Drug uptake, sensitivity, interaction studies and resistance associated mutations in Plasmodium falciparum

Abstract

Evidence is accumulating that polymorphisms in plmdr 1 and plcrt are involved in resistance to the quinoline based blood schizontocides and possibly artemisinin derivatives; and mutations in the cytochrome b gene (cytb) are strongly associated with resistance to atovaquone. With this in mind, we studied sensitivity, drug interactions and uptake in a range of Plasmodium lalciparum lines, including pfmdr 1 transfectants, plcrt mutated lines 106/1 and K76I, and an atovaquone-resistant isolate (NGATVOl) with a unique point mutation (tyr268asn). In vitro susceptibility studies and genetic characterisation supported the role of plmdr 1 and plert polymorphisms in the sensitivity of parasite lines to a range of structurally diverse antimalarials. Mutations in plcrt conferred chloroquine resistance modulated by changes in plmdr 1, while the wild-type genes were associated with reduced sensitivity to mefloquine, halofantrine, lumefantrine and dihydroartemisinin. Drug interaction studies in vitro using a modified isobologram method showed that dihydroartemisinin in combination with chloroquine, amodiaquine or the new bisquinoline piperaquine was antagonistic in all parasite lines examined. The response was synergistic when the drug was combined with mefloquine, halofantrine or lumefantrine against chloroquine-sensitive (wild-type plmdrl and plcrt), but additive against chloroquine-resistant parasite lines. However, in the 7G8-m<fIP10 transfectant (wild-type plmdrl and mutated plert), synergy was shown between each of these three drugs and dihydroartemisinin. These results suggest that the interaction profile of dihydroartemisinin with arylaminoa1cohols depends on mutations in plmdrl in the presence of other chloroquine resistance mutations. In the D 1 0_mdr7G8 transfectant, the introduction of the mutated plmdr 1 converted the synergy between lumefantrine and dihydroartemisinin or artemether to addition; supporting the idea that interactions between artemisinin-derivatives and lumefantrine are dependent on mutations within pfmdr 1 alone. An exception was seen when dihydroartemisinin was combined with arylaminoa1cohol quinine. It showed a strain-specific effect not related to resistance polymorphisms, although a similar change to synergy in the 7G8-md~10 transfectant as for the other arylaminoalcohols was seen. A verapamil-reversible change in the quinine and dihydroartemisinin interaction from additive to synergistic was also noted with the introduction of a mutated PfCRT codon 76 in K76I. An antagonistic effect of dihydroartemisinin with pyrimethamine, was observed in all pyrimethamine-sensitive parasites, but the effect was additive in pyrimethamine-resistant parasites. In contrast to the quinolines, this was not associated with inhibition of eH]-dihydroartemisinin uptake. The interaction between atovaquone and proguanil was strongly synergistic in atovaquone-sensitive lines Kl and T996 and moderately synergistic in the atovaquoneresistant NGATVOI isolate. While the interaction between atovaquone and dihydroartemisinin was additive in the NGATVOI isolate, the interaction was antagonistic in the atovaquone-sensitive strains tested. Mutations in plmdr 1 and plert had no effect on uptake of eH]-dihydroartemisinin, although eH]-chloroquine accumulation was strongly affected by these polymorphisms. Uptake studies in combination with other antimalarials indicated that some of the quinoline-related drugs and arternisinin-derivatives competed with the uptake of the two radiolabelled drugs. This work highlights the important role of drug resistance polymorphisms in sensitivity, interaction and uptake of structurally diverse antimalarials. The unusual pattern of antagonism between dihydroartemisinin and pyrimethamine seen in vitro is supported by observations in vivo with artemisinin in rodent parasites. These and other differing interactions between dihydroartemisinin and antimalarials could have particularly important implications for the design of drug combinations in the future.