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

Abstract

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.

Keywords

ticagrelor dipyridamole adenosine hyperemia animal model

Introduction

Ticagrelor is the first reversibly binding oral P2Y₁₂ receptor antagonist evaluated for the prevention of clinical thrombotic events in patients with acute coronary syndrome (ACS).^1–3 Phase II studies showed that ticagrelor rapidly produced and maintained greater inhibition of adenosine diphosphate (ADP)–induced platelet aggregation compared with clopidogrel without increasing major bleeding risk.^4–6 The phase III Platelet Inhibition and Patient Outcomes (PLATO) study in a broad population of patients with ACS showed that ticagrelor significantly reduced the risk of the primary end point, driven by a significant reduction in both death from vascular causes and myocardial infarction, compared with clopidogrel without increasing major bleeding risk.⁷ In addition, a 22% reduction in all-cause mortality was observed. The reduction in cardiovascular (CV) death and all-cause mortality was remarkable, because the Trial to assess Improvement in Therapeutic Outcomes by optimizing platelet Inhibition with prasugrel Thrombolysis In Myocardial Infarction 38 (TRITON-TIMI 38) study comparing irreversible P2Y₁₂ inhibitor prasugrel with clopidogrel in a patient population with primary percutaneous coronary intervention (PCI) ⁸ did not show a significant reduction in CV death, despite levels of platelet inhibition similar to those seen with ticagrelor.⁹ In addition, dyspnea was reported as an unexpected adverse event finding during the phase II development program and was confirmed in the PLATO study, occurring more frequently in ticagrelor-treated than in clopidogrel-treated patients.^4–7 There was no increased reporting of dyspnea associated with prasugrel use in the TRITON study.

Secondary pharmacology screening showed inhibition of adenosine uptake into human erythrocytes as one of the most potent off target activities of ticagrelor. Adenosine has been suggested to have beneficial effects in patients with ACS.^10,11 The Acute Myocardial Infarction Study of Adenosine II (AMISTAD II) study investigated the potential benefit of a 3-hour adenosine infusion versus placebo in patients with evolving anterior ST segment elevation myocardial infarction, who were receiving thrombolysis or primary angioplasty.¹² Although the study did not show a treatment effect on the primary end point, a post hoc analysis suggested that early initiation of adenosine treatment in combination with reperfusion therapy may result in reduced mortality.¹³ In addition, intravenous infusion of adenosine in healthy volunteers is associated with dyspnea, potentially through stimulation of the sensory receptors in the respiratory system.¹⁴

Inhibition of adenosine uptake by ticagrelor may effectively result in a prolonged elimination half-life and subsequently increased local exposure to adenosine in vivo. Hence, in the current study, we further characterized the inhibitory effect of ticagrelor on adenosine uptake in several in vitro models under conditions that would distinguish between nucleoside transporter families. To assess a possible in vivo relevance of adenosine uptake inhibition by ticagrelor, we developed an ischemic model of adenosine-induced coronary flow regulation in anesthetized dogs. We evaluated the effect of ticagrelor on hyperemic response and on adenosine infusion in this model.

Materials and Methods

Adenosine Uptake in Erythrocytes and Cell Lines

Materials

Cell culture media were purchased from Invitrogen (Paisley, UK). Ticagrelor was synthesized by AstraZeneca (Charnwood, UK). [2-³H]adenosine was purchased from GE Healthcare (Amersham, UK); all other reagents and chemicals were purchased from Sigma (St Louis, Missouri) and were of the highest grade.

Human blood was obtained from a panel of healthy male and female donors using 1:10 volume 3.2% disodium citrate as an anticoagulant. All donors came from a pool of volunteers from within AstraZeneca’s staff who provided written consent and whose blood parameters are monitored annually.

Erythrocyte preparation

Washed erythrocytes were prepared by differential centrifugation of human blood. The red cell fraction was taken after centrifugation to remove platelet-rich plasma (240 g, 15 minutes at room temperature) and was resuspended in phosphate-buffered saline ([PBS] pH 7.4) to a volume equivalent to the original volume of blood. One volume of this suspension was diluted with 4 volumes of ice-cold PBS. The red cells were sedimented by centrifugation (240 g, 10 minutes at 4°C). The supernatant was discarded and the cells were resuspended in PBS (100 mL) and re-centrifuged. This procedure was repeated twice. The final pellet was resuspended in a volume of PBS equivalent to the original blood volume and stored on ice prior to use.

