Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: a new finding that may influence the design of anti-thrombotic agents

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca2+ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca2+ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y(1) receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca2+ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y(1) antagonist) and AR-C69931 (a P2Y(12) antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y(1) but also with partial antagonism at P2Y(12) receptors. Antagonism at P2Y(12) receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X(1) receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.     

Antagonism of P2Y12 reduces physiological thromboxane levels

Antiplatelet therapy for the management of patients with cardiovascular risks often includes a combination therapy of aspirin and clopidogrel, acting through inhibition of thromboxane generation and blockade of G_i-coupled P2Y₁₂ receptor, respectively. We hypothesized that ADP acting through P2Y₁₂ regulates physiological thromboxane levels. The serum thromboxane levels in mice (n?=?3) dosed with clopidogrel and prasugrel were decreased by 83.1?±?5.3% and 94.26?±?1.75% respectively compared to untreated mice. Pre-treatment of human blood (n?=?3) ex vivo with active metabolites of clopidogrel or prasugrel led to a reduction in thromboxane levels to 16.3?±?3.2% and 4.9?±?0.8% respectively, compared to untreated human serum. We also evaluated serum thromboxane levels in P2Y receptor null mice (n?=?4). Whereas serum thromboxane levels in P2Y₁ null mice were similar to those in wild type littermates, those in the P2Y₁₂ null mice were inhibited by 83.15?±?3.8%. Finally, in a pilot study, serum thromboxane levels were reduced by 76.05?±?8.41% in healthy human volunteers (n?=?6) upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y₁₂ antagonism alone can decrease physiological thromboxane levels. Thus, this study could pave way the for newer/modified treatment regimens for the management of patients with thrombotic complications who are allergic or non-responsive to aspirin.     

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y₁₂ and EP3 receptors, act as antagonists at G_i-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G_i-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y₁₂ antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA₂ mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI₂, iloprost, PGD₂, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE₁ and PGE₂ as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y₁₂ antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.     

Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395

The release of ADP from platelet dense granules and its binding to platelet P2Y₁₂ receptors is key to amplifying the initial hemostatic response and propagating thrombus formation. P2Y₁₂ has thus emerged as a therapeutic target to safely and effectively prevent secondary thrombotic events in patients with acute coronary syndrome or a history of myocardial infarction. Pharmacological inhibition of P2Y₁₂ receptors represents a useful approach to better understand the signaling mediated by these receptors and to elucidate the role of these receptors in a multitude of platelet hemostatic and thrombotic responses. The present work examined and compared the effects of four different P2Y₁₂ inhibitors (MRS2395, ticagrelor, PSB 0739, and AR-C 66096) on platelet function in a series of in vitro studies of platelet dense granule secretion and trafficking, calcium generation, and protein phosphorylation. Our results show that in platelets activated with the PAR-1 agonist TRAP-6 (thrombin receptor-activating peptide), inhibition of P2Y₁₂ with the antagonist MRS2395, but not ticagrelor, PSB 0739 or AR-C 66096, potentiated human platelet dense granule trafficking to the plasma membrane and release into the extracellular space, cytosolic Ca²⁺ influx, and phosphorylation of GSK3β-Ser9 through a PKC-dependent pathway. These results suggest that inhibition of P2Y₁₂ with MRS2395 may act in concert with PAR-1 signaling and result in the aberrant release of ADP by platelet dense granules, thus reducing or counteracting the anticipated anti-platelet efficacy of this inhibitor.     

The platelet P2Y12 receptor contributes to granule secretion through Ephrin A4 receptor

The main responses of P2Y₁ ligation are platelet shape change and transient aggregation while P2Y₁₂ ligation amplifies P2Y₁-induced aggregation and accelerates aggregation, secretion and thromboxane A₂ production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y_1/12 inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y_1/12-induced EphA4 phosphorylation, left P2Y₁-induced aggregation unchanged but inhibited with P2Y₁₂-induced secretion, second phase aggregation and thrombus formation on collagen at 1600?s^?1. These results show that platelet EphA4 is an important intermediate in P2Y₁₂-induced granule secretion.     

