J.J.J. van Giezen

Ticagrelor: The First Reversibly Binding Oral P2Y12 Receptor Antagonist

Ticagrelor (AZD6140) is the first reversibly binding oral P2Y12 receptor antagonist that blocks ADP-induced platelet aggregation. Unlike thienopyridines, which irreversibly bind to the P2Y12 receptor for the lifetime of the platelet, ticagrelor binds reversibly to the receptor and exhibits rapid onset and offset of effect, which closely follow drug exposure levels. Animal models indicate greater separation between antithrombotic effects and bleeding effects with ticagrelor than with thienopyridines. Unlike the thienopyridines, ticagrelor does not require metabolic activation. It is quickly absorbed and exhibits a rapid antiplatelet effect, with higher and more consistent levels of inhibition of platelet aggregation (IPA) being maintained across the dosing interval than with clopidogrel. IPA levels decline with plasma drug levels after discontinuation of dosing. In the phase II DISPERSE-2 trial of 990 patients with non-ST-elevation acute coronary syndromes (ACS), ticagrelor treatment with 90 mg and 180 mg twice daily showed comparable rates of major and minor bleeding compared with clopidogrel 75 mg while there were numerically fewer myocardial infarctions. Ticagrelor resulted in greater IPA in clopidogrel-naïve patients and produced substantial additional reductions in platelet aggregation activity in patients pretreated with clopidogrel. Ticagrelor treatment was well tolerated in DISPERSE-2, and discontinuation rates were comparable to those observed for clopidogrel. An increased risk of mild to moderate dyspnea and mostly asymptomatic ventricular pauses were observed in phase II studies. The mechanisms for these effects are currently being investigated. The efficacy and safety of ticagrelor are being further evaluated in the phase III PLATO trial, involving approximately 18,000 patients with ACS, including both ST-elevation and non-ST-elevation ACS.

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 Inhibits Adenosine Uptake In Vitro and Enhances Adenosine-Mediated Hyperemia Responses in a Canine Model

Aims: A routine secondary pharmacology screen indicated that reversibly binding oral P2Y12 receptor antagonist ticagrelor could inhibit adenosine uptake in human erythrocytes, suggesting that ticagrelor may potentiate adenosine-mediated responses in vivo. The aim of this study was to further characterize the adenosine uptake inhibition in vitro and study possible physiological consequences of adenosine uptake inhibition by ticagrelor in an anesthetized dog model of coronary blood flow compared to dipyridamole. Methods and Results: We measured [2-3H]adenosine uptake in purified human erythrocytes and several cell lines in the presence of ticagrelor or the known uptake inhibitor dipyridamole as a comparator. Using an open-chest dog model (beagles), we measured the left anterior descending (LAD) coronary artery blood flow during reactive hyperemia after 1 minute occlusion or intracoronary infusion of adenosine before and after administration of vehicle, ticagrelor, or dipyridamole (each n = 8). Ticagrelor concentration-dependently inhibited adenosine uptake in human erythrocytes and in cell lines of rat, canine, or human origin. In the dog model, ticagrelor and dipyridamole dose-dependently augmented reactive hyperemia after LAD occlusion, as assessed by percentage repayment of flow debt relative to control (both Ps < .05). Ticagrelor and dipyridamole also dose-dependently augmented intracoronary adenosine-induced increases in LAD blood flow relative to control (both Ps < .05). Conclusion: Ticagrelor inhibits adenosine uptake in vitro and subsequently augments cardiac blood flow in a canine model of reactive hypoxia- or adenosine-induced blood flow increases. These findings suggest that ticagrelor may have additional benefits in patients with acute coronary syndrome beyond inhibition of platelet aggregation.

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).

