Conditions: Atrial Fibrillation
Keywords: Ablation
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 95
Healthy volunteers: no

Brief summary:
The proposed studies will test this hypothesis by randomizing patients with AF to 2-HOBA or placebo 7 days prior to AF ablation to allow 2-HOBA to reach steady-state levels. We hypothesize that tissue injury from AF ablation causes a large release of ROS that react with lipids to generate IsoLGs (Figure 2). In the absence of 2-HOBA, IsoLGs will react within seconds to form IsoLG-macromolecule adducts in atrial tissue, promoting early recurrence of AF. In the presence of 2-HOBA, IsoLGs will rapidly react to form IsoLG-macromolecule adducts in atrial tissue, promoting early recurrence of AF. In the presence of 2-HOBA, IsoLG will preferentially bind to and therefore be inactivated by 2-HOBA thereby sparing injury to the atrial tissue caused by oxidative stress and its contribution to early recurrence of AF. Early recurrence of AF will be measured by ECGs that are recorded once per day by a smartwatch (Apple Watch, Apple Inc., Cupertino, CA) with additional ECGs recorded by the participant if they experience symptoms of AF, or if the smartwatch alerts the participant of a possible AF episode via its auto-detection AF monitoring algorithm. The Apple Watch's AF algorithm is based on sampling of heart rate and variability and will give an audible alarm if those parameters indicate a possible episode of AF. The smartwatch records a single-lead ECG if the participant touches the watch with their contralateral hand. The day and time of the episode is also stored by the smartwatch. At the end of the 28-day follow-up period, study personnel will review the stored ECGs. Blood will be drawn prior to ablation and on post-procedure Day 1 for measurement of IsoLG-adduct levels. DNA will be extracted to explore a pharmacogenomic interaction with haplotypes at the chromosome 4q25 AF risk locus, which: 1) is strongly associated with the development of AF and the early recurrence of AF after ablation27; and 2) has been reported to be a regulator of an anti-oxidant gene program in response to cardiac injury.

Detailed description:
The proposed double-blind, randomized, placebo-controlled trial of 2-HOBA in patients undergoing AF ablation is designed to address the following Specific Aims:

Specific Aim 1: To test the hypothesis that treatment with 2-HOBA reduces early recurrence of AF (clinical endpoint)

Specific Aim 2: To test the hypothesis that treatment with 2-HOBA reduces circulating levels of IsoLG-adducts (biochemical endpoint)

Specific Aim 3: To explore the idea that genetic variation at the 4q25 (PITX2) AF susceptibility locus modulates the clinical and biochemical response to 2-HOBA

Arms:
arm 1: 2-Hydroxybenzylamine(2-HOBA) 250 mg three tabs TID (po) for seven days prior to ablation and 28 days post ablation. arm 2: Placebo- three tabs TID (po) for seven days prior to ablation and 28 days post-ablation

Interventions: intervention 1: 2-Hydroxybenzylamine intervention 2: Placebo
Intervention descriptions: intervention 1: 2-HOBA (2-Hydroxybenzlamine) 750mg will be given TID seven days prior to ablation and 28 days post ablation. intervention 2: Placebo will be given TID for seven days prior to ablation and 28 days post ablation.

Protocol document:
Prot_SAP_000.pdf: 
 
 
1 
2/2/2021 
 
 
 
 
2-Hydroxybenzylamine (2-HOBA) to Prevent Early Recurrence 
of Atrial Fibrillation after Catheter-based Ablation  
 
 
 
Principal Investigator: 
 
Greg Michaud, MD 
Professor of Medicine 
Chief, Arrhythmia Section 
Division of Cardiovascular Medicine 
Vanderbilt University Medical Center 
 
 
Co-Principal Investigator: 
 
M. Benjamin Shoemaker, MD, MSCI 
Assistant Professor of Medicine 
Division of Cardiovascular Medicine, Arrhythmia Section 
Vanderbilt University Medical Center 
 
 
 
 


 
 
2 
2/2/2021 
Table of Contents: 
 
Study Schema 
1.0 
Background 
2.0 
Rationale and Specific Aims 
3.0 
Animal Studies and Previous Human Studies 
4.0 
Inclusion/Exclusion Criteria 
5.0 
Enrollment/Randomization 
6.0 
Study Procedures 
7.0 
Risks of Investigational Agents/Devices (side effects) 
8.0 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or Others 
9.0 
Study Withdrawal/Discontinuation 
10.0 
Statistical Considerations 
11.0 
Privacy/Confidentiality Issues 
12.0 
Follow-up and Record Retention 
13.0 
References 


 
 
3 
2/2/2021 
1.0 
BACKGROUND 
 
1.1 
New therapeutic strategies for atrial fibrillation (AF) are needed. Current rhythm control therapies for 
AF include the antiarrhythmic drugs (sodium and potassium channel blockers) and AF ablation (catheter or 
surgical).1 The recurrence rate for antiarrhythmic drugs is ~50%, while the recurrence rate for catheter ablation 
is up to 50% following a single procedure with 20-40% of patients requiring a second procedure.2 The 
antiarrhythmic drugs not only have limited and unpredictable efficacy but also a narrow therapeutic window with 
potentially fatal ventricular arrhythmias as a major concern for all agents,1 and clinical trials of catheter-based 
ablation have reported a rate of major complications of at least 1% including stroke, tamponade, and death.1 The 
limited efficacy and inherent risk of the antiarrhythmic drug class and AF ablation drive the need to develop 
alternative therapeutic strategies for AF.  
 
1.2 
Inflammation and oxidative stress promote AF and its clinical risk factors. Numerous clinical risk 
factors such as obesity, hypertension, diabetes mellitus/metabolic syndrome, and aging are known to increase 
the risk of AF, but the molecular pathways linking these comorbidities to AF are incompletely understood.3-5 It is 
well recognized that many of these comorbidities are associated with inflammation, which is believed to be a 
major shared mechanism.6 Inflammation is the immune response to cellular injury generated by oxidative stress 
resulting in overabundance of reactive oxygen species (ROS) such as superoxide anions (O2˙ˉ) and hydroxyl 
radicals. Inflammation and oxidative stress are closely related, and each promotes the generation of the other. 
Oxidative stress is known to result in damage to lipids, proteins, and DNA, and is implicated in the development 
of a wide range of diseases including cancer, atherosclerotic cardiovascular disease, autoimmune disease, 
neurological disorders, as well as the pathogenesis and progression of AF.  
 
