｋｉｔの発現状態によって」とは、ｃ−ｋｉｔの発現産物であるタンパク質が全くあるいは実質的に認められない状態を言う。 In this fourth invention, “double staining of c-kit and dct” actually means “double staining of c-kit gene product (protein) and dct gene product (protein)”. . “Depending on the expression state of dct” refers to a state in which a protein that is an expression product of dct is recognized. “Depending on the expression state of c-kit” refers to a state where no or substantially no protein which is an expression product of c-kit is observed.
（第５発明）
上記課題を解決するための本願第５発明の構成は、前記第３発明又は第４発明に係る薬剤評価方法において、明度指数L＊の測色値を、この測色値と第３発明又は第４発明に記載の色素幹細胞数の計数値との相関に基づいて色素幹細胞数に換算し、この色素幹細胞数の換算値を評価指標として白毛評価を行う、薬剤評価方法である。
(Fifth invention)
The structure of the fifth invention of the present application for solving the above problem is that, in the medicine evaluation method according to the third or fourth invention, the colorimetric value of the lightness index L * 4 A drug evaluation method in which the number of pigment stem cells is converted based on the correlation with the count value of the number of pigment stem cells described in the invention, and white hair is evaluated using the converted value of the number of pigment stem cells as an evaluation index.
（第６発明）
上記課題を解決するための本願第６発明の構成は、前記第３発明又は第４発明に係る薬剤評価方法において、一定数の体毛の内の白毛の比率を視覚的観察により計数して得られる白毛率を、この白毛率と第３発明又は第４発明に記載の色素幹細胞数の計数値との相関に基づいて色素幹細胞数に換算し、この色素幹細胞数の換算値を評価指標として白毛評価を行う、薬剤評価方法である。
(Sixth invention)
The structure of the sixth invention of the present application for solving the above-mentioned problem is obtained by counting the ratio of white hair in a certain number of body hairs by visual observation in the drug evaluation method according to the third or fourth invention. The white hair rate obtained is converted into the number of pigment stem cells based on the correlation between the white hair rate and the count value of the number of pigment stem cells according to the third or fourth invention, and the converted value of the number of pigment stem cells is used as an evaluation index. It is a drug evaluation method for performing white hair evaluation.
（第７発明）
上記課題を解決するための本願第７発明の構成は、前記第２発明〜第６発明のいずれかに係る薬剤評価方法において、明度指数L＊の測色値を、この測色値と第１発明の（ｃ）に記載のメラニン量測定値との相関に基づいてメラニン量に換算し、このメラニン量の換算値を評価指標として白毛評価を行う、薬剤評価方法である。
(Seventh invention)
The structure of the seventh invention of the present application for solving the above problem is that in the medicine evaluation method according to any one of the second to sixth inventions, the colorimetric value of the lightness index L * is the first colorimetric value and the first colorimetric value. It is a drug evaluation method for converting to a melanin amount based on the correlation with the measured melanin amount described in (c) of the invention, and performing white hair evaluation using the converted value of the melanin amount as an evaluation index.
（第８発明）
上記課題を解決するための本願第８発明の構成は、前記第６発明又は第７発明に係る薬剤評価方法において、明度指数L＊の測色値を、この測色値と白毛率との相関に基づいて白毛率に換算し、この白毛率の換算値を評価指標として白毛評価を行う、薬剤評価方法である。
(Eighth invention)
The structure of the eighth invention of the present application for solving the above-described problem is that, in the medicine evaluation method according to the sixth invention or the seventh invention, the colorimetric value of the lightness index L * is calculated between the colorimetric value and the white hair ratio. This is a drug evaluation method in which the white hair rate is converted based on the correlation and white hair evaluation is performed using the converted value of the white hair rate as an evaluation index.
（第９発明）
上記課題を解決するための本願第９発明の構成は、前記第５発明〜第８発明のいずれかに係る薬剤評価方法において、ファクターＡとしての測色値あるいは白毛率と、ファクターＢとしての色素幹細胞数、メラニン量あるいは白毛率との関係（ファクターＡ、ファクターＢが共に白毛率である場合を除く）を表す一次回帰直線を予め作成し、これらの一次回帰直線を用いて、前記換算値をファクターＡからファクターＢへの換算として求める、薬剤評価方法である。
(9th invention)
The structure of the ninth invention of the present application for solving the above-described problem is that, in the medicine evaluation method according to any of the fifth to eighth inventions, the colorimetric value or white hair ratio as factor A and the factor B A primary regression line representing the relationship between the number of pigment stem cells, the amount of melanin or the white hair ratio (except when both factor A and factor B are white hair ratios) is prepared in advance, and using these primary regression lines, This is a drug evaluation method in which a conversion value is obtained as conversion from factor A to factor B.
（第１０発明）
上記課題を解決するための本願第１０発明の構成は、前記第１発明〜第９発明のいずれかに係る薬剤評価方法において、薬剤評価方法が、
（イ）毛周期（体毛サイクル）が休止期間中の白髪モデル動物の体毛の白毛評価を行う段階と、
（ロ）白髪モデル動物の体毛を除去する段階と、
（ハ）白髪モデル動物に対し、供試薬剤を１回以上適用する段階と、
（ニ）供試薬剤の適用後に発毛した時点で白毛評価を行う段階と、
を含む、薬剤評価方法である。
(10th invention)
The structure of the tenth invention of the present application for solving the above problems is the drug evaluation method according to any one of the first to ninth inventions, wherein the drug evaluation method comprises:
(A) performing a white hair evaluation of a white hair model animal body hair during which the hair cycle (body hair cycle) is resting;
(B) removing the hair of the gray hair model animal;
(C) applying the reagent to the gray hair model animal at least once;
(D) a stage of white hair evaluation at the time of hair growth after application of the reagent;
It is a medicine evaluation method including these.
なお、この第１０発明において、白髪モデル動物の体毛を毛刈りして白毛評価を行う場合には、（イ）の段階及び（ニ）の段階において、（ロ）に規定する「白髪モデル動物の体毛の除去」も行うことになる。 In the tenth aspect of the invention, when white hair evaluation is performed by trimming the body hair of a white hair model animal, the “white hair model animal” defined in (b) is provided in the steps (a) and (d). The removal of body hair ”is also performed.
（第１１発明）
上記課題を解決するための本願第１１発明の構成は、前記第１０発明に係る薬剤評価方法において、（イ）の段階を経た白髪モデル動物に対して、（ロ）〜（ニ）の段階からなるサイクルを複数サイクル行う、薬剤評価方法である。
(11th invention)
The constitution of the eleventh invention of the present application for solving the above-mentioned problem is that, in the drug evaluation method according to the tenth invention, from the stage of (b) to (d) for the gray hair model animal that has undergone the stage of (b) This is a drug evaluation method in which a plurality of cycles are performed.
（第１２発明）
上記課題を解決するための本願第１２発明の構成は、前記第１０発明又は第１１発明に係る薬剤評価方法において、薬剤の白毛制御効果の評価を、（イ）の段階で得られた白毛評価の評価指標と、（ロ）〜（ニ）の段階からなるサイクルを１サイクル又は複数サイクル行う場合における（ニ）の段階で得られた白毛評価の評価指標との比較によって行う、薬剤評価方法である。
(Twelfth invention)
The structure of the twelfth invention of the present application for solving the above-described problem is that, in the drug evaluation method according to the tenth invention or the eleventh invention, the white hair control effect of the drug is evaluated at the stage (a). A drug that is obtained by comparing the evaluation index of hair evaluation with the evaluation index of white hair evaluation obtained in the stage (d) in the case of performing one cycle or a plurality of cycles consisting of the stages (b) to (d) It is an evaluation method.
この第１２発明において、「（ニ）の段階で得られた白毛評価の評価指標」とは、（ロ）〜（ニ）の段階からなるサイクルを複数サイクル行う場合においては、その各サイクルにおける（ニ）の段階で得られた白毛評価の評価指標を包含し、かつ、この場合における「評価指標との比較」とは、複数回の（ニ）の段階で得られた評価指標の相互比較も含まれる。 In the twelfth aspect of the present invention, the “evaluation index for white hair evaluation obtained in the stage (d)” means that when a plurality of cycles consisting of the stages (b) to (d) are performed, The evaluation index of white hair evaluation obtained in the stage (d) is included, and in this case “comparison with the evaluation index” means that the evaluation index obtained in the stage (d) multiple times A comparison is also included.
（第１３発明）
上記課題を解決するための本願第１３発明の構成は、前記第１発明〜第１２発明のいずれかに係る薬剤評価方法において、白髪モデル動物に対する供試薬剤の適用を、白髪モデル動物の皮膚への塗布、皮下注射、皮内注射、吸入投与及び経口投与から選ばれる一つ以上の形態で行う、薬剤評価方法である。
(13th invention)
The structure of the thirteenth invention of the present application for solving the above-described problem is that, in the drug evaluation method according to any one of the first to twelfth inventions, application of a reagent to a white hair model animal is applied to the skin of the white hair model animal. Is a drug evaluation method performed in one or more forms selected from application, subcutaneous injection, intradermal injection, inhalation administration and oral administration.
