Development of a High-Performance Liquid Chromatographic Method for the Analysis of Enatiomer/Enantiomer Interaction in Oxidative Metabolism of Bunitrolol in Rat Liver Microsomes

A high-performance liquid chromatographic method for the assay of enantiomeric 4-hydroxybunitrolol (4-OH-BTL) formed from racemic bunitrolol (BTL) in rat liver microsomes was developed. Racemic bunitrolol was incubated with rat fiver microsomes fortified with an NADPH-generating system. Metabolites extracted with ethyl acetate were converted to acetyl derivatives with acetic anhydride in pyridine. The derivatives of 4-OH-BTL were well separated by the liquid chromatography equipped with a chiral column. Using this method, the metabolic interaction of BTL enantiomers was examined, The 4-OH-BTL-forming activities from enantiomeric BTL were higher than those from racemic BTL in rat liver microsomes, while the formation of ratios of 4-OH-BTL enantiomer to its antipode were the same under the two conditions. The K_i values obtained from kinetic studies using each BTL enantiomer as an inhibitor of its antipode were almost the same (ca 0.9 μM), which were close to their Michaelis constants (K_m values). Oxidative activities of enantiomeric and racemic BTL were almost equally inhibited by debrisoquine and quinidine, a typical substrate and a selective inhibitor of the CYP2D subfamily, respectively. These results indicate that a BTL enantiomer is a mutual metabolic inhibitor of its antipode and BTL enantiomers compete for the same CYP2D isozyme in rat liver microsomes.