1.3 
An optimal antioxidant or anti-inflammatory drug to treat AF has not been identified. As oxidative 
stress became increasingly recognized for its role in AF pathogenesis7-9, numerous studies were conducted 
investigating the use of dietary antioxidants such as vitamins C, E, and omega-3 polyunsaturated fatty acid (n-3 
PUFAs or “fish oil”) for the treatment of AF. The major limitation of treatment with vitamins C and E are that they 
fail to significantly suppress oxidative stress, which is their therapeutic target.10 This is also a problem for n-3 
PUFA.11 In a clinical trial conducted by our research group, 190 patients with AF were randomized to high dose 
n-3 PUFA or matching placebo and followed for 6-months for AF recurrence. The rate of AF recurrence was 59% 
in the n-3 PUFA group compared to 47% in the placebo group with no difference in the primary endpoint of time-
to-AF recurrence (HR=1.2 [95% CI: 0.8-1.8] P=0.39). Furthermore, there was no difference in the concentration 
of inflammatory cytokines (Interleukins 6, 8, 10; tumor necrosis factor alpha) or measures of oxidative stress 
(urinary F2-isoprostane) between n-3 PUFA and placebo groups.11 Given a large amount of ROS can be 
generated by a variety of different cell types (neutrophils, monocytes, myofibroblasts), a major problem with the 
antioxidant drug class appears to be that they 
cannot achieve sufficient in-vivo concentrations to 
reduce ROS levels.12 Anti-inflammatory drugs, 
steroids and colchicine, can both reduce AF after 
ablation or cardiac surgery, but the side-effects 
preclude widespread long-term use. Steroids are 
associated 
with 
immunosuppression, 
glucose 
intolerance, and agitation; and a meta-analysis of 
studies using colchicine for AF found a 21% rate of 
GI intolerance (diarrhea, nausea, abdominal pain) 
in the colchicine group compared to 8% in placebo 
(P<0.001).13 
 
1.4 
Isolevuglandin 
scavengers 
are 
compounds that target oxidative stress but are 
NOT anti-inflammatories or antioxidants.   
Given the failure of the anti-inflammatory and 
antioxidant drug classes to effectively treat AF in 
humans, the discovery of a new group of 
Figure 1: The dicarbonyl motif of isolevuglandin (IsoLG) 
binds rapidly to lysine side chains to form a dysfunctional 
IsoLG-protein adduct. However, in the presence of an 
IsoLG scavenger such as 2-HOBA, IsoLG preferentially 
binds to 2-HOBA resulting in an inactive byproduct. 


 
 
4 
2/2/2021 
compounds that serve as downstream mediators of oxidative stress damage, the isolevuglandins (IsoLGs), 
represents an important conceptual advance in attacking this pathway to AF. IsoLGs are the product of lipid 
peroxidation, which occurs when ROS react with polyunsaturated fatty acids present in cell membranes or freely 
circulating.14 IsoLGs are highly reactive and following their formation bind within seconds to macromolecules 
with lysine residues forming IsoLG adducts (Figure 1).15 IsoLG adducts are irreversible modifications that cause 
dysfunction of protein targets, including structures relevant to cardiomyocyte homeostasis (e.g. ion channel 
proteins, proteasomes, chromatin, mitochondria). IsoLGs are so highly reactive that they cannot be measured 
in their unreacted form and must instead be measured as IsoLG-adducts.15 The discovery of the IsoLGs has 
identified a critical component in the oxidative stress pathway that may be targeted therapeutically.16 IsoLG 
scavenger compounds, such as 2-HOBA, can bind IsoLG orders of magnitude more rapidly than IsoLG can bind 
with the lysine on other macromolecules, thereby generating permanently inactivated IsoLG compounds rather 
than IsoLG-adducts.16 This study aims to test the efficacy of a therapeutic strategy targeting IsoLG for treatment 
of AF using the IsoLG scavenger 2-HOBA.  
 
1.5 
Early recurrence of AF after catheter-based 
ablation can be used to study AF due to inflammation 
and oxidative stress. Over the past decade, post-
operative AF has been studied extensively as a model of 
AF due to inflammation and oxidative stress.17-20 
However, AF research in the post-cardiac surgery setting 
is complicated because the patients often have severe 
comorbid coronary and valvular heart disease, may be on 
a highly variable amount of vasopressors, may remain 
intubated and sedated for days, often have large volume shifts from intravenous fluids and blood products, and 
may have an unknown history of AF prior to surgery. We propose here to study early recurrence of AF in patients 
who have undergone catheter ablation for AF as an alternative to the post-cardiac surgery setting.  
 Early recurrence of AF is conventionally defined as any recurrence of AF, atrial flutter, or atrial 
tachycardia lasting >30 seconds within 90-days following ablation.2 Approximately 50% of patients experience 
early arrhythmia recurrence and the vast majority of those recurrences are within the first 30-days.2,21 Compelling 
evidence supports oxidative stress and inflammation caused by 
ablation-related atrial tissue injury as the main contributor to early 
recurrence.22-26 Up to 75% of patients experience symptoms from 
pericarditis following AF ablation, and numerous studies have 
demonstrated 
that 
the 
use 
of 
anti-inflammatory 
medications 
(corticosteroids, colchicine) reduces early AF recurrence (Table 1).22-26  
Despite these promising results, common side-effects from steroids 
(immunosuppression, agitation, glucose intolerance) and colchicine 
(gastrointestinal intolerance) preclude widespread long-term use of 
these agents for AF.  
 
 
2.0 
RATIONALE AND SPECIFIC AIMS 
 
We hypothesize that oxidative stress causes AF through the 
production of isolevuglandins and that treatment with IsoLG 
scavengers will significantly reduce AF. The proposed studies will 
test this hypothesis by randomizing patients with AF to 2-HOBA or 
placebo 3 days prior to AF ablation to allow 2-HOBA to reach steady-
state levels. We hypothesize that tissue injury from AF ablation causes 
a large release of ROS that react with lipids to generate IsoLGs (Figure 
2). In the absence of 2-HOBA, IsoLGs will react within seconds to form 
IsoLG-macromolecule adducts in atrial tissue, promoting early 
recurrence of AF. In the presence of 2-HOBA, IsoLGs will rapidly react 
to form IsoLG-macromolecule adducts in atrial tissue, promoting early 
Reference 
N 
Study Drugs 
Risk 
Koyama 
128 
Prednisone 
-22% 
Kim 
138 
Methylprednisolone 
-25% 
Deftereos 
161 
Colchicine 
-18% 
Deftereos 
223 
Colchicine 
-18% 
 
Table 1: Studies of early AF recurrence with anti-
inflammatories. 
Figure 2: Tissue injury from AF ablation 
generates oxidative stress resulting in 
IsoLG-adduct formation that contributes 
to early recurrence of AF. 