The active metabolite of prasugrel effectively blocks the platelet P2Y12 receptor and inhibits procoagulant and pro-inflammatory platelet responses

The aim of these studies was to investigate the extent of platelet P2Y₁₂ receptor inhibition by the thienopyridine active metabolite of prasugrel, R-138727. Blood was taken from healthy volunteers and pre-incubated with R-138727 or cangrelor (AR-C66931MX). Platelet aggregation was assessed in platelet rich plasma (PRP) and whole blood (WB). Vasodilator stimulated phosphoprotein (VASP) phosphorylation, platelet procoagulant activity (annexin V binding and microparticle formation) and calcium mobilisation were measured by flow cytometry. Platelet-leukocyte co-aggregate formation and sCD40L release, both pro-inflammatory responses of platelets, were measured by flow cytometry and ELISA, respectively. P2Y₁₂ receptor antagonism was determined using a radioligand binding assay (³³P 2-MeSADP) in resting and stimulated platelets and the effects of clopidogrel administration were also assessed. R-138727 yielded concentration-dependent inhibition of platelet aggregation, VASP phosphorylation inhibition, procoagulant activity and pro-inflammatory responses. In the presence of R-138727 or cangrelor there was increased calcium reuptake following agonist stimulation. R-138727 30 µmol/L and cangrelor 1 µmol/L completely inhibited ³³P 2-MeSADP binding, compared to partial inhibition following clopidogrel administration. Platelet activation and granule secretion did not expose an additional pool of P2Y₁₂ receptors. Prasugrel's active metabolite effectively blocks the P2Y₁₂ receptor with the highest concentrations tested yielding complete inhibition of P2Y₁₂-mediated amplification of several important platelet responses.     

Evolving pattern of platelet P2Y12 inhibition in patients with acute coronary syndromes

Abstract

Dual antiplatelet therapy consisting of clopidogrel in addition to aspirin has previously been the standard of care for patients with acute coronary syndromes (ACS) but international guidelines have been evolving over the last 4 years with the introduction of prasugrel and ticagrelor. In October 2009, prasugrel was approved in the UK by the National Institute of Health and Clinical Excellence (NICE) for use in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI), diabetic patients with non-ST-elevation (NSTE) ACS undergoing PCI and patients with stent thrombosis while other ACS patients were to continue receiving clopidogrel. Ticagrelor was approved in October 2011 by NICE for use in patients with moderate-to-high risk NSTE ACS and STEMI undergoing primary PCI and was recommended in preference to clopidogrel in European guidelines. These recommendations were adopted in our region, constituting a population of 1.8 million. We studied the effect of changing patterns of P2Y₁₂ inhibitor usage on levels of platelet inhibition during maintenance therapy. Patients admitted to Northern General Hospital, Sheffield, with NSTE ACS or STEMI managed with primary PCI were enrolled over two periods of time: May 2010 to November 2011 (T1); and October 2012 to February 2013 (T2). Venous blood samples were obtained at 1 month after the onset of ACS. Light transmittance aggregometry (LTA) was performed and maximum aggregation response to ADP 20?μM was determined. A total of 116 patients were enrolled in T1 of whom 82 were receiving clopidogrel and 34 were receiving prasugrel. Twenty-nine patients were enrolled in T2, all of whom were receiving ticagrelor. Mean LTA results according to treatment with clopidogrel, prasugrel and ticagrelor were 57?±?18%, 41?±?20%, and 31?±?12%, respectively. Prasugrel was associated with significantly lower platelet aggregation responses than clopidogrel (p?<?0.001) and ticagrelor was associated with significantly lower platelet aggregation responses than both prasugrel (p?=?0.015) and clopidogrel (p?<?0.001). We conclude that international guidelines and NICE approval have led to increasing levels of P2Y₁₂ inhibition in ACS patients in this UK centre between May 2010 and February 2013. Ticagrelor was associated with significantly greater P2Y₁₂ inhibition than both clopidogrel and prasugrel during maintenance therapy.     

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.     

Evaluation of the INNOVANCE PFA P2Y test cartridge: Sensitivity to P2Y12 blockade and influence of anticoagulant

Monitoring of platelet ADP receptor P2Y₁₂ inhibition may be performed by a variety of platelet function assays. Given the lack of sensitivity of the existing PFA-100® cartridge formulations to detect P2Y₁₂ inhibition, a new cartridge for the PFA-100 (INNOVANCE® PFA P2Y) has recently been developed. The performance of the new PFA-100 test cartridge was compared with standard collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges, light transmission aggregometry, vasodilator-stimulated phosphoprotein, the VerifyNow® P2Y₁₂ assay and multiple electrode aggregometry. In this study, 20 normal blood samples anticoagulated with either citrate or hirudin were spiked with two different clinically relevant concentrations (1 and 10?µM final concentration) of the prasugrel active metabolite (R-138727, Lilly/Daiichi Sankyo) for 30?min at 37°C. Comparison of the platelet function tests demonstrated that all tests (except CADP and CEPI) were substantially inhibited by 10?µM R-138727. Intermediate results were typically obtained with 1?µM R-138727 in citrated blood. However, both MEA ADP and ADPHS tests were highly sensitive to 1?µM R-138727 in hirudin anticoagulated blood. Further comparison of citrate or hirudin blood samples (N?=?5) revealed that all platelet tests (except CEPI) became more sensitive to 1?µM R-138727 in hirudinized blood. The INNOVANCE PFA P2Y cartridge proved to be sensitive to P2Y₁₂ inhibition and was comparable to other currently available platelet function tests. The sensitivity of all platelet function tests for detecting in vitro inhibition of P2Y₁₂ is markedly different depending on the anticoagulant used.     

Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y12 antagonist both in-vitro and ex-vivo in human volunteers

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E₂ (PGE₂) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3?µM) used alone or in combination with the P2Y₁₂ antagonist cangrelor (1?µM), both without and with aspirin (100?µM). Platelet aggregation and P-selectin expression were measured in whole blood (n?=?10) following stimulation with the thromboxane A₂ (TXA₂) mimetic U46619 (0.3 or 1?µM) in combination with either the EP3 agonist sulprostone (0.1?µM), or PGE₂ (1?µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n?=?42) were randomly assigned to receive no background treatment, clopidogrel (300?mg loading dose plus 75?mg daily) or clopidogrel and aspirin (75?mg daily) for 10 days alongside DG-041 (200?mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y₁₂ antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y₁₂ blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.     

Polymorphisms of MDR1, CYP2C19 and P2Y12 genes in Indian population: Effects on clopidogrel response

Aims/objectiveInfluence of genetic variations on the response of clopidogrel, an antiplatelet drug is implicated. In the present study, the prevalence of single nucleotide polymorphisms of MDR1 (C3435T), CYP2C19 [CYP2C19*2 CYP2C19*3, CYP2C19*17] and P2Y₁₂ (i-T744C) in Indian population and their effects on clopidogrel response was analyzed.Methods and resultsTo analyze the prevalence of polymorphisms, 102 healthy individuals were recruited. Clopidogrel response was assessed by ADP induced platelet aggregation in clopidogrel naïve acute myocardial infarction (AMI) patients (n = 26) screened from 100 AMI cases, before loading dose of 300 mg, at 24 h before next dose and 6 days after on 75 mg per day and platelet aggregation inhibition (PAI) was calculated between these time intervals. Genotyping was carried out by PCR-based restriction enzyme digestion method for C3435T of MDR1 and i-T744C of P2Y₁₂, by multiplex PCR for CYP2C19*2 (G681A) and CYP2C19*3 (G636A) and by nested PCR for CYP2C19*17 (C806T). The effect of the above mentioned genetic variations on PAI was analyzed. Variant allele of CYP2C19*3 was not observed while the prevalence of 3435T of MDR1 (0.524), CYP2C19*2 (681A, 0.352); i-744C of P2Y₁₂ (0.088), as well as wild type allele CYP2C19*17 (C806, 0.897) associated with decrease clopidogrel response were observed. Trend toward poor response to clopidogrel was observed at 24 h with the variant genotypes of CYP2C19*2 and i-T744C of P2Y₁₂ as compared to wild type.ConclusionThe present study did show a trend toward impaired response of clopidogrel to inhibit platelet aggregation with variant genotypes of CYP2C19*2 and iT744C of P2Y₁₂ compared to respective wild type genotype at 24 h.     

Dyspnea in patients treated with P2Y12 receptor antagonists: insights from the GReek AntiPlatElet (GRAPE) registry

In ‘real life’ acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) and receiving contemporary antiplatelet treatment, data on dyspnea occurrence and impact on persistence with treatment are scarce. In a prospective, multicenter, cohort study, ACS patients undergoing PCI were recruited into the GReekAntiPlatElet (GRAPE) registry. During 1-year follow up, overall, 249/1989 (12.5%) patients reported dyspnea, more frequently at 1-month and decreasing thereafter. Multivariate analysis showed that ticagrelor administration (n = 738) at discharge was associated with the occurrence of dyspnea: Odds ratio 2.46 (95% confidence interval, CI, 1.87–3.25), p < 0.001. Older age, lower hematocrit, and prior bleeding event were also associated with dyspnea reports. Persistence, switching, and cessation rates were 68.3%, 20.9%, and 10.8% vs 76.7%, 12.5%, and 10.9% among patients reporting dyspnea compared with those who did not, p for trend = 0.002. In conclusion, in ACS patients undergoing PCI and treated with a P2Y₁₂ receptor antagonist, dyspnea occurs commonly, particularly when ticagrelor is administered. Non-persistence with antiplatelet agents at discharge is more frequently observed among dyspnea-reporters.     

