Clinical trial NCT03161678
Conditions: Myocardial Infarction Thrombosis Platelet Dysfunction
Keywords: Pharmacogenetics Carboxylesterase 1 (CES1) Antiplatelet therapy
Sponsor: University of Maryland, Baltimore
Start date: 2017-08-22; Completion date: 2022-12-12
Enrollment: 111 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The purpose of this investigation is to evaluate when genetic variation in the carboxylesterase 1 (CES1) gene influences antiplatelet therapy response, as assessed by ex vivo platelet aggregometry, in healthy participants treated with clopidogrel and ticagrelor. We hypothesize that genetic variation in CES1 will significantly impact on-clopidogrel platelet aggregation while having a minimal effect in ticagrelor-treated subjects.

Specific Aim: To conduct a prospective randomized crossover study of clopidogrel and ticagrelor in healthy individuals stratified by CES1 genotype. Participants will be recruited by CES1 genotype into a randomized crossover study of clopidogrel (75 mg daily for 7d) and ticagrelor (90 mg twice daily for 7d) with extensive phenotyping including ex vivo platelet aggregometry performed pre- and post-drug administration in order to assess the interaction of genotype and drug choice on on-treatment platelet function.

Detailed description:

Arms: arm 1: Research subjects with wild type CES1 genotypes will be studied before and after oral ingestion of clopidogrel (75 mg/d for 8 days) and ticagrelor (90 mg twice daily for 8 days) treatment. arm 2: Research subjects who carry the CES1 G143E allele (rs71647871) will be studied before and after oral ingestion of clopidogrel (75 mg/d for 8 days) and ticagrelor (90 mg twice daily for 8 days) treatment. arm 3: Research subjects who carry a CES1 rs7498748 minor allele will be studied before and after oral ingestion of clopidogrel (75 mg/d for 8 days) and ticagrelor (90 mg twice daily for 8 days) treatment.
Interventions: intervention 1: Clopidogrel intervention 2: Ticagrelor
Intervention descriptions: intervention 1: Clopidogrel (75 mg/d for 8 days) will be administered. Measures of pharmacodynamics will be assessed pre- and post-drug administration. intervention 2: Ticagrelor (90 mg twice daily for 8 days) will be administered. Measures of pharmacodynamics will be assessed pre- and post-drug administration.

Protocol documents:
Prot_SAP_001.pdf: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CES1 Crossover Trial of Clopidogrel and Ticagrelor 
NCT03161678 
March 5, 2024 
 


STUDY PROTOCOL AND STATISTICAL ANALYSIS PLAN 
 
The purpose of this investigation was to conduct a randomized crossover study of clopidogrel and ticagrelor in 
participants recruited by carboxylesterase 1 (CES1) genotype (G143E and rs7498748). Extensive phenotyping 
including ex vivo platelet aggregometry performed pre- and post-drug administration was performed in order to 
assess the interaction of genotype and drug choice on on-treatment platelet function. Specifically, we enrolled 
89 healthy Amish participants based on CES1 genotype (17 G143E carriers, 38 rs7498748 minor allele 
carriers, and 34 controls who did contain the minor alleles of either of these variants). All of these individuals 
underwent a randomized crossover study of clopidogrel (75 mg per day for 7 d) and ticagrelor (90 mg twice 
daily for 7 d), with at least a 14-day washout period between drug interventions. Platelet aggregation studies 
and other clinical information were recorded before and after each intervention. 
 
Home visit to screen for eligibility: A research nurse and an Amish community liaison visited potential research 
subjects in their homes. The nurse explained the study and obtained informed consent. If a research subject 
consented, the nurse obtained a medical history and screening laboratory tests to assess eligibility. Briefly, 
participants were excluded if they were pregnant, currently breastfeeding, had history of a bleeding disorder or 
major spontaneous bleed, hypertensive, creatinine levels > 2.0 mg/dL, ALT or AST > 2 x the upper limit of 
normal, hematocrit < 28%, TSH < 0.4 or >5.5 mlU/L, platelet count > 500,000 or < 75,000, currently taking 
clopidogrel, ticagrelor, prasugrel, or other anti-coagulant, history of cardiovascular disease or diabetes, was 
taking vitamins or other supplements and was unwilling or could not safely, in the opinion of the study 
physician, discontinue their use at least 1 week prior to protocol initiation. 
 
Subjects were informed that to participate in this study, they had to discontinue for a period starting 7 days 
prior to the initial clinic visit any vitamins, supplements and medications that might affect the results of the 
study. Specifically, if a subject is on medications that may potentially interfere with the planned intervention, 
he/she was instructed to consult with his/her physician regarding discontinuing such therapy prior to study 
enrollment. Alternatively, with the subject's permission, the study physician contacted the subject's physician to 
discuss and obtain permission for medication withdrawal. If in the opinion of the subject's physician and the 
study physician, discontinuation of medications for the duration of the study did not pose any undue health 
risks, medications were discontinued one week before the first clinic visit. If a medication required tapering, this 
commenced two weeks prior to the first clinic visit. Similarly, all vitamins (except daily multivitamins) and other 
supplements were withdrawn for 7 days prior to the first clinic visit. If the research subject was on anti-platelet 
medications, these agents were withdrawn 14 days prior to the first clinic visit.  
 
