Conditions: Barrett's Esophagus Obesity
Keywords: Omega-3 fatty acid
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 60
Healthy volunteers: no

Brief summary:
This study is being done to understand the effect of dietary omega-3 fats in decreasing tissue inflammation in Barrett's esophagus.

Detailed description:

Arms:
arm 1: Subjects with known Barrett's Esophagus (no dysplasia and low-grade dysplasia) will receive a placebo which looks exactly like the study drug, but contains no active ingredient, to be taken orally for six months. arm 2: Subjects with known Barrett's Esophagus (no dysplasia and low-grade dysplasia) will receive Omega-3 free fatty acids supplements to be taken orally for six months.

Interventions: intervention 1: Omega-3 polyunsaturated fatty acids intervention 2: Placebo
Intervention descriptions: intervention 1: 3 capsules a day of Omega 3 polyunsaturated fatty acids taken orally for six months; 2 capsules with breakfast and 1 capsule with their evening meal. Active drug will consist of 1200 mg of a ω3 FFA preparation containing 675 mg EPA and 300 mg DHA. intervention 2: 3 capsules a day of placebo (1200 mg of ethyl oleate 3 capsules a day) taken orally for six months; 2 capsules with breakfast and 1 capsule with their evening meal.

Protocol document:
Prot_SAP_000.pdf: 
 
 
Modulation of Esophageal Inflammation in 
Barrett’s Esophagus by Omega-3 Fatty Acids, a 
Double Blind Placebo Controlled Randomized 
Pilot Study 
 
 
NCT01733147 
 
 
April 25, 2018 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
Page 1 of 28 
Prasad G. Iyer, MD 
 
CONFIDENTIAL 
4/25/2018 
 
Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids, a 
Double Blind Placebo Controlled Randomized Pilot Study 
 
Principal Investigator: Prasad G. Iyer, MD 
 
Co-investigator(s): Jason T. Lewis, MD; Navtej S. Buttar, MD; Harmeet Malhi, MBBS; 
Kenneth K. Wang, MD; Gregory J. Gores, MD; Alan R. Zinsmeister, PhD 
David A. Katzka MD, Purna C. Kashyap MD 
 
 
 
 
 
 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
 
Table of Contents 
 
1 
INTRODUCTION .................................................................................................................................. 5 
1.1 
BACKGROUND ................................................................................................................................ 5 
2 
STUDY OBJECTIVES ....................................................................................................................... 10 
3 
STUDY DESIGN ................................................................................................................................. 10 
3.1 
GENERAL DESIGN ........................................................................................................................ 10 
3.2 
STUDY ENDPOINTS....................................................................................................................... 12 
3.3 
PRIMARY SAFETY ENDPOINTS ....................................................................................................... 13 
4 
SUBJECT SELECTION ENROLLMENT AND WITHDRAWAL ........................................................ 13 
4.1 
INCLUSION CRITERIA .................................................................................................................... 14 
4.2 
EXCLUSION CRITERIA ................................................................................................................... 14 
4.3 
SUBJECT RECRUITMENT, ENROLLMENT AND SCREENING ............................................................... 14 
5 
STUDY DRUG .................................................................................................................................... 15 
5.1 
DESCRIPTION ............................................................................................................................... 15 
5.2 
TREATMENT REGIMEN .................................................................................................................. 15 
5.3 
METHOD FOR ASSIGNING SUBJECTS TO TREATMENT GROUPS ....................................................... 15 
5.4 
SUBJECT COMPLIANCE MONITORING ............................................................................................. 16 
5.5 
PRIOR AND CONCOMITANT THERAPY ............................................................................................. 16 
5.6 
RECEIVING, STORAGE, DISPENSING AND RETURN .......................................................................... 16 
6 
STUDY PROCEDURES ..................................................................................................................... 16 
6.1 
VISIT 1 ........................................................................................................................................ 16 
6.2 
VISIT 2 ........................................................................................................................................ 17 
7 
STATISTICAL PLAN ......................................................................................................................... 18 
7.1 
SAMPLE SIZE DETERMINATION ...................................................................................................... 18 
8 
SAFETY AND ADVERSE EVENTS ................................................................................................... 19 
8.1 
DEFINITIONS ................................................................................................................................ 20 
8.2 
RECORDING OF ADVERSE EVENTS ................................................................................................ 21 
8.3 
REPORTING OF SERIOUS ADVERSE EVENTS AND UNANTICIPATED PROBLEMS ................................. 21 
8.3.1 
Sponsor-Investigator reporting: notifying the Mayo IRB ....................................................... 21 
8.3.2 
Sponsor-Investigator reporting: Notifying the FDA................................................................ 23 
8.4 
STOPPING RULES ......................................................................................................................... 23 
8.5 
MEDICAL MONITORING ................................................................................................................. 24 
8.5.1 
Internal Data and Safety Monitoring Board ........................................................................... 24 
9 
DATA HANDLING AND RECORD KEEPING ................................................................................... 24 
9.1 
CONFIDENTIALITY ......................................................................................................................... 24 
9.2 
SOURCE DOCUMENTS .................................................................................................................. 25 
9.3 
RECORDS RETENTION .................................................................................................................. 25 
10 
STUDY MONITORING, AUDITING, AND INSPECTING .................................................................. 25 
10.1 
STUDY MONITORING PLAN ............................................................................................................ 25 
10.2 
AUDITING AND INSPECTING ........................................................................................................... 26 
11 
ETHICAL CONSIDERATIONS .......................................................................................................... 26 
12 
STUDY FINANCES ............................................................................................................................ 26 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
12.1 
FUNDING SOURCE ........................................................................................................................ 26 
12.2 
SUBJECT STIPENDS OR PAYMENTS ............................................................................................... 26 
13 
REFERENCES ................................................................................................................................... 26 
 
 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
 
 
List of Abbreviations 
 
 
LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
BE 
Barrett’s Esophagus 
CFR 
Code of Federal Regulations 
Cox-2 
Cyclooxygenase-2 
CRF 
Case Report Form 
DSMB 
Data and Safety Monitoring Board 
EAC 
Esophageal adenocarcinoma 
FDA 
Food and Drug Administration 
FFA 
Free fatty acid 
GCP 
Good Clinical Practice 
GEJ 
Gastroesophageal Junction 
HGD 
High grade Dysplasia 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
IL1β 
Interleukin-1 beta 
IL6 
Interleukin-6 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
MetS 
Metabolic syndrome 
NSAIDs 
Nonsteroidal anti-inflammatory drugs 
ω3 FFA 
Omega 3 free fatty acid 
PGE2 
Prostaglandin E2 
PHI 
Protected Health Information 
PI 
Principal Investigator 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SOP 
Standard Operating Procedure 
SQ 
Subcutaneous 
TNFα 
Tumor necrosis factor-alpha 
 
 
 
 
 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
1 Introduction 
Increased visceral abdominal fat is associated with increased risk of Barrett’s esophagus (BE) 
and esophageal adenocarcinoma (EAC). Novel preliminary data gathered by us has revealed a 
reflux independent influence of visceral abdominal fat on esophageal pro-inflammatory and pro-
neoplastic pathways, mediated via visceral fat produced saturated free fatty acids and activated 
macrophages. ω3 free fatty acids have been shown to inhibit epithelial PGE2 production by 
inhibiting COX-2 activity and modulating the macrophage phenotype from proinflammatory to 
anti-inflammatory. In this pilot randomized double blind placebo controlled study we wish to 
assess the ability of oral ω3 free fatty acids to downregulate pro-neoplastic and pro-inflammatory 
pathways in the esophagus. Pilot data from this proposal will enable us to design a larger 
adequately powered study assessing the role of ω3 free fatty acids as chemoprevention agents in 
subjects with BE, who are at substantial risk of developing EAC, a lethal cancer with rapidly 
rising incidence in the United States. 
 
