JP3712428B2

JP3712428B2 - Antifungal evaluation method

Info

Publication number: JP3712428B2
Application number: JP23998494A
Authority: JP
Inventors: 敏郎馬島; 勝久内田; 英世山口
Original assignee: Pola Chemical Industries Inc
Current assignee: Pola Chemical Industries Inc
Priority date: 1994-10-04
Filing date: 1994-10-04
Publication date: 2005-11-02
Anticipated expiration: 2020-11-02
Also published as: JPH08103292A

Description

【０００１】
【産業上の利用分野】
本発明は足皮膚に対する抗真菌剤の評価法に関し、更に詳しくは足の皮膚に於ける真菌数の分布を正確に測定することによる抗真菌剤の評価法に関する。
【０００２】
【従来の技術】
本邦に於ける表在性真菌症患者は１２００から１８００万人と試算されている。患者が多い原因は生活環境、生活形態、生活習慣に起因すると考えられる真菌の伝播力の強さと、繰り返し感染蔓延する根治の困難さ等が考えられる。これは、かかる真菌症の病の部位が皮膚とりわけ足の裏の皮膚であるため、薬剤の効き方が一様ではなく、従って治癒したように思えても病原菌が完全に駆逐されておらず、やがて再発を招いてしまうことが多いためである。又、足の部位によって薬剤の効き方が異なるため、効かない部位に薬剤を投与しても得られる抗真菌効果は少ない。従って、罹患部位によってその部位に適応した薬剤を使用する事が必要であるが、薬剤の部位による効き方の違いを評価する方法は知られていなかった。
【０００３】
従来、抗真菌剤の評価はイン・ビトロでの抗真菌作用をコロニー形成を指標に評価する方法とイン・ビボに於いて、感染動物に抗真菌剤を塗布し、皮膚を採取し裁断し複数の小断片を取り出し、これを培地中に植え、これより真菌の生えてきた断片数を計数し、全断片数で除した値を指標として用いて評価していた。しかしながら、イン・ビトロの評価では抗真菌剤の評価では最も重要な薬物動態を考慮することが出来ず、従来のイン・ビボの方法では数値が１を切片数で除した値の倍数にしかならず、従って不連続であり、更に、少量しか生えてこない断片も、多量に生えた断片も同じ扱いになってしまい、加えて偶然性の要素が強すぎることも否定できず、従って、偶然的に得た皮膚片に於ける真菌の有無は判定できても、正しい定量はできず、正しい、部位差を考慮した評価は出来なかった。
【０００４】
【発明が解決しようとする課題】
従って、本発明は足の皮膚内の真菌の生存分布を定量的に測定し、実状に合わせた抗真菌剤の評価を行う方法を提供することを目的とする。
【０００５】
【課題を解決するための手段】
上記実状を踏まえ、本発明者らは真菌の定量手段を求めて鋭意研究を重ねた結果、実験動物の足の裏の皮膚を感染させた後、抗真菌剤で処理し、足の皮膚を各部位毎に切り出し、切り出した部位毎の真菌数を定量する事により、抗真菌剤の足の各部位に於ける作用の仕方を的確に知り、評価できることを見いだし発明を完成するに至った。
【０００６】
従って、本発明は、非ヒト動物の足の裏全体に真菌を植え付けた後、被検体を投与し、しかる後に足の裏全体の皮膚を、足に対して縦に線状、横に線状又は縦横格子状に切って切り出した後、部位毎にグループ分けし更に小切片にし、得られた切片毎に培養して当該培地上に生育したコロニーの大きさを測定し、得られたコロニーの大きさを別個に作成された検量線と対比して足の裏全体の皮膚各部位における真菌数（ｃｆｕ／ブロック）の分布を定量することを特徴とする足裏の皮膚各部位に対する抗真菌剤の評価法を提供するものである。
【０００７】
