Augmentation of the Intracellular Levels of Polyglutamyl Derivatives of Methotrexate by Vincristine and Probenecid in Ehrlich Ascites Tumor Cells

Abstract

The intracellular accumulation of poly-γ-glutamyl derivatives of methotrexate was evaluated in the presence of vincristine or probenecid (agents which raise the intracellular level of free methotrexate) in Ehrlich ascites tumor cells. The results show that both intracellular methotrexate and its metabolites are increased by these agents and that, in the presence of l-glutamine, polyglutamate derivatives are increased by a higher percentage than is methotrexate. From 1 to 50 µm vincristine increased the levels of polyglutamate derivatives from 25 to over 300%, whereas methotrexate was raised from 25 to 80%. Similarly, 50 to 200 µm probenecid increased methotrexate polyglutamate derivatives from 31 to 88%, whereas methotrexate was raised from 0 to 30%. A determination of the bound fraction of drug indicated that the proportion of dihydrofolate reductase bound with methotrexate polyglutamates increased in the presence of these agents. Efflux studies showed that over 90% of the large pools of intracellular methotrexate polyglutamates produced by these agents was retained for at least 1 hr in the absence of extracellular methotrexate, whereas the majority of intracellular methotrexate exited the cell. These studies (a) indicate that vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells and (b) introduce another basis for synergism observed between alkaloids and methotrexate.