 
 
5 
2/2/2021 
recurrence of AF. In the presence of 2-HOBA, IsoLG will preferentially bind to and therefore be inactivated by 2-
HOBA thereby sparing injury to the atrial tissue caused by oxidative stress and its contribution to early recurrence 
of AF. Early recurrence of AF will be measured by ECGs that are recorded once per day by a smartwatch (Apple 
Watch, Apple Inc., Cupertino, CA) with additional ECGs recorded by the participant if they experience symptoms 
of AF, or if the smartwatch alerts the participant of a possible AF episode via its auto-detection AF monitoring 
algorithm. The Apple Watch’s AF algorithm is based on sampling of heart rate and variability and will give an 
audible alarm if those parameters indicate a possible episode of AF. The smartwatch records a single-lead ECG 
if the participant touches the watch with their contralateral hand. The day and time of the episode is also stored 
by the smartwatch. At the end of the 28-day follow-up period, study personnel will review the stored ECGs. Blood 
will be drawn prior to ablation and on post-procedure Day 1 for measurement of IsoLG-adduct levels. DNA will 
be extracted to explore a pharmacogenomic interaction with haplotypes at the chromosome 4q25 AF risk locus, 
which: 1) is strongly associated with the development of AF and the early recurrence of AF after ablation27; and 
2) has been reported to be a regulator of an anti-oxidant gene program in response to cardiac injury.28  
 
The proposed double-blind, randomized, placebo-controlled trial of 
2-HOBA in patients undergoing AF ablation is designed to address the 
following Specific Aims: 
 
Specific Aim 1: 
To test the hypothesis that treatment with 2-
HOBA reduces early recurrence of AF (clinical 
endpoint) 
 
Specific Aim 2:  
To test the hypothesis that treatment with 2-
HOBA reduces circulating levels of IsoLG-
adducts (biochemical endpoint) 
 
Specific Aim 3: 
To explore the idea that genetic variation at the 
4q25 (PITX2) AF susceptibility locus modulates 
the clinical and biochemical response to 2-
HOBA  
 
 
3.0 
ANIMAL STUDIES AND PREVIOUS HUMAN STUDIES 
 
3.1. 
The IsoLG scavenger, 2-HOBA, is remarkably effective in 
experimental models of AF 
Oxidative stress contributes to the development of numerous diseases 
through oxidative protein modification. Early results in mouse models of 
Alzheimer’s disease and hypertension demonstrated that treatment using 
2-HOBA effectively reduced cognitive impairment in the Alzheimer’s model 
and lowered blood pressure in the hypertension model.29,30 Our research 
group has been investigating the use of IsoLG scavengers to treat AF in 
experimental mouse models.31 Obesity and hypertension are two of the 
strongest clinical risk factors for the development of AF and are well-
established states of high inflammation and oxidative stress.32 In a diet-
induced obesity mouse model, treatment with 2-HOBA significantly 
reduced AF inducibility compared to controls. The control group included 
mice treated with an inactive structural analog, 4-HOBA, which does not 
scavenge IsoLG (Figure 3). In an angiotensin-II hypertension mouse 
model, AF was readily induced and treatment with 2-HOBA successfully 
reduced inducibility compared to 4-HOBA while a group treated with 
antihypertensive medications (hydralazine and hydrochlorothiazide) also 
had less AF (Figure 3).31 AF is also inducible in a new mouse model of 
inflammation without hypertension. Further in the models of inducible AF, IsoLG adducts elevated at baseline in 
Obesity Model
Hypertension Model
Figure 3: Murine models of obesity and 
hypertension have inducible AF with 
rapid atrial pacing. Treatment with an 
IsoLG scavenger, 2-HOBA, reduces AF 
inducibility, but treatment with an 
inactive structural analog, 4-HOBA, 
does not. (LFD=low fat diet, HFD=high 
fat 
diet, 
angII=angiotensin 
II, 
hyd=hydralazine, 
HCTZ=hydrochlorothiazide). 


 
 
6 
2/2/2021 
the atria are lowered by 2-HOBA. Taken together, a therapeutic strategy aimed at reducing reactive IsoLG-
adduct levels through the use of scavenger compounds such as 2-HOBA has yielded positive results in multiple 
mouse models of oxidative-stress induced AF and provides the basis for advancing this line of research to 
humans.  
 
 
3.2. 
A Phase I clinical trial of the IsoLG scavenger 2-HOBA  
A first-in-humans Phase I clinical trial using oral 2-HOBA was recently completed to assess its safety and 
pharmacologic profile. A single dose study (NCT-3176940) and a multidose study (NCT-03555682) were 
conducted. In the single dose escalation study, 6 ascending doses of 2-HOBA were studied. Three participants 
were enrolled at each dose and 18 participants successfully completed the study.33 The highest planned dose 
(825 mg) was reached. There were no serious adverse events. There were 5 total adverse events (AEs). They 
were all determined to be mild in intensity and not dose-dependent. Reported AEs included: frequent urination 
(N=2), headache (N=1), itchy throat (N=1), rash (N=1), sleepiness (N=1) and abdominal bloating (N=1). No 
clinically significant changes in ECG recordings, vital signs, or laboratory parameters were observed.  
 
In the multi-dose randomized, double-blind clinical trial, 18 healthy volunteers were given 2-HOBA or placebo 
for two weeks. Two doses of 2-HOBA were tested, 500 mg or 750 mg. The study assessed the safety, tolerability, 
and pharmacokinetics of dosing over a two week period, and demonstrated a safe and well-tolerated profile with 
three-times daily dosing. 9 subjects were studied at each dose level, with 6 receiving the ADI and 3 receiving 
placebo. These doses were chosen to achieve similar plasma levels at steady state compared with the single 
dose study. No serious AEs were observed. 14 patients reported at least 1 adverse event (Table 2). Reported 
AEs included headache (N=6), GI distress (N=3), rash/itching (N=3), urine odor (N=2), dry mouth (N=2), nasal 
congestion (N=2), lethargy/sleepiness (N=2), hypertension (N=1), and eye irritation (N=1). One volunteer 
experienced a rash of moderate intensity, and was withdrawn from the study, though this AE was not determined 
to be study-related or dose-dependent. 
 
