Abstract
The aim of this study was to explore the potential of recombinant cytochrome P450 (P450) enzymes for human metabolic clearance prediction. The relative abundance and relative activity approaches were compared as methods to bridge the gap between catalytic activities in recombinant P450 enzymes and human liver microsomes (HLMs). Relative activity factors were measured by determining the intrinsic clearance (CLint) of probe substrates (bufuralol-CYP2D6, diclofenac-CYP2C9, midazolam-CYP3A4, and phenacetin-CYP1A2) in recombinant P450s and 16 HLM donors. Simultaneous determination of drug depletion and metabolite formation profiles has enabled a direct comparison of these methods for CLint determination. Of the 110 drugs tested, 66% were metabolized by one or more P450 enzymes; of these 44% of were metabolized by CYP3A4 (0.3–21 μl/min/pmol of P450), 41% by CYP2D6 (0.6–60 μl/min/pmol of P450), 26% by CYP2C19 (0.4–8.1 μl/min/pmol of P450), 9% by CYP1A2 (0.4–2.5 μl/min/pmol of P450), and 4% by CYP2C9 (0.9–6.4 μl/min/pmol of P450). Recombinant enzymes demonstrated improved prediction reliability relative to HLMs and hepatocytes. The most reliable correlations in terms of lowest bias and highest precision were observed by comparing in vivo CLint, calculated using the parallel-tube model and incorporating fraction unbound in blood, with in vitro CLint determined using relative activity factors and adjusted for nonspecific binding. Predictions were less reliable using the relative abundance approach. For these drugs, recombinant P450 enzymes offer improved assay sensitivity compared with HLMs and cryopreserved hepatocytes for CLint determination using the drug depletion method.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.024810.
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ABBREVIATIONS: P450, cytochrome; HLM, human liver microsome; RAF, relative activity factor; WS, well stirred model; PT, parallel-tube model; LC-MS/MS, liquid chromatography-tandem mass spectrometry; AUC, area under the concentration-time curve; rmse, root mean square error; afe, average fold error.
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
- Received September 24, 2008.
- Accepted January 29, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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