Sven Nylander

Adenosine-mediated effects of ticagrelor: evidence and potential clinical relevance.

This review constitutes a critical evaluation of recent publications that have described an additional mode of action of the P2Y12 receptor antagonist ticagrelor. The effect is mediated by inhibition of the adenosine transporter ENT1 (type 1 equilibrative nucleoside transporter), which provides protection for adenosine from intracellular metabolism, thus increasing its concentration and biological activity, particularly at sites of ischemia and tissue injury where it is formed. Understanding the mode of action of ticagrelor is of particular interest given that its clinical profile, both in terms of efficacy and adverse events, differs from that of thienopyridine P2Y12 antagonists.

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

Introduction: 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. Methods: Inhibition of nucleoside transporter-mediated [3H]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. Results: Ticagrelor (and less effectively its metabolites) and the main cangrelor metabolite inhibited [3H]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). Conclusions: 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 Enhances Adenosine-Induced Coronary Vasodilatory Responses in Humans.

OBJECTIVES This study was undertaken to determine if ticagrelor augments adenosine-induced coronary blood flow and the sensation of dyspnea in human subjects. BACKGROUND Ticagrelor is a P2Y(12) receptor antagonist that showed superior clinical benefit versus clopidogrel in a phase III trial (PLATO [Platelet Inhibition and Patient Outcomes]). Ticagrelor has been shown to inhibit cell uptake of adenosine and enhance adenosine-mediated hyperemia responses in a dog model. METHODS In this double-blind, placebo-controlled study, 40 healthy male subjects were randomized to receive a single dose of ticagrelor (180 mg) or placebo in a crossover fashion. Coronary blood flow velocity (CBFV) was measured by using transthoracic Doppler echocardiography at rest after multiple stepwise adenosine infusions given before and after study drug, and again after the infusion of theophylline. RESULTS Ticagrelor significantly increased the area under the curve of CBFV versus the adenosine dose compared with placebo (p = 0.008). There was a significant correlation between ticagrelor plasma concentrations and increases in the area under the curve (p < 0.001). In both treatment groups, the adenosine-induced increase in CBFV was significantly attenuated by theophylline, with no significant differences between subjects receiving ticagrelor or placebo (p = 0.39). Furthermore, ticagrelor significantly enhanced the sensation of dyspnea during adenosine infusion, and the effects were diminished by theophylline. CONCLUSIONS Ticagrelor enhanced adenosine-induced CBFV and the sensation of dyspnea in these healthy male subjects via an adenosine-mediated mechanism. (Study to Assess the Effect of Ticagrelor on Coronary Blood Flow in Healthy Male Subjects; NCT01226602).

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.

Human target validation of phosphoinositide 3‐kinase (PI3K)β: effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor

Nylander S, Kull B, Björkman JA, Ulvinge JC, Oakes N, Emanuelsson BM, Andersson M, Skärby T, Inghardt T, Fjellström O, Gustafsson D. Human target validation of phosphoinositide 3‐kinase (PI3K)β:effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kβ inhibitor. J Thromb Haemost 2012; 10: 2127–36. See also Jackson SP, Schoenwaelder SM. Antithrombotic phosphoinositide 3‐kinase β inhibitors in humans – a ‘shear’ delight! This issue, pp 2123–6.

Anti‐platelet therapy: cyclo‐oxygenase inhibition and the use of aspirin with particular regard to dual anti‐platelet therapy

Aspirin and P2Y(12) antagonists are commonly used anti-platelet agents. Aspirin produces its effects through inhibition of thromboxane A(2) (TXA(2)) production, while P2Y(12) antagonists attenuate the secondary responses to ADP released by activated platelets. The anti-platelet effects of aspirin and a P2Y(12) antagonist are often considered to be separately additive. However, there is evidence of an overlap in effects, in that a high level of P2Y(12) receptor inhibition can blunt TXA(2) receptor signalling in platelets and reduce platelet production of TXA(2). Against this background, the addition of aspirin, particularly at higher doses, could cause significant reductions in the production of prostanoids in other tissues, e.g. prostaglandin I(2) from the blood vessel wall. This review summarizes the data from clinical studies in which dose-dependent effects of aspirin on prostanoid production have been evaluated by both plasma and urinary measures. It also addresses the biology underlying the cardiovascular effects of aspirin and its influences upon prostanoid production throughout the body. The review then considers whether, in the presence of newer, more refined P2Y(12) receptor antagonists, aspirin may offer less benefit than might have been predicted from earlier clinical trials using more variable P2Y(12) antagonists. The possibility is reflected upon, that when combined with a high level of P2Y(12) blockade the net effect of higher doses of aspirin could be removal of anti-thrombotic and vasodilating prostanoids and so a lessening of the anti-thrombotic effectiveness of the treatment.

