Prasugrel Active Metabolite Research Articles

Pharmacodynamic and pharmacokinetic profiles associated with switching from cangrelor to prasugrel in patients undergoing percutaneous coronary intervention: the SWAP-6 study

Abstract Background Cangrelor is an intravenous P2Y12 inhibitor used in patients undergoing coronary intervention (PCI). Preliminary studies have shown the potential for a drug-drug interaction (DDI) when transitioning from cangrelor to oral P2Y12 inhibition with prasugrel. However, to date this has not been prospectively tested in patients undergoing PCI. Purpose To rule out a DDI when cangrelor and prasugrel are concomitantly administered in patients undergoing PCI. Methods SWAP-6 is a prospective, randomized, 3-arm, open-label, pharmacokinetic (PK) and pharmacodynamic (PD) study conducted in patients undergoing PCI (n=77). Patients were randomized to either a) prasugrel only administered at the start of PCI; b) cangrelor plus prasugrel concomitantly administered at the start of PCI; c) cangrelor administered at the start of PCI plus prasugrel administered at the end of the cangrelor infusion. Cangrelor (30 μg/kg bolus followed by 4 μg/kg/min infusion) was maintained for 2 hours and all patients were on a background of aspirin therapy. Prasugrel was administered as a 60 mg loading dose. PK analysis included plasma levels of prasugrel active metabolite, and PD assessments included VerifyNow PRU, total thrombus-formation analysis system (T-TAS), and vasodilator-stimulated phosphoprotein (VASP), at baseline and 6 time points after prasugrel/cangrelor administration. The primary end point was non-inferiority in PRU measured at 4 hours after prasugrel/cangrelor loading (2 hours after stopping cangrelor infusion) of cangrelor plus prasugrel concomitantly administered versus prasugrel only. The noninferiority margin was defined as 45 PRU. Results Cangrelor achieved lower PRU levels than prasugrel only as early as 30 minutes and up to 2 hours after bolus administration (Figure). At 3 hours after prasugrel/cangrelor loading, PRU levels were higher with cangrelor compared to prasugrel only. At 4 hours after loading (2 hours after stopping cangrelor infusion), PRU levels were significantly lower with prasugrel only compared to cangrelor and prasugrel concomitantly administered (LSM difference 130; 95% CI: 85-176), failing to meet the prespecified noninferiority primary end point (Figure). At this time point, cangrelor followed by prasugrel at the end of the infusion had higher PRU than prasugrel only, but lower than cangrelor and prasugrel concomitantly. Platelet reactivity by T-TAS and VASP showed consistent findings. PK analysis tracked PD results (data not shown). Conclusions In patients undergoing PCI, cangrelor is associated with more potent platelet inhibition than prasugrel alone. However, concomitant administration of prasugrel with cangrelor leads to a marked increase in platelet reactivity after stopping cangrelor infusion, supporting the presence of a DDI. This underscores the importance of administering prasugrel not until the end of cangrelor infusion, a strategy which mitigates the increase in platelet reactivity.

Thrombus remodelling by reversible and irreversible P2Y12 inhibitors

Pharmacological inhibition of the platelet ADP-receptor P2Y12 is a cornerstone in the prevention of atherothrombotic events in adult patients with acute coronary syndrome (ACS). Thienopyridines such as clopidogrel and prasugrel exert their antithrombotic effect by means of active metabolites that irreversibly inhibit P2Y12. Due to the short half-life of these metabolites, a subpopulation of ADP-responsive platelets will form in between dosing. With increased platelet turnover rate or poor patient compliance, the fraction of ADP-responsive platelets will increase, potentially increasing the risk for new thrombotic events. In contrast, the reversible P2Y12 inhibition produced by direct-acting ADP blockers such as ticagrelor and cangrelor inhibit the entire platelet population. In this study, we evaluated the impact of these pharmacological differences on thrombus formation in an ex vivo flow chamber model. A customized image analysis pipeline was used for automatized, large-scale identification and tracking of single platelets incorporated into the thrombus, enabling quantitative analysis of the relative contribution of inhibited and uninhibited platelets to thrombus growth and consolidation. Comparative experiments were conducted using the irreversible and reversible P2Y12 inhibitors prasugrel active metabolite (PAM) and ticagrelor, respectively. Our results show that PAM inhibited thrombus platelet recruitment more gradually than ticagrelor, with a slower onset of inhibition. Further, we show that the presence of a small fraction (<10%) of uninhibited platelets did not abrogate the antithrombotic effect of PAM to any significant extent. Finally, we demonstrate a gradual enrichment of inhibited platelets in the thrombus shell due to selective recruitment of inhibited platelets to the thrombus periphery.

