Activity of Oxantel Pamoate Monotherapy and Combination Chemotherapy against Trichuris muris and Hookworms: Revival of an Old Drug

Background It is widely recognized that only a handful of drugs are available against soil-transmitted helminthiasis, all of which are characterized by a low efficacy against Trichuris trichiura, when administered as single doses. The re-evaluation of old, forgotten drugs is a promising strategy to identify alternative anthelminthic drug candidates or drug combinations. Methodology We studied the activity of the veterinary drug oxantel pamoate against Trichuris muris, Ancylostoma ceylanicum and Necator americanus in vitro and in vivo. In addition, the dose-effect of oxantel pamoate combined with albendazole, mebendazole, levamisole, pyrantel pamoate and ivermectin was studied against T. muris in vitro and additive or synergistic combinations were followed up in vivo. Principal Findings We calculated an ED50 of 4.7 mg/kg for oxantel pamoate against T. muris in mice. Combinations of oxantel pamoate with pyrantel pamoate behaved antagonistically in vitro (combination index (CI) = 2.53). Oxantel pamoate combined with levamisole, albendazole or ivermectin using ratios based on their ED50s revealed antagonistic effects in vivo (CI = 1.27, 1.90 and 1.27, respectively). A highly synergistic effect (CI = 0.15) was observed when oxantel pamoate-mebendazole was administered to T. muris-infected mice. Oxantel pamoate (10 mg/kg) lacked activity against Ancylostoma ceylanicum and Necator americanus in vivo. Conclusion/Significance Our study confirms the excellent trichuricidal properties of oxantel pamoate. Since the drug lacks activity against hookworms it is necessary to combine oxantel pamoate with a partner drug with anti-hookworm properties. Synergistic effects were observed for oxantel pamoate-mebendazole, hence this combination should be studied in more detail. Since, of the standard drugs, albendazole has the highest efficacy against hookworms, additional investigations on the combination effect of oxantel pamoate-albendazole should be launched.


Introduction
Infections with the three major soil-transmitted helminth (STH) species, Ascaris lumbricoides, Trichuris trichiura and the hookworms Necator americanus and Ancylostoma duodenale are among the most common parasitic diseases in areas of rural poverty in developing countries [1]. In regions where soil-transmitted helminthiasis is endemic, preventive chemotherapy, i.e. regular anthelminthic drug administration to all people at risk of morbidity, is one of the key strategies [2]. In 2009 it was estimated that 204 million schoolaged children were treated for soil-transmitted helminthiasis [3]. The benzimidazoles, albendazole and mebendazole are the most widely used drugs in preventive chemotherapy programs. At present, two alternative drugs, pyrantel pamoate and levamisole are available but currently have a less prominent role since they require weight-based dosing [4]. Despite their excellent safety profile, these drugs have serious limitations with regard to their efficacy. When delivered as a single dose, as in preventive chemotherapy programs, all four compounds have a limited effect against infections with T. trichiura as shown in a recent metaanalysis [5]. In addition, drug resistance is a concern [4;6]. Efforts are therefore ongoing to discover and develop the next generation of anthelminthic drugs [7]. Promising strategies to identify potential anthelminthic drug candidates are to assess compounds derived from animal health, to re-evaluate forgotten compounds and to thoroughly study drug combinations [7,8].
Oxantel is the meta-oxyphenol analog of pyrantel. It was discovered in the early 1970s by Pfizer and showed high activity in T. muris-infected mice and T. vulpis-infected dogs [9,10]. Subsequent exploratory clinical trials demonstrated that the drug was safe and effective in the treatment of trichuriasis [11][12][13][14]. For example, complete cure was observed in 10 T. trichiura-infected patients treated with 20 mg/kg oxantel pamoate [11]. In veterinary medicine oxantel pamoate was later combined with pyrantel pamoate, which has, with the exception of activity against Trichuris spp., a broad spectrum of activity against different nematodes [15]. Today oxantel-pyrantel is widely available as a dewormer for dogs and cats. The combination of oxantel-pyrantel was also evaluated in a few clinical trials against human STH infections [13,[16][17][18][19]. For example, a decade ago oxantel-pyrantel (10 mg/kg) was tested in school-aged children on Pemba island. The combination achieved cure rates of 38.2% and 12.7% against infections with T. trichiura and hookworms, respectively [19]. To our knowledge, despite the interesting trichuricidal properties of oxantel, combinations of this drug with other recommended anthelminthic drugs have not been evaluated to date.
The aim of the present study was to investigate the trichuricidal potential of oxantel pamoate combined with the four WHO recommended anthelminthic drugs for the treatment of hookworm, T. trichiura and A. lumbricoides infections (albendazole, mebendazole, levamisole or pyrantel pamoate) as well as combinations of ivermectin and oxantel pamoate. Ivermectin, the first line drug for strongyloidiasis, is known to have trichuricidal properties and combinations of albendazole-ivermectin and mebendazole-ivermectin have been tested clinically [20]. In a first step the EC 50 (ED 50 ) values of oxantel pamoate against T. muris were determined in vitro and in vivo. Next to oral administration we also tested the activity of intraperitoneal oxantel pamoate in mice. We then elucidated whether oxantel pamoate combined with albendazole, mebendazole, ivermectin, levamisole or pyrantel pamoate interacts in an additive, antagonistic or synergistic manner in vitro using the combination index equation [21]. Additive and synergistic combinations were followed up in vivo. In addition, the activity of oxantel pamoate was studied against A. ceylanicum and N. americanus in vitro and in vivo.

