Exploratory Double Blind Placebo Controlled Study Investigating the Regulation of Proresolving Mediators and White Blood Cell Responses by Fish Oil Supplements in Healthy Volunteers
Phase: NA
Conditions: Healthy Volunteers
Sponsor: Queen Mary University of London

 
 
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JRMO Non-CTIMP Protocol Template 
 
 
 
1. TITLE PAGE 
 
 
 
 
 
Exploratory Double Blind Placebo Controlled Study 
Investigating the Regulation of Pro-resolving Mediators and 
White Blood Cell Responses by Fish Oil Supplements in 
Healthy Volunteers 
 

 
 
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Full Title  
Exploratory Double Blind Placebo Controlled 
Study Investigating the Regulation of Pro-
resolving Mediators and White Blood Cell 
Responses by Fish Oil Supplements in Healthy 
Volunteers 
 
Short Title/Acronym  
SPM regulation by Fish Oil Supplements in 
Healthy Volunteers 
 
Sponsor 
  
Name the sponsor organisation and contact 
person:  
§ 
Queen Mary, University of London  
 
Contact 
person 
of 
the 
above 
sponsor 
organisations is: 
 
 
 
Head of Research Resources 
Joint Research Management Office 
 
5 Walden Street 
 
London 
 
E1 2EF 
 
Phone: 020 7882 7260 
 
Email: sponsorsrep@bartshealth.nhs.uk 
 
 
REC Reference 
Insert once known 
 
 
IRAS Reference Number  
193174 
 
Chief Investigator 
 
 
Dr. David Collier, 
Barts & The London School of Medicine and 
Dentistry, Queen Mary University of London, 
Clinical Pharmacology, William Harvey Clinical 
Research Centre, William Harvey Research 
Institute, Charterhouse Square, London EC1M 
6BQ  
Email: d.j.collier@qmul.ac.uk 
Phone: 020 7882 5668 
 
Insert as applicable list of  
A) Sites  
Barts and The London School of Medicine 
Queen Mary University of London 
WHRI Heart Centre 
Charterhouse Square 
London EC1M 6BQ 
 
B) Laboratories and/or technical departments 

 
 
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William Harvey Research Institute 
Center for Biochemical Pharmacology 
Charterhouse Square 
London. EC1M 6BQ 
 
William Harvey Research Institute 
Center for Clinical Pharmacology 
Charterhouse Square 
London. EC1M 6BQ 
 
William Harvey Research Institute 
Center for Biochemical Pharmacology  
Lipid Mediator Metabololipidomics Unit  
Charterhouse Square, 
London. EC1M 6BQ 
 
C) Central facilities  
 
 
 
 
 

 
 
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Contents Page 
 
 
 
2. 
GLOSSARY OF TERMS AND ABBREVIATIONS  
3. 
SIGNATURE PAGE 
4. 
SUMMARY/SYNOPSIS  
5. 
INTRODUCTION 
6. 
TRIAL OBJECTIVES  
7. 
METHODOLOGY  
Inclusion Criteria 
Exclusion Criteria  
Study Design / Plan – Study Visits  
Study Scheme Diagram 
8. 
STUDY PROCEDURES 
Schedule of Assessment (in Diagrammatic Format) 
End of Study Definition  
9. 
STATISTICAL CONSIDERATIONS (if applicable) 
10. 
ETHICS 
11. 
SAFETY CONSIDERATIONS (if applicable) 
12. 
DATA HANDLING AND RECORD KEEPING  
Confidentiality  
Record Retention and Archiving 
13. 
LABORATORIES (if applicable) 
14. 
PRODUCTS, DEVICES, TECHNIQUES AND TOOLS (if applicable) 
15. 
SAFETY REPORTING (if applicable) 
16. 
MONITORING &AUDITING (if applicable) 
17. 
TRIAL COMMITTEES 
18. 
FINANCE AND FUNDING  
19. 
INDEMNITY  
20. 
DISSEMINATION OF RESEARCH FINDINGS (if applicable) 
21. 
REFERENCES 
22. 
APPENDICES 

 
 
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2. GLOSSARY  of Terms and Abbreviations 
 
 
AE 
 
 
Adverse Event 
 
 
AR 
 
 
Adverse Reaction 
ASR 
 
 
Annual Safety Report 
CA 
 
 
Competent Authority 
CI 
 
 
Chief Investigator 
CRF 
 
 
Case Report Form 
CRO  
 
Contract Research Organisation 
DMC  
 
Data Monitoring Committee 
EC 
 
 
European Commission 
GAfREC 
Governance Arrangements for NHS Research Ethics 
Committees 
ICF 
 
 
Informed Consent Form 
JRMO  
 
Joint Research Management Office 
NHS REC 
 
National Health Service Research Ethics Committee 
NHS R&D 
 
National Health Service Research & Development   
Participant 
 
An individual who takes part in a clinical trial 
PI 
 
 
Principal Investigator 
PIS 
 
 
Participant Information Sheet  
QA 
 
 
Quality Assurance 
QC 
 
 
Quality Control 
RCT 
 
 
Randomised Controlled Trial 
REC 
 
 
Research Ethics Committee 
SAE 
 
 
Serious Adverse Event 
SDV 
 
 
Source Document Verification 
SOP 
 
 
Standard Operating Procedure  
SSA 
 
 
Site Specific Assessment 
TMG  
 
Trial Management Group 
TSC 
 
 
Trial Steering Committee 
 
 
 
 
 
 
 
 

 
 
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4.    SUMMARY/SYNOPSIS 
 
 
Short Title 
SPM regulation by Fish Oil Supplements in Healthy 
Volunteers  
Methodology 
 
A randomised, double-blind, placebo-controlled study to 
determine whether fish oil supplementation regulates 
peripheral levels of specialized pro-resolving mediators 
and white blood cell responses in healthy volunteers 
Research Sites 
 
Barts and The London School of Medicine 
Queen Mary University of London 
William Harvey Research Institute 
Charterhouse Square 
London EC1M 6BQ 
Objectives/Aims 
 
The objective of this study is to learn more about the 
biology of fish oil supplementation in human healthy 
volunteers by assessing the actions of these supplements 
in healthy volunteers. 
Number of 
Participants/Patients 
40 
 
Main Inclusion Criteria 
 
Able to provide informed consent, willingness to abstain 
from fish for 2 days before each study visit, physical 
examination, laboratory tests.  
Statistical 
Methodology and 
Analysis (if applicable) 
 
The results will be tabulated and assessed for biological 
activity of the fish oil supplement and any relationship 
between dose and primary variables.  Because the study 
is exploratory in nature, there will be no formal statistical 
analysis of the data. 
Proposed Start Date 
 
July 2016 
 
Proposed End Date 
April 2016 
Study Duration 
 
9 months 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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5. INTRODUCTION  
 
5.1 Background 
 
Acute inflammation is the body’s response to injury and/or infection that is 
orchestrated by mediators produced at the site where the inflammatory insult 
occurred and instructs white blood cell and stromal cell behavior (1-3). When this 
response is self-contained it is protective and leads to disposal of the inciting 
stimulus, clearance of white blood cells from the site, repair of damaged tissue(s) and 
regain of function. If the response becomes dysregulated it leads to uncontrolled 
white blood cell activation, tissue damage and malaise. Unbridled inflammation is 
widely appreciated as the unifying component in many chronic conditions including 
metabolic syndrome, vascular disease and rheumatoid arthritis (1-3).  
 
