JP2013099318A - Antifungal property evaluation method - Google Patents
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Abstract
Description
本発明は、爪真菌症の実態に即し、かつ抗真菌剤又は抗真菌剤を含む組成物の効果を定量的に評価できる方法に関する。 The present invention relates to a method capable of quantitatively evaluating the effect of an antifungal agent or a composition containing an antifungal agent in accordance with the actual state of onychomycosis.
爪真菌症は爪を感染の場とする真菌症であり、爪の混濁、肥厚、変形を引き起こす難治性の疾患である。爪真菌症の治療は、通常、内服療法により行われるが、全身性の副作用や薬剤相互作用など安全性の点で内服治療が困難な場合は、外用療法が選択される。外用療法の場合、全身性の副作用や薬剤相互作用などの可能性が極めて小さいので、本来、爪真菌症治療には理想的である。 Onychomycosis is a mycosis that causes nail infection, and is a refractory disease that causes nail turbidity, thickening, and deformation. Treatment of onychomycosis is usually performed by internal therapy, but external therapy is selected when internal therapy is difficult in terms of safety such as systemic side effects and drug interactions. In the case of external therapies, the possibility of systemic side effects and drug interactions is extremely small, so it is inherently ideal for the treatment of onychomycosis.
現在、国外ではロセリル（登録商標）、ペンラック（登録商標）等のネイルラッカー剤が爪真菌症用外用薬として発売されているが、国内では爪真菌症用の外用薬は発売されていない。これは、多くの爪真菌症において、感染力のある真菌は爪甲の下の皮膚（爪床）に存在するため、抗真菌薬を爪甲表面に投与しても、薬剤が厚い爪甲を浸透できず、爪床まで到達しない、あるいは到達しても爪床で十分な抗真菌効果を発揮できないなど、有効な外用製剤の開発が難しいことによる。爪真菌症用の外用製剤を開発する上では、かかる爪真菌症の病態の特殊性を十分に考慮した方法でその効果を評価することが必要である。 At present, nail lacquers such as Loceryl (registered trademark) and Penlac (registered trademark) are marketed as topical drugs for onychomycosis outside Japan, but no topical drugs for onychomycosis have been marketed in Japan. This is because in many onychomycosis, infectious fungi are present in the skin under the nail plate (nail bed), so even if an antifungal agent is applied to the nail plate surface, This is because it is difficult to develop an effective external preparation, such as it cannot penetrate, does not reach the nail bed, or even when it reaches, it cannot exert a sufficient antifungal effect on the nail bed. In developing an external preparation for onychomycosis, it is necessary to evaluate the effect by a method that fully considers the peculiarities of the pathological condition of such onychomycosis.
爪真菌症用外用製剤の評価方法として、従来報告されているものの中には、爪試料に真菌を感染させた後、この爪試料に抗真菌剤又は抗真菌剤を含む組成物を作用させ、ついで、当該爪試料を生体色素で処理した後、爪試料中の生体色素の量を測定することにより、抗真菌剤又は抗真菌剤を含む組成物の評価を行う方法がある（特許文献１）。しかしながら，この方法は爪試料のみにおける抗真菌剤又は抗真菌剤を含む組成物の効果を対象としたものであり、薬剤の爪床への送達を考慮していないと考えられることから爪真菌症の実態に即しているとはいえない。 As a method for evaluating an external preparation for onychomycosis, among those conventionally reported, after a nail sample is infected with a fungus, an antifungal agent or a composition containing an antifungal agent is allowed to act on the nail sample, Next, there is a method for evaluating an antifungal agent or a composition containing an antifungal agent by measuring the amount of the biological pigment in the nail sample after treating the nail sample with the biological pigment (Patent Document 1). . However, this method is intended for the effect of an antifungal agent or a composition containing an antifungal agent only on a nail sample, and since it is considered that delivery of the drug to the nail bed is not considered, onychomycosis It cannot be said that it is in line with the actual situation.
