Anti-malarial ozonides OZ439 and OZ609 tested at clinically relevant compound exposure parameters in a novel ring-stage survival assay.

BACKGROUND: Drug efficacy against kelch 13 mutant malaria parasites can be determined in vitro with the ring-stage survival assay (RSA). The conventional assay protocol reflects the exposure profile of dihydroartemisinin. METHODS: Taking into account that other anti-malarial peroxides, such as the synthetic ozonides OZ439 (artefenomel) and OZ609, have different pharmacokinetics, the RSA was adjusted to the concentration-time profile of these ozonides in humans and a novel, semi-automated readout was introduced. RESULTS: When tested at clinically relevant parameters, it was shown that OZ439 and OZ609 are active against the Plasmodium falciparum clinical isolate Cam3.IR539T. CONCLUSION: If the in vitro RSA does indeed predict the potency of compounds against parasites with increased tolerance to artemisinin and its derivatives, then the herein presented data suggest that following drug-pulses of at least 48 h, OZ439 and OZ609 will be highly potent against kelch 13 mutant isolates, such as P. falciparum Cam3.IR539T.

In vitro activity of anti-malarial ozonides against an artemisinin-resistant isolate.

BACKGROUND: Recently published data suggest that artemisinin derivatives and synthetic peroxides, such as the ozonides OZ277 and OZ439, have a similar mode of action. Here the cross-resistance of OZ277 and OZ439 and four additional next-generation ozonides was probed against the artemisinin-resistant clinical isolate Plasmodium falciparum Cam3.I, which carries the K13-propeller mutation R539T (Cam3.IR539T). METHODS: The previously described in vitro ring-stage survival assay (RSA0-3h) was employed and a simplified variation of the original protocol was developed. RESULTS: At the pharmacologically relevant concentration of 700 nM, all six ozonides were highly effective against the dihydroartemisinin-resistant P. falciparum Cam3.IR539T parasites, showing a per cent survival ranging from <0.01 to 1.83%. A simplified version of the original RSA0-3h method was developed and gave similar results, thus providing a practical drug discovery tool for further optimization of next-generation anti-malarial peroxides. CONCLUSION: The absence of in vitro cross-resistance against the artemisinin-resistant clinical isolate Cam3.IR539T suggests that ozonides could be effective against artemisinin-resistant P. falciparum. How this will translate to the human situation in clinical settings remains to be investigated.