Role of drug concentration, duration of exposure, and endogenous metabolites in determining methotrexate cytotoxicity

The cytotoxic effects of methotrexate for normal and neoplastic tissues are well known to clinicians but the scheduling of drug administration has primarily been based on empiric observation. However, experimental studies in recent years have provided a rational basis for understanding the role of specific drug concentrations, durations of exposure, and physiologic environment in determining the cytotoxicity of methotrexate. The studies reviewed in this paper demonstrate the requirement for free drug in excess of approximately 10^-8 M concentration for inhibiting DNA synthesis in mouse bone marrow in vivo and in vitro and in various cell culture lines in vitro. Rapid reversal of this inhibition is possible by removal of free drug by washing of cells, enzymatic cleavage of drug, or removal of infusion source in vivo. This requirement for free drug is thought to be the result of the incomplete titration of dihydrofolate reductase by stoichiometric amounts of drug, as demonstrable in in vitro competitive binding experiments. Further studies with constant drug infusion in mice have delineated the relative importance of duration of exposure and drug concentration in determining bone marrow toxicity. It was shown that for drug concentrations >10^-8M, the rate of depletion of nucleated bone marrow cells was directly related to plasma methotrexate concentration and reached its nadir of 30% of control within 12 hours at 10^-5M MTX, 24 hours at 2 x 10^-6M MTX, and 48 hours at 2 x 10^-7M MTX. The rate of loss of nucleated cells was directly related to duration of exposure until the nadir in cell count was reached. Depletion of total nucleated cells was mitigated somewhat by probable recruitment of previously uncommitted precursor cells to myeloid colony-forming potential during the second 24 hours of drug infusion. It was also found that 10% fetal calf serum as well as thymidine (10^-5M) plus a purine nucleoside could prevent the cytotoxic effect of methotrexate on bone marrow colony formation in vitro, indicating that endogenous nucleosides may be important in determining cell-kill.