また、真菌を接種した平板培地上に、これに接して爪試料を配置し、当該爪の非接面に、平板培地上に漏れることなく抗真菌剤を含有する組成物をチャージし、爪と培地間の真菌の生育状況を指標として抗真菌剤を評価する方法がある（特許文献２）。この方法は、薬剤の爪甲への浸透を考慮し、爪と培地間の真菌に着目していることから、特許文献１と比較すると爪真菌症の病態を想定したものになっていると考えられるが、爪真菌症における爪床での抗真菌効果については言及していないため、やはり完全に爪真菌症の実態に即した方法とはいい難い。従って、爪真菌症の実態に即し、かつ抗真菌剤又は抗真菌剤を含む組成物の効果を定量的に評価できる方法が望まれていた。 In addition, a nail sample is placed in contact with the plate medium inoculated with the fungus, and a non-contact surface of the nail is charged with a composition containing an antifungal agent without leaking onto the plate medium. There is a method for evaluating an antifungal agent using the growth status of fungi between culture media as an index (Patent Document 2). This method considers the penetration of the drug into the nail plate and focuses on the fungus between the nail and the medium. Therefore, compared to Patent Document 1, it is considered that this method assumes a pathological condition of onychomycosis. However, since the antifungal effect in the nail bed in onychomycosis is not mentioned, it is difficult to say that it is a method that perfectly matches the actual condition of onychomycosis. Therefore, there has been a demand for a method that can quantitatively evaluate the effect of an antifungal agent or a composition containing an antifungal agent in accordance with the actual state of onychomycosis.
本発明は、爪真菌症の病態の特殊性に即し、かつ抗真菌剤又は抗真菌剤を含む組成物の効果を定量的に評価できる方法を提供することを課題とする。 An object of the present invention is to provide a method that can conform to the peculiarity of the pathological condition of onychomycosis and quantitatively evaluate the effect of an antifungal agent or a composition containing an antifungal agent.
本発明者らは、かかる課題を解決するために鋭意検討した結果、爪試料を平板培地上に配置し、爪試料及び平板培地を共に真菌に感染させることにより、この平板培地を爪真菌症における爪床に見立てることが可能となることを見出した。またこの際、従来の方法（特許文献２）にあるように、真菌を接種した平板培地上に爪試料を配置して培養する方法では、爪試料中の真菌が増殖する間に、薬剤を適用していないにも関わらず平板培地中の真菌が減少してしまうが、爪試料を予め真菌に感染させておき、この感染させた爪試料を、真菌を接種した平板培地上に配置して培養することで、爪試料中の真菌と平板培地中の真菌が、薬剤を投与しない限り共に増殖するため、薬剤の抗真菌性効果をより正確に評価できることを見出し、本発明を完成するに至った。 As a result of intensive studies to solve such problems, the present inventors have placed the nail sample on a plate medium, and infecting both the nail sample and the plate medium with a fungus, whereby this plate medium is used in onychomycosis. It has been found that it is possible to make it look like a nail bed. At this time, as in the conventional method (Patent Document 2), the nail sample is placed on the plate medium inoculated with the fungus and cultured, and the drug is applied while the fungus in the nail sample grows. Although the fungus in the plate medium will decrease despite not being done, the nail sample is previously infected with the fungus, and this infected nail sample is placed on the plate medium inoculated with the fungus and cultured. As a result, the fungus in the nail sample and the fungus in the plate medium proliferate together unless the drug is administered, so that the antifungal effect of the drug can be evaluated more accurately, and the present invention has been completed. .
すなわち本発明は、爪試料に真菌を感染させた後、真菌を接種した平板培地上に爪試料を配置し、次いで爪試料上に抗真菌剤又は抗真菌剤を含む組成物を適用して培養した後、爪試料及び平板培地における真菌量をATP生物発光法を利用して評価することを特徴とする抗真菌剤又は抗真菌剤を含む組成物の抗真菌性評価方法である。 That is, in the present invention, after a nail sample is infected with a fungus, the nail sample is placed on a plate medium inoculated with the fungus, and then an antifungal agent or a composition containing an antifungal agent is applied to the nail sample and cultured. Then, it is an antifungal evaluation method of an antifungal agent or a composition containing an antifungal agent, characterized in that the amount of fungus in a nail sample and a plate medium is evaluated using an ATP bioluminescence method.