Adenosine uptake measurement in erythrocytes

Immediately prior to use, the washed erythrocyte suspension was diluted 1:20 in PBS at room temperature. Suitably diluted test compound (25 μL) and 25 μL [2-³H]adenosine (0.83 mmol/L, 20 μCi/mL) in PBS (+5 μmol/L MgCl₂ and 125 μmol/L erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA]) were mixed in a 1.5-mL microcentrifuge tube. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, was used to prevent adenosine metabolism. Uptake was initiated by the addition of diluted erythrocytes (200 μL). The assay tubes were incubated at room temperature for 2 minutes, after which time a stopping solution (250 μL) consisting of adenosine (5 mmol/L), dipyridamole (100 μmol/L), and EHNA (25 μmol/L) in PBS was added. Finally, dibutylphthalate (250 μL) was added to each tube and the contents were centrifuged (approximately 12 000g) for 2 minutes. At this stage, the erythrocytes formed a pellet in the bottom of the tube under the dibutylphthalate layer and the incubation medium formed a separate phase above the dibutylphthalate. Both these layers were carefully removed, and the radioactivity associated with the cell pellet was determined by solubilizing the pellet in 10% Triton X-100 (0.5 mL) and mixing the solution with HiSafe scintillant (4 mL) prior to beta scintillation counting using an LS6500 scintillation counter (Beckman Coulter, UK).

Cell lines

Madin-Darby canine kidney (MDCK) and rat hepatoma H4IIE cell lines were maintained in Minimal Essential Media (MEM)-α medium and human breast carcinoma Michigan Cancer Foundation-7 breast cancer cell line (MCF-7) cell lines were maintained in Roswell Park Memorial Institute (RPMI) medium. All media were supplemented with 10% fetal bovine serum, 1% GlutaMAX, and 1% penicillin/streptomycin. The cells were maintained at 37°C in 5% CO₂:95% air for routine culturing and all experimental procedures. All tissue culture media and PBS were pre-warmed to 37°C unless otherwise stated. The cell density was assessed using the modified Fuchs-Rosenthal hemocytometer, and cell viability was determined by the trypan blue exclusion test. The cell cultures were passaged every 3 to 4 days.

Preparation of cell suspensions

The cells were seeded into culture flasks (1 × 10⁵/cm²) and incubated overnight in appropriate media. After 24 hours, the monolayers were washed in PBS and detached from the vessels by accutase digestion. The cell suspension was pelleted by centrifugation (300g, 3 minutes), resuspended in sodium-free buffer (137 mmol/L N-methyl-D-glucamine, 1.5 mmol/L KH₂PO₄, 6.3 mmol/L K₂HPO₄, 2.7 mmol/L KCl, 0.5 mmol/L MgCl₂·6H₂O, 0.9 mmol/L CaCl₂·2H₂O; pH 7.4.) containing 25 µmol/L EHNA and adjusted to a working suspension of 4 × 10⁶ cells/mL.

Adenosine uptake measurements in cell lines

Individual samples were prepared by combining 5 μL [2-³H]adenosine (5 µCi) and 0.25 μL of the test compound (formulated to 1000× final concentration in dimethyl sulfoxide) with 45 μL sodium-free buffer in the bottom of a 1.5-mL centrifuge tube. The final concentration of [2-³H]adenosine after addition of the cell suspension was 0.84 mmol/L (20 μCi/mL).

The assay was started by the addition of 200 µL cell suspension (∽1 × 10⁶ cells) to the prepared tubes and stopped after 2 minutes by the addition of 250 µL stopping solution (100 µmol/L dipyridamole, 10 mmol/L adenosine, and 50 µmol/L EHNA in sodium-free buffer). Cells were isolated and lysed and radioactivity counted as described above for human erythrocytes.

Data analysis

Data were analyzed by nonlinear regression using the sigmoidal dose—response (variable slope) model in GraphPad Prism version 4.02 for Windows (GraphPad Software). IC₅₀ values for each cell line were calculated from concentration curves generated from at least 3 separate experiments and the mean and range are indicated. Error bars in the figures represent standard error of the mean (standard error of the mean [SEM]).

Dog cardiac blood flow model

Beagle dogs of either sex were anesthetized with propofol (7-10 mg/kg, intravenous) and anesthesia was continued with an infusion of α-chloralose (100 mg/kg bolus infusion followed by a continuous infusion of 40 mg/kg per h) and inhaled isoflurane (1.8% added to the ventilator air). Catheters were placed into the right saphenous artery for blood pressure recording and blood sampling, into the right saphenous vein for administration of study compounds or vehicle, and into the right jugular vein for measurement of central venous pressure. A dual sensor pressure transducer (Millar Micro-Tip SPC-771, Millar Instruments Inc, Houston, Texas) was inserted via the right carotid artery, with the proximal transducer in the ascending aorta and the distal transducer in the left ventricle. Left femoral artery blood flow was measured with a 3.0-mm ultrasonic transit-time volume flow sensor (model T403, Transonic Systems Inc, Ithaca, New York).

The thorax was opened in the fourth intercostal space, the pericardium opened, and the heart suspended in a pericardial cradle. Cardiac output and stroke volume were measured at the ascending aorta with a 14-mm ultrasonic transit-time volume flow sensor (model T403, Transonic Systems Inc). Left anterior descending (LAD) coronary artery blood flow and circumflex coronary artery (CCA) blood flow were measured with 1.5 mm ultrasonic transit-time volume flow sensors (model T403, Transonic Systems Inc) placed on the proximal LAD coronary artery and CCA. A cannula for intracoronary infusions was inserted via a coronary artery branch into the LAD coronary artery, with the tip just distal to the flow probe. A custom balloon occluding device was placed at the LAD coronary artery just proximal to the flow probe. After all surgeries were completed the isoflurane was switched off. Body temperature (37°C) and respiration rate (15 cycles/min) were maintained for the entirety of the experiment and heart rate and blood pressure were continuously monitored for possible signs of distress.

