Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil

Abstract

Aims: The present study was conducted to evaluate metabolism of the enantiomers of verapamil and norverapamil using a broad range of cytochrome P450 isoforms and measure the kinetic parameters of these processes.

Methods: Cytochrome P450 cDNA-expressed cells and microsomes from a P450-expressed lymphoblastoid cell line were incubated with 40 microm concentrations of R- or S-verapamil and R- or S-norverapamil and metabolite formation measured by h.p.l.c. as an initial screening. Those isoforms exhibiting substantial activity were then studied over a range of substrate concentrations (2.5-450 microm ) to estimate the kinetic parameters for metabolite formation.

Results: P450s 3A4, 3A5, 2C8 and to a minor extent 2E1 were involved in the metabolism of the enantiomers of verapamil. Estimated Km values for the production of D-617 and norverapamil by P450 s 3A4 and 3A5 were similar (range=60-127 microm ) regardless of the enantiomer of verapamil studied while the Vmax estimates were also similar (range=4-8 pmol min-1 pmol-1 P450). Only nominal production of D-620 by these isoforms was noted. Interestingly, P450 2C8 readily metabolized both S- and R-verapamil to D-617, norverapamil and PR-22 with only slightly higher Km values than noted for P450s 3A4 and 3A5. However, the Vmax estimates for P450 2C8 metabolism of S- and R-verapamil were in general greater (range=8-15 pmol min-1 pmol-1 P450) than those noted for P450 s 3A4 and 3A5 with preference noted for metabolism of the S-enantiomer. Similarly, P450 s 3A4, 3A5 and 2C8 also mediated the metabolism of the enantiomers of norverapamil with minor contributions by P450 s 2D6 and 2E1. P450s 3A4 and 3A5 readily formed the D-620 metabolite with generally a lower Km and higher Vmax for S-norverapamil than for the R-enantiomer. In contrast, P450 2C8 produced both the D-620 and PR-22 metabolites from the enantiomers of norverapamil, again with stereoselective preference seen for the S-enantiomer.

Conclusions: These results confirm that P450s 3A4, 3A5 and 2C8 play a major role in verapamil metabolism and demonstrate that norverapamil can also be further metabolized by the P450s.

Figure 1

Structures of verapamil and its primary metabolites indicating the major routes of oxidative metabolism. *Also known as D-715

Figure 2

Metabolism of S-verapamil (a), R-verapamil (b), S-norverapamil (c) and R-norverapamil (d) to their respective metabolites by vaccinia-expressed cytochrome P450 3A4 containing cells. Substrate concentrations ranged from 2.5 to 450 μm. ○ D-617, • norverapamil, □ D-620.

Figure 3

Metabolism of S-verapamil (a), R-verapamil (b), S-norverapamil (c) and R-norverapamil (d) to their respective metabolites by vaccinia-expressed cytochrome P450 3A5 containing cells. Substrate concentrations ranged from 2.5 to 450 μm. ○ D-617, • norverapamil, □ D-620.

Figure 4

Metabolism of S-verapamil (a), R-verapamil (b), S-norverapamil (c) and R-norverapamil (d) to their respective metabolites by vaccinia-expressed cytochrome P450 2C8 containing cells. Substrate concentrations ranged from 2.5 to 450 μm. ○ D-617, • norverapamil, □ D-620, ▪ PR-22.