本発明の抗真菌性評価方法は、抗真菌剤又は抗真菌剤を含む組成物の爪真菌症に対する効果を、爪真菌症の実態に即し、かつ定量的に評価することができるものである。また、本発明の評価方法は、抗真菌剤又は抗真菌剤を含む組成物の爪への浸透性及び爪甲での抗真菌効果を、定量的に評価することができるものである。さらに本発明の評価方法は、抗真菌剤又は抗真菌剤を含む組成物の爪床への送達及び爪床での抗真菌効果を、定量的に評価することができるものである。 The antifungal evaluation method of the present invention is capable of quantitatively evaluating the effect of an antifungal agent or a composition containing an antifungal agent on onychomycosis according to the actual condition of onychomycosis. . Moreover, the evaluation method of this invention can evaluate quantitatively the permeability to the nail | claw of the composition containing an antifungal agent or an antifungal agent, and the antifungal effect in a nail plate. Furthermore, the evaluation method of the present invention is capable of quantitatively evaluating the delivery of an antifungal agent or a composition containing an antifungal agent to the nail bed and the antifungal effect on the nail bed.
本発明の抗真菌性評価方法は、爪試料に真菌を感染させた後、真菌を接種した平板培地上に爪試料を配置し、次いで爪試料上に抗真菌剤又は抗真菌剤を含む組成物を適用して培養した後、爪試料及び平板培地における真菌量をATP生物発光法を利用してそれぞれ評価するものである。 In the antifungal evaluation method of the present invention, after a nail sample is infected with a fungus, the nail sample is placed on a plate medium inoculated with the fungus, and then the antifungal agent or the antifungal agent is contained on the nail sample. Is applied, and the amount of fungi in the nail sample and the plate medium is evaluated using the ATP bioluminescence method.
本発明で用いる爪試料としては、一般に真菌が増殖する動物の爪の一部あるいは全部を採取して用いるが、動物種に関しては特に制限なく使用でき、例えばヒト、ブタ、ウシ、モルモット等の爪が例示できる。これらの爪は、真菌に非感染のものを爪試料として使用することが望ましいが、さらに定量的な評価を行うため、真菌を含む雑菌を殺菌してから用いることが望ましい。殺菌の方法としては、アルコール処理、UV照射処理等があるが、滅菌効果の点からオートクレーブ処理を行うことがより望ましい。また、定量的な評価を可能とするため、爪試料を予め一定の径及び厚みなど、所定形状に加工してから用いることが望ましい。 As the nail sample used in the present invention, a part or all of the nail of an animal in which a fungus grows is generally collected and used, but the animal species can be used without particular limitation, for example, nail of human, pig, cow, guinea pig, etc. Can be illustrated. Although it is desirable to use those nails that are not infected with fungi as nail samples, it is desirable to sterilize miscellaneous bacteria including fungi for further quantitative evaluation. As the sterilization method, there are alcohol treatment, UV irradiation treatment, etc., but autoclave treatment is more preferable from the viewpoint of sterilization effect. Moreover, in order to enable quantitative evaluation, it is desirable to use the nail sample after processing it into a predetermined shape such as a certain diameter and thickness in advance.
本発明方法においては、まず上記爪試料に真菌を感染させる。爪試料に感染させる真菌は、通常爪に感染すると認められているものであれば特に限定されるものではなく、例えばトリコフィトン・ルブルム（Trichophyton rubrum）、トリコフィトン・メンタグロファイテス（Trichophyton mentagrophytes）、カンジダ・アルビカンス（Candida albicans）などが例示できる。 In the method of the present invention, first, the nail sample is infected with a fungus. The fungus that infects the nail sample is not particularly limited as long as it is normally recognized to infect the nail. For example, Trichophyton rubrum, Trichophyton mentagrophytes And Candida albicans.