Each experiment consisted of 3 consecutive cycles of blood flow measurements lasting 75 minutes each, with the first determining each animal’s reference value (control) and the following 2 measuring the flow response at increasing doses of ticagrelor, dipyridamole, or vehicle (saline). Each cycle consisted of 3 consecutive measurements of hyperemic blood flow after a 1-minute ischemic period, each separated by a 10-minute recovery period, followed by the measurement of blood flow response to 5 minutes constant intracoronary infusions of adenosine at 2 doses (15 and 30 µg/kg per min), each followed by a 10-minute washout period (Figure 1A). Dogs were randomized to receive 2 doses of either test compound (ticagrelor or dipyridamole) or vehicle. Each treatment consisted of a 1-minute bolus followed by a 74-minute continuous infusion to maintain a stable plasma concentration. Ticagrelor was administered at dose levels of (1) 210 µg/kg bolus plus 30 µg/kg per min infusion and (2) 700 µg/kg bolus plus 100 µg/kg per min infusion. Dipyridamole (MP BIOMEDICALS Inc, Solon, Ohio) was administered at dose levels of (1) 10 µg/kg bolus plus 0.17 µg/kg per min infusion and (2) 30 µg/kg bolus plus 0.5 µg/kg per min infusion. The doses of ticagrelor were chosen to obtain maximum P2Y₁₂ inhibition, with the lower dose aiming at exposure levels of approximately 4 μmol/L, which is similar to the high-end exposure levels observed using the 100-mg dose in humans in the Dose Confirmation Study Assessing Anti-Platelet Effects of AZD6140 versus Clopidogrel in Non-ST-Segment Elevation Myocardial Infarction (DISPERSE) trial,⁴ whereas the dipyridamole doses were based on effective regimens used in pilot experiments. At the end of the experiment, each animal was euthanized using an intravenous overdose of pentobarbital.

Figure 1.

A, Experiment scheme showing the three 75-minute test periods, each consisting of 3 reactive hyperemia cycles, followed by two 5-minute continuous infusions of adenosine into the left anterior descending (LAD) coronary artery. The first test period was used to set each animal’s baseline values, before 2 doses of either ticagrelor or dipyridamole were used. To check for a possible time-dependent effect, a third group received 2 vehicle infusions after the control period instead. B, Typical recording of coronary artery blood flow during 1 reactive hyperemia cycle and calculation of repayment of flow debt.

The effect of 1 minute of ischemia on blood flow was calculated as total blood volume and repayment of flow debt (Figure 1B). Total blood volume (mL) was measured for the period from the start of reperfusion until blood flow returned to 0.1(Q_peak − Q_before), where Q is LAD blood flow, with the result for each individual animal obtained from the mean of 3 measurements during the control period and at each dose level. Repayment of flow debt (%) was calculated as 100 × (total blood volume/calculated absent blood volume during 1 minute ischemia). The increased blood flow to adenosine infusion was calculated at steady state of each dose level as 100 × (Q_Adenosine/Q_before − 1). Dependency on adenosine receptor signaling was confirmed in pilot experiments using the adenosine receptor antagonist theophylline.¹⁵ For statistical analysis, all effects on blood flow were normalized to each animal’s reference value; an unequal variance t test without multiplicity adjustment was used between the control period and each dose level for the different treatments, with a P value of < .05 considered significant. Blood flow in the CCA, heart rate, aortic pressure, and cardiac output were monitored as the reference. All data are presented as mean values and the SEM values are indicated, unless stated otherwise.

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The research protocol was approved by a local ethics committee which falls under the supervision of the ministry of agriculture in Sweden.

Results

Adenosine Uptake in Human Erythrocytes and Human, Canine, and Rat Cell Lines

The potential for ticagrelor to inhibit adenosine uptake into human erythrocytes was determined during the development of ticagrelor as part of a standard selectivity screen.¹⁶ It was demonstrated that ticagrelor dose-dependently inhibited adenosine uptake, with an IC₅₀ of 100 (95% C.I. 79-128) nmol/L (n = 5), whereas the model adenosine transport inhibitor dipyridamole was 10-fold more potent, with an IC₅₀ of 10 (95% C.I. 8-13) nmol/L (n = 5). These findings were further investigated using a human cell line expressing adenosine uptake transporters in sodium-free buffer. In the human MCF-7 breast carcinoma cell line, the mean IC₅₀s were 61 (range 3.0-121) nmol/L for ticagrelor and 2.2 (range 1.3-3.6) nmol/L for dipyridamole (n ≥ 3; Figure 2).