本発明において、抗真菌剤という語は、１）抗真菌作用を有する化合物、２）１）の化合物を含む組成物の両者を意味し、組成物には人為的な組成物である剤形と天然抽出物などの人為的でない組成物とが含まれる。
【０００８】
ここで、本発明の抗真菌作用の評価法の対象となる真菌は、表在性真菌症原因菌であるが、これを具体的に例示するならば、トリコフィトン属（Trichophyton）、ミクロスポーラム属（Microsporum）、エピデルモフィトン属（Epidermophyton）等の不完全糸状菌やキャンディダ属（Candida）、マラセチア属（Malassezia）の不完全酵母、及びこれらの変異株が挙げられる。この様な変異株としては、自然に薬物に対して耐性を獲得した耐性株、栄養依存性を有するようになった栄養依存性変異株、遺伝子導入などを行い人為的に変異させた人工変異株等が例示できる。
【０００９】
本発明方法に用いられる動物としては、哺乳類、例えばラット、モルモット、ウサギ、マウス、ブタ等が挙げられる。これらの動物の足の裏への真菌の移植方法としては、真菌を皮膚上に塗布する方法、真皮を露出させて当該真皮上に塗布する方法、クローズドパッチ法、皮内注射法等が挙げられるが、再現性の点よりクローズドパッチ法が好ましい。ここで真菌の移植は足の裏全体に行う。
【００１０】
被検体の投与は、被検体の種類によって異なり、経皮投与、経口投与、静脈内投与等が挙げられるが、経皮投与が好ましい。経皮投与の場合、真菌を移植した部位全てに塗布するのが望ましい。
【００１１】
本発明における、足の裏全体の皮膚の切り出し方は、足に対して縦に線状に切っても良いし、横に線状に切っても良いし、縦横格子状に切っても良い。例えば、横に線状に切り出した後、部位毎にグループ分けし（図１参照）、その各グループ内の皮膚を更に小切片にして培養に供するのが好ましい。かかる小切片は、例えば１切片あたり１×１〜２０×２０mmに切断すればよい。この小切片は、真菌以外の細菌を除去する目的で塩化ベンザルコニウム、ヒビデングルコネート等の殺菌剤溶液で洗浄するのが好ましい。
【００１２】
得られた切片の培養に用いる培地としては、通常培養や菌分離等に用いているものであれば特に限定はなく、例えば、サブロー培地、改変サブロー培地、ツァペック寒天培地等が例示できる。
【００１３】
培養は、１０〜４０℃、好ましくは２０〜４０℃でコロニーが生育するのに充分な時間、例えば１〜２０日間静置培養すればよい。
【００１４】
次に培養により培地上に生育したコロニーを測定することにより足の裏の皮膚の各部位における真菌数の分布を定量する。コロニーの測定は、コロニーの大きさを測定しその数値を用いる。コロニーの大きさの測定は、長径（ｌ）及び短径（ｓ）を計測し、その積（ｌ×ｓ）を求めて、コントロールと対比するのが簡便で好ましい。
【００１５】
真菌数の定量は、上記の如くして測定したコロニーの大きさを、別個に作成された検量線と対比して行うことができる。この検量線はコロニーの大きさと真菌数との間の相関性を示す検量線であり、例えば所定の菌数の真菌を前記と同一の条件で培養し、生育したコロニーの大きさを測定することにより作成できる。ここで菌数の計測法としては、菌体全体をニュートラルレッドの様な染色剤で染色し、その着色度により菌数を計量しても良いし、同位体などでマークした栄養素の代謝を放射活性より定量して菌数を測定しても良い。最も好適な菌数の計数方法は、菌体と分生子を分離し、血球計数板等で分生子を計数しこれを菌数のコントロールとすることである。この方法によれば、極めて簡易に再現性良く菌数のコントロールを作ることができる。ここで用いるコントロール真菌は、標準株でも臨床分離株でも良い。
【００１６】
各切片毎の真菌数から、足の裏の各部位における真菌の分布、すなわち抗真菌剤の効果を各部位毎に評価できる。
【００１７】
また、本発明において被検体として、組成物を用いた場合には、その組成物としての評価（例えば、基剤の評価、剤形の評価など）ができる。
【００１８】
【実施例】
