Clinical trial NCT02010359
Conditions: Obesity
Sponsor: University of Pennsylvania
Start date: 2014-06-01; Completion date: 2017-07-01
Enrollment: 29 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study is a clinical trial designed to assess whether fish oil treatments are effective in the prevention of obesity-related fat tissue (adipose) inflammation. Specifically it addresses the hypothesis that fish oils treatments will reduce signaling by chemokine pathways (fractalkine and MCP-1) important in adipose tissue for the recruitment and activation of certain white blood cells (macrophages). The study is a double-blind placebo-controlled trial of the fish oil Lovaza from GlaxoSmithKline (omega-3-acid ethyl esters; a combination of ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) in obese non-diabetic adults, to determine if Lovaza decreases markers of inflammation and macrophage activation in adipose and blood and understand the mechanism by which fish oils affect inflammation.

Detailed description:
We propose to conduct a parallel arm, double blind, placebo-controlled trial to understand the anti-inflammatory and metabolic effects of marine derived omega-3 fatty acids (EPA + DHA) on obesity-related inflammation and chemokine signaling in humans. Obese (BMI\>30 kg/m2) but non-diabetic young males and females (n\~44; 25 to 50 years of age) taking no omega-3 fatty acid (EPA + DHA) supplements and consuming a low fish diet (defined as usual intake of high omega-3 fish (tuna and other non-fried fish) \< 3 to 4 servings per month) will be included. A low habitual fish intake is important to control EPA + DHA intake during the supplement period (see exclusion criteria below). Subjects also will be required to maintain their current fish intake throughout the treatment period.

There will be two treatment groups; (1) placebo and (2) omega-3 fatty acids 4 grams/day. The trial will be randomized, double blind, placebo-controlled, with parallel treatment groups; treatment with omega-3 fatty acids 8 weeks. We will enroll participants until at least 22 subjects per study-arm complete all study visits (i.e., at least 44 participants).

Prescription Lovaza (omega-3-acid ethyl esters) from GlaxoSmithKline at 4 grams day (2 1-gram capsules twice daily, with food) will be used. We expect this dose to be safe because it is a dose that is recommended, and routinely used, for management of moderate or worse hypertriglyceridemia (levels \> 400 mg/dL). We will use matching placebo capsules so that all participants are taking 2 capsules twice daily - with food as recommended in clinical practice.

The study duration was chosen in order to provide adequate time for potential anti-inflammatory effects of treatments without requiring maximum lipid modifying effects while also reducing the potential for drop-out and loss to follow up that is associated with longer study durations.

Participants will be recruited from the Preventive Cardiology Research databases, comprised of healthy subjects who have participated in previous research studies and who have asked to be contacted for future studies. Potential participants will also be recruited via local IRB-approved flyers, internet, and newspaper advertisements (see flyer and advertisement attached) targeting the Delaware Valley region.

STUDY PROCEDURES

Informed Consent Before study initiation, the clinical protocol, the Informed Consent Form (ICF), and any advertisements for subject recruitment will be submitted for review and approval to the Institutional Review Board (IRB). The investigator, or his designated research staff, will obtain written informed consent from each subject enrolled in the study, in accordance with the U.S. Food and Drug Administration (FDA) regulations 21 CFR parts 50.20-50.27. It is the responsibility of the investigator to ensure that informed consent is obtained from the subject or his/her guardian or legal representative before any activity or treatment is undertaken which is not part of routine care.

Safety Assessments Safety will be evaluated by the incidence, severity, and relationship to treatment agent of adverse events.

Monitoring for Adverse Events: Details of all AEs that occur after the first dose of study drug will be collected as indicated in Section 12. Any illness or injury that occurs before the first dose of a study drug will be recorded under Medical History and evaluated to determine if the occurrence affects the patient's eligibility to participate in the study. Subjects will be asked the following standard questions by the designated clinical evaluator: At clinic check-ins:

1. "Have you had any medical problems since your last visit?"
2. "Have any medical problems present at your last visit changed, i.e., stopped, worsened, or improved?"
3. "Have you taken any medicines, other than study drug, since your last visit?" Any spontaneous AE information provided by the patient will be recorded. NOTE: An AE may be a new disease(s), any untoward event(s), or an exacerbation of a pre existing condition. See Section 12.0 for complete details.

Adipose biopsy: Approximately 500-1000 mg of subcutaneous adipose tissue will be biopsied from the gluteal region, processed, weighed and frozen until analyzed. Adipose tissue will be obtained through a small (\~5mm) incision under local anesthesia (2% Lidocaine without epinephrine), as has been previously described13-15. A portion (200 mg) of the samples will be flash frozen and analyzed for gene expression and protein assays. Another portion (500-800 mg) will be placed in sterile media and undergo digestion with collagenase, filtration, and centrifugation for separation of the stromal vascular fraction (SVF). SVF cells will be stained with antibodies for flow cytometer analysis. Finally, remaining tissue will be fixed in paraffin for immunohistochemistry.

6.5 Subgingival analysis

Saliva samples will be collected using the OMNIgene DISCOVER (OM-505) kit (DNA Genotek), an all-in-one kit which collects and stabilizes both microbial DNA and RNA in saliva. Samples will be stored at -80C prior to extraction. Nucleic acids will be extracted using the QIAGEN QIAamp MinElute Virus Spin Kit (Qiagen). Subgingival plaque samples will be obtained from 3 oral cavity regions in each subject (2 samples per region) using a soft-bristled microbrush (DenTek). Specimens will be collected from the same anatomical location in all subjects. DNA will be isolated using the PSP Spin Stool DNA kit (Invitec). DNA samples will be amplified using V1-V2 region primers targeting bacterial 16S genes and sequenced using IlluminaHiSeq or 454 Pyrosequencing technology.

Specific Study Procedures (by visit)

Screening (Visit 1) Potential participants will be screened first by a telephone or e-mail interview with the research coordinator (see Appendix B for email interview form; for telephone interviews this information will be verbally exchanged). Participants who meet initial study requirements will be invited for a screening visit (visit 1) at the CTRC. Prior to this visit, participants will receive a detailed health questionnaire (see Appendix C) that has been used in numerous previous studies that addresses questions regarding past medical history, current use of medications and dietary supplements, exercise and social habits. In addition, participants will be asked about fish consumption in order to ensure recruitment of subjects with lower EPA + DHA intake during the study period. Participants will be asked to complete these questionnaires at home and bring the completed forms with them to their screening visit. During the screening visit, they will meet with research staff to review and sign an IRB-approved consent form. Research staff will review the health questionnaire with the participant as well as measure weight, height, waist and hip circumference, sitting blood pressure and heart rate. A brief physical exam will be performed as well as an electrocardiogram (ECG). In addition, participants will have blood drawn (after a 12 hour fast) for evaluating fasting lipids, glucose, serum chemistries and liver function and a complete blood count. Female participants will also be asked to provide a urine sample for a pregnancy test. These laboratory parameters are included to assure the participant meets eligibility criteria and that he/she is generally in good health. Once all clinical data has been reviewed, study personnel will contact each study volunteer to let them know if they are eligible.

Randomization visit (Visit 2) One to four weeks after the screening Visit 1, eligible participants will return to the CTRC after a 12 hour fast for a 2 hour visit. Participants will be asked to brush their teeth after their last meal the day before the visit, or at the start of the 12-hour fasting period, and to refrain from additional brushing or use of mouthwash until after the study visit. Participants will meet with a research nurse / coordinator who will assess any change in medical history and medication use, as well as review any adverse events. Blood pressure, heart rate, height and weight will be measured, blood and urine will be collected for pre-randomization baseline efficacy measures and a urine pregnancy test performed for all females. The simple fish consumption questionnaire will be administered to assess any changes to the level of omega-3 intake in the diet.

They will also be asked to record all food and beverages consumed on dietary records on 3 days prior to the visit, as well as completing a digestive health questionnaire and diet history questionnaire. All nutrient data collected from 3-day dietary records will be analyzed by Food Processor 8.1 (06/03, Escha). Participants will be asked to complete these questionnaires at home and bring the completed forms with them to their study visit. If a subject is unable to print out or complete the forms at home, they will be completed at the visit.

Saliva and subgingival plaque samples will be taken for assessment of oral microbiome. Saliva samples will be collected using a standardized kit. Under the supervision of study personnel, subjects will be given the kit, which consists of a funnel attached to a tube, and instructed to spit into the funnel until the amount of liquid reaches a fill line located on the outside of the attached tube. Once the proper amount has been collected, the lid of the funnel will be closed and the funnel removed from the tube. The tube is then capped and shaken for about 10 seconds. Subgingival plaque samples will be collected by swabbing several regions of the buccal mucosa using microbrushes. A separate brush will be used for each swab and two samples will be collected from each region.

In order to establish effects of treatments on adipose inflammatory parameters, we will obtain gluteal region subcutaneous adipose tissue biopsies under local anesthesia (2% Lidocaine). A detailed description of the adipose collection procedures is given in Section 6.4. Participants will then be randomized to drug or placebo group. Supplies of study drug will be provided to last 8 weeks (4 pills/day of either Lovaza or placebo).

Participants will be provided verbal and written instruction to take the study drugs as follows; 2 fish oil/placebo with food in the morning, 2 fish oil/placebo with food in the evening. A pill diary will be given to all subjects.

Telephone / E-mail Follow up (Visit 3) Participants will be contacted by telephone or e-mail approximately four weeks (visit 3) post-randomization to review medication compliance and pill diary, adverse events and any changes in fish consumption. Participants with \<80% apparent adherence to study medication will be counseled to increase compliance so that their adherence at 8 weeks will be ≥80%.

Completion Visit (Visit 4) Participants will come to the CTRC after a 12 hour fast. Participants will meet with a study coordinator who will assess changes to the medical history and medication use. They will also be asked to record all food and beverages consumed on dietary records on 3 days prior to the visit. Any adverse events will be recorded, pill diary reviewed, and study medications will be counted (pill count) Weight and height will be recorded and BMI calculated. ECG will be performed. Blood will be drawn for post-drug screening of liver functions and chemistries as well as for all outcome-related measures (lipids, insulin/glucose, inflammatory markers). Urine for efficacy measures will also be collected. Urine pregnancy test will be performed in all females. Saliva and subgingival plaque samples will be taken to assess for any change in oral microbiome by the treatment. An adipose biopsy will be performed as above.

Arms: arm 1: Lovaza 4 grams daily (2 1-gram capsules twice daily) will be given for 8 weeks arm 2: An inactive Placebo (2 pills twice daily) will be given for 8 weeks
Interventions: intervention 1: Lovaza intervention 2: Placebo
Intervention descriptions: intervention 1: Lovaza 4 grams per day intervention 2: Placebo pill

Protocol documents:
Prot_SAP_000.pdf: 
 
 
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Title of Study Protocol:   
Fish oils and Adipose Inflammation Reduction (FAIR 
Study) 
 
Principal Investigator: 
Rachana Shah, MD, MsTR 
 
 
Sponsors of Protocol: 
National Institutes of Health 
 
 
Grants: 
1K23DK095913-01A1 (NIDDK): Omega-3 PUFA 
suppress adipochemokines (PI:Shah) 
 
 
 
 
IRB Protocol Number: 
818359 
 
 
Date of Document: 
 
November 4, 2014 
 
 
 


 
 
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Synopsis 
Title 
Fish oils and Adipose Inflammation Reduction (FAIR Study) 
Short Title 
FAIR Study 
Protocol Number 
 818359 
Study Design 
Parallel arm, double blind, placebo-controlled trial 
Study Center(s) 
University of Pennsylvania Medical Center Philadelphia, PA  USA  
Objective 
To examine the effect of omega-3 fish oil supplementation  
Study Population, 
Diagnosis And Main 
Inclusion Criteria 
Obese (BMI>30), non-diabetic men and non-pregnant/non-
lactating women aged 18-50 not currently consuming significant 
amounts of omega 3 fatty acids.    
Study Product, 
Dose, Route, 
Regimen, Duration 
of administration 
Participants will be randomized to placebo of Lovaza 4 grams/day 
orally. Subjects will be treated for 8 weeks.  
Reference therapy 
Placebo 
Pharmacodynamic 
Evaluations 
Inflammatory markers (cytokines, chemokines, acute phase 
proteins), metabolic markers, oral microbiome composition, 
monocyte function and lipoproteins                 
 
Safety Evaluations 
Clinical and laboratory measurements will be taken at screening 
and upon completion of the study.   
Statistical 
Methodology 
Analysis will compare within group and between group differences 
to determine effect of omega 3 fatty acids vs placebo on all 
measures indicated 
 


 
 
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1.0  ETHICAL CONSIDERATIONS 
The study will be conducted in accordance with the University of Pennsylvania’s clinical 
research standards that meet regulations relating to Good Clinical Practice (GCP).  
These standards adhere to the following guidelines: 
• 
Good Clinical Practice:  ICH Consolidated Guideline (International Conference on 
Harmonization of Technical Requirements for the Registration of Pharmaceuticals for 
Human Use, May 1996). 
• 
United States (US) Code of Federal Regulations (21 CFR) dealing with human 
subject protection and conduct of investigational clinical studies (21 CFR parts 50, 
54, 56, 312, and 314). 
• 
Declaration of Helsinki, concerning medical research in humans (“Recommendations 
Guiding Physicians in Biomedical Research Involving Human Subjects,” Helsinki 
1964, amend Tokyo 1975, Venice 1983, Hong Kong 1989, revised version of 
Somerset West, Republic of South Africa, October, 1996 and Scotland, 2000). 
 