爪試料に対する真菌の感染方法は特に限定されるものではなく、例えば真菌の分生子数を一定量に調整して接種した培地と爪試料を接触させ、一定期間培養する方法によって爪に真菌を感染させることができる。さらに望ましくは、真菌の分生子数を一定量に調整して接種した液体培地に爪試料を浸漬し、一定期間培養することにより、効率的かつ定量的に爪の感染を進行させることができる。より具体的には、真菌を予め他の培地で培養し、必要に応じて界面活性剤等を添加した分散媒を加えて分生子を分散させ、ガーゼなどで濾過して菌糸を除去し、さらに分散媒を加えて分生子数を一定にした分生子分散液を、一定容量均一に混合した液体培地に爪試料を浸漬することで、定量的な評価が可能となり、効率的かつ定量的な爪の感染を行うことができる。分散媒としては、生理食塩水やリン酸緩衝生理食塩水などが望ましく、爪試料の感染に液体培地を用いる場合には途中段階からは分散媒として液体培地自体を用いることがより望ましい。爪試料を浸漬する液体培地の真菌濃度は１×１０２個/mL〜１×１０７個/mLが好ましく、１×１０３個/ｍＬ〜１×１０５個/mLがさらに好ましい。感染は、爪試料を液体培地に浸漬した状態で２０〜３０℃、５〜１４日間程度培養することにより行う。例えば２８℃程度の温度条件で、７日間程度培養を行えばよく、感染が十分に行われたことは、例えば爪試料周囲の菌の発育により判断することができる。また参考例１に記載の方法に従ってＡＴＰ発光強度を測定し、その測定値が１×１０３CPS〜１×１０４CPS程度になることにより判断することもできる。上記液体培地としては、液状のものであれば特に限定されずに使用することができ、例えばRPMI1640培地などを用いることができる。 The method of infecting the nail with the fungus is not particularly limited. For example, the nail is infected with the fungus by contacting the inoculated medium with the inoculated medium and adjusting the number of conidia of the fungus to a certain amount and culturing for a certain period. Can be made. More desirably, the nail sample can be efficiently and quantitatively advanced by immersing the nail sample in a liquid medium inoculated with a constant amount of fungal conidia and inoculating it. More specifically, the fungus is cultured in another medium in advance, and if necessary, a dispersion medium to which a surfactant or the like is added is added to disperse the conidia, and the mycelium is removed by filtration with gauze or the like. A nail sample can be quantitatively evaluated by immersing the nail sample in a liquid medium in which a conidia dispersion with a constant number of conidia is added and mixed in a constant volume. Can be infected. As the dispersion medium, physiological saline, phosphate buffered saline, or the like is desirable. When a liquid medium is used for infection of a nail sample, it is more desirable to use the liquid medium itself as a dispersion medium from an intermediate stage. The fungal concentration of the liquid medium in which the nail sample is immersed is preferably 1 × 10 2 cells / mL to 1 × 10 7 cells / mL, more preferably 1 × 10 3 cells / mL to 1 × 10 5 cells / mL. Infection is performed by culturing nail samples in a liquid medium at 20 to 30 ° C. for about 5 to 14 days. For example, the culture may be performed for about 7 days under a temperature condition of about 28 ° C., and the sufficient infection can be determined by, for example, the growth of bacteria around the nail sample. Moreover, it can also be judged by measuring the ATP emission intensity according to the method described in Reference Example 1 and the measured value is about 1 × 10 3 CPS to 1 × 10 4 CPS. The liquid medium can be used without particular limitation as long as it is liquid, and for example, RPMI1640 medium can be used.
このようにして真菌に感染させた爪試料を，爪試料に感染させた真菌と同一の真菌を接種した平板培地上に配置する（以下、「爪真菌症モデル」という）。この際の平板培地としては、平板を形成するものであれば特に限定されることなく、例えば寒天を固化剤として用いた無機塩水のものを用いることができる。さらにはリン酸２カリウム塩、硫酸マグネシウム塩、塩化カルシウム塩等を含有する寒天塩培地が望ましい。かかる培地に真菌を接種する際には、定量的な評価を可能とするため、加熱滅菌した後５０℃程度まで放冷し、かつ固化がまだ始まっていない平板培地に上記爪試料の感染にあたって調製した分生子分散液を一定容量一様に混ぜ込むことにより、定量的な平板培地への接種が行える。平板培地の真菌濃度は、１×１０２個/mL〜１×１０６個/mLが好ましく、さらに１×１０３個/mL〜１×１０５個/mLが好ましい。この範囲であると培養時の真菌濃度のばらつきを小さくすることができる。 The nail sample infected with the fungus in this way is placed on a plate medium inoculated with the same fungus as the fungus infected with the nail sample (hereinafter referred to as “onychomycosis model”). In this case, the flat plate medium is not particularly limited as long as it forms a flat plate, and for example, an inorganic salt water using agar as a solidifying agent can be used. Furthermore, an agar salt medium containing dipotassium phosphate, magnesium sulfate, calcium chloride and the like is desirable. When inoculating fungi on such a medium, in order to allow quantitative evaluation, it is cooled to about 50 ° C. after heat sterilization, and prepared for infection of the nail sample on a plate medium that has not yet solidified. Quantitative inoculation on a flat plate medium can be performed by uniformly mixing a certain volume of the conidia dispersion. The fungal concentration of the plate medium is preferably 1 × 10 2 cells / mL to 1 × 10 6 cells / mL, more preferably 1 × 10 3 cells / mL to 1 × 10 5 cells / mL. Within this range, variation in fungal concentration during culture can be reduced.
