Vascular Effects of Regular Cigarettes Versus electronIc Cigarette Use
Phase: NA
Conditions: Vascular Diseases
Sponsor: University of Dundee

Page 1 of 23 
Study Protocol 
 
Vascular EffectS of regUlar cigarettes Versus electronIc cigarette USe 
Study Acronym 
VESUVIUS  
Sponsor 
University of Dundee – NHS Tayside 
Sponsor R&D Number 
2014CV10 
Funder 
British Heart Foundation 
Chief Investigator 
Dr Jacob George 
REC Number 
16-ES-0087 
ISRCTN Number 
      
Version Number and Date 
V5.0 08/05/2018 
 
 
 
 

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SUMMARY ................................................................................................................ 6 
1 
INTRODUCTION ......................................................................................... 7 
1.1 
BACKGROUND & RATIONALE FOR STUDY .................................... 7 
2 
STUDY OBJECTIVES & OUTCOMES ........................................................ 9 
3 
STUDY DESIGN .......................................................................................... 9 
3.1 
STUDY DESCRIPTION ...................................................................... 9 
3.2 
STUDY FLOWCHART ...................................................................... 10 
3.3 
Study Matrix ..................................................................................... 11 
4 
STUDY POPULATION .............................................................................. 12 
4.1 
NUMBER OF PARTICIPANTS ......................................................... 12 
4.2 
INCLUSION CRITERIA .................................................................... 12 
4.3 
EXCLUSION CRITERIA ................................................................... 12 
5 
PARTICIPANT SELECTION AND ENROLMENT ..................................... 13 
5.1 
IDENTIFYING PARTICIPANTS ........................................................ 13 
5.2 
CONSENTING PARTICIPANTS ....................................................... 13 
5.3 
SCREENING FOR ELIGIBILITY ....................................................... 13 
5.4 
INELIGIBLE AND NON-RECRUITED PARTICIPANTS .................... 14 
5.5 
RANDOMISATION ........................................................................... 14 
5.5.1 
Randomisation ........................................................................ 14 
5.5.2 
Intervention Allocation ............................................................. 14 
5.5.3 
Withdrawal procedures ............................................................ 15 
6 
STUDY & SAFETY ASSESSMENTS ........................................................ 15 
7 
DATA COLLECTION & MANAGEMENT .................................................. 16 
7.1 
Data Collection ................................................................................. 16 
7.2 
Data Management System ............................................................... 16 
8 
STATISTICS AND DATA ANALYSIS ....................................................... 17 
8.1 
SAMPLE SIZE CALCULATION ........................................................ 17 
8.2 
PROPOSED ANALYSES ................................................................. 17 
8.3 
Missing data ..................................................................................... 17 
8.4 
TRANSFER OF DATA ...................................................................... 18 
9 
TRIAL MANAGEMENT AND OVERSIGHT ARRANGEMENTS................ 18 
9.1 
TRIAL MANAGEMENT GROUP ....................................................... 19 
9.2 
TRIAL/STUDY MANAGEMENT ........................................................ 19 
9.3 
INSPECTION OF RECORDS ........................................................... 19 

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10 
GOOD CLINICAL PRACTICE ................................................................... 19 
10.1 
ETHICAL CONDUCT OF THE STUDY ............................................. 19 
10.1.1 
Confidentiality .......................................................................... 19 
10.1.2 
Data Protection ........................................................................ 20 
10.1.3 
Insurance and Indemnity ......................................................... 20 
11 
STUDY CONDUCT RESPONSIBILITIES .................................................. 20 
11.1 
PROTOCOL AMENDMENTS, deviations and breaches ................... 20 
11.2 
STUDY RECORD RETENTION ....................................................... 21 
11.3 
END OF STUDY ............................................................................... 21 
12 
REPORTING, PUBLICATIONS AND NOTIFICATION OF RESULTS ....... 21 
12.1 
AUTHORSHIP POLICY .................................................................... 21 
12.2 
PUBLICATION ................................................................................. 21 
12.3 
PEER REVIEW ................................................................................ 21 
13 
REFERENCES .......................................................................................... 22 
 
 
 

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PROTOCOL APPROVAL 
VESUVIUS: Vascular EffectS of regUlar cigarettes Versus electronIc cigarette Use 
 
Signatures 
By signing this document I am confirming that I have read, understood and approve 
the protocol for the above study. 
 
