This article is available online at http://dmd.aspetjournals.org ABSTRACT: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 K m enzyme, 1 M). CYP2B11 and CYP3A12 effectively catalyzed the N 1 -demethylation and C 3 -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 M) were achieved, suggesting a high K m for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low K m process (K m ‫؍‬ 0.7 M), similar to that in dog liver microsomes (K m ‫؍‬ 2.3 M). CYP2D15 was also capable of metabolizing bufuralol (1-hydroxylation), with a K m of 3.9 M, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16␣-, 2␣/2␤-, and 6␤-positions. Selectivity of CYP3A12 was observed toward testosterone 6␤-(K m ‫؍‬ 83 M) and 2␣/2␤-hydroxylations (K m ‫؍‬ 154 M). However, the 16␣-hydroxylation of testosterone was catalyzed by CYP2C21 also (K m ‫؍‬ 6.4 M for CYP2C21). Therefore, the 6␤-and 16␣-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.

No abstract

No abstract

A hydrophilic interaction liquid chromatographic method with tandem mass spectrometry for the determination of atenolol, a beta-blocking agent, in human plasma has been developed and validated over the curve range of 10--2000 ng/mL. The assay was based on protein precipitation followed by evaporation of the extraction solvent, reconstitution with acetonitrile, and chromatography on an Hypersil silica column (50 x 4.6 mm) using a low aqueous--high organic mobile phase. The mobile phase consists of 85% acetonitrile, 15% water, 0.5% acetic acid and 0.04% trifluoroacetic acid and runs isocratically at a flow rate of 2.0 mL/min. The column ef fluent was split so that 50% of it was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 2.0 min per injection. Atenolol and the internal standard, atenolol-d(7), showed a retention time of 1.0 min. The inter-day and intra-day precision and accuracy of the quality control samples were <5.3% relative standard deviation and <8.0% relative error, respectively. To explore the application of the current method for the analysis of other beta-blocking agents, propranolol and metoprolol were tested under the same chromatographic conditions with retention times of 0.68 and 0.75 min, respectively. The present method could be used for therapeutic drug monitoring, pharmacokinetic and drug--drug interaction studies of beta-blocking agents.

No abstract