Abstract

Antifolates such as methotrexate, raltitrexed, and pemetrexed are among the most effective and widely used anticancer drugs. The antifolates are also among the most unpredictable of anticancer drugs with respect to pharmacokinetics and toxicity. In this study, we assessed the binding of folates and antifolates to the folate receptors (FRs) of human proximal tubules and the effects of pH on binding. Binding of [³H]folic acid was pH-dependent, with maximal binding seen at pH 6. Equilibrium binding experiments with [³H]folic acid showed that K_d values were unaffected, and B_max values increased as the pH was decreased from 8.0 to 6.0. Increasing the osmolarity at pH 6.0 had no effect on intravesicular content, confirming that increased site-specific binding caused the observed changes in B_max values. Enzymatic cleavage of glycosyl-phosphatidylinositol linkages abolished binding of [³H]folic acid to brush-border membrane vesicles, suggesting that [³H]folic acid was bound to FRs. In concentration-effect experiments conducted at different pH values, the antifolates raltitrexed and (2S)-2-[o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-yl-methyl)-N-(prop-2-ynyl)amino]benzamido]-4-(tetrazol-5-yl) butyric acid (ZD9331) bound more tightly as pH increased from 6.0 to 8.0, whereas binding of 10-propargyl-5,8-dideazafolic acid (CB3717) was unchanged. The results obtained when K_i values were converted to binding energies suggested that binding of some, but not all, antifolates and folates to FRs was pH-dependent, further indicating roles of luminal pH in renal reabsorption or secretion processes.