Evaluation of the changes in exposure to thiol compounds in chronic kidney disease patients using the PBPK model

Targeted covalent inhibitors designed to bind covalently to a specific molecular target have recently been a focus of drug development. Among these inhibitors, thiol compounds bind covalently to endogenous thiols in the body through a process involving disulfide bonds. We investigated the predictability of changes in the exposure to captopril, tiopronin, the active form of dalcetrapib and the active metabolite of prasugrel, R-138727, all of which have a sulfhydryl group, in moderate and severe chronic kidney disease (CKD) patients using a constructed PBPK model. The changes in the exposure to captopril, tiopronin and the active form of dalcetrapib under CKD conditions were well predicted. However, the change in exposure to R-138727, which is a secondary metabolite of prasugrel, was overpredicted. Although these thiol compounds covalently bind to endogenous thiols, our study concluded that changes in exposure to these compounds under CKD conditions can probably be predicted, except for compounds with a complicated mechanism whereby the thiol metabolite is generated.

Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti‐platelet therapy

BackgroundEndothelium‐derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO‐sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache. ObjectiveWe investigated using GC‐activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti‐thrombotic effect of both drugs. MethodsIn vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride‐induced arterial thrombosis model were also performed. ResultsThe GC‐activator BAY‐70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra‐platelet cyclic nucleotide levels. Furthermore, mice administered sub‐maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow. ConclusionsUsing in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti‐platelet effects without altering blood flow. Therefore, modulation of intra‐platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti‐thrombotic regimen while minimizing vasodilator side effects.

Influence of cytochrome P450 polymorphisms on the antiplatelet effects of prasugrel in patients with non-cardioembolic stroke previously treated with clopidogrel

This randomized double-blind crossover study aimed to investigate the influence of cytochrome P450 (CYP) 2C19 polymorphisms on the antiplatelet effects of prasugrel in patients with non-cardioembolic stroke treated with clopidogrel. Patients received clopidogrel 75 mg/day for > 4 weeks. Subsequently, patients received prasugrel 3.75 mg/day (group A; n = 64) or 2.5 mg/day (group B; n = 65) for 4 weeks followed by a 4 week switched-dose regimen. To assess the influence of CYP2C19 polymorphisms, patients were classified as extensive metabolizers (EMs), intermediate metabolizers (IMs), and poor metabolizers (PMs). The primary endpoint was P2Y12 reaction units (PRU) at the end of each 4 week treatment. A significant reduction in PRU was noted after treatment with prasugrel 3.75 mg/day compared with the pre-dose value (after treatment with clopidogrel) (p < 0.0001). By CYP2C19 phenotypes, a significant reduction in PRU was noted in IMs and PMs after treatment with prasugrel 3.75 mg/day and in PMs after treatment with prasugrel 2.5 mg/day, as compared with the pre-dose value (p < 0.0001). The plasma concentration of the active metabolite of clopidogrel was relatively low in PMs compared to EMs and IMs; prasugrel was similar across all CYP2C19 phenotypes. No major or clinically significant hemorrhagic adverse events occurred. By CYP2C19 phenotype, the antiplatelet effects of prasugrel were greater with 3.75 mg/day in IMs and PMs, and with 2.5 mg/day in PMs compared with clopidogrel 75 mg/day, without safety concerns. CYP2C19 polymorphisms did not affect the plasma concentration of the active metabolite of prasugrel or its antiplatelet effects. (JapicCTI-101044).

Prasugrel in critically ill patients

SummaryWhile prasugrel is indicated for the treatment of myocardial infarction, its effects in the most severely affected patients requiring intensive care is unknown, so that we measured the antiplatelet effects and sparse pharmacokinetics of prasugrel in critically ill patients. Twenty-three patients admitted to medical intensive care units, who were treated with 10 mg prasugrel once daily, were included in this prospective trial. Critically ill patients responded poorly to daily prasugrel treatment: adenosine diphosphate (ADP)-induced aggregation in whole blood classified 65 % (95 % confidence intervals (CI) 43–84 %) of patients as having high on treatment platelet reactivity, platelet function under high shear rates even 74 % (95 %CI 52–90 %). There was only limited additional inhibition provided 2 hours after the next dose of prasugrel. In contrast, insufficient inhibition of the target was only seen in 26 % (95 %CI 10–48 %) of patients as measured by the vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) assay. Low effective plasma levels of prasugrel active metabolite were measured at trough [0.5 (quartiles 0.5–1.1) ng/ml at baseline], and 2 hours after intake [5.7 (3.8–9.8) ng/ml], but showed coefficients of variation of ~70 %. In sum, inhibition of platelet aggregation by prasugrel is not uniform but highly variable in critically ill patients, similar to clopidogrel in a general population. The pharmacokinetic measurements indicate that poor absorption/metabolism of prasugrel may partly contribute while inflammation induced heightened intrinsic platelet reactivity may also play a role. Supplementary Material to this article is available online at www.thrombosis-online.com . Note: This work was performed at the Medical University of Vienna, Austria.

