The interaction between atovaquone and proguanil has never been studied against liver stage malaria, which is the main target of this drug combination when used for chemoprevention. Using human hepatocytes lacking cytochrome P450 activity, and thus avoiding proguanil metabolizing into potent cycloguanil, we show in vitro that the atovaquone-proguanil combination synergistically inhibits the growth of rodent Plasmodium yoelii parasites. These results provide a pharmacological basis for the high efficacy of atovaquone-proguanil used as malaria chemoprevention. The drug combination atovaquone-proguanil (AP) is an efficient drug for malaria chemoprevention and treatment. The rationale for combining these two drugs originated from the observation that they produce a synergistic inhibitory effect against replicating blood stage parasites in vitro (1-3). In the context of malaria chemoprevention, however, AP exerts its protective effect primarily during the hepatic phase of the parasite infection (4, 5), and whether the AP synergy is operating during this preerythrocytic phase has not been explored.To test this directly, we sought to evaluate in vitro the interaction between atovaquone and proguanil against liver stage Plasmodium infection through a fixed-ratio isobologram method. In hepatocytes, proguanil is partially metabolized by host cytochrome P450 enzymes into cycloguanil (6), which potently inhibits the development of liver stage Plasmodium infection (7). To address this confounding factor, we used HepG2-CD81 human hepatoma cells that retain many liver-specific properties and can be infected by some Plasmodium species (8) while displaying impaired cytochrome P450 activity (9).To confirm the lack of or very minimal proguanil metabolism, HepG2-CD81 cells were incubated in the presence of 100 M proguanil for 2 days, and cell culture supernatants were collected at 24 and 48 h after treatment to quantify drug levels. Proguanil and its metabolite cycloguanil were separated and quantified on a liquid chromatography mass detection mass spectrometer (TSQ Quantum Ultra; Thermo Fisher, France) using an Atlantis dC 18 column (100 by 2.1 mm, 3 m; Waters, France) and a calibration curve. Cycloguanil was not detected in any of the collected samples from the 24-and 48-h time points (n ϭ 8 values) at the 0.04 M limit of our assay (proguanil was detected as expected). In contrast, cycloguanil was detected in the corresponding samples of primary human hepatocytes used as positive controls (median, 0.32 M; minimum, 0.21 M; maximum, 0.97 M; n ϭ 8 values; P Ͻ 0.001, Mann-Whitney test). The same observations regarding cycloguanil production in the two cell types were made when using 20 M instead of 100 M proguanil (P Ͻ 0.001, Mann-Whitney test).Subsequently, Plasmodium-infected hepatocytes were treated with single agents, and then combination treatments were performed to assess drug interaction. Primary human hepatocytes were isolated as previously described (8). HepG2 cells were derived from the liver tissue of a 15-year-old boy wit...
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