Étude randomisée contrôlée Par Placebo de Phase II Visant à Mesurer l'Effet Anti-inflammatoire et Anti-sénescence de la quercétine Lors d'Une Chirurgie Cardiaque
Phase: PHASE2
Conditions: Coronary Artery Disease
Sponsor: Montreal Heart Institute

Numéro du projet : ICM 2020-2761  
 
Version : 4 
Confidentiel 
Page 1 of 14 
Project Number: ICM 2020-2761 
 
PROTOCOL QUERCETIN (Q-CABG) 
 
The Anti-inflammatory and Senolytic Effect of Quercetin During Cardiac Surgery 
 
Sponsors or Funding Organizations 
 
Departmental Chair of the Department of Cardiac Surgery 
 
Protocol Version: Version 5 
 
Date: September 15, 2020 
 
CONFIDENTIAL 
This protocol contains confidential information and is the property of the Montreal Heart 
Institute 
 
 

Table of Contents 
1. Introduction and Definition of the Research Problem  
 
 
 
– 3 
2. Relevant Literature Review   
 
 
 
 
 
 
– 3 
3. Objectives and Evaluation Criteria   
 
 
 
 
 
– 6 
 3.1. Primary Objective  
 
 
 
 
 
 
 
– 6 
 3.2. Secondary Objective   
 
 
 
 
 
 
– 6 
 3.3. Exploratory Objective  
 
 
 
 
 
 
– 7 
 3.4. Primary Evaluation Criterion  
 
 
 
 
 
– 7 
 3.5. Secondary Evaluation Criterion  
 
 
 
 
 
– 7 
 3.6. Tertiary / Exploratory Evaluation Criteria   
 
 
 
– 7 
4. Methodology   
 
 
 
 
 
 
 
 
– 9 
 4.1. Study Design   
 
 
 
 
 
 
 
– 9 
 4.2. Population Description  
 
 
 
 
 
 
– 10 
  4.2.1. Inclusion Criteria  
 
 
 
 
 
 
– 10 
  4.2.2. Exclusion Criteria  
 
 
 
 
 
 
– 10 
  4.2.3. Strategy for Recruiting and Retaining Study Participants  
 
– 11 
 4.3. Study Plan and Course  
 
 
 
 
 
 
– 11 
  4.3.1. Description of Study Progression and Visits  
 
 
 
– 11 
  4.3.2. Description of the Intervention   
 
 
 
 
– 11 
  4.3.3. Description of Randomization   
 
 
 
 
– 12 
 4.4. Measurements and Data Collection Methods  
 
 
 
– 12 
5. Statistical Aspects  
 
 
 
 
 
 
 
 
– 13 
 5.1. Sample Size Calculation  
 
 
 
 
 
 
– 13 
 5.2. Statistical Analyses   
 
 
 
 
 
 
– 13 
  5.2.1. General Considerations   
 
 
 
 
 
– 13 
  5.2.2. Statistical Analyses  
 
 
 
 
 
 
– 14 
6. References  
 
 
 
 
 
 
 
 
 
– 15 
 
7. ANNEX 1 – TIMELINE FOR STUDY MEDICATION ADMINISTRATION AND 
BLOOD SAMPLING – 17 
 
 

1. Introduction and Definition of the Research Problem 
Quercetin is a naturally occurring flavonoid with anti-inflammatory and antioxidant effects. 
Numerous studies have investigated the effects of quercetin in animals. However, few studies 
have addressed the medical use of quercetin in humans. 
The purpose of this study is to evaluate the effect of quercetin on the inflammatory response 
following cardiac surgery, as well as its senolytic effect on endothelial cells, primarily in patients 
with unstable coronary artery disease, such as those undergoing myocardial revascularization 
after a myocardial infarction or with clinical unstable angina. 
 