The acute inflammatory response has classically been divided into two 
phases, initiation and resolution. The initiation phase is catalysed by local acting 
mediators including the essential fatty acid derived autacoids as well as cytokines 
and 
chemokines. 
Among 
these 
autacoids 
the 
arachidonic 
acid 
derived 
prostaglandins (PG; i.e. PGD2 and PGE2) and cysteinyl leukotrienes (LT) mediate 
vascular leakage, LTB4 is a potent leukocyte chemo-attractant and thromboxane A2 
is a potent pro-thrombotic agent (4). On the other hand, the resolution phase is now 
appreciated to be a coordinated cellular and biochemical response that involves 
leukocyte trafficking to and from the site of injury/infection and production of 
autacoids that reprogram the immune response(s) (2). 
 
Given the important role that pro-inflammatory mediators play in the initiation 
of inflammation it was thought that chronic inflammation is a result of unabated 
proinflammatory mediator production both at the site of inflammation and 
systemically (4, 5). Thus, a great deal of effort has been devoted over the last 
decades to identifying therapeutics that inhibit production of proinflammatory 
mediators (4, 5). This has led to the development of nonsteroidal anti-inflammatory 
drugs (NSAIDS), glucocorticoids, and disease-modifying antirheumatic drugs 
(DMARDs) that are widely used in the management and treatment of rheumatoid 
arthritis. The first two classes are primarily used to relieve symptoms, whereas 
DMARDs reduce or even prevent disease progression caused by unregulated 
inflammation when used early on (6). While these approaches are successful at 
controlling inflammation in the short term, the majority of anti-inflammatory 
therapeutics in clinical use to date have demonstrated adverse effects when 
administered over extended periods of time (1-3). Indeed chronic inhibition of these 
pro-inflammatory pathways results in gastro-intestinal barrier breakdown (7) and 
immune suppression amongst others (1-3). There is also significant variability in 
patient responsiveness to these therapeutics and their ability to limit inflammation 
significantly declines with disease severity (8, 9). In addition, these anti-
inflammatories may also delay tissue repair and/or regeneration, thus underscoring 
the importance of developing alternative strategies that are able to reprogram the 
inflammatory response and promote tissue repair and regeneration (10). Given that 
under ideal conditions the body engages protective mechanisms that limit 
inflammation without unwanted side effects (1-3), gaining insights into these 
processes is of great interest.  
 
5.2 Identification and actions of pro-resolving mediators.  
 
Recently efforts to better understand processes that regulate the termination of the 
inflammatory response and the re-establishment of homeostasis, provided 

 
 
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mechanistic evidence that the resolution of inflammation is not simply a passive 
dissipation of signals but a coordinated cellular and biochemical process (11-16). 
Indeed, cells require signals that instruct their behaviour at the site of inflammation. 
Systematic analysis of inflammatory fluids collected from the site of injury/infection 
during self-resolving inflammation and assessment of cell trafficking at site of 
inflammation lead to the identification of a novel genus of local mediators that 
potently and stereospecifically regulate key steps in the resolution of inflammation. 
Thus these novel mediators produced by the body does not simply inhibit 
inflammation but activates resolution responses, thereby displaying both anti-
inflammatory and pro-resolving actions (2, 11-16). Given that these the potent 
biological actions these novel molecules are coined as specialized pro-resolving 
mediators (SPM). The potent biological actions exerted on different components of 
the inflammatory response by these mediators further underscore the notion that 
inhibition of inflammation, or anti-inflammation, may not be the only approach to treat 
uncontrolled and chronic inflammation. Indeed these mediators provide evidence that 
under ideal conditions our body can regulate the inflammatory response by actively 
terminating it rather then inhibiting it, thereby sparing the immune response by 
repurposing the biological actions of leukocytes to re-establish homeostasis.   
 
SPM production occurs via stereospecific conversion of essential fatty acids, 
including eicosapentaenoic acid (EPA), n-3 docosapentaenoic acid (DPA) and 
docosahexaenoic acid (DHA) by leukocyte enzymes, primarily lipoxygenases and 
cyclooxygenases. The omega-3 derived SPM are divided into four main families D- 
and E-series resolvins (Rv), protectins (PD) and maresins (MaR) and display both 
overlapping as well as characteristic biological actions. Each actively limits further 
neutrophil recruitment to the site of inflammation (1-3), they reduce chemo-attractant 
production and adhesion molecule expression, counter-regulate the production of 
pro-inflammatory mediators by regulating their formation and clearance, and promote 
neutrophil apoptosis and their clearance by macrophages as well as pain. In addition 
the maresins actively promote the repair and regeneration of damaged tissues, the 
protectins display potent anti-viral actions the D-series resolvins regulate B-cell 
antibody production and the E-series resolvins regulate T-cell and dendritic cell 
responses (1-3).  
 
Studies investigating the production of these molecules during unresolved or 
chronic inflammatory conditions in experimental settings demonstrate that 
dysregulation in SPM production may be an underlying cause for disease 
propagation. For example in delayed or non resolving Escherichia coli infections 
SPM production becomes dysregulated leading to exacerbated local and systemic 
inflammation, failure of the host response to clear the invading pathogen and 
eventually death. Results with human tissues lend further support to this hypothesis, 
where SPM production is found to be dysregulated in a number of clinical settings 
including Alzheimer’s disease and sepsis.  
 
5.3 Fish oil supplementation to boost endogenous SPM 
 
Evidence that chronic inflammation may result from inability of the body to produce 
adequate levels of SPM to counter regulate the ongoing inflammation suggests that 
increasing endogenous omega-3 levels may help promote SPM formation and 
thereby facilitate the termination of inflammation. A Pubmed search for the term ‘fish 
oils supplementation + inflammation’ yielded over 500 hits highlighting the fact that 
Fish oil supplementation is of general interest to both the academic and clinical fields 
and has be extensively studied. Animal studies using both fish oil as the main source 
of omega-3 essential fatty acids as well as other omega-3 enriched supplements 

 
 
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provide further evidence for the relationship between increased SPM production 
following supplementation and a reduction in inflammation (17-19). Studies with 
humans also indicate that fish oil supplementation may exert protective actions in 
chronic conditions such as rheumatoid arthritis (20) and certain cardiovascular 
diseases (21).  
 
Omega-3 essential fatty acid supplementation has also been associated with 
increase SPM production in humans. In healthy volunteers taking 4 g of fish oil per 
day for three weeks plasma levels of three SPM, RvD1, RvD2 and AT-RvD1 were 
found to increase (22).  In a separate study patients with chronic kidney disease were 
given 4 g of essential fatty acids and the investigators found significant increases in 
markers SPM pathways and RvD1 (23). In patients diagnosed with Alzheimer’s or 
minor cognitive impairment, omega-3 fatty acid supplementation improved or 
stabilized cognitive function, it also lead to an increased ability of peripheral blood 
monocyte derived macrophages to phagocytose Amyloid β, and increase RVd1 
levels (24). We recently developed a new platform that allows for the simultaneous 
measurement of all the pro-resolving mediators in addition to their pathway markers 
(>90 molecules) in human tissues. Using this platform we found that supplementation 
of healthy volunteers of healthy volunteers with 1 g essential fatty acids and aspirin 
increased plasma SPM levels, including RvD1, RvD2, RvD5 and RvE2 ((25), as early 
as 4 hours after intake providing the first evidence in humans that omega-3 
supplementation can rapidly increases plasma SPM levels.  
 
5.4 Rationale for the study  
 
The relationship between omega-3 essential fatty acid supplementation, and 
specifically fish oil supplementation, and SPM production in humans is very poorly 
understood. Given that the body produces SPM from omega-3 essential fatty acids to 
regulate inflammation and also to repair damaged tissues, it is critical to gain further 
insights on how the body utilizes dietary supplementation of omega-3 fatty acids from 
fish oils for SPM formation. With the availability of the platform we recently developed 
(25) we are now in a unique position to better understand the biology of fish oil 
supplementation by monitoring the levels of SPM in plasma. This understanding may 
in turn shed light into the beneficial actions of omega-3 supplementation. It may also 
provide new leads for the control of excessive inflammation, as found in chronic 
inflammatory disorders, via dietary supplementation to exploit the body’s own 
defense systems. 
 