Comparison of ticagrelor and thienopyridine P2Y12 binding characteristics and antithrombotic and bleeding effects in rat and dog models of thrombosis/hemostasis

Ticagrelor (AZD6140), the first reversibly binding oral P2Y(12) receptor antagonist, blocks adenosine diphosphate (ADP)-induced platelet aggregation via a mode of action distinct from that of thienopyridine antiplatelet agents. The latter must be metabolically activated and binds irreversibly to P2Y(12) for the life of the platelet, precluding restoration of hemostatic function without the generation of new platelets. In in vitro studies comparing binding characteristics of ticagrelor and compound 105, a chemical compound indistinguishable from the active metabolite of prasugrel, ticagrelor exhibited 1) an approximately 100-fold higher affinity for P2Y(12) and rapid achievement of equilibrium (vs no equilibrium reached with compound 105) as assessed by radioligand displacement in a receptor filtration binding assay, 2) 48-fold greater potency in a functional receptor assay using recombinant human P2Y(12), and 3) 63-fold greater potency in inhibiting ADP-induced aggregation in washed human platelets. In rat and dog models of thrombosis/hemostasis, there was greater separation between doses that provided antithrombotic effect and those that increased bleeding for ticagrelor compared with clopidogrel and compound 072, a chemical compound indistinguishable from the prasugrel parent compound. The ratio of dose resulting in 3-fold increase in bleeding time to dose resulting in 50% restoration of blood flow in rats was 9.7 for ticagrelor compared with 2.0 for clopidogrel and 1.4 for compound 072. Similar results were observed in dogs. Our findings suggest that reversibility of P2Y(12) binding with ticagrelor may account for the greater separation between antithrombotic effects and increased bleeding compared with the irreversible binding of clopidogrel and prasugrel.

Stabilizing role of platelet P2Y(12) receptors in shear-dependent thrombus formation on ruptured plaques.

Background In most models of experimental thrombosis, healthy blood vessels are damaged. This results in the formation of a platelet thrombus that is stabilized by ADP signaling via P2Y12 receptors. However, such models do not predict involvement of P2Y12 in the clinically relevant situation of thrombosis upon rupture of atherosclerotic plaques. We investigated the role of P2Y12 in thrombus formation on (collagen-containing) atherosclerotic plaques in vitro and in vivo, by using a novel mouse model of atherothrombosis. Methodology Plaques in the carotid arteries from Apoe −/− mice were acutely ruptured by ultrasound treatment, and the thrombotic process was monitored via intravital fluorescence microscopy. Thrombus formation in vitro was assessed in mouse and human blood perfused over collagen or plaque material under variable conditions of shear rate and coagulation. Effects of two reversible P2Y12 blockers, ticagrelor (AZD6140) and cangrelor (AR-C69931MX), were investigated. Principal Findings Acute plaque rupture by ultrasound treatment provoked rapid formation of non-occlusive thrombi, which were smaller in size and unstable in the presence of P2Y12 blockers. In vitro, when mouse or human blood was perfused over collagen or atherosclerotic plaque material, blockage or deficiency of P2Y12 reduced the thrombi and increased embolization events. These P2Y12 effects were present at shear rates >500 s−1, and they persisted in the presence of coagulation. P2Y12-dependent thrombus stabilization was accompanied by increased fibrin(ogen) binding. Conclusions/Significance Platelet P2Y12 receptors play a crucial role in the stabilization of thrombi formed on atherosclerotic plaques. This P2Y12 function is restricted to high shear flow conditions, and is preserved in the presence of coagulation.

A novel series of piperazinyl-pyridine ureas as antagonists of the purinergic P2Y12 receptor

A novel series of P2Y(12) antagonists for development of drugs within the antiplatelet area is presented. The synthesis of the piperazinyl-pyridine urea derivatives and their structure-activity relationships (SAR) are described. Several compounds showed P2Y(12) antagonistic activities in the sub-micromolar range.