2. Relevant Literature Review 
In a recent clinical study involving 47 patients, we observed increased blood levels of high-
sensitivity reactive C protein (hs-CRP) and angiopoietin-like 2 (ANGPTL2)—a pro-
inflammatory adipokine—24 hours after cardiac surgery (1). After surgery, ANGPTL2 levels 
increased in a group of older patients with higher blood pressure, while levels decreased in 
younger patients with lower blood pressure (1) (Table 1). hs-CRP levels remained relatively 
stable and did not mirror the ANGPTL2 secretion profile; however, hs-CRP levels did reflect 
acute inflammation partly caused by extracorporeal circulation and the surgical procedure. 
In a preliminary analysis assessing endothelial dysfunction, single-cell mRNA sequencing of the 
internal mammary artery endothelium identified nine significantly overexpressed genes in 
patients with unstable coronary artery disease (Table 2). These genes were associated with 
targets of senolytic agents—such as quercetin (which eliminates senescent cells)—and with 
cardiovascular diseases, reflecting the severity of endothelial dysfunction and atherosclerotic 
involvement. 
 
Effect of Quercetin 
Quercetin is a flavonoid found in plants, fruits, vegetables, and red wine. It is thus a natural 
compound, regularly consumed through dietary plant products. Dietary intake varies 
considerably but is estimated between 10 and 100 mg per day (2). It has well-demonstrated anti-
inflammatory and antioxidant effects in both animals and humans (3–8). Quercetin slows the 
progression of atherosclerosis in rabbits (4). It also reduces the activity of inflammatory 
mediators such as cyclooxygenase, myeloperoxidase, NOS, and serum CRP levels in animals 
with aortic aneurysms (7), and has antihypertensive effects through renal sodium regulation 
mechanisms (9). It has also been proposed as a potential antidiabetic agent based on animal 
studies (10). 
However, there are still few human studies involving quercetin supplementation for medical 
purposes, particularly for cardiovascular disease prevention (11). 
Surprisingly, quercetin has been extensively studied in sports medicine to evaluate its impact on 
physical performance in athletes and soldiers (2,12,13). A dose of 1 g per day for three weeks did 
not reduce inflammation in young subjects but improved muscle performance (14); it was the 
first study to show that quercetin improves muscle function and attenuates immune alterations 

after exercise. These results were confirmed by McAnulty’s team (13). The absence of an anti-
inflammatory effect may be due to the fact that participants were young and physically active. 
Indeed, a daily dose of 500 mg (15) and 730 mg (16) for 10 and 4 weeks respectively reduced 
blood pressure and inflammation (IL-6, TNF-alpha) in women with type 2 diabetes and reduced  
 
Tableau 1. Comparaison entre les paramètres préopératoires entre les deux groupes de 
patients avec un DANGPTL2 positif (groupe INCREASED) ou négatif (groupe 
DECREASED) (1). 
 
blood pressure in patients with stage 1 hypertension (140–159 mmHg systolic/90–99 mmHg 
diastolic), but not in pre-hypertensive individuals. Thus, the anti-inflammatory effects of 
quercetin may only be apparent in patients with cardiovascular risk factors. It is therefore 

relevant to test a 1000 mg/day dose over a short period to assess its anti-inflammatory effects 
during cardiac surgery in a population already considered “inflamed.” 
Bioavailability varies depending on the oral formulation used in humans (5,17). Notably, no 
adverse effects have been reported from oral administration of quercetin at doses ≤1200 mg/day 
for up to 12 weeks (2,17). 
 
Table 2. Main Genes Differentially Expressed in Endothelial Cells of the Mammary Artery 
from a Patient with Stable Coronary Artery Disease and a Patient with Unstable Coronary 
Artery Disease.  
Gene 
Stable CAD 
Patient 
Unstable CAD 
Patient 
P-value (Benjamini-
Hochberg corrected) 
Associated 
Condition 
TGFB2 
0.0% 
41.7% 
0.00061 
Atrial fibrillation 
SQLE 
0.0% 
41.7% 
0.00061 
Statins 
RPPH1 
2.9% 
58.3% 
0.00090 
Quercetin 
ARL14EP 7.1% 
66.7% 
0.00339 
Systolic blood 
pressure 
RPL17 
25.7% 
100.0% 
0.00914 
Myocardial 
ischemia 
IGFBP7 
17.1% 
83.3% 
0.01556 
Ruptured aneurysm 
TMEM8B 0.0% 
33.3% 
0.01699 
Valvular heart 
disease 
ZNF675 
0.0% 
33.3% 
0.01699 
Congestive heart 
failure 
SELL 
8.6% 
66.7% 
0.04848 
Vaso-occlusive 
crisis 
Percentages represent the number of endothelial cells expressing each gene per patient. 
 