Drugs
Albendazole and levamisole were purchased from Fluka (Buchs, Switzerland), oxantel pamoate, mebendazole, ivermectin and pyrantel pamoate were obtained from Sigma-Aldrich (Buchs, Switzerland). Note that, the pamoate salts of oxantel and pyrantel contain only 35.8% and 34.7% of the active ingredients, oxantel and pyrantel base, respectively.

Animals
Four week-old female C57BL/10 mice and 3 week-old male Syrian golden hamsters were purchased from Charles River (Blackthorn, UK and Sulzfeld, Germany, respectively). Before infection, animals were allowed to acclimatize for one week in our animal facility. They were kept in groups of maximum ten (mice) or three (hamsters) in macrolon cages with free access to water and rodent food pellets (Rodent Blox from Eberle NAFAG, Gossau, Switzerland).

Ethics statement
Experiments were performed in an attempt to comply with the 3R rules for animal experiments. The current study was approved by the cantonal veterinary office Basel-Stadt (Switzerland) based on Swiss cantonal and national regulations (permission no. 2070).

Parasites and infections
Trichuris muris. The life cycle of T. muris has been maintained at the Swiss TPH since January 2010 [22][23][24][25]. Mice were treated with dexamethasone (1 mg/l, dexamethasone-water soluble, Sigma-Aldrich) supplied with the drinking water 2 days before infection onwards and were infected orally with 200 embryonated T. muris eggs.
Ancylostoma ceylanicum and Necator americanus. The A. ceylanicum and N. americanus life cycles have been maintained at the Swiss TPH since June 2009 and April 2011, respectively, as described previously [25][26][27][28]. Hamsters were treated with 0.5 mg/ l dexamethasone in the drinking water, 2 days before infection onwards. They were infected orally with 150 L3 (A. ceylanicum) or subcutaneously with 250 L3 (N. americanus). Hamsters assigned to in vivo studies were not immunosuppressed and were infected with 300 L3.
Combination chemotherapy studies. Drug combination assays were carried out as described for single drug assays, with slight alterations. Three to 4 adult worms were transferred into