2-Hydroxybenzylamine acetate dose 
Total  
(n=18) 
Placebo 
(n=6) 
500 mg 
(n=6) 
750 mg 
(n=6) 
Any event, n (%) 
4 (67) 
6 (100) 
4 (67) 
14 (78) 
Headache 
2 (33) 
2 (33) 
2 (33) 
6 (33) 
GI distress (nausea, bloating, constipation) 
2 (33) 
1 (17) 
0 (0) 
3 (17) 
Rash/itching  
1 (17) 
1 (17) 
1 (17) 
3 (17) 
Urine odor 
0 (0) 
2 (33) 
0 (0) 
2 (11) 
Dry mouth 
1 (17) 
1 (17) 
0 (0) 
2 (11) 
Nasal congestion 
0 (0) 
2 (33) 
0 (0) 
2 (11) 
Lethargy/sleepiness 
0 (0) 
1 (17) 
1 (17) 
2 (11) 
Hypertension 
0 (0) 
1 (17) 
0 (0) 
1 (6) 
Eye irritation 
0 (0) 
1 (17) 
0 (0) 
1 (6) 
 
Table 2. Summary of reported adverse events by dose in multi-dose Phase 1 study of 2-HOBA 
 
The pharmacokinetics observed in the multi-dose trial largely resembled those obtained in the single dose study. 
2-HOBA had a Tmax of 1-2 h and achieved steady-state Cmax in the range of those observed with the highest dose 
in the single dose study - 3177 ± 1993 ng/mL after 15 days of dosing at 500 mg and 2292 ± 913 ng/mL after 15 
days of dosing at 750 mg. Both groups (500 and 750 mg doses) experienced progressive AUC increases over 
the 15 days, yielding accumulation ratios of 1.25-1.50. The half-life in this dosing interval was slightly longer than 
that observed in the single dose study, contributing to the increased accumulation of 2-HOBA. Average systemic 
exposure (AUC and Cmax) was similar for the 500 and 750 mg doses. Response to 2-HOBA revealed considerable 
variability among subjects, as those dosed at the 500 mg regimen showed greater exposure on average than 
volunteers dosed at 750 mg. Oral bioavailability is not currently established in humans, and limits the 
interpretation of these data. Notably, the major metabolite of 2-HOBA, salicylic acid, showed a peak plasma 


 
 
7 
2/2/2021 
concentration of 12.8 ± 3.7 mg/L, considerably lower than the accepted therapeutic range of 150-300 mg/L 
required for anti-inflammatory activity. Consequently, inhibition of cyclooxygenases was not observed, as 
determined by urinary metabolites of 3 major prostaglandins, pGE-M, TxB2-M, and PGI-M.] 
  
No evaluation of the compound in pediatric settings or in patients with reported history of disease has been 
undertaken. A greater range of age and ethnicity should be explored in additional clinical studies to gain a better 
understanding of its clinical profile. 
 
 
3.3. 
The peak plasma concentration of 2-HOBA in humans will reach the level demonstrated to reduce 
IsoLG-adduct formation in experimental models 
Prior studies in mice demonstrated that the plasma concentration of 2-HOBA needed for efficacy was ~2.7 µM.16 
To date, the peak plasma concentration of 2-HOBA in humans has been analyzed from subjects given the 50mg 
and 100mg doses in our Phase I clinical trial. The peak plasma concentration of 2-HOBA from subjects given a 
single 100mg dose was 1.7 µM. The single dose escalation study has administered the 550mg dose without any 
adverse events, which based on estimates using first-order kinetics, is expected to achieve a peak plasma 
concentration (8.5 μM) that significantly exceeds the level found to be efficacious in experimental models. 
Therefore, 2-HOBA is safely tolerated at doses that will produce plasma 
concentrations known to reduce formation of IsoLG-adducts. 
 
3.4. 
A pilot study demonstrates that AF ablation increases IsoLG 
adduct levels 
In preparation for this trial, a pilot study (IRB #171722) was conducted to 
demonstrate the feasibility of the study protocol, demonstrate that IsoLG-
adduct levels increase following AF ablation, and generate preliminary data 
for the power/sample size analysis. These measurements are made using 
a cellular system (since IsoLG adducts are not free in the circulation), and 
has used IsoLG measurements in monocytes and dendritic cells (DC).30 
Over an 8-week period, 11 subjects who met the eligibility criteria for our 
proposed clinical trial were prospectively enrolled and underwent AF 
ablation. The data from our pilot study support the following conclusions: 
 
1. The rate of participant enrollment was sufficient to meet the enrollment 
target of this proposal. We have proposed to enroll 1 participant per 
week to accrue 162 participants over 36 months. We enrolled 1.375 
participants per week in the pilot study. 
 
2. AF ablation resulted in a statistically significant increase in circulating 
IsoLG-adduct levels. IsoLG-adduct levels were measured in blood 
samples collected pre-ablation and on post-procedure day #1. IsoLG-
adduct levels increased in 82% (9/11) of subjects, and the mean 
increase was 23% in DCs (Pre: 41±16% vs. Post: 63±17%; P=0.02; 
Figure 4, top panel) and 21% (Pre: 38±19% vs. Post: 59±18%; 
P=0.04) in monocytes following ablation.  
 
3. There was an early recurrence of AF in 45% of participants (5/11). This 
demonstrates that, similar to other published reports, early recurrence 
of AF is common in our study population and is sufficient to power the 
primary endpoint of Aim 1, which is AF recurrence within 28-days 
following ablation (Figure 4, bottom panel). This is comparable to the 
early recurrence rate of AF from numerous other published reports, 
which is ~50%.21,23 
 
 
0
1
2
3
4
S u b je c ts  (N )
0 -7
8 -1 4
1 5 -2 1
2 2 -2 8
> 2 8
3
1
0
1
0
D a ys  to  A F  R e cu rre n ce
Figure 4: (Top) There is a 
significant increase in the 
proportion of dendritic cells 
containing IsoLG-adducts from 
pre-ablation to post-operative day 
#1 (POD1; P=0.02). (Bottom) 
45% of participants had an early 
recurrence of AF within our 
proposed 28-day monitoring 
period. 


 
 
8 
2/2/2021 
4.0 
INCLUSION/EXCLUSION CRITERIA 
 
 
 
5.0 
ENROLLMENT AND RANDOMIZATION 
 
Recruitment and informed consent: Eligible subjects will be identified through EPIC. A member of the study team 
will request an introduction from the clinical staff. If the patient agrees, a member of the study team will approach 
the patient in person in Arrhythmia Clinic or by phone prior to AF ablation. Information describing the study, why 
the research is being done and what will be learned will be provided. The risks as described in this protocol will 
be delineated. The benefits will be described as general scientific knowledge, along with potential treatment 
advances. No direct immediate benefit to the subjects is anticipated. Informed consent will be documented with 
the subject’s signature using an IRB-approved consent form for this protocol. An option will be available for 
phone consent with electronic signature. 
 
