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 b(5) in the expression system and the differences in the marker activities.
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 b(5) (b(5)) 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 b(5) 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 b(5)/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 6beta-hydroxylation and midazolam 1'-hydroxylation for CYP3A4 were compared. Our results indicated that the differences in the expression levels of OR and b(5) 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 b(5) if required) could be valuable for the prediction of the contribution of each CYP in drug metabolism.
Nakajima
Miki
M
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan. nmiki@kenroku.kanazawa-u.ac.jp
Tane
Kazuhiro
K
Nakamura
Sumika
S
Shimada
Noriaki
N
Yamazaki
Hiroshi
H
Yokoi
Tsuyoshi
T
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015415:Biomarkers / Q000032:analysis; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D015786:Cytochromes b5 / Q000096:biosynthesis* / Q000235:genetics; D005544:Forecasting / Q000379:methods; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000235:genetics / Q000821:virology; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000235:genetics; D010793:Phthalazines / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis
D015415:Biomarkers; D010793:Phthalazines; D011994:Recombinant Proteins; C054114:desmethylazelastine; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C020976:azelastine