Chronic treatment with ticagrelor limits myocardial infarct size: An adenosine and cyclooxygenase-2-dependent effect

Objective— In a phase III clinical trial (PLATelet inhibition and patient Outcomes, PLATO), ticagrelor provided better clinical outcomes than clopidogrel in patients with acute coronary syndromes. In addition to P2Y12-receptor antagonism, ticagrelor prevents cell uptake of adenosine and has proven able to augment adenosine effects. Adenosine protects the heart against ischemia–reperfusion injury. We compared the effects of clopidogrel and ticagrelor on myocardial infarct size (IS). Approach and Results— Rats received oral ticagrelor (0, 75, 150, or 300 mg/kg/d) or clopidogrel (30 or 90 mg/kg/d) for 7 days and underwent 30-minute coronary artery ligation and 24-hour reperfusion. Area at risk was assessed by blue dye and IS by 2,3,5-triphenyl-tetrazolium-chloride. Cyclooxygenase-2 (COX2) enzyme activity was assessed by ELISA and expression by real-time polymerase chain reaction. Mechanism responsible was explored using adenosine-receptor antagonist (CGS15943, an A2A/A1 antagonist) or cyclooxygenase inhibition by either aspirin (5, 10, or 25 mg/kg) or specific cyclooxygenase-1 (SC560) or COX2 (SC5815) inhibitors. Ticagrelor, dose-dependently, reduced IS, whereas clopidogrel had no effect. Adenosine-receptor antagonism blocked the ticagrelor effect and COX2 inhibition by SC5815, or high-dose aspirin attenuated the IS-limiting effect of ticagrelor, whereas cyclooxygenase-1 inhibition or low-dose aspirin had no effect. Ticagrelor, but not clopidogrel, upregulated COX2 expression and activity. Also this effect was blocked by adenosine-receptor antagonism. Ticagrelor, but not clopidogrel, increased Akt and endothelial nitric oxide synthase phosphorylation. Conclusions— Ticagrelor, but not clopidogrel, reduces myocardial IS. The protective effect of ticagrelor was dependent on adenosine-receptor activation with downstream upregulation of endothelial nitric oxide synthase and COX2 activity.Objective— In a phase III clinical trial (PLATelet inhibition and patient Outcomes, PLATO), ticagrelor provided better clinical outcomes than clopidogrel in patients with acute coronary syndromes. In addition to P2Y12-receptor antagonism, ticagrelor prevents cell uptake of adenosine and has proven able to augment adenosine effects. Adenosine protects the heart against ischemia–reperfusion injury. We compared the effects of clopidogrel and ticagrelor on myocardial infarct size (IS). Approach and Results— Rats received oral ticagrelor (0, 75, 150, or 300 mg/kg/d) or clopidogrel (30 or 90 mg/kg/d) for 7 days and underwent 30-minute coronary artery ligation and 24-hour reperfusion. Area at risk was assessed by blue dye and IS by 2,3,5-triphenyl-tetrazolium-chloride. Cyclooxygenase-2 (COX2) enzyme activity was assessed by ELISA and expression by real-time polymerase chain reaction. Mechanism responsible was explored using adenosine-receptor antagonist (CGS15943, an A2A/A1 antagonist) or cyclooxygenase inhibition by either aspirin (5, 10, or 25 mg/kg) or specific cyclooxygenase-1 (SC560) or COX2 (SC5815) inhibitors. Ticagrelor, dose-dependently, reduced IS, whereas clopidogrel had no effect. Adenosine-receptor antagonism blocked the ticagrelor effect and COX2 inhibition by SC5815, or high-dose aspirin attenuated the IS-limiting effect of ticagrelor, whereas cyclooxygenase-1 inhibition or low-dose aspirin had no effect. Ticagrelor, but not clopidogrel, upregulated COX2 expression and activity. Also this effect was blocked by adenosine-receptor antagonism. Ticagrelor, but not clopidogrel, increased Akt and endothelial nitric oxide synthase phosphorylation. Conclusions— Ticagrelor, but not clopidogrel, reduces myocardial IS. The protective effect of ticagrelor was dependent on adenosine-receptor activation with downstream upregulation of endothelial nitric oxide synthase and COX2 activity. # Significance {#article-title-45}