（第１４発明）
上記課題を解決するための本願第１４発明の構成は、前記第１発明〜第１３発明のいずれかに係る薬剤評価方法において、供試薬剤の白毛制御効果の評価が、該供試薬剤の白毛抑制効果を評価するものである、薬剤評価方法である。
(14th invention)
The structure of the fourteenth invention of the present application for solving the above-mentioned problem is that, in the drug evaluation method according to any one of the first to thirteenth inventions, the evaluation of the white hair control effect of the reagent agent is performed. It is a drug evaluation method for evaluating the effect of suppressing white hair.
（第１５発明）
上記課題を解決するための本願第１５発明の構成は、前記第１発明〜第１３発明のいずれかに係る薬剤評価方法において、供試薬剤の白毛制御効果の評価が、該供試薬剤の白毛促進効果を評価するものである、薬剤評価方法である。
(15th invention)
The structure of the fifteenth aspect of the present invention for solving the above-described problem is that, in the drug evaluation method according to any one of the first to thirteenth aspects, the white hair control effect of the reagent is evaluated by the reagent. It is a drug evaluation method for evaluating the effect of promoting white hair.
（第１６発明）
上記課題を解決するための本願第１６発明の構成は、前記第１発明〜第１５発明のいずれかに係る薬剤評価方法において、白髪モデル動物がマウスである、薬剤評価方法である。
(16th invention)
The configuration of the sixteenth invention of the present application for solving the above problem is a drug evaluation method in which the gray hair model animal is a mouse in the drug evaluation method according to any one of the first to fifteenth inventions.
体毛の白毛割合が経時変化する白髪モデル動物を用いて供試薬剤の白毛制御効果を評価するという方法自体は公知である。白髪モデル動物に対する供試薬剤の適用はヒトに適用した場合と類似した評価結果を期待できるし、ヒトに適用する場合に懸念される人体への負担や危険を回避できる。更に、小型で、評価に適切な体毛サイクルを有し、管理が容易な白髪モデル動物を用いることによって、評価時間を短縮できるというメリットもある。本願発明者は、白髪モデル動物を用いて供試薬剤の白毛制御効果を評価するに当たり、白毛評価を第１発明の（ａ）〜（ｃ）の評価指標に基づいて行うことが特に有効であることを見出した。 A method of evaluating the white hair control effect of the reagent using a white hair model animal in which the white hair ratio of body hair changes with time is known. The application of the reagent to the gray hair model animal can be expected to give an evaluation result similar to that applied to humans, and the burden and danger to the human body, which is a concern when applied to humans, can be avoided. Furthermore, there is an advantage that the evaluation time can be shortened by using a white hair model animal that is small, has a hair cycle suitable for evaluation, and is easy to manage. In evaluating the white hair control effect of the reagent using the white hair model animal, the present inventor is particularly effective to perform the white hair evaluation based on the evaluation indices of (a) to (c) of the first invention. I found out.
第１発明における白毛評価の第１の評価指標である「（ａ）白髪モデル動物の体毛の測色値」は、毛刈りした体毛を束にして光学的測色系で測定して、あるいは毛刈りしていない白髪モデル動物の体毛を直接に光学的測色系で測定して、得られる指標である。この測色値のうち、特に、第２発明に規定するL＊a＊b＊表色系の明度指数L＊の測色値が好ましく、かつ非常に有用である。このような測色値は、１検体当たり僅か数秒程度で測定できるので、簡便かつ迅速な評価が可能である。又、検体中の全体毛をマスとして測定する指標であるため、通常の肉眼的な全体観察による白毛化体毛の外観印象と良好に対応した白毛評価が可能となる。しかも、信頼性の高い評価指標である白毛率と非常に高い相関を示す。 The “(a) colorimetric value of the body hair of a white hair model animal”, which is the first evaluation index for the white hair evaluation in the first invention, is measured by an optical colorimetry system with bundled hairs or This is an index obtained by directly measuring the body hair of an unshaved gray hair model animal using an optical colorimetry system. Among these colorimetric values, in particular, the colorimetric value of the lightness index L * of the L * a * b * color system defined in the second invention is preferable and very useful. Since such a colorimetric value can be measured in only a few seconds per sample, simple and quick evaluation is possible. Further, since it is an index for measuring the whole hair in the sample as a mass, it is possible to evaluate the white hair corresponding to the appearance impression of the whitened body hair by normal macroscopic overall observation. Moreover, it shows a very high correlation with the white hair rate, which is a highly reliable evaluation index.
第１発明における白毛評価の第２の評価指標である「（ｂ）白髪モデル動物における体毛発生部分の皮膚組織の観察結果」は、少なくとも毛胞部分を含む皮膚組織の観察により得られるものである。このような体毛発生部分の皮膚組織の観察結果としては、特に、第３発明に規定する色素幹細胞数の計数が好ましい。色素幹細胞数の計数値を得るに当たり、個々の細胞が色素幹細胞であるか否かを認定する方法としては、第４発明に規定する方法が特に好ましい。なぜなら、（１）体毛バルジ領域に存在し、（２）色素細胞であり、かつ（３）未分化細胞（幹細胞）であれば、体毛発生部分の色素幹細胞であると判定できるからである。色素幹細胞数も白毛率と高い相関を示し、白毛評価の評価指標としての信頼性が高い。 “(B) Observation result of skin tissue of hair generation part in white hair model animal” which is the second evaluation index of white hair evaluation in the first invention is obtained by observing skin tissue including at least the hair follicle part. is there. As an observation result of the skin tissue of such a hair generation part, counting of the number of pigment stem cells defined in the third invention is particularly preferable. In obtaining the count value of the number of pigment stem cells, the method defined in the fourth invention is particularly preferable as a method for determining whether or not each cell is a pigment stem cell. This is because it can be determined that a pigment stem cell is a pigmented stem cell of the hair generation part if it is (1) present in the hair bulge region, (2) is a pigment cell, and (3) is an undifferentiated cell (stem cell). The number of pigment stem cells is also highly correlated with the white hair rate, and is highly reliable as an evaluation index for white hair evaluation.
色素幹細胞数は非常にユニークな評価指標である。即ち、色素幹細胞が体毛のメラニンを供給する色素細胞に分化した後、毛母に移動すると考えられるから、色素幹細胞数は、通常の白毛評価だけではなく、評価時点以後の白毛化の進行に対する予測や、外観的に未だ生じていない白毛化の発生時期に対する予測ができる可能性を秘めていると考えられる。 The number of pigment stem cells is a very unique evaluation index. That is, it is considered that pigment stem cells differentiate into pigment cells that supply hair melanin and then move to the hair matrix. Therefore, the number of pigment stem cells is not only evaluated for normal white hair, but also the progress of whitening after the evaluation time point It is considered that there is a possibility that it is possible to predict the occurrence of whitening that has not yet occurred in appearance.
更には、白毛化の亢進は毛周期（体毛サイクル）の回数と相関があるので、色素幹細胞数から体毛年齢が分かる。 Furthermore, since the enhancement of whitening correlates with the number of hair cycles (body hair cycle), the age of the hair is known from the number of pigment stem cells.
一方、色素幹細胞は表皮にも移動して皮膚のメラニンを供給する色素細胞に分化すると考えられるから、白毛評価とは直接の関係はないが、皮膚が外傷を受けた際の皮膚色の再生を予測し、又はメラノーマの発生を予測する指標ともなり得る。 On the other hand, pigment stem cells also move to the epidermis and are thought to differentiate into pigment cells that supply melanin in the skin, so there is no direct relationship with white hair evaluation, but skin color reproduction when the skin is injured , Or an index for predicting the occurrence of melanoma.
第１発明における白毛評価の第３の評価指標である「（ｃ）白髪モデル動物の体毛におけるメラニン量の測定値」は、検体中の全ての体毛に含まれるメラニン量をマスとして測定する指標であるため、通常の肉眼的な全体観察による白毛化体毛の外観と良好に対応した白毛評価が可能となる。その信頼性も高いことが確認されている。この（ｃ）のメラニン量の測定値は、例えば回収した体毛を１０分間アルカリ処理して溶解させ、その溶解液中のメラニン量を吸光度計にて測定するという手順によって測定することができるが、このような測定方法に限定されるものではない。 “(C) Measured value of melanin content in body hair of white hair model animal”, which is the third evaluation index of white hair evaluation in the first invention, is an index for measuring the amount of melanin contained in all body hairs in a specimen as a mass. Therefore, it is possible to evaluate white hair that corresponds well with the appearance of whitened hair by general macroscopic overall observation. It has been confirmed that its reliability is also high. The measured value of the amount of melanin in (c) can be measured by a procedure of, for example, dissolving the collected body hair by alkali treatment for 10 minutes and measuring the amount of melanin in the solution with an absorptiometer, It is not limited to such a measuring method.
