Author + information
- Received March 20, 2018
- Revision received April 13, 2018
- Accepted April 13, 2018
- Published online August 20, 2018.
- Sara Ariotti, MDa,
- Luis Ortega-Paz, MDb,
- Maarten van Leeuwen, MDc,d,
- Salvatore Brugaletta, MD, PhDb,
- Sergio Leonardi, MD, MHSe,
- K. Martijn Akkerhuis, MD, PhDf,
- Stefano F. Rimoldi, MDa,
- Gladys Janssens, MDc,
- Umberto Gianni, MDe,
- Jan C. van den Berge, MDf,
- Alexios Karagiannis, PhDg,
- Stephan Windecker, MD, PhDa,
- Marco Valgimigli, MD, PhDa,∗ (, )
- on behalf of the HI-TECH Investigators
- aSwiss Cardiovascular Center Bern, Bern University Hospital, Bern, Switzerland
- bCardiovascular Clinic Institute, Hospital Clínic, University of Barcelona, IDIBAPS, Barcelona, Spain
- cDepartment of Cardiology, VU University Medical Center, Amsterdam, the Netherlands
- dDepartment of Cardiology, Isala Heart Centre, Zwolle, the Netherlands
- eDepartment of Cardiology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
- fDepartment of Cardiology, Erasmus University Medical Center, Rotterdam, the Netherlands
- gCTU Bern, Institute of Social and Preventive Medicine, University of Bern, Bern, Switzerland
- ↵∗Address for correspondence:
Dr. Marco Valgimigli, Swiss Cardiovascular Center Bern, Bern University Hospital, CH-3010, Bern, Switzerland.
Objectives The study sought to assess whether treatment with ticagrelor, as compared with prasugrel and clopidogrel, improves endothelium-dependent dilation throughout the course of the treatment and other vascular biomarkers, including systemic adenosine plasma levels.
Background The in vivo off-target effects of ticagrelor in post–acute coronary syndrome (ACS) patients remain poorly characterized.
Methods Fifty-four stable post-ACS patients were sequentially exposed to each of the 3 oral P2Y12 inhibitors following a 3-period balanced Latin square crossover design with 4 weeks per treatment in 5 European centers. The primary endpoint was the assessment of endothelial function with pulse amplitude tonometry and expressed as reactive hyperemia index at treatment steady state. Secondary endpoints included reactive hyperemia index after loading or before maintenance regimen, systemic adenosine plasma levels, a wide set of vascular biomarkers, and ticagrelor or AR-C124910XX plasma levels throughout each ticagrelor period. In 9 patients, the evaluation of endothelial function was performed simultaneously by pulse amplitude tonometry and flow-mediated dilation.
Results Reactive hyperemia index did not differ after ticagrelor (1.970 ± 0.535) as compared with prasugrel (2.007 ± 0.640; p = 0.557) or clopidogrel (2.072 ± 0.646; p = 0.685), nor did systemic adenosine plasma levels or vascular biomarkers at any time points. P2Y12 platelet reactivity units were lower after ticagrelor as compared with clopidogrel at all time points and after maintenance dose as compared with prasugrel. Flow-mediated dilation did not differ after the maintenance dose of ticagrelor as compared with clopidogrel and prasugrel.
Conclusions Ticagrelor did not improve endothelial function or increased systemic adenosine plasma levels as compared with prasugrel and clopidogrel in stabilized patients who suffered from an ACS. (Hunting for the Off-Target Properties of Ticagrelor on Endothelial Function in Humans [HI-TECH]; NCT02587260).
Ticagrelor, prasugrel, and clopidogrel inhibit platelet aggregation by inhibiting the adenosine diphosphate P2Y12 receptor, and in combination with aspirin have become a Class I guideline-recommended treatment in patients with acute coronary syndromes (ACS) or percutaneous coronary intervention (1).