Author Summary
The roundworm Ascaris lumbricoides, the whipworm Trichuris trichiura and the two hookworm species Ancylostoma duodenale and Necator americanus are responsible for the most common infections worldwide and place more than 5 billion people at risk. To control these infections, at risk populations are treated regularly with anthelminthic drugs, mostly albendazole and mebendazole. Since both drugs have a low therapeutic effect against T. trichiura, alternative drugs should be discovered and developed. Possible strategies are to re-evaluate forgotten compounds and to thoroughly study drug combinations. We evaluated the activity of the ''old'', veterinary drug oxantel pamoate against T. muris, Ancylostoma ceylanicum and Necator americanus in vitro and in vivo. In addition, we studied the activity of oxantel pamoate combinations with the four standard treatments for soil-transmitted helminthiasis. Our results confirm that oxantel pamoate has excellent trichuricidal properties. We show that the drug lacks activity against hookworms. It is therefore necessary to combine oxantel pamoate with an anti-hookworm drug. Synergistic effects were observed with oxantel pamoate-mebendazole in our study. Additional preclinical studies should be launched with oxantel pamoate-mebendazole as well as oxantel pamoate-albendazole, since albendazole is the most widely used and efficacious anti-hookworm drug.
each well of a 48-well plate containing 500 ml pre-warmed supplemented RPMI medium. Then, 250 ml of the drug solution #1 and 250 ml of the drug solution #2 were added at a constant dose ratio based on the calculated IC 50 values (inhibitory concentration 50%) and 2-fold dilutions were carried out up and down. In more detail, the following combinations were tested: 2IC 50 :2IC 50 , IC 50 :IC 50 , 0.5IC 50 :0.5IC 50 and 0.25IC 50 :0.25IC 50 . Since for albendazole, mebendazole and ivermectin no IC 50 value could be calculated (IC 50 s.200 mg/ml) [29], a concentration of 400 mg/ml was selected as IC 50 value. A combination index (CI) was calculated to characterize the interaction of each combination: synergism (CI,1), antagonism (CI.1) and additive effect (CI = 1) [21].
Two to 3 adult worms, collected from the hamsters intestines (binocular, magnification 166), were incubated per well in 48-well plates for 72 hours in 1 ml supplemented HBSS medium and 10% v/v fetal calf serum containing oxantel pamoate dilutions (ranging from 0.1 to 100 mg/ml) at 37uC, 5% CO 2 . The motility was determined microscopically (magnification 206) using a viability scale ranging from 2 (normal viability, 100% motility) to 0 (death). Control worms were incubated with the highest DMSO concentration used in the test (2% v/v). Assays were conducted in triplicate.

In vivo studies
Trichuris muris. Each animal was checked for the presence of eggs in the stools on day 40 p.i. and assigned to treatment or control groups (n = 4 mice per group) and treated with a single oral drug dose on the following day. Oxantel pamoate was administered at 10 mg/kg, 5 mg/kg, 2.5 mg/kg and 1 mg/kg. Two groups of mice were treated intraperitoneally with 10 mg/kg oxantel pamoate and 10 mg/kg ivermectin. Expelled worms, recovered from stools collected for up to 72 hours after treatment, were counted. At dissection, worms remaining in the gut 7 days posttreatment were collected and counted. Worm burden arithmetic means were calculated for each treatment and control group. Worm burden reductions (WBRs) and worm expulsion rates (WERs) were calculated as described previously [25]. Drug combinations revealing synergism in vitro (CI,1) (oxantel pamoate-albendazole, oxantel pamoate-levamisole, oxantel pamoatemebendazole and oxantel pamoate-ivermectin), were tested in vivo using a constant dose ratio. The ratio of the ED 50 s (effective dose 50%) of each drug was chosen as starting dose (ED 50 :ED 50 ). If the treatment reduced the worm burden by more than 75% (threshold for additivity when the dose effect curves for both drugs are hyperbolic [21]), the drug doses were divided in half. ED 50 values of the partner drugs were 345 mg/kg for albendazole, 79 mg/kg for mebendazole (both values determined in the frame of the present work), 4 mg/kg for ivermectin, and 46 mg/kg levamisole [29].
Briefly, the fecal egg burden was established on days 21 and 22 p.i. (A. ceylanicum) and 46 and 47 (N. americanus) and treatment and control groups formed on the basis of arithmetic mean fecal egg burden. Hamsters were treated with a single oral dose of 10 mg/ kg oxantel pamoate on the following day. Animals left untreated served as controls. The complete stools were collected from each hamster for up to 48 hours posttreatment and searched for expelled worms (binocular, magnification 166). WERs were calculated.