更に本願発明者は、前記したように明度指数L＊の測色値と白毛率との間だけでなく、明度指数L＊の測色値と色素幹細胞数の計数値との間、白毛率と色素幹細胞数の計数値との間、明度指数L＊の測色値とメラニン量測定値との間にもそれぞれ、非常に高い相関があることを見出した。従って第５発明〜第９発明に規定するように、これらの指標間の相関に基づき、より好ましくは、これらの指標間で予め作成した一次回帰直線を用いて、実際に測定され又は算出された測色値又は白毛率（ファクターＡ）から、未知の色素幹細胞数、メラニン量あるいは白毛率を信頼性ある換算値として求め、これらの換算値を評価指標として白毛評価を行うことができる。これにより、目的に応じた多様な白毛評価が簡便に可能となる。 Furthermore, as described above, the inventor not only between the colorimetric value of the lightness index L * and the white hair ratio, but also between the colorimetric value of the lightness index L * and the count value of the number of pigment stem cells. It has been found that there is a very high correlation between the rate and the count value of the pigment stem cell number, and between the colorimetric value of the lightness index L * and the melanin amount measurement value. Therefore, as defined in the fifth to ninth inventions, it is actually measured or calculated based on the correlation between these indices, more preferably using a linear regression line created in advance between these indices. From the colorimetric value or the white hair rate (factor A), the number of unknown pigment stem cells, the amount of melanin, or the white hair rate can be obtained as a reliable conversion value, and white hair evaluation can be performed using these conversion values as an evaluation index. . Thereby, various white hair evaluation according to the objective is simply attained.
次に、第１０発明〜第１２発明に規定するように、以上の第１発明〜第９発明の薬剤評価方法において、第１０発明の（イ）の段階で白髪モデル動物の体毛の白毛評価を開始することは、一般に体毛がその毛周期（体毛サイクル）の回数を重ねるに従い白毛化し易くなる傾向がある点に鑑みれば、検体に含まれる多数の体毛のサイクルを揃えて正確な評価を期する上で合理的である。このことは、同一の白毛評価に係る実施例又は比較例に用いる複数個体の白髪モデル動物間においても言える。更に、（ロ）〜（ニ）の段階からなるサイクルを１サイクル又は複数サイクル行うと共に、そのうちの（ハ）の段階では必要に応じて供試薬剤を２回以上適用し、その中で（イ）の段階で得られた白毛評価の評価指標と（ニ）の段階で得られた白毛評価の評価指標とを比較することは、白髪モデル動物に対する供試薬剤の経時的効果を詳細に分析する上で極めて効果的である。 Next, as defined in the tenth to twelfth inventions, in the pharmaceutical evaluation method of the first to ninth inventions described above, the white hair evaluation of the body hair of the gray hair model animal at the stage (a) of the tenth invention. In view of the fact that body hair generally tends to become white hair as the number of hair cycles (body hair cycle) increases, accurate evaluation is performed by aligning the cycle of many hairs contained in the specimen. Is reasonable. This can also be said among a plurality of individual white hair model animals used in Examples or Comparative Examples relating to the same white hair evaluation. Furthermore, the cycle consisting of the stages (b) to (d) is performed one cycle or a plurality of cycles, and in the stage (c), the reagent agent is applied two or more times as necessary. Comparing the evaluation index for white hair evaluation obtained in step (ii) with the evaluation index for white hair evaluation obtained in step (d) shows the detailed effects of the reagent on the gray hair model over time. It is extremely effective for analysis.
なお、第１３発明〜第１６発明に規定するように、白髪モデル動物に対する供試薬剤の適用方法は限定されないが、例えば、皮膚への塗布、皮下注射、皮内注射、吸入投与及び経口投与から選ばれる一つ以上の適用方法が好ましく、供試薬剤の白毛制御効果の評価としては、白毛予防・治療効果あるいは白毛促進・増加効果の評価が好ましく例示され、白髪モデル動物の種類はヒトを除く他は限定されないが、マウスが好ましく例示される。 As defined in the thirteenth to sixteenth inventions, the method of applying the reagent to the gray hair model animal is not limited. For example, from application to the skin, subcutaneous injection, intradermal injection, inhalation administration and oral administration One or more selected application methods are preferred, and the evaluation of the white hair control effect of the reagent is preferably exemplified by the evaluation of white hair prevention / treatment effect or white hair promotion / increase effect, and the types of white hair model animals are: Although it is not limited except humans, a mouse is preferably exemplified.
次に、本発明を実施するための形態を、その最良の形態を含めて説明する。 Next, modes for carrying out the present invention will be described including the best mode.
〔薬剤評価方法〕
本発明に係る薬剤評価方法は、白髪モデル動物に供試薬剤を適用し、その白髪モデル動物における白毛評価によって前記供試薬剤の白毛制御効果を評価する方法である。
[Drug evaluation method]
The drug evaluation method according to the present invention is a method in which a reagent agent is applied to a gray hair model animal and the white hair control effect of the reagent agent is evaluated by white hair evaluation in the gray hair model animal.
（白髪モデル動物）
白髪モデル動物は、ヒト以外の動物であって、体毛の白毛割合が経時変化するという表現型を持つ各種のモデル動物中から任意に選択することができるが、より好ましくは体毛の白毛化現象についてヒトとの共通性が大きい哺乳動物であり、更に好ましくは、小型、体毛サイクルの適切さ、管理の容易さ等の点からラット、マウス等の齧歯目動物であり、とりわけ好ましくはマウスである。
(White hair model animal)
The gray hair model animal is a non-human animal, and can be arbitrarily selected from various model animals having a phenotype in which the proportion of white hair of body hair changes over time, but more preferably white hair of white hair Mammals that have a great deal in common with humans in terms of phenomenon, more preferably rodents such as rats and mice, particularly preferably mice in terms of small size, appropriate hair cycle, ease of management, etc. It is.
白髪モデルマウス等の白髪モデル動物としては、体毛の白毛割合が経時変化するモデル動物を用いる。白毛割合の経時変化は、長期にわたって徐々に白毛割合が増大するものでも、極めて短期間に急速に白毛割合が増大するものでも使用できる。好ましい白髪モデルマウスの具体例としては、ＰＣＴ／ＪＰ２００６／３０２７８３号に開示されたものや、公知のビチリゴマウス（C57BL/6 Mitfｍｉ−ｖｉｔ）を例示できる。 As a white hair model animal such as a white hair model mouse, a model animal in which the white hair ratio of body hair changes with time is used. As the white hair ratio, the white hair ratio gradually increases over a long period, or the white hair ratio rapidly increases in a very short time. Specific examples of preferable white hair model mice include those disclosed in PCT / JP2006 / 302783 and known vitiligo mice (C57BL / 6 Mitf mi-vit ).
（供試薬剤）
白髪モデル動物に適用する供試薬剤の種類は限定されない。供試薬剤の剤型も限定されず、供試薬剤のみからなり又はこれを主成分として含む液剤、粉剤、錠剤等を、そのまま、あるいは餌料等に混合して用いることができる。
(Reagents)
The type of reagent used for the gray hair model animal is not limited. The dosage form of the reagent is not limited, and liquids, powders, tablets and the like that are composed solely of the reagent or that contain this as a main component can be used as they are or mixed with feed.
白髪モデル動物に対する供試薬剤の適用形態も限定されず、公知の任意の適用形態を採用できるが、好ましくは、皮膚への塗布、皮下注射、皮内注射、吸入投与及び経口投与から選ばれるいずれか一つの適用形態を実施するか、あるいはそれらの内の二つ以上の適用形態を併せ実施することができる。 The application form of the reagent for the gray hair model animal is not limited, and any known application form can be adopted, but preferably any one selected from application to the skin, subcutaneous injection, intradermal injection, inhalation administration and oral administration One application can be implemented, or two or more of them can be implemented together.
（評価項目）
本発明において、薬剤評価方法の評価項目は白毛制御効果である。「白毛制御効果」とは、白毛抑制効果と白毛促進効果とを含む。「白毛抑制効果」とは、体毛の白毛化を予防し、既に生じた白毛化の一層の進行を抑制又は停止させ、あるいは更に既に生じた白毛を黒毛化させる効果をいう。「白毛促進効果」とは、体毛の白毛化を誘発し、又は既に生じた白毛化を一層進行させる効果をいう。
(Evaluation item)
In the present invention, the evaluation item of the drug evaluation method is a white hair control effect. The “white hair control effect” includes a white hair suppressing effect and a white hair promoting effect. The “white hair suppressing effect” refers to an effect of preventing whitening of body hair, suppressing or stopping further progress of whitening that has already occurred, or further blackening white hair that has already occurred. The “white hair promoting effect” refers to an effect of inducing whitening of body hair or further promoting whitening that has already occurred.
