Substrate specificity and kinetic properties of seven heterologously expressed dog cytochromes p450.
Seven dog cytochromes p450 (p450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low Km enzyme, 1 microM). CYP2B11 and CYP3A12 effectively catalyzed the N1-demethylation and C3-hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N- and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 microM) were achieved, suggesting a high Km for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low Km process (Km = 0.7 microM), similar to that in dog liver microsomes (Km = 2.3 microM). CYP2D15 was also capable of metabolizing bufuralol (1'-hydroxylation), with a Km of 3.9 microM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16alpha-, 2alpha/2beta-, and 6beta-positions. Selectivity of CYP3A12 was observed toward testosterone 6beta-(Km = 83 microM) and 2alpha/2beta-hydroxylations (Km = 154 microM). However, the 16alpha-hydroxylation of testosterone was catalyzed by CYP2C21 also (Km = 6.4 microM for CYP2C21). Therefore, the 6beta- and 16alpha-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4'-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the p450s selectively metabolized (S)-mephenytoin 4'-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog p450s and the development of dog p450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.
Shou
Magang
M
Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA. magang_shou@merck.com
Norcross
Ryan
R
Sandig
Grit
G
Lu
Ping
P
Li
Yinghe
Y
Lin
Yuh
Y
Mei
Qin
Q
Rodrigues
A David
AD
Rushmore
Thomas H
TH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D007313:Insecta / Q000166:cytology; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System