Analysis of the cytotoxic determinants for methotrexate (NSC 740): a role for 'free' intracellular drug

Therapeutic regimens which employ high dose methotrexate (MTX) infusions over long intervals are likely to create conditions in which cells are exposed to levels of intracellular MTX in considerable excess of the capacity of high affinity intracellular binding sites. This paper reviews experimental evidence which suggests that the ability of MTX to inhibit nucleic acid and protein synthesis may depend upon the accumulation of free intracellular MTX. The possibility is raised that this may be related to the association of a portion of this intracellular component with a form of dihydrofolate reductase with a reduced affinity for this agent. The therapeutic end points and cytotoxic determinants for MTX are contrasted for conditions in which binding to the target enzyme is stoichiometric versus binding to lower affinity target enzymes. In an accompanying paper, factors which control the intracellular MTX level in excess of the tightly bound fraction are explored, focusing in particular on the roles of membrane transport and cellular energy metabolism.