CYTOCHROMES OF THE P450 2C SUBFAMILY ARE THE MAJOR ENZYMES INVOLVED IN THE O-DEMETHYLATION OF VERAPAMIL IN HUMANS

The calcium channel blocker verapamil hoxyphenyl)-6-methyl-2-isopropyl -6-azaoctanitrile] undergoes extensive biotransformation in man. We ha ve previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 [2-(3,4-dimethoxyphenyl)-5- methylamino-2-isopropylvaleronitrile], and verapamil N-demethylation (formation of norverapamil 4-dimethoxypheny l)-2-isopropyl-6-azaoctanitrile]), while there was no involvement of C YP3A4 and CYP1A2 in the third initial metabolic step of verapamil, whi ch is verapamil O-demethylation. This pathway yields formation of D-70 3 hoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and D-702 ethoxyp henyl)-8-(4-hydroxy-3-methoxyphenyl)-6-meth yl-2-isopropyl-6-azaoctani trile]. The enzymes catalyzing verapamil O-demethylation have not been characterized so far. We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using t he following in vitro approaches: (I) char characterization of O-demet hylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibo dies and selective inhibitors and (III) investigation of metabolite fo rmation in microsomes obtained from yeast strain Saccharomyces cerevis iae W(R), that was genetically engineered for stable expression of hum an CYP2C8, 2C9 and 2C18. In human liver microsomes (n=4), the intrinsi c clearance (CL(int)), as derived from the ratio of V-max/K-m, was sig nificantly higher for O-demethylation to D-703 compared to formation o f D-702 following incubation with racemic verapamil (13.9+/-1.0 vs 2.4 +/-0.6 mlmin(-1)*g(-1), mean+/-SD; p<0.05), S-verapamil (16.8+/-3.3 v s 2.2+/-1.2 ml; min(-1)*g(-1) p<0.05) and R-verapamil (12.1+/-2.9 vs 3.6 +/-1.3 mlmin(-1)* g(-1); p<0.05), thus indicating regioselectivit y of verapamil O-demethylation process. The CL(int) of D-703 formation in human liver microsomes showed a modest but significant degree of s tereoselectivity (p<0.05) with a S/R-ratio of 1.41+/-0.17 Anti-LKM2 (a nti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2 C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maxi mum rate of formation of D-703 by 81.5+/-4.5% and 45%, that of D-702 b y 52.7+/-7.5% and 72.5%, respectively. Both D-703 and D-702 were forme d by stably expressed CYP2C9 and CYP2C18. whereas incubation with CYP2 C8 selectively yielded D-703. In conclusion. our results show that enz ymes of the CYP2C subfamily are mainly involved in verapamil O-demethy lation. Verapamil therefore has the potential to interact with other d rugs which inhibit or induce these enzymes.