The paraoxonase-1 pathway is not a major bioactivation pathway of clopidogrel in vitro

Abstract

Background and purpose: Clopidogrel is a prodrug bioactivated by cytochrome P450s (CYPs). More recently, paraoxonase-1 (PON1) has been proposed as a major contributor to clopidogrel metabolism. The purpose of this study was to assess the relative contribution of CYPs and PON1 to clopidogrel metabolism in vitro.

Experimental approach: Clopidogrel metabolism was studied in human serum, recombinant PON1 enzyme (rePON1), pooled human liver microsomes (HLMs), HLMs with the CYP2C19*1/*1 genotype and HLMs with the CYP2C19*2/*2 genotype. Inhibition studies were also performed using specific CYP inhibitors and antibodies. Clopidogrel and its metabolites were measured using LC/MS/MS method.

Key results: PON1 activity was highest in the human serum and there was no difference in PON1 activity between any of the HLM groups. The production of clopidogrel's active metabolite (clopidogrel-AM) from 2-oxo-clopidogrel in pooled HLMs was approximately 500 times that in serum. When 2-oxo-clopidogrel was incubated with rePON1, clopidogrel-AM was not detected. Clopidogrel-AM production from 2-oxo-clopidogrel was lower in CYP2C19*2/*2 HLMs compared with CYP2C19*1/*1 HLMs, while PON1 activity in HLMs with both genotypes was similar. Moreover, incubation with inhibitors of CYP3A, CYP2B6 and CYP2C19 significantly reduced clopidogrel bioactivation while a PON1 inhibitor, EDTA, had only a weak inhibitory effect.

Conclusion and implications: This in vitro study shows that the contribution of PON1 to clopidogrel metabolism is limited at clinically relevant concentrations. Moreover, CYP2C19, CYP2B6 and CYP3A play important roles in the bioactivation of clopidogrel.

Figure 1

Pathways of clopidogrel metabolism.

Figure 2

Kinetic analysis of OH-mephenytoin formation from mephenytoin in CYP2C19-genotyped human liver microsomes. Data are mean ± SD of three independent data points.

Figure 3

Kinetic analysis of 2-oxo-clopidogrel production from clopidogrel (A) and clopidogrel-AM production from 2-oxo-clopidogrel (B) in human liver microsomes (HLMs) with CYP2C19*1/*1 and CYP2C19*2/*2 genotypes. Data are mean ± SD of three independent data points.

Figure 4

Inhibitory effects of cytochrome P450 (CYP) and paraoxonase (PON) inhibitors on the metabolic pathways from 2-oxo-clopidogrel to clopidogrel-AM (A) and from clopidogrel to clopidogrel-AM (B) in human liver microsomes (HLMs) with 10 µM clopidogrel or 10 µM 2-oxo-clopidogrel. Each column represents the mean ± SD of three independent data points.

Figure 5

Active metabolite production from 2-oxo-clopidogrel 10 µM in human liver microsomes (HLM) incubated with increasing quantities of anti-CYP3A4 and anti-CYP2C19 antibodies. Data are mean ± SD of three independent data points.

Figure 6

Kinetic analysis of 2-oxo-clopidogrel production from clopidogrel (A) and clopidogrel-AM production from 2-oxo-clopidogrel (B) in human serum and pooled human liver microsomes (HLMs) with adjusted paraoxonase 1 (PON1) activity. Data are mean ± SD of three independent data points.

Figure 7

Comparison of the production of clopidogrel-AM in pooled human liver microsomes (HLMs), HLMs with CYP2C19*1/*1 and CYP2C19*2/*2 genotypes, and recombinant PON1 (rePON) at two different concentrations. Data are mean ± SD of three independent data points.