Randomization: This will be a double-blind, randomized study. Eligible subjects will be randomized according to 
a stratified permuted block scheme with a block size of 8. This ensures that the cumulative number of 
assignments to each treatment (2-HOBA or placebo) will be in balance after each block of assignments had 
been made.  Stratification will be made based on: 1) the planned ablation technique, cyroballoon vs. 
radiofrequency, 2) paroxysmal vs. persistent atrial fibrillation, 3) de novo vs. repeat ablation.  A statistician will 
design the randomization table and enable the randomization tool within REDCap. After a patient enrolls for the 
study, the study nurse will determine the treatment assignment using the randomization tool in REDCap.  
 
The study drug will be started 3 days prior to ablation. The dose of 2-HOBA will be 750mg three times 
per day (TID). At least 24 hours in advance, the study nurse will notify IDS to randomize the participant and 
prepare the study drug. MTI will provide 2-HOBA to the IDS as a 250 mg capsule. MTI will also provide matching 
INCLUSION 
 
EXCLUSION 
First time AF ablation with radiofrequency or cryo 
ablation 
Planned surgical or hybrid (surgical + catheter) 
ablation 
Repeat AF ablation if the patient has persistent AF 
and ablation of non-pulmonary vein substrate is 
planned (e.g. posterior wall ablation, mitral or roof 
line, etc) 
Current use of amiodarone or within past 3 months 
Able to provide written, informed consent 
Use of oral steroids or colchicine 
22 years of age or older 
Pro-inflammatory, rheumatologic disorder (e.g. RA, 
SLE, IBD, psoriasis, ankylosing spondylitis) 
 
NYHA Class III/IV Heart Failure 
 
Active ischemia 
 
Hypertrophic Cardiomyopathy 
 
Cardiac or thoracic surgery within 6 months 
 
Expected life span < 1 year 
 
Creatinine clearance <30 ml/min 
 
Prior or planned heart transplantation 
 
Pregnant women 
 
Aspirin allergy 
 
Current use of MAO-I 
RA=rheumatoid arthritis; SLE=systemic lupus erythematosus; IBD=inflammatory bowel disease (Crohn’s, 
Ulcerative Colitis); NYHA=New York Heart Association; LVEF=left ventricular ejection fraction 
 
Table 3: Eligibility Criteria. 
 


 
 
9 
2/2/2021 
placebo capsules. The study nurse will arrange for the study drug to be given to the participant in-person or 
mailed. The total duration of the study drug is 31 days (3 days before ablation + 28 days after ablation).  
 
6.0 
STUDY PROCEDURES 
 
Encounter 1: Enrollment and Informed Consent 
• 
Performed by a member of the study team either in person in the Vanderbilt Arrhythmia Clinic or by phone 
with an electronic signature.  
• 
In person meetings will occur in either: 1) the Vanderbilt Cardiac MRI Suite (if a pre-ablation cardiac MRI is 
scheduled), 2) the Vanderbilt Cardiac CT Suite (if a pre-ablation cardiac CT is scheduled), 3) the VHVI 
Research Room (5th Floor Medical Center East, South Tower), or at their cardiology or imaging appointments 
at the One Hundred Oaks location (if applicable).  
• 
A supply of the study drug will be given to the participant along with dosing instructions either in-person or 
via the mail. 
 
Encounter 2: Day of ablation 
• 
The participant will arrive the morning of ablation as instructed by the clinical team 
• 
Prior to ablation, the participant will be taken to the EP pre-op holding room. A dose of the study drug will be 
taken at this time (~30 minutes prior to the procedure). 
• 
The study team will provide the participant with the smartwatch as well as an accompanying smart phone (if 
the patient does not own their own iPhone).  The study team will initialize the smartwatch and phone with 
configurations to collect data, and instruct patients on how to record ECGs from the smartwatch. 
• 
After the patient arrives in the operating room, they will undergo standard of care preparation for surgery 
including sterile drape and administration of general anesthesia using endotracheal intubation and 
mechanical ventilation. Central venous catheters are placed in the femoral veins as part of the procedure. 
• 
A 30 mL blood sample will be collected from one of the central venous catheters prior to ablation. 10 mL of 
blood (divided between purple and blue top tubes) will be delivered to the Core Laboratory for Cardiovascular 
Translational and Clinical Research, and 20 mL will be put in green top tubes, placed on ice, and delivered 
to the research laboratory for culturing of monoctyes and dendritic cells for measurement of isolevuglandin-
adduct levels. 
• 
AF ablation will then be performed as per standard of care by the primary operator and clinical team.  
• 
A post ablation blood draw will be collected at the conclusion of the AF ablation prior to the central venous 
catheter removal.  We will collect an additional 30 mL blood sample for research, with 10 mL of blood 
delivered to the Core Laboratory for Cardiovascular Translational and Clinical Research and 20 mL put in 
green top tubes and delivered to the research laboratory for culturing of monocytes and dendritic cells for 
measurement of isolevuglandin-adduct levels 
• 
Following ablation, the patient will be extubated while in the operating room and returned to the EP holding 
room awake and alert. 
• 
After removal of central venous catheters and when the patient is ready to begin eating and drinking, another 
dose of the study drug will be given.  
 
Encounter 3: Post-ablation day #28 
• 
The participant will be met in either the: 1) VHVI Arrhythmia Outpatient Clinic at the time of their 1-month 
Post-op Clinical Visit (Medical Center East or One Hundred Oaks locations), or 2) the VHVI Research Room. 
• 
A 10 mL blood sample (divided between purple and blue top tubes) will be delivered to the Core Laboratory 
for Cardiovascular Clinical and Translational Research. 
• 
The study personnel will record any ECGs recorded from the smartwatch from the day of the ablation until 
the end of the 28-day of follow-up period 
• 
The study personnel will record remaining pills of either placebo or 2-HOBA at the conclusion of the study 
• 
Participants will be allowed to keep the smartwatch 
• 
Should the participant have required an iPhone as well as the smartwatch, the phone will be returned to the 
study personnel 


 
 
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Encounter 4: 6 and/or 12 month follow up visits 
• 
Participants undergoing ablation are routinely seen at 6 and 12 months during follow up as part of clinical 
care 
• 
During these visits, study team personnel may review medical records and meet with participants regarding 
clinical outcomes of their ablation procedure 
• 
If 6 or 12 month follow up visits are not planned, study personnel may call patients during the 12 month time 
frame to see if any clinical events have occurred 
 
7.0 
RISKS 
 
Patients undergoing AF ablation are at risk for complications by nature of the procedure. These risks are 
inherent to the patient population studied here. The risks of obtaining blood samples from phlebotomy of 
peripheral veins and existing venous lines are minimal.  
 
One risk of donating samples for genetic research may be the release of information that could link the 
patient to the stored samples and/or the results of the tests run on those samples. The release of this information 
could cause problems with insurance or future employment. We have mechanisms in place to protect against 
such risks.  
 
