Importance of potent P2Y12 receptor blockade in acute myocardial infarction: focus on prasugrel

Introduction: Angiogenesis plays a crucial role in the proliferation and in the metastatic spread of tumour cells. Several agents with anti-angiogenic activity have been tested in advanced non-small-cell lung cancer (NSCLC) patients. Motesanib (AMG 706) is a promising anti-angiogenic multi-targeted tyrosine kinase inhibitor (TKI), which has been investigated as a monotherapy or in combination with chemotherapy, in several types of cancer.

Areas covered: We have reviewed the literature, and we have presented the results of clinical trials that have investigated the administration of motesanib in advanced NSCLC patients.

Expert opinion: Encouraging results have been described with the administration of motesanib as first-line treatment in combination with carboplatin and paclitaxel in Asian patients with non-squamous advanced NSCLC. Further studies are needed in order to identify the predictive biomarkers of response and to select patients who may benefit from this anti-angiogenic treatment.     

Selective blockade of P2Y12 receptors by prasugrel inhibits myocardial infarction induced by thrombotic coronary artery occlusion in rats

We evaluated the effects of prasugrel, a third-generation thienopyridyl prodrug, on P2Y12 receptors, adenosine 5'-diphosphate (ADP)-induced platelet aggregation, and myocardial infarction (MI) in rats. Oral administration of prasugrel (0.3-3 mg/kg) resulted in the dose-related inhibition of washed platelet aggregation induced by ADP (1-10 μM). Ex vivo [H]-2-MeS-ADP binding to platelet P2Y12 receptors was also inhibited by prasugrel in a similar dose range. The antiaggregatory effects of prasugrel correlated strongly with P2Y12 blockade with correlation coefficients of 0.85-0.92, suggesting that the antiaggregatory activity of prasugrel largely reflected P2Y12 blockade achieved in vivo. We further examined the effects of the in vivo P2Y12 inhibition by prasugrel (1-10 mg/kg, po) on MI induced by thrombotic coronary artery occlusion in rats. In surviving rats, infarct size at 24 hours after photoirradiation was evaluated. In the vehicle group, necrosis area/total left ventricular area was 37.9% ± 6.8% (mean ± SE, n = 7). At all prasugrel doses tested (n = 7 for each dose), necrosis area/total left ventricular area was significantly smaller than that in the vehicle group: 14.4% ± 4.0% for 1 mg/kg (P < 0.01), 19.8% ± 4.5% for 3 mg/kg (P < 0.05), and 14.8% ± 3.6% for 10 mg/kg (P < 0.01). At the highest administered dose of prasugrel (10 mg/kg), blood pressure and heart rate were unchanged. Arrhythmia was observed in 5 of 7 animals in the vehicle group at 24 hours after irradiation; in contrast, no arrhythmia was found in the group treated with prasugrel (10 mg/kg). Taken together, these results demonstrate that prasugrel is a selective P2Y12 inhibitor in vivo, providing effective inhibition of platelet aggregation and MI in rats.     

Effects of neuropeptide Y2 receptor blockade on ventricular arrhythmias in rats with acute myocardial infarction

Excessive sympathetic activity is believed to be the key arrhythmogenic factor both in the setting of acute myocardial infarction and during chronic heart failure. The aim of this study was to evaluate the effect of neuropeptide Y2 blockade on malignant ventricular arrhythmias in rats with acute myocardial infarction. Vagotonic dose-finding study for neuropeptide Y2 receptor antagonist, (S)-N2-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl] cylopentyl] acetyl]-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamid (AR-H05359) was conducted in guinea pigs (n=50) and rats (n=3). Induction of postinfarction arrhythmias was conducted in Sprague-Dawley rats that were randomized into 3 groups. Neuropeptide Y2 antagonist treated rats (n=7), placebo group (n=10) and amiodarone treated rats (n=8). Myocardial infarction was induced by ligation of the left coronary artery. Computerized telemetric ECG tracings were obtained continuously before induction of myocardial infarction and up to 120 min postinfarction. Occurrence of ventricular arrhythmias was analyzed according to a 10-point arrhythmia score. There was no difference in the arrhythmia scores between the neuropeptide Y2 and the saline group. The amiodarone treated animals had lower score compared to the neuropeptide Y2 and the placebo group (p<0.05). The blockade of receptors does not reduce ventricular arrhythmias in the rats with acute myocardial infarction. Further studies are needed to evaluate whether increasing vagal tonus during sympathetic activation may be valuable anti-arrhythmic strategy to prevent sudden death in patients with myocardial infarction and heart failure.     

