title: Evaluation of recombinant cytochrome P450 enzymes as an in vitro system for metabolic clearance predictions.
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 (CL(int)) 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 CL(int) 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 microl/min/pmol of P450), 41% by CYP2D6 (0.6-60 microl/min/pmol of P450), 26% by CYP2C19 (0.4-8.1 microl/min/pmol of P450), 9% by CYP1A2 (0.4-2.5 microl/min/pmol of P450), and 4% by CYP2C9 (0.9-6.4 microl/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 CL(int), calculated using the parallel-tube model and incorporating fraction unbound in blood, with in vitro CL(int) 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 CL(int) determination using the drug depletion method.
authors.affiliation: Novartis Institutes for Biomedical Research, Wimblehurst Road, Horsham, West Sussex, RH12 5AB, UK. rowan.stringer@novartis.com
authors.forename: Rowan A
authors.initials: RA
authors.lastname: Stringer
authors.forename: Claire
authors.initials: C
authors.lastname: Strain-Damerell
authors.forename: Paul
authors.initials: P
authors.lastname: Nicklin
authors.forename: J Brian
authors.initials: JB
authors.lastname: Houston
mesh_terms: D000465:Algorithms; D019540:Area Under Curve; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D005544:Forecasting; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008657:Metabolic Clearance Rate; D008862:Microsomes, Liver / Q000378:metabolism; D008954:Models, Biological; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System