Impact of Chronic Kidney Disease on the Pharmacodynamic and Pharmacokinetic Effects of Ticagrelor in Patients With Diabetes Mellitus and Coronary Artery Disease
Phase: PHASE4
Conditions: Coronary Artery Disease, Diabetes Mellitus
Sponsor: University of Florida

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Impact of chronic kidney disease on the pharmacodynamic and pharmacokinetic 
effects of ticagrelor in patients with diabetes mellitus and coronary artery disease 
 
 
 
 
 
 
 
Dominick J Angiolillo, MD, PhD, FACC, FESC, FSCAI 
Director of Cardiovascular Research 
Associate Professor of Medicine 
Division of Cardiology 
University of Florida College of Medicine - Jacksonville 
655 West 8th Street 
Jacksonville, FL – 32209 
Tel: 904-2443933, Fax: 904-2443102 
dominick.angiolillo@jax.ufl.edu 

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Project overview 
Patients with diabetes mellitus (DM) are at increased risk of atherothrombotic events. 
Importantly, DM is a key risk factor for the development of chronic kidney disease (CKD), 
which further enhances atherothrombotic risk. These observations underscore the importance of 
antiplatelet therapy for prevention of atherothrombotic recurrences in these high-risk patients. 
Clopidogrel is the most widely used platelet P2Y12 receptor inhibitor. However, despite its 
clinical benefit, patients with DM and CKD frequently experience recurrent atherothrombotic 
events. This may be in part due to the impaired pharmacokinetic (PK) and pharmacodynamic 
(PD) effects of clopidogrel in patients with DM and CKD. Since both DM and CKD represent 
pandemic public health problems, the prevalence of which will double over the next 20 years, 
identifying antiplatelet agents with more favorable PK/PD profiles is of key importance.  
Ticagrelor is an oral, reversible, non-competitive P2Y12 receptor inhibitor with more potent 
and consistent platelet inhibition than clopidogrel. In large-scale clinical investigation, ticagrelor 
significantly reduced ischemic events to a greater extent than clopidogrel, a finding that was 
consistent also among DM patients. In patients with CKD, ticagrelor led to an even greater 
relative risk reduction of ischemic events, including cardiovascular mortality, compared to 
patients without CKD. However, to date there has been no analysis on the efficacy of ticagrelor 
in DM patients according to CKD status. Moreover, although PD studies showed enhanced 
platelet inhibition associated with ticagrelor, it is unknown how this may be affected by CKD 
status. Ultimately, how PK/PD profiles of different ticagrelor dosing regimens may be affected 
by DM and CKD status is also unknown. The proposed study is aimed to show the impact of 
CKD status among patients with DM and CAD on PD and PK profiles of ticagrelor used at 2 
doses (90mg bid and 60mg bid) in the setting of a prospective, randomized, cross-over trial. 

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Background: 
Diabetes mellitus (DM) and chronic kidney disease (CKD) represent pandemic diseases and 
a major public health concerns because of their prevalence and poor cardiovascular prognosis (1-
3). DM is the most common cause of CKD and up to 40% of diabetic patients develop CKD (4). 
CKD was present in approximately one-third of patients with NSTE-ACS and was a powerful 
independent predictor of subsequent mortality (5). Importantly, patients with DM or CKD have a 
high thrombotic milieu and frequently experience recurrent atherothrombotic events (6-11). 
Multiple mechanisms may contribute to these observations, including accelerated platelet 
turnover rates (12,13). These observations underscore the importance of antiplatelet therapy for 
prevention of atherothrombotic recurrences in these high-risk patients. Clopidogrel is the most 
widely used platelet P2Y12 receptor inhibitor. However, despite its clinical benefit, patients with 
DM and CKD frequently experience recurrent atherothrombotic events. The high 
atherothrombotic recurrence rates in DM and CKD patients are partly due to the inadequate 
response to antiplatelet agents, in particular clopidogrel, which is enhanced with the co-existence 
of these disorders (14-17). Therefore, identifying antiplatelet agents with more favorable 
pharmacokinetic (PK) and pharmacodynamics (PD) profiles is of key importance.  
Ticagrelor represents an attractive strategy especially in high-risk patients, such as those 
with DM and/or CKD (18-20). Ticagrelor, a cyclopentyltriazolopyrimidines, is an oral, 
reversible, non-competitive P2Y12 receptor inhibitor with rapid onset and offset of action and 
provides a stronger and more consistent platelet inhibition than clopidogrel (21-23). Although 
ticagrelor is a direct acting agent, ~30% of its effects are attributed to a metabolite (AR-
C124910XX) derived from hepatic metabolism (23). In the large scale phase III PLATO (Platelet 
Inhibition and Patient Outcomes) trial conducted in ACS patients, ticagrelor, in addition to 

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aspirin, was associated with a significant reduction in ischemic events, driven by a reduction in 
myocardial infarction (MI) and cardiovascular mortality, at 1-year compared with clopidogrel 
(24). Such benefit was irrespective of patient management (invasive vs non-invasive). Of note, 
this higher efficacy was associated with no differences in overall PLATO major bleeding, 
although there was an increased rate of non-coronary artery bypass graft bleeding (24). A 
predefined subgroup analysis of patients with DM (n=4,662) showed a non-significant reduction 
of the rate of the primary end point (14.1% vs. 16.2%; hazard ratio [HR]: 0.88; 95% confidence 
interval [CI]: 0.76-1.03), which however was consistent with the overall trial results (p for 
interaction: 0.49) (19). The benefit of ticagrelor was enhanced in patients with worse metabolic 
control, as defined per HbA1c levels above the median (19). In the subgroup of patients with 
CKD, ticagrelor led to an enhanced benefit, with a 23% relative risk reduction of the primary 
ischemic end point (compared with a nonsignificant 10% reduction in patients without CKD) and 
an even more remarkable 4.0% absolute and 28% relative risk reduction of all-cause mortality 
(20).  
The recently reported PEGASUS-TIMI 54 (Prevention of Cardiovascular Events in Patients 
With Prior Heart Attack Using Ticagrelor Compared to Placebo on a Background of Aspirin-
Thrombolysis in Myocardial Infarction 54) expands upon the findings of the PLATO trial 
showing that prolonging dual antiplatelet therapy up to 3 years with aspirin and ticagrelor (tested 
at two different doses: 90 mg bid and 60 mg bid) among high-risk patients who experienced a MI 
in the prior 1-3 years significantly reduced the ischemic events albeit at the expense of increased 
bleeding complications (25). Patients required enrichment factors to be eligible for study entry 
which included DM and CKD; others were age >65 year, multiple prior MI, and multivessel 
disease which are all common features in patients with DM and/or CKD. Although both 

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ticagrelor doses showed a similar magnitude of clinical efficacy, the 60 mg dose appeared to 
have a more favorable safety profile (25). In patients with DM, the benefits of long-term use of 
ticagrelor was consistent with the overall trial results (HR, 0.85; 95% CI, 0.71-1.03 in DM versus 
HR, 0.85; 95% CI, 0.73-0.99 in non-DM; p for interaction: 0.97). There was also no apparent 
heterogeneity in the efficacy of ticagrelor at either dose with respect to the presence or absence 
of CKD (HR, 0.81; 95% CI, 0.66-0.99 in CKD versus HR, 0.88; 95% CI, 0.76-1.03 in non-CKD; 
p for interaction: 0.48). Indeed more insights on the efficacy of ticagrelor in patients with DM, 
and how this may be affected by CKD status, is being evaluated in the ongoing THEMIS (Effect 
of Ticagrelor on Health Outcomes in DiabEtes Mellitus Patients Intervention) Study 
(NCT01991795).  
 
Significance and Rationale: 
The emerging role of ticagrelor as a key agent for the secondary prevention of ischemic 
events underscores the need for a comprehensive understanding of its PK and PD profiles in 
high-risk settings such as patients with DM and CKD. These assessments will provide important 
mechanistic insights to recently complete and ongoing clinical outcomes studies. Importantly, 
these assessments will allow to better understand the aspects of ticagrelor pharmacology which 
may differ from other P2Y12 inhibiting therapies. DM significantly affects the PK and PD of 
thienopyridine-class P2Y12 receptor blockers (16, 26-28). However, it is unknown if DM also 
modulates the plasma concentrations of ticagrelor and its CYP3A4-derived metabolite 
(ARC124910XX). Platelets from patients with CKD are exposed to higher levels of dinucleoside 
polyphosphates, which can act as an agonists of the P2Y12 receptor (29). Moreover, CKD per se 

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may affect disposition process, efficacy or safety of drugs (30,31). However, ticagrelor does not 
need biotransformation and its clearance depends minimally on renal function (23).  
To date there has not been any analysis on the clinical efficacy of ticagrelor in DM patients 
with CKD. Moreover, although ticagrelor improved heightened platelet reactivity in DM 
patients, it is unknown how this may be affected by CKD status. Ultimately, whether PK/PD 
profiles of different ticagrelor dosing may be affected by CKD status in patients with DM is also 
unknown. 
 
Study aims 
The aim of the study is to assess the impact of CKD status on the PK and PD profile of 
ticagrelor among patients with DM and CAD. Moreover, how DM and CKD status can affect 
PK/PD profiles in patients treated with different dosing regimens of ticagrelor (60mg and 90 mg) 
will also be assessed.  
 
Study Population 
The study will be performed at the University of Florida Health Science Center at UF 
Health Jacksonville - Division of Cardiology. Patients will be recruited in the Cardiology Clinics 
of our institution and will be screened by Cardiology Research staff, who will verify that all 
candidates meet inclusion and exclusion criteria. Results from blood tests performed within the 
last 90 days will be considered valid for screening purposes. If these are not available, a blood 
sample will be collected for the screening phase. The research design will have common 
inclusion and exclusion criteria as described below.  

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Inclusion criteria: 
 
Age >18 years. 
 
Type 2 DM, defined according to WHO definition, on treatment with oral hypoglycemic 
agents and/or insulin for at least 2 months without any changes in treatment regimen;  
 
Angiographically documented CAD.  
 
On treatment with low-dose aspirin (81mg/day) and clopidogrel (75mg/day) for at least 
30 days as part of standard of care.  
Exclusion criteria: 
 
Patients with end-stage renal disease on hemodialysis. 
 
Use of any antiplatelet therapy (except aspirin and clopidogrel) in past 30 days.  
 
Use of parenteral or oral anticoagulation in past 30 day.  
 
Active pathological bleeding.  
 
History of intracranial hemorrhage with prior hemorrhage stroke.  
 
Blood dyscrasia or bleeding diathesis.  
 
Any active malignancy.  
 
Platelet count < 80x106/µl.  
 
Hemoglobin <8 g/dl.  
 
Known hepatic dysfunction (known moderate and severe hepatic dysfunction). 
 
Hemodynamic instability.  
 
Known allergy or hypersensitivity to ticagrelor or any excipients. 
 
Pregnant / lactating females (women of childbearing age must use reliable birth control 
while in the study).  

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 
Strong inhibitors of cytochrome CYP3A4 and potent inducers of cytochrome CYP3A4 
(to avoid interaction with ticagrelor): ketoconazole, itraconazole, voriconazole, 
clarithromycin, nefazodone, ritonavir, saquinavir, nelfinavir, indinavir, atazanavir and 
telithromizycin. 
 
Patients with sick sinus syndrome (SSS) or high degree AV block without pacemaker 
protection. 
 
Research design 
The proposed investigation is a prospective, randomized, cross-over, open-label, PD and PK 
study in which CAD patients with DM on low-dose (81mg) aspirin and standard-dose (75mg) 
clopidogrel will be prospectively recruited. Patients will be screened during their regularly 
scheduled outpatient visits and informed about the study. Patients will be stratified according to 
CKD status into CKD and non-CKD groups. CKD will be defined according to the functional 
definition of the KDIGO (Kidney Disease: Improving Global Outcomes) guidelines (CKD: 
glomerular filtrate rate [GFR] <60ml/min/1.73m2; non-CKD: GFR ≥ 60ml/min/1.73m2) (32,33). 
The rationale for considering the functional classification to initially stratify patients is in line 
with clinical studies showing the increased cardiovascular risk and markedly dysfunctional 
platelets according to GFR strata (10,11,16). For patient stratification, GFR will be estimated 
through the CKD-EPI (chronic kidney disease epidemiology collaboration) equation, as this is 
suggested by guidelines as the most accurate method to calculate GFR, especially for values in 
the normal range (above 60ml/min/1.73m2) (32,33). For comparison, GFR will also be calculated 
with the MDRD (modification of diet in renal disease) equation. In addition, as exploratory 
analysis, renal function will also be classified according to markers of kidney damage, in 

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particular albuminuria, in order to obtain a more comprehensive information about CKD (32,33). 
Albuminuria will be evaluated as albumin-to-creatinine ration (ACR) expressed in mg/g, which 
is approximately equivalent to albumin excretion rate. According to KDIGO guidelines, CKD 
will be defined as ACR >30mg/g and non-CKD as ACR ≤30mg/g (32,33). Albuminuria will be 
measured on random untimed spot urine samples. DM will be defined according to World Health 
Organization criteria and all subjects with DM will need to be on treatment with oral 
hypoglycemic agents and/or insulin for at least 2 months without any changes in their regimen 
(34).  
After providing informed consent, eligible patients will be divided into CKD group and 
non-CKD group as described above. DM patients with CKD or without CKD will be randomized 
in a 1:1 fashion to one of the following treatment arms: 
A. standard dose (90mg twice daily of ticagrelor) for 7-10 days (phase 1) and then cross-
over to low dose (60mg twice daily of ticagrelor) for 7-10 days (phase 2) 
B. low dose (60mg twice daily of ticagrelor) for 7-10 days (phase 1) and then cross-over to 
standard dose (90mg twice daily of ticagrelor) for 7-10 days (phase 2). 
After the 2 phases of treatment (maximal treatment period 20 days) patients will resume their 
standard DAPT with aspirin and clopidogrel (end of study treatment). 
 
After 7-10 days of treatment, patients will come for their blood sampling in the morning 
hours. In particular, patients will not assume their scheduled morning dose of study medication 
to allow assessment of trough levels of platelet reactivity. Patients will be administered the dose 
of the randomized treatment while at the clinic and blood sampling will be collected after 2 hours 

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in order to assess peak levels of platelet reactivity. After completing the first phase of the 
randomized assignment, patients will initiate the second phase and start assuming assigned 
medication 12 hours the last dose.  
 
Blood for sampling will be collected at the following visits (a total of 5 assessments at 3 visits): 
a) baseline (24 hours after last clopidogrel MD): PD assessments 
b) after 7-10 days of ticagrelor therapy (phase 1-pre-crossover): PK and PD assessment will 
be assessed to define trough (12 ± 2 hours after last ticagrelor dose) and peak (2 hours 
after ticagrelor administration) activity 
c) after 7-10 days of ticagrelor therapy (phase 2-post-crossover): PK and PD assessment 
will be assessed to define trough (12 ± 2 hours after last ticagrelor dose) and peak (2 
hours after ticagrelor administration) activity.  
 
A flow diagram of the study is presented below.  

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Blood sampling: Peripheral venous blood samples (20 mL) will be drawn through a short 
venous catheter inserted into a forearm vein and collected in citrate and serum tubes as 
appropriate for assessments. The first 2-4 mL of blood will be discarded to avoid spontaneous 
platelet activation. 
Laboratory assessments 
1. Whole blood vasodilator-stimulated phosphoprotein (VASP) 
2. VerifyNow P2Y12 point of care testing  
3. Light-transmittence aggregometry (LTA) 
4. Thromboelastography 
5. 
 Reticulated Platelets (RPs) 

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6. Serum thromboxane B2 
 
7. PK assessment 
Description of laboratory assays 
1) Whole blood vasodilator-stimulated phosphoprotein (VASP): VASP phosphorylation 
(VASP-P) is a marker of P2Y12 receptor reactivity, which is the target for ticagrelor. VASP 
will be assessed according to standard protocol using labeled monoclonal antibodies by flow 
cytometry with the Platelet VASP-FCM kit (Biocytex Inc., Marseille, France) as previously 
described (16). PGE1 increases VASP-P levels by stimulation of adenylate cyclase. Binding 
of ADP to P2Y12 leads to Gi-coupled inhibition of adenylate cyclase. Therefore, the 
addition of ADP to PGE1-stimulated platelets reduces PGE1-induced VASP-P levels. If 
P2Y12 receptors are successfully inhibited by inhibitors, addition of ADP will not reduce the 
PGE1-stimulated VASP-P levels. The platelet reactivity ratio (PRI) will be calculated after 
measuring VASP-P levels after stimulation with PGE1 (MFI PGE1) and also PGE1 + ADP 
(MFI PGE1 + ADP). The P2Y12 reactivity ratio = ([MFI PGE1] – [MFI PGE1 + 
ADP]/[MFI PGE1]) x 100%. 
2) VerifyNow Point-of-care Testing: The VerifyNow (VN) System is a turbidimetric based 
optical detection system which measures platelet induced aggregation as an increase in light 
transmittance (Accumetrics, San Diego, CA) and will be utilized according to 
manufacturer’s instructions, as previously described (22). The assay is based on microbead 
agglutination and uses specific reagents for the pathways of interest. The VN-P2Y12 assay, 
by combining ADP+PGE1, measures changes in platelet function specific to P2Y12 receptor 
inhibitors. The assay is based upon the ability of activated platelets to bind fibrinogen. 
Fibrinogen-coated microparticles aggregate in proportion to the number of GP IIb/IIIa 

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receptors expressed. Microbead aggregation is more rapid and reproducible if platelets are 
activated; therefore the reagents are incorporated into the assay channel to induce platelet 
activation without fibrin formation. Light transmittance increases as activated platelets bind 
and aggregate fibrinogen-coated beads. The instrument measures this change in optical 
signal and reports results in P2Y12 Reaction Units (PRU). 
3) Light transmittance aggregometry (LTA): Platelet aggregation will be performed using LTA 
according to standard protocols. Blood will be collected in citrated (3.8%) tubes. LTA will 
be assessed using platelet rich plasma (PRP) by the turbidimetric method in a 2-channel 
aggregometer (chrono-Log 490 Model, Chrono-Log Corp., Havertown) as previously 
described (16,22). Platelet agonists will include 5 and 20 µM ADP. PRP will be obtained as 
a supernatant after centrifugation of citrated blood at 1000 rpm for 10 minutes. The isolated 
PRP will be kept at 37° C before use. Platelet poor plasma (PPP) will be obtained by a 
second centrifugation of the blood fraction at 2800 rpm for 10 minutes. Light transmission 
will be adjusted to 0% with the PRP and to 100% for the PPP for each measurement. Curves 
will be recorded for 6 minutes and platelet aggregation will be determined as the maximal 
percent change in light transmittance from baseline using PPP as a reference. 
4) Thromboelastography (CORA® system): The CORA® system (Haemonetics Corporation, 
Braintree, MA, USA) is a new generation portable thrombelastography technology able to 
evaluate all phases of hemostasis, including time to clot formation, rate of clot formation, 
strength of clot and residual clot strength due to antiplatelet drugs, rate of clot lysis. Whole 
blood samples will be collected into non-gel heparin vacutainer tubes with ≥ 14.5 IU 
heparin/ mL blood, completely filled by vacuum. The PlateletMapping Cartridge are used to 
assess platelet function in patients who have received platelet inhibiting drugs. The 

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PlateletMapping assay consists of a set of agonists, ADP and AA platelet agonists together 
with ActivatorF, which can measure the inhibition of platelet function. This assay 
specifically determines the MA (Maximum Amplitude, a measure of clot strength) and the 
reduction in MA due to antiplatelet therapy and reports it as a percentage of reduction in clot 
strength. The HKH reagent, a combination of kaolin and heparinase, generates test data for 
the uninhibited MA resulting from thrombin activation of the blood sample, while the 
heparinase neutralizes the effects of heparin. The HKH test provides measures of R 
(Reaction time; the amount of time between the start of the test and the beginning of 
coagulation), K (the speed of formation of the clot from R time to a specific clot strength), 
Angle (the speed of clot strengthening), LY30 (Percent lysis 30 minutes after MA is 
finalized) and MA parameters; the activatorF test provides the contribution of fibrin to the 
overall strength of the clot. This test value is used in the calculation of aggregation/inhibition 
for MA ADP and MA AA; the AA and ADP test provide measures of MA, percent 
inhibition and percent aggregation. 
5) Reticulated Platelets: Reticulated platelets (PRs) will be measured with a previously 
described flow cytometry assay (35,36), which is an appropriate method of measuring young 
platelets and platelet turnover that has been validated in multiple studies (37,38).  
6) Serum thromboxane B2: The concentration of serum thromboxane B2 (TXB2) will be 
measured by using the TXB2 EIA kit (Cayman Chemical Company, Ann Arbor, MI) 
according to the instructions of the manufacturer (39). Briefly, samples will be diluted with 
EIA buffer to bring their concentrations within the range of the standard curve. No other 
purification will be performed on any of the samples. A standard curve will be established by 
serial dilution of TXB2 between 1000 pg/mL and 7.8 pg/mL using EIA buffer as the matrix. 

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The concentration of TXB2 in the samples will be calculated from a logistic 4-parameter fit 
of the standard concentrations versus percentage bound/maximum bound. 
7) Pharmacokinetic (PK) assay: Determination of plasma concentration of ticagrelor and its 
active metabolite (AR-C124910XX) will be assessed from blood samples taken at the same 
time points of PD analysis as previously described [40]. Blood samples for measurement of 
drug concentrations will be drawn through the same cannula used for PD sampling. The 
blood will be taken into lithium heparin tubes and placed on ice until centrifugation (2800 
rpm, 4°C, 10 min) within 30 min of sampling to collect the plasma, which will be transferred 
to plain polypropylene tubes and frozen upright at or below -20°C until analysis.  
 
Primary and secondary objectives 
The primary end point is the level of on-treatment platelet reactivity as assessed by VASP-
PRI after treatment with ticagrelor 90mg bid on top of aspirin (81mg/day) for 7-10 days. The 
primary hypothesis of our study is that in DM patients the presence of CKD is associated with 
lower platelet inhibition (assessed by PRI determined by VASP-P) compared with those without 
CKD.  
 
Additional objectives include: 
1) The comparison of PD measures following administration of ticagrelor 90 mg between 
CKD and non-CKD groups at each time point assessed with PRU determined by the 
VerifyNow P2Y12 assay, 5 and 20 µM ADP-induced platelet aggregation by LTA and 
clot kinetic assessed by CORA® system.  

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2) The comparisons of level of platelet reactivity as assessed by multiple assays after 
treatment with ticagrelor 60 mg bid (on top of aspirin 81mg/day) for 7-10 days between 
the CKD and non-CKD groups. 
3) The comparison of plasma concentrations of ticagrelor and its metabolite between the 
DM patients with and without CKD after 7-10 days of ticagrelor (60 and 90 mg) 
treatment.  
4) The comparison of platelet inhibition by standard dose (90mg twice daily) versus low 
dose (60mg twice daily) of ticagrelor in DM patients with or without CKD. 
5) The comparison of the rates of high on-treatment platelet reactivity (HPR), defined as 
VASP-PRI ≥50%, PRU >208, 5 µM ADP-induced maximal platelet aggregation ≥46% 
or ADP induced MA >47 (41,42). 
6) To evaluate whether response to ticagrelor varies with the proportion of RPs in DM 
patients according to the CKD status. 
7) To assess the effects of different levels of P2Y12 inhibition with thromboxane levels.  
8) The comparison of PD and PK measures according to the CKD classification based on 
markers of kidney damage (ACR). 
 
Sample Size Computation and Power Analysis 
The primary hypothesis of our study is that in DM patients the presence of CKD is 
associated with lower platelet inhibition compared with those without CKD after administration 
of ticagrelor 90 mg bid. Under the assumption of 10% absolute difference in mean PRI between 
CKD and non-CKD groups, with a common standard deviation of 13.0% a sample size of 37 
patients per group will allow to detect a difference in platelet aggregation between the two 

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cohorts with a 90% power and two-sided alpha=0.05 (22). Considering the two cohorts (CKD 
and non-CKD), 74 patients with a valid primary end point data will be needed. Assuming a 40% 
rate of invalid results due to hemolysis or drop-out, up to 102 patients will need to be 
randomized in the study. We anticipate to consent approximately 115 patients in order to reach 
the target number of patients. We have chosen a 10% absolute increase of PRI by VASP-P as 
reference value since this has been associated with a difference in clinical outcomes (43). 
 
Statistical analysis plan 
Categorical variables will be expressed as frequencies and percentages. Continuous 
variables will be presented as mean ± SD or median [IQR] when appropriate. Continuous 
variables will be analyzed for normal distribution with the Kolmogorov-Smirnov test. Student’s 
t-test will be used for comparison of normally distributed continuous variables. Wilcoxon test or 
Mann-Whitney’s U test will be used for comparisons of continuous variables not following a 
normal distribution. An analysis of covariance (ANCOVA) method with a general linear model, 
using baseline variables significantly different between groups as covariates, will be used to 
evaluate the overall difference between groups and all between-groups comparisons, in line with 
other PK and PD studies (22,24). Analysis of variance (ANOVA) will be used to evaluate 
intragroup comparisons. In line with recommendations for cross-over study designs, treatment 
effects will be evaluated considering the functional parameters observed in the overall patient 
population after the treatment regardless of the sequence in which patients received treatment. 
Patients who will successfully complete at least one treatment phase of the study will be 
considered for comparisons. The absolute between-treatment mean differences and 95% 
confidence intervals will be estimated in the two groups. In order to perform an unbiased 

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estimation of the treatment effect, period and sequence effects, which may occur in crossover 
studies, will be evaluated. A p-value <0.05 will be considered statistically significant. Statistical 
analysis will be performed using a SPSS v22.0 software. 
 
Publication Strategy/Additional Information  
This study will be performed at the University of Florida Health Science Center at UF 
Health - Division of Cardiology. Patients will be recruited and consented at the Cardiology 
clinics in the UF Health system. Patients will be screened by cardiology research, who will 
verify that all candidates meet inclusion criteria. Subjects will be identified with a number and 
data collection sheets will all be stored in a locked area. Data will be kept for 6 years after 
enrollment ends to comply with HIPAA regulations. Patients will receive a handout with the 
names and telephone numbers of the doctors involved in the study. Patients will sign an informed 
consent relating to the study. Study subjects will be identified first (months 1-18): we expect to 
enroll 4-5 subjects monthly and complete enrollment in 18 months (total: 82 subjects enrolled). 
Months 19-20 will be implied for statistical analysis and months 21-22 for manuscript 
preparation. 
We anticipate no major problems with the described protocol since the approach is a 
straight forward prospective study and is based on well-established methods. However, since 
there is limited experience with to define platelet function in this specific subset, variability may 
be higher than expected and we cannot currently perform a detailed sample size calculation. We 
anticipate adding this to the protocol after inclusion of the study population has been completed. 
If the sample size after one year is estimated to be too small, additional patients will be included. 
This approach is in agreement with recommendations for pilot investigations (44). 

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Possible Discomforts and Risk 
In clinical trials, the most common clinical side effects of ticagrelor were dyspnea (13.8%), 
headache (6.5%), cough (4.9%), dizziness (4.5%), nausea (4.3%), bradycardia (4.4%) 
principally. Infrequent events included intracranial hemorrhage (0.3%) and severe bradycardia 
requiring pacemaker insertion (0.9%). Patients considered to be at high risk of bradyarrhythmic 
events will be excluded. The most important adverse effect associated with the use of ticagrelor 
is bleeding. The risk of spontaneous bleeding with ticagrelor is 2.8% (24). However, such 
bleeding prevalence occurred in the setting of long-term (12-15 months) trials, while our study is 
limited to up to 14 ± 4 days of active treatment thus reducing the risk of bleeding complications. 
All clinical events described above, if they were to occur, as well as death, MI, stroke, and urgent 
revascularization procedure with PCI or coronary artery bypass grafting will be recorded. 
Bleeding data will be collected using PLATO definitions (24). Clinical events will be evaluated 
by a local committee, comprised of 2 faculty members (2 cardiologists), not directly involved in 
the research. In the event of a report of a serious adverse event (major bleeding – defined as life-
threatening: fatal, symptomatic intracranial hemorrhage, leading to a drop in hemoglobin of at 
least 5 g/dL, significant hypotension requiring intravenous inotropes, requiring surgical 
intervention, or requiring transfusion of 4 or more units of blood; non–life-threatening: 
substantially disabling, intraocular bleeding leading to vision loss, or requiring at least 2 units of 
blood; thrombocytopenia <50,000) the local committee will meet and antiplatelet treatment 
management will be managed according to physician recommendation. 
 

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Definition of Adverse Events 
An adverse event is any unintended or undesirable experience that occurs during the course 
of the clinical investigation whether or not it is considered to be therapy related. This includes 
any newly occurring event or previous condition that has increased in severity or frequency since 
the initiation of study treatment. Adverse events will be followed until resolution while the 
patient remains on-study. Once the patient is removed from study, events thought to be related to 
the study therapy will be followed until resolution or until the patient starts a new treatment 
regimen. 
Serious adverse events (SAE): An adverse event occurring while on study and considered 
related (reasonable possibility that the study treatment caused the adverse experience) to the 
study treatment that results in the following outcomes: 
- Death 
- A life-threatening adverse experience. 
- A persistent or significant disability, incapacity, or is a congenital anomaly, or birth defect. 
- Requires inpatient hospitalization, or prolongation of existing hospitalization. 
The definition of serious adverse event also includes ‘important medical event’. Medical 
and scientific judgment should be exercised in deciding whether expedited reporting is 
appropriate in other situations, such as important medical events that may not be immediately 
life-threatening or result in death or hospitalization but may jeopardize the patient and/or may 
require medical or surgical intervention to prevent one of the other outcomes listed in the 

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definition above. These should also usually be considered serious. Examples of such events are 
intensive treatment in an emergency room or at home for allergic bronchospasm; blood 
dyscrasias or convulsions that do not result in hospitalization; or development of drug 
dependency or drug abuse. 
 
Reporting of serious adverse events  
A copy of the MedWatch/AdEERs report must be faxed to AstraZeneca at the time the 
event is reported to the FDA. It is the responsibility of the investigator to compile all necessary 
information and ensure that the FDA receives a report according to the FDA reporting 
requirement timelines and to ensure that these reports are also submitted to AstraZeneca at the 
same time. 
Serious adverse events that do not require expedited reporting need to be reported to 
AstraZeneca quarterly either as individual case reports or as line-listings. When reporting to 
AstraZeneca, a cover page should accompany the MedWatch/AdEERs form indicating the 
following: 
 
External Sponsored Research (ESR) 
 
The investigator IND number assigned by the FDA 
 
The investigator’s name and address 
 
The trial name/title and AstraZeneca ESR reference number 
Investigative site must also indicate, either in the SAE report or the cover page, the 
causality of events in relation to all study medications and if the SAE is related to disease 
progression, as determined by the principal investigator. 

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Send SAE report and accompanying cover page by way of fax to AstraZeneca’s 
designated fax line: +1-302-886-4114. 
Serious adverse events that do not require expedited reporting to the FDA need to be 
reported to AstraZeneca preferably using the MedDRA coding language for serious adverse 
events.  
In the case of blinded trials, AstraZeneca will request that the Sponsor either provide a 
copy of the randomization code/ code break information or unblind those SAEs which require 
expedited reporting. 
All SAEs have to be reported to AstraZeneca, whether or not considered causally related 
to the investigational product. All SAEs will be documented. The investigator is responsible for 
informing the IRB and/or the Regulatory Authority of the SAE as per local requirements. 
 
Possible benefits 
The present investigation is aimed to evaluate the differences on the functional impact of 
CKD on platelet inhibition by ticagrelor in DM patients. This study is not designed to evaluate 
differences in clinical benefit. However, differences in antiplatelet profiles will perhaps prompt 
further investigation of the clinical implication of this difference by means of a larger scale 
clinical study. 
 
Ethical and regulatory 
A copy of the protocol, proposed informed consent form, other written subject 
information, and any proposed advertising material must be submitted to the IRB for written 
approval. A copy of the written approval of the protocol and informed consent form must be 

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received by AstraZeneca before recruitment of subjects into the study and shipment of 
AstraZeneca investigational product.  
The investigator must submit and, where necessary, obtain approval from the IRB for all 
subsequent protocol amendments and changes to the informed consent document. The 
investigator is to notify the IRB of deviations from the protocol or serious adverse events or 
unanticipated problems occurring at the site and other adverse event reports received from 
AstraZeneca, in accordance with local procedures.  
The investigator is responsible for obtaining annual IRB approval /renewal throughout the 
duration of the study. Copies of the investigator’s reports and the IRB continuance of approval 
must be sent to AstraZeneca. 
 
Labeling and storage 
The label will include the following information: Facility Name, Patient Name, Dose, 
Prescription Number, and Dispensing Date. In addition, the drug name will be included as 
“Investigational Drug”. The investigational drug will be stored in the Cardiovascular Research 
drug room. This area has limited and restricted access. 
  
Potential Financial Risks or Benefits 
 
None 
 
Conflict of Interest 
Dr. Angiolillo is a consultant for Astra Zeneca, the maker of ticagrelor. 

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