Figure 2.

Adenosine uptake in different cell lines in the presence of dipyridamole (A) or ticagrelor. (B) Data shown are mean ± standard error of the mean (n ≥ 3). MDCK indicates Madin-Darby canine kidney cells; MCF-7, Michigan Cancer Foundation-7 breast cancer cell line; H4IIE rat hepatoma cells.

To verify whether the observed inhibition was specific for human cells, we also investigated adenosine uptake in a rat cell line and a canine cell line. Ticagrelor also inhibited adenosine uptake in the dog MDCK cells (mean IC₅₀s of 34 [range 24-50] nmol/L and 3 [range 1.8-3.5] nmol/L for ticagrelor and dipyridamole, respectively; n ≥ 3), and rat hepatoma H4IIE cells (mean IC₅₀s of 104 [range 70-155] nmol/L and 53 [range 40-71] nmol/L, respectively; n ≥ 3; Figure 2). Using dog MDCK cells, further experiments were performed to determine the K_i values for ticagrelor and dipyridamole, which were 21 and 1.5 nmol/L, respectively (K_m for adenosine was 134 μmol/L).

Coronary Blood Flow in the Canine Model

Reactive hyperemia and effects of intracoronary adenosine in the LAD coronary artery in vehicle-treated animals

A possible biological role of the observed in vitro adenosine uptake inhibition was studied in a dog model of coronary blood flow. Repeatable temporary increases in blood flow through the LAD coronary artery were observed in vehicle-treated animals after repeated 1-minute occlusions as a reactive hyperemia response. Steady-state blood flow was 18.3 ± 1.6 mL/min, with the reactive hyperemia response at 63 ± 7 mL (n = 8; Figure 3). This equates to a repayment of flow debt of 348% ± 37%. Throughout the experimental period, 9 reactive hyperemia responses were measured in each animal in 3 groups of 3, each group separated by 2 infusions of adenosine. Although there was no statistically significant difference in the extent of the response between these periods, there appeared to be a time-dependent reduction, with the second and third groups of hyperemia responses being 2.7% and 10.4% lower, respectively, than those in the first period. A catheter was placed in the LAD coronary artery distal to the flow probe to study the effects of a direct adenosine infusion. At steady state, a continuous infusion of adenosine 15 μg/kg per min increased blood flow through the LAD coronary artery to 41.5 ± 3.1 mL/min (127% increase of control flow, P < .05 vs baseline), and a continuous infusion of 30 μg/kg per min further increased the blood flow to 59.9 ± 3.8 mL/min (227% increase of control flow; P < .05 vs 15 μg/kg per min; Figure 3). These responses were constant throughout the experimental period. No changes were observed in blood flow through the CCA, which was measured throughout each experiment as a control, indicating no systemic effect of the adenosine infusion (data not shown).

Figure 3.

Absolute blood flow in left anterior descending (LAD) coronary artery in vehicle-treated dogs (n = 8) after 1 minute occlusion and during adenosine infusion. Data are mean ± standard error of the mean. *P < .05 versus baseline. **P < .05 versus adenosine 15 µg/kg per min.

Effects of ticagrelor and dipyridamole on LAD reactive hyperemia and intracoronary adenosine-induced blood flow increases

Effects of ticagrelor and dipyridamole on reactive hyperemia following 1 minute occlusion are shown as percentage repayment of flow debt and as relative to that observed during the vehicle period of each experiment (Figure 4). Both ticagrelor (n = 8) and dipyridamole (n = 8) produced dose-related and significant increases in reactive hyperemia response versus the control period, with repayment of flow debt increased by 26% ± 15% and 50% ± 14%, respectively, at the highest dose of each compound (P < .05; Figure 4).

Figure 4.

Repayment of flow debt after 1 minute occlusion in left anterior descending coronary artery normalized to control period (Control) after vehicle or ticagrelor or dipyridamole administration. Data are mean ± standard error of the mean, n = 8. *P < .05 versus control, unequal variance t test.

Results for ticagrelor and dipyridamole following intracoronary adenosine are expressed as percentage of pre-adenosine flow and as flow normalized to the increase observed during the vehicle period. Both ticagrelor (n = 8) and dipyridamole (n = 8) dose-dependently and significantly augmented intracoronary adenosine-induced increases in LAD flow at both adenosine doses with up to 150% ± 19% and 193% ± 17% increased blood flow versus the vehicle period, respectively, for the low adenosine dose and up to 140% ± 13% and 153% ± 30% increased blood flow versus the vehicle period, respectively, for the high adenosine dose (P < .05; Table 1 ).

Table 1.

