JP4452417B2 - Evaluation method of itch caused by ringworm - Google Patents

Description

【００３１】
結果：
２匹にそれぞれ２回及び７回の頭部スクロールを計測した。
以上の結果から、１０分程度の短い時間では掻き行動回数も少なく、又データのバラツキも大きいことがわかった。
【００３２】
実施例１
包帯絆創膏を５日目に除去した参考例１のモルモット６匹を無作為に２群（各Ｎ＝３）に分けて、１群には生理食塩水２ｍｌを塗布し、もう１群には生理食塩水に塩酸ジフェンヒドラミンを溶解させ１％（ｗ／ｖ）とした溶液２ｍｌを塗布した。塗布１０分後までの掻き行動回数を参考例１と同様にビデオカメラにて撮影し、実験終了後、映像を観察することにより掻き行動をカウントした。
【００３３】
【表２】

【００３４】
生理食塩水塗布群では、無処置群の掻き行動回数（表２）と比較して、約１６０倍に掻き行動回数が増加した。この時の1% DPH塗布群の平均掻き行動回数は、３５．３回であり、ＤＰＨが痒みを抑制していることを示している。
【００３５】
以上の結果から、上記白癬菌感染モルモットモデルは、感染によって潜在的に保持している痒みが、生理食塩水を塗布することにより、劇的に惹起されることが明らかとなった。
【００３６】
実施例２
包帯絆創膏を５日目に除去した参考例１のモルモット１５匹を無作為に下記４群（１群３〜４匹）に分け実施例１と同様に各成分塗布後、１０分間の掻き行動回数をカウントした。有意差検定はすべてStudentのt-検定（等分散を仮定した２標本による検定）で計算し、P(T<=t)両値を採用した。
【００３７】
１．無処置群
２．陽性対照群
（無水エタノール-ポリエチレングリコール４００［３０：７０（ｖ／ｖ）］混液を塗布）
３．５％ＣＲＴ塗布群
（無水エタノール-ポリエチレングリコール４００［３０：７０（ｖ／ｖ）］混液にクロタミトン（ＣＲＴ）を臨床投与量である５％（ｖ／ｖ）溶解した液を２ｍｌ塗布）
４．１％ＤＰＨ塗布群
（無水エタノール-ポリエチレングリコール４００［３０：７０（ｖ／ｖ）］混液に塩酸ジフェンヒドラミン（ＤＰＨ）を臨床投与量である１％（ｗ／ｖ）溶解させた液を２ｍｌ塗布）
【００３８】
結果を表３に示す。
【００３９】
【表３】