5.5 Rationale for choice of doses 
 
Given that in a study using a different fish oil source and formulation we found that 1 
g of essential fatty acids gave a mild but significant increase in plasma SPM levels 
(25) we chose the lowest dose in the study to be of 1.5g. We then chose to 
investigate two other doses that were within the European Food Safety Authority’s 
Tolerable Upper Intake Level for supplements containing both EPA and DHA 
(See Appendix 2 and 3). Given that this limit is of 5 g and previous study with both 
healthy volunteers and patients demonstrated that doses up to 4 g are well tolerated 
(22-24), we chose the remaining 2 doses to be 3.0 g and 4.5 g.  In addition, this 
supplement was awarded a Generally Recognized as Safe Status (see appendix 
1) in the for a dose of up to 5 g.  Similar doses of the emulsion from of the fish oil 
supplement are also being used in an ongoing clinical study in the USA 
(ClinicalTrials.gov Identifier: NCT02719665) measuring different outcomes to those 
being investigated in the present study.  
 

 
 
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5.6 Fish oil supplement manufacture  
 
The fish oil and placebo will be provided by Metagenics and will be manufactured as 
detailed below: 
Fish oils are manufactured following current Good Manufacturing Practice (cGMP), 
using semi-refined fish oil (anchovy/sardine oil) or pre-concentrated omega-3 fatty 
acid ethyl ester as a starting material. When semi-refined fish oil is used, the first 
step in the process is chemical esterification followed by a distillation step, in order to 
meet the same specifications as is the case for the pre-concentrated omega-3 fatty 
acid ethyl ester starting material.  Concentration steps follow which are dependent on 
the degree of concentration of the starting material (natural heterogeneous material). 
The intermediates generated in the process are processed using carbon dioxide 
(CO2) in a supercritical fluid extraction processes (SFE). The process benefits from 
the fact that CO2 in supercritical conditions can operate at moderate temperatures 
without stressing the products and avoids further degradation of the products due to 
the inert properties in front of oxidation. A bleaching unit and or a deodorization step 
can be included if applicable. A bleaching step is usually performed to control and 
reduce oxidative parameters in oils, and to reduce the colour. A deodorization step is 
performed to reduce volatile components of the product and consequently its odour. 
The last step is homogenization with an added quantity of natural antioxidants such 
as a mixture of tocopherols, in order to improve the oxidative stability of the final 
product. The product is then packaged in a clean room into steel drums with an 
appropriate internal coating. 
Reagents/processing aids are suitable for use in the production of the fish oil 
supplement.  They are commonly used in food ingredient manufacturing processes 
as described in the table below. 
 
Reagents/Processing Aids 
Reagent/Processing Aid 
CAS Number(s) 
21 CFR/GRN Citation(s) 
Ethanol, food grade 
64-17-5 
184.1293 
Sodium ethylate 
141-52-6 
NA* 
Carbon dioxide 
124-38-9 
184.1240 
Diatomaceous earth 
NA 
GRN No. 87; SCOGS No. 61 
Nitrogen 
7727-37-9 
184.1540 
Mixed natural 
tocopherols 
59-02-9; 16698-35-4; 
54-28-4; 119-13-1 
182.3890; 184.1890 
*Basic catalyst employed for esterification purposes; employed in food and pharmaceutical 
manufacturing processes. 
 
The Specifications for the fish oil supplement (monohydroxylated ethyl ester of omega-3) are 
presented below in the table below. 
 
Specifications for Fish Oil Supplement 
 
Parameter (Assay Method) 
Specification 
General Characteristics 
Appearance (Visual) 
Slightly yellow 
Odour 
Slight fish odor 
Solubility 
Almost insoluble in water. Miscible with acetone, 
ethanol, heptane, and methanol 
Acid value (mg KOH/g) (Eur.Ph 2.5.1) 
Max. 3 
Peroxide index (meq O2/kg) (Eur.Ph 2.5.5) 
Max. 10 

 
 
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Fatty Acid Profile 
EPA (mg/g, as FFA) (Eur.Ph 2.4.29) 
100 – 260 
DHA (mg/g, as FFA) (Eur.Ph 2.4.29) 
300 – 450 
Total omega-3 (mg/g, as FFA)* (Eur.Ph 2.4.29) 
600 – 850 
Heavy Metals 
Lead  (mg/kg) (AOCS Ca18c-91) 
Max. 0.1 
Arsenic (mg/kg) (AOAC 986.15) 
Max. 0.1 
Cadmium (mg/kg) (AOCS Ca18d-01) 
Max. 0.1 
Mercury (mg/kg) (AOAC 971.21) 
Max. 0.1 
Potential Contaminants 
Copper (mg/kg) (AOCS Ca18-79) 
Not specified, but analysis required 
Iron (mg/kg) (AOCS Ca18-79) 
Not specified, but analysis required 
Cesium 134 (Bq/kg) (Gamma SPECTR) 
Max. 3 
Cesium 137 (Bq/kg) (Gamma SPECTR) 
Max. 3 
Iodine 131 (Bq/kg) (Gamma SPECTR) 
Max. 3 
Dioxins and Furans (pg WHO-TEQ/g) (EPA 1613) 
Max. 1.75 
Dioxin-like PCBs (pg WHO-TEQ/g) (EPA 1668) 
Max. 3 
Sum of dioxins and furans + dioxin-like PCBs (pg WHO-
TEQ/g) (EPA 1613 and EPA 1668)  
Max. 3 
PCBs (209 congeners) (mg/g) (EPA 8082) 
Max. 0.09 
PAH (16 compounds) (GC/MS) 
Not specified, but analysis required 
* Sum of 18:3ω3, 18:4ω3, 20:4ω3, 20:5ω3, 21:5ω3, 22:5ω3, and 22:6ω3. 
 
The fish oil product meets the above analytical specifications.  Stability testing of fish 
oil supplement has been conducted at 25°C for up to 18 months.  After a 18-month 
storage period, fish oil supplement was found to be stable in terms of peroxide value, 
acid value, total omega-3, EPA and DHA.  
 
 
 
6. TRIAL OBJECTIVES 
 
6.1 Aim of research 
 
The aim of this research is to investigate whether fish oil supplementation increases 
the peripheral blood levels of SPM and whether fish oil supplementation also 
regulates peripheral white blood cell responses (including neutrophils, monocytes 
and platelets) to inflammatory stimuli. 
 
6.2 Original hypothesis 
 
Given that fish oils are rich in omega-3 essential fatty acids that are precursors in the 
biosynthesis of SPM the hypothesis underlying the present study is: Fish oil 
supplementation increase peripheral blood levels of SPM precursors that may 

 
 
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be converted to bioactive mediators which in turn will regulate white blood cell 
responses. 
 
6.3 Primary Endpoint 
The Primary endpoint of the study will be an increase in peripheral blood SPM levels 
that will be measured using established liquid chromatography tandem mass 
spectrometry based approach (see ref 17 above). 
 
6.4 Secondary Endpoints 
The secondary endpoints of this study are 
1) an increase in ex vivo phagocytosis of Escherichia coli by peripheral white 
blood cells 
2) a decrease in white blood cell activation when cells are incubated with an 
inflammatory stimulus (e.g. Platelet activation factor). This will be achieved by 
assessing the expression of adhesion molecules on peripheral blood cells 
using flow cytometry and fluorescently conjugated antibodies.  
 
 
7. METHODOLOGY  
 
 
7.1 Subject Selection 
 
 
 
This is a single centre trial and Participants will be enrolled via adverts posted 
at each of the three QMUL campuses and on social media. No one with direct 
connections to the management or supervision of this study or any their collaborators 
will be eligible for recruitment. For QMUL student recruitment, permission from the 
Deanery will also be sought. 
 