（薬剤評価のスケジュール）
薬剤評価方法における薬剤評価の具体的なスケジュールは、少なくとも、白髪モデル動物に対して供試薬剤を適用する段階と、供試薬剤の適用後に白髪モデル動物の体毛について白毛評価を行う段階とが含まれる限りにおいて、限定されない。
(Drug evaluation schedule)
The specific schedule of drug evaluation in the drug evaluation method includes at least a step of applying a reagent to a gray hair model animal and a step of performing a white hair evaluation on the body hair of the gray hair model animal after applying the reagent agent. As long as it is included, it is not limited.
好ましくは、白髪モデル動物に対して供試薬剤の適用を開始する時期が、好適に選択される。白髪モデル動物が余りに未成熟で幼いと、体毛が十分に生え揃わず、又は体毛評価のための毛刈りや供試薬剤の適用に耐えない恐れがある。一方、多数の体毛を収集して評価するに当たり、それらの体毛サイクルがバラバラであると、正確な白毛評価を期待し難い。 Preferably, the timing for starting application of the reagent to the gray hair model animal is suitably selected. If the gray hair model animal is too immature and young, there is a risk that the body hair will not be sufficiently grown, or that it will not be able to withstand the application of hair cutting or reagent supply for body hair evaluation. On the other hand, when collecting and evaluating a large number of body hairs, it is difficult to expect an accurate white hair evaluation if the body hair cycles are disjoint.
このような点から、特にマウスにおいては体毛サイクルが休止期間中にバリカンとシェーバーにて毛刈りし、供試薬剤の適用を開始することが特に好ましい。この時点では白髪モデル動物が毛刈りや供試薬剤の適用に耐える程度に成長しており、又、この時点で最初の白毛評価を行って評価結果を初期値とし、この初期値を供試薬剤適用後の白毛評価結果と比較すれば、より正確な白毛評価が可能になる。更に、上記の最初の白毛評価の際、又はその直後に白髪モデル動物の体毛をバリカンとシェーバーにて除去すれば、その後の評価における体毛サイクルを揃えることができ、一層正確な白毛評価が可能になる。なお、休止期間中にバリカンとシェーバーにて毛刈りすることにより、体毛サイクルを白毛制御効果の評価に好適な成長期に揃えることが出来る。 From this point of view, particularly in mice, it is particularly preferred that the hair cycle is cut with a clipper and a shaver during the rest period and application of the reagent is started. At this point, the gray hair model animal has grown to the extent that it can withstand shaving and application of the reagent. At this point, the first white hair is evaluated and the evaluation result is set as the initial value, and this initial value is used as the test value. Compared with the white hair evaluation result after application of the drug, more accurate white hair evaluation becomes possible. Furthermore, if the hair of the white hair model animal is removed with a clipper and a shaver immediately after the first white hair evaluation described above, the hair cycle in the subsequent evaluation can be aligned, and a more accurate white hair evaluation can be performed. It becomes possible. In addition, by trimming hair with a clipper and a shaver during the rest period, the body hair cycle can be aligned with a growth period suitable for evaluating the white hair control effect.
以上の点から、薬剤評価のスケジュールとしては、（イ）体毛サイクルが休止期間中の白髪モデル動物の体毛の白毛評価を行う段階と、（ロ）白髪モデル動物の体毛を除去する段階と、（ハ）白髪モデル動物に対し、供試薬剤を１回以上適用する段階と、（ニ）供試薬剤の適用後に発毛した時点で白毛評価を行う段階と、を含むスケジュールが特に好ましい。その際、白毛評価を毛刈りした体毛について行う場合には、（イ）と（ロ）の段階を同時に行うことができる。 From the above points, the schedule of drug evaluation includes (a) a stage of evaluating white hair of a white hair model animal during a period when the hair cycle is in a rest period, and (b) a stage of removing body hair of the white hair model animal, (C) A schedule including a step of applying the reagent agent to the gray hair model animal at least once and (d) performing a gray hair evaluation at the time of hair growth after application of the reagent agent is particularly preferable. At that time, when the white hair evaluation is performed on the trimmed body hair, the steps (A) and (B) can be performed simultaneously.
又、前記（イ）の段階を経た白髪モデル動物に対して、前記（ロ）〜（ニ）の段階からなるサイクルを複数サイクル行うことも、長期的かつ詳細な評価を行う上で好ましい。この際にも、白毛評価を毛刈りした体毛について行う場合には、前のサイクルの（ニ）の段階と、次のサイクルの（ロ）の段階を同時に行うことができる。更に、前記した白毛評価の初期値を各サイクルの（ニ）の段階の評価値とそれぞれ比較し、かつ、各サイクルの（ニ）の段階の評価値相互間で比較することも好ましい。 In addition, it is also preferable to perform a plurality of cycles composed of the steps (b) to (d) on the gray hair model animal that has undergone the step (a) for long-term and detailed evaluation. Also in this case, when the white hair evaluation is performed on body hair that has been trimmed, the stage (d) of the previous cycle and the stage (b) of the next cycle can be performed simultaneously. Furthermore, it is also preferable to compare the initial value of the white hair evaluation described above with the evaluation value at the stage (d) of each cycle and between the evaluation values at the stage (d) of each cycle.
〔白毛評価の評価指標〕
本発明においては、白髪モデル動物の白毛評価を、以下（ａ）〜（ｃ）のいずれかの評価指標に基づき、又はこれらの内の二つ以上の評価指標に基づいて行う。
[Evaluation index for white hair evaluation]
In the present invention, white hair evaluation of a white hair model animal is performed based on any of the following evaluation indices (a) to (c), or based on two or more of these evaluation indices.
（ａ）白髪モデル動物の体毛の測色値
この評価指標は、白髪モデル動物の一定量の体毛を毛刈りして検体とし、この検体を綿塊状の束のままで、適宜な光学的測色系で測定して得られる数値化された指標である。
(A) Colorimetric value of the hair of a white hair model animal This evaluation index is obtained by shaving a certain amount of hair of a white hair model animal as a specimen, and using this specimen as a lump-like bundle, an appropriate optical colorimetry It is a digitized index obtained by measuring with a system.
又、この評価指標は、毛刈りしていない白髪モデル動物の体毛を直接に光学的測色系で測定して得ることもできる。但し、この手法による場合は、測定中の白髪モデル動物の身動き等に起因してデータがばらつくなどのデメリットを生じ得る。 This evaluation index can also be obtained by directly measuring the body hair of a gray hair model animal that has not been trimmed with an optical colorimetry system. However, with this method, there may be a demerit such as data variation due to the movement of the gray hair model animal being measured.
検体としては、例えば白髪モデル動物の背中の一定面積部分の体毛を用いることができる。光学的測色系としては、例えば、コニカミノルタ社製の色彩色差計「CR-400」等の適宜な色差計を用いることができる。 As the specimen, for example, body hair of a certain area on the back of a white hair model animal can be used. As the optical colorimetry system, for example, an appropriate color difference meter such as a color difference meter “CR-400” manufactured by Konica Minolta can be used.
（ｂ）白髪モデル動物における体毛発生部分の皮膚組織の観察結果。 (B) The observation result of the skin tissue of the hair generation part in the gray hair model animal.
この評価指標は、白髪モデル動物の毛胞部分を含む皮膚組織の観察結果、より具体的には毛胞部分の色素細胞生産組織の観察結果である。その観察結果は、色素幹細胞数の計数値であることが特に好ましい。色素幹細胞数は、単一の毛胞部分についての色素幹細胞数の計数値、より好ましくは、数箇所ないし数十箇所の毛胞部分についての色素幹細胞数の合計計数値又は平均値である。色素細胞生産組織における色素幹細胞の認定に当たり、（１）体毛バルジ領域に存在する細胞であること、（２）ｄｃｔが発現していることによる色素細胞であるとの判定、（３）ｃ−ｋｉｔが発現していないことによる未分化細胞であるとの判定、の３条件を全て満たす細胞を、色素幹細胞と認定することができる。 This evaluation index is an observation result of the skin tissue including the follicle portion of the white hair model animal, more specifically, an observation result of the pigment cell producing tissue of the follicle portion. The observation result is particularly preferably a count value of the number of pigment stem cells. The number of pigment stem cells is a count value of the number of pigment stem cells for a single hair follicle portion, more preferably a total count value or an average value of the number of pigment stem cells for several to several tens of hair follicle portions. In certifying pigment stem cells in a pigment cell-producing tissue, (1) determining that the cells are present in the hair bulge region, (2) determining that the cells are pigment cells by expressing dct, (3) c-kit A cell that satisfies all the three conditions of determining that it is an undifferentiated cell due to the fact that is not expressed can be identified as a pigment stem cell.
