Received for publication June 11, 2007.
Revised August 2, 2007.
Accepted for publication August 24, 2007.

USE OF ISOLATED HEPATOCYTE PREPARATIONS FOR P450 INHIBITION STUDIES: COMPARISON WITH MICROSOMES FOR KI DETERMINATION

Abstract

Abstract Predicting drug-drug interactions (DDIs) requires an assessment of the drug concentration available to the enzyme active site, both in vivo, and within an in vitro incubation. These predictions are confounded when the inhibitor accumulates within the liver, either as a result of active transport processes or intracellular binding (including lysosomal trapping). In theory, hepatocytes should provide a more accurate estimation of inhibitory potency compared to microsomes for those compounds that undergo hepatic accumulation. However they are not routinely used for K_i determination and there is limited comparative information available. Therefore the aims of this study were to compare K_i values determined in rat microsomes and freshly isolated hepatocytes using six P450 inhibitors (miconazole, fluconazole, ketoconazole, quinine, fluoxetine and fluvoxamine) with a range of uptake properties (cell-to-medium concentration ratios 4.2 - 6000). Inhibition studies were performed using four probe substrates for CYP2C, CYP2D and CYP3A enzymes (tolbutamide and phenytoin, dextromethorphan and midazolam, respectively). Comparison of unbound K_i values (range 0.05-30µM) showed good agreement between microsomes and hepatocytes, for inhibition of 18 pathways of metabolism. In addition to this, there was no relationship between the cell-to-medium concentration ratios (covering over three orders of magnitude) and the microsomal to hepatocyte K_i ratio of these inhibitors. These data suggest that the hepatic accumulation of these inhibitors results from intracellular binding rather than the involvement of uptake transporters, and indicate that microsomes and hepatocytes appear to be equivalent for determining the inhibitory potency of the six inhibitors investigated in the present study.

Key words: CYP inhibition, drug clearance, drug-drug interactions, hepatic transport, hepatic uptake, hepatocytes, in vitro-in vivo prediction, in vitro-in vivo scaling, microsomes