このような爪真菌症モデルは、爪試料、平板培地ともに一定の真菌量に達しているため、直ぐに抗真菌剤又はこれを含む組成物（以下、「抗真菌製剤」という）を適用し、抗真菌性評価に用いることが可能である。あるいは２８℃程度の温度条件で一定期間培養し、さらに真菌量を増加させた後に抗真菌性評価に用いることもできる。 In such onychomycosis model, both the nail sample and the plate medium have reached a certain amount of fungus, and therefore, an antifungal agent or a composition containing the same (hereinafter referred to as “antifungal preparation”) is immediately applied. It can be used for fungal evaluation. Alternatively, it can be used for antifungal evaluation after culturing for a certain period of time at a temperature of about 28 ° C. and further increasing the amount of fungi.
次にこの爪真菌症モデルの爪試料上に、抗真菌剤又は抗真菌製剤を適用する。抗真菌剤としては、特に限定されることなく使用でき、ミコナゾール、ミコナゾール硝酸塩、テルビナフィン、テルビナフィン塩酸塩、アモロルフィン、アモロルフィン塩酸塩、ルリコナゾール、ケトコナゾール、リラナフタート等が例示できる。これらの抗真菌剤を適当な担体に溶解ないし分散させて適用することができる。またこれらの抗真菌剤を含有する抗真菌製剤の剤形は、外用剤として爪に適用可能な剤形であれば特に限定されず、例えば、液剤、クリーム剤、ラッカー剤、軟膏剤、貼付剤等が挙げられ、これらを爪試料上に塗布あるいは貼付して適用する。この際、薬剤の爪甲の浸透及び平板培地への送達を定量的に評価するため、抗真菌剤又は抗真菌製剤と平板培地は直接接触しないようにすることが望ましい。このため、例えば液剤を適用する場合には漏出しないよう、漏出防止壁を設けることが望ましい。このような漏出防止壁としては、例えばゴム、樹脂などでできたOリングやシリコンボンドを用いることができる。なお、クリーム剤や貼付剤のように、塗布量あるいは貼付サイズを調整することで漏出を防止できる剤形の場合は、漏出防止壁を設けなくともよい。 Next, an antifungal agent or an antifungal preparation is applied onto the nail sample of this onychomycosis model. Examples of antifungal agents that can be used include, but are not limited to, miconazole, miconazole nitrate, terbinafine, terbinafine hydrochloride, amorolfine, amorolfine hydrochloride, luliconazole, ketoconazole, and rilanaphthalate. These antifungal agents can be applied by dissolving or dispersing in a suitable carrier. The dosage form of the antifungal preparation containing these antifungal agents is not particularly limited as long as it is a dosage form that can be applied to the nail as an external preparation. For example, liquids, creams, lacquers, ointments, patches These are applied or applied on a nail sample. At this time, in order to quantitatively evaluate the penetration of the nail plate into the nail plate and the delivery to the plate medium, it is desirable that the antifungal agent or antifungal preparation and the plate medium are not in direct contact. For this reason, when applying a liquid agent, for example, it is desirable to provide a leakage prevention wall so as not to leak. As such a leakage prevention wall, for example, an O-ring or silicon bond made of rubber or resin can be used. In addition, in the case of a dosage form that can prevent leakage by adjusting the application amount or the pasting size, such as a cream or a patch, it is not necessary to provide a leakage prevention wall.
