Amino ozonides exhibit in vitro activity against Echinococcus multilocularis metacestodes
Introduction
Alveolar echinococcosis (AE), caused by the parasitic tapeworm Echinococcus multilocularis, is a zoonotic disease that occurs mainly in the Northern hemisphere, including central Europe, North America and East Asia. More recently, AE has been reported in Eastern Europe and South Korea [1], [2], areas that had earlier been considered free of this disease. The increased urbanisation and increased numbers of foxes in populated areas in Switzerland have led to a higher infection pressure, and significantly increased infection risk has been reported [3].
The disease is acquired through accidental ingestion of E. multilocularis eggs containing the first larval stage (oncosphere). These are shed with the faeces of adult-stage-infected foxes, dogs or cats. The oncospheres penetrate the intestinal epithelium, are disseminated via blood or lymphatic vessels and reach the liver, where they develop into metacestodes. AE is characterised by infiltrative growth and tumour-like proliferation of E. multilocularis metacestodes, primarily in the liver, but other organs can also be affected. The disease is fatal if not treated appropriately [3], [4].
The current strategy for treating human AE consists of surgical measures complemented by chemotherapy employing benzimidazoles such as mebendazole or albendazole. This strategy inhibits parasite proliferation but is rarely curative, requires a long duration of treatment, incurs high costs and there is an elevated risk of adverse effects [4]. Many compounds have been investigated in the past as treatment alternatives, using either in-vitro-cultured parasites and/or in vivo rodent models [4]. However, few options have been translated into clinical applications. For instance, amphotericin B deoxycholate has been applied as salvage treatment, but this drug cannot be used for extended time periods owing to toxicity [5], and nitazoxanide is less effective than was predicted by in vivo studies in mice [6].
We have previously demonstrated that the antimalarial drug mefloquine significantly decreases parasite burden in mice experimentally infected with E. multilocularis metacestodes [7]. Moreover, the antimalarial semisynthetic artemisinins and synthetic ozonides such as OZ78, OZ209 and OZ288 [8], [9], [10] (Fig. 1) have varying efficacies against trematode species such as schistosomes, Fasciola hepatica, Echinostoma caproni, Clonorchis sinensis and Opisthorchis viverrini [10]. In addition, larval stages of E. multilocularis and Echinococcus granulosus are susceptible to dihydroartemisinin and artesunate at 10–40 μM [11]. However, an artesunate treatment regimen (200 mg/kg/day for 6 weeks) was not effective in mice infected with E. multilocularis metacestodes. Nevertheless, combination treatments of dihydroartemisinin and artesunate with albendazole led to a substantial, but statistically insignificant, reduction in parasite weight compared with treatment with albendazole alone [11].
In this study, a small library of synthetic ozonides was screened for in vitro activity against E. multilocularis metacestodes using a validated E. multilocularis viability assay. The four most active ozonides were further characterised for dose-dependent antiparasitic activity and for cytotoxicity in cells of human, rat and monkey origin. Ultrastructural changes in metacestodes induced by two of these compounds (OZ401 and OZ491) were demonstrated using transmission electronic microscopy (TEM).
Section snippets
In vitro culture of E. multilocularis metacestodes
If not stated otherwise, all culture media and reagents were purchased from Gibco-BRL (Zürich, Switzerland) and biochemical reagents were from Sigma (St Louis, MO). Culture of E. multilocularis (isolate H95) was carried out as previously described [12]. In short, metacestodes dissected from experimentally infected BALB/c mice were pressed through an autoclaved tea sieve. Metacestodes were incubated in antibiotic solutions of 20 mg/L levofloxacin (Aventis, Meyrin, Switzerland) and 20 mg/L
In vitro screening of E. multilocularis metacestodes
A small library of 34 neutral, acidic and basic ozonides was pre-screened against E. multilocularis at a concentration of 20 μM over a period of 5 days. This library contained OZ78, OZ209 and OZ288 (see Fig. 1); nitazoxanide and artesunate [11], [13] were included as positive controls. Other drugs investigated were OZ compounds (99, 165, 180, 277, 288, 323, 401, 406, 429, 436, 443, 444, 445, 465, 466, 491, 493, 494, 535, 536, 539, 545, 554, 608, 612, 621, 634, 635, 660, 674, 675). Albendazole,
Discussion
Thirty-four synthetic ozonides, including OZ78, OZ209 and OZ288, which are ozonides previously demonstrated to have activity against trematodes, were screened for activity against E. multilocularis metacestodes in vitro. Most of the ozonides had none to very weak efficacies. Three ozonides containing an aminopropylether substructure (OZ401, OZ455 and OZ491) had the highest efficacies but were an order of magnitude less efficacious than nitazoxanide. These three ozonides were also cytotoxic at
Acknowledgments
The authors are grateful to Bruno Gottstein and Markus Spiliotis (Institute of Parasitology, Bern, Switzerland) for continuous support and helpful discussions.
Funding: This study was financed through the Swiss National Science Foundation [grant no. 31003A-125990], the Swiss Life Foundation and the Bangerter-Rhyner Foundation.
Competing interests: None declared.
Ethical approval: Not required.
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