Studies on the in Vivo Synthesis of Methotrexate Polyglutamates and their Efflux Properties in Normal, Proliferative, and Neoplastic Mouse Tissues
Synthesis of poly-γ-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 di-hydrofolate 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 poly-glutamate 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.
KeywordsHydrolysis Leukemia Titration Carboxyl Syringe
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