皮膚組織の採取は微量で済むため、動物に苦痛を与えない程度に抑えることができる。ましてやマウスを屠殺する必要もなく、体毛サイクルが回る度に皮膚の一部を回収することで経時変化を観察することも可能なので、皮膚組織の観察により白毛制御効果を評価することはメリットがある。 Since a small amount of skin tissue can be collected, it can be suppressed to a level that does not cause pain to animals. Furthermore, there is no need to sacrifice mice, and it is possible to observe changes over time by collecting a part of the skin each time the body hair cycle rotates, so it is advantageous to evaluate the effect of controlling white hair by observing the skin tissue. is there.
（ｃ）白髪モデル動物の体毛におけるメラニン量の測定値。 (C) The measured value of the amount of melanin in the body hair of a gray hair model animal.
この評価指標は、要するに白髪モデル動物の一定量の体毛に含まれるメラニン量の、適宜な方法による定量値である。具体的なメラニン量の定量方法としては、その目的を達する限りにおいて公知の任意の方法を採用すれば良いが、例えば前記したように回収した体毛を１０分間アルカリ処理し、メラニン量を吸光度計にて測定する方法が例示される。 In short, this evaluation index is a quantitative value by an appropriate method of the amount of melanin contained in a certain amount of body hair of a gray hair model animal. As a specific method for determining the amount of melanin, any known method may be adopted as long as the purpose is achieved. For example, as described above, the collected hair is treated with an alkali for 10 minutes, and the amount of melanin is stored in an absorptiometer. The method of measuring is illustrated.
白髪モデル動物を用いて供試薬剤の白毛制御効果を評価するに当たり、一般的に考えられる評価指標である白毛率、及び本発明に特有の評価指標である（ａ）白髪モデル動物の体毛の測色値（特に、明度指数L＊の測色値）、（ｂ）白髪モデル動物における体毛発生部分の皮膚組織の観察結果（特に、色素幹細胞数）、（ｃ）白髪モデル動物の体毛におけるメラニン量の測定値の各々は、前記のように、それぞれ特徴的な白毛評価を可能とするものである。 In evaluating the white hair control effect of the reagent using a white hair model animal, the white hair rate, which is a generally considered evaluation index, and (a) the hair of the white hair model animal, which is an evaluation index unique to the present invention (Especially the colorimetric value of the lightness index L * ), (b) the observation result of the skin tissue of the hair generation part in the gray hair model animal (especially the number of pigment stem cells), (c) the body hair of the white hair model animal As described above, each of the measured values of the melanin amount enables characteristic white hair evaluation.
これらの内の特定の評価指標間には、実施例欄で後述するように一次回帰直線を作成可能な高い相関が認められることが分かっている。従って、測定値／計数値が既に得られた評価指標をファクターＡとし、測定値／計数値が得られていないが実際に利用したい評価指標をファクターＢとしたとき、ファクターＡとファクターＢの関係を表す一次回帰直線を予め作成しておけば、ファクターＡの測定値／計数値に基づき、ファクターＢの評価指標を信頼性の高い換算値として容易に求めることができる。 It is known that a high correlation capable of creating a linear regression line is recognized between specific evaluation indexes among these as described later in the example column. Therefore, when an evaluation index for which a measured value / count value has already been obtained is a factor A, and an evaluation index for which a measured value / count value is not obtained but is actually desired to be used is a factor B, the relationship between the factor A and the factor B If a linear regression line representing the above is created in advance, the evaluation index of factor B can be easily obtained as a highly reliable conversion value based on the measured value / count value of factor A.
このような高い相関を示す一対の評価指標として、「明度指数L＊の測色値と色素幹細胞数の計数値」、「白毛率と色素幹細胞数の計数値」、「明度指数L＊の測色値とメラニン量」、「明度指数L＊の測色値と白毛率」を挙げることができる。これらの一対の評価指標間では、任意の一方の評価指標を上記のファクターＡとし、他方の評価指標を上記のファクターＢとすることができる。 As a pair of evaluation indexes showing such a high correlation, “colorimetric value of lightness index L * and count value of pigment stem cell number”, “count value of white hair ratio and pigment stem cell number”, “value index of lightness index L * ” Colorimetric value and amount of melanin "," Colorimetric value of lightness index L * and white hair ratio "can be mentioned. Between these pair of evaluation indexes, any one of the evaluation indexes can be the above factor A, and the other evaluation index can be the above factor B.
しかし実用的な見地からは、相対的に測定値／計数値が得られ易い評価指標がファクターＡであり、相対的に測定値／計数値が得られ難い評価指標がファクターＢであることが合理的である。かかる実用的な見地から、以下の１）〜４）の評価指標間の換算が好ましい。 However, from a practical standpoint, it is reasonable that the evaluation index that is relatively easy to obtain the measurement value / count value is Factor A, and the evaluation index that is relatively difficult to obtain the measurement value / count value is Factor B. Is. From such a practical point of view, conversion between the following evaluation indices 1) to 4) is preferable.
１）明度指数L＊の測色値から色素幹細胞数の換算値を求める。 1) A conversion value of the number of pigment stem cells is obtained from the colorimetric value of the lightness index L * .
２）白毛率から色素幹細胞数の換算値を求める。 2) The conversion value of the number of pigment stem cells is determined from the white hair rate.
３）明度指数L＊の測色値からメラニン量の換算値を求める。 3) A conversion value of the melanin amount is obtained from the colorimetric value of the lightness index L * .
４）明度指数L＊の測色値から白毛率の換算値を求める。 4) A conversion value of the white hair ratio is obtained from the colorimetric value of the lightness index L * .
以下に本発明の実施例を比較例と共に説明する。本発明の技術的範囲は以下の実施例及び比較例によって限定されない。
〔実施例１：薬剤評価のスケジュール〕
（白髪モデル動物）
白髪モデル動物として、ＰＣＴ／ＪＰ２００６／３０２７８３号に開示された公知の白髪モデルマウス（以下、単に「マウス」という）を使用した。このマウスは、詳細な説明は省くが、一定の遺伝子型を持ったノックアウト・トランスジェニックマウスであって、生後はじめて生える体毛の色が黒色ないしほぼ黒色であり、加齢と共に白毛を自然発症するという表現型を持っている。
Examples of the present invention will be described below together with comparative examples. The technical scope of the present invention is not limited by the following examples and comparative examples.
[Example 1: Schedule of drug evaluation]
(White hair model animal)
As a gray hair model animal, a known white hair model mouse (hereinafter simply referred to as “mouse”) disclosed in PCT / JP2006 / 302783 was used. This mouse is a knock-out transgenic mouse with a certain genotype, which will not be described in detail. The hair color that grows after birth is black or almost black, and spontaneously develops white hair with age. Has the phenotype.
（薬剤評価のスケジュール）
本実施例における薬剤評価のスケジュールを図１に示す。図１の上部に示すタイムテーブルにおいて右端に「age」の表記で示す段はマウスの月齢を示し、最初の「４Ｍ」との表記は「４ケ月齢の時点で評価を開始した」ことを意味する。本実施例における評価の開始時点は休止期の体毛を持った４ヶ月齢のマウスをバリカンとシェーバーにて毛刈りした時点である。
(Drug evaluation schedule)
The schedule of drug evaluation in this example is shown in FIG. In the time table shown in the upper part of FIG. 1, the stage indicated by “age” at the right end indicates the age of the mouse, and the first notation “4M” means “the evaluation started at the age of 4 months”. To do. The starting point of the evaluation in this example is the time when a 4-month-old mouse with resting body hair was shaved with a clipper and a shaver.
タイムテーブルの右端に「Exp. Period」の表記で示す段は評価の開始時点からの経過月数を示し、その下部の「下方が欠けた丸形状の矢印」様の記号はその時点で「マウスの体毛を毛刈りした」ことを示す。即ち、マウスを評価開始時点である４ケ月齢の時点で初めて毛刈りし、以後、マウスの体毛サイクルがほぼ１ケ月であることから、１ケ月経過ごとに８ケ月齢の時点まで毛刈りを繰り返した。この毛刈りにおいては、マウスの背中部分における長方形のエリアの体毛を刈り取った。このエリアで刈り取った体毛を、以下、「検体毛」という。 The stage indicated by “Exp. Period” at the right end of the timetable indicates the number of months that have elapsed since the start of the evaluation, and the symbol “a round arrow with a lower part” at the bottom is “Mouse” The hair of the body was shaved. " That is, for the first time, the mouse was shaved at the age of 4 months, which is the start of evaluation, and since then, the hair cycle of the mouse is almost 1 month, so the hair cutting is repeated until 8 months of age every month. It was. In this hair cutting, the hair of the rectangular area on the back part of the mouse was cut off. The body hair cut in this area is hereinafter referred to as “specimen hair”.
そして４ケ月齢の時点（評価開始時）から８ケ月齢の時点（評価終了時）に至るまで、実施例に係るマウス（図に「Drug」と表記）の毛刈りした部分には供試薬剤の７０％エタノール溶液を、図に「コントロール（70％EtOH）」と表記したコントロール用のマウスの毛刈りした部分には７０％エタノール（エタノール：水=容積比７：３）を、それぞれ毎日２００μＬ塗布した。 From the time of 4 months of age (at the start of evaluation) to the time of 8 months of age (at the end of evaluation), the portion of the mouse (shown as “Drug” in the figure) according to the example had a reagent agent 70% ethanol solution, and 70% ethanol (ethanol: water = volume ratio 7: 3) was added to the shaved portion of a control mouse labeled “control (70% EtOH)” in the figure, 200 μL each day. Applied.
