FLOW CYTOMETRIC ASSESSMENT OF HYDROXYPYRIDINONE IRON CHELATORS ON IN-VITRO GROWTH OF DRUG-RESISTANT MALARIA

The resurgence of drug-resistant malaria makes urgent the evaluation o f new antimalarial agents. This study describes a flow cytometric meth od (FCM) for testing in vitro drug susceptibility of Plasmodium falcip arum malaria to several orally active hydroxypyridinone (CP) iron chel ators and to the parenteral iron chelator desferrioxamine (DF). After exposure of parasites to various concentrations of iron chelating agen ts, aliquots of cultures were fixed with glutaraldehyde. The fixed sam ples were washed and stained for parasite DNA with propidium iodide an d analyzed by how cytometry, The remaining cells were pulsed with H-3- hypoxanthine, using the microdilution radioisotope method. Both CP and DF showed dose-dependent inhibition of parasite growth. Of the compou nds studied, DF exerted a stronger inhibitory effect. Fifty percent of inhibitory concentrations (IC50) of CP and DF determined by DNA fluor escence profiles in the now cytometer were consistent with those obtai ned from the radioisotope method and by microscopic examination, Moreo ver, the minimum inhibitory concentrations (MIC) of drug required to i nhibit parasite growth, as detected by the decreasing DNA fluorescence intensity of the schizont, correlated with observed abnormal microsco pic morphology, The validity of the MIG, as indicated by deceased fluo rescence intensity, was confirmed by subsequent parasite culture. Our FCM study demonstrated the sensitivity of both chloroquine- and pyrime thamine-resistant malaria parasites to iron chelators, Addition of equ imolar concentrations of ferric ion completely abolished the inhibitor y effect of fron chelators, indicating the importance of iron for para site growth and the primary effect of the compounds as iron (III) chel ating agents. These data demonstrate that FCM provides a simple and re liable means for antimalarial drug susceptibility testing, and suggest that iron chelators have potential for the treatment of drug-resistan t malaria. (C) 1997 Wiley-Liss, Inc.