8.0 
REPORTING OF ADVERSE EVENTS OR UNANTICIPATED PROBLEMS INVOLVING RISK TO 
PARTICIPANTS OR OTHERS 
 
Data and Safety Monitoring Plan 
 
Oversight of the data and safety monitoring plan will be provided by a Data and Safety Monitoring Board (DSMB). 
The DSMB will meet at least twice a year and review data on adverse events, adverse drug reactions, data 
quality, and study recruitment. DSMB reports will be sent to the IRB at least yearly.  
 
Data Safety Monitoring Board (DSMB) will be formed to review safety data, study progress, and data quality 
every 6 months or after enrollment of every 30 subjects, whichever occurs earlier. The DSMB will be chaired by 
Dr. Julia Wattacheril, MD, MPH Associate Professor of Medicine, Director, Nonalcoholic Fatty Liver Disease 
Program, Columbia University – NY Presbyterian Hospital; an external member will be Dr. Christine Albert, MD, 
MPH, Professor of Medicine, Brigham and Women’s Hospital, Director of the Center for Arrhythmia Prevention, 
who has expertise in clinical trials of AF; and a biostatistical member, Gregory Dan Ayers, who has expertise in 
biostatistical review for DSMBs. The DSMB will review all serious adverse events (AEs). Any serious AE will be 
reported to the DSMB, IRB, and FDA as soon as possible, but not more than three days from the investigators’ 
awareness of the event. Any untoward medical event will be classified as an AE, regardless of its causal 
relationship with the study. 
 
Reporting of Adverse Events or Unanticipated Problems involving Risk to Participants or Others: 
 
Definitions of adverse events: an adverse event (AE) is "any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product and which does not necessarily have a causal 
relationship with this treatment". 
 
Adverse events include: 
o Worsening (change in nature, severity or frequency) of conditions present at the onset of the trial 
o Patient / subject deterioration due to the primary illness 
o Intercurrent illnesses 
o Drug interactions 
o Events related or possibly related to concomitant medications 
o Abnormal laboratory values or changes of vital signs, as well as significant shifts from baseline within the 
range of normal, which the Investigator considers clinically significant. 


 
 
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Unexpected Adverse Drug Reaction: an unexpected Adverse Drug Reaction is “an adverse reaction, the nature 
or severity of which is not consistent with the applicable product information (e.g., Investigator’s Brochure for an 
unapproved investigational medicinal product)”. Definitions of serious adverse events or serious adverse drug 
reaction: during clinical investigations, adverse events may occur which, if suspected to be drug-related (adverse 
drug reactions), must be significant enough to lead to important changes in the way the medicinal product is 
developed (e.g., change in dose, population, needed monitoring, consent forms). This is particularly true for 
reactions, which, in their most severe forms, threaten life or function.  
 
A serious adverse event/experience (SAE) or reaction is any untoward medical occurrence that at any dose: 
 
1. results in death 
2. is life-threatening 
3. requires inpatient hospitalization or prolongation of existing hospitalization 
4. results in persistent or significant disability/ incapacity (as per reporter’s opinion) 
5. is a congenital anomaly/birth defect 
6. is another medically important condition 
7. The term "life-threatening" in the definition of "serious" refers to an event in which the patient is at risk of 
death at the time of the event; it does not refer to an event which hypothetically might have caused death 
if it were more severe. 
 
Important medical conditions that may not result in death, be life-threatening, or require hospitalization may be 
considered as SAEs when, based upon appropriate medical judgment, they may jeopardize the patient or may 
require intervention to prevent one of the outcomes listed in the definition above. 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.  
 
Definition of severity of adverse events: 
 
Mild: 
 
Causing no limitation of usual activities; the subject / patient may experience slight discomfort. 
 
Moderate:  
Causing some limitation of usual activities; the subject / patient may experience annoying 
discomfort. 
 
Severe:  
Causing inability to carry out usual activities; the subject / patient may experience intolerable 
comfort or pain. 
 
Definition of adverse event causality: 
 
The Investigator will determine causality of each adverse event by using the classification criteria: unlikely, 
likely, or not assessable. 
 
Unlikely: 
The AE is considered by the Investigator to be due to a pre-existing condition, a known 
manifestation of the target disease, a recurrent condition, or is likely explained by environmental 
or diagnostic therapeutic factors or was pre-existing and did not deteriorate. 
 
Likely: 
The AE occurred during or after administration of the study treatment or a pre-existing event 
worsened within an appropriate period of time, and at least one of the following criteria is 
applicable:  
o the event could not be explained by the clinical condition or history of the subject, 
environmental or toxic factors, or other diagnostic or therapeutic measure; 
o was an expected ADR associated with study treatment or a class-labeled drug effect; 
o AE subsided or disappeared after withdrawal or dose reduction of study treatment; or 
o AE recurred after re-exposure to study treatment. 


 
 
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Not assessable: There is insufficient or conflicting evidence for classifying the causality of the AE as likely or 
unlikely. Lack of information may apply for this situation. 
 
Note: AEs with causality 'likely' or 'not assessable' are considered to be 'possibly drug-related.' 
 
 
Adverse event reporting 
 
Any adverse events (AEs) will be reported to the PI within 72 hours of notification of the event. The PI will notify 
the DSMB of any major adverse events. Any unanticipated problems involving risk to the participants or others 
will be discussed with the PI and DSMB. Non-serious AEs and incidences of noncompliance with the protocol 
will be reported to the IRB at the time of annual review. 
 
Serious Adverse Events (SAEs) will be reported according to the following procedure: 
 
The occurrence of serious adverse events will be reported to the Investigator within 24 hours after notification of 
their occurrence. The Investigator will report SAEs to the DSMB and the Vanderbilt University Medical Center 
Institutional Review Board within 7 days of the Investigator’s notification of the event. 
 
In an unanticipated event of prolonged side effect, requiring prolongation of hospital stay, patients will be 
retained in the hospital until side effects have resolved. For minor side effects, where inpatient care is deemed 
unnecessary, follow- up will be maintained via phone or as outpatient if necessary. Patient and their families will 
be given the PI’s contact number for reporting any other effects of medication following discharge. 
 
Any newly discovered information which may affect the subject or their caregiver’s decision to continue to 
participate in the study will be passed on to them as soon as possible. This may also result in a change to the 
consent form and review by the IRB. 
 