Prasugrel and clopidogrel are thienopyridines, require conversion to an active metabolite, and mediate an irreversible inhibition of the target receptor. Ticagrelor is a nonthienopyridine direct and reversible P2Y12 platelet receptor antagonist and, unlike prasugrel or clopidogrel, concentration-dependently inhibits the sodium-independent equilibrative nucleoside transporter 1 (ENT1) (2). This ticagrelor-mediated off-target effect has potential to increase adenosine levels, which may carry important clinical implications (2,3).
Increased adenosine levels in patients taking ticagrelor may explain some drug-specific side effects such as dyspnea and bradycardia or ventricular pauses (3). In addition, the ticagrelor-mediated increase of adenosine levels might improve endothelial function (4), a possible barometer of the total atherosclerotic risk burden (5) and this effect may contribute to explain the reduced risk of mortality observed with ticagrelor as compared with clopidogrel in the PLATO (Platelet Inhibition and Patient Outcomes) study.
There is limited and inconsistent evidence (6–8) that ticagrelor can increase adenosine plasma levels and subsequently improve endothelial function as compared with prasugrel and clopidogrel. We aimed to assess the effects of ticagrelor compared with other oral P2Y12 inhibitors on the endothelial function, systemic adenosine plasma levels, and circulating vascular biomarkers at currently approved regimens in post-ACS patients.
Study design, procedures, and patients
The HI-TECH (Hunting for the off-target propertIes of Ticagrelor on Endothelial function and other Circulating biomarkers in Humans) trial (NCT02587260) is a randomized, open-label, crossover study conducted at 5 centers in Switzerland, the Netherlands, Spain, and Italy. Detailed inclusion and exclusion criteria were previously reported (9) and are itemized in the Online Appendix. Eligible patients were those who suffered at least 30 days earlier from an ACS, were free from ischemic or bleeding complications, and reported regular intake of dual antiplatelet therapy regimen consisting of aspirin (80 to 160 mg daily) and a clinically indicated P2Y12 inhibitor, including ticagrelor, prasugrel or clopidogrel. After a baseline pre-randomization assessment, each patient was sequentially exposed to each of the 3 oral P2Y12 inhibitors following a 3-period balanced Latin square crossover design with 4 weeks per treatment period. Patients were allocated in a 1:1:1:1:1:1 ratio to 1 of 6 possible treatment sequences (Figure 1). Allocation of study treatment was performed via an Internet-based interactive randomization system and achieved with a computer-generated random sequence with random block size, stratified according to the clinical site and the presence of diabetes mellitus.
Post-randomization measurements were performed 1 to 2 h following the loading dose (LD) of the first assigned oral P2Y12 inhibitor (ticagrelor at 180 mg [T1] or prasugrel at 60 mg [P1] or clopidogrel at 600 mg [C1]). Patients were then requested to come back to each recruiting site 30 ± 5 days thereafter. All measurements were then repeated before (T2, P2, or C2) and 1 to 2 h after (T3, P3, or C3) the witnessed intake of the maintenance dose (MD) of the same P2Y12 inhibitor (90 mg twice a day for ticagrelor; 10 mg/day for prasugrel, or 5 mg/day if >75 years of age or weight <60 kg; and 75 mg/day for clopidogrel). One to 7 days thereafter, patients returned to the referral hospital to receive the LD of the second randomized P2Y12 inhibitor followed by an identical assessment algorithm until the completion of the randomized sequence (Online Appendix). No washout time was allowed before or in-between the randomized treatment sequences. Patients were requested to fast for at least 2 h before each hospital visit; caffeine-containing beverages were not permitted for 12 h before each study visit.
At baseline and each study visit, finger plethysmography assessment (EndoPAT 2000 device, Itamar Medical, Caesarea, Israel) was performed. Blood samples were taken for the assessment of vascular biomarkers (Online Appendix), systemic plasma adenosine, platelet P2Y12 inhibitor, and aspirin functional assays (VerifyNow system, Accriva Diagnostics, San Diego, California). Ticagrelor and AR-C124910XX (ticagrelor active metabolite) plasma levels were measured at baseline and throughout each ticagrelor sequence (Online Appendix). Patients included at Bern University Hospital underwent simultaneous evaluation of endothelial function with ultrasound assessment of flow-mediated dilation (FMD) and finger plethysmography, as previously described (9) (Online Appendix).
