Evaluation of Escherchia coli membrane preparations of canine CYP1A1, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26 with co-expressed canine P450 reductase. Drug metabolism and disposition: the biological fate of chemicals [Drug Metab Dispos] Journal article

The preparation of bacterial membranes ("Bactosomes") containing expressed canine (beagle) hepatic cytochromes P450 (CYPs) is described. cDNAs from seven canine CYPs were subcloned into inducible expression plasmids, and for the first time, co-transformed and expressed with a canine P450 oxidoreductase in E. coli to produce active, full-length, native sequence CYPs. Enzyme expression levels, while 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 CYP substrates, phenacetin deethylation, temazepam demethylation, and bufuralol 1'-hydoxylation were shown to be relatively selective reactions catalyzed by CYPs 1A1, 2B11, and 2D15, respectively. 1'-OH midazolam was formed in higher quantities by CYPs 2B11 and 2C21 than by 3A12 raising questions about the use of midazolam as a CYP3A12 probe in vivo. In summary, a panel of recombinant CYPs was produced to make up for the lack of commercially available canine CYP 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 inter-species differences in CYP-mediated drug metabolism.

Drug metabolism and disposition: the biological fate of chemicals [Drug Metab Dispos]