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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Praziquantel-based morbidity control is the current mainstay of schistosomiasis control in high burden areas with the objective to regularly treat at least 75% of school-age children and other high-risk groups by 2010.5,6 Discovered in the mid-1970s,7 praziquantel has been widely and effectively used against schistosomiasis; >100 million doses have been administered in China and Egypt alone.8,9 Interestingly, unexpectedly low cure rates have been reported from S. mansoni–infected patients in Egypt and Senegal who were treated with recommended single oral doses of 40 mg/kg praziquantel.10,11 The pressing need to develop alternative anti-schistosomal drugs, while praziquantel remains effective, has been stressed.3,12
Triclabendazole is a safe and efficacious drug for the treatment of fascioliasis.13 Triclabendazole has also been tested against other major trematodes, including S. mansoni.13 Exposure of S. mansoni worms in vitro to a concentration of 15 µg/mL triclabendazole resulted in immediate contraction, destruction of the tegument, and death of the worms within 24 hours.14 However, results in the S. mansoni-mouse model are conflicting. On the one hand, a high worm burden reduction of 84% and cessation of oviposition have been reported after oral administration of 120 mg/kg triclabendazole to S. mansoni–infected mice.15 On the other hand, no effect on S. mansoni was observed when 500 ppm triclabendazole was added to standard rodent diet and given for 7 days.16 Interestingly, epidemiologic studies are underway in Egypt to assess the effect of triclabendazole administered to patients co-infected with Fasciola spp. and S. mansoni.17
The aim of this study was to assess the effect of triclabendazole and its two main metabolites, triclabendazole sulphone and triclabendazole sulphoxide, against S. mansoni harbored in mice. We used two different parasite strains. As a benchmark, praziquantel was also administered. Drug efficacy was evaluated on the basis of worm burden reduction, excretion and hatchability of eggs, tissue egg load, and oogram patterns.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice, parasites, and infection. All animal studies presented here had been approved by the local government based on national regulations. Female NMRI mice (N = 25, age: ~5 weeks, weight: ~24 g) and male CD-1 Swiss albino mice (N = 101, age: ~4 weeks, weight: ~20 g) were purchased from RCC (Itingen, Switzerland) and the Schistosome Biologic Supply Center, Theodore Bilharz Research Institute (Giza, Egypt), respectively. Mice were kept in groups of five under environmentally controlled conditions (temperature: ~25°C; humidity: ~70%; 12 hour light/dark cycle) and acclimatized for 1 week. They had free access to water and food.
Cercariae of S. mansoni (Liberian strain) were obtained from infected Biomphalaria glabrata following routine procedures at the laboratories of the Swiss Tropical Institute. Cercariae of S. mansoni (Egyptian/CD strain) were purchased from the Schistosome Biologic Supply Center. Female NMRI mice were infected subcutaneously with 80 S. mansoni cercariae (Liberian strain), and male CD-1 mice were infected with 80 S. mansoni cercariae (Egyptian/CD strain) using the body immersion technique.
Treatment. Two sets of experiments were carried out at the High Institute of Public Health (Alexandria, Egypt) using an Egyptian S. mansoni strain. First, 15 mice were treated with a single oral dose of 120 mg/kg triclabendazole 3 days before S. mansoni infection. Second, mice with a 7-week-old adult S. mansoni infection were divided into four groups of 14 mice each and treated with triclabendazole at a single oral dose of either 120 or 200 mg/kg or a double dose on consecutive days at 120 or 200 mg/kg daily. As a benchmark, two groups of eight mice each received single or double doses of 500 mg/kg praziquantel, respectively. One group of 14 infected mice remained untreated and hence served as a control.
A third set of experiments was carried out at the Swiss Tropical Institute using a Liberian strain of S. mansoni. Seven weeks after infection, three groups of five mice each were treated with single oral doses of triclabendazole, triclabendazole sulphone, and triclabendazole sulphoxide at 400 mg/kg. Another group of five mice received a single oral dose of 400 mg/kg praziquantel, whereas the fifth group of mice remained untreated and served as a control.
Fecal egg counts and hatching of S. mansoni eggs. Five weeks after infection, fecal sampling from mice infected with an Egyptian strain of S. mansoni commenced and lasted until necropsy. Fecal samples were collected from each group of mice over a 2-hour period daily until S. mansoni eggs could be detected. Subsequently fecal sampling was done twice a week. Fecal samples were suspended in 2–4 mL of saline, and six aliquots of 50 µL were examined microscopically. The number of eggs per gram (epg) of feces was determined by multiplying the average number of eggs in each 50-µL sample by the total volume of the saline suspension and dividing this value by the weight of the sample in grams.