Chronic Treatment With Ticagrelor Limits Myocardial Infarct Size

Objective— In a phase III clinical trial (PLATelet inhibition and patient Outcomes, PLATO), ticagrelor provided better clinical outcomes than clopidogrel in patients with acute coronary syndromes. In addition to P2Y12-receptor antagonism, ticagrelor prevents cell uptake of adenosine and has proven able to augment adenosine effects. Adenosine protects the heart against ischemia–reperfusion injury. We compared the effects of clopidogrel and ticagrelor on myocardial infarct size (IS). Approach and Results— Rats received oral ticagrelor (0, 75, 150, or 300 mg/kg/d) or clopidogrel (30 or 90 mg/kg/d) for 7 days and underwent 30-minute coronary artery ligation and 24-hour reperfusion. Area at risk was assessed by blue dye and IS by 2,3,5-triphenyl-tetrazolium-chloride. Cyclooxygenase-2 (COX2) enzyme activity was assessed by ELISA and expression by real-time polymerase chain reaction. Mechanism responsible was explored using adenosine-receptor antagonist (CGS15943, an A2A/A1 antagonist) or cyclooxygenase inhibition by either aspirin (5, 10, or 25 mg/kg) or specific cyclooxygenase-1 (SC560) or COX2 (SC5815) inhibitors. Ticagrelor, dose-dependently, reduced IS, whereas clopidogrel had no effect. Adenosine-receptor antagonism blocked the ticagrelor effect and COX2 inhibition by SC5815, or high-dose aspirin attenuated the IS-limiting effect of ticagrelor, whereas cyclooxygenase-1 inhibition or low-dose aspirin had no effect. Ticagrelor, but not clopidogrel, upregulated COX2 expression and activity. Also this effect was blocked by adenosine-receptor antagonism. Ticagrelor, but not clopidogrel, increased Akt and endothelial nitric oxide synthase phosphorylation. Conclusions— Ticagrelor, but not clopidogrel, reduces myocardial IS. The protective effect of ticagrelor was dependent on adenosine-receptor activation with downstream upregulation of endothelial nitric oxide synthase and COX2 activity.Objective— In a phase III clinical trial (PLATelet inhibition and patient Outcomes, PLATO), ticagrelor provided better clinical outcomes than clopidogrel in patients with acute coronary syndromes. In addition to P2Y12-receptor antagonism, ticagrelor prevents cell uptake of adenosine and has proven able to augment adenosine effects. Adenosine protects the heart against ischemia–reperfusion injury. We compared the effects of clopidogrel and ticagrelor on myocardial infarct size (IS). Approach and Results— Rats received oral ticagrelor (0, 75, 150, or 300 mg/kg/d) or clopidogrel (30 or 90 mg/kg/d) for 7 days and underwent 30-minute coronary artery ligation and 24-hour reperfusion. Area at risk was assessed by blue dye and IS by 2,3,5-triphenyl-tetrazolium-chloride. Cyclooxygenase-2 (COX2) enzyme activity was assessed by ELISA and expression by real-time polymerase chain reaction. Mechanism responsible was explored using adenosine-receptor antagonist (CGS15943, an A2A/A1 antagonist) or cyclooxygenase inhibition by either aspirin (5, 10, or 25 mg/kg) or specific cyclooxygenase-1 (SC560) or COX2 (SC5815) inhibitors. Ticagrelor, dose-dependently, reduced IS, whereas clopidogrel had no effect. Adenosine-receptor antagonism blocked the ticagrelor effect and COX2 inhibition by SC5815, or high-dose aspirin attenuated the IS-limiting effect of ticagrelor, whereas cyclooxygenase-1 inhibition or low-dose aspirin had no effect. Ticagrelor, but not clopidogrel, upregulated COX2 expression and activity. Also this effect was blocked by adenosine-receptor antagonism. Ticagrelor, but not clopidogrel, increased Akt and endothelial nitric oxide synthase phosphorylation. Conclusions— Ticagrelor, but not clopidogrel, reduces myocardial IS. The protective effect of ticagrelor was dependent on adenosine-receptor activation with downstream upregulation of endothelial nitric oxide synthase and COX2 activity. # Significance {#article-title-45}

