Efflux of methotrexate and its polyglutamate derivatives from hepatic cells in vitro.

The efflux of methotrexate and methotrexate polyglutamates was examined with primary monolayer cultures of rat hepatocytes and also with monolayer cultures of a stable hepatocarcinoma cell line (H35 cells). In this study, the polyglutamates were considered as those species with two or more additional glutamate residues. When methotrexate polyglutamates were present in hepatocytes in excess of methotrexate, efflux led to a further increase in the intracellular proportion of polygluta mates and a selective loss of methotrexate. The same was observed when methotrexate was the predominant intracellular species at the onset of efflux. In both cases, there was as low liberation of intact methotrexate polyglutamates. These results demonstrate that both methotrexate and methotrexate polyglu tamates can cross the cell membrane but that methotrexate does so much more rapidly, resulting in selective retention of methotrexate polyglutamate derivatives. Similar studies were conducted with H35 cells. When meth otrexate polyglutamates were the predominant intracellular species (85% of the total methotrexate), there was a slow release of methotrexate polyglutamates. This was accompa nied by approximately two-fold more rapid release of metho trexate. The latter was derived from the methotrexate polyglu tamate pool since, during the course of efflux (six hr), the intracellular methotrexate concentration was constant, and the polyglutamates gradually decreased. This demonstrates that intact methotrexate polyglutamates can leave the cells, but this is probably not as rapid in H35 cells as cleavage to methotrex ate and subsequent efflux. Examination of the initial rate of efflux of methotrexate poly glutamates and methotrexate under similar conditions in H35 cells demonstrates that the rate of methotrexate efflux is nearly 70 times more rapid than the rate of polyglutamate efflux. Similar studies in hepatocytes indicate not less than a 10-fold more rapid efflux of methotrexate. The rate of efflux of the polyglutamate was nearly the same in hepatocytes and H35 cells. Measurements of cell permeability during efflux showed no change in trypan blue uptake with either cell type, indicating that the loss of methotrexate and its polyglutamate derivatives did not appear to be due to cell fragility. Hepatocytes and H35 cells that were made permeable (90% permeable by trypan blue uptake) by lysolecithin treatment lost approximately 85% of the total intracellular methotrexate including polyglutamates within 2 min. There was no retention of any of the methotrexate species, indicating that the selective retention of methotrexate polyglutamates in untreated cells was due to reduced perme ability.