This protocol and any amendments will be submitted to University of Pennsylvania 
institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study conduct.  The decision of the IRB concerning the conduct of the 
study will be made in writing to the 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.  The 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 
IRB-approved consent form, must be obtained before that subject is submitted to any 
study procedure.  This consent form must be signed by the subject or legally acceptable 
surrogate, and the investigator-designated research professional obtaining the consent.  
 
2.0  INTRODUCTION AND BACKGROUND 
Adipose inflammation, induced by inflammatory leukocyte infiltration, is a crucial link 
between obesity and its metabolic complications1-3 2-8 9-12, including insulin resistance, 
type 2 diabetes, and metabolic syndrome.  Chemokine pathways including monocyte 
chemotactic protein (CCL2) and fractalkine (CX3CL1) and their receptors CCR2 and 
CX3CR1 may modulate insulin resistance by regulating adipose macrophage 
recruitment and activation4,13-15. Variable expression of CCR2 and CX3CR1 is a critical 
determinant of monocyte survival and inflammatory state and delineates two 
populations: pro-inflammatory CX3CR1loCCR2+ monocytes associated with classically-
activated, inflammatory (M1) adipose macrophages vs. less inflammatory 
CX3CR1hiCCR2- thought to give rise to alternatively-activated (M2) adipose 
macrophages1-3. Metabolic and inflammatory stimuli associated with obesity induce 
release of chemokines that promote disproportionate migration and adhesion of pro-
inflammatory CX3CR1loCCR2+ monocytes and/or shift of M2 to M1 macrophages16.  


 
 
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Omega-3 polyunsaturated fatty acids: Murine LC n-3 PUFA, from fish and fish oil 
supplements, include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), 
and are used clinically to treat dyslipidemia. Decreased triglycerides with increased HDL 
is seen and adiponectin levels are increased in mice and humans on LC n-3 PUFA17,18.  
Data assessing beneficial effect of LC n-3 PUFA on incidence of cardiovascular disease 
is conflicting19,20. Several large scale studies have reported benefits of LC n-3 PUFA in 
primary 21, 22, 23 and secondary24, 25 prevention of adverse cardiovascular outcomes; 
however, the recently completed ORIGIN trial showed no reduction in cardiovascular 
death in a large cohort of dysglycemic subjects receiving 1 gram LC n-3 PUFA vs. 
placebo26.  Dose, treatment duration, and underlying disease burden may underlie this 
discrepancy27.  
LC n-3 PUFA reduce obesity-related inflammation:. LC n-3 PUFA themselves or anti-
inflammatory metabolites they produce exert pleiotropic anti-inflammatory effects in 
human and animal models28 . In patients with chronic inflammatory diseases, LC n-3 
PUFA reduce systemic inflammation29-32; remarkably, decreases in inflammatory 
markers are also suggested with LC n-3 PUFA in the low-grade inflammatory condition 
of obesity33. In a trial of 25 adolescents with metabolic syndrome, 3 weeks of LC n-3 
PUFA supplementation decreased circulating lymphocytes and monocytes as well as 
levels of cytokines TNF, IL-1, and IL6 despite no improvement in weight or insulin 
sensitivity34.  
LC n-3 PUFA reduce monocyte inflammatory state and ATM infiltration: Dietary studies 
in humans suggest that LC n-3 PUFA reduce monocyte inflammatory mediators35,36, 
monocyte migration and adhesion, and receptor expression (CD44 variants) 37,38. Human 
THP-1 macrophages treated with LC n-3 PUFA in vitro had decreased LPS-stimulated 
cytokine expression (TNF IL-1, IL6) and inflammatory transcription factor nuclear 
factor kappa beta (NF) activity39. In mice, adding LC n-3 PUFA to diet reduced 
circulating Ly6Chi inflammatory monocytes40. In rodents fed a high fat diet (HFD), 
addition of LC n-3 PUFA prevents inflammatory ATM infiltration, adiponectin decrease, 
and inflammatory gene expression (including chemokines CCL2 and CCL5)41,42 43,44 45.  
LC n-3 PUFA may attenuate NF signaling: Though multiple mechanistic models are 
proposed, the NF pathway is consistently implicated in reduced inflammation by LC n-
3 PUFA28. NF signaling is downstream of TLR4 and cytokine receptor activation46. In 
vitro LC n-3 PUFA is shown to inhibit LPS-induced activation of NF and I 
phosphorylation in macrophages, with associated reduction in inflammatory cytokines47 
and increased production of anti-inflammatory marker IL1048. 


 
 
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GPR120 is a LC n-3 PUFA receptor and may inhibit NF signaling: Recent work 
suggests the effects of LC n-3 PUFA on obesity-related inflammation are mediated via 
interactions GRP120, a G-protein-coupled receptor (GPR), which is highly expressed on 
adipocytes and macrophages49. GPR120 is part of the Gq-linked GPR family and 
stimulation results in increased intracellular Ca++ and ERK activation50. Compared to 
wild-type mice GRP120 deficient mice fed a HFD have increased weight, insulin 
resistance, circulating and adipose inflammatory markers—including the chemokine 
CCL2, and ATMs51. Remarkably, humans with a dominant-negative loss-of-function 
variant in GPR120 (R270H) have increased incidence of obesity 51. In 3T3-L1 
adipocytes, siRNA knockdown of GPR120 reduced expression of insulin signaling 
genes52. Highlighting its role as an LC n-3 PUFA receptor, GPR120 deficiency blocks the 
benefits of LC n-3 PUFA supplementation to HFD in mice, agonists to GPR120 mimic 
effects of DHA to block LPS-induced NF activation in macrophages, and knockdown 
of GPR120 abolishes DHA effects49.  Whether GPR120 deficiency modulates EPA 
effects is not reported, notably anti-inflammatory effects of EPA occur via distinct 
changes in plasma cell membranes in immune cells53. 
LC n-3 PUFA may affect the oral microbiome 
Periodontal disease has been associated with increased risk of cardiometabolic diseases 
54,55, There is data that suggests that dietary fat intake, including omega-3 fatty acids affect 
the composition of the intestinal and oral microbiome56,57. The direct effect of LC n-3 PUFA 
supplementation on oral microbiome composition and the relationship between  
inflammatory chemokine pathways and the microbiome have not previously been 
evaluated.  
Overall LC n-3 PUFA modulate chemokine signaling including adipocyte and 
macrophage inflammation, ATM infiltration, and monocyte activation; these actions may 
be mediated, at least partially, via GPR120 and NF However, no human studies 
examining the effect of LC n-3 PUFA on the CCL2 and CX3CL1 adipochemokine 
pathways have been reported and the role of GPR120 and NF on these pathways has 
not been explored. Insight into LC n-3 PUFA action on these functional pathways may 
clarify mechanisms and guide therapeutic developments in human.  
We propose a clinical trial to assess the validity of LC n-3 PUFA as an adjuvant treatment 
in prevention of obesity-related adipose inflammation and metabolic disturbance. While 
clinical studies of LC n-3 PUFA show decreased systemic inflammation, and animal 
models demonstrate decreased adipose inflammation, the effect of LC n-3 PUFA on 
human adipose inflammation chemokine signaling and GPR120/NF activity in obesity 
have not been explored. We will utilize a double-blind placebo-controlled trial of the LC n-3 
PUFA fish oil Lovaza from GlaxoSmithKline (omega-3-acid ethyl esters; a combination of 
ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) in obese 
non-diabetic adults, to determine if LC n-3 PUFA decreases adipose CCL2-CCR2 and 
CX3CL1-CX3CR1, alters circulating monocyte and ATM phenotype, and stimulates 
GPR120 signaling and NF attenuation. We will explore the relationship between the oral 
microbiome and metabolic and inflammatory parameters at baseline, as well as the effect 
of LC n-3 PUFA supplementation on the oral microbiome composition. 


 
 
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We have chosen Lovaza at 4 grams/day (1860 mg EPA and 1500 mg DHA) for 8 weeks 
based upon anti-inflammatory effects noted in our preliminary dataA relatively young and 
healthy population was chosen to remove risk of chronic disease and medication use, 
yet subjects will be obese to enrich for low grade chronic adipose and systemic 
inflammation where LC n-3 PUFA are expected to have greater impact.  Our previous 
work shows elevated CX3CL1 protein levels in obese adipose15, while elevated CCL2 
and macrophage infiltration with obesity is established13-15,58.  
 
 
3.0 STUDY OBJECTIVES 
Hypotheses   
Primary 
1. Omega-3 fatty acid supplementation for eight weeks reduces systemic 
chemokine signaling (plasma levels of CX3CL1).     
Secondary 
1. Omega-3 fatty acid supplementation will attenuate systemic and adipose tissue 
inflammation and leukocyte activation.   
a. Additional circulating inflammatory chemokines and cytokines levels e.g., 
IL-6, MCP-1, CCL5, TNFalpha 
b. Circulating monocyte subpopulations (ratio of inflammatory 
CX3CR1lowCCR2+ to less inflammatory CX3CR1hiCCR2-) 
c. Protein levels and mRNA expression of adipose tissue inflammatory 
chemokines and cytokines (CX3CL1, MCP-1, IL6, TNFalpha, CCL5) 
d. Adipose tissue macrophage phenotype (ratio of inflammatory M1 to 
regulatory M2 macrophages, relative T-cell quantification) 
2. Changes in chemokine signaling will correlate with improvements in metabolic 
parameters  
a. Decreases in plasma lipids 
b. Decreases in fasting glucose, insulin and homeostasis model assessment 
for IR (HOMA-IR), a marker of insulin resistance 
 
Exploratory 
1. Omega-3 fatty acid supplementation will decrease indirect markers of GPR120 
and NF action. Adipose protein levels of I and total/phospo-TAK1 will be 
assessed as well as GPR120 mRNA expression and protein levels will also be 
quantified, 


 
 
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2. Any effects seen on GPR120 and NF action will be correlated to serum and 
urine DHA and EPA levels. 
3. Omega-3 fatty acid supplementation will cause changes in oral microbiome 
(bacterial DNA composition). 
4. Baseline composition and treatment-related change in oral microbiome 
composition will be correlated to dietary history (as recorded in dietary recall and 
food frequency questionnaire) as well as to metabolic parameters  
5. Subject’s genotype at GPR120 and other loci involved in omega-3 action, 
inflammatory signaling, and nutrient sensing will affect response to treatment.  
Objectives:  Compared to placebo, we will examine the effect of omega-3 fish oil 
supplementation in obese subjects on:  
Primary:  
1) Plasma levels of CX3CL1   
 
Secondary:  
2) Additional plasma cytokines and chemokines 
3) Circulating monocyte subpopulations 
4) Adipose tissue cytokine and chemokine levels 
5)Adipose tissue macrophage subpopulations 
6)Plasma lipids, insulin, glucose, and HOMA-IR 
 
Exploratory:  
7) Adipose protein levels of I and total/phospo-TAK1  
8) GPR120 mRNA expression and protein levels 
9) serum and urine DHA and EPA levels 
10) Oral microbiome bacterial DNA composition 
11) GPR120 genotype 
 
4.0  STUDY DESIGN AND RATIONALE  
We propose to conduct a parallel arm, double blind, placebo-controlled trial to 
understand the anti-inflammatory and metabolic effects of marine derived omega-3 fatty 
acids (EPA + DHA) on obesity-related inflammation and chemokine signaling in humans. 
Obese (BMI>30 kg/m2) but non-diabetic young males and females (n~44; 25 to 50 years 
of age) taking no omega-3 fatty acid (EPA + DHA) supplements and consuming a low 
fish diet (defined as usual intake of high omega-3 fish (tuna and other non-fried fish) < 3 
to 4 servings per month) will be included. A low habitual fish intake is important to control 
EPA + DHA intake during the supplement period (see exclusion criteria below). Subjects 
also will be required to maintain their current fish intake throughout the treatment period.  
 
