Polymorphic hydroxylation of perhexiline in vitro

Abstract

AimsThe 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 K m was 3.3 ± 1.5 µ M , the V max was 9.1 ± 3.1 pmol min − 1 mg − 1 microsomal protein and the in vitro intrinsic clearance (V max /K m ) was 2.9 ± 0.5 m l min − 1 mg − 1 microsomal protein in the extensive metabolizer livers. The corresponding values in the poor metabolizer livers were: apparent K m 124 ± 141 µ M ; V max 1.4 ± 0.6 pmol min − 1 mg − 1 microsomal protein; and intrinsic clearance 0.026 µ 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.

CYP2B6, CYP2D6, and CYP3A4 Catalyze the Primary Oxidative Metabolism of Perhexiline Enantiomers by Human Liver Microsomes

“…These K m values are within the range of unbound (ϩ)-and (Ϫ)-PHX concentrations observed in clinical plasma specimens , and are consistent with the saturability of cis-4-monohydroxy metabolite formation at therapeutic concentrations in vivo (Sallustio et al, 2002). Sørensen et al (2003) also reported a high-affinity 4-monohydroxylation reaction by EMs in vitro, although the K m values were larger because they reflected total rather than unbound microsomal PHX concentrations. The mean contribution of cis-4-monohydroxylation to mean total CL int for (ϩ)-PHX was 43% in EMs and 12% in IMs, and for (Ϫ)-PHX it was 99% and 89%, respectively.…”

“…The intrinsic clearance of PHX was approximately 100-fold lower in PMs, presumably mediated by P450 isoforms other than CYP2D6 with a much lower affinity for PHX (Sørensen et al, 2003), although these iso-forms and the 4-monohydroxy metabolites they produced were not identified.…”

“…An investigation of the in vitro enzyme kinetics of PHX 4-monohydroxylation in human liver microsomes (HLMs) determined that it is almost exclusively catalyzed with high affinity by CYP2D6 in EMs, with K m values within the range of therapeutic PHX concentrations in plasma (Sørensen et al, 2003); this is consistent with the nonlinear kinetics observed clinically in EMs (Cooper et al, 1985). The intrinsic clearance of PHX was approximately 100-fold lower in PMs, presumably mediated by P450 isoforms other than CYP2D6 with a much lower affinity for PHX (Sørensen et al, 2003), although these iso-forms and the 4-monohydroxy metabolites they produced were not identified.…”

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity

“…53 Perhexiline has a concentration-related hepatotoxicity and peripheral neuropathy and determination of the CYP2D6 genotype will predict dose requirements and reduce the risk of perhexiline concentration-related toxicity. 54 In a retrospective study, Wuttke et al 55 identified 24 patients treated with metoprolol who had experienced pronounced adverse effects.…”

CYP2D6 Gene Polymorphism in Pakistani Population

“…The enzyme product of CYP2D6*4 genotype is an inactive enzyme as a result of defective splicing. The CY P2D6*4 homozygous individuals are therefore categorized as poor metabolizers [9]. When women carrying wild type allele were compared with those having heterozygous and homozygous variant genotypes, there was a significant association between incidence of host flushes and the genotypes.…”