Statistical analyses
All the data obtained were analyzed by Excel (Microsoft Office, 2007). In vitro data obtained from the individual motility assays were averaged and normalized to the controls. IC 50 s (medianeffect dose), defined as the concentration of a drug required to decrease the mean worm's motility to 50% at the 72 hour time point, were calculated with the CompuSyn software (CompuSyn, version 3.0.1). The combination index (CI) was calculated for the combination chemotherapy data with CompuSyn. To test the significance of the WBRs in vivo, the Kruskal-Wallis (several treatment doses vs. controls) or the Mann-Whitney U test (one treatment dose vs. control) was applied, using StatsDirect (version 2.4.5; StatsDirect Ltd; Cheshire, UK).

In vitro studies with T. muris
Oxantel monotherapy. Temporal drug effects of different oxantel pamoate concentrations over the incubation period of 72 hours are depicted in Figure 1. Exposure of T. muris L4 to 0.15 and 0.3 mg/ml oxantel pamoate achieved only a negligible effect (mean motilities of 76.7% (SD 631.3%) and 83.3% (SD 623.3%), respectively) on the worms 24-72 hours posttreatment. Incubation of T. muris L4 for 24-72 hours with 0.6-600 mg/ml oxantel pamoate resulted in strongly reduced viabilities within 24 hours but did not kill the worms. Control worms showed normal movements over the entire incubation period. We calculated an IC 50 of 2.35 mg/ml for oxantel pamoate (corresponding to 0.78 mg/ml for the free base oxantel) on T. muris L4 (Table 1).
Trichuris muris combination chemotherapy. Oxantel pamoate was combined with albendazole, mebendazole, pyrantel pamoate, ivermectin or levamisole using ratios based on their IC 50 s and T. muris adults were exposed simultaneously to one of these combinations. The results are presented in Table 1 and dose response relationships of the combinations depicted in Figure 2. Synergistic effects were observed for four of the combinations, namely oxantel pamoate-mebendazole (CI = 0.06), oxantel pamoate-ivermectin (CI = 0.27), oxantel pamoate-albendazole (CI = 0.37) and oxantel-pamoate levamisole (CI = 0.46). An antagonistic interaction was found when oxantel pamoate was combined with pyrantel pamoate (CI = 2.53). Worms exposed to this combination were only affected at the two highest concentration ratios (2IC 50 :2IC 50 and IC 50 :IC 50 ) and showed normal viability at the two lowest concentration ratios examined (0.5IC 50 :0.5IC 50 and 0.25IC 50 :0.25IC 50 ).

In vivo studies with Trichuris muris
Oxantel monotherapy. Oxantel pamoate displayed a high activity against T. muris in vivo, with an ED 50 of 4.71 mg/kg. In more detail, a WBR of 92.5% and WER of 88.4% were achieved after administration of 10 mg/kg ( Table 2). Administration of 5 mg/kg resulted in a WBR of 81.1% and a WER of 78.2%. A low activity was observed with oxantel pamoate at 2.5 mg/kg (WER = 24.3%, WBR = 13.5%) and no effect was observed when mice were treated with 1 mg/kg (WER = 1.5%, WBR = 0%). The worm burden in orally oxantel pamoate treated mice was significantly different from untreated mice (P = 0.041). An  intraperitoneal treatment of 10 mg/kg lacked activity against T. muris (both WER and WBR = 0%). For comparison, 10 mg/kg ivermectin given intraperitoneally resulted in a worm burden reduction of 93.5%. Combination chemotherapy. The four drug combinations that displayed synergistic effects in vitro were followed up in vivo ( Table 2). Simultaneous treatment of T. muris-infected mice with a combination of oxantel pamoate and albendazole using the approximate ED 50 doses resulted in a WBR of 76.6%, while combining 0.5ED 50 s was inefficacious (WBR = 0%). The combination was modeled as antagonistic (CI = 1.90). A synergistic interaction was found for the combination oxantel pamoatemebendazole, as illustrated by a combination index of 0.15. A WBR of 88.8% was achieved combining both drugs using the ED 50 doses and a still moderate WBR of 58.2% was observed when doses of 0.63 mg/kg oxantel pamoate and 10 mg/kg mebendazole (1/8 ED 50 s) were administered. Oxantel pamoate combined with ivermectin achieved a WBR of 84.8% at the highest dose tested (ED 50 :ED 50 (5 and 4 mg/kg) of oxantel pamoate and ivermectin, respectively), but the combination at 0.5ED 50 :0.5ED 50 only produced a worm burden reduction of 37.5%. The combination dose-effect analysis yielded antagonistic properties for the oxantel pamoate-ivermectin combination (CI = 1.27). Finally, while the combination of oxantel pamoate and levamisole at the ED 50 :ED 50 removed most of the worms (WBR = 82.0%, WER = 71.7%), using half of the dosage reduced the worm burden by less than 50% (WBR = 34.3%, WER = 31.1%). The overall behavior of the combination of oxantel pamoate and levamisole was found to be antagonistic (CI = 1.27).
In vivo studies with A. ceylanicum and N.
americanus. Oxantel pamoate exerted no effect on A. ceylanicum in vivo following a single dose treatment of 10 mg/kg, illustrated by a WER of 0% (data not shown). The same oral treatment (10 mg/ kg) in the N. americanus model resulted in a very low WER of 10.3% (data not shown).