This document is a protocol for a human research study. This study will be carried out in 
accordance with the applicable United States government regulations and Mayo Clinic research 
policies and procedures.  
1.1 Background 
BE and visceral abdominal fat: Barrett’s esophagus (BE) is characterized by intestinal 
metaplasia of the esophagus in response to esophageal injury and inflammation [1]. BE is the 
strongest risk factor and precursor of esophageal adenocarcinoma (EAC) a malignancy with 
rapidly rising incidence and poor outcomes. In addition to reflux, increased abdominal visceral 
fat has been identified as an independent risk factor for BE and EAC[2, 3]. Abdominal fat is 
composed of visceral and subcutaneous fat. Visceral fat may predispose to BE by mechanically 
increasing reflux and/or by reflux independent mechanisms [4]. We have found that increased 
visceral abdominal and gastroesophageal junction (GEJ) fat are independently associated with 
BE, esophageal inflammation and high grade dysplasia (HGD) [5]. Visceral fat is metabolically 
active, leading to a systemic inflammatory state and the metabolic syndrome.  We have also 
identified Metabolic syndrome as a risk factor for BE, independent of reflux, in a population-
based case control study [6], further supporting a reflux independent pathway by which visceral 
abdominal fat may modulate esophageal inflammation and neoplasia.  
 
Visceral fat, Free Fatty Acids (FFAs) and Macrophage mediated inflammation:  
Abdominal visceral fat releases proinflammatory 
cytokines and saturated FFAs which are known to 
induce a systemic inflammatory insulin resistant state: 
which is strongly associated with BE [7, 8]. FFAs 
function 
as 
ligands 
modulating 
inflammatory 
responses, but with different effects. sFFAs exert 
proinflammatory, 
while 
ω3 
FFAs 
exert 
anti-
inflammatory 
effects 
[9-12]. 
sFFAs 
activate 
macrophages to a proinflammatory (M1) phenotype 
[13]. Preliminary data obtained by us show increased 
levels of serum sFFA in obese BE subjects compared 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
to controls, as well as higher density of activated macrophages (M1 phenotype) and their 
products in BE biopsies compared to controls. M1 macrophage produced proinflammatory 
cytokines (such as TNFα, IL6, IL1β) are known to be overexpressed in BE and are thought to 
contribute to malignant progression to EAC in BE [14-16]. 
 
Anti-inflammatory effects of ω3 FFA: Increased intake of polyunsaturated FFAs and ω3 FFAs in 
particular, is associated with a 50% lower risk of BE [17].  ω3 FFAs have been shown to have 
anti-neoplastic activity in the colon. A major proposed mechanism by which ω3 FFAs exert anti-
inflammatory and antineoplastic effect is by modulation of prostaglandin biosynthesis, by 
providing an alternate substrate for COX-2 (instead of the ω6 FFA : Arachidonic acid), which 
leads to decreased PGE2  production, epithelial proliferation and adenoma/aberrant crypt foci 
number in animal and human trials [9] [18-20]. We have shown in prior studies that increased 
levels of COX2 are seen with increasing grades of dysplasia in BE and that inhibition of COX-2 
activity leads to reduced EAC incidence in an animal model of BE, by reducing PGE2 
production [21, 22]. ω3FFAs have also been shown to modulate macrophage phenotype from 
proinflammatory (M1) to anti-inflammatory (M2). GPR120 has been identified as a likely 
ω3FFA receptor, mediating the anti-inflammatory and insulin sensitizing effects of ω3FFAs on 
macrophages and tissue [13]. Our novel central hypothesis is that oral supplementation with ω3 
FFAs in obese BE subjects will lead to downregulation of pro-neoplastic inflammatory pathways 
in BE by modulating the esophageal macrophage phenotype from proinflammatory to anti-
inflammatory. We propose to test this hypothesis by the following specific aims, using a 
randomized controlled trial study design: 
 
Specific Aim 1: To estimate the magnitude and variation of changes in esophageal inflammation 
and injury in BE as measured by tissue PGE2 and the esophageal inflammation score, 
attributable to ω3 FFA supplementation. 
 
Specific Aim 2: To estimate the magnitude and variation of changes in esophageal macrophage 
infiltration and modulation of esophageal macrophage phenotype attributable to ω3 FFA 
supplementation.  
 
Specific Aim 3:  To estimate the magnitude and variation in changes in esophageal mucosal 
impedance attributable to ω3 FFA supplementation. 
 
Specific Aim 4:  To define the baseline esophageal mucosal microbiome  and the potential 
change in the esophageal mucosal microbiome attributable to ω3 FFA supplementation. 
 
 
Significance and Innovation: 
The prevalence of BE has been estimated to be 1.6-2.0% in the United States. The incidence of 
EA has increased exponentially in the Western world in parallel with the prevalence of obesity, 
now estimated to affect > 30% of the US population. Radiofrequency ablation has emerged as 
an alternative modality to reduce the risk of progression to EAC in dysplastic BE, but is 
expensive, with potential endoscopic complications and a fairly high risk of recurrent IM (30% 
at 2 years) following successful ablation. This study uses a safe compound (ω3 FFA) based on a 
novel biologically plausible, mechanistic hypothesis and strong supportive preliminary data to 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
Fig 1: Serum saturated and ω3 FFA (EPA) levels 
in BE subjects with and without central obesity 
1500
1600
1700
1800
1900
2000
2100
Centrally
obese
Not centrally
obese
Serum Palmitic Acid
100
110
120
130
140
150
Centrally
obese
Not centrally
obese
Serum EPA
attenuate and inhibit proinflammatory and pro-neoplastic pathways in subjects with BE who are 
at high risk of developing EAC, a lethal malignancy with rapidly increasing incidence. While 
used extensively in preclinical and clinical studies in the colon with encouraging results, there is 
no data on their ability to inhibit proinflammatory and neoplastic pathways in subjects with BE. 
If successful, this novel trial will lay the ground work for a larger adequately powered trial to 
demonstrate the use of ω3 FFA as a novel chemoprevention compound in subjects with BE to 
decrease EAC incidence. While inhibition of proinflammatory pathways in BE is possible with 
NSAIDs (and Aspirin), their use is associated with adverse effects including cerebrovascular and 
gastrointestinal bleeds even at low doses. ω3 FFA may provide a safer alternative to NSAIDs for 
chemoprevention in BE. Additionally, given the close association of obesity and esophageal 
injury, ω3 FFAs could also be used as an adjunct or alternative to proton pump inhibitors (PPIs) 
in modulating esophageal inflammation and injury, particularly in those patients with an 
incomplete response to PPIs. This can provide a novel alternative to PPIs in the management of 
patients with obesity and reflux induced esophageal injury, which is a very prevalent problem in 
the United States. 
 
Preliminary data: 
1. Increased visceral and GE junction fat area are independently associated with BE, 
esophageal inflammation and HGD [5]. GE junction, visceral and subcutaneous (SQ) fat area 
were assessed in age and gender matched 50 BE cases and 50 controls, using CT scans. BE was 
strongly associated with visceral and GE junction fat area independent of BMI. There was no 
association with SQ fat area, suggesting a non-mechanical influence of visceral fat on BE 
pathogenesis. Furthermore, increased visceral fat area (but not SQ fat area) was also 
independently associated with severe esophageal inflammation and HGD, further suggesting a 
non- mechanical influence of visceral fat on esophageal inflammation and carcinogenesis. 
 
2. Metabolic Syndrome is a risk factor for BE independent of reflux[6]. We studied the 
association between BE and metabolic syndrome 
in a population based case control study: 309 
subjects from Olmsted County, MN were 
studied (103 BE cases, 103 reflux + controls and 
103 reflux - controls). 64% of cases, 49% of 
reflux + controls and 51% of reflux - controls 
had MetS.  MetS was associated with a 2 fold 
increased risk of BE with reflux (+) controls 
(OR 1.9, 95% CI 1.1, 3.6, p=0.04) and reflux (–) 
controls (OR 1.9, 95% CI 1.1, 3.4 p=0.04) on 
multivariable regression analysis.  
 
