title: Use of isolated hepatocyte preparations for cytochrome P450 inhibition studies: comparison with microsomes for Ki determination.
abstract: Predicting drug-drug interactions 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 with microsomes for those compounds that undergo hepatic accumulation. However, they are not routinely used for Ki determination and there is limited comparative information available. Therefore, the aims of this study were to compare Ki values determined in rat microsomes and freshly isolated hepatocytes using six cytochrome 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 Ki values (range 0.05-30 microM) 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 3 orders of magnitude) and the microsomal to hepatocyte Ki 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.
authors.affiliation: Centre for Applied Pharmacokinetic Research, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Hayley S
authors.initials: HS
authors.lastname: Brown
authors.forename: Anthony
authors.initials: A
authors.lastname: Chadwick
authors.forename: J Brian
authors.initials: JB
authors.lastname: Houston
mesh_terms: D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002478:Cells, Cultured; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003915:Dextromethorphan / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D015725:Fluconazole / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D005473:Fluoxetine / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D016666:Fluvoxamine / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D007654:Ketoconazole / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008825:Miconazole / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D008874:Midazolam / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D010672:Phenytoin / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D011803:Quinine / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D014044:Tolbutamide / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: D065690:Cytochrome P-450 CYP2D6 Inhibitors; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005473:Fluoxetine; D010672:Phenytoin; D003915:Dextromethorphan; D008825:Miconazole; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D011803:Quinine; C507848:CYP2C9 protein, rat; C465260:Cyp3a2 protein, rat; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D016666:Fluvoxamine; D008874:Midazolam; D007654:Ketoconazole