Frequency of drug resistance in Plasmodium falciparum: a nonsynergistic combination of 5-fluoroorotate and atovaquone suppresses in vitro resistance

Abstract

A combination of 5-fluoroorotate and atovaquone eliminated Plasmodium falciparum in long-term cultures more efficiently than either compound alone. The improved potency came not through synergistic activity but through decreased frequency of drug resistance. In support of this finding, it was shown that 5-fluoroorotate and atovaquone do not act in a synergistic fashion, that 5-fluoroorotate-resistant and atovaquone-resistant P. falciparum organisms generated in vitro do not show cross-resistance, and that the frequency of simultaneous resistance to the two compounds approached the product of their individual resistance frequencies. To demonstrate the last finding, and establish proof of principle, an in vitro method was developed for measuring the frequency of drug resistance in P. falciparum. By this method, it was shown that the frequency of resistance to 10(-7) M 5-fluoroorotate was about 10(-6) and the frequency of resistance to 10(-8) M atovaquone was about 10(-5); the frequency of simultaneous resistance to a combination of 10(-7) M 5-fluoroorotate and 10(-8) M atovaquone was less than 5 x 10(-10). On the basis of additional measurements, it was estimated that the frequency of simultaneous resistance to higher, pharmacologically more relevant, concentrations of 10(-6) M 5-fluoroorotate and 10(-7) M atovaquone would be less than 10(-17). Control experiments demonstrated that these drug combinations did not cause increased toxicity to mammalian cells in culture. On this basis, it is predicted that a combination of 5-fluoroorotate and atovaquone will successfully eliminate typical malarial infections in animals and in human patients at doses that are readily tolerated.

Asexual Populations of the Human Malaria Parasite, Plasmodium falciparum, Use a Two-Step Genomic Strategy to Acquire Accurate, Beneficial DNA Amplifications

“…Similar to what has been observed in clinical settings, Plasmodium falciparum malaria parasites are able to acquire resistance under controlled laboratory conditions [16], [17], [18], [19], [20], [21], [22], [23], [24]. Although parasites exposed to potent antimalarials do not show protective, real-time transcriptional responses [25], the targets of novel antimalarials are often definitively revealed in in vitro selected resistant parasites through novel mutations or copy number variations in the parasite genome [20], [21], [22], [24], [26], [27], [28].…”

A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

“…Transfection of the malarial parasite line Dd2 with single copy of the human DHFR originally proved the primary mechanism of action of WR99210 [29]. However, emergence of transformants always involve long delay phases, very similar to those seen during in vitro selection for drug resistance in the laboratory [61],[62]. It is very likely that while human DHFR helps confer resistance to WR99210, additional genetic changes in the hard-wiring of gene expression are necessary to fully realize the WR99210 tolerance and optimum growth in the transformed cell.…”

“…Third, if there is limited flexibility in regulating gene-expression, perturbations by drugs must be balanced by compensating mutations affecting the transcriptome [64]. This would influence frequencies of drug resistance in unpredictable ways [62],[65], as well as leave molecular footprints of prior drug exposure throughout the genome [66],[67].…”

Global Phenotypic Screening for Antimalarials

“…Medium-throughput assays to determine parasite resistance acquisition frequencies under drug pressure (Gassis and Rathod, 1996; Rathod, et al, 1997; White and Pongtavornpinyo, 2003) would be extremely useful.…”