Evaluation of Approach to Predict the Contribution of Multiple Cytochrome P450s in Drug Metabolism Using Relative Activity Factor: Effects of the Differences in Expression Levels of NADPH–Cytochrome P450 Reductase and Cytochrome b5 in the Expression System and the Differences in the Marker Activities

Abstract

The concept of relative activity factor (RAF) to extrapolate data obtained with recombinant cytochrome P450(CYP)s to human liver microsomes has been proposed. To evaluate the approach to predict the contribution of multiple CYPs using RAF, we investigated the effects of the differences in the expression levels of NADPH–cytochrome P450 reductase (OR) and cytochrome b5 (b5) in recombinant CYPs from baculovirus-infected insect cells and the differences in the marker activities. Because we previously clarified that azelastine, an antiallergy and antiasthmatic drug, is N-demethylated by CYP1A2, CYP2D6, and CYP3A4 in humans, the reaction was used as a model. For calculation of RAF, three lots of recombinant CYP1A2, CYP2D6, and CYP3A4 from baculovirus-infected insect cells with different expression levels of OR and b5 were used. The OR/CYP ratios for recombinant CYP1A2, CYP2D6, and CYP3A4 were 3.9–4.8, 5.1–8.7, and 8.0–11.3, respectively. The b5/CYP ratio for recombinant CYP3A4 was 2.1–18.7. As marker activities, ethoxyresorufin O-deethylation and phenacetin O-deethylation for CYP1A2, bufuralol 1′-hydroxylation and debrisoquin 4-hydroxylation for CYP2D6, testosterone 6β-hydroxylation and midazolam 1′-hydroxylation for CYP3A4 were compared. Our results indicated that the differences in the expression levels of OR and b5 coexpressed in baculovirus-infected insect cells would not be a critical factor for the quantitative prediction using RAF. In addition, we confirmed that differences in the marker activities did not significantly affect the calculation of RAF values, when the marker activities are specific for a certain CYP isoform. It was suggested that the RAF approach using recombinant CYPs from baculovirus-infected insect cells coexpressing OR (and b5 if required) could be valuable for the prediction of the contribution of each CYP in drug metabolism. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:952–963, 2002