      
 
 
 
Dr Jacob George 
Chief Investigator 
Signature 
 
Date 
 
 
 
 
      
 
 
 
Dr Daniel Levin 
Individual Responsible for 
Statistical Review 
Signature 
 
Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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LIST OF ABBREVIATIONS 
GCP 
Good Clinical Practice 
IMP 
Investigational Medicinal Product 
ISF 
Investigator Site File 
TMF 
Trial Master File 
SOP 
Standard Operating Procedure 
CRF 
Case Report Form 
AE 
Adverse Event 
SAE 
Serious Adverse Event 
AR 
Adverse Reaction 
UAR 
Unexpected Adverse Reaction 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
CNORIS 
Clinical Negligence and Other Risks Scheme 
SmPC 
Summary of Product Characteristics 
NRES 
National Research Ethics Service 
REC 
Research Ethics Committee 
MHRA 
Medicines and Healthcare Products Regulatory 
Agency 
 
 
 

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SUMMARY 
Electronic cigarettes (EC) are gaining popularity as an alternative to Traditional 
Cigarettes (TC). Despite not containing all the harmful substances seen in TC, EC 
are known to contain impurities that may have a detrimental impact on human health. 
The effects of ECs compared with TC on vascular function, inflammation and 
oxidative stress are unknown. It is increasingly recognised that nicotine itself has 
significant atherothrombotic effects. Therefore, clinicians are unable to confidently 
recommend ECs as a less risky alternative to TC.  
Hypothesis: Endothelial function will be improved on EC compared to TC when 
measured by flow mediated dilatation (FMD). 
Aims: To assess the effects of EC-nicotine and EC-nicotine free on endothelial 
function as compared to TC.  
Design: Single centre, 2-cohort parallel group, randomised controlled trial. with a 
comparator TC arm 
Population: ≥18 years old, current TC smokers. Excluding those with cardiovascular  
disease (hypertension allowed). 
Interventions: 2 groups will be randomised to either EC-nicotine or EC-nicotine free 
for 4 weeks.1 group of TC smokers will be recruited as a comparator arm 
Outcomes: the primary outcome is the change in endothelial stiffness as measured 
by Flow Medicated Dilatation between the TC and EC-nicotine free groups. 
Secondary outcomes are change in endothelial stiffness between EC-nicotine free 
and EC-nicotine groups and change in oxidative stress between all 3 groups. 
Assessments: performed at baseline and 4 weeks. 
Samples size: 135 participants randomised to 2 groups EC-nicotine (n=45) and EC-
nicotine free (n=45) plus a comparator arm of TC (n=45), will have a 80% power to 
detect an improvement in FMD of 2.0% and 1.0% in the EC-nicotine free and EC-
nicotine groups respectively compared to the TC group at 5% significance. This 
allows for a 10%, 15%, and 20% drop-out rates in the TC, EC-nicotine & EC-nicotine-
free groups respectively. 
Statistical Analysis: Change in outcome measures between interventions will be 
assessed by a linear contrast test, and a linear regression including the baseline 
FMD level and experimental group as covariates. 
Potential benefit: This study will provide further information on the potential use these 
devices and the risks and/or benefits associated with them, specifically in relation to 
vascular health. 
 

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1 
INTRODUCTION 
1.1 
BACKGROUND & RATIONALE FOR STUDY 
How safe are Electronic Cigarettes? 
Electronic cigarettes, also known as e-cigarettes (EC), are increasingly widely used 
in an attempt to reduce rates of smoking traditional cigarettes (TC). There have been 
many controversial claims and counter-claims by various experts, social scientists 
and politicians in the media regarding its effectiveness and safety1-3. The Medicines 
Health Regulatory Authority (MHRA) has stated that EC are to be licensed and 
regulated as part of European Union regulations which have imposed tougher 
restrictions on EC sales and marketing. The MHRA also state that although the 
amount of harmful carcinogens is only one thousandth of that contained in a TC, that 
the effects of these toxins, even at much smaller levels are unknown. They are 
known to be modestly effective in helping smokers to quit, with similar achievement 
of abstinence as with nicotine patches4. In one study, sustained 50% reduction in the 
number of cig/day at week-24 was shown in 32.5% of participants and sustained 
smoking abstinence at 24-weeks was observed in 22.5% participants5.  A recent 
study looking at using ECs for smoking cessation, published in the Lancet, has 
concluded that “more research is urgently needed to clearly establish their overall 
benefits and harms at both individual and population levels6 . EC are battery-powered 
devices that simulate tobacco smoking by using a heating element that vaporizes a 
liquid solution containing a mixture of nicotine and flavourings, while others release a 
flavoured vapour without nicotine. They are being marketed as a safer alternative to 
TC.  
The benefits of EC on vascular function in terms of the reduction in tar and other 
contaminants that are present in TC are currently unknown. They are generally 
thought to deliver smaller quantities of nicotine compared with TC. Furthermore, TC 
contain >4,000 chemicals, including high levels of nicotine, carbon monoxide, 
acrolein, and pro-oxidant compounds7. However, recent evidence from the United 
States Food and Drug Administration (FDA) has shown that EC may not be 
completely harmless and in fact, may contain carcinogens such as nitrosamine and 
toxic chemicals such as diethylene glycol8 which are absent from TC. Subsequent 
analysis has also revealed the presence of tobacco-specific impurities suspected of 
being harmful to humans such as anabasine, myosmine, and β-nicotyrine in a 
majority of the samples tested9. Anabasine has been shown to experimentally affect 
adrenomedullary catecholamine excretion in rats10. Myosamine causes DNA damage 
through pyridyloxobutylation similar to the tobacco-specific nitrosamines (TSNA)11. β-
nicotyrine is an inactivator of CYP2A6, which is the Cytochrome P450 pathway for 
nicotine metabolism12. 
The impact of using EC on the cardiovascular system in particular is as yet unknown. 
The Medicines Health Regulatory Authority (MHRA) has stated that EC are to be 
licensed and regulated as part of the European Union regulations which have 
imposed tougher restrictions on EC sales and marketing13. The MHRA also state that 
the amount of harmful carcinogens mentioned in the paragraph above is only one 
thousandth of that contained in a TC but also concede that the effects of these 

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toxins, even at much smaller levels are unknown14. Following this, EC’s are banned 
in health facilities in countries like the Republic of Ireland15 and parts of Wales16.  
 
