Parasitol Res (2008) 102:1227–1234 Among all the FDs, 5-methyl-tetrahydrofolate (5-Me- THF) is the most important one. It is the predominant FD in mammals, representing 80–90% of the total folate content (Belz and Nau 1998; Wagner 1995). The predominance of 5-Me-THF reflects its role as a cofactor in the conversion of homocysteine to methionine, which in turn is the precursor of S-adenosylmethionine, the methylation cofactor involved in almost 100 reactions (Priest and Bunni 1995). Tetrahy- drofolate (THF) is the second most represented FD in mammals (10% of the total content); it is the product of the main folate enzyme dihydrofolate reductase (DHFR; Nzila et al. 2005a). The FDs FA and folinic acid (FNA) are used clinically to lower the toxicity of antifolate drugs in the treatment of cancer and rheumatoid arthritis (RA; Jackson 1999). In Plasmodium falciparum parasites, studies have clearly demonstrated that the addition of FA or FNA decreases the activity of antifolate drugs, both in vitro and in vitro (Kinyanjui et al. 1999; van Hensbroek et al. 1995). Likewise, the lowering of the folate concentration in in vitro culture medium enhances the activity of antifolate antimalarial agents (Wang et al. 1997). By increasing the folate derivative pools, the parasite can bypass the inhibition of folate synthesis, leading to a decrease in antifolate activity. However, the impact of the other FDs on antifolate activity has not been explored in P. falciparum. In the present paper, we explore the impact of 5-Me-THF and THF along with FA and FNA on the standard antifolates pyrimethamine (PM) and chlorcycloguanil (CCG) against P. falciparum and the anticancer antifolates methotrexate (MTX), aminopterin (AMP), and trimetrexate (TMX; Fig. 1). We have included these anticancer drugs because there have been indications that these agents are potent against malaria parasite (Elslager et al. 1983; Fidock et al. 1998; Walter et al. 1991). Our study indicates that these anticancer drugs alone (for MTX) or in combination with 5- Me-THF (for TMX and AMP) could be used to treat malaria. The potential of MTX as an antimalarial has led us to explore the interaction of this antifolate with other antimalarial drugs. Materials and methods FA, FNA, THF, 5-Me-THF, PM, dapsone (DDS), MTX, AMP, chloroquine (CQ), mefloquine (MFQ), primaquine (PRQ), quinine (QN), proguanil (PG), and probenecid (PBN) were purchased from Sigma Chemical Co. (Poole, UK). CCG was a gift from AstraZeneca (Cheshire, UK). Amodiaquine (AQ), desethyl-amodiaquine (DEAQ), dihy- droartemisinin (DHA), piperaquine (PQ), lumefantrine (LM), pyronaridine (PRN), halofantrine (HLF), and chlor- proguanil (CPG) were gifts from Professor Steve Ward, 1229 Liverpool School of Tropical Medicine, Liverpool, UK. Trimetrexate was a gift from Professor Andre Rosowsky, Dana-Farber Cancer Institute, Boston, MA, USA. Antimalarial activity was measured in the presence of varying concentrations of each compound using radioiso- topic incorporation (Sixsmith et al. 1984). Results were expressed as the drug concentration required for 50% inhibition of [3H]hypoxanthine incorporation into parasite nucleic acid (IC50), using nonlinear regression analysis of the dose–response curve. These IC50 values were deter- mined in the presence or absence of increasing concen- trations of folate derivatives. Two reference P. falciparum laboratory isolates were tested: M24, a fully pyrimeth- amine-sensitive isolate, and V1/S, a highly pyrimethamine- resistant isolate. M24 carries a wild-type dhfr gene, but the V1/S isolate has four mutations at codons 108, 51, 59, and 164 in its dhfr gene (Nzila et al. 2003). Cultures were carried out in Roswell Park Memorial Institute (RPMI) 1640 (GIBCO BRL, UK) medium supplemented with 10% (v/v) normal human serum, 25 mM bicarbonate, 2 mM glutamine, 25-mM 4-(2-hydroxyethyl)-1-piperazine-ethane- sulfonic acid buffer, and 3.6 nM para-aminobenzoic acid. Human blood used to culture the parasites in vitro was obtained from healthy subjects and washed three times with RPMI culture medium nonsupplemented with serum. Synergy Synergy analyses were carried out using V1/S isolate in RPMI medium containing folic acid at physio- logical concentration (23 nM). Synergy was assessed by combining MTX with another drug in the following ratios: 1:0, 0.8:0.2, 0.6:0.4, 0.4:0.6, and 0.2:0.8. Synergy was measured in vitro both geometrically by construction of isobolograms (with a minimum of five coordinates) and algebraically by calculating the sum of the minimum fractional inhibitory concentration (FIC; Berenbaum 1978). Synergy is demonstrated when the FIC is <0.5. A FIC value of >4.0 denotes an antagonistic effect and a FIC value between 0.5 and 4.0 indicates either nonsynergistic or nonantagonistic interaction. Results Folate effect We tested the in vitro activity of the anticancer agents MTX, AMP, and TMX and the antimalarials PM and CCG in the presence of increasing concentrations of FA, the commonly used folate derivative, and THF, FNA, and 5- Me-THF, using the multidrug-resistant V1/S and the fully sensitive isolate M24. We used four folate concentrations: 0.023 μM, which is the physiological folate concentration (normal value in the human body), and concentrations