Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial, yeast and human lymphoblastoid cells.
1. The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E. coli), yeast (S. cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers. 2. Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ). The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters K(m) or S(50), V(max) and intrinsic clearance (CL(int)) or maximum clearance (CL(max)) from the kinetic profiles; the latter differing by 100-fold across the three substrates. 3. The K(m) or S(50) for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems. For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the V(max) for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E. coli membrane preparations, resulting in greater CL(int) or CL(max). In the case of 3HDZ formation, non-linear kinetics were observed for E. coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S. cerevisiae were linear. 4. The use of native human liver microsomes for drug metabolic studies will always be preferable. However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities.
Andrews
J
J
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK. julie.andrews@man.ac.uk
Abd-Ellah
M F
MF
Randolph
N L
NL
Kenworthy
K E
KE
Carlile
D J
DJ
Friedberg
T
T
Houston
J B
JB
D000328:Adult; D002851:Chromatography, High Pressure Liquid; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D003915:Dextromethorphan / Q000494:pharmacology; D003975:Diazepam / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005260:Female; D018757:GABA Modulators / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000473:pathology; D008214:Lymphocytes / Q000201:enzymology*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008874:Midazolam / Q000494:pharmacology; D008875:Middle Aged; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013693:Temazepam / Q000494:pharmacology
D018691:Excitatory Amino Acid Antagonists; D018757:GABA Modulators; D011994:Recombinant Proteins; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; D013693:Temazepam; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D003975:Diazepam; D008874:Midazolam