Effect of Ticagrelor and Dipyridamole on Blood Flow in LAD Coronary Artery After Adenosine Infusion^a

	Vehicle			Ticagrelor			Dipyridamole
	Control Period	Vehicle Period 1	Vehicle Period 2	Control Period	30 µg/kg/min	100 µg/kg/min	Control Period	0.17 µg/kg/min	0.50 µg/kg/min
Adenosine 15 μg/kg/min
% increase of pre-adenosine flow	138 ± 17	135 ± 25	149 ± 17	152 ± 25	175 ± 34	210 ± 27	120 ± 17	192 ± 32	229 ± 33
% increase vs normalized control period	≡100%	98 ± 10	110 ± 7	≡100%	114 ± 10	150 ± 19^b	≡100%	160 ± 2^b	193 ± 17^b
Adenosine 30 μg/kg/min
% increase of pre-adenosine flow	250 ± 35	235 ± 27	234 ± 30	224 ± 25	247 ± 40	302 ± 36	208 ± 29	267 ± 36	277 ± 25
% increase vs normalized control period	≡100%	99 ± 6	97 ± 9	≡100%	109 ± 10	140 ± 13^b	≡100%	136 ± 17^b	153 ± 30^b

^aData are mean ± standard error of the mean, n = 8 for each treatment.

^b P < .05 versus control, unequal variance t test.

Heart rate and mean arterial pressure were not affected by ticagrelor, dipyridamole, or vehicle treatment. All 3 treatments were associated with comparable, time-dependent decreases in cardiac contractility, cardiac output, and femoral artery blood flow but not with decreases in central venous pressure, as a result of the duration of the anesthetic period. No effects of treatment were observed on body temperature, tracheal pressure, blood gases, pH, Na⁺, K⁺, Ca²⁺, Cl^–, glucose, lactate, HCO₃ ^–, or hematocrit.

Plasma exposure levels of ticagrelor were 4.1 ± 1.5 μmol/L for the low dose and 13.4 ± 2.1 μmol/L for the high dose. Plasma exposure levels for dipyridamole were not measured.

Discussion

In this study, we investigated the possible in vivo relevance of adenosine uptake inhibition as an off target effect of the reversibly binding P2Y₁₂ receptor antagonist ticagrelor. Data from the preclinical in vitro and in vivo studies presented suggest that ticagrelor may exert additional clinical benefits beyond inhibition of platelet aggregation via this mechanism.

The in vitro studies show that ticagrelor inhibits adenosine uptake by human erythrocytes and by cell lines of human, rat, or canine origin, with potency approximately 10-fold less than that of the reference compound, dipyridamole, in human erythrocytes. Adenosine uptake is mediated by sodium-dependent concentrative nucleoside transporters and sodium-independent equilibrative nucleoside transporters (ENTs).^17,18 The studies in canine MDCK cells and human breast carcinoma MCF-7 cells were performed under sodium-free conditions, suggesting that ticagrelor most likely inhibits sodium-independent ENT activity. Furthermore, the subtype most likely inhibited by ticagrelor is ENT1, since it is the only one expressed in MDCK cells and also is described as regulating the influx and efflux of adenosine in cardiomyocytes.¹⁹ Recent data from a murine model of ischemia—reperfusion injury in ENT1 knockout mice suggest that inhibition of ENT1 may have cardioprotective effects.²⁰ This may be because ENT1 is downregulated under hypoxic conditions in cardiomyocytes,²¹ which would augment extracellular adenosines. However, more experiments are needed to confirm the exact subtypes that are inhibited by ticagrelor. It should also be noted that ticagrelor’s ability to inhibit adenosine uptake was approximately 30- to 50-fold less potent compared with its ability to inhibit the P2Y₁₂ receptor or platelet aggregation.^1,3 However, average exposure levels in humans after a single 200-mg dose were 1224 ng/mL (approximately 2.3 μmol/L) and after repeated 100 mg dosing equaled 798 ng/mL (approximately 1.5 μmol/L),⁴ suggesting that adenosine uptake inhibition may play a contributing role.

Regulation of local blood flow via rapid vasodilatation is an important consequence of adenosine signaling through adenosine receptors on vascular smooth muscle cells during episodes of hypoxia.^22–24 In addition, adenosine may further modulate blood flow via inhibition of vascular sympathetic neuroeffector transmission.²⁵ We chose a dog model of coronary blood flow to study the potential biological relevance of the in vitro observed effects on adenosine clearance. Inhibition of adenosine uptake, that is, prolongation of adenosine’s half-life, should result in an augmented reactive hyperemia response, which is highly dependent on the release of adenosine from the hypoxic tissue.^26,27 To further confirm the proposed mechanism of action, the effect on a direct infusion of adenosine into the LAD was measured as well, and all effects were compared to those of dipyridamole, a known adenosine uptake inhibitor.²⁸ The studies in the canine model indicate that, indeed, both ticagrelor and dipyridamole exhibited dose-dependent augmentation of adenosine-induced increases in coronary blood flow, whether the adenosine was induced endogenously via temporary LAD coronary artery occlusion or introduced via direct infusion into the LAD coronary artery. It should be noted that there was no systemic alteration of blood flow by either of the test compounds or by the infused adenosine, since blood flow in the circumflex artery remained stable and was unaffected by any compound treatment. This result indicates that, at the achieved plasma concentrations of ticagrelor, there is no functional interaction between ticagrelor and adenosine receptors, which has been confirmed by specific receptor pharmacology studies.²⁹ However, a limitation of this study was that local adenosine concentrations were not measured, which may have provided further support of an indirect effect of ticagrelor via increased adenosine levels.

