Exogenous Iron Increases Fasciocidal Activity and Hepatocellular Toxicity of the Synthetic Endoperoxides OZ78 and MT04.

The synthetic peroxides OZ78 and MT04 recently emerged as fasciocidal drug candidates. However, the effect of iron on fasciocidal activity and hepatocellular toxicity of these compounds is unknown. We investigated the in vitro fasciocidal activity and hepatocellular toxicity of OZ78 and MT04 in absence and presence of Fe(II)chloride and hemin, and conducted a toxicological study in mice. Studies were performed in comparison with the antimalarial artesunate (AS), a semisynthetic peroxide. Fasciocidal effects of OZ78 and MT04 were confirmed and enhanced by Fe2+ or hemin. In HepG2 cells, AS reduced cellular ATP and impaired membrane integrity concentration-dependently. In comparison, OZ78 or MT04 were not toxic at 100 µM and reduced the cellular ATP by 13% and 19%, respectively, but were not membrane-toxic at 500 µM. The addition of Fe2+ or hemin increased the toxicity of OZ78 and MT04 significantly. AS inhibited complex I, II, and IV of the mitochondrial electron transport chain, and MT04 impaired complex I and II, whereas OZ78 was not toxic. All three compounds increased cellular reactive oxygen species (ROS) concentration-dependently, with a further increase by Fe2+ or hemin. Mice treated orally with up to 800 mg OZ78, or MT04 showed no relevant hepatotoxicity. In conclusion, we confirmed fasciocidal activity of OZ78 and MT04, which was increased by Fe2+ or hemin. OZ78 and MT04 were toxic to HepG2 cells, which was explained by mitochondrial damage associated with ROS generation in the presence of iron. No relevant hepatotoxicity was observed in mice in vivo, possibly due to limited exposure and/or high antioxidative hepatic capacity.

Isothermal microcalorimetry to study the activity of triclabendazole and its metabolites on juvenile and adult Fasciola hepatica.

Isothermal microcalorimetry (IMC) is an analytical tool that continuously measures the heat flow generated by chemical, physical or biological processes. We have demonstrated that IMC is a useful tool to analyze drug effects on helminths, including adult Fasciola hepatica. Here, we used IMC to examine the activity of triclabendazole and its metabolites triclabendazole sulphone and triclabendazole sulphoxide on juvenile and adult F. hepatica. Worms (one adult or 2-3 juveniles) were placed in 4 or 20 ml glass ampoules containing RPMI 1640 and the test compound (25-100 µg/ml) and the heat flow and motility of worms was examined with TAM48 and TAMIII isothermal microcalorimetry instruments. IMC was found to be precisely document drug effects on juvenile F. hepatica and confirmed the pronounced effect of the benzimidazole derivatives on the motor activity of F. hepatica. Juvenile F. hepatica incubated with 100 µg/ml triclabendazole, triclabendazole sulphone and triclabendazole sulphoxide showed no movements 8.3, 35 and 6h post-incubation (all p<0.001). The metabolic heat of triclabendazole sulphoxide treated worms (100 µg/ml) was reduced by 50% and 76% 24 and 120 h post-incubation, respectively. Limitations of calorimetric measurements were observed using adult F. hepatica as only three worms could be measured simultaneously and also control worms showed a considerable decrease in heat flow. Adult F. hepatica exposed to triclabendazole, triclabendazole sulphone and triclabendazole sulphoxide showed no movements after 31 (p=0.009), 49 (p>0.05) and 88 (p>0.05)h. In conclusion, IMC is useful to document drug effects on juvenile F. hepatica and since rapid technological developments in this field are occurring IMC might also hold promise to study adult F. hepatica in the near future.

Pharmacokinetics of the fasciocidal drug candidates MT04 and OZ78 in uninfected rats and in vitro pharmacodynamic studies.

OZ78 and MT04 are promising drug candidates against fascioliasis (fasciolosis). We determined basic pharmacokinetic (PK) parameters of OZ78 and MT04 in uninfected rats. Rats were treated with single oral doses of 50 mg/kg OZ78 or MT04. Blood samples were withdrawn at selected time points post treatment and the plasma concentrations were quantified by a validated liquid chromatography/mass spectrometry (LC/MS) method. The LC/MS method for MT04 and OZ78, initially developed for sheep plasma analysis, was adapted for rat plasma. In vitro pharmacodynamic studies with Fasciola hepatica incubated in solutions of either test agent complemented our work. The adapted and validated method was precise and accurate to measure OZ78 and MT04 in rat plasma. Accuracies for MT04 ranged from 87.9% to 104.7% with precisions not exceeding 14.3%. Precisions for OZ78 were lower than 9.8% and accuracies were between 88.4% and 105.3%. Following oral administration, maximum plasma concentrations (C max) of MT04 and OZ78 were 49.8 and 70.1 µg/ml after 2.7 h and 1.6 h, respectively (p > 0.05). The estimated area under the plasma time curves (AUCs) were comparable for MT04 and OZ78. Mean elimination half-lives (t1/2) of MT04 and OZ78 covered a range from 1 to 7 h. In vitro studies demonstrated that the fasciocidal activity of MT04 and OZ78 was dependent on the incubation-time, with exposure of flukes for 24 h to the drugs not being sufficient to kill the worms. In conclusion, differences in PK parameters of MT04 and OZ78 were observed in rats. However, further studies (e.g. in infected rats) are necessary to characterize these drugs in greater detail.