The influence of P2Y12 receptor deficiency on the platelet inhibitory activities of prasugrel in a mouse model: evidence for specific inhibition of P2Y12 receptors by prasugrel

Prasugrel is a novel orally active thienopyridine with faster, higher and more reliable inhibition of platelet aggregation than clopidogrel reflecting its metabolism in vivo to an active metabolite with selective P2Y(12) antagonistic activity. Several lines of evidence support the contention that prasugrel provides selective P2Y(12) receptor antagonistic activity. To date, however, direct evidence of P2Y(12) specific action by prasugrel in vivo is limited. In the present study, effects of prasugrel on ex vivo platelet aggregation were examined in wild type (WT) and P2Y(12)(-/-) mice. In WT mice, prasugrel showed platelet inhibition that was 8.2 times more potent than clopidogrel. In P2Y(12)(-/-) mice, ADP induced platelet aggregation was minimal, and its extent was similar to that in prasugrel-treated WT mice. In addition, no further inhibition of platelet aggregation was observed after administration of prasugrel to P2Y(12)(-/-) mice. Furthermore, prasugrel-treated WT mice showed similar aggregation patterns using collagen- and murine PAR-4 agonist peptide to those of P2Y(12)(-/-) mice treated with vehicle or prasugrel. Overall, these results clearly provide additional in vivo evidence that prasugrel has selective P2Y(12) antagonistic activity.     

Purinergic P2Y12 receptor blockade inhibits shear-induced platelet phosphatidylinositol 3-kinase activation

Pathologically elevated shear stress triggers aspirin-insensitive platelet thrombosis. Signaling mechanisms involved in shear-induced platelet thrombosis are not well understood. To investigate these, we examined the hypothesis that functionally important platelet phosphatidylinositol 3-kinase (PI3-K) activity is stimulated by an in vitro shear stress of 120 dynes/cm(2) (shear rate of 6,000 sec(-1)). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) production was examined in washed human platelets subjected to pathological shear stress in a cone-plate viscometer. PIP(3) production peaks 30 s after shear begins and is initiated by von Willebrand factor (VWF) binding to the glycoprotein (Gp) Ib-IX-V complex. Inhibiting PI3-K with wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) results in the inhibition of shear-induced platelet aggregation. In resting platelets, class IA PI3-K associates with the tyrosine kinase Syk. Within 30 s of beginning shear, PI3-K-associated Syk becomes tyrosine phosphorylated. Inhibiting Syk activation with piceatannol results in the inhibition of PIP(3) production and aggregation. Selective blockade of the P2Y(12) receptor results in the inhibition of Syk phosphorylation, PIP(3) production, and aggregation. These results indicate that shear-induced VWF binding to platelet GpIb-IX-V stimulates functionally important PI3-K activity. PI3-K activation is signaled by rapid feedback amplification that involves P2Y(12) receptor-mediated activation of Syk.     

Repeat oral dosing of prasugrel, a novel P2Y12 receptor inhibitor, results in cumulative and potent antiplatelet and antithrombotic activity in several animal species