Incomplete reversibility of platelet inhibition following prolonged exposure to ticagrelor

Introduction Ticagrelor is described as a reversible P2Y12 antagonist. However, residual platelet inhibition persists after discontinuation of ticagrelor when plasma levels are undetectable. We assessed the reversibility of platelet inhibition by ticagrelor and its active metabolite (T-AM) in comparison with cangrelor and prasugrel's active metabolite (P-AM). Methods Whole blood was treated in vitro with ~ 50% inhibitory concentrations of ticagrelor, T-AM, cangrelor, P-AM and assessed for ADP-stimulated activated GPIIb-IIIa and P-selectin and vasodilator-stimulated phosphoprotein (VASP) platelet reactivity index (PRI) before and after 100-fold dilution. Results Platelets exposed for 30 min to ticagrelor, T-AM or cangrelor showed full recovery of activated GPIIb-IIIa but only partial recovery of P-selectin. Longer exposure (24 h) to the drug decreased reversibility of activated GPIIb-IIIa by ticagrelor (65.1% [49.5-80.6], % of vehicle with 95% confidence interval [CI]) and T-AM (88.8% [79.2-98.3]), but not by cangrelor (101.4% [96.4-106.4]). Compared with 30 min exposure, the reversibility of P-selectin further decreased after 24 h exposure to ticagrelor (from 91.8% [82.1-101.5] to 51.8% [45.5-85.0]), but not T-AM (from 79.0% [67.8-90.3] to 77.4% [61.8-93.1]) or cangrelor (from 76.0% [67.6-84.4] to 76.2% [70.6-81.8]). In contrast, 24 h exposure to ticagrelor, T-AM and cangrelor resulted in full recovery of platelet reactivity as measured by PRI. Platelets exposed to P-AM showed no recovery of ADP reactivity. Conclusions Incomplete recovery after prolonged exposure to ticagrelor, observed by activated GPIIb-IIIa and P-selectin but not upstream VASP signaling, suggests that P2Y12 regains functionality and irreversible changes occur independent of VASP signaling.

Analysis of prasugrel active metabolite R-138727 in human plasma: a sensitive, highly selective and fast LC-MS/MS method.

A cost effective, sensitive, simple, and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the prasugrel metabolite in human plasma. Following solid phase extraction, the analyte (prasugrel active metabolite; R-138727) and internal standard (emtricitabine) were separated using a mobile phase in an isocratic elution mode on a reverse phase C18 column and were analyzed by an LC-MS/MS in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 498.3-206.0 for R-138727 and m/z 248.2-130.1 for the internal standard. The assay exhibited a linear dynamic range of 0.2-120 ng/mL. The lower limit of quantification was 0.2 ng/mL with a relative standard deviation of 5.0%. This LC-MS/MS method was validated with intra-batch and inter-batch precision and accuracy. Results for precision and accuracy are in range of 3.9-9.6% and 95.2-102.2% respectively. This validated method is simple and repeatable to use in bioequivalence/pharmacokinetic studies.

Morphine delays the onset of action of prasugrel in patients with prior history of ST-elevation myocardial infarction.

Delays in the onset of action of prasugrel during primary percutaneous coronary intervention (PPCI) have been reported and could be related to the effects of morphine on gastric emptying and subsequent intestinal absorption. The study objective was to determine whether morphine delays the onset of action of prasugrel in patients with a prior history of ST-elevation myocardial infarction (STEMI) treated with PPCI. This was a crossover study of 11 aspirin-treated patients with prior history of STEMI treated with PPCI, for which prasugrel and morphine had been previously administered. Patients were randomised to receive either morphine (5 mg) or saline intravenously followed by 60 mg prasugrel. Blood samples were collected before randomised treatment and over 24 hours after prasugrel administration. The inhibitory effects of prasugrel on platelets were determined using the VerifyNow P2Y12 assay and light transmission aggregometry. Plasma levels of prasugrel and prasugrel active metabolite were measured. Platelet reactivity determined by VerifyNow PRU, VerifyNow % Inhibition and LTA was significantly higher at 30-120 minutes (min) when morphine had been co-administered compared to when saline had been co-administered. Morphine, compared to saline, significantly delayed adequate platelet inhibition after prasugrel administration (158 vs 68 min; p = 0.006). Patients with delayed onset of platelet inhibition also had evidence of delayed absorption of prasugrel. In conclusion, prior administration of intravenous morphine significantly delays the onset of action of prasugrel. Intravenous drugs may be necessary to reduce the risk of acute stent thrombosis in morphine-treated STEMI patients undergoing PPCI.

