Glutaredoxin and Thioredoxin Can Be Involved in Producing the Pharmacologically Active Metabolite of a Thienopyridine Antiplatelet Agent, Prasugrel

Hagihara, Katsunobu; Kazui, Miho; Kurihara, Atsushi; Kubota, Kazuishi; Ikeda, Toshihiko

doi:10.1124/dmd.110.035196

Cited by 11 publications

(18 citation statements)

References 21 publications

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“…In the absence of cytosol, the reduction of the CPT conjugate is approximately one-half as fast, with a rate constant of 0.39 minute 21 for the reduction of the conjugate and 0.35 minute 21 for the formation of the AM. This indicates that cytosol accelerates the reduction of the thiol-disulfide exchange reaction, as observed previously (Hagihara et al, 2011(Hagihara et al, , 2012. We confirmed this observation by use of boiled cytosol, as shown in Supplemental Fig.…”

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confidence: 76%

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Formation, Reactivity, and Antiplatelet Activity of Mixed Disulfide Conjugates of Clopidogrel

et al. 2013

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In this work, we investigated the formation, reactivity, and antiplatelet activity of various mixed disulfide conjugates of clopidogrel. Our results showed that the production of the active metabolite (AM) from 2-oxoclopidogrel by human liver microsomes (HLMs) is greatly affected by the thiol reductants used. Among the 10 thiol compounds tested, glutathione (GSH) is most efficient in producing the AM at a rate of 167 pmoles AM/ min/mg HLM. Interestingly, no AM but only the mixed disulfide conjugates were formed in the presence of 6-chloropyridazine-3-thiol (CPT), 2,5-dimethylfuran-3-thiol, and 3-nitropyridine-2-thiol (NPT). The mass spectrometry (MS) and MS 2 spectra of the conjugates of these thiol compounds confirmed the presence of a mixed disulfide bond linkage between the AM and the thiol reductants. Kinetic studies revealed that the mixed disulfide conjugates were capable of exchanging thiols with GSH to release the AM with second order rate constants ranging from 1.2 to 28 M 21 s 21 . The mixed disulfide conjugates of CPT and NPT showed potent inhibition of platelet aggregation after pretreatment with 1 mM GSH, confirming that the AM is responsible for the antiplatelet activity of clopidogrel. Collectively, our results provide strong support for a cytochrome P450 (P450)-mediated bioactivation mechanism involving the initial formation of a glutathionyl conjugate, followed by thiol-disulfide exchange with another GSH molecule to release the AM. Furthermore, the stable mixed disulfide conjugates identified in this study provide a platform to quantitatively generate the therapeutic AM without the need for P450-mediated bioactivation. This property can be further explored to overcome the interindividual variability in clopidogrel therapy.

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confidence: 76%

“…In addition, it is shown that the thiol-disulfide exchange reactions are facilitated by the cytosolic fraction of human liver. This is likely due to the involvement of glutaredoxin and/or thioredoxin in the thiol-disulfide exchange reaction as previously reported (Hagihara et al, 2011(Hagihara et al, , 2012.…”

Section: Mixed Disulfide Conjugates Of Clopidogrelmentioning

confidence: 65%

Formation, Reactivity, and Antiplatelet Activity of Mixed Disulfide Conjugates of Clopidogrel

et al. 2013

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“…Although the exact mechanism by which ClopNPT is converted to the active metabolite in vivo remains to be elucidated, it is likely that ClopNPT is converted to the active metabolite by a thiol disulfide exchange reaction with glutathione (Zhang et al, 2013). This exchange may also be accelerated by glutaredoxin and thioredoxin (Hagihara et al, 2011(Hagihara et al, , 2012. The rapid conversion of ClopNPT to the active metabolite also occurs after intravenous administration.…”

Section: Discussionmentioning

confidence: 99%

Significant Improvement of Antithrombotic Responses to Clopidogrel by Use of a Novel Conjugate as Revealed in an Arterial Model of Thrombosis

Zhang

Lauver

Wang

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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Clopidogrel is a prodrug that requires bioactivation by cytochrome P450 (P450) enzymes to a pharmacologically active metabolite for antiplatelet action. The clinical limitations of clopidogrel are in large part due to its poor pharmacokinetics resulting from inefficient bioactivation by P450s. In this study, we determined the pharmacokinetics and pharmacodynamics of a novel conjugate of clopidogrel, referred to as ClopNPT, in animal models and we evaluated its potential to overcome the limitations of clopidogrel. Results from pharmacokinetic (PK) studies showed that ClopNPT released the active metabolite with a time to maximal plasma concentration of ,5 minutes in C57BL/6 mice after either oral or intravenous administration, and plasma concentrations of the active metabolite reached C max values of 1242 and 1100 ng/ml after a 10-mg/kg oral dose and a 5-mg/kg intravenous dose, respectively. Furthermore, ClopNPT was highly effective in preventing arterial thrombosis in rabbits and mice after vascular injuries. Formation of occlusive thrombi was prevented by ClopNPT at the 1-mg/kg dose with no significant increase in tongue bleeding time, whereas clopidogrel was ineffective at the same dose. These results suggest that ClopNPT has favorable PK/pharmacodynamic properties that can potentially overcome the attenuated PK properties of clopidogrel and thus significantly improve the efficacy of antiplatelet therapy.

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“…This low level of active metabolite may be prone to changes as a result of factors other than P450-catalyzed reactions, such as the concentrations of GSH or other reductants that may be associated with oxidative stress or inflammation. In addition, a study by Hagihara et al (2011) demonstrated that glutaredoxin and thioredoxin accelerated the conversion of the glutathionyl conjugate of the active metabolite of prasugrel, another thienopyridine antiplatelet agent like clopidogrel, to the active metabolite. It is unclear to what extent glutaredoxin and thioredoxin may contribute to the metabolism of clopidogrel to its active metabolite.…”

Section: Discussionmentioning

confidence: 99%

Formation of the Thiol Conjugates and Active Metabolite of Clopidogrel by Human Liver Microsomes

Zhang

Lau

Hollenberg³

2012

Mol Pharmacol

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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%) Ͻ L-Cys (53%) Ͻ ascorbic acid (61%) Ͻ 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 ϳ0.5 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 ϳ10 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.

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Glutaredoxin and Thioredoxin Can Be Involved in Producing the Pharmacologically Active Metabolite of a Thienopyridine Antiplatelet Agent, Prasugrel

Cited by 11 publications

References 21 publications

Formation, Reactivity, and Antiplatelet Activity of Mixed Disulfide Conjugates of Clopidogrel

Formation, Reactivity, and Antiplatelet Activity of Mixed Disulfide Conjugates of Clopidogrel

Significant Improvement of Antithrombotic Responses to Clopidogrel by Use of a Novel Conjugate as Revealed in an Arterial Model of Thrombosis

Formation of the Thiol Conjugates and Active Metabolite of Clopidogrel by Human Liver Microsomes

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