Abstract 1784: The impact of the natural (6S) isomer of 5-formyltetrahydrofolate on the activities of pralatrexate and methotrexate in vitro
Authors: Visentin Michele , Unal Ersin Selcuk , Goldman I David 
Published: May 2015

Abstract
Introduction: Pralatrexate, the 10-propargyl-10-deaza analog of aminopterin, is a much superior substrate for the reduced folate carrier and folylpolyglutamate synthetase than methotrexate (MTX) resulting in the rapid build-up and retention of high levels of the polyglutamate derivatives of this agent within cells. In HeLa cells in vitro, the growth inhibitory effect of a 6h exposure to pralatrexate is two orders of magnitude greater than that of MTX. Yet the drug can be administered weekly at similar doses than MTX, consistent with a high level of selectivity. Nonetheless, mucosites can result in dosage reductions. This has led to the application of “rescue” regimens with the natural isomer of 5-formyltetrahydrofolate, (6S)5-formylTHF.
Purpose: To investigate the impact of (6S)5-formylTHF on the antitumor activity of pralatrexate in HeLa cells in vitro, simulating the exposures to this folate as occurs in the clinical setting. These properties were compared to those of MTX.
Experimental design: HeLa cells were grown with 25 nM (6S)5-formylTHF as the folate growth source. Experiments evaluated the impact of a 24h exposure to different concentration of (6S)5-formylTHF varying intervals prior to, or after, a 6h exposure to antifolate. The impact on the accumulation of [3H](6S)5-formylTHF and [3H]antifolate was also assessed.
Results: A 24h interval between antifolate, followed by (6S)5-formylTHF, was sufficient to sustain the full activities of the antifolates up to a (6S)5-formylTHF level of 4 µM. A 72h interval was required between (6S)5-formylTHF, followed by the antifolate, to sustain full antifolate activities. HeLa cells were then exposed to 4 µM (6S)5-formylTHF for 24h weekly X 4; a concentration of the folate in excess of what is achieved in “rescue” regimens. This had no significant effect on the pralatrexate IC50; however, the MTX IC50 increased 2.5-fold. The four cycles of exposure to 4 µM [3H](6S)5-formylTHF resulted in a 16% increase in the tritiated intracellular folate level (P=0.02) as compared to cells growing continuously in 25 nM [3H](6S)5-formylTHF.There was no significant decrease in total intracellular pralatrexate; there was a 15% decrease in total intracellular MTX (P=0.004).
Conclusions: Exposure of HeLa cells to (6S)5-formylTHF either before or after pralatrexate or MTX, simulating the sequence of administration in “rescue” regimens, and at concentrations in excess of what is achieved clinically, had no effect on pralatrexate activity but decreased the activity of MTX. This cumulative effect was associated with a small increase in the intracellular (6S)5-formylTHF level and a small decrease in total intracellular MTX. The data suggests that standard “rescue” regimens should have little or no impact on the antitumor activity of pralatrexate.
Citation Format: Michele Visentin, Ersin Selcuk Unal, I David Goldman. The impact of the natural (6S) isomer of 5-formyltetrahydrofolate on the activities of pralatrexate and methotrexate in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1784. doi:10.1158/1538-7445.AM2014-1784