Morphine interaction with prasugrel: a double-blind, cross-over trial in healthy volunteers.

BackgroundMorphine decreases the concentrations and effects of clopidogrel, which could lead to treatment failure in myocardial infarction.ObjectivesTo clarify whether more potent P2Y12-inhibitors may provide an effective alternative, we examined drug–drug interactions between morphine and prasugrel.MethodsTwelve healthy volunteers received 60 mg prasugrel with placebo or 5 mg morphine intravenously in a randomized, double-blind, placebo-controlled, cross-over trial. Pharmacokinetics were determined by liquid chromatography tandem mass spectrometry, and prasugrel effects were measured by platelet function tests.ResultsMorphine neither diminished total drug exposure (AUC), which was the primary endpoint, nor significantly delayed drug absorption of prasugrel. However, morphine reduced maximal plasma concentrations (Cmax) of prasugrel active metabolite by 31 % (p = 0.019). Morphine slightly, but not significantly, delayed the onset of maximal inhibition of platelet plug formation under high shear rates (30 vs. 20 min). Whole blood aggregation was not influenced.ConclusionsAlthough morphine significantly decreases the maximal plasma concentrations of prasugrel active metabolite, it does not diminish its effects on platelets to a clinically relevant degree in healthy volunteers. However, it should be considered that the observed decrease in Cmax of prasugrel active metabolite caused by morphine co-administration may gain relevance in STEMI patients.Clinical Trial Registration: NCT01369186, EUDRA-CT#: 2010-023761-22.

Cangrelor inhibits the binding of the active metabolites of clopidogrel and prasugrel to P2Y12 receptors in vitro

Cangrelor is a rapid-acting, direct-binding, and reversible P2Y12 antagonist which has been studied for use during percutaneous coronary intervention (PCI) in patients with or without pretreatment with an oral P2Y12 antagonist. As cangrelor is administered intravenously, it is necessary to switch to an oral P2Y12 antagonist following PCI, such as the thienopyridines clopidogrel, and prasugrel or the non-pyridine ticagrelor. Previous studies have suggested a negative pharmacodynamic interaction between cangrelor and thienopyridines. This in vitro study evaluated the receptor-level interaction between cangrelor and the active metabolite (AM) of clopidogrel or prasugrel by assessing functional P2Y12 receptor number using a 33P-2MeSADP binding assay. All P2Y12 antagonists studied resulted in strong P2Y12 receptor blockade (cangrelor: 93.6%; clopidogrel AM: 93.0%; prasugrel AM: 97.9%). Adding a thienopyridine AM in the presence of cangrelor strongly reduces P2Y12 receptor blockade by the AM (clopidogrel AM: 7%, prasugrel AM: 3.2%). The thienopyridine AMs had limited ability to compete with cangrelor for binding to P2Y12 (% P2Y12 receptor blockade after co-incubation with cangrelor 1000 nmol/L: 11.7% for clopidogrel AM 3 µmol/L; 34.1% for prasugrel AM 3 µmol/L). In conclusion, in vitro cangrelor strongly inhibits the binding of clopidogrel and prasugrel AMs to the P2Y12 receptor, consistent with the previous observation of a negative pharmacodynamic interaction. Care may need to be taken to not overlap exposure to thienopyridine AMs and cangrelor in order to reduce the risk of thrombotic complications following PCI.

213 Synergy between Endothelial Mediators and P2Y12 Receptor Blockade as a Potential Determinant in Tailoring Anti-Platelet Therapy