To assess the miracidial hatchability of eggs, stool samples were collected from each group of mice over a 2-hour period, suspended in 10 mL of saline, and centrifuged at 1,000 rpm for 1 minute. After washing with saline, the sediment was re-suspended in 10 mL dechlorinated water and pored into a 1-L measuring flask. After adding 1 L of dechlorinated water, egg hatching was stimulated through exposure to artificial light.
Evaluation of the worm burden. Mice from the first two experimental series were necropsied 11 weeks after infection, and worms were recovered from the hepatic and portomesenteric vessels using the perfusion technique.18 The small and large intestines were placed in a Petri dish, and all S. mansoni in the mesenteric veins were removed, sexed, and counted under a stereoscopic microscope.
In the third set of experiments, mice were killed 11 weeks after infection and dissected by removing the liver and small and large intestines. The liver was placed into a 20 x 20-cm transparent plastic folder and gently compressed between two glass plates, and the parenchyma was examined at a 10-fold magnification under a stereoscopic microscope. S. mansoni were sexed and counted. The small and large intestines were put in a Petri dish, and all S. mansoni in the mesenteric veins were removed, sexed, and counted under a stereoscopic microscope.
Oogram pattern and tissue egg load. Three fragments of the small intestine (from the middle part of the small intestine) were cut longitudinally, washed with saline, compressed between two microscope slides, and examined under a low-power microscope. A total of 100 eggs per animal were observed, and the stage of each egg and the mean number of the different stages were recorded.19
The tissue egg load was determined as follows. First, 0.3 g of liver and small intestine was taken from each mouse and digested overnight in 5 mL KOH (5%). Second, after complete digestion, the samples were vortexed, and three aliquots of 100 µL each were examined microscopically. All S. mansoni eggs were counted. The hepatic and intestinal tissue egg loads (expressed as epg) were determined by multiplying the number of eggs in each 100-µL sample by the total volume of KOH and dividing this value by the weight of the sample in grams.20
Statistical analysis. Drug efficacy was assessed by comparing the mean number of total and female worms in any treatment group with that of the respective control groups. Differences were tested for significance using an unpaired two-tailed Student t-test, allowing for unequal variance. The data were considered significant if P < 0.05. Statistical analyses were done with version 9 of the STATA software (Stata-Corp., College Station, TX).
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RESULTS |
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). On the other hand, in S. mansoni–infected mice treated with a single dose of 500 mg/kg praziquantel, immature eggs of the first stage were absent, the number of second to fourth stage immature eggs and the number of mature eggs strongly decreased, whereas a notable increase in dead eggs was recorded compared with the untreated control group. Neither immature nor mature eggs, but dead eggs, were found in mice treated with two doses of 500 mg/kg praziquantel (Table 1).
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Table 2 summarizes the effect of various doses of triclabendazole in mice infected with an Egyptian strain of S. mansoni. There was no correlation between the total or female worm burden reductions and the dose of triclabendazole administered. In mice treated with a single dose of 120 mg/kg triclabendazole 3 days before infection with S. mansoni, low, but statistically significant, total (31.0%) and female (27.6%) worm burden reductions were achieved (P < 0.05). When mice harboring a 7-week-old adult S. mansoni infection were given a single or double dose of 120 mg/kg triclabendazole, moderate, but statistically significant, total worm burden reductions (35.5 and 29.8%, respectively) and insignificant female worm burden reductions (24.5 and 27.6%, respectively) were achieved. In mice receiving a single oral dose of 200 mg/kg triclabendazole, low, but statistically significant, total and female worm burden reductions of 31.3 and 35.9%, respectively, were observed (P < 0.05). Finally, administration of a double dose of 200 mg/kg triclabendazole (i.e., the highest dose given to mice infected with an Egyptian strain of S. mansoni) resulted in total and female worm burden reduction of 18.6 and 28.6%. These reductions were not statistically significant compared with the untreated control group (P > 0.05).
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. Administration of 400 mg/kg of triclabendazole, triclabendazole sulphone, or triclabendazole sulphoxide failed to significantly reduce the S. mansoni worm burden; total and female worm burden reductions were < 10%.
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and 3).
An elevated death rate was observed in mice harboring both the Egyptian and the Liberian strain of S. mansoni after treatment with triclabendazole compared with the praziquantel or control groups (Tables 2 and 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our main finding is that triclabendazole, as well as its two main metabolites, lacks antischistosomal activity. Only low total and female worm burden reductions were observed after oral administration of triclabendazole, triclabendazole sulphone, or triclabendazole sulphoxide at doses as high as 400 mg/kg, regardless of the S. mansoni strain used. In addition, eggs of all developmental stages were present in mice treated with triclabendazole, and no significant reductions in either the hepatic and intestinal tissue egg loads were observed. Furthermore, eggs collected from fecal samples from treated mice were able to hatch and developed active miracidia.
