Differential enantioselectivity and product-dependent activation and inhibition in metabolism of verapamil by human CYP3As.

In vitro studies of enantioselective metabolism of R-(+)- and S-(-)verapamil (VER) were conducted using human cDNA-expressed CYP3A isoforms, CYP3A4, CYP3A5, and CYP3A7. N-dealkylated products nor-VER [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile] and D617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile] were the major metabolites for all CYP3A isoforms regardless of enantiomer. Enantioselectivity of CYP3A4 and CYP3A7 was most similar among the three isoforms. This coincides with the degree of homology of amino acids at the active sites and in the total amino acid sequences of the enzymes. Biphasic substrate inhibition was observed for the formation of nor-VER and D617, whereas simple biphasic kinetics were observed for the formation of O-demethylated products for both enantiomers with CYP3A4. The biphasic substrate inhibition was observed only for nor-VER, and simple biphasic kinetics were observed for D617 and O-demethylated products for both enantiomers with CYP3A5. However, with CYP3A7, D617 and O-demethylated products showed typical Michaelis-Menten kinetics, and only nor-VER displayed substrate (monophasic) inhibition. When metabolic rates of VER were determined in the presence of three different effectors, midazolam, testosterone, and nifedipine, activation, inhibition, or activation and inhibition of VER metabolism was observed depending on the enantiomers, metabolites, effectors, and cytochrome P450 isoforms. Addition of anti-CYP3A4 antibody inhibited formation of all metabolites for both CYP3A4 and CYP3A5. The atypical phenomena (biphasic substrate inhibition, activation, and inhibition depending on product formation) of VER kinetics could be adequately explained by introducing the concept of steric interaction into a two binding-site model.