Assessment of endothelial function
Assessment of endothelial function was obtained using pulse amplitude tonometry (primary and secondary endpoint measures), a collection of vascular biomarkers (secondary endpoint measures), and FMD of the brachial artery in a subset of patients (exploratory endpoint measures) as described (see Online Appendix). Pulse amplitude tonometry is an operator-independent, Food and Drug Administration–approved method to measure the endothelium-dependent dilation in response to reactive hyperemia (10). The pulse amplitude tonometry device records digital pulse wave amplitude using fingertip plethysmography (EndoPAT, Itamar Medical) and quantifies the endothelium-mediated changes in vascular tone, elicited by a 5-min occlusion of the brachial artery. A post-occlusion to pre-occlusion ratio is calculated by the EndoPAT software and expressed as the reactive hyperemia index (RHI). These values are normalized to measurements from the contralateral arm, which serves as a control for non–endothelial-dependent systemic effects. An RHI value of 1.670 or below denotes an abnormal endothelium-dependent dilation (endothelial dysfunction) in response to reactive hyperemia (10).
Study endpoints and statistical considerations
The primary endpoint was defined as RHI at treatment steady state, assessed 1 to 2 h after MD intake of the 3 P2Y12 inhibitors (T3, P3, C3), and consisted of 2 main comparisons: ticagrelor versus prasugrel difference in RHI and ticagrelor versus clopidogrel difference in RHI. Secondary endpoints included RHI assessed 1 to 2 h after LD (T1, P1, C1), before MD (T2, P2, C2) P2Y12 inhibitor administration, and other biomarkers of endothelial function (Online Appendix). For sample size calculation, based on a repeat 2-way analysis of variance (ANOVA) measures, we set a mean RHI of 1.800 with a within-subjects SD of 0.31 (11). With 36 patients completing all sequences (i.e., 6 patients/sequence) the study provided 90% power to detect a 10% RHI relative change in the ticagrelor group (RHI after ticagrelor MD administration) with a 2-sided alpha level at 5%. A final sample size of at least 50 patients was targeted to account for dropouts and incomplete data assessment at all time points.
The primary endpoint only of the HI-TECH trial was previously reported (12). In this paper, we report the full results of the trial including all secondary endpoints and predefined subgroup analyses.
The primary endpoint was analyzed using repeated-measures 1-factorial ANOVA. The treatment factor had 3 levels: ticagrelor, prasugrel, or clopidogrel. The ANOVA yielded the differences between the 2 main comparisons: ticagrelor RHI versus prasugrel RHI and ticagrelor RHI versus clopidogrel RHI. The significance of the 2 main comparisons was combined using the Benjamini-Hochberg method to assess the primary endpoint. The null hypothesis of randomized treatment equivalence comparing the response in RHI after ticagrelor versus prasugrel and ticagrelor versus clopidogrel administration was rejected if significance was achieved for both main comparisons at a 2-sided alpha level of 0.05, or 1 comparison at a 2-sided alpha level of 0.025 (9). Within-subgroup comparisons were performed using a repeated-measures ANOVA. Additionally, p values of the interaction effect between the subgroup and the treatment factor were calculated from the repeated-measures ANOVA including the main effects and the interaction.
Correction for possible intragroup correlation was done by the Greenhouse-Geisser method. Each secondary endpoint was analyzed using nonparametric paired sign tests for the 2 main comparisons: ticagrelor versus prasugrel and ticagrelor versus clopidogrel. All analyses were performed on an intention-to-treat basis using Stata version 14.2 (StataCorp, College Station, Texas) and R version 3.4.0 (R Foundation for Statistical Computing, Vienna, Austria).