There will be two treatment groups; (1) placebo and (2) omega-3 fatty acids 4 
grams/day. The trial will be randomized, double blind, placebo-controlled, with parallel 
treatment groups; treatment with omega-3 fatty acids 8 weeks. We will enroll participants 
until at least 22 subjects per study-arm complete all study visits (i.e., at least 44 


 
 
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participants).  
 
4.1 Drug, Dose Selection and Duration 
Prescription Lovaza (omega-3-acid ethyl esters) from GlaxoSmithKline at 4 grams day (2 
1-gram capsules twice daily, with food) will be used. We expect this dose to be safe 
because it is a dose that is recommended, and routinely used, for management of 
moderate or worse hypertriglyceridemia (levels > 400 mg/dL). We will use matching 
placebo capsules so that all participants are taking 2 capsules twice daily – with food as 
recommended in clinical practice.  
The study duration was chosen in order to provide adequate time for potential anti-
inflammatory effects of treatments without requiring maximum lipid modifying effects 
while also reducing the potential for drop-out and loss to follow up that is associated with 
longer study durations.  
5.0 
SUBJECT POPULATION 
5.1  Number of Subjects 
We will enroll 22 subjects per arm for a total of 44 subjects completing. Based on prior 
experience with similar studies we anticipate a drop-out/failure to adhere rate of ≤20%. 
Therefore, we estimate that we will randomize ~52 (~26 per arm) subjects to reach our 
desired sample size.  
5.2  Source of Subjects 
Participants will be recruited from the Preventive Cardiology Research databases, 
comprised of healthy subjects who have participated in previous research studies and 
who have asked to be contacted for future studies. Potential participants will also be 
recruited via local IRB-approved flyers, internet, and newspaper advertisements (see 
flyer and advertisement attached) targeting the Delaware Valley region. 
5.3  Inclusion Criteria 
To be eligible for enrollment in this study, participants must meet all of the following 
criteria: 
 
1. Men and non-pregnant/lactating women between the ages of 18 and 50.  
2.   BMI ≥30 kg/m2 
3.  Participants who are able to give written informed consent and willing to comply 
with all study-related procedures. 
 


 
 
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5.4  Exclusion criteria 
1. Diabetes Mellitus (glucose fasting >126, or random >200, ,or use of any anti-
diabetic agent) 
2. Self-reported fish or shellfish allergy  
3. Planned usage of any prescription or non-prescription anti-inflammatory, 
antibiotic, glucose-lowering, lipid modulating or otherwise relevant medication 
during the study period.  
4. Recent (within 6 months) use of LC n-3 PUFA or fish oil supplements or self-
reported dietary intake of >3 servings of fish/month 
5. Blood pressure >140/90 
6. Recent (within 6 months) use of statins, niacin, or fenofibrates 
7. Current or planned pregnancy/lactation.  Pre-menopausal women unwilling to 
prevent pregnancy by use of the following approved contraceptive strategies: 
diaphragm, cervical cap, condom with spermicide, surgical sterility, birth control 
pills, Depo-Provera injection, IUD, progestin implant, or abstinence. 
 
8. History of liver disease or abnormal LFTs (AST, ALT, Alk. Phos; bilirubin > 2x 
ULN) at Screening Visit 
9. Men who are unwilling to limit alcohol consumption to < 14 alcoholic drinks per 
week or < 4 alcoholic drinks per occasion (AMA / NIAAA criteria for “at risk” 
usage levels) while participating in the study 
10. Women who are unwilling to limit alcohol consumption to < 7 alcoholic drinks per 
week or < 3 alcoholic drinks per occasion (AMA / NIAAA criteria for “at risk” 
usage levels) while participating in the study.  
11. Hemoglobin less than 11.0 g/dL  
12. Any reported arrhythmia, usage of anti-arrhythmic therapy, or abnormal 
screening electrocardiogram 
13. Known bleeding disorder or coagulopathy 
14. Any major active rheumatologic, pulmonary, hematologic or dermatologic 
disease or inflammatory condition or minor active infection 
15. Self-reported history of HIV positive 
16. Patients who have undergone any organ transplant 


 
 
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17. Individuals who currently use tobacco products or have done so in the previous 
30 days 
18. Treatment with aspirin, NSAIDs, COX-2 inhibitors, steroids or any 
immunomodulatory therapy 2 weeks prior to the Screening Visit 
19. Participants who are unwilling to eliminate omega-3 fatty acid (EPA + DHA) 
supplements and/or fortified food, or have their usual intake of high omega-3 fish 
(tuna and other non-fried fish) be > 3 to 4 servings per month as assessed by a 
simple screening questionnaire 
20. Recent (within 6 months) treatment with coumarin-type anticoagulants  
21. Positive urine pregnancy test result. 
22. Self-reported history of injected recreational drug use. 
23. Any medical condition or abnormal laboratory value that is judged clinically 
significant by an investigator    
 
6.0  STUDY PROCEDURES 
 
6.1  Informed Consent 
Before study initiation, the clinical protocol, the Informed Consent Form (ICF), and any 
advertisements for subject recruitment will be submitted for review and approval to the 
Institutional Review Board (IRB).  The investigator, or his designated research staff, will 
obtain written informed consent from each subject enrolled in the study, in accordance 
with the U.S. Food and Drug Administration (FDA) regulations 21 CFR parts 50.20-
50.27.  It is the responsibility of the investigator to ensure that informed consent is 
obtained from the subject or his/her guardian or legal representative before any activity 
or treatment is undertaken which is not part of routine care. 
6.2  Subject Identification Code 
All subjects who sign an ICF will be assigned a subject code.  The code assigned will not 
contain any identifying information. The subject code will be used to identify the subject for 
the rest of the study. 
 
6.3  Safety Assessments 
Safety will be evaluated by the incidence, severity, and relationship to treatment agent of 
adverse events.   
6.3.1  Monitoring for Adverse Events:  Details of all AEs that occur after the first dose of 
study drug will be collected as indicated in Section 12. Any illness or injury that occurs 


 
 
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before the first dose of a study drug will be recorded under Medical History and evaluated 
to determine if the occurrence affects the patient’s eligibility to participate in the study.   
Subjects will be asked the following standard questions by the designated clinical evaluator: 
At clinic check-ins: 
1. “Have you had any medical problems since your last visit?” 
2. “Have any medical problems present at your last visit changed, i.e., stopped, 
worsened, or improved?” 
3. “Have you taken any medicines, other than study drug, since your last visit?” 
Any spontaneous AE information provided by the patient will be recorded. 
NOTE: An AE may be a new disease(s), any untoward event(s), or an exacerbation of a 
pre-existing condition.  See Section 12.0 for complete details. 
6.4   Adipose biopsy:   
Approximately 500-1000 mg of subcutaneous adipose tissue will be biopsied from the 
gluteal region, processed, weighed and frozen until analyzed.  Adipose tissue will be 
obtained through a small (~5mm) incision under local anesthesia (2% Lidocaine without 
epinephrine), as has been previously described13-15.  A portion (200 mg) of the samples 
will be flash frozen and analyzed for gene expression and protein assays. Another 
portion (500-800 mg) will be placed in sterile media and undergo digestion with 
collagenase, filtration, and centrifugation for separation of the stromal vascular fraction 
(SVF).  SVF cells will be stained with antibodies for flow cytometer analysis. Finally, 
remaining tissue will be fixed in paraffin for immunohistochemistry.  
6.5 Subgingival analysis 
 
Saliva samples will be collected using the OMNIgene DISCOVER (OM-505) kit (DNA 
Genotek), an all-in-one kit which collects and stabilizes both microbial DNA and RNA in 
saliva. Samples will be stored at -80C prior to extraction. Nucleic acids will be extracted 
using the QIAGEN QIAamp MinElute Virus Spin Kit (Qiagen). Subgingival plaque 
samples will be obtained from 3 oral cavity regions in each subject (2 samples per 
region) using a soft-bristled microbrush (DenTek). Specimens will be collected from the 
same anatomical location in all subjects. DNA will be isolated using the PSP Spin Stool 
DNA kit (Invitec). DNA samples will be amplified using V1-V2 region primers targeting 
bacterial 16S genes and sequenced using IlluminaHiSeq or 454 Pyrosequencing 
technology. 
 
6.6  Specific Study Procedures (by visit) 
A detailed study time-table and list of all procedures to be performed at each visit is 
included in Appendix A. 
6.6.1  Screening (Visit 1) 
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research coordinator (see Appendix B for email interview form; for telephone interviews 
this information will be verbally exchanged).  Participants who meet initial study 
requirements will be invited for a screening visit (visit 1) at the CTRC.  Prior to this visit, 
participants will receive a detailed health questionnaire (see Appendix C) that has been 
used in numerous previous studies that addresses questions regarding past medical 
history, current use of medications and dietary supplements, exercise and social habits.  
In addition, participants will be asked about fish consumption in order to ensure 
recruitment of subjects with lower EPA + DHA intake during the study period. 
Participants will be asked to complete these questionnaires at home and bring the 
completed forms with them to their screening visit.  During the screening visit, they will 
meet with research staff to review and sign an IRB-approved consent form.  Research 
staff will review the health questionnaire with the participant as well as measure weight, 
height, waist and hip circumference, sitting blood pressure and heart rate.  A brief 
physical exam will be performed as well as an electrocardiogram (ECG). In addition, 
participants will have blood drawn (after a 12 hour fast) for evaluating fasting lipids, 
glucose, serum chemistries and liver function and a complete blood count.  Female 
participants will also be asked to provide a urine sample for a pregnancy test.  These 
laboratory parameters are included to assure the participant meets eligibility criteria and 
that he/she is generally in good health.   Once all clinical data has been reviewed, study 
personnel will contact each study volunteer to let them know if they are eligible. 
6.5.2  Randomization visit (Visit 2) 
One to four weeks after the screening Visit 1, eligible participants will return to the CTRC 
after a 12 hour fast for a 2 hour visit. Participants will be asked to brush their teeth after 
their last meal the day before the visit, or at the start of the 12-hour fasting period, and to 
refrain from additional brushing or use of mouthwash until after the study visit.  
Participants will meet with a research nurse / coordinator who will assess any change in 
medical history and medication use, as well as review any adverse events.  Blood 
pressure, heart rate, height and weight will be measured, blood and urine will be 
collected for pre-randomization baseline efficacy measures and a urine pregnancy test 
performed for all females.  The simple fish consumption questionnaire will be 
administered to assess any changes to the level of omega-3 intake in the diet.  
They will also be asked to record all food and beverages consumed on dietary records 
on 3 days prior to the visit, as well as completing a digestive health questionnaire and 
diet history questionnaire.  All nutrient data collected from 3-day dietary records will be 
analyzed by Food Processor 8.1 (06/03, Escha).  Participants will be asked to complete 
these questionnaires at home and bring the completed forms with them to their study 
visit.  If a subject is unable to print out or complete the forms at home, they will be 
completed at the visit. 
Saliva and subgingival plaque samples will be taken for assessment of oral microbiome. 
Saliva samples will be collected using a standardized kit. Under the supervision of study 
personnel, subjects will be given the kit, which consists of a funnel attached to a tube, 
and instructed to spit into the funnel until the amount of liquid reaches a fill line located 
on the outside of the attached tube.  Once the proper amount has been collected, the lid 
of the funnel will be closed and the funnel removed from the tube.  The tube is then 


 
 