Thrombin-induced platelet activation and its inhibition by anticoagulants with different modes of action

Thrombin-induced platelet activation involves cleavage of protease-activated receptors (PARs) 1 and 4, and interaction, via glycoprotein (Gp)Ibα, with the platelet GpIb/IX/V complex. This study investigated inhibition of platelet activation by thrombin inhibitors with different modes of action: two reversible direct thrombin inhibitors, melagatran and inogatran; hirudin, a tightly binding direct thrombin inhibitor; and two indirect thrombin inhibitors, heparin and dalteparin. Up-regulation of P-selectin (CD62P) and PAR-1 cleavage was measured in human whole blood, by flow cytometry. The thrombin concentration that induced 50% of maximum (EC50) PAR-1 cleavage was 0.028 nmol/l, while that of platelet activation (CD62P) was over two-fold higher (0.64 nmol/l). The EC50 of a PAR-1-independent component, defined as a further activating effect of thrombin on top of the maximum PAR-1-activating peptide (AP) effect, was 3.2 nmol/l. All anticoagulants were concentration-dependent inhibitors of thrombin-induced platelet activation and PAR-1 cleavage, but none inhibited PAR-1-AP or PAR-4-AP induced activation. Melagatran and inogatran were more potent inhibitors of CD62P up-regulation than of PAR-1 cleavage; conversely, hirudin, heparin and dalteparin were more potent inhibitors of PAR-1 cleavage.Thus, reversible direct thrombin inhibitors, such as melagatran, are potent inhibitors of thrombin-induced platelet activation, acting mainly by inhibition of a PAR-1-independent component.

Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP- and thrombin-induced human platelet activation

The objective of this study was to investigate if there is a synergistic effect of a combination of P2Y12 and P2Y1 inhibition and P2Y12 and thrombin inhibition, on ADP‐ and thrombin‐induced platelet activation, respectively. The rationale being that these combinations will cause a concurrent inhibition of both Gαq and Gαi signalling. Blood from healthy volunteers was preincubated with AR‐C69931MX, a reversible P2Y12 antagonist; MRS2179, a reversible P2Y1 antagonist; or melagatran, a direct reversible thrombin inhibitor; alone or in various combinations prior to activation with ADP or thrombin. Platelet function in whole blood was assessed by flow cytometry using the antibody PAC‐1 to estimate the expression of active αIIbβ3 (the fibrinogen receptor GPIIb/IIIa). A synergistic effect was evaluated by comparing the concentrations in the different combinations with those of corresponding equipotent concentrations of each single inhibitor alone. The equipotent single concentrations were experimentally obtained from concentration response curves performed in parallel. A synergistic effect regarding inhibition of ADP‐induced platelet activation (10 μM) was obtained with different combinations of AR‐C69931MX and MRS2179. Inhibition of thrombin‐induced platelet activation (2 nM) with combinations of AR‐C69931MX and the thrombin inhibitor melagatran did also result in a strong synergistic effect. To our knowledge, this is the first time that data supporting a synergistic effect has been published for the inhibitor combinations described. Whether this synergistic effect in vitro also results in an improved antithrombotic effect in vivo with or without an increased risk of bleeding remains to be studied in well‐conducted clinical studies.