Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Clopidogrel: Involvement of Both Covalent Modification of Cysteinyl Residue 475 and Loss of Heme □ S

Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-5632, USA.
Molecular pharmacology (Impact Factor: 4.13). 08/2011; 80(5):839-47. DOI: 10.1124/mol.111.073783
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We have investigated the mechanisms by which clopidogrel inactivates human cytochrome P450 2B6 (CYP2B6) in a reconstituted system. It was found that clopidogrel and its thiolactone metabolite, 2-oxo-clopidogrel, both inactivate CYP2B6 in a time- and concentration-dependent manner. On the basis of k(inact)/K(I) ratios, clopidogrel is approximately 5 times more efficient than 2-oxo-clopidogrel in inactivating CYP2B6. Analysis of the molecular mass of the CYP2B6 wild-type (WT) protein that had been inactivated by either clopidogrel or 2-oxo-clopidogrel showed an increase in the mass of the protein by ∼350 Da. This increase in the protein mass corresponds to the addition of the active metabolite of clopidogrel to CYP2B6. It is noteworthy that this adduct can be cleaved from the protein matrix by incubation with dithiothreitol, confirming that the active metabolite is linked to a cysteinyl residue of CYP2B6 via a disulfide bond. Peptide mapping of tryptic digests of the inactivated CYP2B6 using electrospray ionization liquid chromatography-tandem mass spectrometry identified Cys475 as the site of covalent modification by the active metabolite. This was further confirmed by the observation that mutation of Cys475 to a serine residue eliminates the formation of the protein adduct and prevents the C475S variant from mechanism-based inactivation by 2-oxo-clopidogrel. However, this mutation did not prevent the C475S variant from being inactivated by clopidogrel. Furthermore, inactivation of both CYP2B6 WT and C475S by clopidogrel, but not by 2-oxo-clopidogrel, led to the loss of the heme, which accounts for most of the loss of the catalytic activity. Collectively, these results suggest that clopidogrel inactivates CYP2B6 primarily through destruction of the heme, whereas 2-oxo-clopidogrel inactivates CYP2B6 through covalent modification of Cys475.

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Available from: Haoming Zhang, Oct 06, 2015
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    • "Recent studies show, however, that both steps of the activation process of clopidogrel are more complicated, leading to many other possibly reactive intermediates [25] [29] [30]. Regarding the potential to form off-target adducts [27] [28], the active metabolite (and possibly also other metabolites formed) could be responsible not only for the pharmacological action of clopidogrel, but also for toxic effects, including hepatotoxicity . In support of a toxic mechanism involving clopidogrel metabolites, we have shown recently that incubation of clopidogrel with CYP3A4 is associated with the formation of metabolites that are toxic for granulocyte precursors [31]. "
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    ABSTRACT: Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of the current study was to find out mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100μM) was cytotoxic only after CYP-induction by rifampicin. Clopidogrel (10 and 100μM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100μM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10µM and 100µM) in HepG2/CYP3A4, but not in HepG2 cells/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10µM and 100µM) in HepG2/CYP3A4 supersome. Co-incubation with 1μM ketoconazole or 10mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 or HepG2/CYP3A4 supersome, respectively. HepG2/CYP3A4 incubated with 100μM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites which are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.