以下に実施例を挙げて更に詳しく本発明について説明するが、本発明がこれら実施例に何等限定されないことは言うまでもない。
【００１９】
実施例１
トリコフィトンＴＩＭＭ２７８９株を改変１／１０サブロ−寒天培地（ペプトン０．２％グルコース０．１％、燐酸２水素カリウム０．１％、硫酸マグネシウム０．１％、寒天１．５％）に接種し２７℃で斜面培養した。２週間後斜面部を０．１％（ｗ／ｖ）ツィーン８０添加滅菌生理食塩水で覆い、振盪により分生子を遊離させた。菌糸塊を滅菌ガ−ゼで除去後、分生子浮遊液中の分生子数を血球計算盤で算定し、１×１０⁸ 分生子／mlの濃度に調整し、接種菌液とした。藤田法の変法である内田らの方法によりモルモットを感染させた。即ち、まず酒精綿でモルモット左右後肢をよく拭き、ガーゼ付き絆創膏（バンドエイドジョンソン＆ジョンソン製）のリント綿部分に上記接種液１００μｌを染み込ませ、同じ絆創膏をモルモット左右後肢プランター部分に縦にあてがい、爪とアキレス健の２カ所で粘着させ、ガムテープを準備し、バンドエイドを装着した後肢外側を覆い、サージカルテープを縦に２分し、このテープでガムテープの上部でモルモット後肢を縛り、固定した。) ３日後、ハサミでテープを切取り、絆創膏を除去し、足を露出させた。感染開始１４日目に病巣を形成しているのを確認した上で動物を群分けした。感染開始１５日目より２８日間１％ブテナフィン含有親水軟膏（Ｄｒｕｇ−Ａ）、１％ビフォナゾール含有ワセリン（Ｄｒｕｇ−Ｂ）、及びベヒクルで処理し、感染後４４日めに動物を屠殺し、足全体を酒精綿で３回拭いた後、足底部の皮膚をカカトから入刀し指付け根部分まで切り取り、各組織を1%消毒剤中で超音波洗浄のあと滅菌水で３回洗浄後、図１に示す実線の如くに、指付け根からカカトにかけ水平にほぼ等間隔に１０個の小片に刻んだ。付け根部分の小切片をＮｏ．１、以下切片順にナンバリングし、カカト部分をＮｏ．１０とした。次に、それぞれの小片をシクロヘキシミド５００μｇ／ml、クロラムフェニコール１００μｇ／ml、シソマイシン５０μｇ／mlを含むサブロー培地平板にのせ、２７℃で培養した。培養した組織切片は、１０日後白癬菌集落が発育したものを陽性とし、薬効を判定し評価した。また同時にコロニーの大きさも長径×短径で測定した。これらとは別途感染実験で用いたのと同じ菌株を希釈し、１０倍希釈列を作成した後、菌液を上記寒天平板に乗せ培養した。培養開始１０日目に長径（ｌ）と短径（ｓ）を測り、掛け合わせた値も測定し検量線を引きこの検量線より、上記の動物実験より得られたコロニーの大きさから皮膚片に生存していた菌数を定量した。生存菌数の部位分布を図２に示す。この図より、抗真菌剤により生存菌数の部位分布が異なること、及び、抗真菌剤が足の皮膚に対してどの様な効き方をするのかが明白に理解できる。従って、本発明の評価法によれば、真菌の治療後の生存の仕方、抗真菌剤を作用させた場合の特性などが的確に評価できる。更にこの方法を応用すれば、病巣の位置によって用いる抗真菌剤を変えたり、ある抗真菌作用のある物質の、適用部位に適応した剤形を探索したりすることも可能である。又２種以上の抗真菌作用を有する物質の組み合わせの効果も明確にすることができる。
【００２０】
【発明の効果】
本発明方法によれば、足の裏の部位毎に、正確かつ詳細に抗真菌剤の薬効が評価できる。
【図面の簡単な説明】
【図１】足の裏の皮膚を細切した例を示す図である。
【図２】足の裏を細切した部位毎の真菌数の分布を示す図である。[0001]
[Industrial application fields]
The present invention relates to a method for evaluating an antifungal agent for foot skin, and more particularly to a method for evaluating an antifungal agent by accurately measuring the distribution of the number of fungi in the foot skin.