Discussion
Since the introduction of albendazole, mebendazole, levamisole, and pyrantel pamoate in the human armamentarium to treat STH infections 3-4 decades ago, successes in the discovery and development of a novel nematocidal drug have been limited. The danger of resistance development therefore raises concern for the availability of effective therapies in the future. Furthermore, all four above-mentioned drugs have a limited activity against Trichuris spp when administered as single oral doses. To accelerate the discovery of novel anthelminthic treatments potential drug candidates have recently been examined in vitro, in vivo and in clinical trials. Disappointingly, nitazoxanide, a potential drug candidate identified through systematic literature searches [7] as well as a combination of albendazole and nitazoxanide revealed low trichuricidal activity in a randomized placebo controlled trial on Pemba [31]. Furthermore, monepantel, a safe nematocidal drug recently marketed for veterinary use showed a very poor activity against Ascaris suum and T. muris in vitro and in vivo [25]. Hence, neither nitazoxanide nor monepantel can be recommended for the treatment of infections with STH.
In the present work, another potential candidate, oxantel pamoate, widely used in veterinary medicine was evaluated against T. muris and hookworms in vitro and in vivo. Note that one limitation of our study (and helminth drug discovery in general), is that in vitro testing relied on motility scoring using microscopy, which is a subjective examination procedure [32].
Oxantel pamoate revealed an excellent trichuricidal activity in mice. We calculated an ED 50 of 4.7 mg/kg in T. muris-infected mice. A similarly low ED 50 of 1.7 mg/kg was reported previously in this model [33]. For comparison, the WHO recommended drugs for the treatment of STH infections are characterized by much higher ED 50 values against T. muris in vivo, namely 345 mg/ kg for albendazole, 79 mg/kg for mebendazole, 46 mg/kg for levamisole and .300 mg/kg for pyrantel pamoate [25,30]. Ivermectin, used in the treatment of strongyloidiasis and filarial infections, displayed a comparable ED 50 value of 4 mg/kg in our T. muris model [29]. A dose of 10 mg/kg oxantel pamoate administered intraperitoneally lacked activity in T. muris-infected mice. For comparison, the same i.p. dose of ivermectin resulted in a high reduction of the worm load (.93%). This demonstrates that in contrast to ivermectin oxantel pamoate does not kill the worm via the blood stream.
Oxantel pamoate lacked in vivo activity against both hookworm species A. ceylanicum and N. americanus. This finding is in line with a previous study in A. caninum-infected mice [34]. Interestingly, N. americanus adults were affected by the drug in vitro while no activity was observed on A. ceylanicum. To our knowledge, the activity of oxantel pamoate against hookworms has not been studied in humans.
Oxantel pamoate showed also no effect against the third major soil-transmitted helminth species, A. lumbricoides in humans (all 53 patients treated with oxantel revealed Ascaris eggs in the stools collected posttreatment regardless of the dose administered) [11]. It is therefore necessary to combine oxantel pamoate with a partner drug with a therapeutic profile that covers roundworms and hookworms. In the present work we have, for the first time, thoroughly evaluated the potential of oxantel pamoate in drug combinations. This work builds on a series of laboratory investigations on the potential of combination chemotherapy for the treatment of STH infections. We have for example recently examined combinations of marketed drugs in in vitro and in vivo studies against T. muris [8].