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capped and shaken for about 10 seconds.  Subgingival plaque samples will be collected 
by swabbing several regions of the buccal mucosa using microbrushes. A separate 
brush will be used for each swab and two samples will be collected from each region.  
In order to establish effects of treatments on adipose inflammatory parameters, we will 
obtain gluteal region subcutaneous adipose tissue biopsies under local anesthesia (2% 
Lidocaine).  A detailed description of the adipose collection procedures is given in 
Section 6.4.  Participants will then be randomized to drug or placebo group. Supplies of 
study drug will be provided to last 8 weeks (4 pills/day of either Lovaza or placebo).  
Participants will be provided verbal and written instruction to take the study drugs as 
follows; 2 fish oil/placebo with food in the morning, 2 fish oil/placebo with food in the 
evening. A pill diary will be given to all subjects. 
6.5.3 Telephone / E-mail Follow up (Visit 3)  
Participants will be contacted by telephone or e-mail approximately four weeks (visit 3) 
post-randomization to review medication compliance and pill diary, adverse events and 
any changes in fish consumption. Participants with <80% apparent adherence to study 
medication will be counseled to increase compliance so that their adherence at 8 weeks 
will be ≥80%.  
6.5.4 Completion Visit (Visit 4) 
Participants will come to the CTRC after a 12 hour fast. Participants will meet with a 
study coordinator who will assess changes to the medical history and medication use. 
They will also be asked to record all food and beverages consumed on dietary records 
on 3 days prior to the visit. Any adverse events will be recorded, pill diary reviewed, and 
study medications will be counted (pill count) Weight and height will be recorded and 
BMI calculated. ECG will be performed. Blood will be drawn for post-drug screening of 
liver functions and chemistries as well as for all outcome-related measures (lipids, 
insulin/glucose, inflammatory markers). Urine for efficacy measures will also be 
collected. Urine pregnancy test will be performed in all females. Saliva and subgingival 
plaque samples will be taken to assess for any change in oral microbiome by the 
treatment. An adipose biopsy will be performed as per Section 6.4.  
6.6  Study Discontinuation/Completion 
 
A subject may be withdrawn from the study at any time at either the investigator’s 
discretion or the subject’s request.  An effort must be made to document the reason(s) 
why a subject fails to return to the study clinic for necessary visits or is discontinued from 
the study.  The primary reason for discontinuing participation in the study must be stated 
and may include, but is not limited to, one of the following: 
 Development of any abnormality in blood counts, blood chemistries, or urinalysis 
because of which the investigator deems continued participation in the study to be ill-
advised 
 Use of unapproved concomitant medications 


 
 
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 Occurrence of intolerable AEs 
 Development of LFT abnormalities (>1.5 ULN), unexplained muscle 
aches/myopathy, kidney dysfunction (Cr>ULN), or significant bleeding (e.g. GI bleed, 
cerebral hemorrhage, major ENT bleed).  
 Withdrawal of consent by patient 
 Noncompliance with protocol, e.g., the patient fails to appear at one or more visits 
 Non adherence with study medications – defined as failure to take >80% of study 
medications (pill count and diaries)  
 Development of an intercurrent illness, injury, or medical condition likely to interfere 
with patient safety, the overall assessment, or the required administration of study 
medication 
 Development of any condition for which the investigator feels treatment withdrawal is 
justified 
 Termination of the study by the sponsor (NIH) 
The procedure to discontinue a subject from the study is as follows: 
 record any AEs since previous visit 
 record any medications  
 record vital signs 
 perform a physical examination (if discontinuation is due to an AE) 
 collect blood for clinical laboratory analysis  
Patients who discontinue from the study because of an AE(s) should be treated and 
followed according to established medical practice. 
The study may also be discontinued at the discretion of the Principal Investigator, if any 
safety concerns are identified during the course of the study. 
7.0  SAFETY MEASURES  
 
A detailed description of all safety related issues including the risks of study, participant 
monitoring, adverse event data collection and reporting is provided in a formal Safety 
Plan (Appendix D) developed also for our Data Safety and Monitoring Board (DSMB). 
Therefore, in this section we briefly describe the safety measures that we will record and 
measure during the execution of the study. Additional safety-related details are provided 
in the Safety Plan. 
 
7.1 Lovaza-study related safety measures  
The most common reported side effects with Lovaza are burping, infection, flu 
symptoms, upset stomach, change in sense of taste, back pain and skin rash.  We will 
monitor for development of these side effects at follow-up Visit 3 and Visit 4 and 
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occur or become bothersome.  There is a risk of an increase in alanine aminotransferase 
(ALT) and/or low-density lipoprotein cholesterol (LDL-C) levels; therefore we will be 
monitoring participants’ ALT and LDC-C levels at Visit 2 (baseline) and at study 
completion. There is a possible association between Lovaza and more frequent 
recurrences of symptomatic atrial fibrillation or flutter in patients with paroxysmal or 
persistent atrial fibrillation. Thus there will be an ECG at visit 1  and subjects with 
arrhythmias will be excluded. ECG will also be done at end of study to assess of any 
potential induction of arrhythmias by use of study drug. Subjects will be discontinued as 
described above in Section 6.6. 
 
If any of the above responses occur, the specific AE will be assessed with the 
circumstance of the occurrence, and discussed with the DSMB, NIH, and University of 
Pennsylvania IRB in order to outline a response / plan that accounts for this occurrence.  
Once the AE has been assessed, dosing will resume according to protocol. 
 
8.0 
DATA COLLECTION AND ANALYSIS 
8.1 Source Documents & Case Report Forms 
All primary primary records, such as original copies of signed consent forms, will be 
stored in a locked filing cabinet and will only be available to study investigators. The 
documents will be stored in files labeled only with a unique research identification 
number. Electronic copies of these documents will be created and stored within the 
REDCap database. These primary records will not be available to any future recipients 
of registry-repository data without separate IRB approval. 
8.2 Computer Systems 
All electronic data will be managed and stored using the research-focused electronic 
data capture system REDCap, under an agreement with the software’s development 
consortium, led by Vanderbilt University. REDCap supports two secure, web-based 
applications designed exclusively to support data capture for research studies. REDCap 
is a PHP web application served by Apache Tomcat over a 128 bit SSL connection using 
a signed certificate. The application relies on a study-specific data dictionary defined in 
an iterative self-documenting process that will be conducted by all members of the 
research team. The data dictionary is the foundation for custom case report form design 
and validated coding of variables. Authentication of research staff will be performed via 
LDAP using CHOP’s enterprise Active Directory service. The application generates a 
complete audit trail of user activity, provides reporting, and has an automated export 
mechanism to common statistical packages (SAS, SPSS, Stata, R/S-Plus).  
The REDCap MySQL database is replicated in real time to a completely redundant 
instance of MySQL.  The redundant instance is available for restoration of the primary 
database or for manual failover in the case of primary database failure.  Time-stamped 
backup files are made from the replicated database daily by CHOP Research Information 
Systems using automated backup routines.  Backup files are encrypted and transferred to 
a secure file server accessible only to designated personnel.  A rolling seven-day window 


 
 
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of backup files is maintained in an immediately available online state, with a larger 
window maintained in a compressed file archive available at a reduced speed of access. 
Daily destructive database backup files are stored on the database server and are deleted 
only after successful backup of the entire database to file. In the event of data error, loss 
or corruption, Research personnel will work with CHOP Research Information Systems 
to determine the most appropriate recovery strategy.   
 
Data and backups are stored in the CHOP Research Information Systems Storage Area 
Network (SAN).  Access to the SAN directories where data are stored will be limited to 
Research Information Systems personnel, with authentication performed using CHOP’s 
enterprise Active Directory service.  
 
REDCap’s extensive security features will be taken advantage of to limit PHI, and any 
links between this PHI and all other data, to only the study investigators listed on this 
IRB protocol. All other users, particularly future recipients of data output from the 
registry-repository, will only have access to data tagged with a unique research ID 
number.  
9.0  
STATISTICAL METHODS: 
9.1  Sample size and Power computation  
Sample size is based on the primary goal of detecting a 26% reduction in plasma 
CX3CL1 levels in drug treated participants compared to placebo.  We believe that such 
a difference in CX3CL1 levels represents a clinically important difference with biological 
implications in setting of obesity, metabolic syndrome and type 2 diabetes.  We have 
chosen TNFα as the primary response variable for the following reasons; (1xxx 
The calculations were performed using our data establishing expected CX3CL1 levels in 
a healthy sample (0.58 +/- 0.17 ng/mL in recent FFAME cohort; n=80). Using a two-
sample Wilcoxon test, for 80% power and =0.05, 22 subjects per group will allow 
detection of an effect size of 0.9σ difference in plasma CX3CL1 levels between Lovaza 
and placebo (0.14 ng/mL difference—26%, using mean/SD above). Allowing for 20% 
dropout, 26 subjects per group will be required. 
9.2  Statistical Analyses  
Analysis will be performed by Dr Shah with support from Penn Statistics faculty with 
expertise in human translational research (Dr. Propert). In the human trial, the primary 
endpoint will be compared between groups using two-sample Wilcoxon test, with 
graphical explorations and repeated measured ANOVA to explore the shape of changes 
over time. Correlations between circulating and adipose CX3CL1/CCL2 and 
monocyte/macrophage markers, systemic inflammatory markers and metabolic 
parameters will utilize Spearman’s correlation. Differences among conditions in cell 
studies will be analyzed by ANOVA. All analysis will use STATA 11 (StataCorp).  


 
 
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All secondary/exploratory variables will be assessed as independent responses because 
they may reflect independent effects of the treatment. The multiple testing of non 
independent responses may result in type I error and false positive findings. Therefore, 
any statistically significant differences will be interpreted in terms of the overall 
significance level, considering multiple comparisons, and ultimately in the context of the 
clinical significance of the difference. 
10.0   STUDY AGENTS 
10.1  Preparation and Labeling 
The Investigational Drug Service (IDS) pharmacist will ensure that all study drugs are 
stored in a secured area, under recommended storage conditions (below 86 F and 
protected from moisture and freezing) in accordance with applicable regulatory 
requirements and will be dispensed by qualified staff members.  The pharmacist will 
maintain accurate records regarding study drug administration and return. 
 
10.2 Omega-3 fish oil formulation 
Prescription grade omega-3 fish oil supplementation will be used in the form of oral 
Lovaza (omega-3-acid ethyl esters) manufactured by GlaxoSmithKline.  Subjects will be 
randomized to a dosage of either 1 or 4 capsules daily. We will use matching  placebo 
capsules provided by the manufacturer so that all participants are taking 2 capsules 
twice daily – with food as recommended in clinical practice.  
10.3  Study Agent Compliance 
Study drug adherence will be monitored by pill count and medication diary and will be 
assessed by telephone/email contact at 4 weeks after randomization and in person at Visit 4 
(8 weeks after randomization) . The final analysis of compliance will be performed at this 
time. 
 
 
11.0  
POTENTIAL RISKS 
 
A complete description of all study related risks is also provided in the Safety Plan 
(Appendix D). Briefly, risks include (1) general study procedure-related risks (e.g., blood 
draws, intravenous lines, adipose biopsies), (2) Lovaza related risks [e.g., increase in 
alanine aminotransferase (ALT) and/or low-density lipoprotein cholesterol (LDL-C) 
levels], and (3) risks from clinical or laboratory abnormalities that emerge during the 
study.  The process of monitoring the study is outlined in the Safety Plan for proposed 
Data Safety and Monitoring Board (DSMB) (Appendix D). Additional safety-related 
details are provided in the Safety Plan  
 
12.0   POTENTIAL BENEFIT OF THE PROPOSED RESEARCH 
 
There is no direct benefit to the participant of this study; however data obtained from this 
study may be very useful in understanding the role of omega-3 fatty acids on obesity-
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temporary improvement in triglyceride levels while in the study. Clinical data relating to 
general health and risk for metabolic disease (diabetes, cholesterol) will be provided to 
them and their primary physician if they so wish. 
 