Our results are in contrast to the previous study of Khalil and others15 reporting a total worm burden reduction of 84% after the administration of a single oral dose of 120 mg/kg triclabendazole to mice harboring an Egyptian strain of S. mansoni. We replicated this experiment, but only found a total worm burden reduction of 35.5%. We have no explanation for this large difference in worm burden reductions because the same S. mansoni strain, route of administration, dose of triclabendazole, and parasitological methods were used to assess the worm burden. Interestingly, subsequent experiments with higher doses of triclabendazole failed to replicate a total worm burden reduction of 35.5%; instead worm burden reductions of only 6.6–31.3% were observed.
It is noteworthy that triclabendazole has a pronounced effect on schistosomes in vitro,14 but the drug and its two main metabolites failed to significantly reduce S. mansoni worms in our experiments in vivo. This finding is surprising because the liver is the target organ of both schistosomes and the biologically related Fasciola spp. Pharmacokinetics in the host might play a key role in the differing susceptibilities to triclabendazole between Fasciola and schistosomes in vivo, and might also explain the differing in vitro and in vivo activities of the drug on schistosomes. For example triclabendazole is highly protein bound in different animal species.13 While F. hepatica is known to be very susceptible to drugs that mainly bound to plasma proteins, schistosomes might be less affected by the protein bound triclabendazole.
Another aspect worth mentioning is the elevated death rate of S. mansoni–infected mice in the triclabendazole groups compared with the corresponding praziquantel or control groups (Tables 2 and 3). The LD50 value of oral triclabendazole is > 8 g/kg in mice, and the LD50 values of the sulphoxide and sulphone metabolites administered orally are > 5 g/kg in rats.23 S. mansoni is known to cause a downregulation of hepatic cytochrome P-450 activities in chronic infections in mice,24 and hence the safety margin of triclabendazole might decrease because of a S. mansoni infection in the mouse. We are not aware of studies that have investigated similarly high doses of triclabendazole against chronic infections of F. hepatica in rodent models, which might have revealed comparable changes of toxicity margins, because F. hepatica is also known to alter cytochrome P-450 activities.25
Based on our findings, we speculate that triclabendazole, when administered as a dose of 10 mg/kg to fascioliasis patients, will have no or only a minimal effect against S. mansoni. This issue is currently under investigation in an epidemiologic study carried out in Egypt, where patients co-infected with F. hepatica and S. mansoni are given triclabendazole.17 We are curious to learn the results of this study. In case our findings of the lack of antischistosomal activity of triclabendazole and its metabolites in the mouse are confirmed in human studies, we are basically left with praziquantel as the only drug for treatment and control of schistosomiasis. The activity of oxamniquine is restricted to S. mansoni.3 The encouraging initial results obtained with the natural product myrrh (Mirazid), claiming a cure rate of 91.7–96.2% in S. mansoni–infected patients in Egypt,26,27 could not be confirmed in follow-up studies in humans or rodents.28,29 In light of these disappointing observations regarding triclabendazole and myrrh and no viable alternative to praziquantel, we conclude that establishment of an anti-schistosomal drug discovery and development portfolio is of pressing urgency.
Received January 23, 2006. Accepted for publication April 1, 2006.
Acknowledgments: We thank G. Büscher, Novartis Animal Health, for the donation of triclabendazole, triclabendazole sulphoxide, and triclabendazole sulphone.
Financial support: J.K. (Project PMPDB-10622) and J.U. (Project PPOOB–102883) are grateful to the Swiss National Science Foundation for financial support.
* Address correspondence to Rashida Barakat, High Institute of Public Health, Alexandria University, Alexandria, Egypt. E-mail: Barakat{at}dataxprs.com.eg 
Authors’ addresses: Jennifer Keiser, Jacques Chollet, and Jürg Utzinger, Swiss Tropical Institute, PO Box, CH-4002 Basel, Switzerland. Nadia Abou El Ela, Engy El Komy, and Rashida Barakat, High Institute of Public Health, Alexandria University, Alexandria, Egypt. Naglaa El Lakkany and Tarek Diab, Theodore Bilharz Research Institute, Giza, Egypt.
Reprint requests: Rashida Barakat, High Institute of Public Health, Alexandria University, Alexandria, Egypt. E-mail: Barakat{at}dataxprs.com.eg.
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REFERENCES |
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  J. Keiser, X. Shu-Hua, J. Chollet, M. Tanner, and J. Utzinger Evaluation of the In Vivo Activity of Tribendimidine against Schistosoma mansoni, Fasciola hepatica, Clonorchis sinensis, and Opisthorchis viverrini Antimicrob. Agents Chemother., March 1, 2007; 51(3): 1096 - 1098. [Abstract] [Full Text] [PDF]
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