3. Centrally obese BE patients have higher serum sFFA levels and lower ω3 FFA levels than 
age, gender and BMI matched controls. Fasting serum from 10 controls without BE and 20 age 
and gender matched BE cases was analyzed for serum FFA profiles, using mass spectroscopy. 
Patients with BE had higher saturated FFA and lower ω3 FFA levels than those without BE 
(figure 1).  
 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
Fig 2: Esophageal PGE2 in 
subjects with and without 
esophageal inflammation 
0
10
20
30
40
50
Inflammation
present
Inflammation
absent
Esophageal PGE2 (pg/mg)
 
Fig 3: Columnar metaplasia is associated with 
increased macrophage infiltration A-C: IF for  
CD68 on distal esophageal biopsies c (panel A), with BE (panel 
B) and for HAM56 in BE tissue (panel C) show increased 
CD68+/HAM56 cell count and stain area  in BE cases than 
controls 
(panel 
D 
is 
graphical 
representation 
of  
immunostaining). 
 
Table 1: Comparison of anthropometric parameters in subjects with 
and without esophageal inflammation and metaplasia. 
Variable, Mean (SEM) 
BE or 
esophagitis 
(N=11) 
No BE or  
esophagitis  
(N=47) 
Waist circumference (cm) 
100.3 (4.3) 
95.8 (2.1) 
Waist hip ratio  
0.94 (0.02) 
0.90 (0.01) 
BMI  
30.9  (1.7) 
29.0 (0.8) 
4. Central obesity increases risk 
of esophagitis and metaplasia. In 
a population based screening 
study [23], 60 age, gender and 
reflux symptom stratified subjects 
were randomized to evaluation by 
sedated 
endoscopy, 
unsedated 
transnasal endoscopy or capsule 
endoscopy. 11 subjects with esophagitis or BE had higher waist circumference and waist hip 
ratios (measures of central obesity) than those without (table 1). 
 
5. Esophageal tissue PGE2 accurately reflects endoscopic and 
histologic esophageal injury. Tissue esophageal PGE2 levels 
were measured in biopsies of 10 subjects with no symptoms of, 
endoscopic or histologic evidence of reflux and 10 subjects with 
esophagitis or BE. Levels in subjects with esophageal injury 
(mean 42 pg/mg) were significantly higher than those with no 
symptoms or signs of esophageal injury (mean 2 pg/mg), p=0.03. 
(Figure 2). 
 
6. BE patients have denser macrophage infiltration than 
controls. IHC for CD68 and HAM56 (macrophage markers) was 
performed on esophageal biopsies from 5 BE cases (without 
endoscopic evidence of esophagitis) and 5 controls.[24] Mean counts of CD68 positive cells in 
BE were higher compared to controls (p=0.06) (Figure 3). 
 
7. Sub-characterization of macrophages in 
BE: Increased M1 like macrophages in 
BE. IHC using double labeling for CD68 
and CD206 was performed on esophageal 
biopsies from subjects with BE [24]. Cells 
double labeling for CD68 and CD206 are 
M2 like cells and those labeling for only 
CD68 are M1 like cells. M2 like 
macrophages constituted a minority (30%) 
of all CD68 positive cells, indicating a 
preponderance of M1 like cells in BE 
tissue (Figure 4)  
 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
Fig 5: Higher CD68 and MCP1 (M1 macrophage marker)  
mRNA in BE compared to squamous esophageal 
biopsies.  
qPCR for CD68 and MCP in squamous and BE biopsies. 
 
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
CD68 mean relative mRNA expression
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
MCP1 mean relative mRNA expression
Fig 4: Columnar metaplasia is associated with 
increased 
M1 
like 
macrophage 
infiltration.  
Immunofluorescence with double labeling for CD68 and 
CD206 on distal esophageal biopsies from subjects with 
BE. 
 
Fig 6A and 6B: Exposure of macrophages to satFFA leads to a 
proinflammatory response which is blunted by DHA (an ω3FFA) and 
induction 
of 
M2 
macrophage 
markers. 
0
2
4
6
8
10
12
14
16
18
TNF-alpha
MCP-1/CCL-2
Il-1beta
Relative mRNA Expression
VC
PA
DHA
PA+DHA
0
0.5
1
1.5
2
2.5
3
3.5
CD206
Arginase
Relative mRNA Expression
VC
PA
DHA
PA+DHA
 
8. M1 Macrophage markers are more 
prevalent in BE compared to squamous 
tissue. Esophageal biopsies were obtained 
from patients with and without BE on 
endoscopy and snap frozen at -800 C. 
RNA was extracted from these biopsies 
using the RNAase easy kit. qPCR was  
performed for macrophage (CD68), M1 
(MCP-1) 
and 
M2 
(IL10, 
arginase) 
markers. 
Results 
show 
increased 
expression of CD68 RNA in BE tissue 
compared to that of normal squamous 
tissue (p=0.02). MCP1, a M1 macrophage 
marker was also greater in BE tissue than 
in squamous tissue (p=0.03) (figure 5). 
 
9. Influence of saturated and ω3 
FFA on macrophages in vitro. THP-
1 cell line, a human monocyte cell 
line 
was 
differentiated 
into 
macrophages 
using 
previously 
described 
methods 
[25]. 
Macrophages were treated with 
palmitic acid (PA), a saturated FA 
or docosahexanoic acid (DHA), a 
ω3 FFA, or both in combination. 
RNA 
was 
isolated 
using 
the 
RNAase easy kit (Qiagen). qPCR 
was then performed for macrophage (CD68), M1 (IL1β, MCP-1) and M2 (CD206, arginase) 
markers. Gene expression analysis was performed by calculating the ΔCt for each target gene 
using β actin as the reference gene. As seen in the results from figure 6a and 6b, exposure of 
macrophages to PA led to a proinflammatory response while administering DHA with PA lead to 
a substantial blunting of the proinflammatory response. Exposure to DHA led to the induction of 
M2 markers (CD206 and Arginase) in macrophages. 
 
 
 
 
 
 
 
 
 
 
 
 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
CONFIDENTIAL 
Hence in these preliminary data we have discovered increased visceral fat, serum saturated 
FFAs and M1 proinflammatory macrophage infiltration in BE and have found that exposure of 
macrophages to ω3FAs is able to blunt the proinflammatory and neoplastic cascade induced by 
saturated FFAs and induce a M2 phenotype. These data help support and justify our hypothesis 
of Specific Aims 1 & 2 and their feasibility. 
2 Study Objectives 
 
Specific Aim 1: To estimate the magnitude and variation of changes in esophageal inflammation 
and injury in BE as measured by tissue PGE2 and the esophageal inflammation score, 
attributable to ω3 FFA supplementation. 
 
Specific Aim 2: To estimate the magnitude and variation of changes in esophageal macrophage 
infiltration and modulation of esophageal macrophage phenotype attributable to ω3 FFA 
supplementation. 
 
Specific Aim 3:  To estimate the magnitude and variation in changes in esophageal mucosal 
impedance attributable to ω3 FFA supplementation. 
 
