Title Establishment of a novel model of onychomycosis in the rabbit for evaluation of antifungal ...

39 Pages·2011·3.74 MB·English

AAC Accepts, published online ahead of print on 9 May 2011
Antimicrob. Agents Chemother. doi:10.1128/AAC.00399-11
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Title
2 Establishment of a novel model of onychomycosis in the rabbit
3 for evaluation of antifungal agents
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5 Running title o
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6 Establishment of onychomycosis model using rabbits //
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8 Tsuyoshi Shimamura1, 3*, Nobuo Kubota1, Saori Nagasaka1, Taku Suzuki2, Hideki Mukai2, and n

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9 Kazutoshi Shibuya3 0
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11 R&D Laboratories, POLA PHARMA INC., 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812,1 t
12 Department of Dermatology, Toho University Ohashi Medical Center, 2-17-6 Ohashi, Meguro-ku,
13 Tokyo 153-8515,2 Department of Surgical Pathology, Toho University Omori Medical Center, 6-11-1
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1 Omori-nishi Ota-ku, Tokyo 143-8541, Japan3
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3 * Corresponding author. Mailing address: Tsuyoshi Shimamura, R&D Laboratories, POLA
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4 PHARMA INC., 560 Kashio-cho, Totsuka-ku, Yokohama 244-081, Japan. lo
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5 TEL: [phone]. FAX: [phone]. E-mail: [email protected]. o
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1 ABSTRACT
2 We developed a novel model of onychomycosis in which we observed the fungi in the deep layer of the
3 nail and used the model to evaluate the efficacy of two topical antifungal drugs. To establish an
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4 experimental, in vivo model of onychomycosis, we applied Trichophyton mentagrophytes TIMM2789 lo
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5 to the nails of the hind limbs of rabbits that underwent steroid treatment. The nails were taken from the o
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6 rabbits' feet at zero, two, and six weeks after a two-week infection. The localization of the fungi was //
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7 evaluated histopathologically. Some fungi were seen to penetrate to the nail bed and infection rate in m
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8 the sample at zero, two and six weeks after infection were 57%, 87% and 93%, respectively. In n

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9 addition, fungi proliferated and moved proximally into the nail plate in a manner that depended on the 0
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10 duration of infection. Secondly, using this model we evaluated antifungal efficacy, both by the culture b
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11 recovery method and histopathological examination. Two topical antifungal drugs, 8% ciclopirox nail t
12 lacquer and 5% amorolfine nail lacquer, were applied to the nail for four weeks in each group. On
13 histopathological examination, two antifungal treatment groups showed no significant difference
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1 against non-treated control group. However, there was a significantly low fungus-positive rate and
2 intensity of recovery of fungi on culture between antifungal treatment and non-treated control group.
3 We therefore suggest that we have established an in vivo model of onychomycosis that is useful for the
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4 evaluation of the efficacy of antifungal agents. lo
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1 INTRODUCTION
2 Onychomycosis is an intractable superficial mycosis, and oral administration of antifungal agents is
3 the main modality for clinical treatment (8). However, some of these oral antifungal drugs have
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4 well-known drug interactions with other medications, and these interactions limit their usage in older lo
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5 people and those living with diabetes or human immunodeficiency virus (2). These patients eagerly o
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6 await topical antifungal agents that are both effective and useful. //
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7 Tatsumi et al. (20) reported the evaluation of some antifungal agents using an in vivo experimental m
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8 model of onychomycosis but detailed histological examination was limited, and they do not describe n