What are the known cardiovascular effects of nicotine? 
While EC are sold on the premise that they are a much cleaner alternative to TC in 
that they do not contain harmful substances like tobacco and tar, they do 
nevertheless contain nicotine. What is increasingly clear is the detrimental effect of 
nicotine itself on vascular health. It is well established that nicotine increases the 
heart rate, blood pressure, platelet aggregation and pathological angiogenesis and 
down-regulation of nitric oxide17,18 Recent studies have shown that nicotine (the 
addictive component of cigarettes) binds to high affinity nicotinic acetylcholine 
receptors (nAChRs) cell–surface receptors. This binding has been shown to 
accelerate the atherogenic process. Unlike free radicals, benzene and tar, nicotine is 
not thought to initiate plaque formation. However, it accelerates the growth of a pre-
existing plaque. This is achieved by vascular smooth muscle cells (VSMC) 
proliferation, transforming growth factors (TGF) α and β, vascular endothelial growth 
factors (VEGF) and fibroblast growth factor (FGF) and concomitant suppression of 
apoptosis19,20 Acute exposure to nicotine alone also worsens endothelial dysfunction 
in long term smokers21 
Further detrimental adverse vascular effects of TC 
The complete adverse patho-biological effects of smoking TC are unknown. Aside 
from containing high amounts of nicotine and the attendant associated effects on 
plaque formation and atherosclerosis as described above, TC smoking is also known 
to impair endothelial function 22,23 because it contains large amounts of harmful 
oxidants that cause oxidative stress damage to vascular endothelium24, even in 
passive smokers25 by nitric oxide (NO) uncoupling as well as arginase activation26. 
Other effects include increasing production and release of endothelin, the up-
regulation of matrix metalloproteinases27 . Finally, cigarette smoking is also 
thrombogenic in that it causes changes in platelet membrane fluidity and Na+/K+ 
ATPase activity28 as well as increases platelet aggregation29. 
Amato et al30 compared the effect of light versus heavy cigarettes on brachial artery 
FMD and found no difference in chronic smokers. This suggests that EC may not be 
the safe alternative that it is being promoted as but as of now, there is no data to 
suggest that this is true, hence this proposed study. 
There are many ongoing debates on the role of EC and whether or not they are a 
legitimate replacement for TC. Therefore a clinical trial examining the cardiovascular 
effects of EC is needed to better inform regulatory authorities and governments 
regarding the possible cardiovascular risks or benefits of these devices. This 
proposed study will provide some preliminary useful information on which further 
definitive studies can later build on. 
 
 

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2 
STUDY OBJECTIVES & OUTCOMES 
Table 1: Primary Objectives and Outcome Measures 
Primary Objective 
Outcome Measure 
Timepoints measured 
To determine the effect of 
EC-nicotine free on vascular 
function as compare to TC. 
Change in FMD between the 
TC group and the EC-
nicotine free groups  
0 and 4 weeks 
 
 
Table 2: Secondary Objectives and Outcome Measures 
Secondary Objectives 
Outcome Measures 
Timepoints measured 
To compare the effects of 
EC-nicotine vs EC-nicotine 
free on vascular function. 
Change in FMD between 
EC-nicotine and EC-nicotine 
free groups  
0 and 4 weeks 
To compare the effects of 
EC-nicotine free vs TC on 
vascular function. 
Change 
in 
Pulse 
Wave 
Velocity between the TC 
group and the EC-nicotine 
free groups. 
Change 
in 
Augmentation 
Index@75bpm between the 
TC group and the EC-
nicotine free groups 
0 and 4 weeks 
To compare the effects of 
TC vs EC vs EC nicotine 
free on oxidative stress.  
Change in total F2-
Isoprostanes between the 
TC and EC groups 
0 and 4 weeks 
 
3 
STUDY DESIGN 
3.1 
STUDY DESCRIPTION 
VESUVIUS is a single-centre, (Tayside) randomized, 2-cohort parallel group of EC-
nicotine and flavouring versus EC- nicotine free and flavouring versus comparator TC 
arm. These three arms will assess the differential benefits between (1) TC vs EC (2) 
the presence of nicotine in EC-nicotine versus EC-nicotine free. 
 