Activity of OZ78 analogues against Fasciola hepatica and Echinostoma caproni.

The rapid spread of triclabendazole resistance in veterinary medicine is an important motivation for the discovery and development of novel fasciocidal drugs. The aim of this study was to characterize the fasciocidal properties of 1,2,4,5-tetraoxane (MT04 and MT14) and 1,2,4-trioxane (ST16 and ST28) analogues of the fasciocidal drug candidate OZ78, a 1,2,4-trioxolane. Dose response relationships were determined against juvenile and adult Fasciola hepatica in rats and Echinostoma caproni in mice. The temporal effects of MT04, MT14, ST16, and ST28 compared to OZ78 on the viability of F. hepatica were tested in vitro. The heat flow of OZ78 and MT04 treated flukes was studied with isothermal microcalorimetry. Finally, surface changes to adult flukes were monitored by scanning electron microscopy (SEM) 18, 24, and 48 h post-treatment of rats with 50 mg/kg MT04. Administration of 50-100 mg/kg of the synthetic peroxides resulted in complete elimination of adult F. hepatica from rats. SEM pictures revealed sloughing and blebbing already 18 h post-treatment with MT04. MT04 (100mg/kg) cured infections with juvenile F. hepatica, whereas MT14, ST16, and ST28 showed only low to moderate worm burden reductions. At 300 mg/kg, MT14 was the only compound to completely eliminate worms from E. caproni infected mice. MT14 showed the highest activity against juvenile F. hepatica in vitro. MT04 was very active against adult F. hepatica in vitro, which was confirmed by heat flow measurements. In conclusion, we have identified MT04 as another lead compound with potential against F. hepatica, hence further preclinical studies are necessary to determine if MT04 can be considered a drug development candidate.

Development and validation of a liquid chromatography/mass spectrometry method for pharmacokinetic studies of OZ78, a fasciocidal drug candidate.

Fascioliasis is a zoonotic disease of considerable public health and great veterinary significance and new drugs are needed. OZ78 is a promising fasciocidal drug candidate. In order to support the development of OZ78, including pharmacokinetic (PK) studies an accurate, precise, and selective liquid chromatography/mass spectrometry (LC/MS) method for OZ78 was developed for sheep plasma and validated in accordance with the US Food and Drug Administration Guidance on Bioanalytical Method Validation. Protein precipitation was used for sample clean up. Separation was performed through a Phenomenex C8(2) analytical column (50.0mm×2.0mm, 5µm) with a mobile phase of acetonitrile (buffer B) and 5mM ammonium formate (buffer A) at a flow-rate of 0.3mL/min and a gradient from 20% to 95% acetonitrile. The mass spectrometer was operated under selected ion monitoring, and orifice voltage set to -4.1kV and ion spray temperature to 400°C. Nitrogen was used as a nebulizer, curtain, and collision gas. OZ78 was monitored at 321.4m/z (deprotonated parent compound, M-). The validated linear dynamic range was between 156.25ng/mL and 5µg/mL and the achieved correlation coefficient (r(2)) was greater than 0.99. The validation results demonstrated that the developed LC/MS method is precise, accurate, and selective for the determination of OZ78 in sheep plasma. The method was successfully applied to the evaluation of the PK profile of OZ78 in sheep.

Efficacy, safety and pharmacokinetics of 1,2,4-trioxolane OZ78 against an experimental infection with Fasciola hepatica in sheep.

The synthetic peroxide OZ78 is an effective flukicide in the rodent model, but the potential of OZ78 in target animals has not been studied to date. In the present study, OZ78 was administered at 50mg/kg orally and subcutaneously to sheep harbouring an experimental Fasciola hepatica infection and the efficacy, tolerability and pharmacokinetic profiles were monitored. OZ78 given orally or subcutaneously revealed no effect neither on faecal egg counts nor on worm burdens. Apart from significant subcutaneous swelling at the injection sites of most of the treated animals, no other treatment related adverse events occurred. OZ78 had no significant effect on any haematological, coagulation or clinical chemistry variables tested. Following oral administration, a mean C(max) of 45.8±13 µg/ml was reached after 1h. An estimated elimination half-life of 1.0 h and a mean AUC of 116.2±47 µg min/ml was calculated for the oral administration. Following subcutaneous treatment with OZ78 C(max) and t(max) were 13.7±6.1µg/ml and 0.9±0.4h, respectively. The α and ß half-lives were 4.5±4.3 h and 56.5±36 h, respectively and the mean AUC was 219.1±74 µg min/ml. Further studies are needed to determine whether the excellent activity observed with OZ78 in the rat model can be translated into efficacy in larger mammals.