In vivo and in vitro antimalarial properties of azithromycin-chloroquine combinations that include the resistance reversal agent amlodipine

Abstract

Evidence of emerging Plasmodium falciparum resistance to artemisinin-based combination therapies, documented in western Cambodia, underscores the continuing need to identify new antimalarial combinations. Given recent reports of the resurgence of chloroquine-sensitive P. falciparum parasites in Malawi, after the enforced and prolonged withdrawal of this drug, and indications of a possible synergistic interaction with the macrolide azithromycin, we sought to further characterize chloroquine-azithromycin combinations for their in vitro and in vivo antimalarial properties. In vitro 96-h susceptibility testing of chloroquine-azithromycin combinations showed mostly additive interactions against freshly cultured P. falciparum field isolates obtained from Mali. Some evidence of synergy, however, was apparent at the fractional 90% inhibitory concentration level. Additional in vitro testing highlighted the resistance reversal properties of amlodipine for both chloroquine and quinine. In vivo experiments, using the Peters 4-day suppressive test in a P. yoelii mouse model, revealed up to 99.9% suppression of parasitemia following treatment with chloroquine-azithromycin plus the R enantiomer of amlodipine. This enantiomer was chosen because it does not manifest the cardiac toxicities observed with the racemic mixture. Pharmacokinetic/pharmacodynamic analyses in this rodent model and subsequent extrapolation to a 65-kg adult led to the estimation that 1.8 g daily of R-amlodipine would be required to achieve similar efficacy in humans, for whom this is likely an unsafe dose. While these data discount amlodipine as an additional partner for chloroquine-based combination therapy, our studies continue to support azithromycin as a safe and effective addition to antimalarial combination therapies.

Figures

Fig. 1.

FIC50 and FIC90 isobolograms of AZ-CQ against five freshly cultured CQR P. falciparum isolates from Mali tested in a 96-h assay. Assays were performed using 5 CQ-AZ combinations on the basis of relative ratios of their IC50s: 5:1, 3:1, 1:1, 1:3, and 1:5. As controls, CQ and AZ were tested alone to define their respective IC50 and IC90 values and derive FIC50 and FIC90 values in the combinations. The line of additivity is shown in light gray. Combinations were tested on two separate occasions, and FIC50 (A) or FIC90 (B) values for each occasion and each ratio are indicated with a line connecting the two separate values. Results show an essentially additive set of interactions at the FIC50 level, except for the 1:1 combinations that displayed evidence of synergy. At the IC90 level, all drug combinations provided evidence of some synergy. For each panel, sum FIC values are listed as means ± SDs (values are provided in Table S1 in the supplemental material).

Fig. 2.

Response modification index of various CQ resistance reversal agents against standard culture-adapted P. falciparum lines. An index of 1 implies no reversal, whereas low values imply substantial reversal. All reversal agents were tested in the presence of CQ. Values were determined from three independent experiments performed in duplicate and are presented as means ± SEMs (values are presented in Table S2 in the supplemental material). VP, verapamil; AMLO, amlodipine; R-AMLO, R enantiomer of AMLO; DILT, diltiazem; DES, desipramine; CHLOR, chlorpheniramine; CYP, cyproheptadine.

Fig. 3.

Day 4 suppressive activity of CQ, AZ, and R-AMLO in CD-1 mice infected with P. yoelii nigeriensis N67. Drug concentrations are in mg/kg, abbreviated MKG. Data were obtained from 5 to 15 mice per group and are presented as mean + SEM suppression (values are listed in Table S3 in the supplemental material).

Fig. 4.

Curves of plasma and RBC concentrations as a function of time, obtained from mice administered R-AMLO orally at time points 0, 24, 48, and 72 h. Values are presented as means ± SDs from the groups of three mice sampled at each time point.