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

Zhang, Haoming; Lauver, D. Adam; Lucchesi, Benedict R.; Hollenberg, Paul F.

doi:10.1124/mol.112.084392

Cited by 15 publications

(45 citation statements)

References 26 publications

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“…29 This was also in complete agreement with the almost identical results previously described for the formation of both cis 5a and trans 5a′ upon microsomal metabolism of 1a or 2a in the presence of GSH, NAC, or ME, 1,6,19,22 and the exclusive formation of 5a in the presence of ascorbic acid instead of GSH. 11,19 Replacement of GSH with the reductant Tris(carboxyethyl)-phosphine (TCEP) led to the exclusive formation of the cis diastereomers 5b (Figure 3E), as when ascorbic acid was used instead of GSH. This result is in agreement with literature data showing that sulfenic acids are efficiently reduced by TCEP with the formation of the corresponding thiols.…”

Section: ■ Introductionsupporting

confidence: 92%

Bioactivation of Clopidogrel and Prasugrel: Factors Determining the Stereochemistry of the Thiol Metabolite Double Bond

Dansette

Levent

Hessani

et al. 2015

Chem. Res. Toxicol.

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The antithrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The second step is a further oxidation with the formation of a thiolactone sulfoxide whose hydrolytic opening leads to a sulfenic acid that is eventually reduced into the corresponding active cis thiol. Very few data were available on the formation of the isomer of the active cis thiol having a trans configuration of the double bond, the most striking result in that regard being that both cis and trans thiols were formed upon the metabolism of clopidogrel by human liver microsomes in the presence of glutathione (GSH), whereas only the cis thiol was detected in the sera of patients treated with this drug. This article shows that trans thiols are also formed upon the microsomal metabolism of prasugrel or its thiolactone metabolite in the presence of GSH and that metabolites having the trans configuration of the double bond are only formed when microsomal incubations are done in the presence of thiols, such as GSH, N-acetyl-cysteine, and mercaptoethanol. Intermediate formation of thioesters resulting from the reaction of GSH with the thiolactone sulfoxide metabolite appears to be responsible for trans thiol formation. Addition of human liver cytosol to the microsomal incubations led to a dramatic decrease of the formation of the trans thiol metabolites. These data suggest that cytosolic esterases would accelerate the hydrolytic opening of thiolactone sulfoxide intermediates and disfavor the formation of thioesters resulting from the reaction of these intermediates with GSH that is responsible for trans isomer formation. This would explain why trans thiols have not been detected in the sera of patients treated with clopidogrel.

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Section: ■ Introductionsupporting

confidence: 92%

Bioactivation of Clopidogrel and Prasugrel: Factors Determining the Stereochemistry of the Thiol Metabolite Double Bond

Dansette

Levent

Hessani

et al. 2015

Chem. Res. Toxicol.

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“…The rapid decrease in ClopNPT and simultaneous increase in the active metabolite in the plasma indicate that oral ClopNPT is rapidly absorbed in the small intestine and subsequently converted to the active metabolite. 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.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, further improvement of the pharmacokinetic (PK)/pharmacodynamic (PD) properties of clopidogrel would provide more efficacious DAPT. We previously reported that ClopNPT, a mixed disulfide conjugate of the active metabolite with 3-nitropyridine-2-thiol (NPT), is readily converted to the active metabolite in the presence of glutathione through a thiol disulfide exchange reaction (Zhang et al, 2013;Scheme 1B). Furthermore, ClopNPT inhibits platelet aggregation in the platelet-rich plasma (PRP) of rabbits upon intravenous administration and its inhibitory effects do not require bioactivation by P450s .…”

Section: Introductionmentioning

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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“…Thiolactone 2 (a 1:1 mixture of 2a and 2b) was incubated for 15 minutes at 37°C with HLMs in the presence of NADPH and GSH (5 mM) to reduce sulfenic acid 4 to the corresponding thiols (H1-H4) (Dansette et al, 2009(Dansette et al, , 2010Zhang et al, 2013). A UPLC-Q/TOF MS study of the incubate showed the formation of four thiol isomers exhibiting a molecular ion (ESI + ) corresponding to [M + H] + that was characterized by two peaks at m/z = 356.073 and 358.071 with the ratio expected for the 35 Cl and 37 Cl isotopes.…”

Section: Chiral Stability Of Thiolactone 2 Isomers (2a and 2b) In Bufmentioning

confidence: 99%

“…The sulfenic acid 4 is highly unstable and can be rapidly reduced by glutathione (GSH) to form a mixed disulfide conjugate 5, which is subsequently reduced by another GSH molecule to form the active thiol 6 (Fig. 1) (Dansette et al, 2009;Zhang et al, 2012Zhang et al, , 2013.…”

Section: Introductionmentioning

confidence: 99%

Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel

et al. 2015

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Clopidogrel, a thienopyridine antiplatelet prodrug, is metabolized by oxidation to 2-oxo-clopidogrel, followed by conversion to its pharmacologically active thiol metabolite. After oral administration of clopidogrel to humans, two thiol isomers (H3 and H4) are observed in plasma, with similar concentrations, and only H4 is active in humans. In this work, the mechanism of stereoselectivity in the formation and S-methylation of H3 and H4 was investigated in vitro. The two diastereomers of 2-oxo-clopidogrel were epimerized rapidly at physiologic pH. The intrinsic clearance (CL int ) for H3 formation from 2-oxo-clopidogrel in human liver microsomes (HLMs) was 3.1-fold higher than that for H4 formation, indicating stereoselective metabolism. Kinetic studies using expressed enzymes demonstrated that the contributions of CYP2B6, CYP2C19, and CYP3A4 to the formation of H4 from 2-oxo-clopidogrel were 18.5%, 26.1%, and 53.5%, respectively. The CL int ratios of H3 formation to H4 formation from 2-oxo-clopidogrel by CYP2B6, CYP2C19, and CYP3A4 were 2.2, 1.0, and 1.7, respectively. In HLMs, H3 and H4 were further S-methylated, and the S-methylation was inhibited by 2,3-dichloromethyl benzylamine, indicating the involvement of thiol S-methyltransferase. The CL int value for the S-methylation of H3 in HLMs was 98.1-fold higher than that for H4. The stereoselective formation of H3 from 2-oxo-clopidogrel and the stereoselective S-methylation of H3 may lead to the similar exposure levels of H3 and H4 previously reported in humans. The epimerization of 2-oxoclopidogrel and the variations of thiol S-methyltransferase may affect the exposure to H4 in humans.

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

Cited by 15 publications

References 26 publications

Bioactivation of Clopidogrel and Prasugrel: Factors Determining the Stereochemistry of the Thiol Metabolite Double Bond

Bioactivation of Clopidogrel and Prasugrel: Factors Determining the Stereochemistry of the Thiol Metabolite Double Bond

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

Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel

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