9.0 
STUDY WITHDRAWAL/DISCONTINUATION 
 
Participants may withdraw from the study at any time by informing the study staff verbally or in writing. If an 
individual withdraws their consent, we will withdraw the participant. Contact information for the PI and study staff 
will be made available to the participant upon enrollment in the consent document.  Any remaining biological 
samples and data will be destroyed. Any data or biological samples that have been used for research prior to 
their withdrawal request will not be withdrawn and destroyed. 
A participant may be withdrawn from the study by the PI if any of the following occurs: 
i. The participant, for any reason, does not undergo AF ablation. 
ii. The participant does not comply with the study protocol, such as failure to attend research encounters, or 
inability to wear the smartwatch.  
iii. The participant experienced an adverse event related to the study drug/protocol necessitating withdrawal. 
  
10.0 
STATISTICAL CONSIDERATIONS 
 
Dr. Fei Ye (Associate Professor of Biostatistics, Vanderbilt University Medical Center) formulated the plan and 
will direct the statistical analysis for this trial.  
Analysis Sets: 1) Full analysis set (FAS): All randomized participants. The FAS will be used in the analysis of all 
efficacy endpoints. Participants will be included in FAS according to the treatment to which they are randomized. 
2) Safety analysis set (SAS): All randomized participants who have received 2-HOBA. The SAS will be used in 
the analyses of all safety endpoints. Participants will be included in the analyses according to the treatment they 
actually received.  


 
 
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General Statistical Strategies: Patient demographics/other baseline characteristics will be listed by patient and/or 
summarized descriptively by treatment group and by stratification factors. Categorical data will be presented as 
frequencies and percentages. Binary and categorical data will be analyzed with Fisher’s exact test and chi-
squared test. For continuous data, summary statistics will be presented. For single time-point data, between-
group differences will be assessed with t-test (2 groups) or analysis of variance (3 or more groups) based on a 
continuous variable. Nonparametric counterparts, Wilcoxon rank sum test and Kruskal-Wallis test, will be used 
when assumptions for parametric methods are not met. For correlated data (e.g., repeated measurements) will 
be analyzed with generalized estimating equation (GEE) or linear mixed models (LMM). Multiple comparison 
issues will be corrected using the Bonferroni’s approach. All statistical analysis will be performed using R 3.4.2 
or a newer version.  
 
10.1. Aim 1 (Clinical Endpoint)  
The primary hypothesis will test whether 2-HOBA reduces the rate of early recurrence of AF, atrial tachycardia, 
or atrial flutter following AF ablation within 28 days follow-up. For simplicity we will refer to this as “AF recurrence”. 
Measurement of Outcome (Aim 1): Participants enrolled in the study will wear a smartwatch linked to an iPhone 
to continually record heart rate, variability and detection of arrhythmias. For detection of the primary endpoint, 
participants will record a daily ECG each morning upon waking via the watch.  In addition, participants will be 
notified by the smartwatch of 1) detection of atrial fibrillation or atrial flutter, 2) persistent high HR (> 110 bpm) 
outside of exercise.  Upon prompting or upon patient self-report of symptoms, participants will utilize the ECG 
capture function of the watch and record the ECG for study personnel.  A continuous tracing of 30 seconds (the 
full duration of a Watch EKG tracing) will be considered as having met the primary endpoint.   
 
Sample size and power (Aim 1): The sample size of this study is based on a logistic regression of the binary 
primary endpoint of early AF recurrence by the end of follow-up. A total sample size of 162 with 81 in each arm 
will provide 81% power to detect a reduction in the rate of early recurrence of AF if the event rate is 29% in the 
treatment arm. This change corresponds to an odds ratio of 0.4 (treatment vs control), which is nearly equivalent 
to the odds ratio observed in similar trials using colchicine (colchicine OR 0.38)26 at the significance level of 
0.046 using a 1-sided Farrington and Manning likelihood score test, and a 48% rate of early recurrence in the 
placebo (control) group.  The estimate 48% rate of early recurrence was extrapolated from our pilot data, which 
found a 45% rate of recurrence, but did not utilize continuous monitoring as will be performed in our trial.  An 
interim analysis will be performed for superiority after half of the patients (n=40 per group) have completed their 
28-day assessment of the primary endpoint at the significance level of 0.006. The final analysis will be performed 
after all patients have completed their 28-day assessment of the primary point at two-sided significance level of 
0.041. Efficacy will be declared early at the interim analysis if the efficacy boundary is crossed (p<0.007). All on-
study patients will be followed for efficacy and safety until the planned end-of-follow-up. If the interim analysis 
indicates that the treatment is not better than the control by 13% (non-binding futility boundary p>0.4), we may 
close the recruitment into study with regard to any recommendations of DSMB.  The O’Brien-Fleming boundaries 
was used for the alpha- and beta-spending functions to preserve the overall Type I and Type II error.    
 
Primary Analysis (Aim 1): All FAS will be included in the primary analysis of AF recurrence, the primary efficacy 
endpoint. Binary recurrence of AF will be compared between treatment and control using a chi-square test. The 
primary analysis will be performed using a multivariable logistic regression model, where the dependent/outcome 
variable is the early AF recurrence (yes/no) by the end of the 28-day follow-up and the primary 
independent/predictor variable is the treatment arm (2-HOBA or placebo). Adjustment will be made for age, 
gender, AF type (paroxysmal versus persistent), method for pulmonary vein isolation (radiofrequency versus 
cryoablation), additional ablation lesions (yes/no), BMI (continuous), and hours of monitoring (0-672 hours). To 
avoid over-fitting our multivariable logistic regression model, a 10:1 ratio for degrees of freedom per the less 
frequent outcome event will be used in our modeling. Nonlinear form of the continuous predictors (age, BMI, and 
hours of monitoring) will be considered when building the model with restricted cubic splines. Models will be 
evaluated for goodness-of-fit, and internally validated for the calibration and discrimination performance using 
the bootstrap method.  
Secondary Analysis (Aim 1): A secondary analysis will analyze a surrogate of AF burden as the endpoint and 
designed to account for the impact of cardioversion on AF burden assessment. AF burden is the percentage of 
time in AF, atrial tachycardia, or atrial flutter over total time monitored.  The secondary analysis will use the data 


 
 
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provided by the continuous HR monitor provided by the smartwatch.  The analysis will be performed using a 
linear mixed models (LMM) in which the AF burden is assessed weekly during the 4-week (28-day) follow-up 
period. Same covariates mentioned in the primary analysis will be considered. Additionally, AF burden will also 
be analyzed in ordinal scale using generalized estimating equation (GEE), ranked based on severity according 
to increasing AF burden and whether or not a cardioversion was performed. For any missing data in the 
covariates, we will use a multiple imputation method that incorporates predictive mean matching and flexible 
additive imputation models as implemented in the aregImpute function in the Hmisc R package. The impact of 
missing data on the validity of results will be examined with a pattern-mixture approach using the R package 
SensMice. This will be done for Aims 1 and 2. Additional secondary analyses will include: 1) multivariable 
modeling of participants who were excluded from the primary analysis due to the use of colchicine or steroids 
during the follow-up period (will examine clinical and biochemical endpoints); 2) multivariable modeling with 
adjustment for 2-HOBA levels for the clinical and biochemical endpoints; 3) multivariable modeling with 
adjustment for duration of smartwatch use.  
Interim Analyses (Aim 1): An interim analysis will be performed by the study team and reviewed by the DSMB. 
When 50% of the target enrollment is reached testing for an association between 2-HOBA or placebo and 
significant adverse events (see Ethical Aspects of the Proposed Research for details), superiority and futility will 
be performed (see above: Sample Size and Power Aim 1). The interim analysis will include early stopping rules 
for superiority, futility, and harm.  
 