13.0   ADVERSE EVENTS 
 
A complete description of all study related risks, adverse events, adverse event 
definitions, recording and reporting is provided in the Safety Plan (Appendix D for 
proposed Data Safety and Monitoring Board (DSMB). Key aspects are summarized 
below. 
13.1 Non-serious Adverse Events 
All other observed or volunteered adverse events regardless of treatment group or 
suspected causal relationship to study drug will be recorded on the adverse event 
source document and transcribed onto the case report form.   Events involving adverse 
drug reactions, illnesses with onset during the study, or exacerbations of pre-existing 
illnesses will be recorded.  In addition, abnormal objective test findings (e.g., 
electrocardiogram changes, abnormal laboratory test results) that result in a change in 
study drug dosage or in discontinuation of the drug, or require intervention or diagnostic 
evaluation to assess the risk to the patient/participant, will be recorded as adverse 
events.  Clinically significant changes in physical examination findings will also be 
recorded as adverse events. 
Thus, any AE that is not designated as Serious, but is not expected as defined in 
appendix D, must be recorded on the non-serious AE page of the CRF.  
13.2  Serious Adverse Event 
 
All serious adverse events (as defined below) regardless of treatment group or 
suspected relationship to study drug will be reported to the DSMB, NIH, FDA and 
University of Pennsylvania IRB as outlined by the Penn Manual for Clinical Research 
(Office of Human Research). 
 
A serious adverse event (SAE) is any AE that: 
 Results in death 
 Is life-threatening (at immediate risk of death from the event as it occurred) 
 Requires inpatient hospitalization (overnight stay) or prolongs a current 
hospitalization 
 Causes a persistent or significant disability/incapacity 
 Is a congenital anomaly/birth defect in the offspring of a patient who received test 
article 
And may be any event that: 
 Requires intervention to prevent one of the outcomes listed above 


 
 
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The investigator should exercise medical and scientific judgment when deciding whether 
expedited reporting is appropriate in other situations not strictly meeting the listed criteria 
above.   
13.3  Intensity   
The investigator is responsible for ensuring that AEs are documented on the appropriate 
page of the CRF according to the following descriptors: 
Mild: associated with no limitation of usual activities or only slight discomfort 
Moderate: associated with limitation of usual activities or significant discomfort 
Severe: associated with inability to carry out usual activities or very marked discomfort 
 
13.4  Relationship to Test Article 
The relationship of AEs to study drug will be assigned by the investigator according to 
the following definitions: 
Definitely:  a reaction that follows a reasonable temporal sequence from administration 
of the test article; that follows a known or expected response pattern to the suspected 
study drug; and that cannot be reasonably explained by the known characteristics of that 
subject/patient’s clinical state. 
Probable:  a reaction that follows a reasonable temporal sequence from administration of 
the test article; that follows a known or expected response pattern to the suspected 
study drug; and that is unlikely explained by the known characteristics of that 
subject/patient’s clinical state. 
Possible:  a reaction that follows a reasonable temporal sequence from administration of 
the test article; that follows a known or expected response pattern to the suspected 
study drug; but that could readily have been produced by a number of other factors. 
Unlikely:  a reaction that does not follow a reasonable temporal sequence from 
administration of the test article.  However, causality from the test article cannot be ruled 
out. 
None:  a reaction for which sufficient data exist to indicate that the etiology is unrelated 
to the study drug. 
13.5  Pretreatment-Emergent Events  


 
 
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Events that occur between the time the informed consent document is signed for the 
study and the time when the patient is first exposed to study drug as “pretreatment-
emergent” events.  These pretreatment-emergent events will not be collected as adverse 
events, unless the event increases in intensity or frequency after study article 
administration. 
13.6  Recording and Documenting AEs and SAEs 
 
The investigator or study team member must completely and promptly record each AE in 
the database on the AE record, even if the relationship of an AE to the study drug is 
assessed by the investigator to be “unlikely” or “none”.  The investigator should attempt, 
if possible, to establish a diagnosis based on the presenting signs and symptoms.   
 
14.0  INVESTIGATOR OBLIGATIONS 
This section reviews each investigator’s obligations according to federal regulations. 
14.1 Adverse Event Reporting Obligations 
 
The investigator is responsible for recording AE’s reported by the subject or discovered 
by any other means during the study.  In addition, AE’s classified as “serious” must be 
reported to the DSMB, NIH, and University of Pennsylvania IRB as outlined by the Penn 
Manual for Clinical Research (Office of Human Research). 
14.2  Study Records and Source Documents  
 
Federal regulations and The University of Pennsylvania policy require that, following 
completion of a clinical study, a copy of all records of that study be maintained by the 
Principal Investigator.  Completed original CRFs and clarification queries, dated, and 
signed by the investigator, will be kept at the study site beyond locked door and key.  
Hard copies of data forms will also be kept on file at The University of Pennsylvania or 
its designee.   
In order to assure the accuracy of data collected in the CRFs, it is mandatory that The 
University of Pennsylvania or its designees, as well as representatives of the FDA or 
other pertinent regulatory agencies, have access to original source documents (e.g., 
patient records, patient charts, laboratory reports).  During the review of these 
documents, the anonymity of the patient will be respected with strict adherence to 
professional standards of confidentiality. 
The University of Pennsylvania reserves the right to terminate the study for refusal of the 
investigator to supply source documentation of work performed in this clinical study. 
 


 
 
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15.0 
REFERENCES 
 
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Thromb 2012;19:194-204. 
22. 
Saito Y, Yokoyama M, Origasa H, et al. Effects of EPA on coronary artery 
disease in hypercholesterolemic patients with multiple risk factors: sub-analysis 
of primary prevention cases from the Japan EPA Lipid Intervention Study (JELIS). 
Atherosclerosis 2008;200:135-40. 
23. 
Oikawa S, Yokoyama M, Origasa H, et al. Suppressive effect of EPA on the 
incidence of coronary events in hypercholesterolemia with impaired glucose 
metabolism: Sub-analysis of the Japan EPA Lipid Intervention Study (JELIS). 
Atherosclerosis 2009;206:535-9. 
24. 
Marchioli R, Schweiger C, Tavazzi L, Valagussa F. Efficacy of n-3 
polyunsaturated fatty acids after myocardial infarction: results of GISSI-
Prevenzione trial. Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto 


 
 
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Miocardico. Lipids 2001;36 Suppl:S119-26. 
25. 
Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E 
after myocardial infarction: results of the GISSI-Prevenzione trial. Gruppo Italiano 
per lo Studio della Sopravvivenza nell'Infarto miocardico. Lancet 1999;354:447-55. 
26. 
Bosch J, Gerstein HC, Dagenais GR, et al. n-3 fatty acids and 
cardiovascular outcomes in patients with dysglycemia. N Engl J Med;367:309-18. 
27. 
Calder PC, Yaqoob P. Marine omega-3 fatty acids and coronary heart 
disease. Curr Opin Cardiol;27:412-9. 
28. 
Calder PC. Omega-3 polyunsaturated fatty acids and inflammatory 
processes: Nutrition or pharmacology? Br J Clin Pharmacol. 
29. 
Mayser P, Mrowietz U, Arenberger P, et al. Omega-3 fatty acid-based lipid 
infusion in patients with chronic plaque psoriasis: results of a double-blind, 
randomized, placebo-controlled, multicenter trial. J Am Acad Dermatol 
1998;38:539-47. 
30. 
Geusens P, Wouters C, Nijs J, Jiang Y, Dequeker J. Long-term effect of 
omega-3 fatty acid supplementation in active rheumatoid arthritis. A 12-month, 
double-blind, controlled study. Arthritis Rheum 1994;37:824-9. 
31. 
Broughton KS, Johnson CS, Pace BK, Liebman M, Kleppinger KM. Reduced 
asthma symptoms with n-3 fatty acid ingestion are related to 5-series leukotriene 
production. Am J Clin Nutr 1997;65:1011-7. 
32. 
Belluzzi A, Brignola C, Campieri M, Pera A, Boschi S, Miglioli M. Effect of an 
enteric-coated fish-oil preparation on relapses in Crohn's disease. N Engl J Med 
1996;334:1557-60. 
33. 
Sijben JW, Calder PC. Differential immunomodulation with long-chain n-3 
PUFA in health and chronic disease. Proc Nutr Soc 2007;66:237-59. 
34. 
Dangardt F, Osika W, Chen Y, et al. Omega-3 fatty acid supplementation 
improves vascular function and reduces inflammation in obese adolescents. 
Atherosclerosis 2010;212:580-5. 
35. 
Vedin I, Cederholm T, Freund Levi Y, et al. Effects of docosahexaenoic 
acid-rich n-3 fatty acid supplementation on cytokine release from blood 
mononuclear leukocytes: the OmegAD study. The American journal of clinical 
nutrition 2008;87:1616-22. 
36. 
Luu NT, Madden J, Calder PC, et al. Dietary supplementation with fish oil 
modifies the ability of human monocytes to induce an inflammatory response. J 


 
 
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Nutr 2007;137:2769-74. 
37. 
Bouwens M, van de Rest O, Dellschaft N, et al. Fish-oil supplementation 
induces antiinflammatory gene expression profiles in human blood mononuclear 
cells. The American journal of clinical nutrition 2009;90:415-24. 
38. 
Madden J, Shearman CP, Dunn RL, et al. Altered monocyte CD44 
expression in peripheral arterial disease is corrected by fish oil supplementation. 
Nutr Metab Cardiovasc Dis 2009;19:247-52. 
39. 
Mullen A, Loscher CE, Roche HM. Anti-inflammatory effects of EPA and 
DHA are dependent upon time and dose-response elements associated with LPS 
stimulation in THP-1-derived macrophages. J Nutr Biochem 2010;21:444-50. 
40. 
Brown AL, Zhu X, Rong S, et al. Omega-3 fatty acids ameliorate 
atherosclerosis by favorably altering monocyte subsets and limiting monocyte 
recruitment to aortic lesions. Arterioscler Thromb Vasc Biol;32:2122-30. 
41. 
Todoric J, Loffler M, Huber J, et al. Adipose tissue inflammation induced by 
high-fat diet in obese diabetic mice is prevented by n-3 polyunsaturated fatty 
acids. Diabetologia 2006;49:2109-19. 
42. 
Baranowski M, Enns J, Blewett H, Yakandawala U, Zahradka P, Taylor CG. 
Dietary flaxseed oil reduces adipocyte size, adipose monocyte chemoattractant 
protein-1 levels and T-cell infiltration in obese, insulin-resistant rats. 
Cytokine;59:382-91. 
43. 
Titos E, Rius B, Gonzalez-Periz A, et al. Resolvin D1 and its precursor 
docosahexaenoic acid promote resolution of adipose tissue inflammation by 
eliciting macrophage polarization toward an M2-like phenotype. J 
Immunol;187:5408-18. 
44. 
Hensler M, Bardova K, Jilkova ZM, et al. The inhibition of fat cell 
proliferation by n-3 fatty acids in dietary obese mice. Lipids Health Dis;10:128. 
45. 
White PJ, Arita M, Taguchi R, Kang JX, Marette A. Transgenic restoration of 
long-chain n-3 fatty acids in insulin target tissues improves resolution capacity 
and alleviates obesity-linked inflammation and insulin resistance in high-fat-fed 
mice. Diabetes 2010;59:3066-73. 
46. 
Kanarek N, Ben-Neriah Y. Regulation of NF-kappaB by ubiquitination and 
degradation of the IkappaBs. Immunol Rev;246:77-94. 
47. 
Novak TE, Babcock TA, Jho DH, Helton WS, Espat NJ. NF-kappa B 
inhibition by omega -3 fatty acids modulates LPS-stimulated macrophage TNF-
alpha transcription. Am J Physiol Lung Cell Mol Physiol 2003;284:L84-9. 


 
 
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48. 
Draper E, Reynolds CM, Canavan M, Mills KH, Loscher CE, Roche HM. 
Omega-3 fatty acids attenuate dendritic cell function via NF-kappaB independent 
of PPARgamma. J Nutr Biochem;22:784-90. 
49. 
Oh DY, Talukdar S, Bae EJ, et al. GPR120 is an omega-3 fatty acid receptor 
mediating potent anti-inflammatory and insulin-sensitizing effects. Cell;142:687-
98. 
50. 
Oh DY, Olefsky JM. Omega 3 fatty acids and GPR120. Cell Metab;15:564-5. 
51. 
Ichimura A, Hirasawa A, Poulain-Godefroy O, et al. Dysfunction of lipid 
sensor GPR120 leads to obesity in both mouse and human. Nature;483:350-4. 
52. 
Liu D, Wang L, Meng Q, Kuang H, Liu X. G-protein coupled receptor 120 is 
involved in glucose metabolism in fat cells. Cell Mol Biol (Noisy-le-
grand);Suppl.58:OL1757-62. 
53. 
McMurray DN, Bonilla DL, Chapkin RS. n-3 Fatty acids uniquely affect anti-
microbial resistance and immune cell plasma membrane organization. Chem Phys 
Lipids;164:626-35. 
54. 
Mattila KJ, Nieminen MS, Valtonen VV, et al. Association between dental 
health and acute myocardial infarction. BMJ (Clinical research ed 1989;298:779-81. 
55. 
Zeigler CC, Persson GR, Wondimu B, Marcus C, Sobko T, Modeer T. 
Microbiota in the oral subgingival biofilm is associated with obesity in 
adolescence. Obesity 2012;20:157-64. 
56. 
Myles IA, Pincus NB, Fontecilla NM, Datta SK. Effects of parental omega-3 
Fatty Acid intake on offspring microbiome and immunity. PloS one 2014;9:e87181. 
57. 
Tabbaa M, Golubic M, Roizen MF, Bernstein AM. Docosahexaenoic acid, 
inflammation, and bacterial dysbiosis in relation to periodontal disease, 
inflammatory bowel disease, and the metabolic syndrome. Nutrients 2013;5:3299-
310. 
58. 
Shah R, Lu Y, Hinkle CC, et al. Gene profiling of human adipose tissue during 
evoked 
inflammation 
in 
vivo. 
Diabetes 
2009;58:2211-9.