実施例及びコントロール用のマウスの体毛の白毛評価は、前記の「下方が欠けた丸形状の矢印」様の記号の傍らに上向きの矢印で示すように、それぞれ４ケ月齢、６ケ月齢及び８ケ月齢の時点で毛刈りした検体毛について行った。評価項目は、後述の実施例で述べるように、「白毛率」、「明度指数L＊の測色値（以下、単に「L＊値」という）」、「色素幹細胞数」及び「メラニン量」である。
〔実施例２：白毛率の視覚的観察による測定〕
白毛率とL＊値との相関を求めるため、検体毛中の白毛の数を視覚的観察による計数によって算出した。バリカン・シェーバーによって刈り取ったマウスの検体毛について、光学顕微鏡（オリンパス社製ＢＸ６１、１０倍〜２０倍）にて１本ずつ白毛か黒毛かを分別し、「白毛率＝白毛本数／全体毛本数」として算出した。白毛か黒毛かの分別においては、毛髪のメジュラー中にメラニンが見られるか否かを基準として判断した。この基準に基づく黒毛の例を図２の「黒毛」と表記した上側の顕微鏡写真（メジュラー中にメラニンが詰まっている）に示し、白毛の例を図２の「白毛」と表記した下側の顕微鏡写真（メジュラー中にメラニンが見られない）に示す。
〔実施例３：検体毛のL＊値の測定〕
刈り取った検体毛を図３で示したように毛束状にした。図３の右側には白毛の毛束を、左側には白毛混じりの黒毛の毛束を示す。色彩色差計（コニカミノルタ社製CR-400）のプローブをこれらの毛束に押し当ててL＊値を測定した。なお、毛刈りせず直接マウスの体毛にプローブを押し当てても測定は可能であるが、マウスが動いたり、データがばらつくなどのデメリットが生じるため、前記の通り毛束状での測定がより好ましい。なお、後述するL＊値はすべて本方法により求めたものである。
〔実施例４：白毛率とL＊値の相関〕
実施例２で測定した白毛率と実施例３で測定したL＊値との相関性を、白毛率が異なる６１種の検体毛を対象に検討した。その実測値を表１に示す。
The evaluation of the white hair of the body hair of Examples and Control mice is 4 months old, 6 months old and The test hairs cut at the age of 8 months were used. The evaluation items are “white hair ratio”, “colorimetric value of lightness index L * (hereinafter, simply referred to as“ L * value ”)”, “number of pigment stem cells”, and “melanin amount” as described in Examples below. Is.
[Example 2: Measurement of white hair ratio by visual observation]
In order to obtain the correlation between the white hair rate and the L * value, the number of white hairs in the sample hair was calculated by counting by visual observation. Mouse hairs cut with a hair clipper shaver were classified into white hair or black hair one by one with an optical microscope (Olympus BX61, 10 to 20 times). "White hair ratio = number of white hairs / total Calculated as “number of hairs”. In the classification of white hair or black hair, it was judged based on whether or not melanin was found in the hair jersey. An example of black hair based on this criterion is shown in the upper micrograph (with melanin clogged up) in FIG. 2 labeled “black hair”, and an example of white hair is labeled “white hair” in FIG. It is shown in the photomicrograph on the side (melanin is not seen in the regular).
[Example 3: Measurement of L * value of specimen hair]
The cut sample hair was made into a hair bundle as shown in FIG. The white hair bundle is shown on the right side of FIG. 3, and the black hair bundle mixed with white hair is shown on the left side. The L * value was measured by pressing a probe of a color difference meter (CR-400 manufactured by Konica Minolta) against these hair bundles. Measurements are possible even if the probe is pressed directly against the body hair of the mouse without shaving, but there are disadvantages such as movement of the mouse and variations in data. preferable. All L * values described later are obtained by this method.
[Example 4: Correlation between white hair ratio and L * value]
The correlation between the white hair ratio measured in Example 2 and the L * value measured in Example 3 was examined for 61 specimen hairs with different white hair ratios. The measured values are shown in Table 1.
この点を図４のグラフに示す。従って、高い信頼性のもとに、L＊値と白毛率を相互に換算することができる。
〔実施例５：色素幹細胞数の計数〕
体毛発生部分の皮膚組織を観察し評価する方法として、色素幹細胞数を以下の（１）〜（９）に述べる手順により計数した。
This point is shown in the graph of FIG. Therefore, the L * value and the white hair ratio can be converted into each other with high reliability.
[Example 5: Counting the number of pigment stem cells]
As a method for observing and evaluating the skin tissue of the hair generation part, the number of pigment stem cells was counted according to the procedures described in (1) to (9) below.
（１）検体毛を毛刈りした部分の皮膚の一部（約１０ｍｍ×５ｍｍ角）を切除後に縫合可能なように切り取る。 (1) A part of the skin (about 10 mm × 5 mm square) obtained by cutting the sample hair is cut out so that it can be sutured after excision.
（２）切り取った皮膚を未固定にて凍結包埋する。 (2) The cut skin is frozen and embedded without fixing.
（３）凍結切片を１２μmの厚さにて作製する。 (3) A frozen section is prepared with a thickness of 12 μm.
（４）スライドグラスに切片をのせ、１０分間冷アセトンで固定する。 (4) Place a section on a slide glass and fix with cold acetone for 10 minutes.
（５）ＰＢＳにて洗浄し、４℃で抗dct抗体（サンタクルーズ社製を１：１０００で使用）および抗c-kit抗体（サンタクルーズ社製を１：１０００で使用）にて免疫染色する。 (5) Wash with PBS and immunostain at 4 ° C. with anti-dct antibody (Santa Cruz used 1: 1000) and anti-c-kit antibody (Santa Cruz 1: 1000 used) .
（６）１２時間後、ＰＢＳにて洗浄する。 (6) After 12 hours, wash with PBS.
（７）Alexa 594 donkey αgoat IgG （サンタクルーズ社製を１：１０００で使用）およびAlexa 488 donkey αRat IgG （サンタクルーズ社を１：１０００）にて室温で１０分間二次抗体反応をさせる。 (7) A secondary antibody reaction is performed at room temperature for 10 minutes with Alexa 594 donkey αgoat IgG (manufactured by Santa Cruz at 1: 1000) and Alexa 488 donkey αRat IgG (Santa Cruz at 1: 1000).
（８）ＰＢＳにて洗浄し、核染色後封入する。 (8) Wash with PBS and enclose after nuclear staining.
（９）光学顕微鏡（オリンパス社製ＢＸ６１ ２０〜４０倍）にて毛包を観察し、下記（I）〜（III）の全ての条件を満たす細胞を色素幹細胞とし、各例で評価するマウス皮膚中の毛包７０本中の色素幹細胞をカウントする。 (9) Mouse skin to be evaluated in each case by observing hair follicles with an optical microscope (Olympus BX61 20 to 40 times), and using cells satisfying all the following conditions (I) to (III) as pigment stem cells Count pigment stem cells in 70 hair follicles.
（I）体毛バルジ領域に存在する細胞である。 (I) A cell present in the hair bulge region.
（II）ｄｃｔの発現産物であるタンパク質が認められる細胞である。 (II) A cell in which a protein which is an expression product of dct is observed.
（III）ｃ−ｋｉｔの発現が、全くあるいは実質的に認められない細胞である。
〔実施例６：白毛率と色素幹細胞数の相関〕
実施例５の手順に従って計数した毛包７０本当たりの色素幹細胞数と、該当する皮膚部分に生えていた検体毛についての実施例２と同じ視覚的観察による計数により測定した白毛率との相関性を、白毛率が異なる２９種の検体毛を対象に検討した。実測値を表２に示す。
(III) A cell in which c-kit expression is not observed at all or substantially.
[Example 6: Correlation between white hair ratio and pigment stem cell number]
Correlation between the number of pigment stem cells per 70 hair follicles counted according to the procedure of Example 5 and the white hair rate measured by the same visual observation as in Example 2 for the sample hair growing on the corresponding skin part Sexuality was examined for 29 types of test hairs with different white hair rates. The measured values are shown in Table 2.