• 
Interim Analysis for Harm: although none of our Phase I pre-clinical or clinical data have raised safety 
concerns, and no adverse events have been observed, we planned the assessment of safety which will 
be based mainly on the frequency of adverse events and on the number of laboratory values that fall 
outside of pre-determined ranges. Other safety data such as vital signs will be considered as appropriate. 
All safety data will be listed. Patients will be summarized by treatment and other covariates. All adverse 
events recorded during the study will be summarized. The incidence of treatment-emergent adverse 
events (new or worsening from baseline) will be summarized by body system, severity, type of adverse 
event, and relation to the investigational regimen. Severe adverse events and non-fatal serious adverse 
events are not expected. 
• 
Interim Analysis for Efficacy and Futility: early stopping rules for efficacy and futility will be based on the 
primary endpoint in Aim 1, which is arrhythmia recurrence during the 28-day follow-up period. There will 
be no early stopping rules for the primary endpoints in Aim 2 (biochemical endpoint) or Aim 3 
(pharmacogenomic analysis). Both of those aims will be considered exploratory.   
  
10.2. Aim 2 (Biochemical Endpoint)  
Primary hypothesis: The primary endpoint for Aim 2 will be the IsoLG-adduct content of dendritic cells expressed 
as a percentage (IsoLG-adducted dendritic cells/total dendritic cells x 100). We will test the hypothesis that 2-
HOBA reduces the change in IsoLG-adduct levels that occurs with AF ablation (as measured on post-procedure 
day #1 minus pre-ablation blood samples).  
Sample size and power (Aim 2): Our pilot data shows that the change in dendritic cell IsoLG-adduct level is 23% 
following ablation in our placebo (control) group and we expect the range of possible observed reduction to be 
20-40%. With n=81 participants in each arm, the study provides at least 80% power to detect a minimal 22% 
reduction in lsoLG-adduct level at two-sided significance level of 5%.  
Primary Analysis (Aim 2): Our primary analysis will use multivariable linear regression to test whether our primary 
determinant (2-HOBA versus placebo), significantly reduces the change in IsoLG-adduct levels (continuous), 
adjusted for baseline pre-ablation IsoLG-adduct level and other potential confounding factors: age, sex, method 
for pulmonary vein isolation (radiofrequency versus cryo), additional ablation lesions (yes/no), and BMI 
(continuous). The IsoLG content of dendritic cells adjusted for baseline level will be presented for both treatment 
arms, together with corresponding 95% confidence intervals (CIs) and p-values will be graphically interpreted 
using boxplots, partial effect plots, residual plots, etc.  
Secondary Analysis (Aim 2): A secondary endpoint will be the IsoLG-adduct level in monocytes. Due to the 
prohibitively labor-intensive process required to measure IsoLG content in dendritic cells and monocytes, only 
the pre-ablation and post-procedure day#1 samples will be analyzed for the analysis. However, blood samples 
from 2 additional time points (baseline and post-procedure day #28) will be available for pharmacokinetic 


 
 
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analysis and secondary analysis of other serological markers. These will include malondialdehyde and other 
biomarkers of oxidative stress.  
 
10.3. Aim 3 (Exploratory Genomics): This aim will use the 162 subjects enrolled in Aims 1 and 2. DNA will be 
extracted and genotyping will be performed on a GWAS chip that includes the 3 SNPs located at chromosome 
4q25 that are independently associated with AF risk (rs2200733, rs17570669, and rs3853445).34 The 4q25 GRS 
will be a weighted score calculated by using the OR for AF multiplied by the number of risk alleles present from 
each of the 3 independent 4q25 SNPs. We hypothesize that a statistically significant interaction will exist between 
the 4q25 GRS and the treatment group (2-HOBA or placebo) for an association with our clinical and biochemical 
endpoints. In Aim 3, we will test for an interaction between the 4q25 GRS and the treatment group for an 
association with the primary clinical endpoint (early recurrence of AF). Adjustment will be made for the covariates 
used in Aim 1. We will then add the 4q25 GRS to the multivariable linear regression model used in Aim 2 and 
test whether there is a statistical interaction between the GRS and the treatment group for an association with 
the primary biochemical endpoint (change in dendritic cell IsoLG-adduct level with ablation) by comparing models 
with and without the interaction term using likelihood ratio test. The added predictive value of 4q25 GRS and the 
interaction of GRS and treatment will be evaluated by comparing nested models with and without the predictor 
with regard to c statistics and the integrated discrimination improvement. Models involving 4q25 GRS will be 
internally validated using the aforementioned bootstrap method for the evaluation and correction of potential 
optimism in the performance of the model by balancing the bias and variance in the prediction error. All statistical 
tests will be considered significant at two-sided 5% level. 
 
11.0 
PRIVACY/CONFIDENTIALITY ISSUES 
 
The conduct of genetic studies raises specific issues with respect to protection of human subjects. We describe 
here mechanisms in place at Vanderbilt University Medical Center through IRB policy to protect against such 
risks; these apply to all studies described below.  All records are retained on password-protected computers 
accessible only to members of the study team. Computers containing these records are only connected to 
networks if they include appropriate firewalls and security measures. Deidentified records and DNA samples 
may be shared with other investigators who have IRB-approved protocols and who agree to comply with the 
protections provided at this institution. These research materials are transferred only by secure methods. The 
identity of any individuals and their families are not to be revealed in any publication without their written informed 
consent. 
 
12.0 
FOLLOW-UP AND RECORD RETENTION 
 
The expected duration of this study is estimated to be 4 years. The study results will be retained for at least six 
years after the study is completed. At that time, the research information, with the exception of genetic 
information, not already in the medical record will be destroyed. Genetic information will be kept for an 
undetermined period of time for future gene research.     
 
 
13.0 
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