The role of alpha-folate receptor-mediated transport in the antitumor activity of antifolate drugs.

Purpose: Raltitrexed, pemetrexed, lometrexol, and ZD9331 are antifolate drugs transported into cells via the ubiquitously expressed reduced-folate carrier. They display also high affinity for the alpha-folate receptor (alpha-FR), a low capacity folate transporter that is highly overexpressed in some epithelial tumors. The role of alpha-FR in the activity of the antifolates has been evaluated in two alpha-FR-overexpressing cell lines grown in a physiological concentration of folate (20 nM R,S-Leucovorin).

Experimental Design And Results: A431-FBP cells (transfected with the alpha-FR) were 3-5-fold more sensitive to the antifolates than A431 cells. KB cells (constitutive alpha-FR overexpression) were less sensitive to the drugs when coexposed to 1 microM folic acid to competitively inhibit binding to the alpha-FR. Raltitrexed, pemetrexed, and lometrexol are polyglutamated in cells leading to drug retention, e.g., the raltitrexed 4- and 24-h IC(50)s in A431 cells were approximately 0.6 and 0.008 microM, respectively, compared with 0.003 microM for 72-h continuous exposure. A431-FBP cells were approximately 3-fold more sensitive to raltitrexed and pemetrexed at all exposure times. ZD9331 is not polyglutamated, and the 4- and 24-h IC(50)s in A431 cells were >100 and approximately 100 microM, respectively, reducing to 2 and 0.1 microM, respectively, in A431-FBP cells. The ZD9331 4- and 24-h IC(50)s in KB cells were 20 and 1 microM, respectively, and reversible by coaddition of 1 microM folic acid. An in situ thymidylate synthase assay demonstrated continued thymidylate synthase inhibition after ZD9331-treated A431-FBP and KB, but not A431, cells were placed in drug-free medium for 16 h. A model is proposed in which the antifolates accumulate in the alpha-FR/endosomal apparatus, leading to slow release into the cytoplasm. In particular, this leads to cellular retention of the nonpolyglutamatable ZD9331.

Conclusions: Antifolate drugs, particularly ZD9331, have the potential for increased efficacy in tumors that highly overexpress the alpha-FR.