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Drug 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 falciparum V1S 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 falciparum clinical 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 berghei and 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 vitro selectivity toward the Plasmodium DHFR enzyme, QN254 does not show the adequate therapeutic index to justify its further development as a single agent.

We have analyzed the in vitro chemosensitivity profiles of 115 Kenyan isolates for chloroquine (CQ), piperaquine, lumefantrine (LM), and dihydroartemisinin in association with polymorphisms in pfcrt at codon 76 and pfmdr 1 at codon 86, as well as with variations of the copy number of pfmdr 1 . The median drug concentrations that inhibit 50% of parasite growth (IC 50 s) 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 ( r 2 = −0.26; P = 0.02). Interestingly, parasites for which LM IC 50 s were higher were wild type for pfcrt -76 and pfmdr 1 - 86 . All isolates had one pfmdr 1 copy. Thus, the decrease in LM activity is associated with the selection of wild-type pfcrt - 76 and pfmdr 1 - 86 parasites, 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.

We tested the effect of probenecid and verapamil in chemosensitizing Plasmodium falciparum to 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.

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 .

The antifolate combination of pyrimethamine (PM) and sulfadoxine (SD) is the last affordable drug combination available for wide-scale treatment of falciparum malaria in Africa. Wherever this combination has been used, drug-resistant parasites have been selected rapidly. A study of PM-SD effectiveness carried out between 1997 and 1999 at Kilifi on the Kenyan coast has shown the emergence of RI and RII resistance to PM-SD (residual parasitemia 7 days after treatment) in 39 out of 240 (16.25%) patients. To understand the mechanism that underlies resistance to PM-SD, we have analyzed the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes of 81 patients. Fifty-one samples were obtained, before treatment, from patients who remained parasite free for at least 7 days after treatment. For a further 20 patients, samples were obtained before treatment and again when they returned to the clinic with parasites 7 days after PM-SD treatment. Ten additional isolates were obtained from patients who were parasitemic 7 days after treatment but who were not sampled before treatment. More than 65% of the isolates (30 of 46) in the initial group had wild-type or double mutant DHFR alleles, and all but 7 of the 47 (85%) had wild-type DHPS alleles. In the paired (before and after treatment) samples, the predominant combinations of DHFR and DHPS alleles before treatment were of triple mutant DHFR and double mutant DHPS (41% [7 of 17]) and of double mutant DHFR and double mutant DHPS (29% [5 of 17]). All except one of the posttreatment isolates had triple mutations in DHFR, and most of these were “pure” triple mutants. In these isolates, the combination of a triple mutant DHFR and wild-type DHPS was detected in 6 of 29 cases (20.7%), the combination of a triple mutant DHFR and a single mutant (A437G) DHPS was detected in 4 of 29 cases (13.8%), and the combination of a triple mutant DHFR and a double mutant (A437G, L540E) DHPS was detected in 16 of 29 cases (55.2%). These results demonstrate that the triply mutated allele of DHFR with or without mutant DHPS alleles is associated with RI and RII resistance to PM-SD. The prevalence of the triple mutant DHFR-double mutant DHPS combination may be an operationally useful marker for predicting the effectiveness of PM-SD as a new malaria treatment.

We have analyzed the in vitro chemosensitivity profiles of 115 Kenyan isolates for chloroquine (CQ), piperaquine, lumefantrine (LM), and dihydroartemisinin in association with polymorphisms in pfcrt at codon 76 and pfmdr1 at codon 86, as well as with variations of the copy number of pfmdr1. The median drug concentrations that inhibit 50% of parasite growth (IC(50)s) 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 (r(2) = -0.26; P = 0.02). Interestingly, parasites for which LM IC(50)s were higher were wild type for pfcrt-76 and pfmdr1-86. All isolates had one pfmdr1 copy. Thus, the decrease in LM activity is associated with the selection of wild-type pfcrt-76 and pfmdr1-86 parasites, 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.

We have analyzed the activities of the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), WR99210, trimethoprim (TMP), methotrexate (MTX), and trimetrexate (TMX) against KenyanPlasmodium 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 50of 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 parasitedhfrgenotypes by the PCR-enzyme restriction technique. No wild-typedhfrparasite 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 againstP. falciparum, and quadruple mutants are now emerging in Africa.

We have tested the hypothesis that 2,4-diamino-6-hydroxymethyl-pteridine (DAP), 2,4-diaminopteroic acid (DAPA), and 2,4 diamino-N10-methyl-pteroic acid (DAMPA) could be converted into aminopterin (from DAP and DAPA) and methotrexate (from DAMPA), both of which are potent inhibitors of dihydrofolate reductase, a proven drug target for Plasmodium falciparum . DAP, DAPA, and DAMPA inhibited parasite growth in the micromolar range; DAMPA was the most active, with 50% inhibitory concentrations in vitro of 446 ng/ml against the antifolate-sensitive strain and 812 ng/ml against the highly resistant strain under physiological folate conditions. DAMPA potentiates the activity of the sulfone dapsone, an inhibitor of dihydropteroate synthase, but not that of chlorcycloguanil, a known inhibitor of dihydrofolate reductase (DHFR). Experiments with a Saccharomyces cerevisiae strain dependent upon the P. falciparum DHFR enzyme showed that DHFR is a target of DAMPA in that system. We hypothesize that DAMPA is converted to methotrexate by the parasite dihydrofolate synthase, which explains the synergy of DAMPA with dapsone but not with chlorcycloguanil. This de novo synthesis will not occur in the host, since it lacks the complete folate pathway. If this hypothesis holds true, the de novo synthesis of the toxic compounds could be used as a framework for the search for novel potent antimalarial antifolates.

The activities of 28 6-substituted 2,4-diaminoquinazolines, 2,4-diamino-5,6,7,8-tetrahydroquinazolines, and 2,4-diaminopteridines against Plasmodium falciparum were tested. The 50% inhibitory concentrations (IC50s) of six compounds were

Resistance 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. falciparum in 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).