3. Objectives and Evaluation Criteria 
3.1 Primary Objective 
The primary objective of this study is to evaluate the effect of quercetin (500 mg BID; 1 g/day) 
on the inflammatory response four days after cardiac revascularization surgery in patients with 
unstable coronary artery disease—such as those undergoing myocardial revascularization 
following a myocardial infarction or experiencing clinical unstable angina. 
 

3.2 Secondary Objective 
The secondary objective is to assess the senolytic effect of quercetin (500 mg BID) on 
endothelial cells following revascularization surgery. 
 
3.3 Exploratory Objective 
The exploratory objective is to study the effect of quercetin (500 mg BID) on the inflammatory 
response at additional time points following revascularization surgery—specifically, 24 hours 
post-surgery and at hospital discharge (or up to a maximum of seven days post-surgery). 
 
3.4 Primary Evaluation Criterion 
The primary evaluation criterion is the change in blood levels of hs-CRP between baseline (t-1) 
and four days after surgery (t2). 
 
3.5 Secondary Evaluation Criteria 
Secondary evaluation criteria include: 
• 
Change in blood levels of ANGPTL2 between baseline (t-1) and four days after surgery 
(t2); 
• 
Endothelium-dependent relaxation function; 
• 
Expression of arterial markers of inflammation and senescence, assessed by: 
o Ex vivo measurement of endothelial relaxation function induced by acetylcholine; 
o mRNA assays in isolated wall cells. 
 
3.6 Tertiary / Exploratory Evaluation Criteria 
Exploratory criteria include changes in hs-CRP and ANGPTL2 levels between baseline (t-1) and 
two other time points: 24 hours after surgery (t1) and at hospital discharge (or up to a maximum 
of seven days post-surgery) (t3). 
 
Objectives – Evaluation Criteria – Rationale Table 
Objectives Evaluation Criteria 
Justification 
Primary 
Change in hs-CRP levels 
between baseline and day 4 
(t2) 
hs-CRP and ANGPTL2 blood levels are expected to 
peak on day 4 post-surgery, based on Figures 1 and 
2. 
 
Change in ANGPTL2 levels 
between baseline and day 4 
(t2) 
Secondary criterion for the same objective. 

Objectives Evaluation Criteria 
Justification 
Secondary 
Endothelial relaxation 
function (acetylcholine, ex 
vivo); mRNA assays 
The nine genes identified via mRNA sequencing 
were associated with senolytic targets including 
quercetin and cardiovascular disease, reflecting 
severity of endothelial dysfunction. 
Exploratory 
Change in hs-CRP and 
ANGPTL2 at t1 (24h) and 
t3 (hospital discharge) 
These exploratory criteria are included to validate 
inflammatory markers at additional time points, as 
shown in Figures 1 and 2. 
 
 
 
 
Figure 1. Mean plasma levels of hs-CRP post-surgery in all patients (All) and in the group 
where ANGPTL2 increased (INCREASED) or decreased (DECREASED) (see Figure 2). 
Data are means ± SEM of 47 patients. Two way ANOVA for repeated measures (Group 
effect: p=0.997; time: p<0.0001; Groupe x time: p=0.375) (1). 
 
 
Figure 2. Circulating levels of ANGPTL2 decreased (group DECREASED, n=21) or 
increased (group INCREASED, n=26) at the end of the follow-up (76 days post-surgery). 
The opposite profils of change are presented. For comparaison, the average profil of 
ANGPTL2 evolution in plasma as a fonction of time post-surgery is  shown (group All). 

Data are means ± SEM of 47 patients. Two way ANOVA for repeated measures 
(interaction between Group and Time p<0.0001); ‡: p<0.05 versus DECREASED; $: 
p<0.05 versus basal (t=0), in each group of patients) (1). 
 
4. Methodology 
4.1 Study Design 
This research is a randomized, placebo-controlled study. Patients undergoing myocardial 
revascularization surgery following a myocardial infarction or presenting with unstable angina 
will be approached to participate in the study. These are patients awaiting surgery in the coronary 
care unit. All patients will undergo coronary artery bypass grafting (CABG) using standard and 
recognized procedures. 
Five milliliters of blood will be collected at four time points: preoperative (t-1), 24 hours post-op 
(t1), four days post-op (t2), and at hospital discharge (or up to a maximum of seven days post-
op) (t3), to measure plasma levels of hs-CRP and ANGPTL2. 
A segment of internal mammary artery discarded during bypass surgery will be retrieved for: 
1. Measurement of ex vivo endothelial relaxation function induced by acetylcholine; 
2. mRNA analysis in cells isolated from the arterial wall. 
 