【００４０】
陽性対照群
無処置群との有意差検定結果：ｐ＜０．０５で有意差有り
５％ＣＲＴ塗布群
無処置群との有意差検定結果：ｐ＝０．５０で有意差無し
陽性対照群との有意差検定結果：ｐ＜０．０５で有意差有り
１％ＤＰＨ塗布群
無処置群との有意差検定結果：ｐ＝０．４７で有意差無し
陽性対照群との有意差検定結果：ｐ＜０．０５で有意差あり
【００４１】
陽性対照群である無水エタノール-ポリエチレングリコール４００［３０：７０（ｖ／ｖ）］混液投与群が無処置群に対し３０倍掻き行動回数を増加させた。ＣＲＴおよびＤＰＨは共に塗布直後から鎮痒作用が発現し、陽性対照群と比較して約１２％まで掻き行動回数を抑制し、有意な差のある結果であった。しかしながら、無処置群との比較では、掻き行動回数において有意な差は認められなかった。
【００４２】
【発明の効果】
本発明の、白癬菌感染由来の痒み測定動物実験モデルを使用することにより、現在市販されている水虫治療薬よりも優れた鎮痒効果（症状の改善）を有する薬物を開発する際に、ヒトの痒みと同一と考えられる試験系で、短期間で数多くの薬物の薬効評価試験を実施することが可能となった。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for evaluating itch caused by human ringworm infection and a method for screening a drug having an antipruritic effect.
[0002]
[Prior art]
Crotamiton (CRT) and diphenhydramine hydrochloride (DPH) are formulated as antipruritic agents in the commercially available athlete's foot treatment to suppress itching of vesicular ringworm (athlete's foot), but these are ideal for athlete's foot itch It is hard to say that it is an antipruritic agent. The reason for this is that these antipruritic agents have not been developed as a remedy for pruritus specific to athlete's foot, so even if administered to athlete's foot patients, long-term itch control is not obtained, and antipruritic drugs are administered frequently. It is the current situation that must be done.
[0003]
Kuraishi et al. Itching based on the scratching behavior of mice caused by administration of compound 48/80 and substance P, substances that cause itching in the back of mice. (European Journal of Pharmacology Vol. 275 (1995) 229-233). There are many reports of irritating substances that cause itching, but most of them still have unclear mechanisms. It is also known that prednisolone, which is a steroid, suppresses allergic reactions and scratching behavior of mice, but does not suppress scratching behavior by Compound 48/80.
[0004]
In this way, the body of the itch is complex, and the sensation of sensation is only subjective, so objective evaluation is difficult. The causes of pruritus are diverse and it has not yet been clarified which experimental model can be used to evaluate athlete's foot itch, and therefore there is no assessment model for these animals, and therefore the search for drugs with antipruritic effects No evaluation system has been established yet.
[0005]
[Problems to be solved by the invention]
It is an object of the present invention to provide a method for evaluating itch caused by ringworm fungus infection and a method for screening a drug having an antipruritic effect.
[0006]
[Means for Solving the Problems]
As a model for evaluating the itch caused by ringworm, the causative agent of athlete's foot, the present inventors have used a guinea pig ringworm fungus infection model (hereinafter referred to as “guinea pig”) that has been used for a long time as a model for evaluating the antibacterial activity of antifungal agents. Measurement of the scratching behavior of the “Rhizobium fungus infection model”) revealed that it was capable of scratching the itching resulting from the infection with ringworm. However, the scratching behavior was largely different among individuals and could not be evaluated stably. Therefore, if an inorganic salt aqueous solution or alcohol that does not itself have a curative effect is applied to the infected site of ringworm, the scratching behavior is surprisingly amplified and the reliability of the itch evaluation experiment system is improved. As a result, the inventors have found that the evaluation time can be remarkably shortened, and that the screening of drugs having an antipruritic effect can be quantitatively performed by combining administration of the test drug in this experimental system, and the present invention has been completed.
[0007]
That is, the present invention is characterized by applying a component selected from an aqueous inorganic salt solution, a lower alcohol, and a polyhydric alcohol to the skin of a guinea pig body part infected with ringworm, and measuring the subsequent scratching behavior of the guinea pig. The present invention provides a method for evaluating itch caused by bacterial infection.
[0008]
Further, the present invention is characterized by applying a test drug and a component selected from an aqueous solution of inorganic salt, a lower alcohol and a polyhydric alcohol to the skin of a guinea pig body infected with ringworm fungus, and specifying the subsequent scratching behavior of the guinea pig. The present invention also provides a method for screening a drug having an antipruritic effect against pruritus caused by ringworm fungus infection.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The “guinea pig ringworm infection model” is known as an experimental animal model for evaluating antifungal activity and is widely used, but has not yet been used as an evaluation model for pruritus (Fungus Magazine Vol. 38, No. 38) No. 1, pages 33-37). This model is obtained by infecting the back skin or legs of guinea pigs or hairless guinea pigs with an eutrophic ringworm, and is widely used for the purpose of observing antifungal activity. Since the purpose of this evaluation system is to observe antifungal activity, the affected area is fixed with gauze to eliminate factors that hinder the evaluation of the drug, such as the subject animal licking the drug or damaging the affected area. For the purpose of suppressing licking and licking, guinea pigs were not used in evaluations related to itching because they were wearing restraints such as necklaces (collar winding) around their necks.
[0010]
The “guinea pig ringworm infection model” is a known model animal and can be prepared by methods described in various literatures (Antimicrob. Agents Chemother., Vol. 42, pp. 967-970). 1997). More specifically, it can be produced by the following method.
[0011]
The hair of the horny skin of the guinea pig body is shaved with a clipper, razor, hair removal wax, etc., and the horny layer of the extracted guinea pig is rubbed with a paper file, etc. After peeling off the stratum corneum by applying glue tape or cellophane tape and peeling off vigorously, immerse the gauze in the spore solution of pre-cultured ringworm, attach gauze to the infected area, and apply adhesive bandage or Cover completely with a bandage and adhere for 3-7 days to infect. The infection site can be any of the back, abdomen, legs, etc., but the back is most preferable for itching evaluation experiments.
[0012]
Trichophyton mentagrophytes, Microsporum canis, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton verum, Trichophyton equinum (Trichophyton equinum) Epitonmophyton floccosum can be used. Human ringworm pathogens are not necessarily pathogenic to animals, and Trichophyton rubrum, the most commonly isolated human ringworm pathogen, is humanoid and most strains that are pathogenic to animals. Trichophyton Mentaglophytes (Trichophyton), which is also less preferred because it is weak and difficult to obtain stable lesions, and is also an aerobic ringworm that is the second most commonly isolated pathogen of Trichophyton rubulum mentagrophytes) or Microsporum canis (Microsporum canis) is particularly preferred because it causes reproducible infection of test animals.
[0013]
The guinea pig used in the present invention may be a guinea pig that can be used as a known “guinea pig ringworm infection model”, that is, a Hartley guinea pig or a hairless guinea pig is preferable. These are supplied by Nippon Charles River Co., Ltd. and are readily available. Males and females can be used, and males are generally used. The body weight is usually 300 to 350 g from the viewpoint of appropriate age (4 to 6 weeks) and ease of handling. These guinea pigs may be bred in a normal environment. Usually, those bred at 23 ± 1 ° C and 50% humidity will be bred alone per cage from the quarantine period of 1 week after arrival until the end of the experiment. To do.
[0014]
