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. 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- or N. americanus-infected hamsters, T. muris- or Ascaris suum-infected mice, and Strongyloides ratti-infected rats were treated with single oral doses of monepantel or with one of the reference drugs. Monepantel showed excellent activity on A. ceylanicum adults (IC50 = 1. 7 µg/ml), a moderate effect on T. muris L3 (IC50 = 78. 7 µg/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 µg/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. 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.
The hookworm species Ancylostoma duodenale and Necator americanus, the whipworm Trichuris trichiura, the threadworm Strongyloides stercoralis, and the roundworm Ascaris lumbricoides are soil-transmitted helminths (STH) of great public health importance. Cumulatively, these parasites affect more than one billion people globally, particularly in developing regions of Asia, Africa, and Latin America [1], [2]. If untreated, infections with STH are present for years and patients suffer from moderate to severe intestinal disturbances, anemia, nutrient loss and profound physical and mental deficiencies [3], [4]. Helminth control relies primarily on the regular administration of anthelmintics, typically carried out within the framework of school-based deworming programs, once or twice a year [5]–[7]. Five drugs are currently available for the treatment of infections with STH (albendazole, mebendazole, pyrantel pamoate, levamisole, and ivermectin), all of which have been registered for human use before or during the 1980' s [8], [9]. No new anthelmintic drug for human use has reached the market since then. Moreover, none of these drugs are efficacious using single doses on all STH species, with particularly low efficacy observed on T. trichiura [10]. Relying on only a handful of drugs is a precarious situation, in the light of a possible emergence of drug resistance [10]. Since drug resistance to nematodes of veterinary importance is widely spread and increasing in frequency, most of the anthelmintic drug research and development efforts are motivated by veterinary needs [11]. For example, albendazole, mebendazole, and pyrantel pamoate were originally developed for livestock and pets [12]. Monepantel (AAD1566) belongs to a new class of veterinary anthelmintics, the amino-acetonitrile derivatives. It has been proposed that monepantel interferes with nematode-specific acetylcholine receptor subunits, leading to body wall muscle paralysis and subsequent death of worms. Due to its unique mode of action, the drug has proven efficacy against nematodes infecting livestock which are resistant to current anthelmintic drugs [13]. Monepantel has been extensively tested on different nematode isolates. Administered at a single oral dose of 2. 5 mg/kg, it was found to be safe, well-tolerated by ruminant hosts, and showed high cure rates on fourth stage larvae and adult worms of 15 nematode species [13]–[16]. Due to its high and broad nematocidal activity, monepantel was considered to be a candidate for a human health directed program. The aim of the present investigation was to study the activity of monepantel, compared with the reference drugs, albendazole, levamisole, and pyrantel pamoate, in five parasite-rodent models, that correspond to important human STH and in vitro. Ancylostoma ceylanicum and Necator americanus were both adapted using eggs from infected dogs or humans to an unnatural host, the golden hamster, and represent robust rodent models for hookworm infections [17], [18]. The Trichuris muris mouse model is an excellent model for trichuriasis [19], [20]. Strongyloides ratti in rats is a commonly used murine model for strongyloidiasis [21]. Finally, murine infection with Ascaris suum is a model which mimics the early infection of A. lumbricoides. The survival of different larval stages and adult worms of A. ceylanicum, N. americanus, and T. muris was evaluated in vitro, following monepantel incubation using a motility assay. The in vitro activity of monepantel on S. ratti has been described recently [22]. In vivo, we studied worm burden reductions and for A. ceylanicum, N. americanus, and T. muris, worm expulsion rates were also measured.