上記のように、爪真菌症モデルに抗真菌剤又は抗真菌製剤を適用し、一定期間培養した後、爪試料と平板培地を分離し、それぞれの真菌量をATP生物発光法を利用して評価する。抗真菌剤又は抗真菌製剤を適用後の培養は、抗真菌剤の種類、用量や真菌の種類等に応じて適宜設定できるが、例えば２０〜３０℃で５〜４０日間程度行えばよい。培養後、爪試料と平板培地のATP発光強度を測定し、それぞれの真菌量を評価することにより、爪試料における抗真菌効果及び平板培地における抗真菌効果を共に評価できる。このように本発明の評価方法は、爪真菌症の病態における爪甲及び爪床での抗真菌剤又は抗真菌製剤の効果を総合的に評価することができるため、爪真菌症の実態に即した方法といえる。ATP（アデノシン三リン酸）は全ての生細胞中に存在する生体エネルギーであり、ルシフェリン・ルシフェラーゼ試薬と反応すると発光し、この発光強度はATP量に比例する。そしてATP発光強度は生存する真菌数に比例するものであることから、ATP発光強度を指標とすることにより、定量的な抗真菌性の評価が可能である。ATP発光強度は、高感度で生菌数のみの測定ができるため優れた指標となる。この際のATP発光強度の測定法としては特に限定されるものではないが、爪試料及び平板培地を、歯科用ルーター、ホモジネーター等を用いて粉砕し、必要に応じて界面活性剤等を添加した生理食塩水等の分散媒を加えて真菌を抽出したところにATP発光測定試薬を加え、ルミノメーター等の測定装置により簡便に測定することができる。 As described above, an antifungal agent or antifungal preparation is applied to an onychomycosis model, cultured for a certain period of time, then the nail sample and the plate medium are separated, and the amount of each fungus is evaluated using the ATP bioluminescence method To do. The culture after application of the antifungal agent or antifungal preparation can be appropriately set according to the type of antifungal agent, the dose, the type of fungus, and the like. For example, the culture may be performed at 20 to 30 ° C. for about 5 to 40 days. After culturing, by measuring the ATP emission intensity of the nail sample and the plate medium and evaluating the amount of each fungus, both the antifungal effect in the nail sample and the antifungal effect in the plate medium can be evaluated. As described above, the evaluation method of the present invention can comprehensively evaluate the effect of the antifungal agent or antifungal preparation on the nail plate and nail bed in the pathology of onychomycosis. It can be said that. ATP (adenosine triphosphate) is bioenergy present in all living cells, and emits light when reacted with a luciferin-luciferase reagent. The intensity of the emitted light is proportional to the amount of ATP. Since ATP emission intensity is proportional to the number of surviving fungi, quantitative antifungal evaluation can be performed by using ATP emission intensity as an index. ATP emission intensity is an excellent index because it can measure only the number of viable bacteria with high sensitivity. The method for measuring the ATP emission intensity at this time is not particularly limited, but the nail sample and the plate medium were pulverized using a dental router, a homogenator, etc., and a surfactant or the like was added as necessary. When a fungal is extracted by adding a dispersion medium such as physiological saline, an ATP luminescence measuring reagent is added to the fungus, and the measurement can be easily performed by a measuring device such as a luminometer.
以下に実施例等を挙げて、本発明について更に詳細を加えるが、本発明がこれら実施例にのみ限定を受けないことは言うまでもない。 Hereinafter, the present invention will be described in further detail with reference to examples and the like, but it goes without saying that the present invention is not limited to these examples.
