Uptake of 6-substituted pyrrolo[2,3-d]
pyrimidine thienoyl
antifolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid
tumors by the
proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the
reduced folate carrier (RFC), the major facilitative
folate transporter, RFC expression may alter
drug efficacies by affecting cellular
tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular
enzyme targets. Human
tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT
protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in
drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [³H]THF cofactors derived from [³H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5
tumors than toward WT
tumors, confirming the collateral
drug sensitivities observed in vitro. Thus, solid
tumor-targeted
antifolates with PCFT-selective cellular uptake should have enhanced activities toward
tumors lacking RFC function, reflecting contraction of THF cofactor pools.