Pharmacological and pharmacokinetic studies on trematocidal drugs : praziquantel and two synthetic peroxide lead candidates

Trematodiases affect more than 250 million people worldwide, while the economic impact through veterinary infections is also colossal. Treatment options are however limited to two main drugs, triclabendazole for the liver fluke Fasciola hepatica, and praziquantel (PZQ) for the rest of the trematodes including schistosomes, the etiological agent of bilharziosis. Furthermore, the kinetic disposition of PZQ is hardly studied in diseased patients and lacking in pre-school children, where the burden of schistosomiasis is already high. The aim of the present thesis is to provide pharmacokinetic and pharmacological studies to understand the modalities of PZQ disposition in patients. Additionally, the efficacy of two novel synthetic peroxide lead candidates are evaluated against F. hepatica in sheep and supported by a pharmacokinetic (PK) study.
We first validated an analytical method to assess the PK profile of the synthetic peroxides OZ78 and MT04 in sheep plasma and bile. After an intramuscular administration of a 100 mg/kg dose to groups of 5 sheep naturally infected with F. hepatica, we observed a good fasciocidal efficacy for the peroxide candidate MT04, while the other candidate, OZ78, failed to display any activity. The PK study did not reveal a higher kinetic disposition for MT04 than for OZ78, but highlights problems in drug absorption. The presence of both peroxide candidates in sheep bile at 6 h post-treatment does not rule out a direct drug action on the worm tegument.
We recorded a high antischistosomal activity for the R enantiomer of PZQ against S. mansoni adults and NTS in vitro with IC50 values below 0.04 μg/ml. The S form displayed a very limited effect in vitro. It produced in vivo an important and transient shift of the worms to the liver but at 24 h after S-PZQ administration, the worms had returned to the mesenteric veins. The R forms of the main metabolites in mice and humans, cis- and trans-4-OH, exhibited a moderate activity. In vivo, mice harbouring adult S. mansoni were cured with a 200 mg/kg dose, half of the curative dose required with racemic PZQ. Treatment with a 800 mg/kg dose of S-PZQ failed to display a significant effect.
In order to undertake PK studies, we validated an enantioselective method for PZQ and its main metabolite, R- trans-4-OH-PZQ, in blood, plasma and dried blood spots (DBS) using an analytical range from 0.01 to 2.5 μg/ml for R- and S-PZQ and from 0.1 to 25 μg/ml for the metabolite. The comparison of blood, plasma and DBS samples simultaneously withdrawn from 9 patients infected with the liver fluke Opisthorchis viverrini and treated with 3x 25 mg/kg PZQ allowed us to evaluate the potential of DBS as unique sampling method in future PK trials. Due to the high affinity of PZQ for plasma albumin, samples using whole blood displayed slightly lower concentrations of the parent enantiomers than plasma measurements. This difference is however small and within an acceptable range. For the metabolite, plasma concentrations did not display significant differences to blood or DBS levels. Therefore, DBS was validated as an alternative to plasma sampling. The PK profile of the 9 patients investigated in this study was discussed on the basis of plasma PK data. We observed plasma AUCs of 1.1, 9.0 and 188.7 μg/ml*h and half-lives of 1.1, 3.3 and 6.4 h for R-PZQ, S-PZQ and R-trans-4-OH, respectively. Maximal plasma concentrations of 0.2, 0.9 and 13.9 μg/ml for R-PZQ, S-PQZ and R-trans-4-OH peaked at 7 h for PZQ enantiomers and 8.7 h for the metabolite. The high levels of the R form of the main metabolite of PZQ do not rule out its participation in the opisthorchicidal effect of the drug.
In conclusion, we identified MT04 as a potential fasciocidal candidate. We also generated precise in vivo and in vitro data on the sensitivity of S. mansoni to PZQ enantiomers and confirmed that the eutomer of PZQ is its R- form. The LC-MS/MS method for praziquantel enantiomers allows to use DBS in future clinical trials, therefore enabling the sampling of children and the planning of larger PK trials. Finally, we presented the first PK values of PZQ enantiomers and its main metabolite in patients suffering from opisthorchiasis.