 
7.2 Inclusion Criteria 
 
 
 
For participants to be included in the study they will need to meet the 
following criteria: 
 
1) 
Able to provide informed consent 
 
2) 
Men and women between the age of 18 and 45 
 
3) 
Declare not to be taking aspirin, other NSAIDS, other form of 
medication or omega-3 fatty acid supplements for more than 2 weeks prior to 
screening and the duration of the participation.  
 
4)    
Willingness to abstain from eating fish for 2 days before each study 
visit 
 
5)   
Willingness to abstain from alcohol consumption for at least 24h prior 
to each study visit 
 
6)         Willingness to abstain from caffeine as directed before and during 
study  
 
 
 
 
7.3 Exclusion Criteria 
 
 
 
1)  
History of, chronic disorders, cardiovascular disease (e.g., heart 
disease, stroke), cancer, or diabetes or significant genetically inherited 
conditions. 
 
2)  
Pregnancy or breast-feeding. 
 
3)  
Hypothyroidism in the opinion of the investigator. 
 
4)  
Liver disease in the opinion of the investigator. 

 
 
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5)  
Any abnormality or pre-existing disease which, in the opinion of the 
investigator, might either expose the subject to risk, or influence the validity of 
the results. 
 
6)  
Women of childbearing potential not taking adequate methods of 
contraception  
 
7)  
Inability to read and write in English 
 
8) 
Participation in a clinical study of a new chemical entity, biological 
product or a prescription medicine, or loss of more than 400 mL blood, within 
the previous 3 months 
 
9) 
Smoking 
 
10) 
Presence or history of drug or alcohol abuse or intake of more than 
the amount of alcohol in the current guidelines on alcohol consumption  
 
 
7.4 Study Design / Plan – Study Visits 
 
 
 
This is an open label double-blind, placebo controlled, crossover study that 
aims to determine the dose of omega-3 fatty acids that leads to an increase in 
peripheral blood SPM levels and the kinetics of this regulation.  
 
• 
Each participant will give a baseline blood sample then they will be 
given one of the randomly allocated three doses of fish oil supplement, or a matching 
placebo in one of 8 study groups [this will be on a 1:1:1:1:1:1:1:1 ratio]. Blood will be 
collected again at 2h, 4h, 6h and 24h after supplementation/placebo.  
 
• 
This design allows to compare the levels of SPM pre and post 
supplementation and therefore to account for inter participant variation in circulating 
SPM levels as well as for any differences in white blood cell responses ex vivo.  
 
• 
Using a crossover design and a placebo, diurnal variation in the levels 
of SPM (currently not known) will be checked. In addition, using each volunteer as 
their own control for comparison with each dose of supplement will further improve 
the power of the study.  
 
• 
Each participant is expected to be involved in the study for a 
maximum of 11 weeks from initial consent and screening.  
 
• 
Participants will be asked to refrain from consuming caffeine 
containing drinks at least 2h prior to their visit.  
 
• 
Participants will be expected to refrain from alcohol intake for at least 
24h before screening for the study, and for at least 24h before each cycle of the 
study.  
 
• 
Participants will also be asked not to take other supplements, 
medications for the duration of the study, and to avoid fish containing meals for 2 
days before each study cycle. 
 
• 
For each cycle, the participants will make two visits, the first visit will 
be of a maximum of 8hrs. Here baseline (0hrs) blood sample will be obtained and the 
supplement/placebo given.  
 
• 
Blood will be collected 2hrs, 4hrs and 6hrs following 
supplement/placebo intake and the participant will be free to leave after the 6hrs 
blood collection. 
 
• 
During this time participants will be asked to refrain from drinking 
caffeine containing drinks such as coffee or tea. 
 
• 
Participants will then be asked to return the next day (24h after fish oil 
supplement/placebo intake). 
 
• 
The 24h blood draw should take a maximum of 30 min. 
 
• 
After a minimum of 48 hours, and not more than two weeks 
participants will be asked to return, their eligibility criteria re-assessed and then 
administered the next supplement/placebo dose in the cycle as determined by the 
group they were assigned to at the beginning of the study (see Section 13.5.1) 

 
 
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• 
A first follow-up phone-call to the volunteer will be made to check well-
being at 2 weeks (+/- 1 week) after the end of the volunteers last cycle 
 
• 
A Second follow-up phone-call to the volunteer will be made to check 
well-being at 4 weeks (+/- 1 week) after the end of the volunteers last cycle 
 
• 
If any adverse effects are reported by the volunteer they will be asked 
return for further evaluation. 
 
 
 
The fish oil supplements that will be used in the present studies are isolated 
from Peruvian anchovies. These are then packaged as emulsions and supplied by 
Metagenics with whom we will have contractual agreement in place before the start 
of the study. Metagencis will also supply the emulsion that does not contain fish oil 
supplements to be used as placebo. Both of these produces are not considered to be 
drugs or medicines and classified as foodstuffs.  
 
7.5 Study Scheme Diagram 

 
 
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8. STUDY PROCEDURES   
 
8.1 Recruitment 
 
Randomiza)on to one of 8 study groups 
Blood Draw 
Witnessed supplementa)on with omega-3/placebo 
Blood Draws a@er 2h, 4h, 6h and 24h 
Healthy volunteer screening/enrollment 
Healthy volunteer wriGen informed consent 
A@er at least 48h healthy volunteers are re-assessed for eligibility 
Blood Draw 
Witnessed supplementa)on of omega-3/placebo 
Blood Draws a@er 2h, 4h, 6h and 24h 
A@er at least 48h healthy volunteers are re-assessed for eligibility 
Blood Draw 
Witnessed supplementa)on of omega-3/placebo 
Blood Draws a@er 2h, 4h, 6h and 24h 
A@er at least 48h healthy volunteers are re-assessed for eligibility 
Blood Draw 
Witnessed supplementa)on of omega-3/placebo 
Blood Draws a@er 2h, 4h, 6h and 24h 

 
 
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Participants will be enrolled via adverts posted at each of the three QMUL campuses 
and social media. No one with direct connections to the management or supervision 
of this study or any their collaborators will be eligible for recruitment. For QMUL 
student recruitment, permission from the Deanery will also be sought. Adverts will 
also be posted on social media such as Facebook and Twitter as well as 
newspapers. A PDF copy of the poster will be added to the William Harvey website. 
 
Potential participants will be asked to contact the research team via email or over the 
phone. Then they will be invited to attend screening thereafter. 
 
All potential participants will be given the Participant Information Sheet (PIS) by a 
member of the study team which they can take away for consideration. The 
participant will be given at least 48 hours to decide whether or not to take part in the 
study following being given the PIS. 
 
All participants who demonstrate interest in enrolling will be invited to attend the 
William Harvey Clinical Research Centre (CRC) one week later where they will have 
the opportunity pose any questions to the research team and then will have the 
opportunity to formally enrol.  
 
Up to a maximum payment of £400 will be organised for participation in the study to 
reimburse London travel expenses within Greater London. 
 
8.2 Screening and Consent 
 
The consenting procedure will follow the William Harvey Heart Centre, Clinical 
Research Centre's SOP 011.  
 
The doctors obtaining informed consent will have current GCP training. They will be 
familiar with the study, the fish oil supplement and will use the current version of the 
Patient Information Sheet (PIS) and Consent Form, which has been approved by 
ethics.  
 
The doctors obtaining consent will ensure that the participant is able to understand 
the information given and will explain the nature, purpose, procedures, risks and 
benefits of the study to the participant. The doctor will answer any questions the 
participant may have to the participant's satisfaction. The participant will be given at 
least 48 hours to decide whether or not to take part in the study following being given 
the PIS. The date the PIS is given to the participant will be documented to ensure 
that sufficient time is given for consideration. 
 