Lead Optimization of Ethyl 6-Aminonicotinate Acyl Sulfonamides as Antagonists of the P2Y12 Receptor. Separation of the Antithrombotic Effect and Bleeding for Candidate Drug AZD1283

Synthesis and structure-activity relationships of ethyl 6-aminonicotinate acyl sulfonamides, which are potent antagonists of the P2Y12 receptor, are presented. Shifting from 5-chlorothienyl to benzyl sulfonamides significantly increased the potency in the residual platelet count assay. Evaluation of PK parameters in vivo in dog for six compounds showed a 10-fold higher clearance for the azetidines than for the matched-pair piperidines. In a modified Folts model in dog, both piperidine 3 and azetidine 13 dose-dependently induced increases in blood flow and inhibition of ADP-induced platelet aggregation with antithrombotic ED50 values of 3.0 and 10 μg/kg/min, respectively. The doses that induced a larger than 3-fold increase in bleeding time were 33 and 100 μg/kg/min for 3 and 13, respectively. Thus, the therapeutic index (TI) was ≥ 10 for both compounds. On the basis of these data, compound 3 was progressed into human clinical trials as candidate drug AZD1283.

Evaluation of ticagrelor pharmacodynamic interactions with reversibly binding or non-reversibly binding P2Y12 antagonists in an ex-vivo canine model

INTRODUCTION As ticagrelor, clopidogrel and cangrelor therapies may be used in the same clinical setting, their potential pharmacodynamic interactions are of interest. Hence, we investigated possible interactions between these agents in dogs using a variety of switching protocols. METHODS Six male dogs all received 7 different dosing regimens separated by 1-5week washout periods: cangrelor (1μg/kg/min, intravenous infusion); ticagrelor (0.8mg/kg, oral); clopidogrel (3mg/kg, intravenous injection); cangrelor together with ticagrelor initiated 10minutes after cangrelor infusion start or clopidogrel given 30minutes after cangrelor infusion start; ticagrelor followed by clopidogrel given 3 or 7hours after ticagrelor dosing. ADP-induced whole blood platelet aggregation was measured by impedance aggregometry. RESULTS Mean maximum inhibition of platelet aggregation (IPA) was 81-87% at 6minutes (cangrelor), 3hours (ticagrelor) and 4hours (clopidogrel) postdosing and platelet function recovered after 1.5hours, 12hours, and 9days, respectively. IPA at 2hours post clopidogrel was reduced to 39% when clopidogrel was given during cangrelor infusion versus 69% for clopidogrel alone. With clopidogrel dosed 3hours after ticagrelor, IPA was reduced after washout of ticagrelor to 38% at 24hrs vs. 68% for clopidogrel alone, but an interaction was not seen when clopidogrel was dosed 7hours after ticagrelor. No pharmacodynamic interaction occurred between ticagrelor and cangrelor. CONCLUSIONS The extent of the pharmacodynamic drug-drug interactions observed between clopidogrel and cangrelor or ticagrelor apparently depends on the level of receptor occupancy when clopidogrel is administered. Importantly, no significant pharmacodynamic interaction occurred between ticagrelor/clopidogrel when clopidogrel was given at clinical trough IPA levels with ticagrelor. No significant pharmacodynamic interaction occurred with cangrelor and ticagrelor.

Synthesis, structure–property relationships and pharmacokinetic evaluation of ethyl 6-aminonicotinate sulfonylureas as antagonists of the P2Y12 receptor

The present paper describes the development of a new series of P2Y12 receptor antagonists based on our previously reported piperazinyl urea series 1 (IC50 binding affinity = 0.33 μM, aq solubility <0.1 μM, microsomal CLint (HLM) ≥300 μM/min/mg). By replacement of the urea functionality with a sulfonylurea group we observed increased affinity along with improved stability and solubility as exemplified by 47 (IC50 binding affinity = 0.042 μM, aq solubility = 90 μM, microsomal CLint (HLM) = 70 μM/min/mg). Further improvements in affinity and metabolic stability were achieved by replacing the central piperazine ring with a 3-aminoazetidine as exemplified by 3 (IC50 binding affinity = 0.0062 μM, aq solubility = 83 μM, microsomal CLint (HLM) = 28 μM/min/mg). The improved affinity observed in the in vitro binding assay also translated to the potency observed in the WPA aggregation assay (47: 19 nM and 3: 9.5 nM) and the observed in vitro ADME properties translates to the in vivo PK properties observed in rat. In addition, we found that the chemical stability of the sulfonylureas during prolonged storage in solution was related to the sulfonyl urea linker and depended on the type of solvent and the substitution pattern of the sulfonyl urea functionality.