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ABSTRACTThe mechanisms of drug resistance development in the Plasmodium falciparumparasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P= 0.04). The high frequency of Pfmspdbl2codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem).

ABSTRACTThe Plasmodium falciparumgenome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitroresponses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P= 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P= 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6were significantly more susceptible to piperaquine than those with 8 (P= 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P= 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC50) (P= 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitroantimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparumare required.

ABSTRACTResistance to the amino alcohol quinine has been associated with polymorphisms in pfnhe, a sodium hydrogen exchanger. We investigated the role of this gene in quinine resistance in vitroin isolates from Kenya. We analyzed pfnhewhole-gene polymorphisms, using capillary sequencing, and pfcrtat codon 76 (pfcrt-76) and pfmdr1at codon 86 (pfmdr1-86), using PCR-enzyme restriction methodology, in 29 isolates from Kilifi, Kenya, for association with the in vitroactivities of quinine and 2 amino alcohols, mefloquine and halofantrine. In vitroactivity was assessed as the drug concentration that inhibits 50% of parasite growth (IC50). The median IC50s of quinine, halofantrine, and mefloquine were 92, 22, and 18 nM, respectively. The presence of 2 DNNND repeats in microsatellite ms4760 of pfnhewas associated with reduced susceptibility to quinine (60 versus 227 nM for 1 and 2 repeats, respectively; P< 0.05), while 3 repeats were associated with restoration of susceptibility. The decrease in susceptibility conferred by the 2 DNNND repeats was more pronounced in parasites harboring the pfmdr1-86 mutation. No association was found between susceptibility to quinine and the pfcrt-76 mutation or between susceptibility to mefloquine or halofantrine and the pfnhegene and the pfcrt-76 and pfmdr1-86 mutations. Using previously published data on the in vitroactivities of chloroquine, lumefantrine, piperaquine, and dihydroartemisinin, we investigated the association of their activities with pfnhepolymorphism. With the exception of a modulation of the activity of lumefantrine by a mutation at position 1437, pfnhedid not modulate their activities. Two DNNND repeats combined with the pfmdr1-86 mutation could be used as an indicator of reduced susceptibility to quinine.

ABSTRACTDrug resistance against dihydrofolate reductase (DHFR) inhibitors—such as pyrimethamine (PM)—has now spread to almost all regions where malaria is endemic, rendering antifolate-based malaria treatments highly ineffective. We have previously shown that the di-amino quinazoline QN254 [5-chloro-N′6′-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine] is active against the highly PM-resistant Plasmodium falciparumV1S strain, suggesting that QN254 could be used to treat malaria in regions with a high prevalence of antifolate resistance. Here, we further demonstrate that QN254 is highly active against Plasmodium falciparumclinical isolates, displaying various levels of antifolate drug resistance, and we provide biochemical and structural evidence that QN254 binds and inhibits the function of both the wild-type and the quadruple-mutant (V1S) forms of the DHFR enzyme. In addition, we have assessed QN254 oral bioavailability, efficacy, and safety in vivo. The compound displays favorable pharmacokinetic properties after oral administration in rodents. The drug was remarkably efficacious against Plasmodium bergheiand could fully cure infected mice with three daily oral doses of 30 mg/kg. In the course of these efficacy studies, we have uncovered some dose limiting toxicity at higher doses that was confirmed in rats. Thus, despite its relative in vitroselectivity toward the PlasmodiumDHFR enzyme, QN254 does not show the adequate therapeutic index to justify its further development as a single agent.

ABSTRACTWe have analyzed the in vitro chemosensitivity profiles of 115 Kenyan isolates for chloroquine (CQ), piperaquine, lumefantrine (LM), and dihydroartemisinin in association with polymorphisms in pfcrtat codon 76 and pfmdr1at codon 86, as well as with variations of the copy number of pfmdr1. The median drug concentrations that inhibit 50% of parasite growth (IC50s) were 41 nM (interquartile range [IQR], 18 to 73 nM), 50 nM (IQR, 29 to 96 nM), 32 nM (IQR, 17 to 46 nM), and 2 nM (IQR, 1 to 3 nM) for CQ, LM, piperaquine, and dihydroartemisinin, respectively. The activity of CQ correlated inversely with that of LM (r2= −0.26; P= 0.02). Interestingly, parasites for which LM IC50s were higher were wild type for pfcrt-76and pfmdr1-86. All isolates had one pfmdr1copy. Thus, the decrease in LM activity is associated with the selection of wild-type pfcrt-76and pfmdr1-86parasites, a feature that accounts for the inverse relationship between CQ and LM. Therefore, the use of LM-artemether is likely to lead to the selection of more CQ-susceptible parasites.