The “guinea pig ringworm infection model” produced in this way shows scratching behavior such as scratching the affected area in order to avoid itching as the infection with ringworm bacteria progresses. However, the scratching behavior is sporadic, has large individual differences, and requires a long observation time to accumulate data that can withstand evaluation, and thus cannot be used as a quantitative evaluation or screening method. On the other hand, if an aqueous solution of inorganic salt, lower alcohol and / or polyhydric alcohol is applied to the infected site, the number of scratching actions will increase several tens of times, and the variation among individuals will be significantly reduced. Highly reliable evaluation can be performed in the significant difference test. These application components are different from conventionally known raising substances, and do not have a function of causing scratching behavior even when applied to normal animals.
[0015]
Here, the inorganic salt aqueous solution is an aqueous solution of weakly acidic, neutral, or weak alkaline inorganic salts that are components existing in the living body, for example, alkali metals such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or alkaline earth. Halide halides, sodium carbonate, sodium hydrogen carbonate, phosphoric acid, sodium hydrogen phosphate aqueous solution and a mixed aqueous solution of one or more of these are preferable, and are preferably used at a concentration isotonic with a living body. Saline and Ringer's solution are particularly preferred because they are easy to prepare and obtain.
[0016]
As a lower alcohol, C1-C4 alcohol, such as methanol and ethanol, is preferable, and especially ethanol is preferable. As the polyhydric alcohol, glycerin, ethylene glycol, polyethylene glycol and the like are preferable. Among these, polyethylene glycol having an average molecular weight of 200 to 1000 is particularly preferable. Further, it is more preferable to use a lower alcohol and a polyhydric alcohol in combination.
[0017]
The application amount of these components is sufficient to be about 1 to 4 mL, and it is preferable to apply the solution by dripping and lightly spreading or soaking in cotton.
[0018]
In screening for a test drug, the test drug is applied to the infected site immediately before, simultaneously with, or immediately after the application of the above components, but simultaneous administration (application) is preferred. For simultaneous administration, when the test drug is water-soluble, it is preferably dissolved and applied in physiological saline. When the test drug is poorly water-soluble or water-insoluble, lower alcohol and / or polyhydric alcohol It is preferable to dissolve and apply in the solution.
[0019]
Since the scratching behavior is shown immediately after application of the component, the scratching behavior is measured. There are two types of scratching behavior, one is a head scroll that eliminates itching by biting the infected site with an incisor or rubbing the tooth. Another action is a scratching action by a rear leg stroke. Among these two kinds of scratching behavior, in the model animal of the present invention, the behavior by the incisor accounts for 90% or more of the number of measurements.
[0020]
The peak of the scratching behavior is within 10 minutes after the application of the component, and in about 1 hour, it settles to a level that causes an occasional scratching behavior that is not different from that of the non-application group. The number of scratching actions may be counted for minutes, more preferably for about 10 minutes.
[0021]
Guinea pigs are very sensitive to the presence of human beings, and if they are observed from the guinea pigs, they do not start all actions including scratching, but affect the evaluation results. Record them with a video camera, etc. It is preferable to measure the scratching behavior.
[0022]
In addition, since the model animal of the present invention evaluates itching by measuring the number of scratching actions, which is an action to avoid itching, the scratching action is suppressed. Be ready to act.
[0023]
The evaluation of itching is based on the fact that each animal holds a potentially equivalent itch due to infection with ringworm fungus before application of the test drug. Randomly divided into non-application groups, summed the number of scratching behaviors observed in each group at a certain time, calculated the difference in the number of scratching behaviors, and conducted a significant difference test The difference in the degree can be evaluated as an index.
[0024]
【Example】
Hereinafter, the present invention will be described in more detail with reference examples and examples.
[0025]
Reference Example 1 (Preparation of guinea pig ringworm infection model)
<Adjustment of infected bacterial solution>
Spores were formed by culturing Trichophyton mentagrophytes in potato dextrose agarten (PDA) medium (medium for fungal growth) at 25 ° C. for 7 days. By suspending these spores in sterile physiological saline containing 1% Tween 80 and measuring them under a microscope with a white blood cell counter, the same solution is used so that the target number of spores is about 10 ⁷ cells / mL. Combined by diluting, etc. to obtain an infectious bacterial solution
[0026]
<Guinea pig infection>
The infection method was carried out by modifying the report of Niwano et al. (Antimicrob. Agents Chemother. Vol. 42, 967-970, 1997).
The back hair of the guinea pig under anesthesia (Nembutal, manufactured by Dynabot Co., Ltd., 20 mg / kg, intraperitoneal administration) is clipped with a clipper and further removed with a commercially available hair remover (product name: feather hair removal foam, # HR-150). I got hair. The hair remover adhering to the body was wiped off with a paper towel soaked with 70% ethanol or sterilized physiological saline, and then moisture was removed with a dry paper towel and dried. Next, using a sandpaper (# 240), the back horny part was shallowly damaged so as not to bleed, and this part was disinfected with 70% ethanol and air-dried.
[0027]
First-aid adhesive plaster (Big Bang G, Nichiban Co., Ltd.) obtained by cutting the spore solution of Trichophyton mentagrophytes (T. mentagrophytes) prepared to about 10 ⁷ cells / mL into 1/3 to 1/5 length of gauze length 35mm x width 250mm In the gauze part of the company, immerse it in a sufficient amount (usually 1/3 length, 2 mL), apply it to the treated back, and then apply a bandage type adhesive bandage (Elastopore, made by Nichiban Co., Ltd. [50 mm width roll [Length 5m] Adhesive bandage]) completely covering Big Bang G applied part, infecting by adhering the spore solution to the infected part for a long time, leaving it for 3-7 days, removing the adhesive bandage, After 3-5 days, it was subjected to the experiment.
[0028]
In addition, the non-infected group was subjected to the same application using sterile physiological saline instead of the spore solution. The infection was established by observing the degree of inflammation at the infected site, peeling the skin at the infected site with surgical scissors, and dividing it roughly into approximately 10 sections, which were prepared with PDA medium (containing 100 μg / mL tetracycline hydrochloride). Place the exfoliated skin sections in close contact with the surface of the Petri dish plate medium, and the number of sections in which trichophyton T. mentagrophytes grew after incubation at 25 ° C for 7 days and the total number tested It confirmed from the ratio of the section number.
[0029]
Reference example 2
Using three guinea pigs of Reference Example 1 from which the bandage bandage was removed on the fifth day, the number of scratches was measured for 10 minutes after the start of the experiment. Each animal was photographed with a digital video camera, and the scratching behavior was counted by observing the video after the experiment. The scratching behavior was counted as one scratching action by the head scroll and the rear leg stroke to eliminate itching by biting the infected site with an incisor or rubbing the teeth.
[0030]
[Table 1]