 
 

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3.2 
STUDY FLOWCHART 
 
(  
BASELINE & RANDOMISATION 
Visit 1 Baseline – week 0 Inclusion/Exclusion 
criteria 
 
Visit assessments 
Demographics 
Past Medical History 
Smoking History 
Blood pressure 
Flow Mediated Dilatation 
Vascular Stiffness 
Blood tests 
CO breath test 
IDENTIFY PARTICIPANTS 
 
 
 
TREATMENT PHASE  
4 weeks 
Excluded  
All participants aged 18+: 
 
local newspaper adverts 
 
posters in public areas 
 
twitter campaigns etc 
Participant Information Sheet sent out  
Excluded  
PHM, Smoking history 
Participant contacts study team  
END OF STUDY 
Discontinued treatment 
(n=23) 
Discontinue study treatment 
Analysis 

Intention to treat  

Completed 4 weeks of treatment (n=112) 
Visit 2 - 4 Weeks 

Visit assessments 
Flow Mediated Dilatation 
Vascular Stiffness  
Blood tests 
CO breath test
(n=45) 
Traditional 
cigarettes 
(n=45) 
e-cigarettes 
nicotine free 
(n=45) 
e-cigarettes 
with nicotine 
Randomised to treatment arm (n=90) 
Comparator TCarm (n=45) 

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3.3 
STUDY MATRIX 
Time 
Baseline Week 4^ 
Informed Consent 
X 
 
Inclusion/exclusion criteria 
X 
 
Demographics 
X 
 
Past Medical History 
X 
 
Smoking history 
X 
 
Blood pressure 
X 
X 
*Pregnancy test (if appropriate) 
X 
 
Flow Mediated Dilatation 
X 
X 
Vascular Stiffness 
X 
X 
Blood test 
X 
X 
CO Breath test 
X 
X 
∞Smoking Diary 
X 
X 
Randomisation 
X 
 
Training on use of EC (if allocated) 
X 
 
Supply EC (if allocated) 
X 
 
Serious Adverse Events  
 
X 
Adherence 
 
X 
Provision of smoking cessation support details 
 
X 
^ The week 4 visit will be carried out at 4 weeks ± up to 10 days. 
* Urine pregnancy testing on females of childbearing potential who do not abstain from sexiary will be 
given out at baseline and collected at the 4 week visit 
Patients will also be supported as required by regular phone or text to encourage 
adherence.  
Participants who complete the study will receive a monetary incentive in the form of 
shopping vouchers. vouchers will be payable at the end of the study to the maximum 
value of £40. this incentive is purely for those who complete both visits as a way to 
try and minimise dropouts. this will only work if payment is made on completion of 
study. however if the study team have to withdraw a participants a £20 of shopping 
voucher could be paid per visit attended 

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4 
STUDY POPULATION 
4.1 
NUMBER OF PARTICIPANTS 
Number: 135 participants. 
Recruitment period: 22 months 
Dropout rate: it is anticipated that the dropout rates will vary depending on which 
group they have been randomised to. Assuming 10%, 15%, and 20% drop-out rates 
in the TC, EC-nicotine & EC-nicotine free groups respectively, we will require 135 
participants to provide a final evaluable sample of 112 participants. 
4.2 
INCLUSION CRITERIA 
 
Aged 18 years and over 
 
Currently smoking ≥15 tobacco cigarettes per day for at least 2 years, or roll-
up tobacco equivalent (cigar or pipe smokers will not be included). 
 
Willing to stop tobacco cigarettes for period of study if required 
 
Willing not to use electronic cigarettes if required 
 
Able to give informed consent 
4.3 
EXCLUSION CRITERIA 
 
Pregnant or lactating. 
 
Women of childbearing potential who do not abstain from sex or use effective 
contraception. 
 
On current prescribed medication for cardiovascular disease. 
 
History of cardiovascular disease (excluding hypertension), diabetes, active 
malignance or chronic renal disease. 
 
Nut allergy  
 
Participation in another clinical trial (other than observational trials and 
registries) with an investigational product and/or intervention within 30 days 
before visit 1. 
 
Use of electronic cigarettes within the last month. 
Contraceptive advice to participants 
Women of child-bearing potential, who do not abstain from sex, must be willing to 
have pregnancy testing prior to study entry. In addition, women of child bearing 
potential, who are sexually active, must be willing to use a form of a medically 
approved birth control method eg; 
 
Combined Oral Contraceptive Pill 
 
Placement of a intrauterine device - ‘ coil’  
 
Barrier methods of contraception: male condom only 
 
Established use of oral, injected, transdermal or implanted hormonal methods 
of contraception 
 
Male partner sterilisation (followed by the appropriate post-vasectomy 
documentation of the absence of sperm in the ejaculate). 

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5 
PARTICIPANT SELECTION AND ENROLMENT 
5.1 
IDENTIFYING PARTICIPANTS 
Participants will be recruited via a publicity campaign using a range of methods: 
 
Posters/leaflets distributed in various places which may include: 
Football/Bingo halls/ Bowling      
Smoking shelters 
Pubs 
Hospital main entrances/ hospital clinics  
Shopping Centres/Supermarkets/Pubs/etc. 
Benefits offices/Post offices etc. 
Sheltered Housing /Housing Associations 
Community and charitable outreach programs 
GP waiting rooms 
Local company newsletters/payslips 
 
Advertisements in local newspapers and on local radio stations. 
 
Social media including twitter and Facebook. 
 