4.2 Description of the Population 
As mentioned previously, patients undergoing myocardial revascularization surgery following a 
myocardial infarction or who present with unstable angina will be considered for study 
enrollment. These patients will be hospitalized in either the coronary care unit or the cardiac 
surgery unit, possibly transferred from a referring center. 
 
4.2.1 Inclusion Criteria 
• 
Aged 18 years or older; 
• 
Able to give free and informed consent; 
• 
Able to communicate in French or English; 
• 
Hospitalized and awaiting cardiac revascularization surgery; 
• 
Have had a myocardial infarction in the past 30 days or present with unstable angina 
preoperatively. 
4.2.2 Exclusion Criteria 
• 
Stable condition without myocardial infarction in the past 30 days; 
• 
Undergoing concomitant cardiac surgery along with revascularization; 

• 
History of infection in the past 30 days; 
• 
Renal failure (GFR < 30); 
• 
Liver impairment (AST, ALT, or bilirubin > 2× normal values); 
• 
Known cirrhosis; 
• 
History of breast cancer or other estrogen-dependent tumors; 
• 
Intolerance to flavonoids, niacin, or ascorbic acid; 
• 
Requiring a quinolone in the postoperative period; 
• 
Inability to give informed consent; 
• 
Inability to communicate in French or English. 
4.2.3 Strategy for Recruiting and Retaining Study Participants 
The study will be presented to patients hospitalized in the relevant units who meet the 
inclusion/exclusion criteria. The research team will present the study and provide the information 
and consent form to potential participants for thorough review. Any questions will be answered 
by the research staff. 
One hundred (100) patients will be recruited and randomly assigned into the two study groups 
using a randomization table (quercetin 500 mg BID vs. placebo). 
 
4.3 Study Plan and Conduct 
4.3.1 Description of Study Progress and Visits 
At the screening visit—ideally 3 days (or at minimum 2 days) prior to surgery—patients will be 
approached. After signing the consent form, demographic and medical history questionnaires 
will be completed. Patients will then be randomized. 
Two days before or the day before surgery (t-1), the first blood sample (hs-CRP and ANGPTL2) 
will be taken, followed by administration of the intervention (quercetin 500 mg BID or placebo). 
On the day of surgery (t0), the study medication will be administered before and after surgery. 
On the day after surgery (t1), blood tests (hs-CRP and ANGPTL2) will be repeated, as well as on 
day 4 post-op (t2), and at hospital discharge or up to a maximum of 7 days post-op (t3). 
Annex 1 presents a schedule of procedures, study drug administration, and planned lab analyses. 
 
4.3.2 Description of the Intervention 
Details of the intervention are provided in Annex 1. 
Note: Study medication must be discontinued if a quinolone is prescribed postoperatively. 
However, to follow an intent-to-treat (ITT) approach, blood tests for hs-CRP and ANGPTL2 
should still be performed during hospitalization even if the patient has stopped taking the study 
medication. 
 
4.3.3 Description of Randomization 
Patients will be randomly assigned to one of the two study groups using a randomization table 
(quercetin 500 mg BID vs. placebo). 

 
4.4 Measurements and Data Collection Methods 
To characterize the study population, a demographic questionnaire will be administered. The 
following data will be collected: 
• 
Age 
• 
Sex 
• 
Preoperative weight 
• 
Height 
• 
Risk factors: 
o Dyslipidemia 
o Hypertension 
o Obesity 
o Diabetes 
o Active smoking 
o Chronic obstructive pulmonary disease 
o Family history of atherosclerotic heart disease 
• 
Date of prior myocardial infarction 
• 
Acute renal failure 
Preoperative medications: 
• 
Beta-blockers 
• 
ACE inhibitors 
• 
Angiotensin II receptor blockers 
• 
Diuretics 
• 
Antidiabetic drugs 
• 
Anticoagulants 
• 
Aspirin 
• 
Statins 
Hemodynamic values: 
• 
Blood pressure 
• 
Heart rate 
Blood analyses: 
• 
Total cholesterol 
• 
LDL (low-density lipoproteins) 
• 
HDL (high-density lipoproteins) 

• 
Triglycerides 
• 
HbA1c (glycated hemoglobin) 
• 
Creatinine 
• 
Estimated glomerular filtration rate (eGFR) 
In addition, at specified time points: 
• 
hs-CRP measurement 
• 
ANGPTL2 measurement 
These lab tests may be combined with standard pre- and post-operative bloodwork protocols. 
 