Monepantel was kindly provided by Novartis Animal Health, St-Aubin, Switzerland. Albendazole and pyrantel pamoate were purchased from Sigma-Aldrich (Buchs, Switzerland), and levamisole-hydrochloride from Fluka (Buchs, Switzerland). For the in vitro studies, stock solutions of the drugs were prepared in 100% DMSO (Fluka, Buchs, Switzerland) and stored at 4°C. For the in vivo studies, drugs were suspended in 7% (v/v) Tween 80% and 3% (v/v) ethanol or DMSO/PEG shortly before treatment. Three-week-old male Syrian Golden hamsters were purchased from Charles River (Sulzfeld, Germany). Four-week-old female NMRI mice and 3-week-old female C57Bl/6J mice were purchased from Harlan (Horst, The Netherlands). Three-week-old female Wistar rats were purchased from Harlan (Horst, The Netherlands). All animals were kept in macrolon cages under environmentally-controlled conditions (temperature: 25°C, humidity: 70%, light/dark cycle 12 h/12 h) and had free access to water and rodent food (Rodent Blox from Eberle NAFAG, Gossau, Switzerland). They were allowed to acclimatize in the animal facility of the Swiss Tropical and Public Health Institute (Swiss TPH) for 1 week before infection. The current study was approved by the local veterinary agency based on Swiss cantonal and national regulations (permission no. 2070). Ancylostoma ceylanicum third-stage larvae (L3) were kindly provided by Prof. J. M. Behnke (University of Nottingham). The A. ceylanicum life cycle [23] had been maintained at the Swiss TPH since June 2009 [17]. To maintain the life cycle, hamsters were treated orally 1 day before infection and then twice weekly with 3 mg/kg hydrocortisone (Hydrocortone®, MSD) or with 1 mg/l dexamethasone (dexamethasone water-soluble, Sigma-Aldrich) in the drinking water. They were orally infected with 150 A. ceylanicum L3, which had been harvested less than 1 month before infection and had been assessed microscopically for viability. Animals assigned to in vivo studies were not treated with hydrocortisone and were infected with 300 L3. Infective N. americanus L3 were the gift of Prof. S. H. Xiao (National Institute for Parasitic Diseases, Shanghai). Hamsters were immunosuppressed with dexamethasone as described above and were infected subcutaneously with 250 viable N. americanus L3. Embryonated T. muris eggs were kindly obtained from Prof. J. M. Behnke and Prof. H. Mehlhorn. The life cycle had been maintained at the Swiss TPH since January 2010 as described elsewhere [19]. Briefly, T. muris eggs were evaluated for embryonation under the microscope (magnification 80–160×, Carl Zeiss, Germany). NMRI mice were orally infected with 400 embryonated eggs. Mice were treated either subcutaneously (s. c.) 1 day before infection and then every second day between days 5 and 15 with 15 mg hydrocortisone (Hydrocortisone 21-hemisuccinate sodium salt, Sigma-Aldrich) in 0. 9% NaCl solution, or with 8 mg/l dexamethasone in the drinking water until the end of the experiment. The S. ratti life cycle had been maintained over decades at the Swiss TPH, by serial passage through rats. Rats were infected subcutaneously with 735 freshly harvested S. ratti L3. Infective A. suum eggs were obtained from Prof. S. M. Thamsborg, University of Copenhagen and Prof. G. Cringoli, University of Naples. Briefly, C57Bl/6J mice were orally infected with 500 embryonated eggs, according to a procedure described elsewhere [24]. The larval or adult motility assay is currently the method of choice to evaluate drug sensitivity of different nematode species [25]–[27]. Non-motile worms were considered as dead and the percent viability or survival in each well was calculated. The average of motility scores for one drug was calculated for each concentration and normalized into percentage, relative to control. IC50 values were expressed based on the median effect principle using CompuSyn (version 1. 0). The r value represents the linear correlation coefficient of the median-effect plot, indicating the goodness of fit, hence the accuracy of the IC50 [33]. Variance analysis in the ovicidal activity studies was performed with the Fisher' s exact test, using StatsDirect (version 2. 4. 5; StatsDirect Ltd; Cheshire, UK). The worm burden reductions were determined by comparing the mean number of adult worms in the intestine of a treated group with the mean numbers of worms in the control group. Means and standard deviations were calculated using Microsoft® Excel 2003. The worm expulsion rates were calculated by dividing the number of expelled worms of a treatment group by the group' s total worm burden. The Kruskal-Wallis test and the Mann-Whitney U test were used to assess the statistical significance of the worm burden reduction, using StatsDirect.
The effects of monepantel and reference drugs on L3 and adult worms of A. ceylanicum and T. muris after 72 h of exposure in vitro are presented in Table 1.
To date, only five drugs are included in the WHO model list of essential medicines to treat infections with human STH. Most of these anthelmintics were discovered before the 1980s. Though there is no evidence yet for emerging resistance to any of these drugs in human helminth populations, there are worrying signs that anthelminthic efficacy may be declining [34], [35]. In addition, the increased frequency of reported low cure rates, in particular against T. trichiura and hookworm infections, highlight the need to find alternative drugs [10]. A. ceylanicum, N. americanus, T. muris, S. ratti, and A. suum are five well-established laboratory parasite-rodent models of relevance to human STH. The aim of the present study was to determine their sensitivities to monepantel, a broad spectrum and safe drug used for livestock which recently entered the market for veterinary use. It is one of the few available drug candidates eligible for rapid transitioning into development for human STH infections [36]. Monepantel activates signaling via nematode-specific DEG-3 subtype nicotinic acetylcholine receptors (nAChRs), causing a hypercontraction of the body wall muscles leading to paralysis and hence, death of the worm [13]. ACR-23 protein, a member of the DEG-3 group in Caernohabtitis elegans, and its homolog MPTL-1 in Haemonchus contortus, another model for gastrointestinal nematodes, are major targets of monepantel. The absence of MPTL-1, observed in some nematode species resulted in reduced drug sensitivity [22]. Ancylostoma