<h3>Introduction</h3> Following ACS or PCI, P2Y12 receptor blockers such as clopidogrel or prasugrel are standardly prescribed alongside aspirin as dual anti-platelet therapy in a “one size fits all” approach, however recurrent thrombotic events occur. Despite the logical hypothesis that thrombotic risk should be associated with level of P2Y12 platelet inhibition, randomised control trials have repeatedly failed to show any clinical benefit of guiding anti-platelet therapy by platelet function tests (PFT). Whilst traditional PFT measure reactivity to ADP, they crucially ignore that P2Y12 receptor antagonists also produce powerful anti-thrombotic effects by potentiating the actions of the inhibitory endothelial mediators prostacyclin (PGI2) and nitric oxide (NO). <h3>Methods</h3> In vitro, IC50 curves produced from light transmission aggregation (LTA) traces for PGI2, NO and prasugrel active metabolite (PAM: 3 μM) were used to generate isobolograms. <i>Ex vivo</i>, healthy male volunteers (n = 8 each) received either prasugrel (10 mg) or aspirin (75 mg) for 7 days. Platelet-rich plasma (PRP) was obtained by centrifugation before and after treatment. Platelet responses to TRAP-6 amide (25 μM) or collagen (4 μg/ml) in the presence of PGI2 (1 nM) and/or the NO donor, DEA/NONOate (100 nM), or vehicle, were assessed by LTA, p-selectin binding by flow cytometry, and downstream cyclic nucleotide and VASP phosphorylation by immunoassay. <h3>Results</h3> Isobolographic analysis of <i>in vitro</i> studies showed P2Y12 blockade powerfully enhanced the synergy between NO and PGI2 (10 fold for TRAP-6). Ex-vivo platelet aggregation responses to TRAP-6 were unaffected by the addition of PGI2+NO prior to therapy. Aspirin monotherapy produced minimal inhibition of TRAP-6, though these became stronger in the presence of PGI2+NO (58%±8 to 28%±9; p &lt; 0.05). Inhibitory effects were observed with prasugrel in the presence of PGI2 and NO individually (control, 63 ± 3%; PGI2, 43 ± 6%; NO, 50 ± 5%), and these became much greater with the combination (PGI2+NO, 7%±3). P-selectin expression was reduced from pre-treatment (vehicle, 32 ± 6%) to (PGI2+NO, 11 ± 4%) post aspirin, and from (vehicle, 25 ± 6%) to (PGI2+NO, 2 ± 4%) following prasugrel. cAMP levels increased with PGI2+NO in the aspirin group (0.97 ± 0.06 vs. 1.50 ± 0.19) compared to (1.41 ± 0.07 vs. 3.4 ± 0.58) in the prasugrel group. <h3>Conclusions</h3> PGI2 and NO synergise with P2Y12 blockade to cause powerful platelet inhibition suggesting that an individual’s endothelial function and production of NO and PGI2 are central determents of the anti-thrombotic protection from P2Y12 receptor blocker therapy. Whilst a therapeutic window of platelet reactivity is an attractive concept our research suggests that endothelial function testing alongside PFT would permit better risk stratification. Our research also suggests therapeutically that enhancing endothelial-derived mediators or optimising downstream cyclic nucleotide signalling could reduce both thrombotic and bleeding risk.

158 The Anti-Platelet Effectiveness of P2Y12Receptor Blockade is Strongly Influenced by the Endothelium-Derived Mediators Nitric Oxide and Prostacyclin

Introduction P2Y12 receptor antagonists that block the pro-aggregatory effects of ADP on platelets are commonly prescribed alongside aspirin, as dual antiplatelet therapy (DAPT) for secondary prevention of atherothrombotic events. It is emerging that these agents exert part of their effects through potentiation of the endogenous, endothelial-derived platelet inhibitors, nitric oxide (NO) and prostacyclin (PGI2), which constantly bathe circulating platelets reducing their reactivity through elevating levels of cyclic nucleotides. Thus, if the in vivosynergy between NO and PGI2 amplifies the anti-thrombotic effects of P2Y12 inhibitors, within the circulation variances in the levels of endothelial-derived mediators will be an important determinant of the efficacy of DAPT, suggesting that for the individual patient, endothelial function influences the therapeutic potential of these powerful agents. Here, we characterised the interplay of PGI2, NO and P2Y12 blockade on platelet function. Methods 8 healthy male volunteers received aspirin (75 mg) and prasugrel (10 mg) for 7 days. Platelet responses to TRAP-6 (25 iM) or collagen (4 ig/ml) in the presence of PGI2 (1 nM), NO (100 nM), PGI2+NO or vehicle were assessed by LTA and lumi-aggreggometry. Experiments were replicated in vitro (n = 4) utilising aspirin (30 iM) and prasugrel active metabolite (PAM; 6, 3 or 1.5 iM) representing maximal and partial P2Y12 blockade. Results Ex vivo, prior to DAPT, PGI2, NO, or PGI2+NO had little effect upon platelet aggregation. TRAP-6 (25 iM): vehicle, 74 ± 3%; PGI2+NO, 66 ± 3%. Following DAPT, PGI2 or NO alone caused minor inhibition: vehicle, 57 ± 4%; PGI2, 47 ± 6%; NO 49 ± 6%. Conversely, the combination of PGI2+NO produced strong inhibitory effects, 19 ± 6% (p In vitro, with maximal P2Y12 inhibition (PAM, 6 iM) aggregation to TRAP-6 (25 iM) was reduced: vehicle, 56 ± 5%; PGI2, 30 ± 12%; NO, 46 ± 7%; PGI2+NO, 15 ± 8% (p Conclusion We demonstrate that PGI2 and NO synergise with P2Y12 blockade to produce the greatest platelet inhibition and that platelet reactivity, despite DAPT is powerfully influenced by the presence of NO and PGI2. This highlights that in vivo platelet reactivity will be a function of the level of P2Y12 receptor blockade and levels of extrinsic inhibitory endothelial mediators. This may be relevant to the efficacy of anti-platelet therapies in patients with endothelial dysfunction and introduces the concept of assessing endothelial function in patients undergoingex vivo platelet function testing to help bridge the disconnect between results of platelet testing and thrombotic outcomes.

