title: Flow cytometric assessment of hydroxypyridinone iron chelators on in vitro growth of drug-resistant malaria.
abstract: The resurgence of drug-resistant malaria makes urgent the evaluation of new antimalarial agents. This study describes a flow cytometric method (FCM) for testing in vitro drug susceptibility of Plasmodium falciparum malaria to several orally active hydroxypyridinone (CP) iron chelators and to the parenteral iron chelator desferrioxamine (DF). After exposure of parasites to various concentrations of iron chelating agents, aliquots of cultures were fixed with glutaraldehyde. The fixed samples were washed and stained for parasite DNA with propidium iodide and analyzed by flow cytometry. The remaining cells were pulsed with 3H-hypoxanthine, using the microdilution radioisotope method. Both CP and DF showed dose-dependent inhibition of parasite growth. Of the compounds studied, DF exerted a stronger inhibitory effect. Fifty percent of inhibitory concentrations (IC50) of CP and DF determined by DNA fluorescence profiles in the flow cytometer were consistent with those obtained from the radioisotope method and by microscopic examination. Moreover, the minimum inhibitory concentrations (MIC) of drug required to inhibit parasite growth, as detected by the decreasing DNA fluorescence intensity of the schizont, correlated with observed abnormal microscopic morphology. The validity of the MIC, as indicated by decreased fluorescence intensity, was confirmed by subsequent parasite culture. Our FCM study demonstrated the sensitivity of both chloroquine- and pyrimethamine-resistant malaria parasites to iron chelators. Addition of equimolar concentrations of ferric ion completely abolished the inhibitory effect of iron chelators, indicating the importance of iron for parasite growth and the primary effect of the compounds as iron (III) chelating agents. These data demonstrate that FCM provides a simple and reliable means for antimalarial drug susceptibility testing, and suggest that iron chelators have potential for the treatment of drug-resistant malaria.
authors.affiliation: Thalassemia Center and Office for Research and Development, Mahidol University University, Thailand.
authors.forename: K
authors.initials: K
authors.lastname: Pattanapanyasat
authors.forename: S
authors.initials: S
authors.lastname: Thaithong
authors.forename: D E
authors.initials: DE
authors.lastname: Kyle
authors.forename: R
authors.initials: R
authors.lastname: Udomsangpetch
authors.forename: K
authors.initials: K
authors.lastname: Yongvanitchit
authors.forename: R C
authors.initials: RC
authors.lastname: Hider
authors.forename: H K
authors.initials: HK
authors.lastname: Webster
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D004396:Coloring Agents; D016054:DNA, Protozoan / Q000032:analysis; D003676:Deferoxamine / Q000494:pharmacology; D004351:Drug Resistance; D004912:Erythrocytes / Q000469:parasitology; D005434:Flow Cytometry / Q000379:methods*; D007501:Iron / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000254:growth & development; D011419:Propidium; D011728:Pyridones / Q000494:pharmacology*
chemical_list: D000962:Antimalarials; D004396:Coloring Agents; D016054:DNA, Protozoan; D007502:Iron Chelating Agents; D011728:Pyridones; D011419:Propidium; D007501:Iron; D003676:Deferoxamine