The doctor will explain the consent form and obtain the participant's initials and dated 
signature on the consent form. They will then countersign and date the consent form 
after the participant has signed it. The original signed consent and PIS will be kept in 
the subject record in the CRC, a copy will be filed in the Site Investigator File and the 
participant given a copy. 
 
Volunteers will also be asked, on the patient information sheet, if they are willing to 
have their RNA extracted and stored for subsequent analysis, on the understanding 
that they will not be informed of any genetic information, and that the team dealing 
with them in the trials unit will also never be made aware of any genotyping 
information. These samples will be stored for a maximum of 2 years. These samples 
will be used to investigate the expression of SPM biosynthetic enzymes, the SPM 
receptors and pathways that these molecules may activate. 

 
 
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Basic screening will performed, which will include- clinical history taking, vital signs- 
Blood pressure, pulse rate, ECG, physical examination and blood tests; full blood 
count, biochemistry, liver function tests, glucose and C reactive protein. The identity 
of healthy volunteers will be documented by taking a photocopy of an original photo 
ID card (passport, driving license, TFL photocard), and archived with the study 
records. Before each dosing the identity of the volunteers will be confirmed against 
the identity records on file. 
 
8.3 Intervention 
 
After determining eligibility participants will be randomly assigned to one of the eight 
groups outlined in the table below where each participant will receive all three doses 
and the placebo but in a different sequence to minimise any interaction effects.  
In order to ensure adequate recruitment into all eight cycles prior to interim analysis 
group allocation will be divided into two blocks with 20 volunteers per block. Each of 
the supplement capsule contains 1.5g of essential fatty acids. So administration of 1 
supplement capsule and 2 placebo capsules will deliver 1.5g of omega-3 fatty acids, 
administration of 2 supplement capsules and 1 placebo capsule will deliver 3 g of 
omega-3 essential fatty acids and administration of 3 supplement capsule will deliver 
4.5g essential fatty acids.  
 
Group 
Treatment 
 
Cycle 1 
Cycle 2 
Cycle 3 
Cycle 4 
Group 1 
3 capsules Placebo  
1 capsules 
Supplement  & 2 
capsules Placebo 
2 capsules 
Supplement  & 
1 capsules Placebo 
3 capsules 
Supplement   
Group 2 
1 capsules 
Supplement & 2 
capsules Placebo 
2 capsules 
Supplement  & 1 
capsules Placebo 
3 capsules 
Supplement   
3 capsules Placebo 
Group 3 
2 capsules  
Supplement  & 
1 doses Placebo 
3 capsules 
Supplement   
3 capsules Placebo   
1 capsules  
Supplement  & 2 
capsules Placebo 
Group 4 
3 capsules 
Supplement 
3 capsules Placebo   1 capsules 
Supplement & 2 
capsules Placebo 
2 capsules 
Supplement  & 
1 capsules Placebo 
Group 5 
3 capsules 
Supplement 
2 capsules 
Supplement & 1 
capsules Placebo 
1 capsules IMP & 
2 capsules Placebo 
3 capsules Placebo 
Group 6 
2 capsules 
Supplement  & 
1 capsules Placebo 
1 capsules 
Supplement  & 2 
capsules Placebo 
3 capsules Placebo 
3 capsules 
Supplement 
Group 7 
1 capsules 
Supplement  & 2 
capsules Placebo 
3 capsules Placebo 
3 capsules 
Supplement 
2 capsules 
Supplement & 
1 capsules Placebo 
Group 8 
3 capsules Placebo  
3 capsules 
Supplement 
2 capsules 
Supplement  & 
1 capsules Placebo 
1 capsules 
Supplement  & 
2 capsules Placebo 
 
 
This above protocol will be followed until all forty participants are recruited (unless 
halted after the interim analysis).  
 
 
8.4 Randomisation Process 
 

 
 
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Patients will be block randomised on a 1:1:1:1:1:1:1:1 basis to receive either one of 
the three doses of fish oil or placebo in one of 8 study groups, using a binary random 
number sequence (www.random.org).   
 
 
8.5 Blinding Process 
 
The study treatment will be prepared and labelled by the WHRI Heart Centre 
Pharmacy, according to the randomisation schedule.  Active and placebo treatments 
will be labelled such that it is not possible to distinguish between them. Each 
subject’s treatment will be given a unique code number, that is traceable to study 
group and batch numbers of placebo and supplement. 
Investigators and research team including CI, PI, trial coordinator, research nurses 
and lab staff, will be blinded to the treatment being provided. A sealed copy of the 
randomisation code will be kept in a locked file in the WHRI Heart Centre 
Dispensary.  The investigator will be supplied with sealed envelopes, each one 
containing the treatment allocation for the subject whose number appears on the 
outside of the envelope.  Those envelopes will be kept in the study master file, 
readily accessible to clinical staff.  Emergency procedures for revealing treatment 
codes are specified in Section 7.6 below.  The investigator, sponsor’s medical expert 
and clinical monitor will remain blinded throughout the study, unless safety concerns 
necessitate unblinding. 
 
 
8.6 Un-blinding Process 
 
In the event of an emergency PI or medically qualified delegate can request 
unblinding from the dispensing team. 
  
The a member of the research team will notify the CI in writing as soon as possible 
following the code break detailing the necessity of the code break. Given that the 
nature of the placebo and supplement, where the supplement is a commercially 
available supplement that will be taken within the recommended daily doses and that 
the participants will all be healthy volunteers it is not anticipated that any 
emergencies will arise as a direct consequence of consumption of either the placebo 
or the fish oil supplement. Therefore 24h unblinding is not required.  
 
The PI must document the breaking of the code and the reasons for doing so on the 
CRF/data collection tool in the site file and medical notes. All unblinding will also be 
documented at the end of the study in any final study report. 
 
At the end of the trial once all the analysis has been conducted the codes will be 
broken by the CI. 
 
 
8.7 Experimental Methods  
 
After collection samples will be processed William Harvey Centre for Biochemical 
Pharmacology/Clinical Pharmacology Laboratories within one hour of following 
standard operating procedures listed in the Lipid Mediator Unit Laboratory Manual. 
 
White Blood cell phagocytosis of Escherichia coli: 
• 
20 µl of blood will be incubated with 2x107 C.F.U. of fluorescently labeled E. 
coli 

 
 
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• 
Cells will be incubated at 37oC for 1h 
• 
Cells will then be processed for flow cytometry using established protocols 
• 
The extent of phagocytosis will be assessed using flow cytometry 
 
Peripheral blood cell activation  
• 
20µl of whole blood will be incubated with or without 100µM of PAF for 1h at 
37oC 
• 
Cells will the be washed 2x with PBS containing 5% fetal calf serum 
• 
Cells will then be incubated with the following fluorescently conjugated 
antibodies for 15 min at room temperature 
• 
Cells will then be processed for flow cytometry  
• 
Adhesion molecule expression as well as platelet-leukocyte aggregates will 
be assessed using flow cytometry 
 
Lipid mediator profiling 
 
• 
Plasma (2ml) will be placed in 8 ml of ice-cold methanol containing deuterium 
labeled internal standards 
• 
Samples will be stored at temperatures of -80oC  
• 
Samples will be processed in batches of 20-24.  
• 
Here samples will be centrifuged at 3000xg for 10 min at 4oC 
• 
Supernatants will be collected  
• 
Lipid mediators (LM) will be extracted using solid phase extraction techniques 
• 
Isolated LM will be profiled using liquid chromatography tandem mass 
spectrometry (see ref 17)  
• 
At the end of the study any excess or unused samples will be disposed of 
unless written consent is obtained from participant for longer term storage. 
Sample disposal will be conducted following standard operating procedures 
as outlined in the Biochemical Pharmacology laboratory manual. 
 
