Functional characteristics of CYP3A4 allelic variants on the metabolism of loperamide in vitro

Abstract

Background: Cytochrome P450 3A4 (CYP3A4) appears to be genetically polymorphic, which in turn contributes to interindividual variability in response to therapeutic drugs. Loperamide, identified as a CYP3A4 substrate, is prone to misuse and abuse and has high risks of life-threatening cardiotoxicity.

Methods: Thus, this study is designed to evaluate the enzymatic characteristics of 29 CYP3A4 alleles toward loperamide in vitro, including the 7 novel CYP3A4 variants (*28-*34). The incubation system (containing CYP3A4 enzyme, cytochrome b5, 0.5-20 μM loperamide, potassium phosphate buffer and nicotinamide adenine dinucleotide phosphate) was subject to 40-mins incubation at 37°C and the concentrations of N-demethylated loperamide were quantified by UPLC-MS/MS.

Results: As a result, CYP3A4.6, .17, .20 and .30 showed extremely low activity or no activity and the rest of CYP3A4 variants presented varying degrees of decrements in catalytical activities when compared with CYP3A4.1.

Conclusion: As the first study to identify the properties of these CYP3A4 variants toward loperamide metabolism, our investigation may establish the genotype-phenotype relationship for loperamide, predict an individual's capability in response to loperamide, and provide some guidance of clinical medication and treatment for loperamide.

Keywords: CYP3A4; cardiotoxicity; genetic polymorphism; interindividual variability; loperamide; misuse and abuse; personalized treatment.

Figure 1

The transformation of loperamide to its main metabolite N-demethylated loperamide (DLOP).

Figure 2

UPLC-MS/MS chromatographs of N-demethylated loperamide and midazolam (10 μg/mL midazolam) in the 200-μL incubation system: (A) without loperamide and midazolam; (B) with activity-abolished microsomes and spiked with 0.25 μM N-demethylated loperamide; (C) incubating with 20 μM loperamide and 1 pmol CYP3A4.1. Abbreviations: CYP3A4, cytochrome P450 3A4; MRM, multiple reaction monitoring.

Figure 3

Michaelis–Menten curve of the enzymatic activities of the wild-type CYP3A4 and other CYP3A4 variants on loperamide metabolism. Data are presented as mean ± SD of 3 parallel experiments. The variants with designated allele names have been arranged into 6 groups (A–F). Abbreviation: CYP3A4, cytochrome P450 3A4.

Figure 4

Relative clearance of CYP3A4 variants toward loperamide metabolism compared with the wild type, arranged in the order. Notes: Significant differences between the wild-type CYP3A4 and CYP3A4 variants analyzed by the mean of one-way ANOVA with Dunnett’s test, *P<0.05, **P<0.01. Abbreviation: CYP3A4, cytochrome P450 3A4.