To evaluate the senolytic effect of quercetin on endothelial cells: 
• 
Ex vivo acetylcholine-induced endothelial relaxation measurement; 
• 
mRNA quantification in isolated arterial wall cells. 
 
5. Statistical Aspects 
5.1 Sample Size Calculation 
The primary evaluation criterion for this study is the change in hs-CRP blood levels between 
baseline (t-1) and four days after surgery (t2). A logarithmic transformation of the data is 
planned prior to analysis, and the sample size calculation was based on this assumption. 
Given this log transformation, the change between baseline and t2, as well as the difference 
between groups, is expressed as a ratio. Based on data from a previous study, the assumed 
standard deviation for the log-transformed hs-CRP ratio (hs-CRP at t2 / hs-CRP at t-1), common 
to both groups, is 1.215 (1). 
A sample of 50 subjects per group (100 subjects total) will allow detection of a 50% relative 
reduction in the change in hs-CRP in the quercetin group compared to the placebo group, with a 
two-sided significance level of 0.05 and a power of 80%. 
A 50% reduction in hs-CRP change would represent a clinically meaningful improvement. 
 
5.2 Statistical Analyses 
5.2.1 General Considerations 
Descriptive statistics will be provided for all study variables for the total sample and by 
intervention group. Means, standard deviations, medians, quartiles, minimums, and maximums 
will be used for continuous variables, while categorical variables will be described using 
frequencies and percentages. 
All assumptions underlying the planned analyses will be verified first. All statistical tests will be 
two-tailed with a significance threshold of 0.05. No imputation will be performed for missing 

values, and no adjustments will be made for multiple testing. Statistical analysis will be 
conducted using SPSS software version 25. 
 
5.2.2 Statistical Analyses 
A logarithmic transformation is planned for hs-CRP values prior to analysis. The change in hs-
CRP levels from baseline (t-1) will be compared between groups using repeated measures 
ANCOVA including terms for: 
• 
Intervention group (quercetin vs. placebo), 
• 
Time, 
• 
Group × Time interaction, 
• 
Baseline adjustment (t-1). 
The comparison between groups at day 4 post-surgery (t2) will address the primary objective. 
Comparisons at other time points will be considered exploratory. 
The change in ANGPTL2 levels will be analyzed in the same way as hs-CRP: repeated measures 
ANCOVA including group, time, interaction, and baseline adjustment. 
The endothelial relaxation function induced by acetylcholine (ex vivo) and mRNA 
expression assays in arterial wall cells will be compared between groups using Student's t-
tests. 
 
6. References 
1. Noly PE, Labbe P, Thorin-Trescases N et al. Reduction of plasma angiopoietin-like 2 
after cardiac surgery is related to tissue inflammation and senescence status of patients. J 
Thorac Cardiovasc Surg 2019;158:792–802.e5. 
2. Andres S, Pevny S, Ziegenhagen R et al. Safety Aspects of the Use of Quercetin as a 
Dietary Supplement. Mol Nutr Food Res 2018;62. 
3. Ou Q, Zheng Z, Zhao Y, Lin W. Impact of quercetin on systemic levels of inflammation: 
a meta-analysis of randomized controlled human trials. Int J Food Sci Nutr 2020;71:152–
163. 
4. Bhaskar S, Kumar KS, Krishnan K, Antony H. Quercetin alleviates hypercholesterolemic 
diet-induced inflammation during progression and regression of atherosclerosis in 
rabbits. Nutrition 2013;29:219–29. 
5. Egert S, Wolffram S, Schulze B et al. Enriched cereal bars are more effective in 
increasing plasma quercetin compared with quercetin from powder-filled hard capsules. 
Br J Nutr 2012;107:539–46. 
6. Boots AW, Haenen GR, Bast A. Health effects of quercetin: from antioxidant to 
nutraceutical. Eur J Pharmacol 2008;585:325–37. 