RNA storage 
If participant consent, blood will also collected and stored in RNA stabilizing reagent 
for later processing. These samples will be used to assess the expression of genes 
involved in the initiation and resolution of inflammation. This sample collection will be 
conducted with the understanding that the participants will not be informed of any 
genetic information, and that the team dealing with them in the trials unit will also 
never be made aware of any genotyping information (a one-way donation). These 
samples will be stored for a maximum of 2 years. RNA will be used to investigate the 
expression of SPM biosynthetic enzymes, the SPM receptors and pathways that 
these molecules may activate. 
 
8.8 Clinical Protocol 
 
At least 24h prior to consent, participants will be provided an information sheet by an 
appropriate method including email, post or by attending the clinic, detailing the 
rationale and hypothesis of the study and on attending the screening visit the study 
physician will address any questions that the participants may have. They will be 
asked to sign the informed consent and then their eligibility criteria will be assessed 
based on the inclusion criteria set out for the study.  Basic observations, blood 
pressure, pulse, resting supine ECG, Blood samples for full blood count, 
biochemistry and glucose and CRP, then a physical examination will be performed.   
 
Volunteers passing screening will then: 
 

 
 
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• 
Will give a baseline blood sample (12ml) 
• 
Be given one of the randomly allocated three doses of fish oil supplement, or 
a matching placebo [this will be on a 1:1:1:1 ratio].  
• 
Give blood (12ml/time interval) will be collected again at 2h, 4h, 6h  
• 
Be allowed to leave and they will be asked to return the following day for the 
collection of the 24h blood sample. 
• 
Be asked to return after a minimum of 48h but no later then 2 weeks  
• 
Identity will be verified  
• 
Blood will be obtained (12ml) 
• 
Be given the next supplement dose/placebo that is listed on their assigned 
study group and the injection witnessed by a member of the study team. 
• 
Blood will be obtained after 2h, 4h and 6h. 
• 
Be free to leave and will be asked to return after 23h from supplementation   
• 
The 24h blood sample will be collected and the participants will be asked to 
return again after a minimum of 48h and no later then 2 weeks for their next 
supplement dose/placebo cycle 
• 
Identity will be verified  
• 
Blood will be collected (12ml) 
• 
Supplement/placebo will be administered and ingestion witnessed. 
• 
Blood will be collected at the same intervals outlined above 
• 
Participants will be asked to return for the last supplement dose/placebo listed 
on their study group at least 48h after the last blood donation (24h blood 
sample) but no later then 2 weeks. 
• 
Essentially the same protocol outlined above will be followed for the last 
cycle. 
• 
A first follow-up phone-call to the volunteer will be made to check well-being 
at 2 weeks (+/- 1 week) after the end of the volunteers last cycle 
• 
A Second follow-up phone-call to the volunteer will be made to check well-
being at 4 weeks (+/- 1 week) after the end of the volunteers last cycle 
 
Participants can withdraw from the study at any time. 
 
 

 
 
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8.9 Schedule of Assessment (in Diagrammatic Format) 
 
 
Assessment 
Scree
ning 
Study 
Visit 1 
(Week 
1) 
Study 
Visit 2 
(Week 
1+24h) 
Study 
Visit 3 
(Week 
2) 
Study 
Visit 4 
(Week 
2+24h) 
Study 
Visit 1 
(Week 
3) 
Study 
Visit 2 
(Week 
3+24h) 
Study 
Visit 3 
(Week 
4) 
Study 
Visit 4 
(Week 
4+24h) 
Follow up 
telephon
e call 
 
End of 
Study 
telepho
ne call  
Informed 
written 
consent 
Inclusion/Excl
usion criteria 
x 
 
 
 
 
 
 
 
 
 
 
ID Asessment 
using Photo id 
x 
x 
x 
x 
x 
x 
x 
x 
x 
 
 
Weight 
x 
 
 
 
 
 
 
 
 
 
 
Height 
x 
 
 
 
 
 
 
 
 
 
 
Screening 
bloods* 
x 
 
 
 
 
 
 
 
 
 
 
Pregnancy 
test  
x 
 
 
 
 
 
 
 
 
 
 
Resting ECG 
x 
 
 
 
 
 
 
 
 
 
 
Physical 
examination 
x 
 
 
 
 
 
 
 
 
 
 
Assess 
adverse 
events 
 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
Dosing 
 
x 
 
x 
 
x 
 
x 
 
 
 
Document any 
medication 
x 
x 
 
x 
 
x 
 
x 
 
 
 
Blood draw** 
@ 0hrs 
 
x 
 
x 
 
x 
 
x 
 
 
 
Blood draw @ 
2hrs 
 
x 
 
x 
 
x 
 
x 
 
 
 
Blood draw @ 
4hrs 
 
x 
 
x 
 
x 
 
x 
 
 
 
Blood draw @ 
6hrs 
 
x 
 
x 
 
x 
 
x 
 
 
 
Blood draw @ 
24hrs 
 
 
x 
 
x 
 
x 
 
x 
 
 
 
 
8.10 End of Study Definition 
 
The CI is delegated the responsibility of submitting the EOT notification to REC once 
reviewed by sponsor. The EOT notification must be received by REC and within 90 
days of the end of the trial. 
If the study is ended prematurely, the Chief Investigator will notify the Sponsor & 
REC including the reasons for the premature termination (within 15 days). 
 
The trial will end after the last healthy volunteer’s last visit and the analysis of all 
sample analysis has been completed.  
 
8.11 Subject Withdrawal 
PI may/must withdraw participants from the study if during any point they intake any 
medication, alcohol, other supplements or oily fish prior to the last blood draw (24h). 
If participants are not able to make their next visit within the stipulate window (2 
weeks + 48h from the supplement/placebo dose) they will be re-screened prior to re-
enrolling. 

 
 
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If blood cannot be obtained at the 0h, 2h, 4h or 6h time intervals then the participant 
will be withdrawn from the primary analysis. If the blood cannot be withdrawn at the 
24h interval the participant can still be included for primary and secondary endpoint 
analysis. 
 
If a participant is withdrawn from the study they may re-enroll in the study after being 
>14 days free of medication. 
 
If the participant withdraws before the completion of the 4th visit and dosing of 
supplement/placebo the samples and information will be still be analysed, but the 
study analysis will be primarily by per-protocol (complete treatment cycles only), 
although all data will also be analysed for Intention-to-treat as a secondary analysis.  
 
Withdrawn subjects may be replaced at the discretion of the investigator.   
 
9. STATISTICAL CONSIDERATIONS  
 
9.1 Primary Endpoint 
The Primary endpoint of the study will be an increase in peripheral blood SPM levels 
that will be measured using established liquid chromatography tandem mass 
spectrometry based approach. 
 
9.2 Secondary Endpoints 
The secondary endpoints of this study are 
1) an increase in ex vivo phagocytosis of Escherichia coli by peripheral white 
blood cells 
2) a decrease in white blood cell activation when cells are incubated with an 
inflammatory stimulus (e.g. Platelet activation factor). This will be achieved by 
assessing the expression of adhesion molecules on peripheral blood cells 
using flow cytometry and fluorescently conjugated antibodies.  
 