Adenosine is an important mediator of the reactive hyperemia response to temporary ischemia from coronary artery occlusion. Ischemia in myocardial infarction, congestive heart failure (CHF), or atherosclerosis produces cardiac hypoxia; hypoxia induces the release of adenosine by cardiomyocytes into the extracellular environment, which, in turn, activates a receptor-mediated compensatory mechanism, resulting in cardioprotection.^26,27 Beneficial effects of exogenously administered adenosine on coronary blood flow, including improvement of flow and prevention of the no reflow phenomenon during PCI in patients with ACS, have been observed in a number of clinical settings.^10,11,30 The AMISTAD-II study investigated the potential benefit of a 3-hour adenosine infusion versus placebo in patients with evolving anterior STEMI who were receiving thrombolysis or undergoing primary angioplasty.¹² The study did not show a significant treatment effect on the primary end point, new CHF more than 24 hours postrandomization, first rehospitalization for CHF, or death from any cause within 6 months. However, a post hoc analysis suggested that early initiation of adenosine treatment in combination with either form of reperfusion therapy may result in reduced mortality.¹³ Hence it is possible that a compound that potentiates the protective effect of adenosine at the site where it is generated, by prolonging its half-life, could exhibit a clinical benefit via such mechanism.

Recently published findings by Wang et al in a canine coronary thrombosis model are consistent with the hypothesis that ticagrelor may exert its beneficial effects via mechanisms other than P2Y₁₂ blockade.³¹ Wang and colleagues compared the effects of ticagrelor versus clopidogrel, in conjunction with thrombolytic therapy, on platelet aggregation, thrombus formation, and myocardial perfusion. In this study, the doses of both compounds were chosen so as to provide maximum P2Y₁₂ blockade and platelet inhibition. In addition to showing that both compounds blocked ADP-induced platelet activation and prevented platelet-mediated thrombosis, the study found that adjunctive ticagrelor resulted in prolonged reperfusion times, reduced rates of re-occlusion and cyclic flow variation, and rapid restoration of myocardial tissue perfusion, all beyond the results observed with clopidogrel. Furthermore, a marked 60% reduction in infarct size was observed in the ticagrelor group but not in the clopidogrel group, despite similar platelet inhibition levels and significantly more prolonged bleeding times in the clopidogrel group.³¹ These findings support activity beyond inhibition of platelet aggregation for ticagrelor, which may also have contributed to the unexpected mortality benefit seen in, for example, the PLATO CABG substudy.³²

The inhibition of adenosine uptake by ticagrelor may also contribute to the excess of dyspnea reported with ticagrelor treatment. The frequency of dyspnea appeared to be dose related in 2 phase II studies of ticagrelor,^4,5 and the recently reported phase III PLATO trial showed an absolute 6% excess of dyspnea in ticagrelor patients compared with clopidogrel patients.⁷ In these studies, the observed dyspnea was not associated with worsening heart condition or bronchoconstriction. Dyspnea is unlikely to be associated with P2Y₁₂ blockade itself, since increased blockade of the receptor by prasugrel did not seem to result in increased reporting of dyspnea.^8,33

Dyspnea is recognized as a side effect of intravenous adenosine administration in humans.^14,34,35 The mechanisms underlying this effect remain unclear, although some studies have shed light on the question. A recent study by Burki and colleagues¹⁴ in healthy participants showed that intravenous adenosine produced transient dyspnea in the absence of bronchoconstriction, perhaps through pulmonary C-fiber activation. In animal models, intravenous adenosine was found to stimulate pulmonary vagal C fibers through activation of A₁ receptors.³⁶ Although dyspnea is not commonly associated with dipyridamole,^37,38 it should be noted that dipyridamole is predominantly used for secondary stroke prevention, not for ACS. However, it is recognized that treatment with dipyridamole can lead to reports of dyspnea when used, for example, during cardiac stress imaging procedures.^39–41 In addition, postmarketing reports of adverse events associated with the use of Aggrenox (aspirin/extended-release dipyridamole, Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield, Connecticut) do include dyspnea.⁴² Taking all this together, it could be possible that the inhibition of adenosine uptake by ticagrelor may contribute to the increased reporting of dyspnea. However, both the CHAMPION PCI and CHAMPION PLATFORM studies also reported an increase in dyspnea associated with reversible P2Y₁₂ inhibitor cangrelor.^43,44 No published data are available indicating whether cangrelor affects adenosine uptake mechanisms. Hence, a link between dyspnea and blockade of nonplatelet P2Y₁₂ receptors cannot be excluded.

