Interference of 7-hydroxymethotrexate with the determination of methotrexate in plasma samples from children with acute lymphoblastic leukemia employing routine clinical assays.

The accuracy of two clinical assays, the enzyme-multiplied immunoassay (EMIT) and fluorescence polarization immunoassay (FPIA2), universally employed for measurement of plasma levels of methotrexate (MTX) in children administered a high dose of this drug for treatment of acute lymphoblastic leukemia was evaluated here. Because of its superior specificity, sensitivity, and precision, high performance liquid chromatography (HPLC) was selected as the reference method with which the other two procedures were compared using approximately 420 different plasma samples for method comparison. 7-Hydroxymethotrexate (7-OHMTX), the major plasma metabolite of MTX, that can be detected in plasma at relatively high concentrations for long periods following infusion of a high dose of MTX, was also quantitated by HPLC.

Disparate mechanisms of antifolate resistance provoked by methotrexate and its metabolite 7-hydroxymethotrexate in leukemia cells: implications for efficacy of methotrexate therapy.

Methotrexate (MTX) is one of the leading drugs in the treatment of leukemia, but extensive metabolism to 7-hydroxymethotrexate (7-OHMTX) can limit its therapeutic efficacy. In this study we investigated whether 7-OHMTX itself can provoke anti-folate resistance that may further disrupt MTX efficacy. For this purpose, we developed resistance to 7-OHMTX as well as MTX in 2 human leukemia cell lines (CCRF-CEM and MOLT-4) by stepwise exposure to increasing concentrations of 7-OHMTX and MTX.