Antiplatelet and antithrombotic activity of multiple oral dosing of prasugrel were evaluated in several animal species. Prasugrel's active metabolite concentration-relatedly inhibited in vitro ADP-induced aggregation of rat, rabbit, dog, monkey and human platelets. Oral administration of prasugrel to dogs (0.03-0.3 mg/kg/day) and monkeys (0.1 and 0.3 mg/kg/day) once a day for 14 days resulted in potent, dose-related and cumulative inhibition of ADP-induced platelet aggregation. The inhibitory effects reached a plateau on days 3 to 5 and thereafter were maintained during dosing. Inhibition decreased gradually after cessation of dosing with near full recovery by 7 days after last dose. Antiplatelet and antithrombotic activity of prasugrel and clopidogrel were further examined in rats. Multiple oral dosing of prasugrel (0.3-3 mg/kg/day) to rats resulted in more potent inhibition of platelet aggregation compared to clopidogrel (3-30 mg/kg/day) and ticlopidine (30-300 mg/kg/day). Separate experiments confirmed that platelet inhibition was associated with inhibition of [(3)H]-2-methylthio-ADP binding to rat platelets. In a rat model of electrically-induced arterial thrombosis, prasugrel (0.1-1 mg/kg/day, p.o.) significantly prolonged the time to arterial occlusion and increased the duration of arterial patency. The inhibition of platelet aggregation of prasugrel was about 10 and 300 times more potent than clopidogrel and ticlopidine, respectively. Overall these results show that in several species multiple oral administration of prasugrel results in more potent inhibition of platelet aggregation and thrombus formation than clopidogrel and ticlopidine, and that these effects are mediated by inhibition of platelet ADP receptors.     

Pharmacogenetics of P2Y12 receptor inhibitors

Oral P2Y₁₂ inhibitors are commonly prescribed for cardiovascular disease and include clopidogrel, prasugrel, and ticagrelor. Each of these drugs has its strengths and weaknesses. Prasugrel and ticagrelor are more potent inhibitors of platelet aggregation and were shown to be superior to clopidogrel in preventing major adverse cardiovascular events after an acute coronary syndrome and percutaneous coronary intervention (PCI) in the absence of genotyping. However, both are associated with an increased risk for non-coronary artery bypass-related bleeding. Clopidogrel is a prodrug requiring bioactivation, primarily via the CYP2C19 enzyme. Approximately 30% of individuals have a CYP2C19 no function allele and decreased or no CYP2C19 enzyme activity. Clopidogrel-treated carriers of a CYP2C19 no function allele have decreased exposure to the clopidogrel active metabolite and lesser inhibition of platelet aggregation, which likely contributed to reduced clopidogrel efficacy in clinical trials. The pharmacogenetic data for clopidogrel are most robust in the setting of PCI, but evidence is accumulating for other indications. Guidance is available from expert consensus groups and regulatory agencies to assist with integrating genetic information into P2Y₁₂ inhibitor prescribing decisions, and CYP2C19 genotype-guided antiplatelet therapy after PCI is one of the most common examples of clinical pharmacogenetic implementation. Herein, we review the evidence for pharmacogenetic associations with clopidogrel response and outcomes with genotype-guided P2Y₁₂ inhibitor selection and describe guidance to assist with pharmacogenetic implementation. We also describe processes for applying genotype data for P2Y₁₂ inhibitor therapy selection and remaining gaps in the field. Ultimately, consideration of both clinical and genetic factors may guide selection of P2Y₁₂ inhibitor therapy that optimally balances the atherothrombotic and bleeding risks.     

Cangrelor: a novel P2Y12 receptor antagonist

Antiplatelet therapy is critical in the prevention of thrombotic complications of acute coronary syndrome and percutaneous coronary interventions. Current antiplatelet agents (aspirin, clopidogrel and glycoprotein IIb/IIIa antagonists) have demonstrated the capacity to reduce major adverse cardiac events. However, these agents have limitations that compromise their clinical utility. The platelet P2Y₁₂ receptor plays a central role in platelet function and is a focus in the development of antiplatelet therapies. Cangrelor is a potent, competitive inhibitor of the P2Y₁₂ receptor that is administered by intravenous infusion and rapidly achieves near complete inhibition of ADP-induced platelet aggregation. This investigational drug has been studied for use during coronary procedures and the management of patients experiencing acute coronary syndrome and is undergoing evaluation for use in the prevention of perioperative stent thrombosis.     

