Modulation of anthracycline accumulation and metabolism in rat hepatocytes in culture by three revertants of multidrug resistance

Abstract

The aim of this study was to compare the action of three multidrug resistance (MDR) modulators, cyclosporine A, S 9788, and verapamil, on the efflux of two anthracyclines, doxorubicin and daunorubicin, and of daunorubicinol, the C-13 alcohol metabolite of daunorubicin. Rat-hepatocyte primary cultures have been used as a model of P-glycoprotein (Pgp) expression. This model allows the study of MDR at different levels of Pgp expression, which increases in parallel with the time in culture; furthermore, the hepatocytes are capable of metabolizing drugs, which enables the determination of the role of Pgp on metabolite efflux. All modulators tested were incubated for 6 h at concentrations of 1, 5, and 15 μM with doxorubicin (0.5 μM) and at 1 and 15 μM with daunorubicin (0.5 μM) on hepatocytes grown for 4 and 48 h in culture. Daunorubicinol (0.5 μM) was tested with modulators at 48 h of culture. In fresh hepatocytes, the three MDR modulators did not induce an increase in the intracellular retention of anthracycline as compared with controls (no MDR modulator). At 48 h of culture, the three test drugs increased doxorubicin intracellular accumulation. In contrast, daunorubicin rubicin retention was not modified, but that of its metabolites was increased. Within the concentration range tested, cyclosporine was the most potent modulator without dose-dependent activity. The activity rank order was cyclosporine > S 9788 > verapamil. Cyclosporine and S 9788 were as active in coincubation as in preincubation with anthracyclines. Verapamil had no action when incubated before the addition of anthracyclines. Cyclosporine and S 9788 had an effect on the intracellular retention of daunorubicinol used alone whereas verapamil did not. The action of cyclosporine and S 9788 on the retention of daunorubicinol proves that at least a part of the efflux of C-13 alcohol metabolites of anthracyclines is mediated by Pgp. This study shows that S 9788, cyclosporine, and verapamil are MDR modulators in hepatocytes with high-level Pgp expression. This study also demonstrates that hepatocytes are a potent tool for the study of the action of new MDR modulators on cytostatic drugs as well as on their metabolites.