[0002]
[Prior art]
The number of superficial mycosis patients in Japan is estimated to be 1200 to 18 million. The cause of the large number of patients is considered to be the strength of fungal transmission that is thought to be due to the living environment, lifestyle, and lifestyle, and the difficulty of radical cure that causes repeated infections. This is because the site of such mycotic disease is the skin, especially the skin on the soles of the foot, so the way the drug works is not uniform, so even if it seems to have healed, the pathogen has not been completely eliminated, This is because it often leads to recurrence. Moreover, since the effect of the drug differs depending on the site of the foot, there is little antifungal effect obtained even if the drug is administered to a site where it does not work. Therefore, it is necessary to use a drug adapted to the affected part depending on the affected part, but a method for evaluating the difference in the effect depending on the part of the drug has not been known.
[0003]
In the past, antifungal agents were evaluated by in vitro antifungal activity using colony formation as an indicator, and in vivo, antifungal agents were applied to infected animals, and the skin was collected and cut. The small fragments were taken out, planted in a medium, the number of fragments from which fungi grew were counted, and the value divided by the total number of fragments was used as an index for evaluation. However, in the in vitro evaluation, the most important pharmacokinetics cannot be considered in the evaluation of the antifungal agent, and in the conventional in vivo method, the numerical value is only a multiple of the value obtained by dividing 1 by the number of intercepts. Therefore, it is discontinuous, and even fragments that grow only in small quantities and fragments that grow in large quantities are treated the same, and in addition, it cannot be denied that the element of contingency is too strong. Although it was possible to determine the presence or absence of fungi in the skin piece, it was not possible to perform a correct quantification, and it was not possible to make a correct evaluation considering the site difference.
[0004]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a method for quantitatively measuring the fungal survival distribution in the skin of the foot and evaluating an antifungal agent in accordance with the actual situation.
[0005]
[Means for Solving the Problems]
Based on the above situation, the present inventors conducted extensive research seeking fungus quantification means.As a result, after infecting the skin of the sole of the foot of the experimental animal, the present invention was treated with an antifungal agent, and each skin of the foot was treated. By cutting out each part and quantifying the number of fungi per cut out part, it was found that the antifungal agent can accurately know how to act on each part of the foot and can be evaluated and completed the invention.
[0006]
Therefore, in the present invention, the fungus is planted on the entire sole of the non-human animal, and then the subject is administered. Thereafter, the skin on the entire sole of the foot is linear with respect to the foot and linear with respect to the foot. Or, after cutting into vertical and horizontal grids, grouping into parts and further dividing into small sections, cultivating each obtained section and measuring the size of the colonies grown on the medium, An antifungal agent for each skin region of the foot, wherein the distribution of the number of fungi (cfu / block) in each skin region of the entire sole is quantified by comparing the size with a separately prepared calibration curve An evaluation method is provided.
[0007]
In the present invention, the term antifungal agent means both 1) a compound having an antifungal action, and 2) a composition containing the compound of 1). And non-artificial compositions such as natural extracts.
[0008]
Here, the fungus that is the target of the antifungal activity evaluation method of the present invention is a superficial mycosis-causing bacterium, and specific examples thereof include Trichophyton, Microsporum. Incomplete filamentous fungi such as genus (Microsporum), Epidermophyton (Epidermophyton), Candida, Malassezia (Malassezia), and mutants thereof. Such mutants include resistant strains that have naturally acquired resistance to drugs, nutrient-dependent mutants that have become nutritionally dependent, and artificial mutants that have been artificially mutated through gene transfer etc. Etc. can be exemplified.
