In Vitro and In Vivo Efficacy of Monepantel (AAD 1566) against Laboratory Models of Human Intestinal Nematode Infections Lucienne Tritten1,2, Angelika Silbereisen1,2, Jennifer Keiser1,2* 1 Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland, 2 University of Basel, Basel, Switzerland Abstract Background: Few effective drugs are available for soil-transmitted helminthiases and drug resistance is of concern. In the present work, we tested the efficacy of the veterinary drug monepantel, a potential drug development candidate compared to standard drugs in vitro and in parasite-rodent models of relevance to human soil-transmitted helminthiases. Methodology: A motility assay was used to assess the efficacy of monepantel, albendazole, levamisole, and pyrantel pamoate in vitro on third-stage larvae (L3) and adult worms of Ancylostoma ceylanicum, Necator americanus and Trichuris muris. Ancylostoma ceylanicum-orN. americanus-infected hamsters, T. muris-orAscaris suum-infected mice, and Strongyloides ratti-infected rats were treated with single oral doses of monepantel or with one of the reference drugs. Principal Findings: Monepantel showed excellent activity on A. ceylanicum adults (IC50 = 1.7 mg/ml), a moderate effect on T. muris L3 (IC50 = 78.7 mg/ml), whereas no effect was observed on A. ceylanicum L3, T. muris adults, and both stages of N. americanus. Of the standard drugs, levamisole showed the highest potency in vitro (IC50 = 1.6 and 33.1 mg/ml on A. ceylanicum and T. muris L3, respectively). Complete elimination of worms was observed with monepantel (10 mg/kg) and albendazole (2.5 mg/kg) in A. ceylanicum-infected hamsters. In the N. americanus hamster model single 10 mg/kg oral doses of monepantel and albendazole resulted in worm burden reductions of 58.3% and 100%, respectively. Trichuris muris, S. ratti and A. suum were not affected by treatment with monepantel in vivo (following doses of 600 mg/kg, 32 mg/kg and 600 mg/kg, respectively). In contrast, worm burden reductions of 95.9% and 76.6% were observed following treatment of T. muris- and A. suum infected mice with levamisole (200 mg/kg) and albendazole (600 mg/kg), respectively. Conclusions/Significance: Monepantel reveals low or no activities against N. americanus, T. muris, S. ratti and A. suum in vivo, hence does not qualify as drug development candidate for human soil-transmitted helminthiases. Citation: Tritten L, Silbereisen A, Keiser J (2011) In Vitro and In Vivo Efficacy of Monepantel (AAD 1566) against Laboratory Models of Human Intestinal Nematode Infections. PLoS Negl Trop Dis 5(12): e1457. doi:10.1371/journal.pntd.0001457 Editor: James S. McCarthy, Queensland Institute for Medical Research, Australia Received March 9, 2011; Accepted November 16, 2011; Published December 27, 2011 Copyright: ß 2011 Tritten et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Jennifer Keiser is grateful to the Swiss National Science Foundation for financial support (project no. PPOOA-114941) (http.www.snf.ch). The work on Necator was supported by the Institute of One World Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction isole, and ivermectin), all of which have been registered for human use before or during the 1980’s [8,9]. No new anthelmintic drug The hookworm species Ancylostoma duodenale and Necator for human use has reached the market since then. Moreover, none americanus, the whipworm Trichuris trichiura, the threadworm of these drugs are efficacious using single doses on all STH species, Strongyloides stercoralis, and the roundworm Ascaris lumbricoides are with particularly low efficacy observed on T. trichiura [10]. Relying soil-transmitted helminths (STH) of great public health impor- on only a handful of drugs is a precarious situation, in the light of a tance. Cumulatively, these parasites affect more than one billion possible emergence of drug resistance [10]. people globally, particularly in developing regions of Asia, Africa, Since drug resistance to nematodes of veterinary importance is and Latin America [1,2]. If untreated, infections with STH are widely spread and increasing in frequency, most of the present for years and patients suffer from moderate to severe anthelmintic drug research and development efforts are motivated intestinal disturbances, anemia, nutrient loss and profound by veterinary needs [11]. For example, albendazole, mebendazole, physical and mental deficiencies [3,4]. and pyrantel pamoate were originally developed for livestock and Helminth control relies primarily on the regular