新型血小板P2Y12受体拮抗药

抑制ADP诱导的血小板聚集的药物(如氯吡格雷)已成为目前心血管领域内最重要的抗血小板药物。但氯吡格雷起效较慢,抗血小板作用相对较弱,尚有一定的低反应者和无反应者。普拉格雷是一个较新的血小板P2Y12受体拮抗药。TRITON-TIMI 38研究表明普拉格雷可使主要终点发生率(心血管死亡、非致死性心肌梗死、非致死性卒中)降低19%,但使出血并发症增加。坎格雷洛,替格雷洛,依诺格雷是更新的血小板P2Y12受体拮抗药。     

The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.     

P2 Y12受体拮抗剂对脓毒症大鼠血小板功能和急性肾损伤影响的实验研究

目的：观察血小板P2Y12受体拮抗剂氯吡格雷对脓毒症大鼠血小板功能和急性肾损伤（AKI）的影响。方法36只雄性Wistar大鼠随机分为正常对照组（NC组）、急性肾损伤组（AKI组）、氯吡格雷组（CL组）,每组12只。AKI组、CL组分别腹腔注射内毒素（LPS）10 mg·kg－1建模;CL组在LPS注射后立即给予氯吡格雷800 mg·kg－1灌胃;建模后3 h,测定各组血小板CD62P的阳性表达水平及血小板聚集率,并检测血肌酐、尿素氮、TNF-α和IL-6的水平,取肾组织HE染色进行病理组织学观察。结果 AKI组血小板CD62P的表达水平明显高于NC组（P＜0．05）,而CL组则明显低于AKI组（P＜0．05）。AKI组血小板聚集率明显高于NC组,CL组血小板聚集率较AKI组明显降低且低于NC组（P＜0．05）。AKI组、CL组大鼠血清TNF-α和IL-6、血肌酐、尿素氮水平均高于NC组（P＜0．05）,而CL组上述指标均明显低于AKI组（P＜0．05）。AKI组大鼠肾组织病理损伤显著,CL组肾损伤程度较AKI组明显减轻。结论血小板参与AKI的发病机制,血小板P2Y12受体拮抗剂氯吡格雷可减轻内毒素诱导的肾组织的损伤,改善肾功能,其机制可能与抑制脓毒症大鼠血小板的过度活化和聚集,下调炎症因子的表达有关。     

P2Y12 receptor blockade synergizes strongly with nitric oxide and prostacyclin to inhibit platelet activation

AimsIn vivo platelet function is a product of intrinsic platelet reactivity, modifiable by dual antiplatelet therapy (DAPT), and the extrinsic inhibitory endothelial mediators, nitric oxide (NO) and prostacyclin (PGI₂), that are powerfully potentiated by P2Y₁₂ receptor blockade. This implies that for individual patients endothelial mediator production is an important determinant of DAPT effectiveness. Here, we have investigated this idea using platelets taken from healthy volunteers treated with anti‐platelet drugs.MethodsThree groups of male volunteers (n = 8) received either prasugrel (10 mg), aspirin (75 mg) or DAPT (prasugrel + aspirin) once daily for 7 days. Platelet reactivity in the presence of diethylammonium (Z)‐1‐(N,N‐diethylamino)diazen‐1‐ium‐1,2‐diolate (DEA/NONOate) and PGI₂ was studied before and following treatment.ResultsEx vivo, PGI₂ and/or DEA/NONOate had little inhibitory effect on TRAP‐6‐induced platelet reactivity in control conditions. However, in the presence of DAPT, combination of DEA/NONOate + PGI₂ reduced platelet aggregation (74 ± 3% to 19 ± 6%, P < 0.05). In vitro studies showed even partial (25%) P2Y₁₂ receptor blockade produced a significant (67 ± 2% to 39 ± 10%, P < 0.05) inhibition when DEA/NONOate + PGI₂ was present.ConclusionsWe have demonstrated that PGI₂ and NO synergize with P2Y₁₂ receptor antagonists to produce powerful platelet inhibition. Furthermore, even with submaximal P2Y₁₂ blockade the presence of PGI₂ and NO greatly enhances platelet inhibition. Our findings highlight the importance of endothelial mediator in vivo modulation of P2Y₁₂ inhibition and introduces the concept of refining ex vivo platelet function testing by incorporating an assessment of endothelial function to predict thrombotic outcomes better and adjust therapy to prevent adverse outcomes in individual patients.     

