Plasmodium falciparum resistance to cytocidal versus cytostatic effects of chloroquine

Abstract

With one exception (Gligorijevic et al., Mol Biochem Parasitol 2008;159:7-23.) all previous quantification of chloroquine (CQ) potency vs. P. falciparum has been by growth inhibition assays, meaning potency is defined as cytostatic potential and quantified by IC(50) values. In this study we investigate the cytocidal potency of CQ and other common quinoline antimalarial drugs (quantified as LD(50)). Similar to results from assays for cytostatic potency, we are able to readily distinguish drug resistant from drug sensitive P. falciparum parasites as well as different degrees of resistance. However, we find that fold-resistance to CQ and other quinoline drugs quantified via LD(50) ratios differs quite dramatically from fold resistance calculated via IC(50) ratios. Also, importantly, we find that verapamil chemoreversal of CQ resistance differs when quantified via cytocidal vs. cytostatic assays, as do patterns of "multidrug" resistance in well-known laboratory strains of P. falciparum. The results have important implications for development of new antimalarial drugs and for fully defining the genetic loci that confer clinically relevant antimalarial drug resistance phenomena.

FIG. 1

A. Representative survival curves for asynchronous HB3 (squares, dotted line), Dd2 (circles, straight line), and 7G8 (diamonds, dashed line) iRBC parasites incubated vs variable bolus CQ (x axis) for 6 hours. Fluorescence data of CQ-treated parasites were converted to % parasitemia using standard curves (see Methods) and then expressed as percent survival relative to parasites not exposed to drug. Data points shown were averaged from three independent experiments, each done in triplicate (9 determinations in total), and then fit to a sigmoidal function [y = y₀ + a/(1 + (x/x₀)^b)] using SigmaPlot 9.0. CQ LD₅₀ values were calculated to be 126 nM (HB3), 15,669 nM (Dd2), and 3,991 nM (7G8) (see Table 1). B. Measured CQ LD₅₀ decreases with increasing drug exposure time. While Dd2 (filled symbols) did not show large LD₅₀ differences between the different stages, at short bolus times HB3 trophozoites (open squares) are more sensitive to CQ than are rings (open triangles) or asynchronous culture (open circles).

FIG. 1

A. Representative survival curves for asynchronous HB3 (squares, dotted line), Dd2 (circles, straight line), and 7G8 (diamonds, dashed line) iRBC parasites incubated vs variable bolus CQ (x axis) for 6 hours. Fluorescence data of CQ-treated parasites were converted to % parasitemia using standard curves (see Methods) and then expressed as percent survival relative to parasites not exposed to drug. Data points shown were averaged from three independent experiments, each done in triplicate (9 determinations in total), and then fit to a sigmoidal function [y = y₀ + a/(1 + (x/x₀)^b)] using SigmaPlot 9.0. CQ LD₅₀ values were calculated to be 126 nM (HB3), 15,669 nM (Dd2), and 3,991 nM (7G8) (see Table 1). B. Measured CQ LD₅₀ decreases with increasing drug exposure time. While Dd2 (filled symbols) did not show large LD₅₀ differences between the different stages, at short bolus times HB3 trophozoites (open squares) are more sensitive to CQ than are rings (open triangles) or asynchronous culture (open circles).

FIG. 2

Verapamil does not chemosensitize the CQR strains Dd2 and 7G8 to CQ as indicated by no statistically significant change in CQ LD₅₀ (open bars) upon addition of 5 µM VPL (filled bars). CQ LD₅₀ for HB3 was found to be significantly different from Dd2 (^**, p < 0.001) and 7G8 (^##, p <0.001). Dd2 and 7G8 CQ LD₅₀ values were also significantly different from each other (⁺, p < 0.01), but there were no significant differences for either of the 3 strains +/− VPL (see also Table 1)