    Free Radical Biology and Medicine 06/2013; 65. DOI:10.1016/j.freeradbiomed.2013.06.007 · 5.74 Impact Factor
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    • "The thienopyridine derivatives clopidogrel and ticlopidine are prodrugs that selectively inhibit platelet aggregation and have been in clinical use for the prevention of atherothrombotic events for several years. Both of them are potent mechanism-based inhibitors of CYP2B6 (Richter et al., 2004; Zhang et al., 2011a). The established anticancer agent, thioTEPA (N,N′,N′′-triethylenethiophosphoramide) was also found to be a highly selective and mechanism-based CYP2B6 inhibitor (Rae et al., 2002; Harleton et al., 2004; Richter et al., 2005). "
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    ABSTRACT: Cytochrome P450 2B6 (CYP2B6) belongs to the minor drug metabolizing P450s in human liver. Expression is highly variable both between individuals and within individuals, owing to non-genetic factors, genetic polymorphisms, inducibility, and irreversible inhibition by many compounds. Drugs metabolized mainly by CYP2B6 include artemisinin, bupropion, cyclophosphamide, efavirenz, ketamine, and methadone. is one of the most polymorphic CYP genes in humans and variants have been shown to affect transcriptional regulation, splicing, mRNA and protein expression, and catalytic activity. Some variants appear to affect several functional levels simultaneously, thus, combined in haplotypes, leading to complex interactions between substrate-dependent and -independent mechanisms. The most common functionally deficient allele is [Q172H, K262R], which occurs at frequencies of 15 to over 60% in different populations. The allele leads to lower expression in liver due to erroneous splicing. Recent investigations suggest that the amino acid changes contribute complex substrate-dependent effects at the activity level, although data from recombinant systems used by different researchers are not well in agreement with each other. Another important variant, [I328T], occurs predominantly in Africans (4-12%) and does not express functional protein. A large number of uncharacterized variants are currently emerging from different ethnicities in the course of the 1000 Genomes Project. The polymorphism is clinically relevant for HIV-infected patients treated with the reverse transcriptase inhibitor efavirenz, but it is increasingly being recognized for other drug substrates. This review summarizes recent advances on the functional and clinical significance of CYP2B6 and its genetic polymorphism, with particular emphasis on the comparison of kinetic data obtained with different substrates for variants expressed in different recombinant expression systems.
    Frontiers in Genetics 03/2013; 4:24. DOI:10.3389/fgene.2013.00024
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    • "The EIC at m/z 661.0 (equivalent to MH ϩ of the AM-GSH conjugate) exhibited a peak at 5.5 min (Fig. 2A, trace b). The MS 2 spectrum of this peak (Fig. 2D) was in excellent agreement with findings we reported previously for the glutathionyl conjugate of the AM observed in a reconstituted system (Zhang et al., 2011). The two major MS 2 peaks, at m/z 531.83 and 353.92, could be attributed to a neutral loss of 129 from the precursor ion m/z 661.0 and the AM fragment cleaved from the mixed disulfide bond of the glutathionyl conjugate, respectively. "
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    ABSTRACT: We reported previously the formation of a glutathionyl conjugate of the active metabolite (AM) of clopidogrel and the covalent modification of a cysteinyl residue of human cytochrome P450 2B6 in a reconstituted system (Mol Pharmacol 80:839-847, 2011). In this work, we extended our studies of the metabolism of clopidogrel to human liver microsomes in the presence of four reductants, namely, GSH, l-Cys, N-acetyl-l-cysteine (NAC), and ascorbic acid. Our results demonstrated that formation of the AM was greatly affected by the reductant used and the relative amounts of the AM formed were increased in the following order: NAC (17%) < l-Cys (53%) < ascorbic acid (61%) < GSH (100%). AM-thiol conjugates were observed in the presence of NAC, l-Cys, and GSH. In the case of GSH, the formation of both the AM and the glutathionyl conjugate was dependent on the GSH concentrations, with similar K(m) values of ~0.5 mM, which indicates that formation of the thiol conjugates constitutes an integral part of the bioactivation processes for clopidogrel. It was observed that the AM was slowly converted to the thiol conjugate, with a half-life of ~10 h. Addition of dithiothreitol to the reaction mixture reversed the conversion, which resulted in a decrease in AM-thiol conjugate levels and a concomitant increase in AM levels, whereas addition of NAC led to the formation of AM-NAC and a concomitant decrease in AM-GSH levels. These results not only confirm that the AM is formed through oxidative opening of the thiolactone ring but also suggest the existence of an equilibrium between the AM, the thiol conjugates, and the reductants. These factors may affect the effective concentrations of the AM in vivo.
    Molecular pharmacology 05/2012; 82(2):302-9. DOI:10.1124/mol.112.079061 · 4.13 Impact Factor
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