 
 
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APPENDIX A 
 
 
 
Study Visits and Procedures 
 
 


 
 
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Procedure 
Visit 1 
Screen 
Visit 2 
Randomization 
Visit 3 
Contact 
Follow-
up 
Visit 4 
Completion 
Week  
-2 to -4 
Week 0 
Week 4 
Week 7-9 
Participant Questionnaire completion1 
X 
 
 
 
Informed consent 
X 
 
 
 
History & physical 
X 
 
 
 
3 
day 
diet 
record, 
Food 
History 
questionnaire 
 
X 
X 
 
Electrocardiogram 
X 
 
 
X 
Blood pressure, heart rate, weight, 
height & waist measures 
X 
X 
 
X 
Glucose and insulin measurement 
X 
 
 
X 
Urine pregnancy test2 
X 
X 
 
X 
Review medications 
X 
X 
X 
X 
Dispense study medication/placebo pills 
 
X 
 
 
Comprehensive Metabolic Lab Panel 3  
X 
 
 
X 
Complete blood count (CBC) 
X 
 
 
X 
Lipid profile4 
X 
 
 
X 
Blood and urine collected for efficacy 
measures 
 
X 
 
X 
Dietary assessment of high omega-3 fish 
consumption (questionnaire) 
X 
X 
X 
X 
Saliva Collection 
 
X 
 
X 
Adipose biopsy 
 
X 
 
X 


 
 
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1 The questionnaire along with a copy of the consent form will be mailed to subjects for completion prior to the 
study visit in order to save time on the day of their screening visit.  After consent is obtained, a coordinator will 
review the questionnaire with the participant for completeness. 
2 Standard urine pregnancy test performed on females of child-bearing potential only  
3 Comprehensive Metabolic panel includes: sodium, potassium, chloride, carbon dioxide, glucose, blood urea 
nitrogen, creatinine, calcium, total protein, albumin, AST, ALT, alkaline phosphatase, and total bilirubin  
4 Includes TC, LDL-C, VLDL, HDL-C, TG 
 
 


 
 
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APPENDIX B 
 
 
 
Pre-screening E-mail Questionnaire  
 
 
 
 
 
 
 


 
 
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Hello,  
 
Thank you for your interest in the FAIR study.  We are looking for healthy volunteers to participate in a study to 
better understand the anti-inflammatory benefits of prescription fish oil supplements that are currently used to help 
people with cholesterol problems.  If you qualify, your participation would last about 3 months during which time 
there are 3 visits to the Clinical and Translational Research Center (CTRC) at the Hospital of the University of 
Pennsylvania.   
 
Visit 1 is an outpatient screening visit which is about 2 hours long. 
Visit 2 is a baseline visit which lasts about 2 hours 
Visit 3 is a follow-up visit conducted via e-mail and/or telephone (you do not need to come to the CTRC) 
Visit 4 is a completion visit which lasts about 2 hours  
 
Visits may include blood sampling, urine collection, blood pressure measurements, fat-tissue sampling, fasting 
and brief physical exams.   
 
Volunteers who complete all study visits will be compensated $180 for time and travel. 
 
If you would like more information and to see if you may qualify, please answer the questions below and reply 
back to me.  A member of our study team will be in touch with you as soon as possible. 
 
1) 
Name:   
2) 
Gender:   
3) 
Phone Number:  
4) 
Email:  
5) 
Age: 
6) 
How did you hear about the study?   
7) 
Do you have a history of diabetes (type 1 or type 2) or are you on any medications to lower your blood   
         sugar? 
 


 
 
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32 
8) 
Do you have any history of abnormal heart rhythm or other heart problems? 
1.  If yes, what are they? 
 
9) 
Do you have any other medical conditions for which you are under a doctor’s care? 
1. If yes, what are they? 
10) 
What is your height?  
11) 
What is your weight? 
12) 
How often do you eat-- as a main course-- tuna or other non-fried fish?  (One serving size is 3 oz = in size 
 
to a deck of cards. Note: One serving of non-fried fish could include: 1, 3 oz. filet of salmon OR 6 pieces of 
 
sushi OR 8 sushi rolls OR ~4 tuna fish (salad) sandwiches, etc.) 
 
 
A) Once or less per month  
 
 
B) 2-3 times per month  
 
 
C) Once per week  
 
 
D) Twice per week  
 
E) More than twice per week  
13) 
Have you taken any fish oil supplements in the past 3 months?  
 
14) 
What medicines, including supplements (like Omega-3 or fish oil) and vitamins, do you currently take and 
how often do you take them?  
1.  
 
 
 
 
 
2.  
3.  
4.  
5.  
 
 
 
 
 
15) 
Do you have any history of gall bladder disease? 
16) 
Do you have any allergies to fish? 
17) 
Do you currently smoke or use any other tobacco product (snuff, chew, dip, etc.)? 
1. If no, have you used tobacco in the last 30 days? 
18) 
On average, how many days per week do you drink alcohol? 
19) 
On a day when you drink alcohol, how many alcoholic drinks do you have? 
20) 
When you drink alcohol, is it beer, wine or liquor? 
21) 
How many ounces are in each drink?  
22) 
Do you have high blood pressure?   
 
 
 
23) 
Do you have a history in your immediate family of pre-mature heart disease or stroke (i.e., mother/sister 
before age 65 or father/brother before age 55)? 
1. If yes, who and at what age?   
 
 
24) 
Have you been treated for cancer? 
1. If yes, are you still being treated?   
i. If you are not still being treated, when was your last treatment?   
25) 
Are you HIV + or do you have AIDS? 
 
26) 
Females only:  Are you currently pregnant or breast-feeding? 


 
 
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33 
27) 
Are you currently enrolled in another research study? 
1. If yes, when will your participation end? 
 
28) 
Have you participated in a clinical research study within the last 6 weeks?  
 
29) 
In terms of race, do you consider yourself a Caucasian, African American, Asian, Pacific Islander, 
 
Hawaiian Native, American Indian or Alaskan Native?   
 
I look forward to your reply and thank you for your time. 
 
Sincerely, 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
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34 
 
 
 
 
 
 
 
APPENDIX C 
 
 
Health Screening Questionnaire  


 
 
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35 
 
 
 
 
 
Screening Health Questionnaire 
 
Personal Information:  
 
 
 
 
 
 
 
 
 
 
 
Name:______________________________________ 
Home 
phone:______________________________ 
Address:____________________________________ 
Work 
phone: 
(optional):______________________ 
City, State, Zip:_______________________________ 
Cell phone (optional):  _______________________ 
Occupation:__________________________________
 
Email:____________________________________ 
 
 
 
 
 
 
 
 
Fax 
#: 
____________________________________ 
 
Contact 
Person:______________________________
 
Phone:____________________________________ 
 
Physicians:  
 


 
 
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36 
1) Primary MD :____________________________ 
 
Address:__________________________________ 
 
 
 
 
 
 
 
     
_________________________________________ 
 
Phone:_________________________________ 
 
Fax #:    __________________________________ 
 
2) Other MD:_____________________________ 
 
Address:__________________________________ 
 
Specialty: ____________________________ 
 
_________________________________________ 
 
Phone:_______________________________   
 
Fax #: ____________________________________ 
 
 
 
Are you currently enrolled or have you participated in any other research studies within the past 6 weeks? 
 
Yes / No 
If yes, what date did the most recent study end?  ______/______/______ 
 
 
 


 
 
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37 
Demographics: 
Gender:   
 M / F   
 
In terms of ethnicity, do you consider yourself Hispanic or Latino?  Yes/No 
 
                                                                                  In terms of race, do you consider yourself: 
Date of Birth:_____________  
 
1-American Indian/Alaska Native 
Yes /  No 
Age:____________________  
 
2-Asian 
 
 
 
Yes /  No 
Height:__________________  
 
3-Black or African American  
Yes /  No 
Weight:_________________    
 
4-Native Hawaiian/Pacific Islander     Yes / No 
 
 
 
 
 
 
5-White 
 
 
 
 Yes / No 
 
 
 
 
 
 
6-other______________________ 
Dietary Assessment of Omega-3 Intake 
1) How often do you eat-- as a main course-- tuna or other non-fried fish?  (One serving size is 3 oz = in size to a 
deck of cards. Note: One serving of non-fried fish could include: 1, 3 oz. filet of salmon OR 6 pieces of sushi OR 8 
sushi rolls OR ~4 tuna fish (salad) sandwiches, etc.) 
 
 
 
 
A) Once or less per month  
 
 
 
 
B) 2-3 times per month  
 
 
 
 
C) Once per week  
 
 
 
 
D) Twice per week  
 
 
 
 
E) More than twice per week  
 


 
 
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38 
2) Do you currently take fish oil (Omega-3) supplements? 
 
Yes / No 
 
3) Do you currently eat foods and/or drink beverages that are fortified with Omega-3 fatty acids? 
 
Yes / No 
 
Heart Risk Assessment: 
1) Do you currently smoke cigarettes/cigars/pipe or use other tobacco products (snuff, chew, etc.)? Yes / No 
           If yes, how many/much per day or week? 
Number/amount:  _________   Per:  day  /  week (circle) 
 
If no, have you ever smoked or used tobacco products (snuff, chew, etc.)? 
Yes / No 
 
 
If yes, when did you quit?  Month:______________ Day:_________Year:__________ 
 
For how many years did you/have you smoked or used tobacco products (snuff, chew, etc.)? __________ 
2)  Do you drink alcohol?   
 
Yes / No 
If yes, How much do you drink? 
 
 
 
a) >1 drink  per day 
 
 
 
b) 1 drink per day 
 
c) <1 drink per day but > 1 drink per week   
d) < 1 drink per week 
 
3) 
How often do you exercise?  
 
 
a) every day 
(for example: walking, running, 
 
 
 
b) three times a week 
biking, swimming or gym work-out)  
 
c) once a week 
       
 
 
d) once a month or less 


 
 
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39 
e) not at all 
 
4)  Do you have or did you ever have High Blood Pressure?  Yes / No 
 
5)  Do you have or did you ever have High Cholesterol? 
Yes / No 
 
6)  Do you have diabetes?    
 
 
 
Yes / No  
 
7) Do you have heart rhythm abnormalities?      
 
Yes/  No 
 
 
8) If Female: 
Are you pregnant? 
Yes / No  
Are you breast-feeding?  
Yes / No  
When was your last menstrual period? __________ 
Postmenopausal? 
Yes / No / don’t know  
 
If no, Contraception being used:  
 
 
Abstinence 
 
 
Barrier: condoms, cervical cap, diaphragm  
 
 
Surgical (tubes tied, partner vasectomy) 
 
 
Hormonal contraception (pill/patch/shot) 
 
 
Intrauterine device (IUD) 


 
 
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40 
 
 
Have you had both ovaries surgically removed?   Yes/No  
 
If post-menopausal, are you taking hormone replacement therapy?      Yes / No  
 
 
Family History: 
Do you have a first degree (mother, father, sister, or brother) family history of heart attack or stroke?  
 Yes / No 
Please answer below for each member of your family. 
 