ceylanicum adult worms were found to be highly sensitive to monepantel in vitro, in contrast to the third-stage larvae, but the drug lacked ovicidal activity. Hamsters harboring adult A. ceylanicum were cleared from the worms following a 10 mg/kg single oral dose of monepantel. N. americanus was not affected by the drug in vitro and only moderately susceptible in vivo to 10 mg/kg or higher doses. These findings suggest a relative stage and species specificity, which might be explained by the absence of a functional MPTL-1 homolog in A. ceylanicum L3 and possibly in L3 and adult N. americanus. Trichuris muris third-stage larvae were only moderately sensitive to monepantel after incubation for 72 h in vitro, whereas adult stages were not affected, neither in vitro nor in vivo. Monepantel had already been reported to lack activity against T. ovis, a minor parasite of sheep [15], [16], a finding that is in accordance with our data. In addition, monepantel lacked activity in S. ratti-infected rats, a result in line with a recent investigation, which revealed that S. ratti third-stage larvae were not affected by monepantel after 72 h of incubation [22]. For comparison, a complete elimination of adult worms was achieved with ivermectin (0. 5 mg/kg) in S. ratti-infected rats [32]. Strongyloides ratti has a remote homolog of DES-2 and ACR-23/MPTL-1 only, which is not targeted by monepantel [22]. Finally, although only one high dosage was tested, our data indicate that A. suum is not affected by treatment with monepantel in vivo, whereas albendazole reduced the worm burden of A. suum in mice at the same dose. Like H. contortus, A. ceylanicum and N. americanus are members of the nematode clade V, whereas S. ratti belongs to clade IV, A. suum, to clade III, and T. muris to clade I [37]. One could hypothesize that only clade V species exhibit sensitivity to monepantel, whereas those that diverged from this lineage of the evolutionary tree earlier (clades I to IV) might not have evolved homologous receptors. As available for A. suum [38], further genome sequencing of A. ceylanicum, N. americanus and Trichuris spp. remains to be performed in order to extend current knowledge about evolutionary and functional relationships of receptors involved in sensitivity to monepantel. In the present investigation, albendazole, levamisole, and pyrantel pamoate have been extensively studied in vitro and in vivo. The results obtained are in agreement with earlier in vivo [29], [39]–[41] and in vitro [41] work using A. ceylanicum and N. americanus. In addition, to our knowledge, the in vitro and in vivo sensitivities of these three drugs against T. muris are presented for the first time. In line with human efficacy data [10], albendazole showed highly potent activity against A. ceylanicum, N. americanus and A. suum, yet much less pronounced activity against T. muris in vivo. A similar trend was observed for pyrantel pamoate, which achieved a moderate effect against A. ceylanicum but lacked activity in T. muris-infected mice. These results on pyrantel pamoate are compatible with cure rates reported in clinical trials [10]. On the other hand, levamisole was highly efficacious in our ancylostomiasis and trichuriasis rodent models, while low to moderate cure rates have been recently reported in humans [10], [42]. Interestingly, contradictory results were obtained with albendazole, levamisole, and pyrantel pamoate against adult A. ceylanicum in vitro and in vivo. In addition, albendazole showed excellent activity in the N. americanus hamster model but lacked activity in vitro. This finding might be partially explained by the presence of active metabolites, since for example albendazole and levamisole are rapidly metabolized in vivo [43]–[45]. In addition, large differences in sensitivity between larval and adult hookworm stages were observed with levamisole (and albendazole for A. ceylanicum). It is commonly accepted that the benzimidazoles tend to be lethal to developing stages but not always to adult worms. Developing cells are obviously more harmed by the benzimidazoles, as the utilization of tubulin in the mitotic cycles is affected [46]. In conclusion, to our knowledge, we have for the first time analyzed the efficacy of monepantel in animal models corresponding to human intestinal helminthiases. A recently developed target product profile suggested that a drug development candidate for the treatment of infections with STH should ideally target all stages (at least adult and ova) and species of the major geohelminths such as Ascaris, Trichuris, both hookworm species and Enterobius [36]. Hence, based on our results, established in nematode-rodent models, monepantel does not fulfill the required minimal product characteristics for a new intestinal anthelmintic.
Soil-transmitted helminthiases affect more than one billion people among the most vulnerable populations in developing countries. Currently, control of these infections primarily relies on chemotherapy. Only five drugs are available, all of which have been in use for decades. None of the drugs are efficacious using single doses against all soil-transmitted helminths (STH) species and show low efficacy observed against Trichuris trichiura. In addition, the limited availability of current drug treatments poses a precarious situation should drug resistance occur. Therefore, there is great interest to develop novel drugs against infections with STH. Monepantel, which belongs to a new class of veterinary anthelmintics, the amino-acetonitrile derivatives, might be a potential drug candidate in humans. It has been extensively tested against livestock nematodes, and was found highly efficacious and safe for animals. Here we describe the in vitro and in vivo effect of monepantel, on Ancylostoma ceylanicum, Necator americanus, Trichuris muris, Strongyloides ratti, and Ascaris suum, five parasite-rodent models of relevance to human STH. Since we observed that monepantel showed only high activity on one of the hookworm species and lacked activity on the other parasites tested we cannot recommend the drug as a development candidate for human soil-transmitted helminthiases.
medicine
infectious diseases
soil-transmitted helminths
trichuriasis
hookworm infection
parasitic diseases
helminth infection
In Vitro and In Vivo Efficacy of Monepantel (AAD 1566) against Laboratory Models of Human Intestinal Nematode Infections