Enhanced polyglutamylation of aminopterin relative to methotrexate in the Ehrlich ascites tumor cell in vitro.

The polyglutamylation of aminopterin and methotrexate (N10-methylaminopterin) was compared in the Ehrlich ascites tumor in vitro. Three poly-gamma-glutamyl conjugates of methotrexate and aminopterin were detected, although at an equal (1 microM) extracellular drug concentration, the net accumulation of aminopterin polyglutamates exceeded that for the methotrexate polyglutamyl derivatives by a factor of 9. When compensation was made for transport differences between these compounds by adjusting the extracellular drug concentrations to achieve equivalent intracellular monoglutamyl substrate levels, the polyglutamylation of aminopterin was still 2.8-fold greater than that for methotrexate, suggesting that aminopterin is a better substrate for the folylpolyglutamate synthetase as well as the transport carrier. An additional metabolite of aminopterin was detected within seconds following drug exposure. This derivative did not bind tightly to dihydrofolate reductase, yet it was rapidly converted to a polyglutamate. The formation of both aminopterin polyglutamates and these novel derivatives was enhanced by increases in the free intracellular level of aminopterin. Aminopterin polyglutamates were bound tightly to dihydrofolate reductase and were retained intracellularly relative to unaltered aminopterin when Ehrlich cells containing these forms were suspended in drug-free medium. These findings support a role for the polyglutamylation of aminopterin as a critical element in drug action and as a factor in addition to membrane transport in the disparate antifolate potencies of aminopterin and methotrexate.