 
 
Age at time of heart   
 
 
History of heart attack or stroke?   
 
attack or stroke?             Current Age? 
Alive? 
Biological mother  
Yes / No 
 
  _____________            ____________ 
 
Yes / No / don’t know 
Biological father  
Yes / No 
 
  ______________          ____________  
Yes / No / don’t know  
Biological brothers 1) Yes / No / not applicable ______________          ____________  Yes / No / don’t know 
or half-brothers     2) Yes / No / not applicable ______________          ____________  Yes / No / don’t know   
 
3) Yes / No / not applicable ______________          ____________  Yes / No / don’t know  
  
 
4)  Yes / No / not applicable ______________          ____________ 
Yes / No / don’t know 
Biological sisters    1) Yes / No / not applicable ______________          ____________ 
Yes / No / don’t know 
or half-sisters         2) Yes / No / not applicable______________          ____________ 
Yes / No / don’t know   
 
 
       3) Yes / No / not applicable______________           ____________ 
Yes / No / don’t know  
       4)  Yes / No / not applicable______________           ____________  Yes / No / don’t know 
*if  >8 siblings, list on back of this sheet  


 
 
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41 
 
General Health / Medical History: 
1) Do you have any allergies to fish? 
Yes / No 
2) Have you ever had cancer? Yes / No 
a. If yes, what type?  ______________________________________ 
b. If yes, do you still have cancer?  Yes / No 
i. If no, when did you go into remission (no more cancer symptoms)?  Month: ____________ 
 
Year: _____________ 
3) Are you HIV+ or do you have AIDS?  Yes / No 
4) Do you have problems with your kidneys?  Yes / No 
 
 
 
5) Have your doctors ever diagnosed with any of the following: 
a) Asthma (including childhood or exercise induced asthma) 
Yes / No 
b) Rheumatoid Arthritis 
Yes / No 
c) Inflammatory Bowel Disease (e.g., Ulcerative Colitis or Crohn's disease)? 
Yes / No 
6) List any other medical conditions you have been diagnosed with. 
_________________________________________________________________________ 
_________________________________________________________________________ 
_________________________________________________________________________ 
_________________________________________________________________________ 
 
Cardiovascular History: 
 
1. Have your doctors ever diagnosed you with:   


 
 
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42 
 
Heart disease  
 
 
 
 
 
 
Yes / No 
   
 
What?___________________________________________________________ 
 
Heart attack 
 
 
 
 
 
 
 
Yes / No 
    
Angioplasty or stent of a heart (coronary) artery 
 
 
Yes / No 
Heart bypass surgery  
 
 
 
 
 
Yes / No 
 
 
        
Stroke  
 
 
 
 
 
 
 
Yes / No 
 
Ministroke or TIA 
 
 
 
 
 
 
Yes / No 
Neck (carotid) artery blockage/narrowing/bypass surgery 
 
Yes / No 
 
 
 
Blocked/narrowed arteries in your legs 
 
 
 
Yes / No 
 
Bypass surgery of your leg arteries  
 
 
 
Yes / No 
 
Angioplasty/stent in arteries in your legs 
 
 
 
Yes / No 
 
 
 
 
 
 
 
 
    
 
 
2. a) Has your doctor ever told you that you have angina?   
 
Yes / No 
 
b) Do you have a history of chest pain/discomfort brought on  
     by exertion and relieved by rest? 
 
 
 
 
Yes / No 
 
   c) Do you have a history of chest pain relieved by medication? 
 
Yes / No 


 
 
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43 
 
What medication?  
 
1- nitroglycerine  
2- other -name________________ 
 
 
 
 
3.  Have you ever had: 
a)  Heart stress test?  
 
 
 
 
Yes / No 
 
 
 
Was the result abnormal?   
 
 
 
Yes / No  
b)  Have you ever had a cardiac catheterization?  
 
Yes / No 
 
 
 
Was the result abnormal?   
 
 
 
Yes / No 
4.  Have you ever had any surgical procedures? 
 
 
Yes / No 
 
 
If yes, what type: 
 
 
 
 
 
 
Date/Year: 
 
 
_____________________________________________________________________________ 
 
 
_____________________________________________________________________________ 
 
Medications and dietary supplements you are currently taking: 
 
Medication or Dietary Supplement: 
 
 
 
Dose, How Often, and Start Date: 
 
 
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 


 
 
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44 
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 
    
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 
 
________________________________ 
 
 
 
______________________________ 
 
Other Past Medical History Not Covered: 
  
Condition 
 
 
 
 
 
 
 
Year of diagnosis 
      ____________________________________________________________________________ 
____________________________________________________________________________ 


 
 
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45 
APPENDIX D 
 
Safety Plan 
 
Title of Study Protocol:   
Fish oils and Adipose Inflammation Reduction (FAIR Study) 
 
Principal Investigator: 
Rachana Shah, MD, MsTR 
 
 
Sponsors of Protocol: 
National Institutes of Diabetes, Digestive, and Kidney Disease  
 
 
Grant Number:  
1K23DK095913-01A1 (NIDDK) 
Title of Grant: 
Omega-3 PUFA Suppress Adipochemokines (PI:Shah)  
 
 
 
IRB Protocol Number: 
818359 
 
 
 
Date of Document:  
June 14, 2013 
 


 
 
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46 
SAFETY PLAN OVERVIEW 
It is recognized that there is some inherent risk in this study because of 1) the outpatient treatment with 
prescription omega-3 fatty acids and 2) multiple blood draws and two adipose biopsies. An important perspective, 
however, is that the study population, while obese, will be relatively young and free of cardiovascular and other 
significant disease. Moreover, the study drug is used for a short duration in an FDA-approved formulation, 
dosage, and population. 
 
It will be the responsibility of the PI (Dr. Shah) and these collaborative investigators to plan, implement, and 
continuously monitor patient safety for the FAIR study. Furthermore, a Data Safety Monitoring Board (DSMB) has 
been established by the PI and approved by the NIH-NIDDK, which will include expertise in Endocrine, and 
Cardiovascular disease, clinical research and clinical trials as well as biostatistics. The DSMB will meet at least bi-
annually in order to review the overall conduct of the study, patient enrollment, and non-serious and serious 
adverse events (AEs and SAEs). The study team will prepare pre-specified reports and an analysis of the 
statistical significance of these events.  In order to further ensure the safety of study patients, the study team will 
implement a safety plan, divided into five components: 1) staff safety training, 2) patient baseline health and 
safety assessment, 3) patient monitoring, 4) patient safety education and 5) adverse event and serious adverse 
event collection and reporting, 6) participant discontinuation and stopping criteria. 
 
1.0  Staff Safety Training  
All study staff will be required to complete Protection of Human Research Subjects Training (e.g., Collaborative 
IRB Training Initiative (CITI) Training) and Health Insurance Portability and Accountability Act (HIPAA) Training, 
as mandated by the University of Pennsylvania.  In addition to these courses, as part of the initial training for the 
FAIR Study, all staff members (e.g. research coordinators and assistants) will be trained to identify specific and 
extensive exclusion criteria for the protocol and to identify situations or circumstances that may be associated with 
a heightened safety concern (e.g., specific monitoring criteria during the outpatient treatment protocol). This 
training will take place before contact with study patients, and will be documented in the regulatory binder. This 
will entail a formal PI-directed in-service training sessions first for all sub-investigators, project managers, 
research coordinators and assistants.       
 
2.0  Participant Baseline Health and Safety Assessment  


 
 
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Baseline Health Assessment: Prior to participation in the FAIR Study, each participant will undergo a baseline 
health assessment at the screening visit composed of the following: a history and physical examination, an 
electrocardiogram, safety laboratory testing (fasting glucose, electrolytes, BUN, creatinine, hematocrit, 
hemoglobin, and platelet count, AST, ALT), and a urine pregnancy test for women of child-bearing potential. The 
purpose of these baseline measures are: (1) to evaluate baseline health; (2) to obtain relevant data that may 
include or exclude a participant from the study; and (3) to provide a baseline to identify and interpret potential 
changes in patient health during the patient’s participation in the study. According to enrollment criteria (Section 
5.4 of Protocol), participants with any inter-current illness, significant current or past health history, regular 
medication use (except contraception), any abnormal physical exam findings or laboratory findings outside of the 
pre-specified range will be excluded from the study.  
3.0 Participant Monitoring 
The regular monitoring of the status of each participant is an essential component of the FAIR Study safety plan. 
Each study staff member will be trained to identify exclusion criteria and situations during the study protocol that 
may be associated with increased risk of adverse events.  
3.1  General Risk Considerations  
The risks associated with blood draws include mild discomfort, occasional bruising and a small risk (< 1%) of 
infection.  The total amount of blood that will be drawn at the screening visit is approximately 20ml.  The total 
amount of blood that will be drawn at the randomization visit (Visit 2) is 50ml and again 50 mL at the completion 
visit (Visit 4).  Overall, this is significantly less than 1 pint of blood which is well within the recommended 
allowance over a span of 2 months. The risks associated with subcutaneous fat aspirations include bleeding, 
bruising, infection and pain.  These will be minimized by using a trained physician to perform the procedures, 
administration of anesthesia to reduce pain, and careful monitoring of the aspirated sites by nursing.    
3.2 Risks for Adverse Events During Treatment with LOVAZA (omega-3-acid ethyl esters) Capsules 
Please refer to Appendix E for complete prescribing information. 
Description:  LOVAZA, a lipid-regulating agent, is supplied as a liquid-filled gel capsule for oral administration. 
Each 1-gram capsule of LOVAZA (omega-3-acid ethyl esters) contains at least 900 mg of the ethyl esters of 
omega-3 fatty acids. These are predominantly a combination of ethyl esters of eicosapentaenoic acid (EPA - 
approximately 465 mg) and docosahexaenoic acid (DHA - approximately 375 mg).  LOVAZA capsules also 
contain the following inactive ingredients: 4 mg α-tocopherol (in a carrier of partially hydrogenated vegetable oils 
including soybean oil), and gelatin, glycerol, and purified water (components of the capsule shell). 
 
Mechanism of Action:  The mechanism of action of LOVAZA is not completely understood. Potential mechanisms 
of action include inhibition of acyl CoA:1,2-diacylglycerol acyltransferase, increased mitochondrial and 
peroxisomal β-oxidation in the liver, decreased lipogenesis in the liver, and increased plasma lipoprotein lipase 
activity. LOVAZA may reduce the synthesis of triglycerides (TGs) in the liver because EPA and DHA are poor 


 
 
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substrates for the enzymes responsible for TG synthesis, and EPA and DHA inhibit esterification of other fatty 
acids.  
 
Pharmacokinetic and Bioavailability Studies: In healthy volunteers and in patients with hypertriglyceridemia 
(HTG), EPA and DHA were absorbed when administered as ethyl esters orally. Omega-3-acids administered as 
ethyl esters (LOVAZA) induced significant, dose–dependent increases in serum phospholipid EPA content, 
though increases in DHA content were less marked and not dose-dependent when administered as ethyl esters. 
Uptake of EPA and DHA into serum phospholipids in subjects treated with LOVAZA was independent of age (<49 
years versus ≥49 years). Females tended to have more uptake of EPA into serum phospholipids than males. 
 
Adverse Reactions:  Treatment-emergent adverse events reported in at least 1% of patients treated with LOVAZA 
4 g per day or placebo during 8 randomized, placebo-controlled, double-blind, parallel-group studies for HTG are 
listed in Table 3 of Appendix E.  Adverse events led to discontinuation of treatment in 3.5% of patients (n=226) 
treated with LOVAZA and 2.6% of patients (n=228) treated with placebo. 
In some patients, increases in alanine aminotransferase (ALT) levels without a concurrent increase in 
aspartate aminotransferase (AST) levels were observed. ALT levels will be monitored before and after LOVAZA 
treatment. If a subject’s ALT level is greater than 1.5 x ULN, then the subject will be excluded from the study.   
In some patients, LOVAZA increased low-density lipoprotein cholesterol (LDL-C) levels. This tends to be 
most notable in patients with more severe hypertriglyceredemia in whom Lovaza causes the greatest 
redistribution of cholesterol from VLDL to LDL particles coincident with reduction in triglyceride rich VLDL 
particles. In such patients, Lovaza increases the size and composition of LDL from small, dense particles (which 
are thought to be atherogenic due to their susceptibility to oxidation), to large buoyant particles.  These larger 
particles have a greater affinity for cholesterol receptors and are catabolized rapidly.  Indeed, total cholesterol in 
atherogenic apoB lipoproteins (e.g., estimated by calculation of non-HDL cholesterol) tends to fall on Lovaza 
consistent with redistribution of cholesterol from VLDL to LDL with increased size of LDL particles rather than any 
overall increase in atherogenic apoB-cholesterol containing particles [10-13]. Thus we will exclude from 
participation any subject with LDL-C at screening of greater than 190 mg/dl and LDL-C will be repeated at 
completion of the study (visit 4).  
The most common adverse effects reported with LOVAZA are burping, infection, flu symptoms, upset 
stomach, a change in sense of taste, back pain and skin rash.  Monitoring for these adverse events will be 
conducted at the follow-up Visit 3 (telephone / email visit at 4 weeks post randomization).  If these events worsen 
and/or become extremely bothersome over time, the subject will be discontinued from the study as outlined below 
in Section 6.1. 
There is a possible association between LOVAZA and more frequent recurrences of symptomatic atrial 
fibrillation or flutter in patients with paroxysmal or persistent atrial fibrillation, particularly within the first months of 
initiating therapy. We will exclude from the study any subject with diagnosis of cardiac arrhythmias or abnormal 
ECG on screening. ECG will be repeated at completion of study (visit 4) and any new arrhythmia noted as an 
adverse event.  
 
