Schistosoma japonicum: In vitro effects of artemether combined with haemin depend on cultivation media and appraisal of artemether products appearing in the media.

We recently found that the exposure of schistosomes in vitro to artemether plus haemin can lead to parasite death, while exposure to each compound singly had no effect. Since these observations might be relevant to understanding the mechanism of action of artemether against schistosomes, we conducted additional experiments. First, we performed a comparative appraisal of Schistosoma japonicum survival after incubation in two media, namely Hanks' balanced salt solution (HBSS) and RPMI 1640, supplemented with inactivated calf serum, antibiotics and different concentrations of artemether and/or haemin. Worm mortalities were consistently higher and occurred faster in HBSS when compared to RPMI 1640. Second, we investigated the behaviour of artemether in different chemical systems, including reduced glutathione or cysteine, in the presence of haemin or ferrous sulfate. Cleavage of the endoperoxide bridge of artemether occurred in all experiments, consistently forming five different products, of which one has not been described previously. Third, RPMI 1640 and HBSS media were supplemented with artemether and haemin in the presence of S. japonicum. The consumption of artemether in RPMI 1640 was much faster than in HBSS. Trace amounts of artemether and five free radical reaction products of artemether could be detected in RPMI 1640 after 24-48 h. In contrast, large amounts of artemether remained without the formation of free radical products in HBSS under the same conditions. These findings coincide with significantly higher schistosome mortalities employing HBSS instead of RPMI 1640. Our results suggest that it is artemether or an active metabolite thereof (most likely a carbon-centred free radical), rather than any free radical reaction product that is harmful for the worms. We speculate that schistosomes ingest artemether and cleave it in their gut. This cleavage is induced by haemin or another iron-containing molecule. These findings might be a further step forward in elucidating the mechanism of action of artemether against schistosomes.