1232 on purified plasmodial DHFR have demonstrated that MTX binds efficiently to P. falciparum and Plasmodium vivax DHFR (Tahar et al. 2001; Toyoda et al. 1997), and transfection of malaria parasite with human DHFR has further demonstrated that the antimalarial activity of MTX is primarily borne by the inhibition of DHFR (Fidock et al. 1998). Thus, we would expect that addition of folate derivative would decrease the activity of these anticancer drugs in P. falciparum. In addition, there is evidence that MTX uses folate receptor–transporters to enter into the cell (Wang et al. 2007); thus, the addition of FD would lower MTX transport, leading to a decrease in MTX activity. However, even at high concentrations, FDs do not decrease MTX activity. Clearly, further studies are required to understand the absence of the “folate effect” against these anticancer antifolates. The most unexpected effect was observed with 5-Me- THF. The use of this folate derivative did not change the activity of any of the drugs we tested, including the antimalarials. 5-Me-THF is utilized to synthesize methio- nine, and this reaction generates THF, the product of DHFR reaction and this THF would modulate the activity of antifolates as has been clearly demonstrated in mammalian cells (Dudman et al. 1982; Etienne et al. 1993; Hilton et al. 1983; Mini et al. 1984; Reggev and Djerassi 1986). Our data indicate that either 5-Me-THF is not transported into the parasite cell or that the de novo methionine pathways may not efficiently exist in the parasite. It is well established that the parasite obtains its amino acid supply, including methionine, from hemoglobin degradation. Thus, under these conditions, the parasite may not need to synthesize it de novo, though studies have indicated that the methionine de novo pathway may exist in P. falcipa- rum, (Asawamahasakda and Yuthavong 1993; Krungkrai et al. 1989). However, because the parasite can obtain this amino acid from hemoglobin degradation or exogenous medium (Lew et al. 2003), this pathway can be of relative importance, a possibility that has already been proffered (Nzila et al. 2005a). Because the addition of FA, FNA, and especially 5-Me- THF does not decrease the activity of the anticancer drugs, these folate derivatives could be used as adjuvants to increase the antifolate therapeutic index. For instance, we propose to use 5-Me-THF in combination with TMX to treat malaria. This folate derivative would protect the host against drug toxicity while it will not negate the antimalar- ial activity of these anticancer drugs. For instance, the same rational has been developed with the combination TMX + FNA in the treatment of Pneumocystis jiroveci infection (an opportunistic infection commonly found with human immunodeficiency virus infection). TMX is a potent drug against P. jiroveci and this microorganism cannot transport folate derivatives; as a result, the combination of TMX + Parasitol Res (2008) 102:1227–1234 FNA is as potent as TMX alone (Walzer et al. 1992). These observations led scientists to propose the use of TMX + FNA to treat P. jiroveci infection. This combination is safe, and it is now the mainstay of P. jiroveci treatment (Amsden et al. 1992; Fulton et al. 1995). In fact, TMX was discovered as an antimalarial drug (Elslager et al. 1983) but was developed as an anticancer because it is also active against human cells. Because TMX is potent against the malaria parasite (with IC50 < 15 nM), including highly PM- resistant isolates (Table S1), and because TMX + 5-Me- THF is as potent as TMX alone, thus, TMX + 5-Me-THF could be used to treat malaria. It is interesting to note that, based on the pharmacokinetics of TMX, the dose that would be required to treat malaria would be lower than that used in the treatment of cancer or P. jiroveci infection (Marshall and DeLap 1994). We also demonstrated that MTX is potent against P. falciparum isolates with IC50 < 50 nM; thus, we propose this drug could be used to treat malaria. MTX is used at high doses up to 5–12 g/m2 per week (130–300 mg/kg) for several weeks for the treatment of cancer (Barnhart et al. 2001). This dose can yield concentrations of >1,000 μM MTX, which can have life-threatening toxicity (Barnhart et al. 2001). On the other hand, a low dose of MTX (LD- MTX; 0.1 to 0.35 mg/kg (7.5 to 25 mg per adult)) is used for the treatment of RA on a chronic basis, for up to 5 years. At this dose, MTX is safe and increasingly becoming the mainstay in the treatment of RA in the Western world (Borchers et al. 2004; Cronstein 2005; Ostor 2005; Suzuki et al. 2005; Swierkot and Szechinski 2006). LD-MTX is also becoming the drug of choice for the treatment of arthritis in children and multiple sclerosis, and under this regime, MTX is safe and well tolerated, (Niehues and Lankisch 2006; Gray et al. 2004, 2006; Krishna Sumanth et al. 2007). All this information has led us to propose the use of LD- MTX in the treatment of malaria. The proof of the concept that LD-MTX can be used to treat malaria in humans has already been provided. Indeed, two small clinical trials have demonstrated that doses as low as 2.5 mg per day for 3–5 days are effective to treat malaria infection in humans (Sheehy and Dempsey 1970; Wildbolz 1973). However, MTX has not come into widespread use because of concerns over toxicity (Ferone 1971; Laing 1972). At the time the aforementioned clinical trials were carried out (which was in the 1970s), no information was available on the safety of LD-MTX. LD-MTX has been widely used for the treatment of arthritis since the 1980s, and its safety has now been proven. Thus, this drug could be revived as an antimalarial. Plans for clinical trials of this drug are under way in our Unit. We have also provided evidence that MTX does not synergize with the DHPS inhibitor dapsone, yet all DHFR inhibitors described as far synergize with inhibitor of DHPS