Synthesis of poly-gamma-glutamyl metabolites of
methotrexate was demonstrated in mouse small intestine, liver and bone marrow, and in
L1210 leukemia,
Sarcoma 180 and Ehrlich
tumor cells after sc
injections of [3H]
methotrexate to
tumor bearing mice. Ion exchange chromatography of
tissue extracts resolved six peaks of radioactivity believed to represent
methotrexate and metabolites with up to 4 additional glutamyl residues.
Polyglutamate formation in L1210 cells and small intestine was shown to be independent of dose at least to 400 mg/kg as long as intracellular levels of
drug in excess of the
dihydrofolate reductase binding capacity (exchangeable) were maintained. Both the total amount of polyglutamates and the average length of the polyglutamyl chain increased with time as long as exchangeable level of
drug were present intracellularily. The results also showed differences in the extent of metabolism of
methotrexate polyglutamates among the tissues examined. Although, these differences were at times very large, there was no consistent correlation between these differences and other pharmacologic parameters or cytoxicity.
Tumor cells appeared to synthesize more polyglutamates than the normal tissues examined. However, differences in total
drug persistence and sensitivity to
drug among
tumor cells and among normal tissues did not reflect the relative extent of
polyglutamate synthesis in each group. We observed no selective retention of polyglutamates as compared to
methotrexate by L1210 cells in vitro as indicated by the extracellular accumulation during efflux of
methotrexate and the polyglutamates. This could only be demonstrated by allowing efflux of intracellular
drug in the presence of extracellular
dihydrofolate reductase, which averted hydrolysis of the polyglutamates. It is concluded that the extent of
polyglutamate synthesis per se may not be a determinant of
drug sensitivity in murine tissues. However, the accumulation of these metabolites may contribute in some way to overall therapeutic response or relative cytotoxicity.