Female research participants were informed that they could not participate in this study if they are pregnant. All 
female subjects had an assessment of child-bearing potential. Pregnancy status was determined by self-report 
at the screening visit. A urine pregnancy test was performed at the clinic visits #1 and #3 prior to drug 
administration. 
 
Following review of screening information and lab results, eligibility was confirmed. Eligible participants 
completed studies during the course of four visits to the Amish Research Clinic in Lancaster, PA. 
 
Randomization: Prior to Clinic Visit #1, enrolled subjects were randomized (1:1) to either the clopidogrel or 
ticagrelor intervention. Clinical staff, blinded to CES1 genotype, were notified of the order of drug intervention 
though the delivery of a sealed, opaque envelope from a research team member who was also blinded to 
participant genotype. Assignment envelopes were shuffled prior to study initiation and delivered sequentially.  
Participants who started with the clopidogrel intervention underwent the ticagrelor intervention after the 
prerequisite washout period and vice versa. 
 
Clinic Visit #1 (Baseline Platelet Function): Research subjects were transported to the Amish Research Clinic 
after an overnight fast where heights, weights, and vital signs were measured. Women of childbearing age 
completed a pregnancy test. A catheter was placed, and fasting blood samples were obtained to assess 
circulating lipid levels (LDL, HDL, and triglycerides), evaluate baseline measures of agonist-stimulated platelet 
aggregation, and for sample banking. Participants were then observed while taking their first dose of 
clopidogrel or ticagrelor (based on randomization procedure), discharged to home with additional tablets, and 


instructed to take the appropriate dose (75 mg/ daily for clopidogrel, 180 mg/d for ticagrelor) at the same time 
each day. A participant logbook was given to each participant to record their daily doses 
 
Clinic Visit #2 (On-Clopidogrel Platelet Function): On the 7th day, subjects returned to the clinic, observed 
taking their last dose, and fasting blood samples were drawn 1-hour post-dose for measurement of agonist-
stimulated platelet aggregation and sample banking. Drug adherence was assessed by pill count and 
participant logbook. Each participant then underwent a 14-day washout period to restore basal platelet 
function, consistent with the average lifespan of a platelet (7 – 10 days). 
 
Clinic Visit #3 (Repeat Measures of Baseline Platelet Function): After the 14-day washout period, research 
subjects again were transported to the Amish Research Clinic in the fasting state where they were weighed 
and their vital signs measured. Reproductive age women underwent a 2nd pregnancy test.  Blood samples 
were obtained for repeat measures of baseline platelet function and sample banking.  Participants were then 
observed while taking their first dose of clopidogrel or ticagrelor (whichever one was not administered during 
the first intervention period), discharged to home with additional tablets, and instructed to take the appropriate 
dose at the same time each day. A participant logbook was given to each participant to record their daily 
doses. 
 
Clinic Visit #4 (On-Ticagrelor Platelet Function): On the 7th day, subjects returned to the clinic, observed taking 
their last dose, and fasting blood samples were drawn 1-hour post-dose for measurement of agonist-stimulated 
platelet aggregation and sample banking.  Drug adherence was assessed by pill count and participant logbook. 
 
Assessment of Platelet Function: at each clinic visit, platelet-rich plasma (PRP) was isolated from fasting blood 
samples drawn into 3.2% citrate-anticoagulated tubes (Becton-Dickinson, Franklin Lakes, NJ) and platelet 
counts were adjusted to 200,000 platelets/μl using platelet-poor plasma. Platelet function was assessed by 
optical aggregometry using a PAP8E Aggregometer (Bio/Data Corporation, Horsham, PA) according to the 
manufacturer’s instructions after stimulation with 20 μM ADP and expressed as the maximal percentage 
change in light transmittance using platelet-poor plasma as a referent. 
 
Distributions of summary statistics (e.g. age, sex, change in platelet function) were generated using SAS 
(Cary, NC). Change in clopidogrel-induced platelet aggregation in response to ADP was calculated by 
subtracting observed post-clopidogrel maximal platelet aggregation values from baseline (i.e. pre-drug) values. 
Similarly, change in ticagrelor-induced platelet aggregation in response to ADP was calculated by subtracting 
observed ticagrelor maximal platelet aggregation values from baseline (i.e. pre-drug) values. 
 
The primary endpoint of this investigation was to determine within each drug group if CES1 genotype is 
associated with change in platelet aggregation. Change in platelet aggregation, defined as the relative 
differences in platelet aggregation between baseline and day 7 of a given drug intervention, was calculated as: 
Change = [(MPA baseline – MPA day 7), where MPA = maximal platelet aggregation. Consideration of relevant 
biological variables that influence platelet aggregation and/or drug response were explored in our statistical 
models.  Specifically, the effect of CES1 genotype on agonist-stimulated change in platelet aggregation was 
calculated using a variance component method under an additive or dominant model that simultaneously 
adjusted for age, sex, body mass index (BMI), and relatedness among study participants. Relatedness among 
participants were accounted for by including a polygenic component as a random effect. Briefly, a model for 
the polygenic component was established by constructing a relationship matrix derived from the complete 
Amish pedigree structure available through published genealogical records maintained by the church.