Specific Aim 4:  To define the baseline esophageal mucosal microbiome  and the potential 
change in the esophageal mucosal microbiome attributable to ω3 FFA supplementation. 
3 Study Design 
3.1 General Design 
 
In this pilot randomized double blind placebo controlled study we wish to assess the ability of 
oral ω3 free fatty acids to downregulate pro-neoplastic and pro-inflammatory pathways in the 
esophagus. Pilot data from this proposal will enable us to design a larger adequately powered 
study assessing the role of ω3 free fatty acids as chemoprevention agents in subjects with BE, 
who are at substantial risk of developing EAC, a lethal cancer with rapidly rising incidence in the 
United States. Our plan is to complete the study on 80 participants, 40 in each group randomized 
to omega-3 or placebo. Subjects between 18-85 years of age, with known BE (no dysplasia and 
low-grade dysplasia) will be identified using an existing GI database for participation in this 
study. Invitation letters will be mailed to eligible subjects. Subjects meeting inclusion and 
exclusion criteria will be screened and consented. Once consent has been obtained baseline 
assessment and endoscopy with biopsy/brushings, and endoscopic mucosal impedance 
measurement will be completed. A single slice CT scan of the abdomen will also be obtained at 
baseline for each subject to quantify visceral and subcutaneous fat area in the abdomen.  Subjects 
will then begin treatment and follow-up for the next 6 months. A final visit for evaluation and 
collection of tissue samples will be conducted at the end of the study. No risks of any particular 
seriousness or severity are anticipated. Blood and tissue samples will be obtained and used in this 
study as outlined in section 3.2. Any remaining samples will be stored in Dr. Iyer -80C freezer 
located at Joseph M97 and used for future research in Barrett’s esophagus. 


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Prasad G. Iyer, MD 
 
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Prasad G. Iyer, MD 
 
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3.2 Study Endpoints 
The primary analysis objective in this pilot study is to estimate response magnitude and variation 
in esophageal inflammation and injury in BE as measured by tissue PGE2 , the esophageal 
inflammation score, esophageal macrophage infiltration and modulation of esophageal 
macrophage phenotype attributable to ω3 FFA supplementation. This will be the information 
needed to design a larger adequately powered study to study the efficacy of w3FFA as a 
chemoprevention agent in BE. 
 
Aim 1: The primary response outcome is tissue PGE2 levels and the main secondary response 
outcome is the histologic score of 3 or 4 (severe grades of inflammation). Other secondary 
outcomes include pre and post supplementation serum and tissue ω3 FFA levels.  
 
Aim 2: The primary response outcome is the cytokine expression profile in esophageal biopsies 
as measured by qPCR [IL1β, IL6 (M1 markers) versus IL1, IL10 (M2 markers)]. Secondary 
response outcomes include change in esophageal macrophage counts and phenotype proportions 
(total, %M1, %M2).  
 
Aim 3: The primary response outcome is change in esophageal mucosal impedance  (baseline-
post treatment) which is a physiologic correlate of the epithelial barrier function, with ω3 FFA 
supplementation. Secondary outcomes include the association of esophageal mucosal impedance 
with measures of central obesity (waist circumference, waist hip ratio and abdominal visceral fat 
area on CT) at baseline and following treatment. 
 
Aim 4: The primary response outcome is the change in the esophageal microbiome in BE 
subjects following ω3 FFA supplementation. Secondary outcomes will be the association of the 
microbiome pattern with measures of central obesity ((waist circumference, waist hip ratio and 
abdominal visceral fat area on CT). 
 
 
Assays performed at baseline and at 6 months will include:  
Aim 1: Primary outcome: Tissue PGE2 in esophageal biopsies. This will be performed by 
previously described methods [21] on esophageal biopsies snap frozen at -800 C in Dr.Buttar’s 
laboratory. 
 
Secondary outcomes: 1. Tissue inflammation score (grade 0-3). This will be performed on 
esophageal H&E stained sections as previously described [5], by Dr.Lewis, an experienced 
gastrointestinal pathologist. 2. Esophageal tissue EPA levels: this will be performed on 
esophageal biopsies obtained at baseline and at 6 months, in the Metabolomics Core laboratory. 
3. Serum ω3 FFA levels: will be performed using mass spectroscopy/liquid chromatography in 
the Immunochemistry Core laboratory at baseline and 6 months.  
 
Aim 2: Primary outcome: 1. Quantification of esophageal macrophage infiltration (and subtypes) 
by qPCR for CD68 (macrophage marker) TNFά and MCP1 (M1 markers) and CD206,IL1, IL10, 
as M2 markers). This will be performed in collaboration with Dr. Gores (see preliminary data 
and letter of support) using previously described standard methodology [29]. 


Modulation of Esophageal Inflammation in Barrett’s Esophagus by ω3 Fatty Acids 
 
 
 
 
 
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Prasad G. Iyer, MD 
 
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Secondary outcomes : Esophageal macrophage counts (using CD68 and HAM 56 as markers) 
and proportion of M1 and M2 (CD206) using immunofluorescence  will be performed using 
previously described methods [24]. Cells double labeled for CD206 and CD68 will be 
categorized as M2 cells and those staining only with CD68 will be categorized as M1 cells. Cell 
counts will be performed as previously described. 
 
Aim 3: Esophageal mucosal impedance measurements will be obtained using a esophageal 
mucosal impedance catheter at 2 cm, 5 cm and 7 cm above the GEJ. At least one measurement 
will be obtained in the squamous mucosa at 3 cm above the BE segment [30]. 
 
Aim 4:  The esophageal microbiome will be characterized from biopsies and brushings from the 
squamous and columnar epithelium using standard methods.  
 
Sequencing : Biopsy samples and brushings will be collected in cryo-vials and flash frozen in 
liquid nitrogen. All samples with be stored at -80 till they are ready for processing.  
Next Generation sequencing:  
Bacterial DNA will be enriched in biopsy and brushings using NEBNext microbiome DNA 
enrichment kit if concentrations are low. Amplicons spanning the variable region 4 (V4) of 
bacterial 16SrRNA will be generated using a barcoded reverse primer (515F, 806R). Samples 
will sequenced using the MiSeq platform at Mayo Genome Facility to obtain 300bp paired end 
reads. Based on our previous experience we will sequence at most 100 samples in one 
sequencing run and we expect ~30,000 high quality reads per sample.  
 
3.3 Primary Safety Endpoints 
ω3 FFAs have an excellent safety and tolerability profile. Most common side effects associated 
with their use in human trials include diarrhea, indigestion, nausea, fishy taste and belching. 
Total incidence of GI disturbances in phase 2 and 3 clinical trials has varied between 1.4 – 4.9%, 
with a very low rate of study termination [27]. The rate of study withdrawal was 3.4% in a phase 
3 study using EPA [20]. 
We will specifically assess the prevalence of adverse effects in subjects randomized to drug or 
placebo.  Adverse effects which have been reported in the literature, may include: 
1. Gastrointestinal adverse effects such as: abdominal pain, nausea, vomiting, diarrhea. 
2. Altered (fishy) taste in the mouth. The preparation used in this trial using orange oil to 
mask this effect. 
3. Allergic reactions. 
4. ω3 FFAs have antiplatelet activity, however increased rates of bleeding have not been 
seen in large clinical cardiovascular trials [28]. We will specifically assess rates of any 
mucosal or skin bleeds in monthly telephone calls (1,2,3,4,5 months) and at the 6 month 
visit. 
4 Subject Selection Enrollment and Withdrawal 
Male and female patients between 18-85 years of age, with known BE (no dysplasia and low-
grade dysplasia) will be identified using an existing GI database for participation in this study. 


