Characterization of mixtures of recombinant human cytochrome p450s as a screening model for metabolic stability in drug discovery.
1. Recombinant human cytochrome p450 (rhCYP) has become an important screening model in drug metabolism studies due to the high cost of human and animal hepatic tissue. Until now, rhCYPs have been evaluated and used as separate forms, but a mixture of CYP forms comparable with the human liver could be of value in early drug discovery. 2. In the present study, rhCYP2C9, rhCYP2D6 and rhCYP3A4 co-expressed with reductase in Escerichia coli were mixed and evaluated with regards to kinetic properties (K(m) and V(max)). Furthermore, antioxidant was added to investigate whether a free radical scavenger would affect the kinetic parameters. Results were compared with data obtained in human liver microsomes (HLM). 3. Results showed a good correlation between mixed rh CYP data and HLM data for K(m) and V(max). K(m) varied < 3-fold between matrices for CYP2C9 and CYP3A4, whereas the K(m) for CYP2D6 varied up to 4.5-fold. V(max) differed up to 3-fold between matrices for the CYP forms investigated. However, the discrepancy in V(max) may depend on the anticipated level of each form in HLM. The addition of antioxidant increased V(max) for CYP2C9 and CYP2D6 by 75 and 50%, respectively, whereas V(max) for CYP3A4 was unchanged. 4. In conclusion, the rhCYP mixture shows promising results as a predictor of CYP kinetic parameters. Furthermore, addition of antioxidant can in certain cases increase catalytic activity.
Hagen
N
N
Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, DK-2100 Copenhagen, Denmark. nhag@novonordisk.com
Olsen
A K
AK
Andersen
J V
JV
Tjørnelund
J
J
Hansen
S H
SH
D000975:Antioxidants / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology; D002384:Catalysis; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry* / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D010090:Oxidoreductases, O-Demethylating / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D000975:Antioxidants; D016166:Free Radical Scavengers; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010090:Oxidoreductases, O-Demethylating; C052861:dextromethorphan O-demethylase; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase; C510163:CYP3A4 protein, human; D001205:Ascorbic Acid