[0009]
Examples of animals used in the method of the present invention include mammals such as rats, guinea pigs, rabbits, mice, pigs and the like. Examples of the method for transplanting fungi to the soles of these animals include a method of applying fungi on the skin, a method of exposing the dermis and applying it on the dermis, a closed patch method, and an intradermal injection method. However, the closed patch method is preferable from the viewpoint of reproducibility. Here at the implantation of fungi intends line to the entire sole of the foot.
[0010]
Administration of the subject varies depending on the type of the subject and includes transdermal administration, oral administration, intravenous administration and the like, and transdermal administration is preferred. In the case of transdermal administration, it is desirable to apply to all sites where fungi have been transplanted.
[0011]
In the present invention, the method of cutting the skin of the entire sole of the foot may be cut linearly with respect to the foot, may be cut linearly horizontally, or may be cut vertically and horizontally. For example, it is preferable to cut out horizontally in a line and then group into regions (see FIG. 1), and further cultivate the skin in each group into smaller sections for culturing. Such small slices may be cut into 1 × 1 to 20 × 20 mm per slice, for example. The small section is preferably washed with a bactericide solution such as benzalkonium chloride or hibdengluconate for the purpose of removing bacteria other than fungi.
[0012]
The medium used for culturing the obtained slice is not particularly limited as long as it is usually used for culturing or bacterial isolation, and examples thereof include Sabouraud medium, modified Sabouraud medium, and Czapec agar medium.
[0013]
The culture may be performed by static culture at 10 to 40 ° C., preferably 20 to 40 ° C., for a time sufficient for the colonies to grow, for example, for 1 to 20 days.
[0014]
Next, the distribution of the number of fungi in each part of the skin on the sole of the foot is quantified by measuring colonies grown on the medium by culture. Measurements of colony, Ru using the numerical measure the size of the colony. Measurement of the size of the colonies measures the longer diameter (l) and the minor axis (s), seeking the product (l × s), preferably a simple and easy comparison with the control.
[0015]
Quantification of the number of fungi can be performed by comparing the colony size measured as described above with a separately prepared calibration curve. The calibration curve is a calibration curve showing a correlation between the magnitude Sato fungal number of colonies, for example, a predetermined fungal count of bacteria was cultured in the same conditions as above, to measure the size of the grown colonies Can be created. Here, as a method for measuring the number of bacteria, the whole cells may be stained with a stain such as neutral red, and the number of bacteria may be measured by the degree of coloration, or the metabolism of nutrients marked with isotopes is emitted. The number of bacteria may be measured by quantification based on the activity. The most preferable method for counting the number of bacteria is to separate the cells and conidia, and count the conidia with a blood cell counter or the like, and use this as a control for the number of bacteria. According to this method, the control of the number of bacteria can be made very easily with good reproducibility. The control fungus used here may be a standard strain or a clinical isolate.
[0016]
From the number of fungi for each section, the distribution of fungi at each site on the sole of the foot, that is, the effect of the antifungal agent can be evaluated for each site.
[0017]
In the present invention, when a composition is used as a subject, the composition can be evaluated (for example, evaluation of a base, evaluation of a dosage form, etc.).
[0018]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to these examples.