9.3 Sample Size 
Power calculations were conducted using data obtained from published studies 
where healthy volunteers were given 1 mg essentially (from a different source to that 
being investigated in the present study) and peripheral blood lipid mediator levels 
were evaluated pre-essential fatty acid and 4 hours after supplement intake (Colas et 
al Reference 17). Using pre and post values of 10 mediators values from these 
studies and Wilcoxon matched-pairs signed rank test a sample size of 33 participants 
is required to detect a threefold odds of an increase in post value when compared 
with the pre value with a power of 80% at the 5% level of statistical significance using 
the formulae from reference 18.  Fifty mediators will be examined and it is recognized 
that at the 5% level of statistical significance around 2-3 mediators will falsely be 
identified as having significant changes. However the functional effect of all 
significant mediators will be subsequently examined by assessing white blood cell 
function, including the ability of peripheral white blood cells to phagocytose bacteria 
and the response of peripheral white blood cells and platelets to an inflammatory 
stimulus. If we do not observe increases in the ability of peripheral white blood cells 
to phagocytose bacteria and/or decreases in the activation of peripheral white blood 
cells and platelets then these mediators will be assumed to be false positive results. 
For these analysis the white blood cell function and peripheral blood lipid mediator 
levels at the 2h and 4h intervals post essential fatty acid/placebo administration will 
be compared to pre-supplementation values. The time point and dose that give the 
greatest change in all the mediators and the greatest regulation of peripheral white 
blood responses will be judged to be the main outcome measure for future trials. 

 
 
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9.4 Statistical Analysis Plan 
Wilcoxon matched-pairs signed rank test will be used to analyse the pre and post 
values of 10 mediators as well as white blood cell function (at 2 and 4h). The time 
point corresponding to the greatest number of the mediators being statistically 
significantly different from their pre-test values at the 5% level of statistical 
significance will be identified as the time point of interest. At the 5% level of 
significance testing 50 mediators will result in at least 2-3 associations being falsely 
statistically significant. Thus, the functional effect of all significant mediators will be 
subsequently examined by assessing white blood cell function, including the ability of 
peripheral white blood cells to phagocytose bacteria and the response of peripheral 
white blood cells and platelets to an inflammatory stimulus to determine if the 
identified associations are likely to be true changes. If we do not observe increases in 
the ability of peripheral white blood cells to phagocytose bacteria and/or decreases in 
the activation of peripheral white blood cells and platelets then these mediators will 
be assumed to be false positive results. Values at different times will not be 
compared just the value at each time point will be compared to the same person's 
pre test values. 
 
10. ETHICS  
 
This protocol and any subsequent amendments, along with any accompanying 
material provided to the patient in addition to any advertising material will be 
submitted by the Investigator to an independent Research Ethics Committee (REC). 
Written Approval from the Committee must be obtained and subsequently submitted 
to the JRMO to obtain Final Sponsorship approval. According to the algorithm on the 
MHRA website, the proposed study is a non-clinical trial of an investigational 
medicinal product (non-CTIMP), and does not require prior approval by the MHRA 
(https://www.gov.uk/guidance/clinical-trials-for-medicines-apply-for-authorisation-in-
the-uk#clinical-trial-phases). The study will be conducted at the WHRI Institute, in 
compliance with ICH GCP, the principles of good manufacturing practice and the 
SOPs issued by NRES for RECs in the UK.  
All subjects must give written consent to participate in this study.  Consent for 
screening evaluations may be obtained using the information and consent form that 
will also be submitted by the Investigator to REC prior to the start of the study. The 
study-specific information and consent form will be signed by the participant before 
any screening evaluation.  Before giving consent, participants must read the 
information sheet about the study.  They must also read the consent form.  They will 
then discuss the study with the investigator or his deputy and be given the 
opportunity to ask questions.  The study-specific information sheet and the consent 
form must be approved by the REC. 
 
Participants are free to withdraw from the study at any time, without providing a 
reason.  
 
The CI will ensure that the REC is informed promptly of any serious adverse event 
that occurs during this study and that is both related and unexpected (see section 
13), in line with NRES SOPs, and will provide the REC with annual progress reports 
of the study, if it lasts longer than a year.   
 
11. SAFETY CONSIDERATIONS: 
Very high (< 10mg) omega-3 doses have been described to lead to impaired blood 
clotting. However this should not be of concern in the present study since the highest 

 
 
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dose to be employed will be of 4.5 g. In addition, the highest does being employed in 
the present study is also below the Generally Recognized as Safe (GRAS) status as 
well as the recommendations of the European Food and Safety Authority of 5 g per 
day 
 
12. DATA HANDLING AND RECORD KEEPING:  
12.1 Confidentiality The Principal Investigator has a responsibility to ensure that 
participant anonymity is protected and maintained. They must also ensure that their 
identities are protected from any unauthorised parties. Information with regards to 
study participants will be kept confidential and managed in accordance with the Data 
Protection Act, NHS Caldicott Guardian, The Research Governance Framework for 
Health and Social Care and Research Ethics Committee Approval. All trial data will 
be stored in line with the Medicines for Human Use (Clinical Trials) Amended 
Regulations 2006 and the Data Protection Act and archived in line with the Medicines 
for Human Use (Clinical Trials) and all subsequent amendments as defined in the 
JRMO SOP 20 Archiving. 
The Chief Investigator and the study team will adhere to these parameters to ensure 
that the Participant’s identity is protected at every stage of their participation within 
the study. To ensure this is done accordingly, at time of consent each participant will 
be allocated a unique screening number by either the PI or a member of the study 
team before undergoing any screening procedures. 
Personal information will be collected upon enrolment, this will be stored in 
accordance with QMUL Data Protection regulations. 
In order to maintain confidentiality, participants will be identified by a unique trial 
identifier only. This number will be randomly generated and will be assigned to each 
participant. In addition to relating to the participant’s dataset the code will also be 
associated with the specific study cycle that the participant is enrolled to.   
12.2 Record Retention and Archiving 
Data will be managed with reference to the Barts CTU SOP 'Data Management' 
Reference: SOP CPTU GEN TM 06, Version 2.0. 
Data will be recorded using dedicated software and computers for each of the 
analyses conducted. The data will then be kept anonymised by investigators. 
Samples will be stored and coded using the coding system in 14.3 and the key to the 
code will be held by the PI. 
During the course of research, all records are the responsibility of the Chief 
Investigator and must be kept in secure conditions. When the research trial is 
complete, it is a requirement of the Research Governance Framework and Trust 
Policy that the records are kept for a further 20 years. 
Destruction of essential documents will require authorisation from the Sponsor. 
The documents will be archived in accordance with the Sponsor’s SOP (JRMO 
SOP). Both electronic and paper documentation will be retained for archiving. These 
will be stored at Barts Health corporate records.  
 

 
 
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13. LABORATORIES (if applicable) 
 
13.1 Adverse Events (AE) 
An AE is any untoward medical occurrence in a subject to whom a medicinal product 
has been administered, including occurrences which are not necessarily caused by 
or related to that product. An AE can therefore be any unfavourable and unintended 
sign (including an abnormal laboratory finding), symptom or disease temporarily 
associated with study activities. 
 
13.2 Notification and reporting Adverse Events or Reactions 
If the AE is not defined as serious, the AE is recorded in the study file and the 
participant is followed up by the research team. The AE is documented in the 
participants’ medical notes (where appropriate) and the CRF. 
 
13.3 Serious Adverse Event (SAE) 
A serious adverse event (SAE) is defined as an untoward occurrence that: 
(a) results in death; 
(b) is life-threatening; 
(c) requires hospitalisation or prolongation of existing hospitalisation; 
(d) results in persistent or significant disability or incapacity; 
(e) consists of a congenital anomaly or birth defect; or 
(f) is otherwise considered medically significant by the investigator. 
 
An SAE occurring to a research participant should be reported to the Sponsor and 
main REC where in the opinion of the Chief Investigator the event was: 
 
• Related – that is, it resulted from administration of any of the research procedures, 
and 
• Unexpected – that is, the type of event is not listed in the protocol as an expected 
occurrence. 
 
13.4 Notification and Reporting of Serious Adverse Events 
Serious Adverse Event (SAEs) that are considered to be ‘related’ and  ‘unexpected’ 
will be recorded in the participants’ notes, the CRF, the sponsor SAE form and 
reported to the JRO within 24 hours of research staff being notified and to the Main 
REC within 15 days in line with require the timeframe. The co-investigators listed in 
this protocol will be authorized to sign the SAE forms in the absence of the PI.  
 