In conclusion, our study shows that ticagrelor inhibits adenosine uptake into human erythrocytes and subsequently potentiates adenosine-mediated blood flow increases in a dog cardiac blood flow model. This suggests that ticagrelor may have beneficial effects in patients with ACS beyond inhibition of platelet aggregation. Such effects may, at least in part, contribute to the observed mortality benefit associated with ticagrelor treatment in the PLATO trial⁷ or to the observed increase in dyspnea in the DISPERSE and PLATO studies. However, specifically designed clinical studies are needed to confirm this potential mechanism in humans.

Footnotes

Acknowledgments

We thank Joe Hirsch, from BioScience Communications, who provided medical writing support funded by AstraZeneca.

The work reported herein was conducted at AstraZeneca R&D Mölndal, Sweden (in vivo work); AstraZeneca R&D Charnwood and AstraZeneca R&D Alderly Park, UK (in vitro experiments).

The author(s) declared the following potential conflicts of interest with respect to research, authorship, and/or publication of this article: All authors on this manuscript are employees of AstraZeneca.

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by AstraZeneca.

References

van Giezen

Humphries

RG.

Preclinical and clinical studies with selective reversible direct P2Y12 antagonists. Semin Thromb Hemost. 2005;31(2):195–204.

van Giezen

Tomlinson

Berntsson

. AZD6140 (ticagrelor) binds to the human P2Y12 receptor independently from ADP, but antagonizes ADP-induced receptor signaling and platelet aggregation. J Thromb Haemost. 2009;7(9):1556–1565.

Husted

van Giezen

JJ.

Ticagrelor: the first reversibly binding oral P2Y12 receptor antagonist. Cardiovasc Ther. 2009;27(4):259–274.

Husted

Emanuelsson

Heptinstall

Sandset

Wickens

Peters

Pharmacodynamics, pharmacokinetics, and safety of the oral reversible P2Y12 antagonist AZD6140 with aspirin in patients with atherosclerosis: a double-blind comparison to clopidogrel with aspirin. Eur Heart J. 2006;27(9):1038–1047.

Cannon

Husted

Harrington

. Safety, tolerability, and initial efficacy of AZD6140, the first reversible oral adenosine diphosphate receptor antagonist, compared with clopidogrel, in patients with non–ST-segment elevation acute coronary syndrome: primary results of the DISPERSE-2 trial. J Am Coll Cardiol. 2007;50(19):1844–1851. Correction in J Am Coll Cardiol. 2007;50:2196

Storey

Husted

Harrington

. Inhibition of platelet aggregation by AZD6140, a reversible oral P2Y12 receptor antagonist, compared with clopidogrel in patients with acute coronary syndromes. J Am Coll Cardiol. 2007;50(19):1852–1856.

Wallentin

Becker

Budaj

. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2009;361(11):1045–1057.

Wiviott

Braunwald

McCabe

. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2007;357(20):2001–2015.

Michelson

Frelinger

3rd Braunwald

. Pharmacodynamic assessment of platelet inhibition by prasugrel vs clopidogrel in the TRITON-TIMI 38 trial. Eur Heart J. 2009;30(14):1753–1763.

10.

Vijayalakshmi

Whittaker

Kunadian

. Prospective, randomised, controlled trial to study the effect of intracoronary injection of verapamil and adenosine on coronary blood flow during percutaneous coronary intervention in patients with acute coronary syndromes. Heart. 2006;92(9):1278–1284.

11.

Marzilli

Orsini

Marraccini

Testa

Beneficial effects of intracoronary adenosine as an adjunct to primary angioplasty in acute myocardial infarction. Circulation. 2000;101(18):2154–2159.

12.

Ross

Gibbons

Stone

Kloner

Alexander

RW.

A randomized, double-blind, placebo-controlled multicenter trial of adenosine as an adjunct to reperfusion in the treatment of acute myocardial infarction (AMISTAD-II). J Am Coll Cardiol. 2005;45(11):1775–1780.

13.

Kloner

Forman

Gibbons

Ross

Alexander

Stone

GW.

Impact of time to therapy and reperfusion modality on the efficacy of adenosine in acute myocardial infarction: the AMISTAD-2 trial. Eur Heart J. 2006;27(20):2400–2405.

14.

Burki

Dale

Lee

LY.

Intravenous adenosine and dyspnea in humans. J Appl Physiol. 2005;98(1):180–185.

15.

AstraZeneca R&D Mölndal, data on file, study number 26709, experiment number 26709033.

16.

AstraZeneca data on file, document SC-103269.

17.

Baldwin

Beal

Yao

King

Cass

Young

JD.

The equilibrative nucleoside transporter family, SLC29. Pflugers Arch. 2004;447(5):735–743.

18.

Gray

Owen

Giacomini

KM.

The concentrative nucleoside transporter family, SLC28. Pflugers Arch. 2004;447(5):728–734.

19.

Hammond

Stolk

Archer

McConnell

Pharmacological analysis and molecular cloning of the canine equilibrative nucleoside transporter 1. Eur J Pharmcol. 2004;491(1):9–19.

20.

Rose

Naydenova

Bang

. Equilibrative nucleoside transporter 1 plays an essential role in cardioprotection. Am J Physiol Heart Circ Physiol. 2010;298(3):H771–H777.