Invitation to those eligible on registered research volunteer databases held by 
the research team and the Scottish Health Research Register (SHARE). 
 
Potential participants will be identified via the Scottish Primary Care Research 
Network (SPCRN).   
 
Existing data-bases of smokers where permission has been granted to be 
approached for research purposes 
5.2 
CONSENTING PARTICIPANTS 
Obtaining consent will be carried out by the PI, researcher or other local delegated 
individual in accordance with TASC SOP07: Obtaining Informed Consent from 
Potential Participants in Clinical Research 
Potential participants who have seen the publicity campaign will be invited to contact 
the study team via phone or email. The researcher or other delegated individual will 
discuss the study via a phone call and assess for suitability. If the participant is 
thought to be suitable a participant information sheet (PIS) will be posted to the 
participant and a baseline visit arranged, all participants will be given at least 24 
hours to consider their participation. If they decide to participate, informed consent 
will be taken by the researcher or other delegated individual and eligibility by all the 
criteria confirmed. 
Where a participant requests to speak with a physician from the study team the 
consent process will not be completed until the participant had spoken to the 
physician and had all their questions answered to their satisfaction. 
5.3 
SCREENING FOR ELIGIBILITY 
Participants who contact the research team expressing their interest in the study will 
be contacted and their suitability will be assessed as above prior to consent and 
booking a screening visit. Eligibility will be confirmed with the participant during the 
screening visit. The study visits will be performed within the Division of 

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Cardiovascular and Diabetes Medicine, University of Dundee, Ninewells Hospital 
Dundee. 
 
5.4 
INELIGIBLE AND NON-RECRUITED PARTICIPANTS  
The reason(s) for ineligibility will be explained to participants and any questions they 
have will be answered. They will be thanked for their participation and any relevant 
information from this will be added to their hospital notes and be communicated to 
their GP where the participant consents for this to happen. 
 
5.5 
RANDOMISATION  
5.5.1 Randomisation  
Randomisation will be carried out in a 1:1 fashion for the 2 randomised arms via a 
centrally controlled web-based GCP compliant randomisation system, TRusT, run by 
Tayside Clinical Trials Unit (TCTU). To ensure balanced assignment across critical 
variables, a minimisation algorithm will be employed, using baseline age (≤40 years; 
>40 years), sex (male; female) and smoking pack years (≤20 pack years; >20 pack 
years). Randomisation will be performed by the PI, researcher or other local 
delegated individual.  
Recruitment will stop when the required number is reached in each arm (maximum 
45 per arm) 
The TC arm will not be randomised. 
To facilitate recruitment and minimise dropouts we propose to recruit the TC smoking 
arm as a separate non randomised group. Potential participants will be identified and 
approached in exactly the same ways as for the 2 EC arms. All participants will be 
asked if they wish to continue to smoke TC or to be randomised to one of the EC 
arms. This will enable us to recruit volunteers who would find it too difficult to stop 
their TC for 4 weeks. It will also avoid disappointment for those volunteers who are 
ready to stop smoking and do not wish to continue with their TC, which has the 
potential to lead to further dropouts. This is an unblinded study with the exception of 
the measurement of endothelial function and vascular stiffness which will be 
performed by a single blinded operator. 
5.5.2 Intervention Allocation 
Participants will initially be asked if they wish to continue smoking TC or if they would 
like to be randomised to one of the EC arms. Participants who wish to be randomised 
to one of the EC arms will be randomised to one of two groups in a 1:1 fashion. The 
groups will be: 
a) Switch to electronic cigarettes containing nicotine plus flavour 
b) Switch to electronic cigarettes containing flavour alone 
Participants wishing to join the TC arm will be asked to continue their usual daily 
smoking habits, however, they will be asked not to smoke more than 30 cigarettes 
per day. Participants in this group will be asked not to use electronic cigarettes for 
the four week period of the trial.  

