Polymorphic hydroxylation of perhexiline in vitro

Abstract

Aims: The aims of this study were to examine the in vitro enzyme kinetics and CYP isoform selectivity of perhexiline monohydroxylation using human liver microsomes.

Methods: Conversion of rac-perhexiline to monohydroxyperhexiline by human liver microsomes was assessed using a high-performance liquid chromatography assay with precolumn derivatization to measure the formation rate of the product. Isoform selective inhibitors were used to define the CYP isoform profile of perhexiline monohydroxylation.

Results: The rate of perhexiline monohydroxylation with microsomes from 20 livers varied 50-fold. The activity in 18 phenotypic perhexiline extensive metabolizer (PEM) livers varied about five-fold. The apparent Km was 3.3 +/- 1.5 micro m, the Vmax was 9.1 +/- 3.1 pmol min-1 mg-1 microsomal protein and the in vitro intrinsic clearance (Vmax/Km) was 2.9 +/- 0.5 micro l min-1 mg-1 microsomal protein in the extensive metabolizer livers. The corresponding values in the poor metabolizer livers were: apparent Km 124 +/- 141 micro m; Vmax 1.4 +/- 0.6 pmol min-1 mg-1 microsomal protein; and intrinsic clearance 0.026 micro l min-1 mg-1 microsomal protein. Quinidine almost completely inhibited perhexiline monohydroxylation activity, but inhibitors selective for other CYP isoforms had little effect.

Conclusions: Perhexiline monohydroxylation is almost exclusively catalysed by CYP2D6 with activities being about 100-fold lower in CYP2D6 poor metabolizers than in extensive metabolizers. The in vitro data predict the in vivo saturable metabolism and pharmacogenetics of perhexiline.

Figure 1

Effect of CYP isoform selective inhibitors on perhexiline monohydroxylation with three phenotypic perhexiline extensive metabolizer livers. The perhexiline concentration was 5 µm. The inhibitor concentrations and incubation conditions are given in Methods and the values are the means of duplicate determinations., H12; □, H25; , H27.

Figure 2

Kinetic plots for perhexiline monohydroxylation with (a) two phenotypic perhexiline extensive metabolizer livers (H25 and H27) and (b) two phenotypic perhexiline poor metabolizer livers (H8 and H30). The solid lines are those fitted to the data by nonlinear regression analysis. The data points are the mean of duplicate determinations.