図５に示すグラフから、色素幹細胞数と白毛率に相関があることも認められるので、色素幹細胞数から白毛率を求めることも可能であることが分かる。
〔実施例７：メラニン量の測定〕
検体毛中のメラニン量を以下の方法により測定した。即ち、メラニン量を、検体毛を溶解した溶液の吸光度により測定をおこなうため、試薬として入手できるメラニンを用い、常法により検量線を作成し、それを用いてメラニン量Ｍ（μｇ／ｍｌ）を求めた。吸光度は、バイオラッドラボラトリー社製のプレートリーダー（４５０ｎｍ）を用いて測定した。検体毛１０ｍｇを精秤し、ＮａＯＨ０．６ｇ、２００ｍＭのＥＤＴＡ２５０μｌ、蒸留水４９．７５ｍｌを混合し調製したアルカリ溶液（ｐＨ＞１３）に１０分間浸漬させ、メラニンを抽出した後、吸光度を測定し、検量線を用いてメラニン量Ｍに変換した。
〔実施例８：L＊値とメラニン量の関係〕
実施例７において測定した一定量の検体毛当たりのメラニン量と、その検体毛からなる毛束について実施例７に先立って測定しておいたL＊値との相関性を、白毛率が異なる８種の検体毛を対象に検討した。実測値を表３に示す。
From the graph shown in FIG. 5, since it is recognized that there is a correlation between the number of pigment stem cells and the white hair rate, it can be seen that the white hair rate can also be obtained from the number of pigment stem cells.
[Example 7: Measurement of melanin content]
The amount of melanin in the sample hair was measured by the following method. That is, in order to measure the amount of melanin by the absorbance of the solution in which the sample hair is dissolved, a calibration curve is prepared by a conventional method using melanin available as a reagent, and the amount of melanin M (μg / ml) is calculated using it. Asked. Absorbance was measured using a plate reader (450 nm) manufactured by Bio-Rad Laboratories. 10 mg of sample hair was precisely weighed, 0.6 g of NaOH, 250 μl of 200 mM EDTA, 49.75 ml of distilled water were mixed for 10 minutes in an alkaline solution (pH> 13), extracted for melanin, and then the absorbance was measured. The amount of melanin was converted to M using a calibration curve.
[Example 8: Relationship between L * value and melanin amount]
The white hair ratio differs in the correlation between the amount of melanin per sample hair measured in Example 7 and the L * value measured prior to Example 7 for the hair bundle composed of the sample hair. Eight kinds of specimen hairs were examined. The measured values are shown in Table 3.
また、この結果から、前記の図５のグラフを踏まえれば、メラニン量と白毛率に相関があることも認められるので、メラニン量から白毛率を求めることも可能である。 From this result, it is also recognized that there is a correlation between the amount of melanin and the white hair rate based on the graph of FIG. 5 described above, and it is also possible to obtain the white hair rate from the amount of melanin.
実施例６の結果も併せれば、本実施例で用いたマウスについて、白毛率、L＊値、色素幹細胞数及びメラニン量の全てに相関性があることが認められたので、このうち１つのパラメーターが測定できれば、残り３つのパラメータを求めることができる。例えば、メラニン量から色素幹細胞数を求めることも可能である。
〔実施例９：皮膚への塗布による薬剤の白毛予防効果の評価〕
実施例１の方法に基づく薬剤評価を、ポリフェノールの一種であるルテオリンを用いて行った例を示す。
In combination with the results of Example 6, it was confirmed that all of the white hair ratio, L * value, pigment stem cell number, and melanin amount were correlated for the mice used in this example. If one parameter can be measured, the remaining three parameters can be determined. For example, the number of pigment stem cells can be determined from the amount of melanin.
[Example 9: Evaluation of white hair prevention effect of drug by application to skin]
The example which performed the chemical | medical agent evaluation based on the method of Example 1 using the luteolin which is 1 type of polyphenol is shown.
ルテオリンとして、ＬＫＴ社製ルテオリンを用い、７０％エタノールにて１％（ｖ／ｖ）希釈した溶液を用いｎ＝１３にて白毛予防・治療効果を評価した結果を図７に示す。図７に示す通り、ルテオリンは、皮膚への塗布による４ケ月間の適用によってマウスの白毛化を有意に抑制し、白毛を制御する効果を有すると評価することができた。
〔実施例１０：経口投与による薬剤の白毛予防効果の評価〕
次に、上記の供試薬剤であるルテオリンの適用形態を皮膚への塗布から経口投与に変更し、それ以外の点は実施例１の薬剤評価スケジュールに従って、ルテオリンの白毛予防効果を評価した。
FIG. 7 shows the results of evaluating white hair prevention / treatment effects at n = 13 using a solution diluted with 1% (v / v) with 70% ethanol using luteolin manufactured by LKT as luteolin. As shown in FIG. 7, luteolin could be evaluated as having an effect of controlling white hair by significantly suppressing whitening of mice by application for 4 months by application to the skin.
[Example 10: Evaluation of white hair prevention effect of drug by oral administration]
Next, the application form of luteolin, which is the aforementioned reagent agent, was changed from application to the skin to oral administration, and the white hair prevention effect of luteolin was evaluated according to the drug evaluation schedule of Example 1 for other points.
具体的には、実施例のマウスにはルテオリン６．２５ｍｇ、精製水２５０ｍｌ、５規定水酸化カリウム２．５ｍｌ、アスパルテーム３１２．５ｍｇおよびチョコレートフレーバー５００μｌを混合した溶液の形態で毎日５０ｍｇ経口投与し、コントロールのマウスには前記溶液からルテオリンのみを除いた溶液の形態で毎日同量経口投与し、投与開始から４ケ月間にわたり実施例及びコントロールに係るマウス検体毛の白毛率を実施例２の方法で測定した。実施例は５個体、コントロールは３個体を供試し、測定値としてはこれらの複数個体の測定値の平均値を採用した。 Specifically, the mouse of the example was orally administered daily in the form of a solution in which 6.25 mg of luteolin, 250 ml of purified water, 2.5 ml of 5 N potassium hydroxide, 312.5 mg of aspartame and 500 μl of chocolate flavor were mixed, The control mice were orally administered daily in the form of a solution in which only luteolin was removed from the solution, and the white hair ratio of the mouse specimen hairs of the examples and controls was measured over the course of 4 months from the start of administration. Measured with In the examples, 5 individuals were used, and 3 individuals were used as controls, and the average value of the measured values of these multiple individuals was adopted as the measured value.
結果を図８に示す。図８において例えば「１Ｍ」とある表記は「投与の開始から１ケ月経過時」を意味する。 The results are shown in FIG. In FIG. 8, for example, the notation “1M” means “when one month has elapsed since the start of administration”.
図８に示すように、「■」のプロットで示すルテオリン投与マウスは、「◆」のプロットで示すコントロールのマウスに比較して、投与の開始から４ケ月経過時においては白毛率の増大を抑制することについて有意な差が認められる。従って、ルテオリンは、投与経路に関わらず、マウスの白毛化を有意に抑制し、白毛を制御する効果を有すると評価することができた。なお、この実験期間中、マウスに有意な体重変化は認められなかった。
〔実施例１１：皮膚への塗布による薬剤の白毛促進効果の評価〕
実施例１の薬剤評価スケジュールに基づいて行った薬剤評価の他の一例を示す。植物抽出液として市販されているタイムエキス（一丸ファルコス株式会社社製 ファルコレックスタイムＢ）を１％含有する７０％エタノール溶液を実施例に係るマウス４個体に適用し、これとは別に７０％エタノール溶液をコントロールに係るマウス４個体に適用して、タイムエキスの白毛促進効果を評価した。
As shown in FIG. 8, the luteolin-administered mouse indicated by the “■” plot shows an increase in the white hair rate after 4 months from the start of administration, as compared to the control mouse indicated by the “♦” plot. There is a significant difference in suppression. Therefore, it was possible to evaluate that luteolin significantly suppresses whitening of mice and controls white hair regardless of the administration route. During this experiment, no significant change in body weight was observed in the mice.
[Example 11: Evaluation of white hair promoting effect of drug by application to skin]
The other example of the medicine evaluation performed based on the medicine evaluation schedule of Example 1 is shown. A 70% ethanol solution containing 1% thyme extract (Falco Rex thyme B manufactured by Ichimaru Falcos Co., Ltd.) commercially available as a plant extract was applied to 4 mice according to the examples, and 70% ethanol separately. The solution was applied to 4 control mice to evaluate the white hair promoting effect of thyme extract.
評価の結果を図９に示す。図９において横軸に表記した、例えば「６Ｍ」とある表記は、「投与の開始から６ケ月経過時」を意味する。又、白毛率の測定値としては、上記の複数個体の測定値の平均値を採用した。 The evaluation results are shown in FIG. In FIG. 9, for example, “6M” written on the horizontal axis means “when 6 months have passed since the start of administration”. In addition, as the measurement value of the white hair ratio, the average value of the measurement values of the above-described plural individuals was adopted.
図９に示すように、「■」のプロットで示すタイムエキス適用マウスは、「◆」のプロットで示すコントロールのマウスに比較して、投与の開始から６ケ月経過時においては白毛率の増大を促進することについて有意な差が認められる。従って、タイムエキスは、マウスの白毛化を有意に促進し、白毛を制御する効果を有すると評価することができた。 As shown in FIG. 9, the time extract-applied mice indicated by the “■” plot show an increase in the white hair rate after 6 months from the start of administration, as compared to the control mice indicated by the “♦” plot. There is a significant difference in promoting Therefore, it was possible to evaluate that the time extract has an effect of significantly promoting whitening of the mouse and controlling white hair.