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4.1 Inclusion Criteria 
• Presence of BE defined as ≥ 1 cm of visible columnar mucosa in the distal esophagus 
with intestinal metaplasia on histology.  
• Absence of high grade dysplasia or EAC on baseline histology.  
• BMI > 30 kg/m2 (this has been shown to correlate strongly with increased abdominal 
visceral fat)[26] or Waist circumference > 102 cm in men, > 88 cm in women. 
• Ability to give informed consent. 
4.2 Exclusion Criteria 
• Allergy to ω3 FFAs, fish or shellfish.  
• Presence of high grade dysplasia or cancer on histology.  
• Pregnant and or breastfeeding women 
• Presence of esophagitis on initial endoscopy or symptoms of refractory GERD (heartburn 
or regurgitation ≥ 2 times a week) indicative of uncontrolled gastroesophageal reflux.  
• Inability to give informed consent.  
• Currently taking ω3 FFA as prescription. 
• Anti-coagulant therapy (Plavix, Warfarin, Coumadin) 
• AST or ALT level > three times upper limit of normal at baseline 
• LDL > 200 mg/dl at baseline. 
• INR > 2 
4.3 
Subject Recruitment, Enrollment and Screening 
Subjects between 18-85 years of age, with known BE (no dysplasia and low-grade dysplasia) 
will be identified using an existing GI database for participation in this study. Invitation letters 
will be mailed to eligible subjects. Subjects will be called to discuss study and scheduled for a 
screening visit. Subjects taking ω3 FFA as a supplement will discontinue use at least four weeks 
prior to visit 1.  Subjects will report fasting 12 hours for this visit. Subjects meeting inclusion 
and exclusion criteria will be screened and consented. Baseline assessment will include the 
measurement of height, weight, waist circumference, hip circumference and waist/hip ratio using 
standard methods of anthropometric measurements. A comprehensive medication list will be 
obtained. Patients will also fill out the Reflux Symptom Questionnaire (RSQ)/GER 
Questionnaire a validated instrument to assess reflux symptoms All patients will continue their 
baseline PPI regimen to control gastroesophageal reflux. 20cc of blood will be drawn after 12 
hours of fasting to obtain a baseline serum FFA profile . Clinical baseline blood will be drawn 
for inclusion/exclusion: INR, LDL, AST and ALT. Endoscopy will then be performed in a 
standard manner.  Six biopsies will be obtained from the GE junction, and seven biopsies from 
the BE mucosa 1 cm above the GE junction.  Two biopsies from each site will be formalin fixed 
and paraffin embedded (for inflammatory score assessment) and the remainder frozen at -80 C 
for subsequent analysis. An additional seven biopsies will be obtained from the squamous 
mucosa at least 3 cm above the proximal end of the BE segment. Two will be placed in formalin 
(or other fixative) and paraffin embedded (IHC for macrophage cell counts) and the remainder 
will be snap frozen at -800 C (PGE2 levels). Endoscopic brushings of the Barrett’s segment as 
well as squamous tissue will be obtained and frozen for each subject.   Endoscopic mucosal 
impedance measurement will be completed during endoscopy.  A single slice CT scan of the 


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abdomen will also be obtained at baseline for each subject to quantify visceral and subcutaneous 
fat area in the abdomen. 
 
 
Patients will be randomized to either a ω3 FFA preparation or a matching placebo for six months. 
A randomization schedule will be generated in the Division of Biomedical Statistics and 
Informatics and sent to the research pharmacy. Investigators and patients will be blinded to drug 
versus placebo assignment and only research pharmacy personnel and statisticians will have 
access to the assignments until the study is completed. Medications (drug and matching placebo) 
will be dispensed through the Mayo research pharmacy. Phone calls will be made by the study 
coordinator every month to the patients to assess for any adverse effects (presence and grade) 
and reinforce compliance. Compliance will also be assessed by pill counts during monthly call. 
 
Patients will undergo repeat assessment at six months following randomization. This will include 
anthropometry (similar to baseline), a fasting blood draw for serum FFA profile, LDL, AST, 
ALT, INR and endoscopy with research biopsies as outlined at baseline. Subjects will also fill 
out the Reflux Symptom Questionnaire (RSQ)/GER Questionnaire. Investigators and patients 
will be blinded to drug versus placebo assignment. Both endoscopic procedures will be 
conducted in the outpatient CTSA unit on Charlton 7. 
5 Study Drug  
5.1 Description 
 
Drug and placebo: 
The DHA/EPA and placebo softgels will be supplied by Sancilio and Company, Inc. (Riviera 
Beach, FL) and stored in the Mayo Clinic Research Pharmacy at room temperature. Active drug 
will consist of 1200 mg of a ω3 FFA preparation containing 675 mg EPA and 300 mg DHA. 
Patients randomized to active drug will be placed on 3 capsules a day of this preparation. A 
matching placebo (1200 mg of ethyl oleate 3 capsules a day) will be provided to subjects 
randomized to placebo. Both preparations will contain orange oil to mask the gustatory effect of 
fish oils. Both drug and placebo will be provided by Sancilio & Company 
5.2 Treatment Regimen 
Investigators, patients and statisticians will be blinded to drug versus placebo assignment.  
Patients randomized to active drug will be placed on 3 capsules a day of this preparation taken 
orally for six months. A matching placebo (1200 mg of ethyl oleate 3 capsules a day) will be 
provided to subjects randomized to placebo taken orally for six months. Both groups will take 2 
capsules with breakfast and 1 capsule with their evening meal. 
5.3 Method for Assigning Subjects to Treatment Groups 
A randomization schedule will be generated in the Division of Biomedical Statistics and 
Informatics and sent to the research pharmacy. Investigators and patients will be blinded to drug 
versus placebo assignment and only research pharmacy personnel and statisticians will have 
access to the assignments until the study is completed. Medications (drug and matching placebo) 
will be dispensed through the Mayo research pharmacy. 


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5.4 Subject Compliance Monitoring 
Phone calls will be made by the study coordinator every month to the patients to assess for any 
adverse effects (presence and grade) and reinforce compliance. Compliance will also be assessed 
by pill counts during this telephone call. Adverse effects mentioned in section 3.3 will be 
specifically assessed in the phone calls (see phone call script). 
5.5 Prior and Concomitant Therapy 
A complete list of medications which the patient is currently consuming (both prescription and 
over the counter) will be obtained at the baseline visit. 
Patients currently taking ω3 FFA s as prescription medications will be excluded from the study. 
5.6 Receiving, Storage, Dispensing and Return 
The research pharmacy will maintain the double-blind status of the study until all of the main 
outcomes have been measured. The DHA/EPA and placebo softgels will be supplied by Sancilio 
and Company, Inc. (Riviera Beach, FL) and stored in the Mayo Clinic Research Pharmacy at 
room temperature. The research pharmacy will assign participants to treatment or placebo based 
on a randomization table prepared by a statistician. Each participant will be instructed to swallow 
2 softgels in the morning and 1 in the evening with meals (morning and evening) for a total of 3 
softgels per day. The DHA/EPA softgels will each contain 1200mg of DHA/EPA for a total daily 
dosage of 3.6g/day. The placebo softgel will contain 1200mg of oleate. The pharmacy will 
maintain records of receipt, dispensation, and return pill counts for compliance.  
 
At the completion of the study, there will be a final reconciliation of drug shipped, drug 
dispensed, drug returns, and drug remaining.  This reconciliation will be logged on the drug 
reconciliation form, signed and dated.  Any discrepancies noted will be documented and 
investigated, prior to return or destruction of unused study drug.  Drug destroyed on site will be 
documented in the study files. 
6 Study Procedures 
6.1 Visit 1 
Subjects taking ω3 FFA as a supplement will discontinue use at least four weeks prior to visit 1.  
Subjects will report fasting 12 hours to the Charlton CRU and have a brief consultation with a 
gastroenterologist specializing in Barrett’s esophagus.  Baseline assessment will include the 
measurement of height, weight, waist circumference, hip circumference and waist/hip ratio using 
standard methods of anthropometric measurements. A comprehensive medication list will be 
obtained. Subjects will also fill out the Reflux Symptom Questionnaire (RSQ)/GER 
Questionnaire a validated instrument to assess reflux symptoms. All subjects will continue their 
baseline PPI regimen to control gastroesophageal reflux. 20cc of blood will be drawn after 12 
hours of fasting to obtain a baseline serum FFA profile. Clinical baseline blood will be drawn for 
inclusion/exclusion: INR, LDL, AST and ALT. Endoscopy will then be performed in a standard 
manner.   Brushings will be obtained from the Barrett’s segment as well as squamous tissue.  
Biopsies will be obtained from the GE junction, from the BE mucosa 1 cm above the GE 
junction and from the squamous mucosa 3cm above the proximal Barrett’s segment. Endoscopic 
mucosal impedance measurement will be completed as described in the methods section above.  
A single slice CT scan of the abdomen will also be obtained at baseline for each subject. 