Prostaglandin E1 potentiates the effects of P2Y12 blockade on ADP-mediated platelet aggregation in vitro: Insights using short thromboelastography

In addition to adenosine diphosphate (ADP), a number of platelet function tests including the VerifyNow P2Y12 assay (VN-P2Y12) employ prostaglandin E1 (PGE1) to improve specificity for P2Y12 blockade by mitigating the contribution of the P2Y1 pathway on ADP-mediated platelet aggregation. Using short thromboelastography (s-TEG), we have previously shown that VN-P2Y12 overestimates the functional effect of clopidogrel in some individuals. We investigated whether PGE1 systematically increases the inhibitory effects of P2Y12 blockade on ADP-mediated platelet aggregation in an in vitro model. Using s-TEG, we measured ADP-induced platelet aggregation either in the presence or absence of PGE1 (11 or 22 nM) in blood samples taken from healthy volunteers pre-incubated with prasugrel active metabolite (PAM; 0, 1, 3 or 10 µM). Individually, both PGE1 (p < 0.02) and PAM (p < 0.0001) inhibited ADP-mediated platelet aggregation in a dose-dependent manner, as expected. Furthermore, inclusion of PGE1 augmented inhibition of ADP-mediated platelet aggregation in response to PAM (p < 0.02) in a dose-dependent manner such that a 10-fold higher dose of PAM was required to attain equivalent inhibition of ADP-mediated platelet aggregation to that achieved by 1 µM PAM in the presence of 11 nM PGE1. In conclusion, PGE1 potentiates the anti-aggregatory effects of P2Y12 blockade on ADP-mediated platelet aggregation. Assays that employ PGE1 with ADP may therefore overestimate therapeutic response to prasugrel in a proportion of individuals, potentially making them unsuitable candidates for guiding delivery of personalized antiplatelet therapy.

Pharmacokinetics and Pharmacodynamics of Ticagrelor and Prasugrel in Healthy Male Korean Volunteers

PurposeA combination of clopidogrel and aspirin is the standard treatment for patients with acute coronary syndrome and those undergoing percutaneous coronary intervention. Two novel antiplatelet agents, ticagrelor and prasugrel, have been shown to rapidly and more effectively inhibit the P2Y12 receptor compared with clopidogrel. The aim of this study was to evaluate and compare the pharmacokinetics (PK) and pharmacodynamics (PD) of ticagrelor and prasugrel in healthy male Korean volunteers. MethodsTwo separate studies were conducted. One study was performed by using a single-sequence, open-label, crossover design in 12 volunteers who received a single oral dose of ticagrelor (180 mg) and then a single oral dose of prasugrel (60 mg for 4 volunteers and 30 mg for 8 volunteers) with a 7-day washout period. The other study was a randomized, open-label, parallel-group investigation in which 8 volunteers received a single oral dose of prasugrel (10 mg for 4 volunteers and 30 mg for 4 volunteers). In each study, blood samples for PK and platelet aggregation inhibition analysis were serially collected after the administration of each dose. Plasma concentrations of ticagrelor, AR-C124910XX (the active metabolite of ticagrelor), R-95913 (the inactive metabolite of prasugrel), and R-138727 (the active metabolite of prasugrel) were measured by using a validated LC-MS/MS method. PK was analyzed by using a noncompartmental method. Maximal platelet aggregations were assessed with light transmission aggregometry after induction with 20 μmol/L of adenosine diphosphate. FindingsTwenty healthy male Korean volunteers participated in the 2 studies. Plasma concentrations of ticagrelor and AR-C124910XX were obtained from 12 subjects, R-95913 from 20 subjects, and R-138727 from 8 subjects. Both ticagrelor and prasugrel were rapidly absorbed, with the shortest median Tmax of 2.0 and 2.25 hours for ticagrelor and AR-C124910XX, respectively, and a Tmax of 0.5 hour for both R-95913 and R-138727. Strong inhibition of platelet aggregation was shown after administration of both ticagrelor and prasugrel, with slightly stronger and more rapid inhibition with prasugrel in the tested doses. Inhibitory activities of prasugrel lasted longer than those of ticagrelor, reflecting the difference in binding kinetics between the 2 drugs. ImplicationsPrasugrel 30 and 60 mg exhibited slightly stronger, more rapid, and sustainable platelet inhibitory effects compared with ticagrelor 180 mg. These differing effects should be considered when determining the efficacy and adverse effects of ticagrelor and prasugrel. ClinicalTrials.gov identifier: NCT01876797 and NCT02075268.