3.5  Risk for Development of Clinical or Laboratory Abnormalities during the Study 


 
 
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Subjects will have screening laboratories done at Visit 1 to assess for any safety concerns that would 
prompst exclusions from the study and as a baseline for drug effect. These clinical and laboratory parameters are 
measured to assure the participant meets eligibility criteria and that he/she is generally in good health prior to 
enrollment in the study.  The development of significant clinical or laboratory abnormalities (based on repeat 
laboratory evaluation at the completion visit, Visit 4) would prompt an Adverse Event Report and a notification of 
the participant and his or her personal physician.  
At the end of the inpatient endotoxemia visit (Visit 5), at the Day 2, 6am time point, blood will be drawn to 
determine if any safety issues have arisen during the inpatient visit. For example, the presence of persistent 
anemia, biochemical abnormalities (e.g., elevated LFTs (>1.5 x ULN) or hemoglobin < 10g/dL), or clinical 
abnormalities (e.g., IV line or adipose biopsy site inflammation, infection or hematoma) would prompt a Adverse 
Event Report, a formal review of the safety for other participants as well as notification of the participant and his or 
her personal physician for appropriate follow up care.    
 
3.6  DSMB Participant Monitoring  
The PI has assembled an NIDDK-NIH approved DSMB comprised of investigators independent of the study. The 
primary responsibilities of the DSMB will be to approve the final safety plan (i.e. this document), to decide whether 
the study is being conducted in accordance with the agreed upon safety plan, to review AEs, and SAEs, to decide 
whether modifications to the protocol should be recommended, and ultimately to recommend whether 
participation can continue. The DSMB will likely perform its first formal review of the conduct, progress, and 
participant safety aspects of the study within three-six months after the first participant is enrolled. Thereafter, the 
DSMB will meet to perform a similar review periodically while there is active participant participation in the study.  
4.0  Patient Safety Education 
During the initial enrollment phase, all prospective participants will have the entire protocol described to them in 
detail and will be informed of all risks and potential benefits of participation. This explanation is a part of the 
informed consent process. Once enrolled, subjects will be educated about the risks of LOVAZA responses, , 
adipose biopsies and other study procedures. All patients will be given practical advice on identifying and 
reporting any adverse events including infection, inflammation or bleeding at biopsy sites. Patients will be asked 
to notify the research study investigator, coordinator or CTRC nursing staff in if they are experience symptoms 
related to the above-mentioned risks or any other adverse events. 
 
5.0  Adverse Event and Serious Adverse event data collection and reporting 
Participants will be closely monitored for all non-serious and serious adverse events. Proper recording and 
reporting of all adverse events is essential to maintaining and evaluating participant safety. All research staff will be 
trained on the proper method of recording adverse events. All non-serious and serious adverse events will be 
reviewed by the PI and included in the Open Reports submitted to the DSMB prior to scheduled meetings.  
5.1 Adverse Event (AE)  


 
 
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An adverse event is described as any untoward medical event that occurs during a clinical study which results in 
the participant experiencing a new and significantly abnormal symptom, physical sign, or laboratory value, or the   
clinically significant worsening of an already existing symptom, physical sign, abnormal laboratory value, whether 
or not the event is considered to be related to the therapy. Significant laboratory abnormalities are defined 
according to screening criteria. Importantly, all screened participants with any significant medical history, physical 
exam abnormality or safety laboratory abnormality that could potentially be associated with increased risk of 
adverse events are excluded in the protocol. An Adverse Event Report must be completed for all AEs.  
5.2  Serious Adverse Event (SAE) 
A serious adverse event is described as any event that is 1) life threatening or fatal, 2) results in significant or 
persistent disability, 3) requires or prolongs hospitalization, 4) results in a congenital anomaly or birth defect, or 5) 
represents other significant hazards or potentially serious harm to research participants or others, in the opinion of 
the investigators. 
5.3.  Serious Adverse Event Categories 
Serious Unexpected Adverse Event: Development or identification of a sudden catastrophic or life-threatening 
illness (e.g., myocardial infarction, stroke, pulmonary embolism, sustained hypotension or significant bradycardia) 
or an injury resulting in intensive care unit hospitalization, persistent disability, and/or death. Given the absence of 
baseline medical conditions at enrollment, all serious adverse events will be categorized as unexpected.  
5.4  Serious Adverse Event Reporting 
In the event of a serious adverse event, notification within 24 hours of learning of the event must be provided to 
the Institutional Review Board, CTRC Research Subject Advocate and Directorship, DSMB, and the NIDDK 
Program Scientist. As a precaution, at baseline all participants will be instructed to notify the study staff as soon 
as possible after a serious adverse event. The study staff will develop a report detailing the event. It should 
include details such as a thorough description of the event, an estimated severity of the event, the frequency or 
the event, any prior indications that a problem existed, as well as time patterns.  In an effort to fully document the 
SAE, a copy of all corresponding progress notes, physical exams, laboratory test results, and diagnostic tests will 
be requested, as supporting documentation. 
5.5  Adverse Event and Serious Adverse Event Attribution and Grading 
The PI will evaluate all AEs and SAEs for attribution based on the following reporting guidelines: 
Reporting Guidelines for Adverse Event Attribution Description 
1. Unrelated: The AE is clearly not related to the intervention 
2. Unlikely: The AE is doubtfully related to the intervention 
3. Possible: The AE may be related to the intervention 
4. Probable: The AE is likely related to the intervention 
5. Definite: The AE is clearly related to the intervention 
 
In addition to attribution, the following FDA reporting guideline for AE grading will be used: 


 
 
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FDA Reporting Guidelines for Adverse Event Grade Description 
1. Grade 1: Mild AE  
2. Grade 2: Moderate AE 
3. Grade 3: Severe AE 
4. Grade 4: Potentially Life-threatening AE 
Action taken in response to the adverse events will be classified as: 
1) No action taken 
2) Study Intervention suspended (specify length of suspension) 
3) Study Intervention terminated and the patient has been withdrawn 
Finally, the study team will work with the IRB, DSMB and other regulatory organization in applying the most up-to-
date and appropriate Reporting Guidelines for participants in this study protocol. 
 
6.0 Participant Discontinuation and Stopping Criteria 
 
6.1  Individual Participant Study Discontinuation/Completion 
A subject may be withdrawn from the study at any time at either the investigator’s discretion or the participant’s 
request.  An effort must be made to document the reason(s) why a subject fails to return to the study clinic for 
necessary visits or is discontinued from the study.  The primary reason for discontinuing participation in the study 
must be stated in the CRF and may include, but is not limited to, one of the following: 
 
 Development of any abnormality in blood counts, blood chemistries, or urinalysis because of which the 
investigator deems continued participation in the study to be ill-advised 
 Use of unapproved concomitant medications 
 Occurrence of intolerable AEs 
 Withdrawal of consent by patient 
 Development of an intercurrent illness, injury, medical condition or medication use likely to interfere with 
patient safety, the overall assessment, or the required administration of study medication 
 Failure to follow the protocol including non-compliance with the study medications (less than 80% by pill 
count at visit 4) 
 Development of any condition for which the investigator feels withdrawal is justified 


 
 
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The procedure to discontinue a subject from the study is as follows: 
 record any AEs since previous visit 
 record any medications  
 record vital signs 
 perform a physical examination (if discontinuation is due to an AE) 
 collect blood for clinical laboratory analysis  
Patients who discontinue from the study because of an AE(s) will be treated and followed according to 
established medical practice. 
 
6.2  Stopping Criteria  
Participant visits and participant enrollment may be discontinued if any clinically significant unexpected adverse 
events occur in participants that are deemed possibly, probably or definitely related to the study procedures.  
 
RISK BENEFIT RATIO 
We would rate the risks associated with this study to be moderate.  
 
There is no direct benefit to the participant of this study, however data obtained from this study may be very 
useful in expanding scientific knowledge. The primary risks have been highlighted in this document, and reflect in 
minor or rare study drug-related symptoms, multiple blood draws and several adipose biopsies. Overall, we 
consider the risk-benefit to be acceptable for the scientific knowledge gained.  
 
 
REFERENCES 
1. 
Michie, H.R., et al., Tumor necrosis factor and endotoxin induce similar metabolic responses in human 
beings. Surgery, 1988. 104(2): p. 280-6. 
2. 
Revhaug, A., et al., Inhibition of cyclo-oxygenase attenuates the metabolic response to endotoxin in 
humans. Arch Surg, 1988. 123(2): p. 162-70. 
3. 
Martich, G.D., A.J. Boujoukos, and A.F. Suffredini, Response of man to endotoxin. Immunobiology, 1993. 
187(3-5): p. 403-16. 


 
 
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4. 
Suffredini, A.F., et al., The cardiovascular response of normal humans to the administration of endotoxin. 
N Engl J Med, 1989. 321(5): p. 280-7. 
5. 
Barber, A.E., et al., Glucocorticoid therapy alters hormonal and cytokine responses to endotoxin in man. J 
Immunol, 1993. 150(5): p. 1999-2006. 
6. 
Lehrke, M.R., M.P.; Millington, S.C.; Iqbal, N.; Rader, D.J.; Lazar, M.A., An Inflammatory Cascade Leading 
to Hyperresistinemia in Humans. PLOS Medicine, 2004. [in press]. 
7. 
Shah, R., et al., Gene profiling of human adipose tissue during evoked inflammation in vivo. Diabetes, 
2009. 
8. 
McGillicuddy, F.C., et al., Inflammation impairs reverse cholesterol transport in vivo. Circulation, 2009. 
119(8): p. 1135-45. 
9. 
Anderson, P.D., et al., Innate immunity modulates adipokines in humans. J Clin Endocrinol Metab, 2007. 
92(6): p. 2272-9. 
10. 
Davidson, M.H., et al., Efficacy and tolerability of adding prescription omega-3 fatty acids 4 g/d to 
simvastatin 40 mg/d in hypertriglyceridemic patients: an 8-week, randomized, double-blind, placebo-
controlled study. Clin Ther, 2007. 29(7): p. 1354-67. 
11. 
Sadovsky, R., et al., Dispelling the myths about omega-3 fatty acids. Postgrad Med, 2008. 120(2): p. 92-
100. 
12. 
Bays, H.E., et al., Prescription omega-3 fatty acids and their lipid effects: physiologic mechanisms of 
action and clinical implications. Expert Rev Cardiovasc Ther, 2008. 6(3): p. 391-409. 
13. 
Bays, H., Clinical overview of Omacor: a concentrated formulation of omega-3 polyunsaturated fatty acids. 
Am J Cardiol, 2006. 98(4A): p. 71i-76i. 
14. 
Mullington, J., et al., Dose-dependent effects of endotoxin on human sleep. Am J Physiol Regul Integr 
Comp Physiol, 2000. 278(4): p. R947-55. 
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Jilma, B., Safety of endotoxin challenge in healthy volunteers: bradycardia. Intensive Care Med, 2005. 
31(3): p. 496. 
16. 
van Eijk, L.T., et al., Severe vagal response after endotoxin administration in humans. Intensive Care Med, 
2004. 30(12): p. 2279-81. 
 
 
 


 
 
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APPENDIX E 
 
 
LOVAZA PRESCRIBING INFORMATION