Advances in the monitoring of anti-P2Y12 therapy

Although there are many new and effective anti-P2Y₁₂ drugs available, clopidogrel in its original or generic forms will probably remain the most widely used and cheaper option. As clopidogrel is a pro-drug, there is marked heterogeneity in drug responsiveness between individuals and lack of responsiveness is associated with poorer clinical outcomes. Various platelet function and genetic tests are now available for potentially measuring whether clopidogrel effectively inhibits platelet function. Monitoring of P2Y₁₂ inhibition and/or identification of loss of function cytochrome P-450 genotypes could, therefore, offer the potential of tailoring therapy by identifying poor responders to clopidogrel and optimizing the levels of platelet inhibition using, for example, alternative drugs such as prasugrel or ticagrelor. The question remains whether any of these tests have prognostic utility with a defined therapeutic window to reliably identify hypo or hyper-responsive patients who may have an increased risk of thrombosis or bleeding, respectively? Once such patients are identified, can the tests then be subsequently used to demonstrate a change or improvement in platelet reactivity by using alternative therapies and equate this with improved clinical outcome? In this review, we describe an overview of the current platelet and genetic tests available and discuss whether these tests will ever become used routinely.     

Rapid and reversible modulation of platelet function in man by a novel P2Y12 ADP-receptor antagonist,INS50589

P2Y₁₂ receptors participate in ADP-induced activation and aggregation of human platelets. INS50589, a selective P2Y₁₂ receptor antagonist, is being developed for use where controlled, reversible modulation of the platelet hemostatic function is needed. The tolerability, pharmacokinetics, and pharmacodynamics of INS50589 were tested in healthy human volunteers. Thirty-six subjects received intravenous infusions of placebo or INS50589 at 0.1–3?mg/kg/h for four hours. Platelet function, clotting parameters, bleeding time, safety assessments, and plasma concentrations of INS50589 and its major metabolite were monitored for 24 hours. Near-steady state plasma concentrations of INS50589 and effects on platelet function were achieved rapidly. The average maximal plasma concentration of INS50589 was linearly related to the dose administered. Intravenous INS50589 produced dose-dependent inhibition of platelet activation and aggregation in response to ADP in vitro until nearly full inhibition was achieved at the higher doses. Bleeding time was correspondingly increased, without any effect on activated clotting time, prothrombin time, or activated partial thromboplastin time. Platelet response to ADP had returned to at least 75% of the baseline value within 0.25–4?h after cessation of the intravenous infusion of INS50589, depending upon the dose and ADP challenge concentration. Infusions were well tolerated up to the highest dose tested. There was no evidence that the principal metabolite (INS51088) contributed to these effects. INS50589 is a well-tolerated, reversible, competitive antagonist of ADP at the P2Y₁₂ human platelet receptor, and its potential therapeutic utility in various cardiovascular settings is discussed.     

Meta-Analysis of Short vs. Prolonged Dual Antiplatelet Therapy after Drug-Eluting Stent Implantation and Role of Continuation with either Aspirin or a P2Y12 Inhibitor Thereafter

Aim: The optimal duration of dual antiplatelet therapy (DAPT) after drug-eluting stent (DES) implantation is an ongoing debate and novel data has emerged. The aim of this meta-analysis was to assess outcomes of short vs. control DAPT duration. In addition, the role of single antiplatelet therapy (SAPT) after DAPT with either aspirin or P2Y₁₂ inhibitor monotherapy was analyzed. Methods: The authors searched MEDLINE and Cochrane databases and proceedings of international meetings for randomized controlled trials (RCT) comparing ≤ 3 months with ≥ 6 months DAPT after DES implantation. The primary and co-primary outcomes of interest were definite or probable stent thrombosis (ST) and bleeding. In addition, we performed an analysis on studies who continued with either aspirin or P2Y₁₂ monotherapy after DAPT. Results: 9 RCTs comprising 41,864 patients were included and we analyzed a short DAPT duration of median 1.5 months vs. 12.1 months in the control group. The risk for ST was similar with short vs. control DAPT duration (0.5 vs. 0.5%; hazard ratio 1.17[95% CI 0.89-1.54];p=0.26). Bleeding was significantly reduced with short vs. control DAPT duration (1.9 vs. 3.0%; 0.65[0.54-0.77];p＜0.0001). ST was not different between short vs. control DAPT duration in the analysis of the 4 RCTs who continued with aspirin after DAPT and the 5 P2Y₁₂ RCTs, respectively, and no heterogeneity was detected (p=0.861). Bleeding was also reduced with short vs. control DAPT in both the aspirin (1.2 vs. 1.7%; 0.71[0.51-0.99];p=0.04) and P2Y₁₂ inhibitor studies (2.1 vs. 3.4%; 0.62[0.47-0.80];p=0.0003) and no heterogeneity was detected (p=0.515). Conclusions: Our meta-analysis shows that short DAPT ≤ 3 months followed by SAPT reduces bleeding and is not associated with an increase in ST. The results were consistent within the aspirin and P2Y₁₂ SAPT studies.     