Abstract 19437: Elevated Platelet Turnover in Patients Receiving Dual Antiplatlet Therapy Could Permit the Formation of Uninhibited Platelet Clusters That Act as Seeds for Thrombus Formation

Introduction: ACS patients who also have conditions associated with increased platelet turnover, such as diabetes or chronic kidney disease, are at increased risk of atherothrombotic events despite receiving dual-antiplatelet therapy (DAPT). It may well be that aspirin and thienopyridines, such as clopidogrel or prasugrel, prescribed once daily as pharmacologically short-lived but irreversibly acting agents, are less effective in these patients because of the daily emergence of a significant subpopulation of uninhibited platelets. Hypothesis: Here we investigated the potentially crucial roles of subpopulations of uninhibited platelets in overcoming DAPT and driving aggregation. Methods: Aliquots of platelet rich plasma were incubated with aspirin, prasugrel active metabolite (PAM) plus aspirin, or vehicle and platelets were differently labelled with membrane dyes. Aliquots were then combined in various proportions and platelet responses measured in standard light transmission aggregometry. Aggregates formed in response to platelet agonists were then fixed and detailed structural analyses made by advanced imaging techniques, including confocal microscopy and ImageStream flow cytometry, to determine the interactions of different platelet populations. Results: Platelet aggregation responses and images of platelet aggregates demonstrated complex, dynamic relationships between platelet subpopulations. Summary analysis indicated that aggregates containing aspirin-treated (90-60%) and drug-free (10-40%) platelets had random distributions of drug-free platelets in response to ADP or AA. In contrast, aggregates containing DAPT-inhibited (90-60%) platelets and drug-free (10-40%) platelets showed marked clustering of drug-free platelets at aggregate cores surrounded by recruited DAPT-inhibited platelets. Conclusions: We show that a subpopulation of drug-free platelets can provide seeds for the formation of aggregates that then recruit DAPT-inhibited platelets. Such interaction between platelet subpopulations are of direct relevance to ACS patients with underlying conditions associated with elevated platelet turnover and may well provide an explanation for the failure of DAPT with aspirin and thienopyridines.

Characterization of the Adenosine Pharmacology of Ticagrelor Reveals Therapeutically Relevant Inhibition of Equilibrative Nucleoside Transporter 1

Studies have shown that ticagrelor has a further adenosine-mediated mechanism of action in addition to its potent inhibition of the P2Y12 receptor, which may explain some of ticagrelor's clinical characteristics. This study aimed to further characterize the adenosine pharmacology of ticagrelor, its major metabolites, and other P2Y12 receptor antagonists. Inhibition of nucleoside transporter-mediated [(3)H]adenosine uptake by ticagrelor, its major metabolites, and alternative P2Y12 antagonists was examined in recombinant Madin-Darby canine kidney (MDCK) cells. The pharmacology of ticagrelor and its major metabolites at adenosine A1, A2A, A2B, and A3 receptor subtypes was examined using in vitro radioligand binding and functional assays and ex vivo C-fiber experiments in rat and guinea pig vagus nerves. Ticagrelor (and less effectively its metabolites) and the main cangrelor metabolite inhibited [(3)H]adenosine uptake in equilibrative nucleoside transporter (ENT) 1-expressing MDCK cells, whereas cangrelor and the active metabolites of prasugrel or clopidogrel had no effect. No significant inhibitory activity was observed in MDCK cells expressing ENT2 or concentrative nucleoside transporters 2/3. Ticagrelor demonstrated high affinity (inhibition constant [Ki] = 41 nmol/L) for ENT1. In adenosine receptor-binding experiments, ticagrelor and its major circulating metabolite, AR-C124910XX, had low affinity (Ki > 6 µmol/L) for each of the adenosine A1, A2A, and A2B receptors, whereas ticagrelor had a submicromolar (Ki = 190 nmol/L) affinity for the adenosine A3 receptor. However, in functional assays, at high concentrations (10 µmol/L) ticagrelor only partially inhibited 3 mmol/L adenosine-induced depolarizations in the guinea pig and rat vagus nerve preparations (by 35% and 49%, respectively). Ticagrelor inhibits cellular adenosine uptake selectively via ENT1 inhibition at concentrations of clinical relevance. However, the low-binding affinity and functional inhibition of adenosine receptors observed with ticagrelor or its metabolites indicate that they possess a negligible adenosine-like activity at clinically relevant concentrations.

