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Pemetrexed (PMX) is a novel antifolate with polyglutamate derivatives that are potent inhibitors of thymidylate synthase (TS) and to a lesser extent glycinamide ribonucleotide formyltransferase (GARFT). Conditions that might modulate relative suppression of these sites were assessed by the pattern of hypoxanthine and thymidine protection. When grown in medium containing 2 μM folic acid, thymidine alone had a similar protective effect for wild-type HeLa cells and a reduced folate carrier (RFC)-null subline exposed to PMX. However, when grown with 25 nM racemic 5-formyltetrahydrofolate, thymidine alone fully protected wild-type HeLa cells to at least 1 μM PMX, but protection of RFC-null HeLa cells required both thymidine and hypoxanthine above a concentration of 30 nM PMX. As medium 5-formyltetrahydrofolate was decreased, protection by thymidine alone decreased and was further diminished when wild-type HeLa cells were grown in dialyzed serum. There was little protection by thymidine of RFC-null HeLa cells under the latter conditions. Thymidine alone was not protective, and hypoxanthine alone produced only a small (2-fold) increase in IC50, in a Hela-derived line eight-fold resistant to PMX due to a modest increase in TS. Thymidine plus hypoxanthine fully protected these cells. Finally, in MCF-7 breast cancer cells there was greater protection with thymidine alone than in HeLa cells when cells were grown in medium containing a low concentration of 5-formyltetrahydrofolate. These observations indicate that as intracellular folates decrease in HeLa cells, due to decreased extracellular reduced folate, or loss of RFC function, PMX inhibition of GARFT increases. These data support the concept that PMX inhibition of GARFT, particularly at low folate levels, is a contributing factor to drug activity but relative inhibition of TS and GARFT may vary among human tumors and cell lines. This work was supported by a grant from the National Institutes of Health, CA-82621, along with a grant from the Eli Lilly Company.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]