抗血小板膜P2Y12受体拮抗剂的研究进展

抗血小板药物是治疗急性冠脉综合征的必备用药,其中抗血小板膜P2Y12受体拮抗剂已成为急性冠脉综合征药物治疗研究中最活跃的一个领域,从基础到临床研究,不断地开发出系列新药.本文就对P2Y12受体拮抗剂作用机制、药代动力学、急性冠脉综合征临床研究、临床指南等进行分析,为临床合理用药提供参考.     

P2Y12 receptor antagonists in acute coronary syndrome: clinical implications of pharmacologic and pharmacogenetic differences

Platelet activation and aggregation are key events in the pathophysiological process of thrombosis, and vascular occlusions. Antiplatelet therapy has proven to be crucial for managing patients with acute coronary syndromes, coronary artery disease and in patients undergoing percutaneous coronary interventions. However, residual platelet reactivity on antiplatelet treatment confers a five-fold increased risk of major adverse cardiovascular events which indicates a need for more effective antiplatelet medications to address the substantial burden of cardiovascular disease. This article reviews the P2Y(12) receptor antagonists with regards to pharmacologic and pharmacogenetic differences and their clinical implications along with the discussion of recent patents.     

Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor

Human platelets express two P2Y receptors: G(q)-coupled P2Y(1), and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC(50) of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist.     

Effects of dual endothelin receptor blockade on sympathetic activation and arrhythmogenesis during acute myocardial infarction in rats

The effects of dual (ETA and ETB) endothelin receptor blockade on ventricular arrhythmogenesis during acute myocardial infarction are not well defined. We randomly allocated Wistar rats to bosentan (100 mg/kg daily, n=24), a dual endothelin receptor antagonist, or vehicle (n=23). After 7 days of treatment, myocardial infarction was induced by permanent coronary ligation. Ventricular tachyarrhythmias were evaluated for 24 h following ligation, using a miniature telemetry electrocardiogram recorder. Action potential duration was measured from monophasic epicardial recordings and sympathetic activation was assessed by heart rate variability and catecholamine serum level measurements. Compared to controls (1012+/-185 s), bosentan (59+/-24 s) markedly decreased (P<0.00001) the total duration of ventricular tachyarrhythmias during the delayed (1-24 h) phase post-ligation, with a modest effect during the early (0-1 h) phase (132+/-38 s, versus 43+/-18 s, respectively, P=0.053). Treatment did not affect infarct size or total mortality. Action potential duration at 90% repolarization prolonged in controls (from 93.1+/-4.7 ms to 117.6+/-6.9 ms), displaying increased temporal dispersion (from 4.14+/-0.45 ms to 10.42+/-2.51 ms, both P<0.001), but was preserved in treated animals. Bosentan decreased norepinephrine, but increased epinephrine levels 24 h post-ligation. Low frequency spectra of heart rate variability, an index of net sympathetic tone, were lower in bosentan-treated rats. Dual endothelin-1 receptor blockade decreases ventricular tachyarrhythmias during myocardial infarction without reperfusion, by preventing repolarization inhomogeneity. Diverse treatment effects on sympathetic activation may ameliorate the antiarrhythmic action.     

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.     

Angiotensin receptor blockers in acute myocardial infarction

It has been established from multiple randomised, placebo-controlled trials that angiotensin-converting enzyme inhibitors reduce the risk of death and major non-fatal cardiovascular events after acute myocardial infarction, especially when these agents are used for long-term treatment in high-risk patients. Angiotensin receptor blockers represent a theoretically appealing class of drugs for use in combination with or as alternatives to angiotensin-converting enzyme inhibitors in such patients. The Valsartan in Acute Myocardial Infarction (VALIANT) trial compared the effects of valsartan, captopril and the combination of both in a population of high-risk patients with evidence of left ventricular dysfunction after acute myocardial infarction. This article discusses this important study and its implications for the clinical management of these high-risk patients.