[0031]
result:
Two heads were measured twice and 7 times, respectively.
From the above results, it was found that in a short time of about 10 minutes, the number of scratching actions was small and the data variation was large.
[0032]
Example 1
Six guinea pigs of Reference Example 1 from which the bandage bandage was removed on the fifth day were randomly divided into two groups (each N = 3), one group was applied with 2 ml of physiological saline, and the other group was physiological. 2 ml of a 1% (w / v) solution of diphenhydramine hydrochloride dissolved in saline was applied. The number of scratching behaviors up to 10 minutes after application was photographed with a video camera in the same manner as in Reference Example 1, and the scratching behavior was counted by observing the video after the experiment was completed.
[0033]
[Table 2]

[0034]
In the physiological saline application group, the number of scratching actions increased about 160 times compared to the number of scratching actions in the untreated group (Table 2). The average number of scratching actions in the 1% DPH application group at this time is 35.3 times, indicating that DPH suppresses itching.
[0035]
From the above results, it was clarified that the itch that is potentially retained by infection in the tinea parasite-infected guinea pig model is dramatically induced by applying physiological saline.
[0036]
Example 2
The 15 guinea pigs of Reference Example 1 from which the bandage bandage was removed on the 5th day were randomly divided into the following 4 groups (3 to 4 animals per group), and the number of scratching actions for 10 minutes after application of each component as in Example 1. Counted. All significant differences were calculated by Student's t-test (two-sample test assuming equal variance), and both P (T <= t) values were adopted.
[0037]
1. Untreated group2. Positive control group (applied with absolute ethanol-polyethylene glycol 400 [30:70 (v / v)] mixture)
3.5% CRT application group (2 ml of a solution obtained by dissolving 5% (v / v) of clinical dose of crotamiton (CRT) in a mixed solution of absolute ethanol-polyethylene glycol 400 [30:70 (v / v)]
4.1% DPH application group (anhydrous ethanol-polyethylene glycol 400 [30:70 (v / v)] 2 ml of a solution of 1% (w / v) of diphenhydramine hydrochloride (DPH) dissolved in clinical dose Application)
[0038]
The results are shown in Table 3.
[0039]
[Table 3]