参 考 例 １
爪真菌症モデルの作製と評価：
約５ｍｍ×５ｍｍに加工したヒト爪をオートクレーブ滅菌して爪試料とした。０．２％リン酸水素二カリウム、０．００５％硫酸マグネシウム、０．００５％塩化カルシウムを添加した１Ｌの水にバクト・アガー１５ｇを加えて寒天塩培地を調製した。この寒天塩培地でトリコフィトン・メンタグロファイテス（TIMM1189株）をスラント培養し、０．０５％Tween80を加えた生理食塩水で分生子を掻き取り、ガーゼで濾過して菌体を除去し、分生子分散液とした。この分生子分散液を、分生子の分散量が1×１０４個/mLになるようRPMI１６４０液体培地で調整したものに上記爪試料を浸漬して約２８℃で７日間培養し、爪真菌症モデル用爪試料とした。また、オートクレーブ滅菌した後約５０℃に冷却した上記寒天塩培地に、上記分生子分散液を分生子の分散量が１×１０５個/mL（平板培地での最終濃度）になるように添加して、爪真菌症モデル用平板培地とした。上記爪真菌症モデル用爪試料を取り出し、表面に菌糸等が付着していた場合は軽く拭き取ってから、上記爪真菌症モデル用平板培地上に配置して爪真菌症モデルとした。この爪真菌症モデルを約２８℃で３５日間培養した。培養開始から２１日、２８日、３５日後に爪と平板培地を分離した。爪はミニルータを用い、平板培地はホモジナイザーを用いてそれぞれ粉砕し、０．０５%Tween80を加えた生理食塩水を爪および平板培地と同量加え、攪拌混合した。それぞれの懸濁液１００μLにATP発光測定試薬（BacTiter-GloTM Microbial Cell Viability Assay（Promega））１００μLを加え、ルミノメーターによりATP発光強度測定を行った。結果を表１に示す。
Reference example 1
Preparation and evaluation of onychomycosis model:
A human nail processed to about 5 mm × 5 mm was autoclaved to obtain a nail sample. An agar salt medium was prepared by adding 15 g of Bacto agar to 1 L of water supplemented with 0.2% dipotassium hydrogen phosphate, 0.005% magnesium sulfate and 0.005% calcium chloride. Slant culture of Trichophyton Mentagrophytes (TIMM1189 strain) on this agar salt medium, scrape the conidia with physiological saline added with 0.05% Tween 80, filter with gauze to remove the cells, A conidia dispersion was obtained. The above nail sample was immersed in RPMI 1640 liquid medium so that the conidia dispersion amount would be 1 × 10 4 cells / mL, and this nail sample was immersed and cultured at about 28 ° C. for 7 days. A model nail sample was used. In addition, the above conidia dispersion is added to the above-mentioned agar salt medium which has been sterilized by autoclave and cooled to about 50 ° C. so that the amount of conidia dispersed is 1 × 10 5 pieces / mL (final concentration in a plate medium). A plate medium for onychomycosis model was obtained. The nail sample for onychomycosis model was taken out, and when mycelium or the like adhered to the surface, it was gently wiped, and then placed on the above plate medium for onychomycosis model to obtain an onychomycosis model. This onychomycosis model was cultured at about 28 ° C. for 35 days. The nails and the plate medium were separated 21 days, 28 days and 35 days after the start of the culture. The nail was crushed using a mini-router, and the plate medium was homogenized using a homogenizer, and the same amount of physiological saline supplemented with 0.05% Tween 80 was added to the nail and the plate medium and mixed with stirring. 100 μL of ATP luminescence measurement reagent (BacTiter-Glo ™ Microbial Cell Viability Assay (Promega)) was added to 100 μL of each suspension, and ATP luminescence intensity was measured with a luminometer. The results are shown in Table 1.
比 較 参 考 例 １
オートクレーブ滅菌した約５ｍｍ×５ｍｍに加工したヒト爪を、参考例１で調製した爪真菌症モデル用平板培地上に配置し、約２８℃で３２日間培養した。培養開始から７，１４，２０，３２日後に爪と平板培地を分離し、参考例１と同様にしてそれぞれを粉砕して０．０５％Tween80を加えた生理食塩水及びATP発光測定試薬を加え、ルミノメーターによりATP発光強度測定を行った。結果を表２に示す。
Comparative Reference Example 1
The human nail processed into autoclave-sterilized about 5 mm × 5 mm was placed on the plate medium for onychomycosis model prepared in Reference Example 1 and cultured at about 28 ° C. for 32 days. After 7, 14, 20, and 32 days from the start of the culture, the nail and the plate medium were separated, and each sample was crushed in the same manner as in Reference Example 1, and then added with physiological saline and 0.05% Tween 80 and ATP luminescence measuring reagent. The ATP emission intensity was measured with a luminometer. The results are shown in Table 2.
予め爪試料を真菌に感染させた参考例１のモデルでは、爪及び平板培地におけるATP発光強度が培養期間にわたって増加傾向にあった。一方、爪試料を真菌に感染させていない比較参考例１のモデルでは、培養期間内に平板培地におけるATP発光強度の減少が認められた。このように参考例１のモデルでは、抗真菌製剤を投与しない限りATP発光強度が維持されることから、比較参考例１のモデルと比較して抗真菌剤又は抗真菌製剤を投与した際の薬剤の抗真菌効果をより正確に評価できることが明らかとなった。 In the model of Reference Example 1 in which the nail sample was previously infected with the fungus, the ATP emission intensity in the nail and the plate medium tended to increase over the culture period. On the other hand, in the model of Comparative Reference Example 1 in which the nail sample was not infected with fungus, a decrease in ATP emission intensity in the plate medium was observed within the culture period. As described above, in the model of Reference Example 1, ATP emission intensity is maintained unless an antifungal preparation is administered. Therefore, compared with the model of Comparative Reference Example 1, the drug when the antifungal agent or the antifungal preparation is administered. It became clear that the antifungal effect of can be evaluated more accurately.
