Pemetrexed (PMX) is a new-generation antifolate recently approved for cancer treatment. Current regimens require coadminstration of 350 -1000 μgm of folic acid +vitamin B12 to minimize toxicity. Previous studies have established that 5-methyltetrahydrofolate (5-methylTHF), the major blood folate, increases with oral folate supplementation. The current study evaluates the impact of extracellular (EC) 5-methylTHF or 5-formylTHF levels on the antitumor activity of PMX in vitro and whether the preservation of PMX activity with loss of reduced folate carrier (RFC) function, observed in human carcinoma cell lines grown with 5-formylTHF, also occurs in cells grown with 5-methylTHF. The HCT-15 colon cancer cell line, and its RFC-deficient subline PT1, were adapted for at least one week to either 5-formylTHF or 5-methylTHF. In the latter case 0.1 mM β-mercaptoethanol was present in the growth medium and half the initial concentration of 5-methylTHF was added qd to compensate for oxidative degradation (estimated half -life of 5-methylTHF in growth medium is about 24 hours). The IC50 of trimetrexate (TMQ), a lipophilic antifolate that is very sensitive to levels of intracellular folate pools, was utilized to estimate changes in cellular folate levels. The IC50s for TMQ were similar when cells were grown in either 5-formylTHF or 5-methylTHF, ∼70 nM and ∼65 nM for HCT-15 and ∼1.2 and ∼1.8 nM for RFC-null PT1 cells, respectively. This supports the equivalence of 5-methylTHF and 5-formylTHF in replenishing intracellular folate pools, whether RFC is present or absent. IC50s for PMX were similar in HCT-15 (∼110 and ∼100 nM), and nearly similar in PT1 cells (∼70 nM and ∼110 nM) with 5-formylTHF or 5-methylTHF, respectively, confirming preservation of PMX activity despite loss of RFC function. Next, EC 5-formylTHF was varied from 1.6 nM to 62.5 nM in 2.5 fold increments and effects on intracellular folate and sensitivity to PMX was assessed in HCT-15 colon and A549 lung cancer cell lines. While TMQ IC50s increased to a similar extent in both cell lines from ∼3 nM to ∼85 nM over this EC folate range, the PMX IC50 increased in A549 cells to a greater extent (from ∼12 nM to ∼300 nM) than in HCT-15 cells (from ∼10 nM to ∼180 nM). Hence, as observed previously in murine leukemia cells, PMX antitumor activity in vitro is markedly decreased as EC and intracellular folates are increased, even over the range of physiological folate concentrations, although different cancer cell-lines are affected to different extents. These observations suggest that (1) excessive folate supplementation in clinical regimens with PMX may decrease chemotherapeutic efficacy, (2) no more than the lowest recommended level of folate supplementation (350-400 μgm) should be utilized and (3) other non-dietary sources of folate should be avoided.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]