administration pets [12]. of anthelmintics, typically carried out within the framework of Monepantel (AAD1566) belongs to a new class of veterinary school-based deworming programs, once or twice a year [5–7]. anthelmintics, the amino-acetonitrile derivatives. It has been Five drugs are currently available for the treatment of infections proposed that monepantel interferes with nematode-specific with STH (albendazole, mebendazole, pyrantel pamoate, levam- acetylcholine receptor subunits, leading to body wall muscle www.plosntds.org 1 December 2011 | Volume 5 | Issue 12 | e1457 Nematocidal Activity of Monepantel Author Summary For the in vivo studies, drugs were suspended in 7% (v/v) Tween 80% and 3% (v/v) ethanol or DMSO/PEG shortly before Soil-transmitted helminthiases affect more than one billion treatment. people among the most vulnerable populations in developing countries. Currently, control of these infections Animals and Parasites primarily relies on chemotherapy. Only five drugs are Three-week-old male Syrian Golden hamsters were purchased available, all of which have been in use for decades. None from Charles River (Sulzfeld, Germany). Four-week-old female of the drugs are efficacious using single doses against all NMRI mice and 3-week-old female C57Bl/6J mice were soil-transmitted helminths (STH) species and show low purchased from Harlan (Horst, The Netherlands). Three-week- efficacy observed against Trichuris trichiura. In addition, the old female Wistar rats were purchased from Harlan (Horst, The limited availability of current drug treatments poses a Netherlands). precarious situation should drug resistance occur. There- fore, there is great interest to develop novel drugs against All animals were kept in macrolon cages under environmental- infections with STH. Monepantel, which belongs to a new ly-controlled conditions (temperature: 25uC, humidity: 70%, class of veterinary anthelmintics, the amino-acetonitrile light/dark cycle 12 h/12 h) and had free access to water and derivatives, might be a potential drug candidate in rodent food (Rodent Blox from Eberle NAFAG, Gossau, humans. It has been extensively tested against livestock Switzerland). They were allowed to acclimatize in the animal nematodes, and was found highly efficacious and safe for facility of the Swiss Tropical and Public Health Institute (Swiss animals. Here we describe the in vitro and in vivo effect of TPH) for 1 week before infection. The current study was approved monepantel, on Ancylostoma ceylanicum, Necator amer- by the local veterinary agency based on Swiss cantonal and icanus, Trichuris muris, Strongyloides ratti, and Ascaris suum, national regulations (permission no. 2070). five parasite-rodent models of relevance to human STH. Since we observed that monepantel showed only high Parasites and Infections activity on one of the hookworm species and lacked Ancylostoma ceylanicum third-stage larvae (L3) were kindly activity on the other parasites tested we cannot recom- provided by Prof. J. M. Behnke (University of Nottingham). The mend the drug as a development candidate for human A. ceylanicum life cycle [23] had been maintained at the Swiss TPH soil-transmitted helminthiases. since June 2009 [17]. To maintain the life cycle, hamsters were treated orally 1 day before infection and then twice weekly with paralysis and subsequent death of worms. Due to its unique mode 3 mg/kg hydrocortisone (HydrocortoneH, MSD) or with 1 mg/l of action, the drug has proven efficacy against nematodes infecting dexamethasone (dexamethasone water-soluble, Sigma-Aldrich) in livestock which are resistant to current anthelmintic drugs [13]. the drinking water. They were orally infected with 150 A. Monepantel has been extensively tested on different nematode ceylanicum L3, which had been harvested less than 1 month before isolates. Administered at a single oral dose of 2.5 mg/kg, it was infection and had been assessed microscopically for viability. found to be safe, well-tolerated by ruminant hosts, and showed Animals assigned to in vivo studies were not treated with high cure rates on fourth stage larvae and adult worms of 15 hydrocortisone and were infected with 300 L3. nematode species [13–16]. Due to its high and broad nematocidal Infective N. americanus L3 were the gift of Prof. S. H. Xiao activity, monepantel was considered to be a candidate for a human (National Institute for Parasitic Diseases, Shanghai). Hamsters health directed program. were immunosuppressed with dexamethasone as described above The aim of the present