7. Wang L, Wang B, Li H et al. Quercetin, a flavonoid with anti-inflammatory activity, 
suppresses the development of abdominal aortic aneurysms in mice. Eur J Pharmacol 
2012;690:133–41. 
8. Mariee AD, Abd-Allah GM, El-Beshbishy HA. Protective effect of dietary flavonoid 
quercetin against lipemic-oxidative hepatic injury in hypercholesterolemic rats. Pharm 
Biol 2012;50:1019–25. 
9. Marunaka Y, Marunaka R, Sun H et al. Actions of Quercetin, a Polyphenol, on Blood 
Pressure. Molecules 2017;22. 
10. Eid HM, Haddad PS. The Antidiabetic Potential of Quercetin: Underlying Mechanisms. 
Curr Med Chem 2017;24:355–364. 
11. Patel RV, Mistry BM, Shinde SK, Syed R, Singh V, Shin HS. Therapeutic potential of 
quercetin as a cardiovascular agent. Eur J Med Chem 2018;155:889–904. 
12. Sharp MA, Hendrickson NR, Staab JS, McClung HL, Nindl BC, Michniak-Kohn BB. 
Effects of short-term quercetin supplementation on soldier performance. J Strength Cond 
Res 2012;26 Suppl 2:S53–60. 
13. McAnulty SR, McAnulty LS, Nieman DC et al. Chronic quercetin ingestion and 
exercise-induced oxidative damage and inflammation. Appl Physiol Nutr Metab 
2008;33:254–62. 
14. Nieman DC, Henson DA, Davis JM et al. Quercetin's influence on exercise-induced 
changes in plasma cytokines and muscle and leukocyte cytokine mRNA. J Appl Physiol 
(1985) 2007;103:1728–35. 
15. Zahedi M, Ghiasvand R, Feizi A, Asgari G, Darvish L. Does Quercetin Improve 
Cardiovascular Risk Factors and Inflammatory Biomarkers in Women with Type 2 
Diabetes: A Double-blind Randomized Controlled Clinical Trial. Int J Prev Med 
2013;4:777–85. 
16. Edwards RL, Lyon T, Litwin SE, Rabovsky A, Symons JD, Jalili T. Quercetin reduces 
blood pressure in hypertensive subjects. J Nutr 2007;137:2405–11. 
17. Dabeek WM, Marra MV. Dietary Quercetin and Kaempferol: Bioavailability and 
Potential Cardiovascular-Related Bioactivity in Humans. Nutrients 2019;11. 
 

Numéro du projet : ICM 2020-2761  
 
Version : 4 
Confidentiel 
Page 14 of 14 
 
ANNEX 1 – Timeline for Study Medication Administration and Blood Sampling 
t-2 (2 days before surgery)  - Screen hospitalized patients for eligibility 
Present study and obtain consent 
Administer demographic questionnaire 
Randomization  
t-1 (Day before surgery) 06:00 Blood tests (hs-CRP, ANGPTL2)  
  09:00  Study drug administration 
  21:00  Study drug administration  
t0 (Day of surgery)  06:00 or 09:00  Study drug (depending on surgical schedule)  
  During surgery  Collection of mammary artery segment  
  21:00  Study drug administration  
 t1 (Post-op Day 1)  09:00  Study drug administration  
  18:00  Blood tests (hs-CRP, ANGPTL2)  
  21:00  Study drug administration  
 Day 2  09:00 / 21:00  Study drug administration  
 Day 3  09:00 / 21:00  Study drug administration  
 t2 (Post-op Day 4)  06:00  Blood tests (hs-CRP, ANGPTL2)  
  09:00 / 21:00  Study drug administration  
 Day 5  09:00 / 21:00  Study drug administration  
 Day 6  09:00 / 21:00  Study drug administration  
 t3 (Discharge or max Day 7)  06:00  Blood tests (hs-CRP, ANGPTL2)  
  09:00  Final dose of study drug  
Note: Study medication will be administered daily throughout hospitalization, up to a maximum of 7 days post-surgery or until 
discharge.