Denufosol: A review of studies with inhaled P2Y2 agonists that led to Phase 3

Among the most promising of the new therapies being developed for the treatment of Cystic Fibrosis (CF) are those targeted at increasing mucosal hydration on the surface of the airways. One of these therapies, P2Y₂ receptor agonists, bypasses the defective CFTR chloride channel, and activates an alternative chloride channel. This activation results in an increase in airway surface epithelial hydration, and through these actions and effects on cilia beat frequency, increases mucociliary clearance. The pharmacology of P2Y₂ agonists has been confirmed in several preclinical and clinical studies. Denufosol tetrasodium is a novel second-generation, metabolically stable, selective P2Y₂ receptor agonist currently in Phase 3 clinical development. In radiolabelled deposition studies of P2Y₂ agonists in healthy non-smokers and smokers, approximately 7 mg of a 40-mg nebulizer (PARI LC Star) load was deposited in the lungs. In a pharmacokinetic study in healthy volunteers, very limited systemic exposure was observed when doses of 200 mg of denufosol were nebulized. Thus, it appears that high concentrations of denufosol can be achieved in the airways with very low systemic absorption. Denufosol has been generally well-tolerated in healthy volunteers and patients with CF. The most common adverse events were in the respiratory system, with cough having the highest frequency. Doses of 20–60 mg have been evaluated in Phase 2 trials of up to 28 days duration, and superiority relative to placebo on FEV1 has been observed in patients with relatively normal lung function (FEV1 greater than or equal to 75% of predicted). The first Phase 3 trial is a comparison of denufosol 60 mg and placebo in 350 patients with CF with FEV1 at study entry greater than or equal to 75% of predicted.     

Poor agreement between light transmission aggregometry,Verify Now P2Y12 and vasodilatator-stimulated phosphoprotein for clopidogrel low-response assessment: A potential explanation of negative results of recent randomized trials

Abstract

Clopidogrel low response as assessed by several different biological tests correlates with poor prognosis after percutaneous coronary intervention (PCI). However, recent randomized clinical trials (RCT) testing the strategy of individual antiplatelet therapy tailoring based on one sole test have all shown negative results. Poor correlation between the different tests may explain the difficulties of patient selection and identification of “true poor responders” to clopidogrel. In this prospective study, clopidogrel response was assessed in 100 consecutive patients between 18 and 24 hours after a 600?mg clopidogrel loading dose using three different tests: light transmission aggregometry with 10?μmol ADP (LTA, results expressed as platelet aggregation percentage: PAP), Verify Now P2Y₁₂ (VN, results expressed as P2Y₁₂ reaction unit: PRU) and vasodilatator-stimulated phosphoprotein (VASP, results expressed as platelet reactivity index: PRI). Patients under chronic clopidogrel therapy were excluded. The mean PAP, PRU and PRI values were 38.6%, 176.1 PRU and 52.4%, respectively. When clopidogrel response was analyzed as continuous variable, there was a good correlation between the different tests: LTA/VN (R^2?=?0.642, p?<?0.001), LTA/VASP (R^2?=?0.409, p?<?0.001) and VN/VASP (R^2?=?0.616, p?<?0.001). However, when clopidogrel response was analyzed as pre-specified cut-off points to define patients as “poor or good responders” (according to the literature: 50% PAP for LTA, 235 PRU for VN and 50% PRI for VASP), only 47% of the patients were defined as “good” or “poor responders” by the three tests. Altogether, 33% of the patients were defined as “poor responders” by only one test, 20% by two tests and only 16% by the three tests. The correlation between the different tests is good when clopidogrel response is analyzed as continuous variable. Each individual is however rarely (less than 50%) defined as “poor or good responder” by all the three tests when pre-specified cut-off values are used. A sole test might not be sufficient to manage antiplatelet therapy in an individual patient and these results may explain the results of recent RCT showing the lack of benefit of systematic antiplatelet therapy monitoring strategy.