Since this study will be blinded, the treatment code for the patient will be broken in 
the reporting of an ‘unexpected and related’ SAE. This will be performed by the Barts 
CTU, who is independent of the study and will allow the rest of the research team to 
remain blinded. The unblinding of single cases by the PI in the course of this study 
will only be performed if necessary for the safety of the trial subject.  
 
13.5 Urgent Safety Measures  
The CI may take urgent safety measures to ensure the safety and protection of the 
clinical trial participants from any immediate hazard to their health and safety, the 
measures should be taken immediately. In this instance, the approval of the REC 
prior to implementing these safety measures is not required. However, it is the 
responsibility of the CI to inform the sponsor and Research Ethics Committee (via 
telephone) of this event immediately.  
 
The CI has an obligation to inform the REC in writing within 3 days, in the form of a 
substantial amendment. The sponsor (Joint Research Management Office [JRMO]) 

 
 
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must be sent a copy of the correspondence with regards to this matter.  
 
13.6 Annual Safety Reporting  
The CI will send the Annual Progress Report to the REC using the NRES template 
(the anniversary date is the date on the REC “favourable opinion” letter from the 
MREC) and to the sponsor.  
 
13.7 Overview of the Safety Reporting responsibilities  
The CI has the overall pharmacovigilance oversight responsibility. The CI has a duty 
to ensure that safety monitoring and reporting is conducted in accordance with the 
sponsor’s requirements.  
 
13.7.1 Pharmacovigilance responsibilities  
All treatment will be carried out at The William Harvey Research Institute & SAE’s will 
be reported by staff to the JRMO as per their SOPs. The Trial Master File and Site 
file will be kept at The William Harvey Research Institute. 
 
 
14.  PRODUCTS, DEVICES, TECHNIQUES AND TOOLS  
 
14.1 Summary Monitoring Plan  
The trial will be monitored in accordance with sponsor SOPs and is open to both 
monitoring and/or audit from the sponsor. Study teams will be initiated and monitored 
in accordance with the sponsor SOPs led by the lead center study team/coordinator.  
 
14.2 Audit and Inspection  
Quarterly meetings will be held with by the research team to review the progress of 
the study and discuss and issues that may arise. 
 
14.3 Serious Breaches in GCP or the Trial Protocol  
The sponsor of the Clinical Trial is responsible for notifying the licensing authority in 
writing of any serious breach of:  
·  The conditions and principles of GCP in connection with that trial; or  
·  The protocol relating to the trial, within 7 days of becoming aware of that breach.  
 
For the purposes of this protocol, a ‘serious breach’, is a breach which is likely to 
effect to a significant degree:  
·  The safety or physical or mental integrity of the participants of the trials; or  
·  The scientific value of the trial.  
 
The CI is responsible for reporting any serious breaches to the sponsor (JRMO) 
within 24 hours. The sponsor will notify and report to REC within 7 working days of 
becoming aware of the serious breach.  
 
These non-compliances may be captured from a variety of different sources 
including monitoring visits, CRFs, communications and updates. The sponsor 
will maintain a log of the non-compliances reported to them and the CI will 
maintained a site Deviation log. The sponsor will assess the non-compliances 
and action a timeframe in which they need to be dealt with. Each action will be 
given a different timeframe dependent on the severity. If the actions are not dealt 
with accordingly, the JRMO will agree an appropriate action, including an on-site 
audit  
 
 

 
 
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15. SAFETY REPORTING  
 
 
Adverse Events (AE) 
An AE is any untoward medical occurrence in a subject to whom a medicinal 
product has been administered, including occurrences which are not 
necessarily caused by or related to that product.  An AE can therefore be any 
unfavourable and unintended sign (including an abnormal laboratory finding), 
symptom or disease temporarily associated with study activities. 
 
Notification and reporting Adverse Events or Reactions 
If the AE is not defined as SERIOUS, the AE is recorded in the study file and 
the participant is followed up by the research team. The AE is documented in 
the participants’ medical notes (where appropriate) and the CRF. 
 
Serious Adverse Event (SAE) 
In other research other than CTIMPs, a serious adverse event (SAE) is 
defined as an untoward occurrence that: 
(a) results in death; 
(b) is life-threatening; 
(c) requires hospitalisation or prolongation of existing 
hospitalisation; 
(d) results in persistent or significant disability or incapacity; 
(e) consists of a congenital anomaly or birth defect; or 
(f) is otherwise considered medically significant by the 
investigator. 
 
An SAE occurring to a research participant should be reported to the main 
REC where in the opinion of the Chief Investigator the event was: 
• Related – that is, it resulted from administration of any of the research 
procedures, and 
• Unexpected – that is, the type of event is not listed in the protocol as an 
expected occurrence.  
 
Notification and Reporting of Serious Adverse Events  
Serious Adverse Event (SAEs) that are considered to be ‘related’ and 
‘unexpected’ are to be reported to the sponsor within 24 hours of learning of the 
event and to the Main REC within 15 days in line with the required timeframe. 
For further guidance on this matter, please refer to NRES website and JRMO 
SOPs 
 
Please note in the case of a blinded study, it is recommended the treatment 
code for the patient is broken in the reporting of an ‘unexpected and related’ 
SAE. Please seek advice on how this can be achieved whilst maintaining the 
team blind. The unblinding of single cases by the PI/CI in the course of a clinical 
trial should only be performed if necessary for the safety of the trial subject. 
 
Urgent Safety Measures 
The CI may take urgent safety measures to ensure the safety and protection of 
the clinical trial subjects from any immediate hazard to their health and safety,. 
The measures should be taken immediately. In this instance, the approval of 
the REC prior to implementing these safety measures is not required. However, 
it is the responsibility of the CI to inform the sponsor and Main Research Ethics 
Committee (via telephone) of this event immediately.  

 
 
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The CI has an obligation to inform both the Main REC in writing within 3 days, 
in the form of a substantial amendment. The sponsor (Joint Research 
Management Office [JRMO]) must be sent a copy of the correspondence with 
regards to this matter. For further guidance on this matter, please refer to 
NRES website and JRMO SOPs. 
 
Annual Safety Reporting  
The CI will send the Annual Progress Report to the main REC using the NRES 
template (the anniversary date is the date on the MREC “favourable opinion” 
letter from the MREC) and to the sponsor. Please see NRES website and 
JRMO SOP for further information 
 
Overview of the Safety Reporting responsibilities 
The CI/PI has the overall pharmacovigilance oversight responsibility. The CI/PI 
has a duty to ensure that safety monitoring and reporting is conducted in 
accordance with the sponsor’s requirements.  
 
Please outline the process/organisation within the study team to ensure that all 
SAE reporting is conducted in accordance with the sponsor’s timelines.  
 
 
 
16. MONITORING &AUDITING 
 
Quarterly meetings will be held with by the research team to review the progress of 
the study and discuss and issues that may arise. 
 
 
 
17. TRIAL COMMITTEES 
 
N/A. 
 
 
18. FINANCE AND FUNDING 
Funding for this study will be provided by Metagenics. 
 
 
19. INDEMNITY  
 
The sponsor is Queen Mary, University of London, who provide insurance for this 
study.  
 
 
20. DISSEMINATION OF RESEARCH FINDINGS: 
 
All relevant data from this study will be submitted to peer review journals for 
publication following the termination of the study in line with sponsor and trust 
publication policy.  
 
 

 
 
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21. REFERENCES 
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lease use a standard referencing system. 
 
 
22. APPENDICIES 
Please do not include the Participant Information Sheet and Consent Form; these 
should be stand alone documents