Atovaquone

After isolation, the P. falciparum or P. cynomolgi sporozoites were suspended in complete medium and added to their respective host's primary hepatocyte cultures. For drug assays, 3×10⁴ sporozoites were added to each well, or 1×10⁵P. cynomolgi sporozoites for cultures initiated in Lab-Tek 8-chambers slides. Infected cultures were fixed with cold 100% methanol on Days 1, 3, 5, 7, 9 and 11 post-infection in order to count and measure the size of the P. cynomolgi or P. falciparum pre-erythrocytic (PE) forms obtained. For the drug assays, the antimalarial drugs were added at different concentrations to complete medium. Infected cells were exposed to the drug for 3 days from Day 5 until fixation at Day 8 post-infection. In a second set of experiments, PE-uni selection was included by treatment of P. cynomolgi hepatic cultures with atovaquone at 67 ng/ml (182 nM) between Day 5 and Day 8. This was followed by a 2 days treatment with either the same concentration of ATO or with PQ at 10 µg/ml (22 nM), until fixation at Day 10 post-infection. In all cases, the medium containing the drug was renewed daily during treatment. Assays for each drug were generally conducted in two or three independent experiments. In an individual experiment, each concentration was tested in duplicate or triplicate wells.
Hepatic parasites were enumerated by immunofluorescence analysis. Briefly, following fixation, the parasites were specifically labelled with a mouse polyclonal serum raised against the P. falciparum heat shock protein 70.1 (PfHSP70.1) obtained after immunization with the recombinant protein. This serum also cross-reacts with P. cynomolgi HSP70. The labelled parasites were visualized with Alexa 488-conjugated goat anti-mouse immunoglobulin (Invitrogen). Parasite and host cell nuclei were stained with 1 µg/ml of diamidino-phenylindole (DAPI; Sigma). Parasites were enumerated by examination of the cultures under a fluorescence microscope at 200× magnification (Leica DMI 4000 B). Photomicrographs were obtained using a confocal Olympus BX61 microscope at 600× magnification on cells cultures in Lab-Tek 8-chambers slides.
The cytotoxicity of the anti-malarial drugs was evaluated on M. fascicularis hepatocytes treated as for the drug activity assays, through the colorimetric methylthiazolyldiphenyl-tetrazolium bromide test (MTT) [27] (link). Briefly, the primary hepatocytes were exposed to various concentrations of drug and three days later, a 100 µl MTT solution (500 µg/mL) was added in each well. After 4 h of incubation at 37°C, the plates were read in a spectrophotometer (540 nm absorbance wavelength) and the results were expressed as percentage of cellular viability compared to the non-treated control.

In vitro testing of drug susceptibility was performed by the standard 42-hour ³H-hypoxanthine uptake inhibition method [5 (link)]. Susceptibility to dihydroartemisinin, artesunate, and ten standard or new anti-malarial drugs, ie chloroquine, quinine, mefloquine, lumefantrine, monodesethylamodiaquine (biologically active metabolite of amodiaquine), pyronaridine, piperaquine, atovaquone, doxycycline and pyrimethamine, was assessed. The laboratory-adapted clone W2, tested on the same day, was used as a reference. Isolates from imported malaria, tested on the same batch of plates, were used as comparators.
Polymorphisms of pfcrt, pfmdr1, pfmrp and pfnhe-1, involved in quinoline resistance, and in pfATPase6, postulated to be involved in artemisinin resistance, and the copy number of pfmdr1 were assessed [6 ].
The French malaria consensus [7 (link)] and the WHO [8 ] recommend to clinically examine patient and control parasitaemia at D0, D3, D7 and D28 to evaluate anti-malarial efficacy. Blood controls were performed at D0, D4, D7 and D43. The genotyping of parasites was assessed at D0, D4 and D7 using six microsatellite loci (microsatellites 7A11, pf2689, pf2802, C4M79, TRAP, C4M69) [9 (link)], msp1 and msp2 [10 (link)].

The principle of pLDH assay is shown in Figure 12a. Red blood cell (RBC) type O+ was obtained from the local Red Cross. P. falciparum 3D7 was maintained in a RPMI-1640 medium (supplemented with Albumax II (Gibco-Thermo Fisher Scientific, Waltham, MA, USA), and contained RBC (3% hematocrit). P. falciparum cell was synchronized into the ring form stage using 5% sorbitol, and then adjusted so the parasitemia was 0.3%. The sample was added to 96-well microplate, followed by the addition of 100 μL of the P. falciparum cell culture. The cell was incubated for 3 days at 37 °C, 5% CO₂, 5% O₂. Cold PBS was added to the cell culture, as much as 200 μL, then centrifuged at 1700× g for 10 min. After discarding the supernatant, 100 µL of LDH reaction mixture (100 mM Tris-HCl pH 8.0, 50 mM sodium-L-lactate, 0.25% (v/v) Triton X-100), 0.2% nitro blue tetrazolium (NBT), 50 µg/mL 3-acetyl pyridine NAD (APAD), and 0.05 U diaphorase) was added into each well and incubated at 37 °C for 30 min before the absorbance was measured at 650 nm using a microplate reader. Inhibition activity was calculated using formula in Figure 12b. Cell cultures with the addition of DMSO (final concentration 0.4%) and atovaquone (final concentration 1 µM) were regarded as negative and positive controls, respectively.

Atovaquone was purchased from Sigma (A7986) for in vitro work and purchased from Amneal Pharmaceuticals for in vivo studies. Fluorochrome-conjugated antibody to pS6-AF647 (Ser240/244) was from Cell Signaling Technologies, CD130-AlexaFluor 488 was from Abcam, pY-STAT3-PE and isotype control were from BD Biosciences.

Statistical analysis was performed using GraphPad Prism software (version 9.1.1; GraphPad Software, La Jolla, CA, USA), R (version 3.6.3, R Core Team, 2021). Pearson correlation was used to compare plasma concentrations, ease of administration scores, AE burden and weight loss. Heatmaps were made using Heatmap.2 of the gplots package available in R [34 ]. In order to account for age-based variation in plasma concentrations and ease of administration scores, patients were divided into 3 age groups of approximately similar size (0 < age ≤ 3 years (6 patients), 3 < age ≤ 15 years (10 patients), and 15 < age ≤ 21 years (8 patients)) (Figure 1B). The significance of observed differences between atovaquone and vehicle treated samples in vitro and in vivo was determined by repeated measures ANOVA to compare all samples at all tested time points. For in vivo survival analyses, Kaplan–Meier survival curves were generated and compared for significant differences by the log-rank test.