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Subjects will be randomized to either a ω3 FFA preparation or a matching placebo for six 
months. A 3 month supply of study medication will be given to subjects at randomization. 
Telephone calls will be made by the study coordinator every month to the subjects to assess for 
any adverse effects (presence and grade) and reinforce compliance. Compliance will also be 
assessed by pill counts during this call. An additional 3 month supply of study medication will be 
mailed to subject by research pharmacy after the 2 month telephone call. 
6.2 Visit 2 
Subjects will undergo repeat assessment at six months following randomization. This will 
include anthropometry (similar to baseline), a fasting blood draw and endoscopy with research 
biopsies and brushings as outlined at baseline. Subjects will also fill out the Reflux Symptom 
Questionnaire (RSQ)/GER Questionnaire. Remaining medication will be counted and recorded.   
 
If the participant is to be scheduled for a clinically indicated upper endoscopy and procedures 
that are ordered cannot be done on the Clinical Research Unit, (i.e. radiofrequency ablation, 
endoscopic mucosal resection, esophageal dilation, endoscopic ultrasound) the research portion 
of the procedure will be done during the clinically indicated upper endoscopy.  These research 
procedures will include biopsies and brushings of the esophagus as well as mucosal impedance.  
 
 
 
 
 
Schedule of Events 
 
 
 
Study Activity 
Month 0 
Month 1 
Month 2 
Month 3 
Month 4 
Month 5 
Month 6 
Month 6 
+ 7 
days 
Informed consent 
X 
  
  
  
  
  
  
  
History 
X 
  
  
  
  
  
  
  
Concurrent meds 
X 
  
  
  
  
  
  
  
Physical exam 
(ht, wt, hip/waist, 
VS) 
X 
  
  
  
  
  
X 
  
Blood draw (FFA, 
LDL, AST, ALT) 
INR ) 
X 
  
  
  
  
  
X 
  
Endoscopy with 
bx/brushing and 
mucosal 
impedance 
measurement 
X 
  
  
  
  
  
X 
  
Randomization 
X 
  
  
  
  
  
  
  
Adverse event 
evaluation 
  
X 
X 
X 
X 
X 
X 
X 
Pill count 
  
X 
X 
X 
X 
X 
X 
  
Single slice CT 
scan of abdomen 
X 
  
  
  
  
  
  
  
Questionnaire 
X 
  
  
  
  
  
 X 
  


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7 Statistical Plan 
7.1 Sample Size Determination 
Statistical Analysis 
Up to 90 patients with Barrett’s will be identified and randomized to either drug or placebo. The 
primary analysis objective in this pilot study is to estimate response magnitude and variation 
which will be the information needed to design a larger adequately powered study to study the 
efficacy of w3FFA as a chemoprevention agent in BE. The data will be summarized as mean, 
standard deviation, as well as median, and interquartile range, overall (baseline) and by treatment 
group (post-supplementation at 6 months). In addition, 95% confidence intervals for response 
mean (or median) treatment group values and 95% confidence intervals for treatment group 
differences in means (or medians) will also be calculated. Exploratory descriptive summaries of 
the response data will also be generated after stratifying on ASA intake. 
 
Aim 1: The primary response outcome is tissue PGE2 levels and the main secondary response 
outcome is the histologic score of 3 or 4 (severe grades of inflammation). Other secondary 
outcomes include pre and post supplementation serum and tissue ω3 FFA levels.  
 
Aim 2: The primary response outcome is the Cytokine expression profile in esophageal biopsies 
as measured by qPCR [IL1β, IL6 (M1 markers) versus IL1, IL10 (M2 markers)]. Secondary 
response outcomes include change in esophageal macrophage counts and phenotype proportions 
(total, %M1, %M2).  
 
Aim 3: The esophageal mucosal impedance of the squamous epithelium and columnar 
epithelium will be summarized as mean (SD) or median (IQR) depending on data normality. The 
data will be summarized (baseline and post-supplementation) by treatment group. Change in 
impedance (Baseline – post supplementation) will be compared using appropriate statistical tests. 
Secondary outcome analysis will include testing the association between esophageal mucosal 
impedance and measures of central obesity (including waist circumference, waist hip ratio and 
abdominal visceral fat area on CT scan). 
 
Aim 4: 
We will align the paired end reads using PANDASeq given our small amplicon size (~392bp) 
which allows for significant overlap in the two reads. Data will be processed using QIIME1.8.0 
(Quantitative Insights Into Microbial Ecology) analysis pipeline. OTUs (Operational Taxonomic 
Units) will be determined at 97% sequence similarity using uclust, taxonomy will be assigned 
using RDP classifier against the GreenGenes database, and a phylogenetic tree will be built using 
FastTree. The OTU table will be rarified at a single sequencing depth. We will compare alpha 
diversity using both phylogenetic (PD whole tree) and non-phylogenetic measures (Shannon, 
observed species and Chao1) to assess evenness and richness of distribution. We will compare 
beta diversity using both phylogenetic (UniFrac) and count based metrics (Bray-Curtis).    
 
Statistical analysis of differences between groups and changes within the microbiome will be 
performed such as using PERMANOVA to compare beta diversity. Differences in individual 
taxa between groups will be assessed using ANOVA with correction for multiple hypotheses 
testing using FDR.  A p-value of <0.05 will be considered statistically significant. Additional 


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analysis to impute potential changes in gene content (PICRUSt) and pathways (HUMAnN) will 
be done depending on the results of initial analysis. 
 
 
 
Pitfalls and Alternative approaches: It is possible that recruitment to this trial may be 
challenging. However, more than 400 patients with BE are seen annually in Rochester and the PI 
has successfully led and participated in many prospective studies which have recruited BE 
patients for clinical trials. It also possible, though unlikely, that the in-vitro effects of ω3 FFAs 
may not translate into clinical effects in terms of alterations in tissue PGE2 and macrophage 
counts. We will also consider assessing the influence of ω3 FFA on BE cell survival, a known 
biological correlate to neoplastic progression in BE, using Ki-67-Caspase dual staining to assess 
other biologic effects of ω3 FFA. Concomitant ASA use may confound the effects on PGE2 
levels. Given that this is a preliminary study, we will collect data on ASA and NSAID use and 
perform analysis stratified on ASA and NSAID use. The randomized study design will however 
mitigate this confounding effect to a large extent. It is possible that the dose of ω3 FFA used in 
this proposal may be inadequate for a clinical effect in vivo. We have chosen the 3.6 grams/day 
dose as described in prior human clinical trials: this dose has been shown to be well tolerated and 
able to produce meaningful clinical response in human trials. We also have proposed to target 
subjects with a BMI>30 given that these subjects with increased visceral fat are most likely to 
benefit from ω3 FFAs. 
 
Feasibility and Timeline: We anticipate recruitment to be complete in 6-9 months following 
IRB approval (which has been initiated). Assays and analysis should be completed in 3 months 
following completion of the trial and publication thereafter. Assays have been previously 
performed and validated in multiple published manuscripts by collaborators and Core 
laboratories. Preliminary data obtained from this trial will be crucial in supporting an aim of a 
R01 application focused on assessing the mechanistic relationship between BE and central 
obesity. 
8 Safety and Adverse Events 
 
Adverse event grading 
Attribution scale. An adverse event is defined as both an expected side effect that is of a serious 
nature, or an unexpected side effect/event regardless of severity. All events will be graded as to 
their attribution (unrelated to protocol, or possibly, probably, or definitely related to protocol). 
Any event that is reported to either the principal investigator or his designated research 
associates by the subject or medical staff caring for the subject and which meets the criteria will 
be documented as such. 
 
