In vitro approach to 7-hydroxymethotrexate interaction on methotrexate metabolism as tool of pharmacokinetic study.

Methotrexate (MTX), a widely used antineoplastic agent, is metabolized to a non-active derivative, 7-OH-MTX, and to some active poly-gamma-glutamyl derivatives (MTX-Gn) which are retained within cells. Pharmacokinetic studies in humans indicate (i) a higher concentration of 7-OH-MTX than of MTX in plasma after a 24-h infusion and (ii) a time-dependent relationship for MTX and 7-OH-MTX kinetics in plasma and urine which might be explained by the variation of MTX metabolism. The intracellular metabolism of MTX and 7-OH-MTX has been investigated using a specific ion-paired method of high-performance liquid chromatography (HPLC) which permits the simultaneous determination of DAMPA, MTX, 7-OH-MTX and their respective polyglutamate derivatives. The formation of 7-OH-MTX polyglutamates and the possible effect of 7-OH-MTX on the transport and/or metabolism of the unchanged MTX in a human acute lymphoblastic leukaemia cell line (Molt 4) have been studied. After incubation of the cells to 1 microM (3H) 7-OH-MTX, four radiolabelled peaks, representing 75% of the intracellular 3H, were converted to 7-OH-MTX upon exposure to hog kidney conjugase indicating the formation of 7-OH-MTX polyglutamyl derivatives. The effects of 7-OH-MTX on MTX-PG formation were analysed after simultaneous incubation of the cells to 1 microM (3H) MTX and 10 microM 7-OH-MTX. The formation of the higher glutamyl derivatives, MTX-G3 and MTX-G4 was completely inhibited and the total intracellular accumulation of the MTX-PG's decreased by 35% compared to control. These data suggest that the 7-OH-MTX and the 7-OH-MTX-PG might modify the chemotherapeutic activity of this agent in vivo.