21.

Chaudary

Naydenova

Shuralyova

Coe

IR.

Hypoxia regulates the adenosine transporter, mENT1, in the murine cardiomyocyte cell line, HL-1. Cardiovasc Res. 2004;61(4):780–788.

22.

Belardinelli

Shryock

Snowdy

. The A2A adenosine receptor mediates coronary vasodilation. J Pharmacol Exp Ther. 1998;284(3):1066–1073.

23.

Feoktistov

Ryzhov

Zhong

. Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype. Hypertension. 2004;44(5):649–654.

24.

Fredholm

Ijzerman

Jacobson

Klotz

Linden

International Union of Pharmacology, XXV: nomenclature and classification of adenosine receptors. Pharmacol Rev. 2001;53(4):527–552.

25.

Husted

Nedergaard

OA.

Inhibition of adrenergic neuroeffector transmission in rabbit pulmonary artery and aorta by adenosine and adenine nucleotides. Acta Pharmacol Toxicol. Copenh). 1981;49(5):334–353.

26.

Berne

RM.

The role of adenosine in the regulation of coronary blood flow. Circ Res. 1980;47(6):807–813.

27.

Mustafa

Morrison

Teng

Pelleg

Adenosine receptors and the heart: role in regulation of coronary blood flow and cardiac electrophysiology. Handb Exp Pharmacol. 2009;193:161–188.

28.

Gresele

Arnout

Deckmyn

Vermylen

Mechanism of the antiplatelet action of dipyridamole in whole blood: modulation of adenosine concentration and activity. Thromb Haemost. 1986;55(1):12–18.

29.

AstraZeneca, data on file, study report numbers SC-103208, SC-103260, SC-103370, 0355SY, and 1805KV.

30.

Wilson

Wyche

Christensen

Zimmer

Laxson

DD.

Effects of adenosine on human coronary arterial circulation. Circulation. 1990;82(5):1595–1606.

31.

Wang

Zhou

Huang

. Adjunctive infusion of AZD6140, but not clopidogrel, with t-PA enables sustained coronary artery recanalization with recovery of myocardium perfusion in a canine model of myocardial infarction. Thromb Haemostas. 2010;104(3):609–617.

32.

Held

Åsenblad

Bassand

. Ticagrelor versus clopidogrel in patients with acute coronary syndromes undergoing coronary artery bypass surgery. Results from the PLATO (Platelet Inhibition and Patient Outcomes) Trial. J Am Coll Cardiol. 2011;57(6):672–684.

33.

Montalescot

Wiviott

Braunwald

. Prasugrel compared with clopidogrel in patients undergoing percutaneous coronary intervention for ST-elevation myocardial infarction (TRITON-TIMI 38): double-blind, randomised controlled trial. Lancet. 2009;373(9665):723–731.

34.

Rankin

Brooks

Ruskin

McGovern

BA.

Adenosine and the treatment of supraventricular tachycardia. Am J Med. 1992;92(6):655–664.

35.

Balan

Critchley

Is the dyspnea during adenosine cardiac stress test caused by bronchospasm?. Am Heart J. 2001;142(1):142–145.

36.

Hong

Kwong

Lee

LY.

Activation of pulmonary C fibres by adenosine in anaesthetized rats: role of adenosine A1 receptors. J Physiol. 1998;508(pt 1):109–118.

37.

Diener

Sacco

Yusuf

. Effects of aspirin plus extended-release dipyridamole versus clopidogrel and telmisartan on disability and cognitive function after recurrent stroke in patients with ischaemic stroke in the Prevention Regimen for Effectively Avoiding Second Strokes (PRoFESS) trial: a double-blind, active and placebo-controlled study. Lancet Neurol. 2008;7(10):875–884.

38.

Sacco

Diener

Yusuf

. Aspirin and extended-release dipyridamole versus clopidogrel for recurrent stroke. N Engl J Med. 2008;359(12):1238–1251.

39.

Beller

GA.

Pharmacologic stress imaging. JAMA. 1991;265(5):633–638.

40.

Ranhosky

Kempthorne-Rawson

The safety of intravenous dipyridamole thallium myocardial perfusion imaging. Intravenous Dipyridamole Thallium Imaging Study Group. Circulation. 1990;81(4):1205–1209.

41.

Perper

Segall

GM.

Safety of dipyridamole-thallium imaging in high risk patients with known or suspected coronary artery disease. J Nucl Med. 1991;32(11):2107–2114.

42.

Aggrenox [package insert]. Ridgefield, CT: Boehringer Ingelheim Pharmaceuticals, Inc; 2009.

43.

Harrington

Stone

McNulty

. Platelet inhibition with cangrelor in patients undergoing PCI. N Engl J Med. 2009;361(24):2318–2329.

44.

Bhatt

Lincoff

Gibson

. Intravenous platelet blockade with cangrelor during PCI. N Engl J Med. 2009;361(24):2330–2341.