Adverse Events 
Safety of ω3 FFA: ω3 FFAs have an excellent safety and tolerability profile. Most common side 
effects associated with their use in human trials include diarrhea, indigestion, nausea, fishy taste 
and belching. Total incidence of GI disturbances in phase 2 and 3 clinical trials has varied 
between 1.4 – 4.9%, with a very low rate of study termination [27]. The rate of study withdrawal 


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Prasad G. Iyer, MD 
 
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was 3.4% in a phase 3 study using EPA [20]. ω3 FFAs have antiplatelet activity, however 
increased rates of bleeding have not been seen in large clinical cardiovascular trials [28]. 
 
Risk Monitoring/Risk Reduction: 
The risk of anaphylactic reactions will be reduced by excluding any potential participants with a 
history of allergies to fish or shellfish. Participants will be instructed to immediately discontinue 
the study medication and contact the study team if they experience any signs of allergic reaction 
(i.e., rash, swelling, itching, cramps, wheezing, upset stomach, loose stool, nasal congestion, 
fainting, dizziness). If the investigators suspect that such symptoms are due to the EPA/DHA, the 
participant will be excluded from the study and referred to the appropriate health care provider. 
8.1 Definitions 
Unanticipated Problems Involving Risk to Subjects or Others (UPIRTSO) 
Any unanticipated problem or adverse event that meets the following three criteria:  
• Serious: Serious problems or events that results in significant harm, (which may be 
physical, psychological, financial, social, economic, or legal) or increased risk for the 
subject or others (including individuals who are not research subjects). These include: (1) 
death; (2) life threatening adverse experience; (3) hospitalization - inpatient, new, or 
prolonged; (4) disability/incapacity - persistent or significant; (5) birth defect/anomaly; (6) 
breach of confidentiality and (7) other problems, events, or new information (i.e. 
publications, DSMB reports, interim findings, product labeling change) that in the opinion 
of the local investigator may adversely affect the rights, safety, or welfare of the subjects 
or others, or substantially compromise the research data, AND 
• Unanticipated: (i.e. unexpected) problems or events are those that are not already 
described as potential risks in the protocol, consent document, not listed in the 
Investigator’s Brochure, or not part of an underlying disease. A problem or event is 
"unanticipated" when it was unforeseeable at the time of its occurrence. A problem or 
event is "unanticipated" when it occurs at an increased frequency or at an increased 
severity than expected, AND 
• Related: A problem or event is "related" if it is possibly related to the research procedures. 
 
Adverse Event 
An untoward or undesirable experience associated with the use of a medical product (i.e. drug, device, 
biologic) in a patient or research subject. 
Serious Adverse Event 
Adverse events are classified as serious or non-serious.  Serious problems/events can be well 
defined and include; 
• death 
• life threatening adverse experience 
• hospitalization 
• inpatient, new, or prolonged; disability/incapacity 
• persistent or significant birth defect/anomaly 
 


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and/or per protocol may be problems/events that in the opinion of the sponsor-investigator 
may have adversely affected the rights, safety, or welfare of the subjects or others, or 
substantially compromised the research data.  
 
In case of a serious adverse event, the PI or a member of the study team will contact the research 
pharmacy for unblinding of the randomization code. 
 
All adverse events that do not meet any of the criteria for serious, should be regarded as non-
serious adverse events.  
 
Adverse Event Reporting Period 
For this study, the study treatment follow-up period is defined as 7 days following the last 
administration of study treatment.  
8.2 Recording of Adverse Events 
The study team will assess subjects for adverse events monthly via telephone. Information on all 
adverse events will be recorded immediately in the source document, and also in the appropriate 
adverse event section of the case report form. The clinical course of each event will be followed 
until resolution, stabilization, or until it has been ultimately determined that the study treatment 
or participation is not the probable cause. Serious adverse events that are still ongoing at the end 
of the study period will be followed up, to determine the final outcome.  Any serious adverse 
event that occurs after the study period and is considered to be at least possibly related to the 
study treatment or study participation will be recorded and reported immediately. 
8.3 Reporting of Serious Adverse Events and Unanticipated Problems 
When an adverse event has been identified, the study team will take appropriate action necessary 
to protect the study participant and then complete the Study Adverse Event Worksheet and log.  
The sponsor-investigator will evaluate the event and determine the necessary follow-up and 
reporting required. 
8.3.1 Sponsor-Investigator reporting: notifying the Mayo IRB 
All adverse events will be reported to the Mayo Clinic IRB. 
Serious adverse events are defined as: 
•  Death 
•  Life Threatening 
•  Inpatient hospitalization or prolongation of existing hospitalization 
•  Persistent or significant disability/incapacity 
 
The PI will report serious adverse events to the chair of the IRB using the Serious Adverse Event 
(SAE) Reporting Form. The PI will sign each Serious Adverse Event form. A copy of the current 
consent form will be submitted with the actual risk highlighted in the current consent form. Or, if 
the PI recommends any changes to the consent form document, a printed and electronic revised 
consent form will be attached. 
 
The IRB office will review the incoming reports and triage according to the nature of the event: 


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Prasad G. Iyer, MD 
 
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• Reports of serious adverse events that resulted in taking immediate action will be given 
first priority. 
• Reports of serious adverse events for which the PI recommends changes to the consent 
form document will be given 2nd priority. 
• All other reports will then be prioritized. 
 
The members will review SAE form and supporting materials. If additional information is 
necessary, the IRB will contact the PI (or study coordinator if the PI is unavailable) by 
conference call. After review of the information, the IRB will make an initial determination of 
the seriousness of the event and determine what actions, if any will be required. 
 
FDA regulations do not require non-serious adverse events (those that do not fall into the 
categories outlined in step 3 above) to be reported to the IRB. If non-serious adverse events are 
reported to the IRB, these reports will be signed by a member of the Subcommittee and returned 
to the investigator. The IRB will not retain a copy of these materials in the IRB office or files. 
 
For serious adverse events that the IRB determines to be unrelated to the study 
drug/device/intervention, the original report and supporting materials will be kept in the IRB file. 
The IRB will review such reports, and if the IRB agrees with the determination that the event 
was unrelated to study drug/device/intervention, then a copy of the report will be returned to the 
PI. The IRB office will retain a list of the studies for which unrelated serious adverse events are 
reported. Serious but unrelated adverse event reports will not be included in the minutes of the 
meeting. 
 
For all serious adverse event reports that are determined by the IRB to be definitely, 
probably, or possibly related to the study drug/device/intervention, or if it is unknown what the 
relationship is at the present time, the IRB will review the reports and related materials and 
include in the minutes of the meeting. The IRB meeting minutes will be referred to the Full 
Board for final action. For these adverse events which are unexpected and require a change in the 
consent form, the Chair or a Vice-Chair will be the primary reviewer upon referral to the Full 
Board. 
 
The convened IRB shall take whatever action(s) it deems appropriate. These actions may include 
but are not limited to: 
• modification of the protocol 
• modification of the consent form document 
• modification to the timetable for continuing review requirements, 
• suspension of new enrollment into the study 
• suspension of the study, or 
• termination of the study. 
Any studies that are suspended or terminated will be promptly reported to NIH that has 
provided funding for the study and/or to the FDA if the study involves an IND or an IDE. 
 
All other events not requiring suspension or termination shall be reported to the FDA through the 
normal reporting channel (notification from the investigator to the sponsor to the FDA). 
 


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Prasad G. Iyer, MD 
 
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The convened IRB will generate a subsequent minute excerpt only if additional action is taken 
(i.e. approval of revised consent form, revisions to the protocol, etc.). 
 
For this protocol, only directly related SAEs/UPIRTSOs will be reported to the IRB. 
8.3.2 Sponsor-Investigator reporting: Notifying the FDA 
The sponsor-investigator will report to the FDA all unexpected, serious suspected adverse 
reactions according to the required IND Safety Reporting timelines, formats and requirements. 
 
