The folate derivatives folic acid (FA) and folinic acid (FNA) decrease the in vivo and in vitro activities of antifolate drugs in Plasmodium falciparum. However, the effects of 5-methyl-tetrahydrofolate (5-Me-THF) and tetrahydrofolate (THF), the two dominant circulating folate forms in humans, have not been explored yet. We have investigated the effects of FA, FNA, 5-Me-THF, and THF on the in vitro activity of the antimalarial antifolates pyrimethamine and chlorcycloguanil and the anticancer antifolates methotrexate (MTX), aminopterin, and trimetrexate (TMX), against P. falciparum. The results indicate that these anticancers are potent against P. falciparum, with IC 50 <50 nM. 5-Me-THF does not significantly decrease the activity of all tested drugs, and none of the tested folate derivatives significantly decrease the activity of these anticancers. Thus, malaria folate metabolism has features different from those in human, and the exploitation of this difference could lead to the discovery of new drugs to treat malaria. For instance, the combination of 5-Me-THF with a low dose of TMX could be used to treat malaria. In addition, the safety of a low dose of MTX in the treatment of arthritis indicates that this drug could be used alone to treat malaria.

We have analyzed the activities of the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), WR99210, trimethoprim (TMP), methotrexate (MTX), and trimetrexate (TMX) against Kenyan Plasmodium falciparum isolates 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 (IC 50 ) of <0.075 nM, followed by TMX, with a median IC 50 of 30 nM. The median IC 50 of CCG was 37.80 nM, and that of MTX was 83.60 nM. PM and TMP were the least active drugs, with median IC 50 s of 733.26 nM and 29,656.04 nM, respectively. We analyzed parasite dhfr genotypes by the PCR-enzyme restriction technique. No wild-type dhfr parasite 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). IC 50 s 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 IC 50 and 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.

The combination therapy of the Artemisinin-derivative Artemether (ART) with Lumefantrine (LM) (Coartem®) is an important malaria treatment regimen in many endemic countries. Resistance to Artemisinin has already been reported, and it is feared that LM resistance (LMR) could also evolve quickly. Therefore molecular markers which can be used to track Coartem® efficacy are urgently needed. Often, stable resistance arises from initial, unstable phenotypes that can be identified in vitro. Here we have used the Plasmodium falciparum multidrug resistant reference strain V1S to induce LMR in vitro by culturing the parasite under continuous drug pressure for 16 months. The initial IC50 (inhibitory concentration that kills 50% of the parasite population) was 24 nM. The resulting resistant strain V1SLM, obtained after culture for an estimated 166 cycles under LM pressure, grew steadily in 378 nM of LM, corresponding to 15 times the IC50 of the parental strain. However, after two weeks of culturing V1SLM in drug-free medium, the IC50 returned to that of the initial, parental strain V1S. This transient drug tolerance was associated with major changes in gene expression profiles: using the PFSANGER Affymetrix custom array, we identified 184 differentially expressed genes in V1SLM. Among those are 18 known and putative transporters including the multidrug resistance gene 1 (pfmdr1), the multidrug resistance associated protein and the V-type H+ pumping pyrophosphatase 2 (pfvp2) as well as genes associated with fatty acid metabolism. In addition we detected a clear selective advantage provided by two genomic loci in parasites grown under LM drug pressure, suggesting that all, or some of those genes contribute to development of LM tolerance – they may prove useful as molecular markers to monitor P. falciparum LM susceptibility.

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.The modulation of chloroquine (CQ) resistance with the calcium channel blocker verapamil (VPM), antipsychotic drugs, histamine receptor antagonists, and antidepressant agents among others was the first case of resistance modulation to be reported (reviewed in references 6, 8, and 28).Clinical evaluation of some of these agents has been carried out in areas where CQ resistance is moderate (22,(24)(25)(26). However, these agents have the disadvantage of being pharmacologically active, with systemic effects that may result in a variety of side effects. In addition, the minimum concentrations of these agents needed to chemosensitize parasites to CQ (usually more than 1 M of free drug) (1, 4, 11-13) are not achievable in vivo when normal doses are used; therefore, high doses have to be used, with all of the attendant risks of toxicity. All of these limitations may explain why the reversal of CQ resistance has never attained widespread application.We have demonstrated that the uricosuric drug probenecid (PBN) chemosensitizes parasites to antifolate and CQ (16,18).In this study, we tested the effect of PBN against the aminoquinolines CQ, piperaquine (PPRQ), primaquine (PMQ), desethylamodiaquin (DEAQ), and amodiaquin (AQ); the amino alcohols lumefantrine (LM), mefloquine (MFQ), halofantrine (HLF), and quinine (QN); the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), and methotrexate (MTX); the benzonaphthyridine pyronaridine (PRN); and the sesquiterpene dihydroartemisinin (DHA). We used the chemosensitizer VPM as a comparator.CQ, PMQ, AQ, MFQ, QN, MTX, PBN, and VPM were purchased from Sigma (Poole, Dorset, United Kingdom). PPRQ was a gift from Universal Corporation Limited, Kikuyu, Kenya. DEAQ, DHA, LM, PRN, and HLF were gifts from Steve Ward, Liverpool School of Tropical Medicine, Liverpool, United Kingdom. Drugs were dissolved as suggested by manufacturers. For PBN, after dilution in dimethyl sulfoxide (500 mg/ml), a subsequent serial dilution was carried out; this consisted of fivefold dilution in absolute ethanol followed by twofold dilution in 2% sodium bicarbonate and then dilution in RPMI 1640 medium (PBN crystallizes when diluted directly from dimethyl sulfoxide to RPMI 1640 medium).We employed two reference P. falciparum laboratory strains: V1S, a multidrug-resistant strain (resistant to CQ, PM, and QN) and 3D7, a strain fully sensitive to all tested antimalarials, except LM and PMQ. Cultures were carried out in RPMI 1640 (GIBCO BRL, United Kingdom), and antimalarial activities were expressed as the drug concentration required for 50% inhibition of [ 3 H]hypoxanthine incorporation (IC 50 ) during the 66-h assay (19).Chemosensit...