In vitro drug interaction studies.

In vitro interactions between plasmodione and clinical antimalarials were investigated using the fixed-ratio isobologram method established by Fivelman et al. (47). These assays were performed in laboratory A (allopurinol, fosmidomycin, cycloguanil, and proguanil) or laboratory B (amodiaquine, chloroquine, quinine, ferroquine, artemisinin, dihydroartemisinin, atovaquone, methylene blue, and pyrimethamine). Briefly, asynchronous cultures of P. falciparum strain 3D7 or K14 were incubated for 48 h in the presence of decreasing drug concentrations in a microtiter plate (1% parasitemia, 1.5 to 2% hematocrit). Drugs were applied alone or in combination at fixed concentration ratios (A:B ratios [vol/vol] of 1:4, 2:3, 2.5:2.5, 3:2, and 4:1; the ratio of 2.5:2.5 was used in laboratory A only) and analyzed in 3-fold serial dilutions. IC₅₀s of drugs A and B alone were determined on the same plate to ensure accurate calculation of drug interactions. The inherent variability of the experimental setup was estimated by combining two individual working solutions of the plasmodione lead. Parasite growth was assessed in laboratory A by using the SYBR green protocol as described above and in laboratory B by using the [³H]hypoxanthine incorporation assay (32), adding [³H]hypoxanthine after 24 h of incubation, and the remainder of the experiment was conducted as previously described (48).

In vitro drug interactions were interpreted based on arithmetical and graphical analyses of the results. For the arithmetical analysis, the fractional inhibitory concentration (FIC) of each drug in a given combination was calculated according to the following definitions: FIC A = IC₅₀ of A in combination/IC₅₀ of A alone, and FIC B = IC₅₀ of B in combination/IC₅₀ of B alone. The sum of FIC A and FIC B (∑FIC) was established for each combination, and then the mean value for the four or five ∑FICs was calculated. A mean ∑FIC of ≤0.7 was assumed to reflect a synergistic interaction between the tested drugs and a mean ∑FIC of ≥1.3 an antagonistic interaction. Interactions with a mean ∑FIC value of 0.7 to 1.3 were considered indifferent (in other words, there was no interaction between the drugs, leading to what was formerly described as an additive effect). The graphical presentation (isobolograms) was established by plotting pairs of FIC values for drug A and drug B for each combination. A straight line indicates an indifferent interaction, a curve toward the origin of the axes represents synergy, and a curve in the opposite direction indicates antagonism.