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For the groups receiving the electronic cigarettes, the Researcher or delegated 
individual will “dispense” the electronic cigarettes to participants. Instructions and 
explanation of how to use the electronic cigarettes will be given. Participants will be 
asked not to smoke tobacco cigarettes during the trial period. 
5.5.3 Withdrawal procedures 
When participants withdraw from active treatment the study team will ask them if they 
are willing to remain in the study and complete the study assessments. 
If participants withdraw from the study and do not wish to return for study visits, 
contact with them will be maintained by the PI, trial manager, researcher or research 
assistant to ensure resolution of adverse event(s). If withdrawal is due to an AE it will 
be logged as such on the Adverse Event Log. 
Participants are free to withdraw from the study at any time.  The reasons, if known, 
will be recorded in the medical casenotes and CRF. Although a participant is not 
obliged to give reason(s) for withdrawing prematurely, if the participant appears lost 
to follow up, the CI will make a reasonable effort to ascertain the reason(s), while 
fully respecting the individual’s rights, and will demonstrate that everything possible 
was done in an attempt to find any participant lost to follow-up. Those lost to follow-
up or withdrawn will be identified and a descriptive analysis of them provided, 
including the reasons for their loss and its relationship to treatment and outcome. 
6 
STUDY & SAFETY ASSESSMENTS 
See Study Matrix, Section 3.3. 
Flow Mediated Dilatation  
Participants will be asked to fast from midnight the night before their visits, with the 
visit being performed in the morning. Participants who are electing to take part in one 
of the 2 randomized arms will also be asked not to smoke for 4 hours prior to their 
visits. Particpants electing to take part in the tobacco cigarette arm do not have to 
withhold cigarettes for 4 hours but they will be asked to keep their smoking practises 
consisted before both visits. Endothelial function will be measured non-invasively at 0 
and 1 month using the standard technique of flow mediated dilatation (FMD) of the 
brachial artery in response to hyperemia and to sublingual GTN.  Brachial artery 
diameter and flow are determined by M mode and Doppler ultrasound as described 
by us previously31.  FMD measurements are represented as change in arterial 
diameter from baseline.  We will follow established protocols 32. FMD is the technique 
we used recently to show a beneficial effect of allopurinol on endothelial function in 
subjects with chronic kidney disease33 and in coronary artery disease34.  Our in 
house CV for FMD measurements is 3%. The FMD will be measured by a single 
experienced operator who will be blinded to treatment arms. 
Vascular Stiffness 
Pulse wave velocity (PWV) and pulse wave analysis (PWA) measurements including 
augmentation index corrected for heart rate (AIx@75) will be performed at baseline 
and 4 weeks using Sphygmocor™ as previously detailed35.  The FMD, PWV and 
PWA will be measured by a single experienced operator who will be blinded to 
treatment arms. These two measures have been validated as markers of 
cardiovascular risk36. 

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Blood tests 
The following blood tests will be carried out: 
1) Oxidised LDL  
2) High-sensitivity CRP 
3) Platelet reactivity 
4) Peripheral blood mononuclear cells for Oxidative stress NRf2 expression only 
(30 patients only, to be randomly selected). 
Blood will also be stored, with patient consent, for future biomarker analysis. 
7 
DATA COLLECTION & MANAGEMENT 
7.1 
DATA COLLECTION 
Data will be recorded at each visit (baseline and 4 weeks) on a paper CRF by the 
Researcher or delegated person and subsequently transcribed into the Data 
Management System database. The CRF will be used as source data but data 
relevant to a participant’s general medical history will be recorded also in their 
medical casenotes. Data from the FMD and vascular stiffness will be entered into the 
Excel database as below. 
7.2 
DATA MANAGEMENT SYSTEM 
Microsoft Excel will be used for data management and data management will be 
conducted as per TASC SOP48. The computer running EXCEL will be password 
protected and kept in a locked room. To prevent unauthorised access, computers will 
be accessible only to named individuals in the delegation log. Each individual will be 
given a password to log onto the computer. EXCEL will be used for the results, which 
are transcribed from paper CRFs. A spread sheet will be opened in EXCEL and will 
record only outcomes that are specified in the protocol. This spreadsheet will be 
saved on the University of Dundee secure server, backed up daily. A Disaster 
Recovery Plan is in place. No scripts or macros will be added to the EXCEL 
spreadsheet The PI/Statistician responsible for data analysis will check the 
spreadsheet template for accuracy and format before any entry begins. Modification 
of the spreadsheet may occur until it is fit for purpose. The trial spreadsheet will be 
password protected and a copy of this password kept with the division secretary. 
Data entry will be a delegated responsibility. In order to be GCP compliant, data is 
entered the same day as visit or as soon as possible afterwards. The spreadsheet 
will be saved each time with date of entry thus an audit trail will be created on the 
EXCEL file. Subjects will be identified only by subject number(s) and age at consent. 
No participant identifiable information will be stored on the results spread sheet. 
Subjects will be cross checked with the Subject Log and/or Screening Log. As the 
paper CRF is completed, it will be checked for empty fields and fields outside of 
normal ranges. Data checking will include looking for fields outside of the subjects’ 
normal ranges, which may lie within the normal expected general range. These will 
be highlighted by the Insert Comment facility available on EXCEL which also includes 
the name of the person highlighting the comment. All queries will be clarified with the 
PI or CI and any actions recorded. Any changes to the data in the CRF will be made 
a single line drawn through, initialled and dated. The original data will be clearly 
visible. The data on the EXCEL file will be always verifiable against the paper CRFs.  
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Data entry and checking will be undertaken by a single entry with a second look or a 
Single entry with a second individual reading aloud. The data entry and checking 
process will be decided according to risk. Data that is recorded in the CRF that is not 
source document in itself will be consistent with the source documents or the 
discrepancies explained. Checks will be made on all missing values and values out 
with normal or expected ranges and that values entered are of the correct type: i.e. 
numerical instead of text. Logical checks will be performed to ensure consistent 
reporting between relevant fields and that there are no differences between fields. 
Data checking will continue until all missing data and/or inconsistent values have 
been corrected or clarified. When data checking is complete, with no outstanding 
data queries, the database will be locked, using the Protect Worksheet function of 
EXCEL, as FINAL RESULTS. The EXCEL spreadsheet will remain archived on the 
Dundee University secure server for 10 years for the retention of essential 
documents, thereafter it will be deleted. 
8 
STATISTICS AND DATA ANALYSIS 
8.1 
SAMPLE SIZE CALCULATION 
The primary endpoint measurement is change in brachial artery FMD, which is 
expressed as the maximum FMD percentage change from baseline. Based on our 
existing data for this measure (mean response 7.8%, SD=3.5%), and assuming 
mean FMD reductions of 2%, 1% and 0% in the EC-Nicotine-free, EC-Nicotine, and 
TC groups respectively with a SD of the change in FMD =3.0% (based on our 
previous study31), and a using a sample size calculation using Linear Contrast 
Tests37 a sample size of 36 subjects in each group will have 80% power to detect an 
improvement in FMD of 2.0% and 1.0% in the EC-nicotine-free and EC-nicotine 
groups respectively compared to the TC group at 5% significance. Assuming 10%, 
15%, and 20% drop-out rates in the TC, EC-nicotine & EC-nicotine-free groups 
respectively, we will require 135 completed subjects. All drop-outs will be replaced to 
achieve 36 completed subjects in each group. 
8.2 
PROPOSED ANALYSES 
Descriptive statistics in the form of means and standard deviations for continuous 
variables and percentages and denominators will be tabulated for baseline and at 
each visit. The dependent variable will be assessed for approximation to a normal 
distribution and transformed if necessary. Pre-specified subgroup analyses will be 
completed by fitting the appropriate interaction term in the regression model and if 
significant outcomes will be presented separately by level of subgroup. The FMD 
response relationship will be assessed by a linear contrast test, and a linear 
regression including the baseline FMD level and experimental group as covariates. 
The statistical analysis will be carried out in accordance to TASC SOP “Statistical 
Analysis Plans for Clinical Trials of Investigational Medicinal Products” by the study 
team statistician. 
8.3 
MISSING DATA 
The primary analysis will be on a per protocol basis. We will perform an intention to 
treat analysis as a secondary analysis..The extent of missing data will be examined 