Unexpected fatal or life threatening suspected adverse reactions where there is evidence to 
suggest a causal relationship between the study drug/placebo and the adverse event, will be 
reported as a serious suspected adverse reaction.  This will be reported to the FDA on FDA Form 
3500A, no later than 7 calendar days after the sponsor-investigator’s initial receipt of the 
information about the event. 
 
Other unexpected serious suspected adverse reactions where there is evidence to suggest a causal 
relationship between the study drug/placebo and the adverse event, will be reported as a serious 
suspected adverse reaction.  This will be reported to the FDA on FDA Form 3500A, no later than 
15 calendar days after the sponsor-investigator’s initial receipt of the information about the 
event. 
 
Any clinically important increase in the rate of serious suspected adverse reactions over those 
listed in the protocol or product insert will be reported as a serious suspected adverse reaction.  
This will be reported to the FDA on FDA Form 3500A no later than 15 calendar days after the 
sponsor-investigator’s initial receipt of the information about the event. 
 
Findings from other studies in human or animals that suggest a significant risk in humans 
exposed to the drug will be reported.  This will be reported to the FDA on FDA Form 3500A, no 
later than 15 calendar days after the sponsor-investigators initial receipt of the information about 
the event. 
8.4 Stopping Rules  
Definite termination criteria: 
• Request by subject to leave study. 
• Evidence of deliberate non-compliance. 
• Pregnancy 
• Alcohol abuse; illicit drug abuse. 
 
Potential termination criteria include: 
• Development of acute or chronic condition that may impact on metabolic variables or 
requiring medications likely to impact on metabolic variables or likely to result in subject 
being unable to participate. Subjects will be reviewed on a subject-by-subject basis and 
all subjects reported to the Safety-Monitoring Panel. 
 
Specific Action plans are pre-assigned for: 
• Depression or low mood. Here, all patients will be asked to be evaluated by their primary 


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Prasad G. Iyer, MD 
 
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care physician and if appropriate referred to a psychiatrist. The opinion of the psychiatrist 
will be used to determine continuance or termination in the protocol.  
 
This does not represent a comprehensive listing of criteria or causes. All subjects who withdraw 
or whom are withdrawn from the study or whom are considering/being consider for withdrawal 
will be referred to the Safety-Monitoring Panel. These subjects data will be scrutinized (whether 
terminated or not), separately to assess for association of intervention with a specific adverse 
outcome(s). 
 
8.5 Medical Monitoring 
It is the responsibility of the Principal Investigator to oversee the safety of the study at his/her 
site.  This safety monitoring will include careful assessment and appropriate reporting of adverse 
events as noted above, as well as the construction and implementation of a site data and safety-
monitoring plan (see section 10  “Study Monitoring, Auditing, and Inspecting”).  Medical 
monitoring will include a regular assessment of the number and type of serious adverse events. 
8.5.1 Internal Data and Safety Monitoring Board 
This will consist of Dr. Elizabeth Rajan and Dr. Michael Levy (both Staff Gastroenterologists at 
Mayo Clinic, Rochester). They will meet with the PI every 6 months to review recruitment and 
any adverse effects in subjects recruited into the study. 
9 Data Handling and Record Keeping 
9.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  
Those regulations require a signed subject authorization informing the subject of the following:  
• What protected health information (PHI) will be collected from subjects in this study 
• Who will have access to that information and why 
• Who will use or disclose that information 
• The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by 
regulation, retains the ability to use all information collected prior to the revocation of subject 
authorization.  For subjects that have revoked authorization to collect or use PHI, attempts 
should be made to obtain permission to collect at least vital status (long term survival status that 
the subject is alive) at the end of their scheduled study period. 
 
The following study specific measures will be taken: 
• Protection of subject privacy: Medical history and physical examination are performed, 
and a brief questionnaire is administered. Blood is drawn for screening purposes. All of 
these materials are obtained for research purposes only, and data are kept in strict 
confidence. No information will be given to anyone without permission from the subject. 
Our consent form includes the Informed Consent statement required by Mayo for these 
types of studies. This statement guarantees the confidentiality. 


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• Database protection: The database is secured with password protection behind the Mayo 
Firewall and accessible only to study team. Electronic communication with our outside 
collaborators involves only coded, unidentifiable information. Subject folders are kept in 
institutionally secured office rooms and laboratory. 
• Confidentiality during adverse event reporting: Adverse event reports and annual 
summaries will not include subject-identifiable material. 
 
9.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other 
activities in a clinical trial necessary for the reconstruction and evaluation of the trial.  Source 
data are contained in source documents.  Examples of these original documents, and data records 
include: hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ 
diaries or evaluation checklists, pharmacy dispensing records, recorded data from automated 
instruments, copies or transcriptions certified after verification as being accurate and complete, 
microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject files, and 
records kept at the pharmacy, at the laboratories, and at medico-technical departments involved 
in the clinical trial. 
9.3 Records Retention 
The sponsor-investigator will maintain records and essential documents related to the conduct of 
the study.  These will include subject case histories and regulatory documents. 
 
All subject-specific data and Case Report Forms will be coded using a unique study number for 
each individual subject. Confidentiality of all medical records is strictly maintained by 
established procedures. The original study data are kept in the principal investigators’ 
laboratory/office and are entered into a secure computer database password protected under a 
secure server space behind the Mayo firewall allocated for use by only the study team. Subject 
names or other directly identifiable information will not appear on any reports, publications, or 
other disclosures of clinical study outcomes. 
 
The sponsor-investigator will retain the specified records and reports for; 
1. Up to 2 years after the marketing application is approved for the drug; or, if a marketing 
application is not submitted or approved for the drug, until 2 years after shipment and 
delivery of the drug for investigational use is discontinued and the FDA has been so 
notified. OR 
2. As outlined in the Mayo Clinic Research Policy Manual –“Access to and Retention of 
Research Data Policy” Whichever is longer. 
10 Study Monitoring, Auditing, and Inspecting 
10.1 Study Monitoring Plan 
This study will be monitored on a routine basis during the conduct of the trial.  The Mayo Clinic 
Office of Research Regulatory Support will provide clinical monitoring for the trial as a service 
for the sponsor-investigator.  Clinical trial monitoring requires review of the study data generated 
throughout the duration of the study to ensure the validity and integrity of the data along with the 


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protection of human research subjects.  This will assist sponsor-investigators in complying with 
Food and Drug Administration regulations. 
 
The investigator will allocate adequate time for such monitoring activities.  The Investigator will 
also ensure that the monitor or other compliance or quality assurance reviewer is given access to 
all the study-related documents and study related facilities (e.g. pharmacy, diagnostic laboratory, 
etc.), and has adequate space to conduct the monitoring visit. 
10.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, the 
sponsor, and government regulatory agencies, of all study related documents (e.g. source 
documents, regulatory documents, data collection instruments, study data etc.).  The investigator 
will ensure the capability for inspections of applicable study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by 
government regulatory authorities and applicable compliance offices. 
11 Ethical Considerations 
This study is to be conducted according to United States government regulations and 
Institutional research policies and procedures. 
 
This protocol and any amendments will be submitted to a properly constituted local Institutional 
Review Board (IRB), in agreement with local legal prescriptions, for formal approval of the 
study.  The decision of the IRB concerning the conduct of the study will be made in writing to 
the sponsor-investigator before commencement of this study. 
 
All subjects for this study will be provided a consent form describing this study and providing 
sufficient information for subjects to make an informed decision about their participation in this 
study.  This consent form will be submitted with the protocol for review and approval by the IRB 
for the study.  The formal consent of a subject, using the Approved IRB consent form, must be 
obtained before that subject undergoes any study procedure.  The consent form must be signed 
by the subject or the subject’s legally authorized representative, and the individual obtaining the 
informed consent. 
12 Study Finances 
12.1 Funding Source 
This study is financed through the Mayo Foundation Division of Gastroenterology. 
12.2 Subject Stipends or Payments 
Subjects completing the study will be given $250 remuneration. 
13 References 
 


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