Determinants of the activities of antifolates delivered into cells by folate-receptor-mediated endocytosis

Purpose: Elements in the endocytic process that are determinants of the activities of antifolates delivered by folate-receptor alpha (FRα) were explored. Methods: Antifolate growth inhibition was assessed with a 1- or 5-day exposure in reduced folate carrier-null HeLa cell lines that express a high level of FRα in the presence or absence of the proton-coupled folate transporter (PCFT). pH-dependent rates of dissociation from FRα were also determined. Results: With a 1-day drug exposure which is relevant to the pulse clinical administration of these drugs, FRα expression enhanced raltitrexed activity and modestly enhanced ZD9331 activity, but did not significantly augment the activity of pemetrexed or lomotrexol. With a 5-day drug exposure, FRα-mediated growth inhibition was increased for raltitrexed and ZD9331 and emerged for lomotrexol. While the FRα-augmented activity of lomotrexol and raltitrexed did not require PCFT, augmentation of ZD9331 activity required the co-expression of PCFT with both 1- and 5-day exposures. In contrast, there was no augmentation of pemetrexed activity by FRα under any condition. The activities of these agents correlated with their rate of dissociation from the receptor at acidic pH: raltitrexed > ZD9331 > lomotrexol > pemetrexed consistent with insufficient pemetrexed release from FRα for export from the endosomes. Conclusions: FRα is unlikely to contribute to the pharmacological activity of antifolates, such as pemetrexed, that bind tightly to, and dissociate slowly from, the receptor particularly when the exposure time is brief. While PCFT was required for FRα-mediated ZD9931 activity, the activities of the other antifolates was independent of PCFT.