A total of 54 patients were allocated to 1 of the 6 randomization sequences from December 17, 2015, to October 25, 2016 (Online Figure 1). Of these, 50 (92.6%) patients completed the randomized P2Y12 inhibitor sequence, and the primary endpoint measure was available for 47 (87.0%) (Figure 2).
The baseline features were similar across groups (Table 1). The mean time from index ACS to baseline visit was 233 ± 189 days, ranging from 38 to 1,023 days (Figure 2, Table 1). At the baseline visit, all patients were on dual antiplatelet therapy with aspirin (100.0%) and ticagrelor (69.0%), clopidogrel (24.0%), or prasugrel (7.0%). All patients but 2 (96.2%) were on statins and 47 (87.0%) fulfilled high-intensity criteria (Table 1). No ischemic or bleeding event was noted throughout the study.
Reactive hyperemia index
RHI after MD assessment (primary endpoint) did not differ after ticagrelor (n = 51; mean 1.970 ± 0.535) as compared with prasugrel (n = 50; mean 2.007 ± 0.64; difference: −0.048; 95% confidence interval: −0.212 to 0.115; p = 0.557) or clopidogrel (n = 49; mean 2.072 ± 0.646; difference: −0.034; 95% confidence interval: −0.200 to 0.132; p = 0.685) (Figure 3, Table 2). A matched and per-protocol analysis (sensitivity analysis) restricted to 45 within-subject comparisons across the 3 P2Y12 inhibitors provided identical results (Table 2). The proportion of patients with on-treatment endothelial dysfunction (RHI ≤1.670) after MD assessment did not differ either across P2Y12 inhibitors (n = 15 [29.4%] after ticagrelor; n = 17 [34%] after prasugrel; n = 11 [22.4%] after clopidogrel). RHI after LD or before MD of ticagrelor was also similar compared with prasugrel or clopidogrel (Figure 3, Table 2). Results for the primary endpoint were consistent across multiple subgroups (Online Figure 2). There was no significant interaction between the primary endpoint and the randomized sequence (p = 0.492).
Adenosine, platelet reactivity, vascular biomarkers, and FMD assessment
Systemic adenosine plasma levels did not differ after ticagrelor as compared with prasugrel or clopidogrel at any time point (Figure 3, Table 2). These results remained consistent at multiple subgroup analyses (Online Figure 3). In ticagrelor-treated patients, there was no correlation between adenosine plasma levels and ticagrelor or AR-C124910XX levels. P2Y12 platelet reactivity units were lower after ticagrelor as compared with clopidogrel at all time points, and it was lower after MD, but not after LD as compared with prasugrel (Figure 3, Table 2). None of the vascular biomarkers differed after ticagrelor as compared with prasugrel or clopidogrel at any time point (Figures 3 and 4). In a subset of 9 patients, FMD of the brachial artery was greater after the MD of ticagrelor (median 5.00%; interquartile range: 4.20% to 6.60%) as compared with clopidogrel (median 3.20%; interquartile range: 1.98% to 4.35%; p = 0.004), but not as compared with prasugrel (median 4.04%; interquartile range: 3.65 to 5.71; p = 0.18), nor did it differ at any other time point (Table 2, Online Table 1).
We conducted a randomized, open-label, balanced Latin square crossover study at 5 European centers to assess whether treatment with ticagrelor improves endothelium-dependent dilation. We also measured systemic adenosine plasma levels and several markers of endothelial function. Our findings do not support a measurable effect of ticagrelor as compared with prasugrel and clopidogrel on endothelium function, assessed by pulse amplitude tonometry, or other circulating vascular biomarkers in post-ACS patients. Systemic adenosine plasma levels did not differ at any time points. Also, none of the assessed vascular biomarkers was affected by treatment with ticagrelor as compared with prasugrel and clopidogrel. There was no signal of heterogeneity across the pre-specified subgroups for the primary endpoint or adenosine plasma levels. FMD of the brachial artery performed in a subset of patients did not provide evidence for a differential effect of ticagrelor as compared with prasugrel and clopidogrel (Figure 5).
Several lines of research have suggested that ticagrelor may exert an adenosine-mediated P2Y12–independent mechanism of action. During the clinical development program of ticagrelor, dyspnea and ventricular pauses were observed in some patients and confirmed at a pivotal approval study (13). Ticagrelor was shown during in vitro experiments to inhibit adenosine uptake via inhibition of ENT1, especially if levels exceeded 1.0 μmol/l (2). In an in vitro experiment, ticagrelor added to whole blood, a high-protein binding medium, concentration-dependently conserved added adenosine. The effect was significant at ≥1.0 μmol/l, suggesting a potential effect in a clinical setting (14). In patients with ACS who received ticagrelor (90 mg twice a day) for 4 weeks, the steady-state mean of maximum plasma concentration was 1.5 μmol/l (15), suggesting that ticagrelor may exert a measurable inhibition of the ENT1 pathway in humans. However, ticagrelor and AR-C124910XX are extensively plasma protein bound in vivo (>99.7%) (2), meaning that the unbound concentration is in the low nanomolar range. Based on the affinity of ticagrelor for the ENT1 transporter (Ki = 41 nmol/l) and the strong plasma protein binding, it was therefore anticipated that at clinically approved doses, ticagrelor could only partially inhibit ENT1 (2).
Circulating systemic plasma adenosine levels are very low in humans. However, it has been reported that they can increase locally at ischemic tissues (16). It was therefore suggested that ticagrelor can increase extracellular adenosine at tissue level through inhibition of ENT1 in erythrocytes and platelets, rather than increase systemic plasma levels (17). We did not find any measurable effect of ticagrelor on systemic adenosine plasma levels compared with the other oral P2Y12 inhibitors, although acknowledging that adenosine tissue levels were not explored.
Ours is the third randomized trial to assess the off-target effects of ticagrelor on endothelial function, yet the first extending the comparison of ticagrelor to both prasugrel and clopidogrel, and the first multicenter trial being executed in a chronic setting, focusing on stabilized post-ACS patients. Ticagrelor was associated with an increase in adenosine plasma levels and an improvement of endothelial function at digital peripheral artery tonometry in 60 ACS patients randomized at the time of the index event to receive ticagrelor or clopidogrel (6). No crossover to the other treatment was foreseen in that study to confirm the findings. More recently, it was observed that compared with prasugrel, ticagrelor decreased inflammatory cytokines such as interleukin 6 and tumor necrosis factor alpha and increased circulating endothelial progenitor cells, contributing to improved arterial endothelial function (evaluated by FMD) in 62 diabetic ACS patients (7). In prior investigations, the duration of treatment with ticagrelor was either 4 or 5 weeks, and therefore comparable to the 4-week duration of each P2Y12 inhibitor in our study. We recruited patients after a mean time from index ACS of 233 ± 189 days, ranging from 38 to 1,023 days. In both previous studies, patients were recruited at index admission for ACS without prior exposure to dual antiplatelet therapy. We focused on stabilized post-ACS patients to minimize the risks that the natural course of the disease (i.e., the acute inflammatory phase of ACS and tissue ischemia) may confound the comparison across P2Y12 inhibitors and systemic adenosine plasma levels. Consistent with our results, Xanthopoulou et al. (8) observed no changes in peripheral arterial tonometry assessment before and after cessation of ticagrelor therapy in a small group of patients with stable coronary artery disease. Furthermore, our findings are also consistent with the DISPERSE-2 (Dose Confirmation Study Assessing Anti-Platelet Effects of AZD6140 vs Clopidogrel in NSTEMI 2) trial, which compared ticagrelor with clopidogrel and found no significant differences in the inflammatory biomarkers in 990 ACS patients recruited across 152 participating centers (18).
