Pemetrexed (
LY231514) is a new-generation
antifolate that, in its polyglutamyl forms, is a potent inhibitor of
thymidylate synthase and
glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to
pemetrexed in L1210 murine
leukemia cells using chemical mutagenesis with
5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to
pemetrexed with comparable cross-resistance to
ZD1694 (
Tomudex) and lesser cross-resistance (5-fold) to
ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-
butyric acid],
DDATHF (dideazatetrahydrofolate) (3.5-fold), and
methotrexate (MTX) (2.7-fold) but comparable sensitivity to
trimetrexate. Influx of
pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells.
Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of
pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]
pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]
DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The
reduced folate carrier (RFC) message and
protein were decreased by 50%, and folypolyglutamate
synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either
protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in
thymidylate synthase mRNA, without mutations in the
protein, and there was no change in
GAR transformylase message. The data indicate that resistance to
pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two
proteins that act in tandem to regulate polyglutamation of folates and
antifolates in cells, resulting in cellular depletion of these active
pemetrexed congeners.