Parasitol Res (2008) 102:1227–1234 Table 11 AAccttiivviittyy ooffMMTTXXininthteheprpersesnecneceofoifnhiinbhiitboirtsorosf omfalmarailaardiaihydrdorfoulgaste (rCedQu,ctPasQe, (CPCRGQ,anLdMW,RM9F92Q1,0)H;LinFh,ibDitoErAsQof, dainhdydrQoNpt)e;rotahte sdyihnythdarsoefo(lDatDeSre),dturciatazsinee(-CbaCsGedacnodmWpoRu9n9d2s1,0s)y;nienrhgibzietrosrsofofatdoivhayqduronpe- (PGseasnqduiCtePrpGe)n, equdiinhoyldinroea,ratnemdiasriynlina;mtihneo-aanlctiofohloalterealantteidcadnrcuegrsT(MCQX,; PtQhe, tPeRroQa,teL Msy,nMthFasQe, H(DLDF,SD), EtAriQaz,inaen-dbaQsNed); cthoemspeosuqnuditse,rpseynneedrgi hizyedrrsoaorftemisbineinnz;otnhaepahnthtiyfroildaitneeaPntRicNanacnedr TthMe Xna;pthethboeqnuzinoonnapehdtheryirviadtiinvee,PARTNVand tahtoevnaaqpuhotnheoq(uPiGnoanneddCerPivGa)t,ivqeu,inAoTlVine, and aryl amino-alcohol related Combination CCG–DDS MTX–DDS ATV–PG MTX–PG MTX–CPG MTX–CCG MTX–WR99210 MTX–CQ MTX–DHA MTX–LM MTX–QN MTX–TMX MTX–PRQ MTX–HLF MTX–DEAQ MTX–MFQ MTX–ATV MTX–PRN MTX–PQ Drug ratio 1:1 Sum FIC 0.36±0.18 1.0±0.1 ND 1.16±0.23 ND 1.1±0.3 ND ND ND 1.57±0.18 ND ND ND ND ND ND ND ND ND 0.8:0.2 0.46±0.0 1.17±0.36 0.25±0.0 1.48±0.64 0.92±0.45 1.09±0.29 1.10±0.3 1.40±0.34 1.47±0.2 1.07±0.0 1.47±0.41 1.0 ±0.1 1.23±0.2 2.36±0.0 1.39±0.9 1.90±0.7 1.26±0.2 3.52±1.2 2.40±0.3 0.6:0.4 0.40±0.0 1.37±0.12 0.22±0.1 1.84±0 1.49±0.0 1.06±0.16 1.20±0.3 1.48±0.44 1.33±0.7 1.77±0.25 1.73±0.24 1.33±0.0 1.34±0.1 2.32±1.1 1.14±0.3 1.67±0.8 1.63±0.0 3.24±1.6 3.00±0.3 0.4:0.6 0.38±0.0 1.82±0.72 0.24±0.0 1.55±0.30 1.57±1.0 1.18±0.12 1.37±0.4 1.08±0.18 1.20±0.30 1.89±0.19 1.56±0.31 1.35±0.0 1.61±0.4 1.59±0.5 0.87±0.5 1.40±0.9 1.15±0.3 3.14±0.4 3.33±0.1 0.2:0.8 0.48±0.1 1.78±0.27 0.28±0.0 1.32±0.41 .98 ±0.0 1.81±0.81 1.40±0.5 1.17±0.0 0.84±0.22 1.82±0.0 1.36±0.0 1.43±0.0 1.14±0.1 1.03±0.2 1.21±0.1 1.38±1.2 1.58±0 3.36±1.0 1.58±0.1 1231 Score Synergistic Additive Synergistic Additive Additive Additive Additive Additive Additive Additive Additive Additive Additive Additive Additive Additive Additive Additive? Additive? CCG–DDS and ATV–PG were used as control synergistic combinations. Data are represented as the total fractional inhibitory concentration. 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 indicates either nonsynergistic or nonantagonistic interaction CCG Chlorcycloguanil, DDS dapsone, MTX methotrexate, ATV atovaquone, PG proguanil, CPG chlorproguanil, WR99210 Walter Reed compound 99210, CQ chloroquine, DHA dihydroartemisinin, LM lumefantrine, QN quinine, TMX trimetrexate, PRQ primaquine, HLF halofantrine, DEAQ desethyl-amodiaquine, MFQ mefloquine, PRN pyronaridine Fig. 2 Effect of probenecid (PBN) on the activity of PM, MTX, and TMX. Y axis represents the percentage decrease in IC50 in the presence of PBN. One hundred percent (100%) represents the IC50 in the absence of PBN. In the absence of PBN, the MTX, TMX, and PM IC50 values were 30, 7, and 1,200 nM, respectively of the four total FIC values of MTX with PQ (at ratios of 0.4:0.6 and 0.6:04) were 3.00 and 3.15. These values are closer to 4, the cutoff point used to identify antagonistic combinations, an indication that PNR and PQ may antagonize MTX activity. Discussion Our data clearly indicate that malaria folate metabolism has features different from those in human. Indeed, the antimalarial activity of the anticancer drugs were not decreased by FA, FNA, and THF. All of these forms of folate increase the concentrations of dihydrofolate and THF, the substrate and the product of DHFR reactions, respec- tively. Under these conditions, one would expect the activity of the inhibitors of DHFR to decrease. This is what we observed with the standard antimalarial antifolates PM and CCG but not with the anticancers MTX, AMP, and TMX. Yet, evidence indicates that these anticancer drugs, at least MTX, target malaria DHFR. Indeed, kinetics studies