Interestingly, antagonistic effects were observed in the present work with oxantel pamoate-pyrantel pamoate against T. muris in vitro, hence this combination was not pursued further. However, we cannot exclude a better trichuricidal effect in vivo for this combination, in particular as a pharmacodynamic interference at  Table 2. Activity of oxantel pamoate monotherapy or combination chemotherapy against T. muris in vivo.  the target is unlikely. Oxantel is classified as an N-subtype AChR agonist, while pyrantel is considered an L-subtype suggesting differences in drug action [35]. The combination of oxantel pamoate-pyrantel pamoate is widely used in veterinary medicine and has also been studied in several human clinical trials. For example, in Korea oxantel pamoate-pyrantel pamoate at 20 mg/ kg achieved a cure rate of 75% and egg reduction rate of 97% against T. trichiura infections and cleared A. lumbricoides infections [13]. A high egg reduction rate against T. trichiura following oxantel pamoate-pyrantel pamoate at 20 mg/kg was also reported in a Malaysian study [18]. A lower effect of this combination administered at 10 mg/kg was observed on Pemba with cure rates of 96.3, 38.2 and 12.7% against A. lumbricoides, T. trichiura and hookworm, respectively [19]. In two Korean trials both oxantel monotherapy as well as an oxantel-pyrantel combination were used, however since different formulations were used (syrup versus tablets), different dosages applied and sample sizes were small no conclusion can be drawn whether the combination was superior to oxantel monotherapy [13,14]. Antagonistic effects were observed in vivo using combinations of oxantel pamoate-albendazole, oxantel pamoate-levamisole and oxantel pamoate-ivermectin. Since the molecular basis for the actions of these drugs is not yet fully elucidated it is impossible to explain the antagonistic interaction profile observed for these combinations. On the other hand, the oxantel pamoate-mebendazole combination revealed highly synergistic effects against T. muris in vivo. It is striking that the two benzimidazole derivates behave so differently when administered as partner drugs in oxantel pamoate combinations to T. muris infected mice given that both drugs, despite their differences in pharmacokinetics [36], have identical targets. However, our results should be interpreted with caution. First of all, drug scheduling and drug vehicle, solubility, host behavior, environmental factors and genetic variations might influence the level of activity [37]. In addition, though the median effect method used in the present work is the most commonly used, our data are based on a single method only and one could have considered applying another method, such as the isobologram method to re-analyze the data [38]. Finally, note that these findings are based on a single ratio of the combined agents (ED 50 values) and it might be worthwhile to assess other ratios of the drug dosages.
In conclusion, our study confirms that oxantel pamoate has excellent trichuricidal properties. In the T. muris mouse model oxantel pamoate showed a higher activity than the standard drugs albendazole, mebendazole, levamisole and pyrantel pamoate. Since the drug has no activity against hookworms it is necessary to combine oxantel pamoate with a partner drug revealing antihookworm properties. Synergistic effects were observed for oxantel pamoate-mebendazole. Despite of our results pointing to an antagonistic behavior of oxantel pamoate-albendazole additional investigations on the effect of this combination might be considered (e.g. evaluation of a different dosing ratio or schedule) since of the standard drugs albendazole has the highest efficacy against hookworms [5]. Systemic drug interactions between oxantel pamoate and partner drugs are unlikely given that the absorption of oxantel pamoate is very poor [11]. Nonetheless, preclinical studies should carefully elucidate metabolic and pharmacokinetic interactions of oxantel pamoate and the benzimidazoles.