本発明によって、有効な白毛制御剤をスクリーニングできる新たな薬剤評価方法が提供される。 The present invention provides a new drug evaluation method capable of screening for an effective white hair control agent.
Claims (16)
前記白毛評価を以下（ａ）〜（ｃ）のいずれか１以上の評価指標に基づいて行うことを特徴とする薬剤評価方法。
（ａ）白髪モデル動物の体毛の測色値。
（ｂ）白髪モデル動物における体毛発生部分の皮膚組織の観察結果。
（ｃ）白髪モデル動物の体毛におけるメラニン量の測定値。 A method of applying a reagent to a gray hair model animal in which the white hair ratio of body hair changes over time, and evaluating the white hair control effect of the reagent by white hair evaluation of the white hair model animal,
The drug evaluation method, wherein the white hair evaluation is performed based on any one or more of the following evaluation indices (a) to (c).
(A) Colorimetric values of body hair of gray hair model animals.
(B) The observation result of the skin tissue of the hair generation part in the gray hair model animal.
(C) The measured value of the amount of melanin in the body hair of a gray hair model animal.
（１）体毛バルジ領域に存在する細胞である。
（２）ｄｃｔの発現状態によって色素細胞であると判定できる。
（３）ｃ−ｋｉｔの発現状態によって未分化細胞であると判定できる。 In counting the number of pigment stem cells, the skin tissue of a gray hair model animal is collected and stained with immunohistochemistry by double staining of c-kit and dct, and cells satisfying all the following conditions (1) to (3) 4. The method for evaluating a drug according to claim 3, wherein the drug is identified as a pigment stem cell.
(1) Cells present in the hair bulge region.
(2) It can be determined that it is a pigment cell by the expression state of dct.
(3) It can be determined that the cell is an undifferentiated cell depending on the expression state of c-kit.
（イ）毛周期（体毛サイクル）が休止期間中の白髪モデル動物の体毛の白毛評価を行う段階と、
（ロ）白髪モデル動物の体毛を除去する段階と、
（ハ）白髪モデル動物に対し、供試薬剤を１回以上適用する段階と、
（ニ）供試薬剤の適用後に発毛した時点で白毛評価を行う段階と、
を含むことを特徴とする請求項１〜請求項９のいずれかに記載の薬剤評価方法。 The drug evaluation method comprises:
(A) performing a white hair evaluation of a white hair model animal body hair during which the hair cycle (body hair cycle) is resting;
(B) removing the hair of the gray hair model animal;
(C) applying the reagent to the gray hair model animal at least once;
(D) a stage of white hair evaluation at the time of hair growth after application of the reagent;
The method for evaluating a drug according to any one of claims 1 to 9, further comprising:
The drug evaluation method according to any one of claims 1 to 15, wherein the gray hair model animal is a mouse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010145179A JP5534200B2 (en) | 2010-06-25 | 2010-06-25 | Drug evaluation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010145179A JP5534200B2 (en) | 2010-06-25 | 2010-06-25 | Drug evaluation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2012008045A true JP2012008045A (en) | 2012-01-12 |
| JP5534200B2 JP5534200B2 (en) | 2014-06-25 |
Family
ID=45538752
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2010145179A Expired - Fee Related JP5534200B2 (en) | 2010-06-25 | 2010-06-25 | Drug evaluation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5534200B2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114580915A (en) * | 2022-03-07 | 2022-06-03 | 南京新生医疗科技有限公司 | Intelligent evaluation method and system for hair-planting effect of novel microneedle technology |
| US11413321B2 (en) | 2017-12-26 | 2022-08-16 | Hirotaro FUKUOKA | Pharmaceutical composition for use in increasing hair, modifying scalp or skin, healing a wound, promoting osteogenesis, or modifying hair |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001048754A (en) * | 1999-08-03 | 2001-02-20 | Lion Corp | External preparation for hair, improvement and prevention of grey hair and improvement of grey hair to black hair |
| JP2004217589A (en) * | 2003-01-16 | 2004-08-05 | Shiseido Co Ltd | Permanent hair treatment agent for dyed hair and method for preventing fading of dyed hair |
| JP2004315442A (en) * | 2003-04-17 | 2004-11-11 | Lion Corp | Gray hair-preventing/improving agent |
| WO2007000433A2 (en) * | 2005-06-28 | 2007-01-04 | S.A. Lhoist Recherche Et Developpement | Pulverulent lime composition, method for the production thereof, and use of the same |
| JP2009035514A (en) * | 2007-08-02 | 2009-02-19 | Kanazawa Univ | Abnormal differentiation inhibitor for xvii-type collagen |
-
2010
- 2010-06-25 JP JP2010145179A patent/JP5534200B2/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001048754A (en) * | 1999-08-03 | 2001-02-20 | Lion Corp | External preparation for hair, improvement and prevention of grey hair and improvement of grey hair to black hair |
| JP2004217589A (en) * | 2003-01-16 | 2004-08-05 | Shiseido Co Ltd | Permanent hair treatment agent for dyed hair and method for preventing fading of dyed hair |
| JP2004315442A (en) * | 2003-04-17 | 2004-11-11 | Lion Corp | Gray hair-preventing/improving agent |
| WO2007000433A2 (en) * | 2005-06-28 | 2007-01-04 | S.A. Lhoist Recherche Et Developpement | Pulverulent lime composition, method for the production thereof, and use of the same |
| JP2009035514A (en) * | 2007-08-02 | 2009-02-19 | Kanazawa Univ | Abnormal differentiation inhibitor for xvii-type collagen |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11413321B2 (en) | 2017-12-26 | 2022-08-16 | Hirotaro FUKUOKA | Pharmaceutical composition for use in increasing hair, modifying scalp or skin, healing a wound, promoting osteogenesis, or modifying hair |
| CN114580915A (en) * | 2022-03-07 | 2022-06-03 | 南京新生医疗科技有限公司 | Intelligent evaluation method and system for hair-planting effect of novel microneedle technology |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5534200B2 (en) | 2014-06-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tsuboi et al. | Guidelines for the management of androgenetic alopecia (2010) | |
| Breitkopf et al. | The basic science of hair biology: what are the causal mechanisms for the disordered hair follicle? | |
| US20070196344A1 (en) | Methods for identifying materials that can help regulate the condition of mammalian keratinous tissue | |
| Sundberg et al. | Skin and adnexa | |
| JP7005244B2 (en) | Hair restorer | |
| Trüeb et al. | A comment on the science of hair aging | |
| Shapiro et al. | Hair loss and restoration | |
| EP2764091B1 (en) | Diagnostic method for connective tissue and its application | |
| JP5534200B2 (en) | Drug evaluation method | |
| Trautman et al. | Melanocytes: a possible autoimmune target in alopecia areata | |
| Refat et al. | Lesional CD8+ T-Cell Number Predicts Surgical Outcomes of Melanocyte–Keratinocyte Transplantation Surgery for Vitiligo | |
| Light | Histological study of human scalps exhibiting various degrees of non-specific baldness | |
| JP6833193B2 (en) | Pigment stem cell differentiation inhibitor, gray hair preventive agent, and gray hair evaluation method | |
| Guarrera | Additional methods for diagnosing alopecia and appraising their severity | |
| JP3953709B2 (en) | Method for evaluating candidate substances for hair restorer or hair nourishing ingredient and alopecia model | |
| Semalty et al. | Techniques for the discovery and evaluation of drugs against alopecia | |
| Radhakrishnan et al. | Exfoliative cytology in age estimation | |
| Shapiro | Hair Disorders: Current Concepts in Pathophysiology, Diagnosis and Management, An Issue of Dermatologic Clinics | |
| Menon et al. | A clinical evaluation on the effect of astringent on keratinization of oral mucosa before and after the insertion of complete denture | |
| JP4674147B2 (en) | Evaluation method of hair restorer | |
| Bergfeld et al. | Proceedings of the Ninth World Congress for Hair Research (2015) | |
| Williams | The progression of interface dermatitis by longitudinal examination of the histopathological features of at three-time intervals histopathology techniques in Fitzpatrick skin types III? VI | |
| Abdelkader et al. | ANDROGENETIC ALOPECIA: AN OVERVIEW | |
| Bhandary | Trichoscopic Findings in Alopecia Areata and their Relation to Disease Activity, Severity and Clinical Subtype | |
| Yamasaki et al. | Effect of polyphenols from water chestnut pericarp on hair-related quality of life in healthy middle-aged, and older subjects: A randomized, placebo-controlled, double-blinded, parallel-group, comparison study |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20130620 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20130621 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20131126 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20131129 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140127 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20140325 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20140415 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5534200 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |