Clinical trial NCT03774394
Conditions: Chronic Kidney Disease (CKD) Type 2 Diabetes Mellitus (T2DM) Coronary Artery Disease (CAD)
Keywords: pharmacodynamics (PD) pharmacokinetic (PK) clopidogrel
Sponsor: University of Florida
Start date: 2019-08-22; Completion date: 2022-05-31
Enrollment: 61 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Patients with diabetes mellitus (DM) and chronic kidney disease (CKD) are at increased risk of atherothrombotic events. Clopidogrel is the most widely used platelet P2Y12 receptor inhibitor in patients with coronary artery disease (CAD). However, despite its benefits, many patients still experience recurrent atherothrombotic events. The proposed study will test the central hypothesis that in DM patients the presence of CKD reduces clopidogrel-mediated P2Y12 inhibitory effects through synergistic mechanisms, which include upregulation of the P2Y12 signaling pathway and impaired clopidogrel metabolism.

Detailed description:
Patients with diabetes mellitus (DM) and coexisting chronic kidney disease (CKD) are at increased risk of atherothrombotic events, underscoring the importance of secondary prevention antiplatelet therapy in these high-risk patients. Clopidogrel is the most widely used platelet P2Y12 receptor inhibitor in patients with coronary artery disease (CAD). However, despite its clinical benefits, many patients still experience recurrent atherothrombotic events. This is in part due to the impaired effects of clopidogrel in DM patients, particularly among those with coexisting CKD. However, underlying mechanism(s) leading to magnification of impaired clopidogrel response among DM patients with CKD remain unexplored. The ever growing prevalence of CKD in patients with DM and their high risk of recurrent events underscores the need to define such mechanism(s) as this may set the basis for identifying treatment regimens leading to more effective platelet inhibition and cardiovascular protection in these high-risk patients. The proposed study will test the central hypothesis that in DM patients the presence of CKD reduces clopidogrel-mediated P2Y12 inhibitory effects through synergistic mechanisms, which include upregulation of the P2Y12 signaling pathway and impaired clopidogrel metabolism. Comprehensive pharmacokinetic and pharmacodynamic assessments, including ex vivo and in vitro experiments, evaluating the impact of CKD on antiplatelet drug response in DM patients are proposed.

Arms: arm 1: Patients with CKD will be administered a 600-mg LD of Clopidogrel followed by a single 75-mg MD administered after 24 hours.

Blood samples collected at baseline will be incubated with clopidogrel active metabolite. arm 2: Patients without CKD will be administered a 600-mg LD of Clopidogrel followed by a single 75-mg MD administered after 24 hours.

Blood samples collected at baseline will be incubated with clopidogrel active metabolite.
Interventions: intervention 1: Clopidogrel intervention 2: Clopidogrel active metabolite
Intervention descriptions: intervention 1: Both CKD and Non-CKD patients will be administered a 600-mg LD of clopidogrel followed by a single 75-mg MD administered after 24 hours. intervention 2: In both CKD and Non-CKD patients, blood samples collected at baseline only (before clopidogrel LD administration) will be incubated with escalating concentrations of clopidogrel active metabolite (1, 3 and 10 μM)

Protocol documents:
Prot_001.pdf: 
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Protocol 
1. Project Title:  Impact of chronic kidney disease on pharmacodynamic profiles of the 
P2Y12 receptor inhibitor clopidogrel in the setting of type 2 diabetes mellitus and coronary 
artery disease 
2. Investigator(s):  Francesco Franchi, MD, Dominick Angiolillo, MD, Phd, and Fabiana 
Rollini, MD  
3. Abstract:  Patients with diabetes mellitus (DM) and coexisting chronic kidney disease 
(CKD) are at increased risk of atherothrombotic events, underscoring the importance of 
secondary prevention antiplatelet therapy in these high-risk patients. Clopidogrel is the 
most widely used platelet P2Y12 receptor inhibitor in patients with coronary artery disease 
(CAD). However, despite its clinical benefits, many patients still experience recurrent 
atherothrombotic events. This is in part due to the impaired effects of clopidogrel in DM 
patients, particularly among those with coexisting CKD. However, underlying 
mechanism(s) leading to magnification of impaired clopidogrel response among DM 
patients with CKD remain unexplored. The ever growing prevalence of CKD in patients 
with DM and their high risk of recurrent events underscores the need to define such 
mechanism(s) as this may set the basis for identifying treatment regimens leading to 
more effective platelet inhibition and cardiovascular protection in these high-risk patients. 
The proposed study will test the central hypothesis that in DM patients the presence of 
CKD reduces clopidogrel-mediated P2Y12 inhibitory effects through synergistic 
mechanisms, which include upregulation of the P2Y12 signaling pathway and impaired 
clopidogrel metabolism. Comprehensive pharmacokinetic and pharmacodynamic 
assessments, including ex vivo and in vitro experiments, evaluating the impact of CKD on 
antiplatelet drug response in DM patients are proposed. This investigation is part of our 
long-term goal of defining optimized antiplatelet treatment regimens in high-risk CAD 
patients. The REDCap system will be used to store study related data, and all patients will 
be identified using a subject identifier.  
4.  Background:  Patients with DM are at increased risk of atherothrombotic events [1,2]. 
Importantly, DM is a key risk factor for the development of CKD, which further enhances 
atherothrombotic risk [3-5]. These observations underscore the importance of antiplatelet 
therapy for secondary prevention of atherothrombotic recurrences in these high-risk 
patients. Clopidogrel is the most widely used platelet P2Y12 receptor inhibitor which, in 
adjunct to aspirin, reduces ischemic event rates in high-risk patients with CAD [6,7]. 
However, despite the clinical benefit of adjunctive clopidogrel therapy, DM patients 
frequently experience recurrent atherothrombotic events [2,8]. This may be in part due to 
the impaired PK and PD effects of clopidogrel in DM patients which leads to increased 
platelet reactivity compared with non-DM patients [8-10]. Moreover, studies have also 
shown patients with CKD to have reduced clinical benefit with clopidogrel [11-14]. 
However, PD studies provide mixed results on the independent role of CKD as a 
determinant of impaired clopidogrel effects [15-19]. This may be attributed to confounders 
intrinsic to the heterogeneous study populations in which these studies have been 


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performed. In fact, assessments conducted selectively in type 2 DM patients have 
consistently shown that the presence of impaired renal function markedly reduces 
clopidogrel-induced antiplatelet effects [15,17]. These PD findings may explain why DM 
patients with CKD are particularly at increased risk of atherothrombotic recurrences [20]. 
However, the underlying mechanism(s) leading to magnification of impaired clopidogrel 
response among DM patients with CKD remains unexplored. The increased 
cardiovascular (CV) risk of DM patients is in part attributed to increased platelet reactivity 
and inadequate response to oral antiplatelet agents, including clopidogrel [1,2,6]. 
Reduced clopidogrel-induced antiplatelet effects among DM patients may in part account 
for their poor outcomes after percutaneous coronary intervention (PCI) [1,2,8,9,17,21-23]. 
This is mainly due to an attenuation of clopidogrel’s PK profile, characterized by lower 
active metabolite levels [9,24]. Notably, patients with high platelet reactivity (HPR) while 
on clopidogrel therapy are at increased risk of ischemic recurrences [25-26]. CKD is a 
pandemic public health problem [27-29] and patients with all stages of CKD are at 
increased CV risk, including increased mortality [30-37]. In CAD patients, the presence of 
either DM or CKD increases long-term CV events to a similar extent. However, when DM 
and CKD coexist, the risk is further amplified [20]. Mechanisms explaining the enhanced 
CV risk carried by CKD include lower likelihood to receive evidence-based therapies [38], 
endothelial dysfunction, platelet activation and reduced response to antiplatelet therapies 
[14,39]. Subgroup analysis of clinical trials showed a reduced benefit of clopidogrel in 
CKD patients [14]. High levels of circulating procoagulant factors, increased thrombin 
generation, abnormalities of nitric oxide synthesis, upregulation of the P2Y12 signaling 
pathway and poor bioavailiability of C-AM have been proposed to explain platelet 
hyperreactivity among CKD patients [14,39]. Platelets from patients with CKD are 
exposed to higher levels of dinucleoside polyphosphates, which can up-regulate the 
platelet P2Y12 signaling pathway [40-42]. Further, CKD is associated with reduced activity 
of the hepatic CYP system, a key mediator of clopidogrel metabolism, which can lead to 
reduced generation of C-AM [43-44]. Indeed, high platelet reactivity in patients with DM 
and CKD may be due to a synergistic effect of the two clinical disorders, which contribute 
to the enhanced risk of CV events when they coexist [20].  
Understanding the mechanisms leading to the different pharmacological profiles of P2Y12 
inhibitors in patients with DM and CKD represents a critical knowledge gap, given the 
prognostic implications associated with profiles of antiplatelet drug response. Defining the 
underlying mechanism leading to magnification of impaired clopidogrel response among 
DM patients with CKD is critical to set the basis of improved secondary prevention in 
these patients at the highest risk of CV events. The prognostic implications associated 
with inadequate antiplatelet drug response are noteworthy, given that 30-40% of ACS 
patients have DM associated with CKD [45] and ischemic recurrences, which are higher 
in the presence of DM and CKD, represent a major health care burden. In the US more 
than $150 billion is spent on ACS care, and 60% of the costs are a result of ischemic 
recurrences [46,47]. The results of the proposed investigation will have the potential to 
help interpret findings from large scale clinical trials as well as help optimize and 
personalize antiplatelet treatment in this high-risk setting. 


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We propose unique and wide-ranging PK and PD assessments with both ex vivo and in 
vitro experiments using a broad range of assays to define mechanisms of impaired 
clopidogrel effects in DM patients with CKD. This is noteworthy because clopidogrel is the 
most utilized P2Y12 receptor inhibitor across the globe and this is the first study 
specifically and prospectively designed to comprehensively address this area of unmet 
need.  
5. Specific Aims:  The proposed study will test the central hypothesis that in coronary 
artery disease (CAD) patients with diabetes mellitus (DM), the presence of chronic kidney 
disease (CKD) reduces clopidogrel-mediated P2Y12 inhibitory effects through synergistic 
mechanisms, which include upregulation of the P2Y12 signaling pathway and impaired 
clopidogrel metabolism. Our approach is innovative since to date there has not been any 
comprehensive pharmacodynamics (PD) and pharmacokinetic (PK) study, involving both 
ex vivo and in vitro experiments, evaluating the impact of CKD on antiplatelet drug 
response in DM patients. The study proposed is significant since it advances our 
understanding on therapeutic approaches aimed to improve outcomes in a high-risk 
patient population representing a pandemic health care problem such as those with DM 
and CKD. Two study aims have been elaborated to reach our goals: 
- AIM 1. To assess the functional status of the platelet P2Y12 signaling pathway in DM 
patients with and without CKD. Study hypothesis: DM patients with CKD have 
upregulation of the platelet P2Y12 signaling pathway compared to DM without CKD. 
Rationale: Platelets from patients with CKD are exposed to higher levels of dinucleoside 
polyphosphates, which can act as agonist of P2Y12 causing a decrease in VASP 
(vasodilator-stimulated phosphoprotein) phosphorylation and therefore lead to 
upregulation of the platelet P2Y12 signaling pathway. The investigational sequence to 
pursue this study aim will imply serial and comprehensive PD assessments in DM 
patients with and without CKD as assessed ex vivo following treatment with clopidogrel 
and in vitro following exposure to P2Y12 antagonism, in the setting of a prospective study 
design. 
- AIM 2. To assess profiles of clopidogrel metabolism in DM patients with and without 
CKD. Study hypothesis: DM patients with CKD have impaired clopidogrel metabolism. 
Rationale: CKD is associated with reduced activity of the hepatic cytochrome P450 (CYP) 
system, which is a key mediator of clopidogrel metabolism. The investigational sequence 
to pursue this study aim will imply comprehensive and serial PK assessments of 
clopidogrel pro-drug and clopidogrel’s active metabolite (C-AM; R-130964) after 
administration of a clopidogrel loading dose (LD) in DM patients with and without CKD. 
6. Research Plan:  Study Aims 1 and 2 will be pursued through the conduct of a 
prospective open-label design in which CAD patients with DM on low-dose (81 mg) 
aspirin will be prospectively recruited. 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) [53,54]. The 
rationale for considering the functional classification to initially stratify patients is in line 
with clinical studies showing the increased CV risk and markedly dysfunctional platelets 


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according to GFR strata [12,13,15]. 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) [53,54]. For comparison, kidney 
function will also be estimated with the MDRD (modification of diet in renal disease) and 
Cockcroft-Gault equations. In addition, as exploratory analysis, renal function will also be 
classified according to markers of kidney damage, in particular albuminuria, in order to 
obtain a more comprehensive information about CKD [53,54]. 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 [53,54]. Albuminuria will 
be measured on random untimed spot urine samples. Patients’ metabolic status and 
glycemic control will be assessed at 
baseline by measuring fasting 
plasma glucose, HbA1c and lipid 
profile.  
After providing informed consent, in 
the ex vivo experimental component 
of our study, eligible patients will be 
administered a 600-mg LD of 
clopidogrel followed by a single 75-
mg MD administered after 24 hours; 
blood samples for PD (Study Aim 1) 
and PK (Study Aim 2) assessments 
will be collected at a total of 8 time 
points: baseline (before LD 
administration) and 30 minutes, 1 
hour, 2 hours, 4 hours, 6 hours and 
24 hours after LD (before administration of MD), and 2 hours after administration of the 75 
mg MD of clopidogrel. In the in vitro experimental design of our study (Study Aim 1), 
blood samples collected at baseline only (before LD administration) will be used; PD 
testing will be performed before and after incubation (for 30 minutes at 37°C) with 
escalating concentrations of C-AM (1, 3 and 10 μM). This will allow to specifically explore 
the functional status of the P2Y12 signaling pathway. A flow diagram of the study design is 
presented in Figure 4. In order to account for possible confounders, CYP2C19 genetic 
status (the major genetic determinant of clopidogrel metabolism) will be assessed through 
a rapid assay or by central lab at baseline. Laboratory personnel will be blinded to group 
assignment.  
b)  Study population: Patients will be recruited at the cardiology clinics of the University of 
Florida-Jacksonville. Patients will be approached by the physician and research staff and 
given information on the study. Consent will be reviewed and the patient will be given time 
to ask any questions and have all concerns addressed.  
 
 
Figure 4. Study design for Aims 1 and 2 
Patients with CAD 
and DM
CKD
Non-CKD
Clopidogrel 
600 mg LD/ 
75 mg MD
Clopidogrel 
600 mg LD/ 
75 mg MD
Baseline 
(time 0)
30’ 1h
2h
4h
6h
2
PD and PK analysis
PD and PK analysis
In vitro escalating 
concentrations of Clop-AM
PD analysis


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Inclusion criteria: 
1) Type 2 DM, defined according to ADA definition [55], on treatment with oral 
hypoglycemic agents and/or insulin 2) age >18 years old; 3) angiographically documented 
CAD; 4) on treatment with low-dose aspirin (81mg/day) for ≥30 days as part of standard 
of care. 
Exclusion criteria: 
1) Use of any antiplatelet therapy (except aspirin) in prior 30 days; 2) use of parenteral or 
oral anticoagulation; 3) active bleeding; 4) high risk of bleeding; 5) clinical indication to be 
on a P2Y12 receptor inhibitor; 6) end-stage renal disease on hemodialysis; 7) any active 
malignancy; 8) platelet count < 100x106/µl; 9) hemoglobin <9 g/dl; 10) severe known liver 
disease; 11) hemodynamic instability; 13) known allergy to clopidogrel; 13) pregnant / 
lactating females (women of childbearing age must use reliable birth control). 
c) Laboratory assessments 
Our group has been deeply invested in conducting translational investigations assessing 
P2Y12 receptor inhibitors. This has allowed us to have ample experience with the 
methodologies described to conduct our study aims, as supported by our description in 
the Preliminary Studies section and summarized below.  
Blood sampling: blood sampling for the ex vivo component of our experiment will be 
performed at baseline and at 7 time points, as described above. For the in vitro 
component of our experiment, blood will be collected at baseline and will be incubated 
with escalating concentrations (1, 3 and 10 μM) of C-AM (Figure 4).  
PD assessments: 
(1) VerifyNow PRU: The VerifyNow System is a turbidimetric based optical detection 
system which measures platelet aggregation as an increase in light transmittance 
(Accriva, San Diego, CA, USA) [56]. The assay is based on microbead agglutination and 
uses specific reagents for the pathways of interest. The change in optical signal is 
reported in PRU. HPR will be defined using consensus definitions, based on cut-off levels 
associated with an increased risk of recurrent ischemic events, as PRU> 208 [26]. 
(2) Whole blood vasodilator-stimulated phosphoprotein (VASP):  VASP phosphorylation 
(VASP-P) is a marker of P2Y12 receptor activity which will be assessed according to 
standard protocols [56]. VASP-P levels will be quantified with ELISA method with the 
Platelet VASP-FCM kit (Biocytex Inc., Marseille, France) and the platelet reactivity index 
(PRI) calculated. HPR will be defined using consensus definitions as PRI> 50% [26]. 
(3) Light transmittance aggregometry (LTA): LTA will be assessed using platelet rich 
plasma (PRP) by the turbidimetric method in a 2-channel aggregometer (Chrono-Log 
Corp., Havertown, PA, USA) as previously described [56]. Platelet agonists will include 5 
and 20 μmol/L ADP. HPR will be defined using consensus definitions as maximal 
aggregation >59% and >46% with ADP 20 μmol/L and 5 μmol/L, respectively) [26]. 


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PK assessment: determination of C-AM and clopidogrel pro-drug concentrations in 
plasma will be performed using liquid chromatography with tandem mass spectrometry 
according to standard protocols as previously described [9]. The geometric mean area 
under the concentration-time curve through the sampling time of the last quantifiable C-
AM concentration (AUC[0-tlast]), the maximum observed plasma concentration (Cmax) of C-
AM, and the time to reach maximum concentration (Tmax) will be calculated. 
Genetic testing: 
Spartan RX rapid genotyping: Spartan RX (Spartan Bioscience Inc., Ontario, Canada) is 
a rapid genotyping platform able to determine the CYP2C19 (*1,*2,*3,*17) allele status 
within 1 hour. This test consists of four separate steps: acquisition of a buccal swab, 
insertion of the swab into the cartridge, insertion of the reaction solution into the device, 
and analysis of CYP2C19 genotype triggered by a button on the device [57-58]. 
Central lab genotyping: genetic testing to determine the CYP2C19 (*1, *2, *3, *17) allele 
status will be performed at baseline by central pathology lab by processing a whole blood 
sample using standard techniques if rapid genotyping is not available.  
Dinucleoside polyphosphates levels: Diadenosine polyphosphate (Ap(n)A) content in the 
supernatant and in intact platelets will be determined at baseline using a chromatographic 
assay established on the basis of affinity- and reversed-phase chromatographic methods, 
as previously described [42]. 
Assessment of metabolic control: Fasting plasma glucose, HbA1c and lipid panel (total 
cholesterol, HDL, LDL and triglycerides) will be measured at baseline by our central 
laboratory. 
7. Possible Discomforts and Risks: The most common clinical side effects of 
Clopidogrel are pruritis (itching), purpura (purple colored spots), diarrhea, rash, and 
bleeding that can be minor, life-threatening or fatal. The risks of the blood draw may 
include faintness, inflammation of the vein, pain, bruising or bleeding at the site of the 
puncture.There is also a slight risk of infection from the blood draw. 
8. Possible Benefits: There is no guarantee that you will personally benefit by 
participating in this research study. We hope to improve our knowledge about the effects 
of Clopidogrel in diabetic patients with and without kidney disease. 
9. Conflict of Interest: None