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and the reason for drop-out ascertained. Multiple imputation will be used to impute 
missing values if the assumptions for missing at random (MAR) data are met.  
8.4 
TRANSFER OF DATA 
Transfer of data out with the study site will not occur. 
9 
SERIOUS ADVERSE EVENTS 
9.1 
RECORDING AND REPORTING SAES 
Adverse events will not be recorded in this non-CTIMP study unless they are deemed 
as serious (SAE). All SAE will be recorded in a SAE Log. 
A serious adverse event (SAE) is defined as an untoward occurrence that: 
 
results in death; 
 
is life-threatening; 
 
requires hospitalisation or prolongation of existing hospitalisation; 
 
results in persistent or significant disability or incapacity; 
 
consists of a congenital anomaly or birth defect;  
 
or is otherwise considered medically significant by the investigator 
Note: Hospitalisations for treatment planned prior to randomisation and 
hospitalisation for elective treatment of a pre-existing condition will not be considered 
as an SAE. However any serious adverse events occurring during such 
hospitalisation will be recorded. 
An SAE occurring to a research participant will be reported to the main REC and 
Sponsor where in the opinion of the Chief Investigator (CI) 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. 
Other SAE will not be reported. 
SAEs will be reported as per HRA guidelines to the REC and Sponsor: 
 
Who 
When 
How 
To whom 
SAE 
Chief 
Investigator 
(CI)  
Within 15 days of 
the CI becoming 
aware 
of 
the 
event  
SAE report form 
for non-CTIMPs  
Main REC for the 
trial. 
Sponsor 
Urgent 
safety 
measures 
Chief 
Investigator 
(CI) or sponsor  
Or exceptionally by 
local 
principal 
investigator (PI) 
(i) Immediately 
(ii) Within 3 days 
(i) By telephone 
(ii) Notice 
in 
writing setting 
out 
the 
reasons 
for 
urgent safety 
measures and 
the plan for 
further action 
Main REC,trial 
REC co-ordinator 
will acknowledge 
within 30 days 
Sponsor 
 
 