ABSTRACTWe have analyzed the activities of the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), WR99210, trimethoprim (TMP), methotrexate (MTX), and trimetrexate (TMX) against Kenyan Plasmodium falciparumisolates adapted in vitro for long-term culture. We have also assessed the relationship between these drug activities and mutations in dihydrofolate reductase (dhfr), a domain of the gene associated with antifolate resistance. As expected, WR99210 was the most potent drug, with a median 50% inhibitory concentration (IC50) of <0.075 nM, followed by TMX, with a median IC50of 30 nM. The median IC50of CCG was 37.80 nM, and that of MTX was 83.60 nM. PM and TMP were the least active drugs, with median IC50s of 733.26 nM and 29,656.04 nM, respectively. We analyzed parasite dhfrgenotypes by the PCR-enzyme restriction technique. No wild-type dhfrparasite was found. Twenty-four of 33 parasites were triple mutants (mutations at codons 108, 51, and 59), and only 8/33 were double mutants (mutations at codons 108 and 51 or at codons 108 and 59). IC50s were 2.1-fold (PM) and 3.6-fold (TMP) higher in triple than in double mutants, though these differences were not statistically significant. Interestingly, we have identified a parasite harboring a mutation at codon 164 (Ile-164-Leu) in addition to mutations at codons 108, 51, and 59. This quadruple mutant parasite had the highest TMP IC50and was in the upper 10th percentile against PM and CCG. We confirmed the presence of this mutation by sequencing. Thus, TMX and MTX are potent against P. falciparum, and quadruple mutants are now emerging in Africa.

ABSTRACTWe tested the effect of probenecid and verapamil in chemosensitizing Plasmodium falciparumto 14 antimalarials using the multidrug-resistant strain V1S and the drug-sensitive 3D7. Verapamil chemosensitizes V1S to quinine and chloroquine. Interestingly, probenecid profoundly chemosensitizes V1S to piperaquine. Thus, probenecid could be used to increase piperaquine efficacy in vivo.

ABSTRACTResistance to drugs can result from changes in drug transport, and this resistance can sometimes be overcome by a second drug that modifies the transport mechanisms of the cell. This strategy has been exploited to partly reverse resistance to chloroquine in Plasmodium falciparum. Studies with human tumor cells have shown that probenecid can reverse resistance to the antifolate methotrexate, but the potential for reversal of antifolate resistance has not been studied in P. falciparum.In the present study we tested the ability of probenecid to reverse antifolate resistance in P. falciparumin vitro. Probenecid, at concentrations that had no effect on parasite viability alone (50 μM), was shown to increase the sensitivity of a highly resistant parasite isolate to the antifolates pyrimethamine, sulfadoxine, chlorcycloguanil, and dapsone by seven-, five-, three-, and threefold, respectively. The equivalent effects against an antifolate-sensitive isolate were activity enhancements of approximately 3-, 6-, 1.2-, and 19-fold, respectively. Probenecid decreased the level of uptake of radiolabeled folic acid, suggesting a transport-based mechanism linked to folate salvage. When probenecid was tested with chloroquine, it chemosensitized the resistant isolate to chloroquine (i.e., enhanced the activity of chloroquine). This enhancement of activity was associated with increased levels of chloroquine accumulation. In conclusion, we have shown that probenecid can chemosensitize malaria parasites to antifolate compounds via a mechanism linked to reduced folate uptake. Notably, this effect is observed in both folate-sensitive and -resistant parasites. In contrast to the activities of antifolate compounds, the effect of probenecid on chloroquine sensitivity was selective for chloroquine-resistant parasites (patent P407595GB [W. P. Thompson & Co., Liverpool, United Kingdom] has been filed to protect this intellectual property).

ABSTRACTSixty-nine Kenyan Plasmodium falciparumfield isolates were tested in vitro against pyrimethamine (PM), chlorcycloguanil (CCG), sulfadoxine (SD), and dapsone (DDS), and their dihydrofolate reductase (DHFR) genotypes were determined. The in vitro data show that CCG is more potent than PM and that DDS is more potent than SD. DHFR genotype is correlated with PM and CCG drug response. Isolates can be classified into three distinct groups based on their 50% inhibitory concentrations (IC50s) for PM and CCG (P< 0.01) and their DHFR genotypes. The first group consists of wild-type isolates with mean PM and CCG IC50s of 3.71 ± 6.94 and 0.24 ± 0.21 nM, respectively. The second group includes parasites which all have mutations at codon 108 alone or also at codons 51 or 59 and represents one homogeneous group for which 25- and 6-fold increases in PM and CCG IC50s, respectively, are observed. Parasites with mutations at codons 108, 51, and 59 (triple mutants) form a third distinct group for which nine- and eightfold increases in IC50s, respectively, of PM and CCG compared to the second group are observed. Surprisingly, there is a significant decrease (P< 0.01) of SD and DDS susceptibility in these triple mutants. Our data show that more than 92% of Kenyan field isolates have undergone at least one point mutation associated with a decrease in PM activity. These findings are of great concern because they may indicate imminent PM-SD failure, and there is no affordable antimalarial drug to replace PM-SD (Fansidar).

We have analyzed the in vitro activities of pyronaridine and methylene blue against 59 Plasmodium falciparum isolates from Kenya in association with polymorphisms in Pfcrt (codon 76), Pfmdr1 (codon 86), and Pfnhe (full sequence). The median inhibitory concentrations that kill 50% of parasites were 13.5 and 3.3 nM for pyronaridine and methylene blue, respectively. Their activities were not associated with polymorphisms in these genes. The drugs' high in vitro activities indicate that they would be efficacious against Kenyan isolates in vivo.

ABSTRACTWe have analyzed the in vitroactivities of pyronaridine and methylene blue against 59 Plasmodium falciparumisolates from Kenya in association with polymorphisms in Pfcrt(codon 76), Pfmdr1(codon 86), and Pfnhe(full sequence). The median inhibitory concentrations that kill 50% of parasites were 13.5 and 3.3 nM for pyronaridine and methylene blue, respectively. Their activities were not associated with polymorphisms in these genes. The drugs' high in vitroactivities indicate that they would be efficacious against Kenyan isolates in vivo.