Antischistosomal properties of Mefloquine : from in vitro studies to drug target discovery

Schistosomiasis is a chronic helminthic disease caused by fluke worms of the genus Schistosoma spp. The disease belongs to the so-called neglected tropical diseases (NTDs) and is endemic in approximately 78 countries of the world, but mainly in sub-Saharan Africa. It has been estimated that more than 207 million people worldwide are infected, the annual mortality rate in sub-Saharan Africa might be as high as 280,000.
Overall, there are five schistosome species parasitizing humans, with Schistosoma haematobium, S. mansoni and S. japonicum being the three main species with the widest geographical distribution. The disease is transmitted by contact with infested fresh water, containing the infectious stage of the parasites, the cercariae, which were released by infected aquatic snails and penetrate human skin. Chronic pathology is not due to the worms itself, but due to inflammatory reactions provoked by the eggs.
The WHO recommended strategy for control of schistosomiasis is morbidity control through regular treatment with praziquantel. Praziquantel is active against the adult stages of all five human schistosome species, however, it has only little or no effect on eggs and immature worms. Hence re-treatment is necessary. Overall, treatment is regarded as safe and generally well-tolerated. However, to date praziquantel is the only drug available for treatment and control of schistosomiasis. The dependence on only one drug for such an important disease and the massive use of praziquantel is a risk factor for the development of praziquantel resistant schistosome strains. Furthermore, to date no vaccines against schistosomiasis are available. Therefore there is a need to develop new drugs against schistosomiasis.
Recently the antimalarial drug mefloquine was found to have interesting antischistosomal activities in vitro and in the mouse model. For example, single oral doses of 200 or
400 mg/kg mefloquine administered to mice infected with S. mansoni and S. japonicum resulted in high or complete total and female worm burden reduction. A clinical trial conducted with school children in Côte d´Ivoire showed that a mefloquine/artesunate combination cured 11 out of 18 children infected with S. haematobium.
Based on these findings, the aim of my PhD thesis was to further assess the newly discovered antischistosomal properties of the antimalarial drug mefloquine and to get a first insight into the mode of action and the pharmacodynamics of mefloquine in schistosomes. Therefore different in vitro and in vivo studies, including drug target discovery studies, were conducted with S. mansoni.
By assessing the time-dependent drug effect of mefloquine on schistosomes we showed that mefloquine has a rapid onset of action on newly transformed schistosomula (NTS; artificially produced, non hematophagous) and a slightly slower onset of action on adult worms (recovered from infected mice) in vitro: 10 µg/ml killed adult worms within 24 h of incubation (LC50= 1.9 µg/mL). In the presence of hemin drug activities against adult S. mansoni were enhanced. In vivo 72 h post treatment more than 90% of schistosomes were shifted to the liver, a criterion for drug activity, which shows how quick after drug treatment worms are forced to migrate from the mesenteric veins to the liver (ED50= 62 mg/kg). With scanning electron microscopy (SEM) studies, we confirmed our previous observations and the differences in the onset of action between in vitro and in vivo studies: SEM revealed extensive tegumental damages including blebbing, shrinking and sloughing on the tegument of NTS and adult worms, in particular following in vitro incubation and on the tegument of female worms.
Conducting a structure-activity relationship study we showed that the optical isomers and the racemic forms of the chiral drug mefloquine have only moderate stereoselectivity, in particular in vivo, since prominent activities were observed with all derivates in the S. mansoni mouse model.
To evaluate the potential of a mefloquine/praziquantel drug combination against S. mansoni we conducted combination studies and achieved encouraging results, since synergistic effects in vitro and in the S. mansoni mouse model were seen, when both drugs were given simultaneously or when praziquantel treatment followed mefloquine. The latter administration regime achieved the best results.
Finally, to get a better knowledge about the mechanism of action of mefloquine in schistosomes, we launched drug target discovery studies using affinity chromatography. We identified enolase, a glycolytic enzyme, as a major mefloquine- binding protein in S. mansoni schistosomula and we demonstrated that mefloquine interacts with glycolysis in schistosomula.
To strengthen the field of antischistosomal drug discovery we assessed a new technique, isothermal microcalorimetry (IMC), to examine in vitro drug effects against S. mansoni. Results were compared to microscopic readouts, the actual “gold standard”. Both methods showed a good correlation, however, IMC identified drug effects that were not visible by microscopic evaluation and precisely determined the onset of action of the test drugs. Furthermore, we compared drug effects on NTS and adult schistosomes, because NTS are increasingly used in antischistosomal drug screening assays.
In conclusion, my work has confirmed that mefloquine possesses excellent antischistosomal properties and that the drug might be an interesting partner drug in combination chemotherapy.
We demonstrated that IMC is a useful tool for antischistosomal drug discovery and should therefore be assessed also for studying drug effects against other helminths. In addition, our data support the use of NTS in in vitro antischistosomal drug assays, since we observed similar drug sensitivities on both stages.