[0040]
Significant difference test result with positive control group untreated group: p <0.05 Significant difference with 5% CRT application group untreated group result: p = 0.50 no significant difference with positive control group Significant difference test result: p <0.05 Significant difference with 1% DPH application group untreated group: Significant difference test result with p = 0.47 and no significant difference positive control group: p <0.05 significant difference [0041]
The absolute ethanol-polyethylene glycol 400 [30:70 (v / v)] mixed solution administration group, which is a positive control group, increased the number of scratching behaviors by 30 times compared to the untreated group. Both CRT and DPH exhibited an antipruritic effect immediately after application, and the number of scratching behaviors was suppressed to about 12% compared to the positive control group, and the results were significantly different. However, there was no significant difference in the number of scratching behaviors compared with the untreated group.
[0042]
【The invention's effect】
In developing a drug having an antipruritic effect (improvement of symptoms) superior to currently marketed athlete's foot treatment drugs by using the animal model of itch measurement derived from ringworm fungus infection of the present invention, In a test system that is considered to be identical to pruritus, it has become possible to carry out drug efficacy evaluation tests for many drugs in a short period of time.

Claims (6)

Evaluation of itchiness derived from ringworm fungus infection, characterized by applying a component selected from aqueous mineral salt solution, lower alcohol and polyhydric alcohol to the skin of guinea pig body infected with ringworm fungus, and measuring the subsequent scratching behavior of guinea pig Method. Infecting ringworm fungus, characterized by applying a test drug and an inorganic salt solution, a lower alcohol and a polyhydric alcohol to the skin of a guinea pig body infected with ringworm, and then identifying the scratching behavior of the guinea pig A method for screening a drug having an antipruritic effect against itching of origin. Trichophyton mentagrophytes, Microsporum canis, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton equinum and Trichophyton equinum The method according to claim 1 or 2, which is selected from Epidermophyton floccosum). The method according to any one of claims 1 to 3, wherein the component to be applied is a component selected from physiological saline, ethanol and polyethylene glycol. The method according to claim 1, wherein the body skin is back skin. The method according to claim 1, wherein the measurement of the scratching behavior is measurement of the number of strokes of the head and the rear legs.