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10 TRIAL MANAGEMENT AND OVERSIGHT ARRANGEMENTS 
10.1 
TRIAL MANAGEMENT GROUP 
The trial will be co-ordinated by a Trial Management Group, consisting of the grant 
holders, Trial Manager, Research Nurse and Senior Trial Manager (TCTU). The Trial 
Management Group will also act as the Trial Steering Committee (TSC). A Data 
Monitoring Committee is not considered necessary as this is a relatively small study 
involving a marketed intervention, this role will be undertaken by the TMG also. 
10.2 
TRIAL/STUDY MANAGEMENT 
A Trial Coordinator will oversee the study and will be accountable to the CI. The Trial 
Coordinator or Research Nurse will be responsible for checking the CRFs for 
completeness, plausibility and consistency. However, this remains the overall 
responsibility of the CI. Any queries will be resolved by the CI or delegated member 
of the study team.  
A study-specific Delegation Log will be prepared, detailing the responsibilities of each 
member of staff working on the study.  
10.3 INSPECTION OF RECORDS 
The CI, PIs and all institutions involved in the study will permit study related 
monitoring, audits, and REC review. The CI agrees to allow the Sponsor or, 
representatives of the Sponsor, direct access to all study records and source 
documentation. 
11 GOOD CLINICAL PRACTICE 
11.1 ETHICAL CONDUCT OF THE STUDY 
The study will be conducted in accordance with the principles of good clinical practice 
(GCP). 
In addition to Sponsorship approval, a favourable ethical opinion will be obtained 
from an appropriate REC. Authorisation from an appropriate competent authority(s) 
and appropriate NHS R&D permissions(s) will be obtained prior to commencement of 
the study. 
11.1.1 Confidentiality 
All laboratory specimens, evaluation forms, reports, and other records will be 
identified in a manner designed to maintain participant confidentiality. All records will 
be kept in a secure storage area with limited access to study staff only. Clinical 
information will not be released without the written permission of the participant, 
except as necessary for monitoring and auditing by the Sponsor, its designee or 
Regulatory Authorities. The CI and study staff involved with this study will not 
disclose or use for any purpose other than performance of the study, any data, 
record, or other unpublished, confidential information disclosed to those individuals 
for the purpose of the study. Prior written agreement from the Sponsor or its 
designee will be obtained for the disclosure of any said confidential information to 
other parties. 

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11.1.2 Data Protection 
The CI and study staff involved with this study will comply with the requirements of 
the Data Protection Act 1998 with regard to the collection, storage, processing and 
disclosure of personal information and will uphold the Act’s core principles. The CI 
and study staff will also adhere, if appropriate, to the current version of the NHS 
Scotland Code of Practice on Protecting Patient Confidentiality.  Access to collated 
participant data will be restricted to the CI and appropriate study staff. 
Computers used to collate the data will have limited access measures via user 
names and passwords. 
Published results will not contain any personal data that could allow identification of 
individual participants. 
11.1.3 Insurance and Indemnity 
The University of Dundee and Tayside Health Board are Co-Sponsoring the study. 
Insurance –The University of Dundee will obtain and hold a policy of Public Liability 
Insurance for legal liabilities arising from the study. 
Tayside Health Board will maintain its membership of the Clinical Negligence and 
Other Risks Insurance Scheme (“CNORIS”) which covers the legal liability of Tayside 
in relation to the study. 
Where the study involves University of Dundee staff undertaking clinical research on 
NHS patients, such staff will hold honorary contracts with Tayside Health Board 
which means they will have cover under Tayside’s membership of the CNORIS 
scheme. 
Indemnity The Co-Sponsors do not provide study participants with indemnity in 
relation to participation in the Study but have insurance for legal liability as described 
above. 
12 STUDY CONDUCT RESPONSIBILITIES 
12.1 PROTOCOL AMENDMENTS, DEVIATIONS AND BREACHES 
The CI will seek approval for any amendments to the Protocol or other study 
documents from the Sponsor, REC and NHS R&D Office(s) as per TASC SOP 30 
for: Amendments to Clinical Research Studies. Amendments to the protocol or other 
study docs will not be implemented without these approvals.  
In the event that a CI needs to deviate from the protocol, the nature of and reasons 
for the deviation will be recorded in the CRF, documented and submitted to the 
Sponsor. If this necessitates a subsequent protocol amendment, this will be 
submitted to the Sponsor for approval and then to the appropriate REC and lead 
NHS R&D Office for review and approval.  
In the event that a serious breach of GCP is suspected, this will be reported to the 
Sponsor immediately using the form “Notification to Sponsor of Serious Breach or 
Serious Deviation”.  

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12.2 STUDY RECORD RETENTION 
Archiving of study documents will be carried out as specified in TASC SOP 13: 
Archiving Clinical Research Documentation and Data. All study documentation will be 
kept for at least 10 years.  
12.3 END OF STUDY 
The end of study is defined as last patient last visit (LPLV). The Sponsor, CI and/or 
the TSC have the right at any time to terminate the study for clinical or administrative 
reasons.  
The end of the study will be reported to the Sponsor and REC within 90 days, or 
15 days if the study is terminated prematurely. The CI will ensure that any 
appropriate follow up is arranged for all participants. 
A summary report of the study will be provided to the Sponsor and REC within 1 year 
of the end of the study 
13 REPORTING, PUBLICATIONS AND NOTIFICATION OF RESULTS 
13.1 AUTHORSHIP POLICY 
Ownership of the data arising from this study resides with the study team and their 
respective employers. On completion of the study, the study data will be analysed 
and tabulated, and a clinical study report will be prepared.  
13.2 PUBLICATION 
The clinical study report will be used for publication and presentation at scientific 
meetings. Investigators have the right to publish orally or in writing the results of the 
study. 
Summaries of results will also be made available to Investigators for dissemination 
within their clinical areas (where appropriate and according to their discretion). 
13.3 PEER REVIEW 
This study has been funded by the British Heart Foundation who have reviewed the 
grant application. 
Additional peer review of the protocol occurs via the Sponsorship Committee. 
Resulting publications will be reviewed by the referees of the journal to which the 
paper (and its protocol) will be submitted. 
 
 

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