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9 whether fungal infection occurred near the nail bed, as occurs in a clinical setting. Drug efficacy tests 0
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10 that used this model were useful in the evaluation of orally administered drugs, but not for topical b
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11 agents. Agents that are active following oral administration reach the nail bed via the circulation, and t
12 then diffuse towards the dorsal surface of nail. In consequence, if the fungal infection that occurs in this
13 animal model is only superficial, where the lowest concentration of active agent is thought to be
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1 following oral administration, drug efficacy can be evaluated adequately. In order to evaluate topical
2 drugs using this model, we need to confirm efficacy in the deeper layers of the nail.
3 Although there are reports that describe histological findings in human onychomycosis and that
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4 confirm the value of histopathological examination in making the diagnosis (3, 10, 16, 18), the route of lo
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5 infection and how the fungi behave in the nail plate have not been determined in detail. o
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6 The rate of morbidity in onychomycosis may be affected by age, smoking, peripheral arterial disease, //
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7 diabetes, smoking, and immunodeficiency (5, 14, 22). In particular, a multicenter survey (7) reports m
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8 that the administration of immunosuppressive agents to people with diabetes may be an important n

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9 factor that predisposes to onychomycosis. 0
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10 In this article, we here attempt to establish an animal model of onychomycosis in immunosuppressed b
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11 rabbits using Trichophyton mentagrophytes, a well-recognized and widely identified pathogen in t
12 rabbit (1, 9, 21, 24). We used histopathological examination and the culture recovery method to
13 evaluate the efficacy of 8% ciclopirox nail lacquer (Penlac®, Dermik Laboratories, Sanofi-Aventis,
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1 Bridgewater, NJ, USA) and 5% amorolfine nail lacquer (Loceryl®, Galderma, Lausanne, Switzerland)
2 known as topical onychomycosis treatment in our model.
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1 MATERIALS AND METHODS
2 In this article, the terminology we use in describing the histology of the nail tissue of the rabbit is
3 identical to that used in humans.
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5 Animals o
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6 Male Japanese white rabbits aged 14 weeks were purchased from KITAYAMA labes and used in this //
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7 study. The experiment to establish the animal onychomycosis model was performed in three groups m
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8 with five rabbits in each group, while the experiment that determined therapeutic efficacy was n

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9 performed in three groups with four rabbits in each group. The nails examined were on the first to third 0
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10 toes of the right and left hind paws, that is, six nails were used per animal. All experimental procedures b
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11 were evaluated and approved in accordance with the Institutional Animal Care and Use Committee t
12 (IACUC) of POLA.
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1 Test organism
2 T. mentagrophytes TIMM2789, isolated from the guinea pig, was purchased from Teikyo University
3 Institute of Medical Mycology, Tokyo, Japan.
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5 Preparation of inocula o
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6 Freeze-dried T. mentagrophytes was suspended in 1 ml of saline containing 0.05% Tween 80. A //
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7 volume of 0.05 ml of this suspension was seeded onto Fluid Sabouraud Dextrose agar, and T. m
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8 mentagrophytes was cultured at 28°C for two weeks in order to prepare microconidia. After n

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9 subculturing the fungi, microconidia of T. mentagrophytes were taken from the fungi in saline, and the 0
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10 suspension of microconidia was adjusted to give a concentration of 108 conidia/ml by counting with a b
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11 hemocytometer. t
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13 Production of onychomycosis
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1 Four mg/kg of methylprednisolone acetate was injected intramuscularly into the hind limb of each
2 rabbit once a week for four weeks until the end of the infection period. In some cases, the dosage of
3 steroid was decreased depending on the condition of the animal. Two weeks after starting steroid
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4 treatment, 0.2 ml of microconidia suspension was dripped onto the nail at a site between the lunula and lo
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5 the proximal nail fold. A gauze patch was then used to wrap together the nail plates of the first to third o
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6 toes of the hind paw. The treated toe nails were covered with a finger cot (that contained the first to //
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7 third toes) and 0.5 ml of sterile water was then injected into the finger cot to produce a culture m
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8 environment around the nail that was suitable for fungal growth. This condition was maintained for a n

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9 period of infection of two weeks, without any other intervention. The finger cot and the gauze patch 0
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10 were removed after two weeks of exposure, and this condition was maintained for zero, two or six b
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11 weeks without finger cot and gauze patch, as the “post-infection period”. After each post-infection t
12 period was completed, the animals were sacrificed and the nails were taken from the treated paw for
13 histopathological and microbiological examination.
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