Dissecting the Activation of Thienopyridines by Cytochromes P450 Using a Pharmacodynamic Assay In Vitro

Abell, Lynn M.; Liu, Eddie C.‐K.

doi:10.1124/jpet.111.184895

Cited by 17 publications

(14 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The procedures used to determine ex vivo antiplatelet activity were similar to those as reported by Abell and Liu (2011) Male New Zealand white rabbits (2.2-2.9 kg) were used as blood donors. Whole blood was drawn from a central ear artery into a plastic syringe containing 3.7% sodium citrate as the anticoagulant (1:10 volume ratio of citrate to blood).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2013

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

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

et al. 2013

View full text Add to dashboard Cite

show abstract

“…Washed platelets were prepared from fresh blood taken from healthy male subjects as described elsewhere (Abell and Liu, 2011). Final isolated platelets were responded in modified Tyrode's buffer containing 2 mM calcium chloride and 1 mg/ml human fibrinogen with cell density between 200,000 and 250,000 platelets/mL.…”

Section: Methodsmentioning

confidence: 99%

Multiple Cytochrome P450 Isoforms Are Involved in the Generation of a Pharmacologically Active Thiol Metabolite, whereas Paraoxonase 1 and Carboxylesterase 1 Catalyze the Formation of a Thiol Metabolite Isomer from Ticlopidine

et al. 2013

View full text Add to dashboard Cite

Ticlopidine is a first-generation thienopyridine antiplatelet drug that prevents adenosine 59-diphosphate (ADP)-induced platelet aggregation. We identified the enzymes responsible for the two-step metabolic bioactivation of ticlopidine in human liver microsomes and plasma. Formation of 2-oxo-ticlopidine, an intermediate metabolite, was NADPH dependent and cytochrome P450 (CYP) 1A2, 2B6, 2C19, and 2D6 were involved in this reaction. Conversion of 2-oxo-ticlopidine to thiol metabolites was observed in both microsomes (M1 and M2) and plasma (M1). These two metabolites were considered as isomers, and mass spectral analysis suggested that M2 was a thiol metabolite bearing an exocyclic double bond, whereas M1 was an isomer in which the double bond was migrated to an endocyclic position in the piperidine ring. The conversion of 2-oxo-ticlopidine to M1 in plasma was significantly increased by the addition of 1 mM CaCl 2 . In contrast, the activity in microsomes was not changed in the presence of CaCl 2 . M1 formation in plasma was inhibited by EDTA but not by other esterase inhibitors, whereas this activity in microsomes was substantially inhibited by carboxylesterase (CES) inhibitors such as bis-(p-nitrophenyl)phosphate (BNPP), diisopropylphosphorofluoride (DFP), and clopidogrel. The conversion of 2-oxo-ticlopidine to M1 was further confirmed with recombinant paraoxonase 1 (PON1) and CES1. However, M2 was detected only in NADPHdependent microsomal incubation, and multiple CYP isoforms were involved in M2 formation with highest contribution of CYP2B6. In vitro platelet aggregation assay demonstrated that M2 was pharmacologically active. These results collectively indicated that the formation of M2 was mediated by CYP isoforms whereas M1, an isomer of M2, was generated either by human PON1 in plasma or by CES1 in the human liver.

show abstract

“…Recently, a significant association was reported between the hepatic cytochrome P450 (CYP2C19) × 2 loss-of-function allele and diabetic status from Mexican population [28]. Clopidogrel requires conversion into an active thiol metabolite by the CYP enzyme to exert its antiplatelet effect [29]. A single nucleotide genetic polymorphism of CYP2C19 has been shown a decreased activation of clopidogrel and antiplatelet effect [30].…”

Section: Discussionmentioning

confidence: 99%

Impact of clopidogrel treatment on platelet function and thrombogenecity in diabetic patients undergoing elective coronary stenting

Singla¹,

Bliden²,

Jeong³

et al. 2012

JDM

View full text Add to dashboard Cite

High platelet reactivity (HPR) and suboptimal response to dual antiplatelet therapy (DAPT) may explain high recurrent rates of ischemic events in Type II diabetes mellitus (DM) patients undergoing percutaneous coronary intervention (PCI). The aim of this study was to examine the effect of clopidogrel on platelet reactivity, and thrombogenecity between DM and non-DM PCItreated patients (n = 138). Patients were categorized according to clopidogrel treatment and DM status. Patients received a maintenance-dose clopidogrel of 75 mg/d (C75 group, n = 72) or a 600 mg clopidogrel loading-dose in clopidogrel naïve patients (C600 group, n = 66). Platelet function was assessed by thrombelastography, flow cytometry, VerifyNow™ aspirin assay and light transmittance aggregometry (LTA). Aspirin response was similar between treatments and DM status. In the C75 group, DM patients had higher 5 and 20 M ADP, 2 g/ml collagen-induced LTA; and p-selectin expression compared to non-DM patients (p ≤ 0.05 for all). DM patients in the C600 group had higher 5 and 20 M ADPinduced LTA post dosing (p < 0.04) and higher maximum induced platelet-fibrin clot strength at baseline and post dosing timepoints (p < 0.05 for all). The present study provides further evidence that DM patients on DAPT are characterized by persistently higher platelet reactivity and thrombogenecity, making them potential candidates for more potent P2Y 12 receptor antagonists.

show abstract

Dissecting the Activation of Thienopyridines by Cytochromes P450 Using a Pharmacodynamic Assay In Vitro

Cited by 17 publications

References 25 publications

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

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

Multiple Cytochrome P450 Isoforms Are Involved in the Generation of a Pharmacologically Active Thiol Metabolite, whereas Paraoxonase 1 and Carboxylesterase 1 Catalyze the Formation of a Thiol Metabolite Isomer from Ticlopidine

Impact of clopidogrel treatment on platelet function and thrombogenecity in diabetic patients undergoing elective coronary stenting

Contact Info

Product

Resources

About