[0019]
Example 1
Trichophyton TIMM2789 strain was inoculated into a modified 1/10 Sabro-agar medium (peptone 0.2% glucose 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, agar 1.5%). Slope culture at 27 ° C. Two weeks later, the slope was covered with sterile physiological saline supplemented with 0.1% (w / v) Tween 80, and the conidia was released by shaking. After removing the mycelial mass with a sterile gauze, the number of conidia in the conidial suspension was calculated with a hemocytometer, adjusted to a concentration of 1 × 10 ⁸ conidia / ml, and used as an inoculum. Guinea pigs were infected by the method of Uchida et al., A modification of the Fujita method. First, wipe the left and right hind limbs of guinea pigs thoroughly with alcoholic cotton, soak 100 μl of the above inoculum into the lint cotton part of gauze adhesive bandage (manufactured by Bandaid Johnson & Johnson), and apply the same adhesive bandage vertically to the left and right hind limb planter parts. The nail and Achilles Ken were adhered to each other, a gum tape was prepared, the outside of the hind limb on which the band aid was attached was covered, the surgical tape was divided into two vertically, and the guinea pig hind limb was tied and fixed on the top of the gum tape with this tape. 3 days later, the tape was cut with scissors, the bandage was removed, and the feet were exposed. The animals were divided into groups after confirming the formation of lesions on the 14th day from the start of infection. Treated with 1% butenafine-containing hydrophilic ointment (Drug-A), 1% bifonazole-containing petrolatum (Drug-B), and vehicle for 28 days from the 15th day after the start of infection, the animals were sacrificed 44 days after the infection, After wiping 3 times with alcoholic cotton, cut the skin of the bottom of the foot from the kakato and cut it to the base of the finger. Each tissue was ultrasonically washed in 1% disinfectant and then washed 3 times with sterilized water. As shown by the solid line in Fig. 10, it was cut into 10 pieces horizontally at almost equal intervals from the finger base to the heel. A small slice of the root part is 1. Numbered in the order of the following sections. It was set to 10. Next, each piece was placed on a Sabouraud medium plate containing 500 μg / ml cycloheximide, 100 μg / ml chloramphenicol, and 50 μg / ml sisomycin, and cultured at 27 ° C. The cultured tissue section was evaluated as positive when 10 days later the growth of ringworm fungus colonies was positive, and the efficacy was determined. At the same time, the size of the colony was also measured by major axis × minor axis. Separately from these, the same strain as used in the infection experiment was diluted to prepare a 10-fold dilution series, and then the bacterial solution was cultured on the agar plate. On the 10th day from the start of culture, the major axis (l) and the minor axis (s) are measured, the multiplied value is also measured, and a calibration curve is drawn. The number of bacteria that survived was quantified. The site distribution of the number of viable bacteria is shown in FIG. From this figure, it can be clearly understood that the site distribution of the number of viable bacteria varies depending on the antifungal agent, and how the antifungal agent works on the foot skin. Therefore, according to the evaluation method of the present invention, it is possible to accurately evaluate the way of survival after fungal treatment, the characteristics when an antifungal agent is allowed to act, and the like. Furthermore, by applying this method, it is possible to change the antifungal agent used depending on the position of the lesion, or to search for a dosage form of a substance having an antifungal action that is adapted to the application site. Moreover, the effect of the combination of two or more substances having antifungal activity can also be clarified.
[0020]
【The invention's effect】
According to the method of the present invention, the efficacy of the antifungal agent can be evaluated accurately and in detail for each part of the sole of the foot.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example in which skin on the sole of a foot is cut into pieces.
FIG. 2 is a diagram showing the distribution of the number of fungi for each part obtained by chopping the sole of the foot.

Claims

After inoculating fungi on the entire sole of a non-human animal, administer the subject, and then cut the skin on the entire sole of the foot in a line vertically, horizontally in a line, or vertically and horizontally in a lattice pattern. Cut out, grouped into parts, further divided into small sections, cultured on the obtained sections, measured the size of the colonies grown on the medium, and prepared the size of the colonies separately A method for evaluating an antifungal agent for each part of the skin on the foot, wherein the distribution of the number of fungi (cfu / block) in each part of the skin on the entire sole of the foot is compared with a standard curve obtained.

The evaluation method according to claim 1, wherein a part of the fungus is a spore or an aerial mycelium.

The evaluation method according to claim 1 or 2, wherein the fungus is one or more selected from the genus Trichophyton or Epidermophyton.