Efficacy and pharmacokinetics of OZ78 and MT04 against a natural infection with Fasciola hepatica in sheep

Abstract

Fasciolosis is a parasitosis caused by the food-borne trematode Fasciola spp. of major veterinary significance. Triclabendazole is the first line treatment in humans and animals but cases of resistance are spreading worldwide. The synthetic peroxides OZ78 and MT04 are lead compounds for the treatment of fasciolosis. In the present study we investigated the efficacy and drug disposition following a single intramuscular dose of 100 mg/kg OZ78 and MT04 in sheep harbouring a natural Fasciola hepatica infection. A liquid chromatography and tandem mass spectrometry (LC–MS/MS) method was developed and validated to quantify plasma and bile concentrations of both compounds. Plasma samples were analysed with an accuracy for OZ78 and MT04 from 91 to 115% and a precision lower than 8.9%. Bile samples displayed an accuracy between 92 and 101% and a precision up to 12.7%. Bile samples were collected at 0 and 6 h post-administration. Plasma mean peak concentration was 11.1 μg/ml at 1.5 h for OZ78 and 4.8 μg/ml at 4.2 h for MT04. Mean AUC of OZ78 and MT04 was 6698 and 3567 μg min/ml, respectively. Bile concentration at 6 h post-treatment was 1.0 μg/ml for OZ78 and 1.4 μg/ml for MT04. Treatment with OZ78 showed no effect on egg burden and adult worm counts in vivo, whereas MT04 displayed a significant egg count reduction of 98.5% and a worm burden reduction of 92%. In conclusion, our study reveals an excellent activity of MT04 against F. hepatica in naturally infected sheep and a first insight into its PK behaviour.

Introduction

Fasciolosis is a major zoonosis of livestock in temperate regions and is responsible of an annual estimated economical loss of US$3 billion (Fairweather, 2005). This parasitosis is caused by the liver flukes Fasciola hepatica and Fasciola gigantica, and is responsible for morbidity and mortality, including weight loss, jaundice, hepatomegaly and biliary cholic. Triclabendazole is the drug of choice in both livestock and humans and is highly active against all the parasite stages (Keiser and Utzinger, 2009). Resistance against triclabendazole has been observed in Australia since 1995 and is now recorded in several European countries (Fairweather, 2009). This situation is critical and new fasciocidal drugs are urgently needed.

Synthetic peroxides, such as ozonide (1,2,4-trioxolane) OZ277, have been synthesized in the late 1990s in the frame of antimalarial research as alternatives to artemisinin derivatives (Vennerstrom et al., 2004). The screening of libraries of structural analogues has generated other interesting antimalarial candidates and ozonide OZ439 successfully completed phase I trials in healthy human volunteers (Moehrle et al., 2013). In the past few years, the ozonides have also been studied against a range of helminths and OZ78 revealed activity against diverse trematode species, such as F. hepatica, Echinostoma caproni (Keiser et al., 2006, Keiser et al., 2007), Clonorchis sinensis (Keiser et al., 2007), Schistosoma mansoni and Schistosoma japonicum (Xiao et al., 2007, Xiao et al., 2011). Additionally, its toxicological profile has been proven acceptable in vitro and in rats and the drug has good pharmacokinetic properties (Vennerstrom et al., 2004). In F. hepatica infections, OZ78 achieved elimination of juvenile and adult worms in rats with a single oral dose of 100 mg/kg (Keiser et al., 2006). In vitro, adult F. hepatica was affected at concentrations of 10–100 μg/ml OZ78 (Halferty et al., 2009, Keiser et al., 2006). Incubation for 48 h at a concentration of 100 μg/ml of OZ78 produced significant tegumental damages and displayed an impact on vitelline and testis follicles on adult worms (Halferty et al., 2009).

Recently, additional dispiro 1,2,4-trioxanes and 1,2,4,5-tetraoxanes were synthesized and tested in F. hepatica infected rats. Among those, the tetraoxane MT04 displayed a higher activity than OZ78 against F. hepatica, especially against adult worms (Wang et al., 2011). A single 50 mg/kg oral dose of MT04 achieved a complete burden reduction in F. hepatica infected rats. Furthermore, the onset of action for MT04 was observed to occur between 18 and 24 h, in contrast to 24–72 h for OZ78 (Kirchhofer et al., 2011).

The goal of the present study was to test the efficacy of MT04 in sheep harbouring a natural F. hepatica infection. For comparison, one group of sheep was treated with OZ78. In previous experiments (Keiser et al., 2010), OZ78 showed no activity in sheep at a single dose of 50 mg/kg given orally and subcutaneously. Since a recent investigation on the fasciocidal properties of artemether and artesunate demonstrated an increased efficacy when the drugs were administered intramuscularly to sheep (Keiser et al., 2008), we opted for a single intramuscular dose of 100 mg/kg for both compounds. We also refined and validated an existing LC–MS/MS method for OZ78 and MT04 (Kirchhofer et al., 2010) with the purpose to study their drug disposition in plasma and bile.