Ticagrelor inhibits human platelet aggregation via adenosine in addition to P2Y12 antagonism

Ticagrelor, a P2Y12 antagonist, is an antiplatelet agent approved for the treatment of acute coronary syndromes; it also inhibits adenosine uptake by erythrocytes and other cells. To test whether ticagrelor inhibits platelet aggregation (PA) in whole blood (WB) by increasing the extracellular levels of adenosine, which inhibits PA via the A2A receptor. Collagen-induced PA was measured in WB or platelet-rich plasma (PRP) from 50 healthy subjects and two patients with inherited P2Y12 deficiency, in presence/absence of adenosine concentrations that by themselves marginally affected PA in WB, and ZM241385 (A2A antagonist). The effects of ticagrelor, the active metabolite of prasugrel (PAM) (P2Y12 antagonist), and dipyridamole (adenosine uptake inhibitor) on PA and on adenosine clearance in WB were compared. For PA in WB, adenosine contributed to drug-induced inhibition of PA; the adenosine contribution was similar for dipyridamole and ticagrelor but was significantly greater for ticagrelor than for PAM (P<0.01). For PA in PRP (no adenosine uptake by erythrocytes), adenosine contributed to inhibition of PA in the presence/absence of all tested drugs. ZM241385 reversed the inhibition by adenosine in WB and PRP. Similar results were obtained with WB and PRP from P2Y12 -deficient patients. Adenosine (7.1μmol L(-1) ) added to WB, was detectable for 0.5min in the presence of vehicle or PAM, for 3-6min in the presence of ticagrelor, and for >60min in the presence of dipyridamole. This study provides the first evidence of an additional antiplatelet mechanism by ticagrelor, mediated by the induced increase of adenosine levels.

Pharmacokinetic and Pharmacodynamic Effects of Prasugrel in Healthy Korean Males

Prasugrel is a thienopyridine for treatment of acute coronary syndromes in patients undergoing percutaneous coronary intervention. Higher concentrations of prasugrel's active metabolite (R-138727) have been observed in Asian than white subjects. The primary objective was to investigate pharmacokinetics of R-138727 in healthy Korean males. Thirty subjects were randomized (1:2) to a 60 or 30 mg loading dose, subsequently (1:1:1) to 10-, 7.5-, or 5-mg maintenance doses. R-138727 plasma concentrations were analyzed with liquid chromatography/mass spectrometry. Platelet aggregation was measured with Accumetrics VerifyNow. Mean (coefficient of variation) exposure to R-138727 was 600 ng·h/mL (16%) after 60 mg prasugrel and 283 ng·h/mL (17%) after 30 mg. After 10, 7.5, and 5 mg, mean exposures were 78.1 (24%), 58.4 (21%), and 38.3 ng·h/mL (24%). Pharmacokinetics were linear over this range. Daily 5 mg doses maintained a 65% (SD = 14.5%) inhibition of adenosine diphosphate-induced platelet aggregation; all other doses produced ≥90%. Prasugrel was well tolerated with no serious adverse events. Results are consistent with other studies of Asian subjects administered prasugrel. Although further guidance will be provided by a recently completed phase 3 study, these preliminary data suggest that dosing strategies approved for white patients with acute coronary syndromes are applicable to Asian patients.

P2Y 12 Receptor Blockade Augments Glycoprotein IIb‐IIIa Antagonist Inhibition of Platelet Activation, Aggregation, and Procoagulant Activity

BackgroundNew antiplatelet agents that provide greater, more consistent inhibition of the platelet ADP receptor P2Y12 may be used in combination with glycoprotein (GP) IIb‐IIIa antagonists, but their combined effect on platelet function and procoagulant activity is not well studied. Therefore, the objective of this study was to evaluate the independent and complementary effects of P2Y12 and GPIIb‐IIIa inhibition on platelet function and procoagulant activity.Methods and ResultsHealthy donor blood was treated with the active metabolite of prasugrel (R‐138727 5 μmol/L), GPIIb‐IIIa antagonists (abciximab 3 μg/mL or eptifibatide 0.9 μg/mL), and combinations thereof, exposed to physiologically relevant agonists (collagen and ADP) and then evaluated for markers of platelet activation and procoagulant activity. Significant interactions between R‐138727 and GPIIb‐IIIa antagonists were observed. R‐138727 and the GPIIb‐IIIa antagonists had additive inhibitory effects on collagen‐stimulated platelet aggregation and on the collagen plus ADP–stimulated level of activated platelet surface GPIIb‐IIIa. R‐138727 and abciximab each inhibited collagen plus ADP–stimulated platelet phosphatidylserine expression and prothrombin cleavage, and the combination produced greater inhibition than achieved with abciximab alone. In contrast, eptifibatide did not inhibit, but instead enhanced, collagen plus ADP–stimulated prothrombin cleavage. Addition of R‐138727 reduced prothrombin cleavage in eptifibatide‐treated samples, suggesting a novel mechanism for potential benefit from combined prasugrel and eptifibatide treatment.ConclusionsThe complementary effects of abciximab and R‐138727 on platelet activation, aggregation, and procoagulant activity suggest their combined use may, to a greater degree than with either agent alone, reduce thrombus formation in vivo.