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 μM) 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.
Footnotes
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doi:10.1124/dmd.107.017095.
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This work was partially funded by a consortium of pharmaceutical companies (Bristol-Myers Squibb, GlaxoSmithKline, F. Hoffmann La Roche, Lilly, Novartis, Pfizer, and Servier) within the Centre for Applied Pharmacokinetic Research at the University of Manchester. H.S.B was financially supported by a Bristol-Myers Squibb studentship. A.C. was financially supported by AstraZeneca BBSRC CASE studentship. Part of this study was presented at the 8th European ISSX Meeting (Dijon, France) 2003 and the 16th International Symposium on Microsomes and Drug Oxidations (Budapest, Hungary) 2006.
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ABBREVIATIONS: DDI, drug-drug interaction; P450, cytochrome P450; LC-MS/MS, liquid chromatography-tandem mass spectrometry; AZT, 3′-azido-2′,3′-dideoxythymidine; HPPH, 5-(4-p-hydroxyphenyl)-5-phenylhydantoin; TBME, tert-butyl methyl ether; MPPH, 5-(4-methylphenyl)-5-phenylhydantoin; CRM, Chow Rat Mouse.
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↵1 Current affiliation: Department of Metabolism, Covance Laboratories Ltd., Otley Road, Harrogate, HG3 1PY, UK.
- Received June 8, 2007.
- Accepted August 24, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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