実 施 例 １
参考例１で作製した爪真菌症モデルの爪試料上に、爪真菌症用ネイルラッカー剤であるロセリル（登録商標ガルデルマ）を、平板培地に漏れることのないよう適量塗布し、約２８℃で７日間培養した後、爪と平板培地を分離して、参考例１と同様にしてそれぞれ粉砕し、０．０５％Tween80を加えた生理食塩水及びATP発光測定試薬を添加して、ルミノメーターによりＡＴＰ発光強度測定を行った。結果を表３に示す。
Example 1
On the nail sample of the onychomycosis model prepared in Reference Example 1, an appropriate amount of loceryl (registered trademark Galderma), a nail lacquer agent for onychomycosis, was applied so as not to leak into the plate medium, and was applied at about 28 ° C. After culturing for a day, the nail and the plate medium were separated, ground in the same manner as in Reference Example 1, added with physiological saline supplemented with 0.05% Tween 80 and a reagent for measuring ATP luminescence, and ATP was obtained using a luminometer. Luminescence intensity was measured. The results are shown in Table 3.
比 較 例 １
実施例１の方法で作製した爪真菌症モデルを約２８℃で７日間培養した後、爪と平板培地を分離して、参考例１と同様にしてそれぞれ粉砕し、０．０５％Tween80を加えた生理食塩水及びATP発光測定試薬を添加して、ルミノメーターによりATP発光強度測定を行った。結果を表３に示す。
Comparative Example 1
After culturing the onychomycosis model produced by the method of Example 1 at about 28 ° C. for 7 days, the nail and the plate medium were separated and ground in the same manner as in Reference Example 1, and 0.05% Tween 80 was added. The physiological saline and the ATP luminescence measuring reagent were added, and ATP luminescence intensity was measured with a luminometer. The results are shown in Table 3.
実施例１では、爪真菌症の臨床上効果のある製剤を塗布した際に、比較例１に対してATP発光強度が顕著に減少しており、実態とよく適合していた。また定量的な比較が可能であった。 In Example 1, when a clinically effective preparation for onychomycosis was applied, the ATP emission intensity was significantly reduced compared to Comparative Example 1, and was well matched with the actual situation. In addition, a quantitative comparison was possible.
本発明の方法は、爪真菌症の実態に即し、かつ抗真菌剤の効果を定量的に評価できるため、爪真菌症に対する抗真菌剤の評価方法として有用なものである。
The method of the present invention is useful as a method for evaluating an antifungal agent against onychomycosis because the effect of the antifungal agent can be quantitatively evaluated in accordance with the actual state of onychomycosis.
Claims (3)
The antifungal evaluation method according to claim 2, wherein the fungal concentration of the liquid medium inoculated with the fungus is 1 x 10 2 to 1 x 10 7 cells / mL.
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| JP2001128696A (en) * | 1999-11-02 | 2001-05-15 | Pola Chem Ind Inc | Method for evaluating antifungal agent for nail |
| JP2007292481A (en) * | 2006-04-21 | 2007-11-08 | Tsumura & Co | Antifungal property evaluation method |
| US20100272783A1 (en) * | 2009-04-24 | 2010-10-28 | Novabay Pharmaceuticals, Inc. | Methods of Treating Infections of the Nail |
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| JP2001128696A (en) * | 1999-11-02 | 2001-05-15 | Pola Chem Ind Inc | Method for evaluating antifungal agent for nail |
| JP2007292481A (en) * | 2006-04-21 | 2007-11-08 | Tsumura & Co | Antifungal property evaluation method |
| US20100272783A1 (en) * | 2009-04-24 | 2010-10-28 | Novabay Pharmaceuticals, Inc. | Methods of Treating Infections of the Nail |
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