FMD is currently the gold standard for noninvasive assessment of endothelial function in humans. We used the digital pulse amplitude tonometry as the primary endpoint of our multicenter trial because it is operator independent, reproducible, and highly correlated with FMD assessment. The pulse amplitude tonometry measures of vascular function more closely reflect basal blood flow in the brachial artery than reactive hyperemia–induced changes in the arterial diameter or flow velocity (19). The implementation of washout periods after each P2Y12 inhibitor sequence might have allowed the assessment of carryover effects, if any, after each investigated drug. In this regard, we evaluated the interaction of the primary endpoint with the sequence, which was not significant. Patient exposure to a nonrandomly selected P2Y12 inhibitor before the inclusion in our study has hampered the assessment of ticagrelor off-target effects when started in P2Y12 inhibitor naïve patients during index ACS. However, despite not being randomly allocated, no effect of ticagrelor treatment versus other P2Y12 inhibitors was noted even at baseline in our study. Our findings do not confirm previous observations that ticagrelor increases systemic adenosine plasma levels and question the existence of a measurable ticagrelor’s effect on endothelial function or vascular biomarkers in stabilized post-ACS patients at currently approved regimen.
We found no evidence that ticagrelor exerts measurable adenosine-mediated off-target effects on endothelial function at currently approved regimen in stabilized patients who suffered from an ACS.
WHAT IS KNOWN? Unlike prasugrel or clopidogrel, ticagrelor is a nonthienopyridine direct and reversible P2Y12 platelet receptor inhibits, at least partially, the sodium-independent ENT1. This ticagrelor-mediated off-target effect has potential to increase adenosine plasma levels, which may carry important clinical implications and may explain ticagrelor-specific side effects, such as dyspnea and bradycardia or ventricular pauses.
WHAT IS NEW? In the HI-TECH trial, endothelial-dependent dilatation, assessed with the RHI, or in a subset of patients with FMD of the brachial artery, did not differ after ticagrelor as compared with prasugrel or clopidogrel in stabilized post-ACS patients at the currently approved regimen, nor did systemic adenosine plasma levels or vascular biomarkers differ at any time points.
WHAT IS NEXT? Further research is needed to assess the effect of ticagrelor on tissue adenosine plasma levels in humans compared with other oral P2Y12 inhibitors and its relationship with clinical outcomes.
The authors thank Jurgen Ligthart and Karen Witberg for technical assistance with the EndoPAT measurements.
This work was supported by a research grant from AstraZeneca. The study was designed by the principal investigator (Dr. Valgimigli), and sponsored by the Erasmus Medical Center and a nonprofit organization. The study sponsor and supporting company had no role in study design, data collection, data monitoring, analysis, interpretation, or writing of the report. CTU Bern, University of Bern, has a staff policy of not accepting honoraria or consultancy fees. However, CTU Bern is involved in the design, conduct, or analysis of clinical studies funded by not-for-profit and for-profit organizations. In particular, pharmaceutical and medical device companies provide direct funding to some of these studies. Drs. van Leeuwen and Janssens have received institutional research grant support from AstraZeneca. Dr. Brugaletta has received institutional research grant support from AstraZeneca; and speaker fees from Abbott Vascular and Boston Scientific. Dr. Leonardi has received consulting fees from AstraZeneca, Ely Lilly, The Medicines Company, and Chiesi. Dr. Rimoldi has served on the Speakers Bureau for Servier and Menarini. Dr. Windecker has received institutional research grant support from Abbott, Bracco, Biotronik, Boston Scientific, St. Jude Medical, Medtronic, and Terumo. Dr. Valgimigli has received grant support from AstraZeneca and Terumo; and personal fees from AstraZeneca, Terumo, Abbott Vascular, Bayer, Amgen, Cardinal Health, Biosensors, Abbott Vascular, and Daiichi-Sankyo. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- acute coronary syndrome
- analysis of variance
- equilibrative nucleoside transporter 1
- flow-mediated dilation
- loading dose
- maintenance dose
- reactive hyperemia index
- Received March 20, 2018.
- Revision received April 13, 2018.
- Accepted April 13, 2018.
- 2018 American College of Cardiology Foundation
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