Evaluation of Escherichia coli membrane preparations of canine CYP1A1, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 with coexpressed canine cytochrome P450 reductase.
The preparation of bacterial membranes ("Bactosomes") containing expressed canine (beagle) hepatic cytochromes P450 (P450s) is described. cDNAs from seven canine P450s were subcloned into inducible expression plasmids and, for the first time, cotransformed and expressed with a canine P450 oxidoreductase in Escherichia coli to produce active, full-length, native sequence P450s. Enzyme expression levels, although variable, were generally sufficient to enable short incubation times and to limit the total protein present in enzyme incubations. Steady-state kinetics of CYP1A1, 2C21, and 2D15 Bactosomes demonstrated similarities with dog liver microsomes or Baculosomes. However, 3A12 lacked substrate inhibition in the formation of 1'-OH midazolam, and 2B11 displayed non-Michaelis-Menten kinetics, suggesting possible differences in protein interaction effects. In monitoring the metabolites of common P450 substrates, phenacetin deethylation, temazepam demethylation, and bufuralol 1'-hydroxylation were shown to be relatively selective reactions catalyzed by CYP1A1, 2B11, and 2D15, respectively. 1'-OH midazolam was formed in higher quantities by CYP2B11 and 2C21 than by 3A12, raising questions about the use of midazolam as a CYP3A12 probe in vivo. In summary, a panel of recombinant P450s was produced to make up for the lack of commercially available canine P450 isoforms. The Bactosomes are expected to facilitate reaction phenotyping and metabolic drug-drug interaction assessment in canine drug development and to enable the study of interspecies differences in P450-mediated drug metabolism.
Locuson
Charles W
CW
Pfizer Animal Health, Pfizer, Inc., Veterinary Medicine Research and Development, Metabolism and Safety, 333 Portage Street, KZO-300-413, Kalamazoo, MI 49001, USA. charles.w.locuson@pfizer.com
Ethell
Brian T
BT
Voice
Michael
M
Lee
David
D
Feenstra
Kenneth L
KL
D000818:Animals; D001483:Base Sequence; D002853:Chromatography, Liquid; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase