An Open Label Parallel Group Study to Investigate the Optimum Methodology for the Use of LPS or GM-CSF as Challenge Agents on Healthy Participants by Assessing Inflammatory Biomarkers in Cantharidin-induced Skin Blisters, Peripheral Blood, and Urine
Phase: PHASE1
Conditions: Arthritis, Rheumatoid
Sponsor: GlaxoSmithKline

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GlaxoSmithKline group of companies
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TITLE PAGE
Protocol Title: An open label parallel group study to investigate the optimum 
methodology for the use of LPS or GM-CSF as challenge agents on healthy participants 
by assessing inflammatory biomarkers in cantharidin-induced skin blisters, peripheral 
blood, and urine.
Protocol Number: 207654 /01
Short Title: Skin blisters with systemic LPS or GM-CSF challenge
Compound
Number:
None
Sponsor Name and Legal Registered Address: 
GlaxoSmithKline Research & Development Limited
980 Great West Road
Brentford
Middlesex, TW8 9GS
UK
Medical Monitor Name and Contact Information can be found in the Study Reference 
Manual
Regulatory Agency Identifying Number(s): N/A
Approval Date: 07-AUG-2017
Copyright 2017 the GlaxoSmithKline group of companies.  All rights reserved.  
Unauthorised copying or use of this information is prohibited.

PPD
PPD
PPD

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PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE
DOCUMENT HISTORY
Document
Date
Amendment 1
07-Aug-2017
Original Protocol
08-May-2017
Amendment 1 07-Aug-2017
Overall Rationale for the Amendment: Change of GM-CSF (LEUKINE) 
administration from subcutaneous to IV infusion. This is due to liquid LEUKINE no 
longer being available from the manufacturer Sanofi-Aventis, however lyophilised 
LEUKINE is available. The guidelines for reconstitution of lyophilised LEUKINE 
prevent us from administering by subcutaneous as the volume would be too high. In order 
to remain consistent with clinical practice in dosing GM-CSF therapeutically the dose 
will be calculated using body surface area.

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Section # and 
Name
Description of Change
Brief Rationale
1 Synopsis
Clarification to primary objective 
to remove the wording “in 
cantharidin-induced skin blisters”
To clarify that the primary objective was 
based on blood and urine samples.
2 Schedule of 
Activities
Removal of Session 2 Day-1 
blood samples for mediators and 
flow cytometry
These were not required as the pre-
challenge blood samples will be 
collected on Day 1
2 Schedule of 
Activities
Clarification of footnote for
mediators blood sample time-
points for pre challenge and pre 
blister
Given the change of administration of 
GM-CSF (LEUKINE) from subcutaneous 
to IV infusion the minus 30 minutes pre-
blister induction was changed to pre 
challenge agent. The minus 5 minute 
time-point was clarified to mean pre-
blister induction.  
2 Schedule of 
Activities
Removal of injection site 
inspection assessment.  
GM-CSF dose now being given as an IV 
infusion and no longer as an injection 
and therefore not required.
2 Schedule of 
Activities
Clarification of footnote for flow 
cytometry sample time-points 
and addition of a 9hour 40 
minutes (10 hours post end of 
GM-CSF infusion) time-point for 
the GM-CSF group only. 
Given the change of administration of 
GM-CSF (LEUKINE) from subcutaneous 
to IV infusion over 2 hours the minus 30 
minutes pre-blister induction was no 
longer valid and so was changed to say
pre challenge agent. The minus 5 
minute time-point was clarified to mean 
pre-blister induction. The  9hour 
40minutes post blister induction time-
point was added as it is anticipated that 
the peak leukocyte count will occur 
between 8-12 hours and this will be 
required to make an accurate decision
at dose escalation.
2 Schedule of 
Activities
Session 2 Day 2 urine collection 
time-point removed and footnote 
12 moved to Day 1.
This was an error in the previous 
protocol as the urine collection was only 
until 12 hours post challenge agent.
4 Objectives and 
Endpoints 
Clarification to primary objective 
to remove the wording “in 
cantharidin-induced skin blisters”
To clarify that the primary objective was 
based on blood and urine samples.
5 Study Design
Change of dose for GM-CSF in 
the study design schematic.
Given the change of administration of 
GM-CSF (LEUKINE) from subcutaneous 
to IV infusion the minimum and 

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Section # and 
Name
Description of Change
Brief Rationale
maximum doses needed to change, as 
mentioned above. 
5.1 Overall 
Design
Study design schematic session 
2 changed to include pre-
challenge blood draw instead of 
minus 30 minutes and 09:40 
blood draw time-point added for 
GM-CSF only. 
Given the change of administration of 
GM-CSF (LEUKINE) from subcutaneous 
to IV infusion over 2 hours the minus 30 
minutes pre-blister induction was no 
longer valid. 
5.5.3 GM-CSF 
(Dose 
Justification)
Initial dose amended and
maximum dose amended. 
Removed the increments of 
5μg/kg.
Initial dose amended to reflect change 
of GM-CSF administration from 
subcutaneous to IV infusion dose and to 
remain consistent with clinical practice 
in dosing GM-CSF therapeutically the 
dose will be calculated by body surface 
area. Increments of 5 μg/kg was now far 
too high with the change from 
subcutaneous injection to IV infusion
and so the increments was removed. 
7.1 Treatment 
Administration
GM-CSF section changed: liquid 
LEUKINE details removed. Dose 
changed, route of administration 
changed to IV infusion and 
dosing instructions amended.
Liquid Leukine no longer available to 
buy from manufacturer. Lyophilised 
LEUKINE is available and will be given 
as an IV infusion.
7.3.3 GM-
CSF(Preparation/ 
Handling/ 
Storage/ 
Accountability)
Removal of dose injected as 
subcutaneous sentence. 
IV infusion dose now being used.
9.3.3 Inspection 
of blister and 
injection sites
Removal of inspection of 
injection site
No longer required due to change in 
administration of GM-CSF.

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TABLE OF CONTENTS
PAGE
PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE.....................................3
1.
SYNOPSIS...............................................................................................................8
2.
SCHEDULE OF ACTIVITIES (SOA).......................................................................11
3.
INTRODUCTION....................................................................................................14
3.1.
Cantharidin-induced skin blister assay........................................................14
3.2.
Study Rationale ..........................................................................................14
3.3.
Background ................................................................................................15
3.3.1.
LPS challenge .............................................................................15
3.3.2.
GM-CSF Challenge .....................................................................16
3.3.3.
Pharmacokinetics and pharmacodynamics of GM-CSF ...............17
3.4.
Benefit/Risk Assessment ............................................................................17
3.4.1.
Risk Assessment .........................................................................18
3.4.2.
Benefit Assessment.....................................................................24
3.4.3.
Overall Benefit : Risk Conclusion.................................................24
4.
OBJECTIVES AND ENDPOINTS...........................................................................24
5.
STUDY DESIGN ....................................................................................................27
5.1.
Overall Design ............................................................................................27
5.2.
Number of Participants ...............................................................................29
5.3.
Participant and Study Completion...............................................................29
5.4.
Scientific Rationale for Study Design ..........................................................30
5.5.
Dose Justification........................................................................................30
5.5.1.
Cantharidin ..................................................................................30
5.5.2.
LPS..............................................................................................31
5.5.3.
GM-CSF ......................................................................................31
6.
STUDY POPULATION...........................................................................................31
6.1.
Inclusion Criteria.........................................................................................32
6.2.
Exclusion Criteria........................................................................................32
6.3.
Lifestyle Restrictions...................................................................................34
6.3.1.
Caffeine and Alcohol....................................................................34
6.3.2.
Activity .........................................................................................34
6.4.
Screen Failures...........................................................................................34
7.
TREATMENTS.......................................................................................................34
7.1.
Treatments Administered............................................................................35
7.2.
Method of Treatment Assignment ...............................................................37
7.3.
Preparation/Handling/Storage/Accountability..............................................37
7.3.1.
Cantharidin ..................................................................................38
7.3.2.
LPS..............................................................................................38
7.3.3.
GM-CSF ......................................................................................38
7.4.
Treatment Compliance................................................................................39
7.5.
Concomitant Therapy..................................................................................39
8.
DISCONTINUATION CRITERIA.............................................................................39

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8.1.
Withdrawal from the Study..........................................................................39
8.2.
Lost to Follow Up........................................................................................40
9.
STUDY ASSESSMENTS AND PROCEDURES .....................................................40
9.1.
Adverse Events...........................................................................................41
9.1.1.
Time Period and Frequency for Collecting AE and SAE 
Information...................................................................................41
9.1.2.
Method of Detecting AEs and SAEs.............................................41
9.1.3.
Follow-up of AEs and SAEs.........................................................42
9.1.4.
Regulatory Reporting Requirements for SAEs .............................42
9.2.
Treatment of Overdose...............................................................................42
9.3.
Safety Assessments ...................................................................................43
9.3.1.
Height and body weight ...............................................................43
9.3.2.
Physical Examinations.................................................................43
9.3.3.
Assessment of blister sites ..........................................................44
9.3.4.
Vital Signs....................................................................................44
9.3.5.
Electrocardiograms......................................................................44
9.3.6.
Participant Diary Card..................................................................44
9.3.7.
Pain scale....................................................................................45
9.3.8.
Clinical Safety Laboratory Assessments ......................................45
9.4.
Genetics .....................................................................................................45
9.5.
Pharmacokinetics .......................................................................................45
9.6.
Pharmacodynamics ....................................................................................45
9.7.
Biomarkers .................................................................................................45
9.7.1.
Biomarkers in blood.....................................................................46
9.7.1.1.
Mediators blood sample .............................................46
9.7.1.2.
Flow cytometry blood sample .....................................46
9.7.1.3.
Ex vivo stimulation blood sample................................46
9.7.1.4.
Transcriptome blood sample ......................................46
9.7.2.
Biomarkers in urine......................................................................46
9.7.3.
Biomarkers in blisters...................................................................47
9.7.3.1.
Flow cytometry blister sample ....................................47
9.7.3.2.
Mediators blister sample.............................................47
9.8.
Health Economics OR Medical Resource Utilization and Health 
Economics..................................................................................................47
10. STATISTICAL CONSIDERATIONS........................................................................47
10.1.
Sample Size Determination ........................................................................47
10.2.
Populations for Analyses ............................................................................50
10.3.
Statistical Analyses.....................................................................................50
10.3.1.
Interim Analyses ..........................................................................51
11. REFERENCES.......................................................................................................52
12. APPENDICES........................................................................................................56
12.1.
Appendix 1: Abbreviations and Trademarks................................................56
12.2.
Appendix 2: Clinical Laboratory Tests.........................................................59
12.3.
Appendix 3: Study Governance Considerations..........................................60
12.4.
Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting .......................................64
12.5.
Appendix 5: Contraceptive Guidance and Collection of Pregnancy 
Information..................................................................................................69

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1.
SYNOPSIS
Protocol Title: An open label parallel group study to investigate the optimum 
methodology for the use of LPS or GM-CSF as challenge agents on healthy participants 
by assessing inflammatory biomarkers in cantharidin-induced skin blisters, peripheral 
blood, and urine.
Short Title: Skin blisters with systemic LPS or GM-CSF challenge
Rationale: This study aims to assess 2 models of systemic inflammatory response: 
exposure of healthy participants to systemic challenge with either LPS or GM-CSF. This 
will be done by measuring inflammatory mediators and cellular activation markers both 
in circulation and in skin blisters induced by exposure to cantharidin (as a model of local 
inflammatory focus). More specifically, the study will assess the time course of up-
regulation of circulating markers of inflammation (both cellular and soluble), in response 
to the systemic challenges and whether the challenges accentuate the inflammatory 
milieu of the blisters. The study will also assess urinary biomarkers of inflammation, and 
more specifically metabolites of prostaglandins, such as tetranor-PGDM. The literature 
reports that these stable urinary metabolites are elevated in patients with Duchenne 
muscular dystrophy and they are mechanistically relevant to treatments currently being 
evaluated at GSK. Data generated in this study will provide the foundation for future 
studies testing the effect of compounds that target key pathways in inflammation and 
autoimmunity.
Objectives and Endpoints:
Objective
Endpoint
Primary

To compare and define the time 
course of soluble and cellular 
circulating inflammatory 
biomarkers (and urinary 
prostaglandins for LPS only)
following systemic challenge with 
LPS or GM-CSF in healthy 
participants

For LPS only: time course and 
magnitude of upregulation of 
circulating TNF-α and IL-6 as well 
as urinary tetranor PGDM.

For GM-CSF only: time course and 
magnitude of upregulation of 
circulating total leukocyte numbers.

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Secondary

To compare soluble and cellular 
inflammatory biomarkers in 
cantharidin-induced skin blisters 
at baseline versus systemic 
challenge with LPS or GM-CSF 
in healthy participants

Soluble inflammatory biomarkers 
in skin blisters (may include but 
not limited to IL-1b, IL-2, IL-6, 
IL-8, IFNg, TNF-α, MCP-1, GM-
CSF, CRP) 

Blister volumes and differential 
cell counts (cellular activation 
markers may include, but not 
limited to expression of CD16, 
CD86, CD80, CD163, CD206, 
CD83, CD40, CD209, HLA-DR 
in blister leukocytes)

To define the time course of 
circulating soluble and cellular 
inflammatory biomarker 
upregulation following systemic 
challenge with LPS or GM-CSF 
in healthy participants

Time course of regulation of 
circulating soluble inflammatory 
biomarkers (may include but is 
not limited to IL-1b, IL-2, IL-6, 
IL-8, IFNg, TNF-α, MCP-1, GM-
CSF, CRP) 

Time course of regulation of 
circulating leukocyte numbers 
and cellular activation markers 
(may include, but not limited to 
expression of CD16, CD86, 
CD80, CD163, CD206, CD83, 
CD40, CD209, HLA-DR in 
circulating leukocytes)
Overall Design:
This is an exploratory parallel design study comprising of 2 parts. In both parts of the 
study, participants will have 2 sessions. Session 1 will include a blister induction
followed by blood draws and a blister harvest on each forearm. After a minimum of 14 
days to allow for blister healing, participants will return for Session 2 where they will 
receive either a LPS or GM-CSF in vivo challenge (4 completed participants in each 
group). Participants will then have another blister induction, followed by multiple, timed, 
blood draws and blister harvests.
For Part I, a dose exploration design is used to find a dose that provides a robust 
inflammatory response for both LPS and GM-CSF. Up to 6 cohorts of 4 participants will 
be tested and all cohorts will have 2 sessions, as described above. During this dose 
exploration phase the response to each challenge agent will be assessed by dose 
escalation meeting after 2 participants have been exposed to that agent. The response to 

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LPS will be primarily assessed based on IL-6, TNF-α and urinary tetranor PGDM. The 
response to GM-CSF will primarily be based on leukocyte counts. The decision whether 
to adjust the dose will be made by the Principal Investigator in consultation with the 
medical monitor based on the response outlined above and in the context of the safety 
and tolerability at that dose.
For Part II, an additional cohort of up to 8 participants may be enrolled if an interim 
analysis indicates that this would provide further precision on estimates of primary 
endpoints. The same 2-session design, as described above, would be used and 
participants will be dosed with LPS and GM-CSF at the same dose as the 8 evaluable 
participants from Part I . 
Number of Participants: It is estimated that up to 8 participants will be evaluated at the 
optimal dose for each challenge agent. Accounting for dose-findings, an estimated 24-30 
(maximum of 40, including replacement of any drop-outs) participants will be enrolled.
Treatment Groups and Duration: Participants will be randomised to receive either a 
LPS or GM-CSF in vivo challenge. The total study duration for each participant is 
approximately 13 weeks from screening to the final follow-up.

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2.
SCHEDULE OF ACTIVITIES (SOA)
Any changes in the timing or addition of time points for any planned study assessments 
must be documented and approved by the relevant study team member and then archived 
in the sponsor and site study files, but will not constitute a protocol amendment. The 
IRB/IEC will be informed of any safety issues that require alteration of the safety 
monitoring scheme or amendment of the ICF.

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PROCEDURE
Screening
SESSION 1
Interim 
inspectio
n
SESSION 2 
1st 
follow-up
2nd  
follow-up
Day:
Within 30 
days of 
Day 1
Day 
1
Day 
2
Day 
3
Minimum 
2 wks 
(max 4 
weeks) 
after end 
of session 
1
Day 
-1
Day 
1
Day 
2
Day 
3
Minimum 
2 wks (max. 4 
weeks) after 
end of 
session 2 
Approx. 
5wks after 
end of 
session 2
Attend Unit
X
X
X
X
X
X3
X3
X
X
X
X
1.
Brief physical exam only
2.
Within an hour before 
cantharidin application  
3.
Overnight stay in the unit
4.
Additional clinical lab 
assessments may be 
performed, if necessary  
5.
Before blister fluid sampling
6.
After the last blister fluid 
sampling at each session
7.
Multiple blood draws at the 
following time points: Pre 
challenge, ,
5 mins (i.e. Pre-blister
induction), 10 min, 25 min, 40 
min, 1hr:10mins, 1hr:40mins, 
2hr:40mins, 5hr:40mins with 
respect to start of blister 
induction
8.
Every half hour for the first 4 
hours after challenge, hourly 
until 8 hours and then 8 hourly 
until discharge. Frequency can 
be increased if symptomatic
9.
For flow cytometry and 
transcriptomic blood samples: 
Multiple blood draws at the 
Informed Consent
X
Inclusion/Exclusion checklist
X
X
Medical /medication history
X
Demographics
X
Body weight 
X
X
Drug/alcohol test
X
X
X
Physical Exam
X
X1
X
X1
X1
Vital Signs and temperature
X
X2
X
X8
X
X
X
ECG
X
X
X
Telemetry
X
Visual forearm check 
(including blister healing and 
cosmetic assessment)
X
X2
X
X
X2
X
X
Cantharidin application 
(Session 2 only: 20 minutes 
post end of LPS or GM-CSF 
challenge)
X
X
AE assessment/Con Meds
      «----------------------------------------------------------------------------------------------------------------------»
SAE
«----------------------------------------------------------------------------------------------------------------------»
Clinical Chemistry, 
Haematology, and Urinalysis
4
X
X
X
X
X
X
X
X
Mediators blood sample
X2
X5
X5
X7
X5
X5
Flow cytometry blood sample
X2
X5
X5
X9
X5
X5
For LPS only: Ex vivo
X10

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PROCEDURE
Screening
SESSION 1
Interim 
inspectio
n
SESSION 2 
1st 
follow-up
2nd  
follow-up
Day:
Within 30 
days of 
Day 1
Day 
1
Day 
2
Day 
3
Minimum 
2 wks 
(max 4 
weeks) 
after end 
of session 
1
Day 
-1
Day 
1
Day 
2
Day 
3
Minimum 
2 wks (max. 4 
weeks) after 
end of 
session 2 
Approx. 
5wks after 
end of 
session 2
stimulation blood sample  
following time points: Pre 
challenge, 40 mins, 2hr:40mins
and 5hr:40mins (and 9hr 
40mins for GM-CSF for flow 
cytometry only) with respect to 
start of blister induction 
10. Ex vivo stimulation blood 
sample: 2 samples (one null 
and one LPS tube) to be taken 
pre-dose and 2 samples (one 
null and one LPS tube) to be 
taken at 5hrs:40mins post 
blister induction
11. Pre-challenge urine sample will 
be collected in session 1. For 
the post-challenge samples in 
session 2, participants will be 
encouraged to pass urine 
immediately before LPS dosing 
and each urine void will be 
collected after LPS until 12 
hours post-LPS
12. From 4 hours prior to LPS until 
8 hours after LPS  
Transcriptomic blood sample
X9
Urine sampling for PD
X11
X11
For LPS only: Intravenous 
hydration with normal Saline 
at a rate of 250 mL / hr
X12
In vivo LPS or GM-CSF 
challenge
X2
Blister sample for biomarkers
X
X
X
X
Pain rating6
X
X
Participant diary card given 
to participant to record blister 
healing
X
X

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3.
INTRODUCTION
3.1.
Cantharidin-induced skin blister assay
The cantharidin blister model was chosen to permit easy access to an acute in vivo 
inflammatory compartment, in which response to inflammatory challenges might be 
investigated directly by analysing cellular and soluble components.
Cantharidin is a vesicant (an agent that causes blisters) and a strong inhibitor of protein 
phosphatases type 1 and 2A (Honkanen, 1993). Following contact with the skin, it is 
absorbed into the epidermis and activates neutral serine proteases that cause dissolution 
of desmosomes, leading to acantholysis (the disruption of intercellular bridges between 
keratinocytes) and intra-epidermal blistering (Bertaux, 1988). The pathology is limited to 
the suprabasal epidermis and lesions heal without scarring (Moed, 2001).  Topical 
cantharidin solution (0.7%) is used in clinical dermatological practice in Canada for the 
treatment of warts and molluscum contagiosum and has been demonstrated as an 
effective treatment, including in children (Noda-Cabrera, 2015). Skin blister induction by 
cantharidin has been utilised in clinical pharmacology for more than 50 years either as a 
tool for pharmacokinetic studies or as a model of acute inflammation in human 
participants. The blister fluid formed after 24 hours of exposure to cantharidin contains 
inflammatory cells, predominantly neutrophils and monocytes/macrophages, that may be 
counted (to evaluate leukocyte trafficking and cellular accumulation), analysed by flow 
cytometry (to perform phenotypic profiling), or used for ex-vivo assays. In parallel, 
various cytokines, chemokines and other mediators of inflammation may be measured in 
blister fluid (Day, 2001, Evans, 2006, Harbord, 2006, Evans, 2008). It has been 
demonstrated that the inflammatory response measured in the cantharidin-induced blister 
fluid is sensitive to pharmacological manipulation using anti-inflammatory treatment 
(Morris, 2009).  Furthermore, prolonged exposure to cantharidin (40+ hours) allows 
insight into the resolving phase of acute inflammation (Yagnik, 2000, Philippidis, 2004). 
In healthy volunteers this phase includes polarisation of monocyte-derived macrophages 
largely towards a resolving phenotype (Yagnik, 2000, Day, 2001), with enhanced 
expression of markers such as CD163 and CD206 (Philippidis, 2004) GlaxoSmithKline 
Document Number (GM2008/00294/00); GlaxoSmithKline Document Number 
(AM2010/00011/01).
3.2.
Study Rationale
This study aims to assess 2 models of systemic inflammatory response: exposure of 
healthy participants to systemic challenge with either LPS or GM-CSF. This will be done 
by measuring inflammatory mediators and cellular activation markers both in circulation 
and in skin blisters induced by exposure to cantharidin (as a model of local inflammatory 
focus). More specifically, the study will assess the time course of up-regulation of 
circulating markers of inflammation (both cellular and soluble), in response to the 
systemic challenges and whether the challenges accentuate the inflammatory milieu of 
the blisters. The study will also assess urinary biomarkers of inflammation, and more 
specifically metabolites of prostaglandins, such as tetranor-PGDM. The literature reports 
that these stable urinary metabolites are elevated in patients with Duchenne muscular 
dystrophy and they are mechanistically relevant to treatments currently being evaluated at 

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GSK. Data generated in this study will provide the foundation for future studies testing 
the effect of compounds that target key pathways in inflammation and autoimmunity.
LPS is often used to induce inflammation to model disease in animal models and in in 
vitro systems.  Human in vivo LPS challenges have been used as a methodology to induce 
systemic inflammation and produce many of the immunological (changes in leukocyte 
numbers and induction of inflammatory mediators) and physical signs of acute and 
chronic disease (including fever, pain, and tachycardia).  The human LPS model of 
systemic inflammation has been applied to clinical pharmacology studies to assess 
therapeutic interventions for analgesics, asthma, effective adjuvants, sepsis, trauma, 
Type-2 diabetes, Alzheimer’s disease and others.  This study will characterize the 
phenotype of peripheral leukocytes and the production of a plethora of inflammatory 
mediators and acute phase proteins over a time course.  With regards to prostaglandins, 
this pathway has been reported in the literature to be disregulated in patients with 
Duchenne muscular dystrophy (Nakagawa, 2013).  However, in normal healthy 
individuals, these inflammatory mediators and their metabolites have very low circulating 
and urine concentrations, respectively.  Therefore, artificial stimulation of this pathway 
by LPS in normal healthy volunteers would be required for early clinical evaluations of 
target engagement for therapeutics target this pathway. 
GM-CSF has a broad range of activities across innate and adaptive immune cells and is 
recognised as a key mediator in a number of inflammatory diseases, such as arthritis, 
multiple sclerosis, colitis, pain and interstitial lung disease (Wicks, 2016).  5 μg / kg 
Leukine (Sargramostim) given subcutaneously in the abdominal area results in a systemic 
GM-CSF challenge that mobilises neutrophils, eosinophils and monocytes from the bone 
marrow.  Changes in leukocyte numbers have previously been characterised (van 
Pelt, 1996) with only minor efforts in detecting secreted inflammatory mediators.  This 
study will extend these observations over a defined time course to include a plethora of 
soluble inflammatory mediators as well as phenotype peripheral leukocytes, particularly 
monocytes.  
3.3.
Background
3.3.1.
LPS challenge
Endotoxin lipopolysaccharide (LPS) is a major component of the outer membrane of 
Gram negative bacteria. It is one of the pathogen-associated molecular patterns that will 
trigger inflammation by the binding to pattern recognition receptors (PRRs), initiating 
downstream intracellular signalling pathways that result in the activation of the nuclear 
transcription factor, nuclear factor kB (NF-kB) (Zhang, 2001), which in turn stimulates 
the transcription of genes coding for pro-inflammatory cytokines such as tumour necrosis 
factor (TNF)-α and interleukin (IL)-1β (May, 1998). These cytokines activate an 
assortment of inflammatory cascades including the complement system, the coagulation
system and the production of nitric oxide, all of which participate in eliminating invading 
microorganisms (Janeway, 2002). 
Systemic inflammation is a pathogenic component in a vast number of acute and chronic 
diseases such as sepsis, trauma, type 2 diabetes, atherosclerosis, and Alzheimer’s disease, 

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all of which are associated with a substantial morbidity and mortality. Human models of 
systemic inflammation have been developed with the purpose of mimicking the changes 
in inflammatory mediators encountered in acute as well as chronic inflammatory disease, 
but in a controlled, standardised experimental setting. These also allow the study of the 
molecular mechanisms and physiological significance of the systemic inflammatory 
response. The most widespread model of systemic inflammation is probably the human 
endotoxin model, in which purified LPS from Escherichia coli or other Gram-negative 
bacteria is administered intravenously to healthy volunteers, inducing an acute systemic 
inflammatory response, which, at least partially, mimics the inflammatory response of 
early sepsis as well as other acute inflammatory conditions (Andreasen, 2008).
After injection, LPS can be measured in plasma within a few minutes, and is quickly 
transferred (plasma levels drop sharply within the next 15 minutes) to the liver to be 
metabolised (van Deventer, 1990). Usually within one hour of LPS administration, 
volunteers will experience varying degrees of flu-like symptoms (e.g. headache, chills, 
myalgia and nausea), with the symptoms slowly disappearing after 4 to 6 hours, in 
parallel with the diminishing inflammatory response. The most reproducible clinical 
findings among participants include an increase in core temperature and tachycardia
(Dinarello, 2004).
Administration of LPS causes a quick decline in neutrophil numbers in circulation in the 
first 15-30 minutes, probably from the margination of these cells due to upregulation of 
vascular adhesion factors, followed by a 3-4 fold increase in the following 4 to 6 hours 
and a return to baseline within 24 hours after exposure (Richardson, 1989, van Deventer,
1990, Jilma, 1999). At the same time, monocytopenia and lymphopenia are observed 
upon LPS challenge with monocytes having a more rapid decline (trough at around 90 
minutes) and return to baseline (6-8 hour) (Thaler, 2016) whereas lymphocyte counts are 
most reduced at 4 hours and only normalise after 8-12 hours (Krabbe, 2001). Monocyte 
subsets are differentially affected by LPS. After 24 hours, distribution was skewed 
towards the intermediate (CD16+ CD14+) subset. These monocytes also displayed the 
largest increase of CD11b expression after 2 hours and the highest increase in IL-6 and 
IL-8 mRNA levels, whereas these mRNA levels in classical monocytes (CD14+ CD16-) 
change only marginally. (Thaler, 2016).
3.3.2.
GM-CSF Challenge
Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a cytokine, belonging 
to the colony-stimulating factors (CSF), crucial for survival, proliferation, differentiation, 
maturation and functional activation of haematopoietic cells. It is often used to treat 
leukopenia, but as other haematopoietins may increase the number of circulating 
leukocytes with higher efficiency, GM-CSF has additional effects that may be far more 
relevant than its haematopoietic activity. It induces differentiation, proliferation and 
activation of macrophages and dendritic cells which are necessary for subsequent T
helper cell type 1 and cytotoxic T lymphocyte activation (Francisco-Cruz, Aguilar-
Santelises et al. 2014).
GM-CSF is used in the clinical setting for the treatment of bone marrow dysfunction. 
Healthy donors may be stimulated with the growth factor to induce generation of 

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peripheral blood progenitor cells (PBPC) or granulocyte for donation. 
(Fischmeister, 1999)
3.3.3.
Pharmacokinetics and pharmacodynamics of GM-CSF
When GM-CSF (also known as sargramostim) is administered to patients by intravenous 
infusion over 2 hours, it reaches a mean beta half-life of approximately 60 minutes. The 
peak concentrations of GM-CSF are observed in blood samples obtained during or 
immediately after completion of the sargramostim infusion, while minor concentrations 
are still detected in blood 6 hours after the beginning of the infusion. GM-CSF is also 
detected in serum first after 15 minutes of subcutaneous injection of sargramostim to 
healthy volunteers. Then, the mean beta half-life is approximately 162 minutes, and peak 
levels are reached after one to 3 hours post-injection and remain detectable for up to 6 
hours.
Sargramostim is usually well tolerated by healthy participants, who have not shown 
clinical alterations in their clinical analysis, as compared to placebo-treated individuals. 
(Francisco-Cruz, 2014)
3.4.
Benefit/Risk Assessment
More detailed information about the known and expected benefits and risks and 
reasonably expected adverse events of cantharidin may be found in the Participant 
Information Leaflet.

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3.4.1.
Risk Assessment
The challenge agents and cantharidin used in this study are established in healthy volunteer studies and are expected to be well tolerated.  
The information in this section is provided in the interests of full disclosure of all potential risks.
Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Cantharidin
Risk of lymphangitis and/or Lymphoedema
Three cases of lymphangitis and lymphoedema 
following high exposure to cantharidin (0.7% 
solution and application on multiple warts) have 
been described in the literature (Dilaimy, 1975, 
Stazzone, 1998).
Participants with a history of lymphangitis and/or 
lymphoedema or any participant who has 
undergone surgery resulting in loss of tissue 
associated with lymphoid drainage (e.g. certain 
breast surgery procedures) are excluded.
Concentration of cantharidin is reduced in this 
study from 0.7% to 0.2 %.
Discomfort and/or pain at the blister site
Forearm blister induction with cantharidin has 
been associated with local discomfort and/or 
pain in previous studies.
Participants are advised to wear loose clothing at 
the site of blistering to minimise hyperaesthesia 
and discomfort that may occur.
Pain will be assessed by asking participants to 
rate their pain using an 11-point scale (0 – no 
pain to 10 –worst pain imaginable) (Farrar,
2001).
For immediate relief of any discomfort, 
paracetamol, at doses of 2 grams/day will be 
given to the participant, unless, in the opinion of 
the investigator and sponsor, the medication will 
interfere with the study.
Risk of dyspigmentation and hypertrophic scar 
After blister healing, temporary post-
Participants with history of keloids, skin allergy, 

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
formation
inflammatory dyspigmentation and hypertrophic 
scar may occur and persist for several months.  
hypersensitivity, or contact dermatitis, including 
previous reactions to dressings to be used in this 
study or any chronic skin disorder (e.g. psoriasis, 
atopic dermatitis, vitiligo) excepting isolated 
lesions remote from intended site of application
of cantharidin are excluded. Appropriate skin 
care to avoid dyspigmentation and hypertrophic 
scar will be implemented.
Risk of infection at the blister site
During the blister fluid harvesting, there is 
potential for infection at the site. To date there 
are no reports of skin infection from clinical 
studies evaluating cantharidin-induced skin 
blister model.
To minimize the risk, appropriate clean
procedures will be applied.
Participants are instructed to visually inspect 
blister sites on a daily basis and are informed 
what signs of inflammation/infection to watch for 
and contact the investigator with any concerns.
Accidental cantharidin ingestion or contact with 
eyes and mucus membranes
Fatal poisoning may occur if cantharidin is 
swallowed. However, no cases caused by 
application of cantharidin solution by a physician 
(or supervised by) have been reported to date.  
There are also no reports of accidental contact 
with eyes or mucus membranes from 
experimental use of cantharidin.
Cantharidin will not be dispensed to participants 
and all applications will be done at the research 
centre by qualified personnel.  
LPS
Risk of exaggerated physiological response to 
intravenous LPS 
Intravenous LPS can cause short-lived 
inflammatory response in healthy volunteers.
Previous studies have shown that single 
intravenous doses of 0.6ng/kg LPS were well 
tolerated in healthy male participants. Observed 
The dose of LPS in this study will be carefully 
titrated to identify a dose that is associated with a 
robust inflammatory response but is well 
tolerated. The administered dose will be 
informed by previous published clinical 

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
adverse events (AEs) were of mild severity and 
self-limiting without therapeutic intervention. The 
most frequent occurring AEs were headache, 
and feeling cold. No clinically relevant changes 
or unexpected treatment-related trends were 
observed in supine systolic and diastolic blood 
pressure, body temperature, or ECG-derived 
parameters following administration of LPS. LPS 
can dose-dependently increase body 
temperature and heart rate, with a maximal 
increase amounting approximately 1.5 °C and 28 
± 13.2 bpm for LPS dose of 2 ng/kg (10 EU/ng) 
tested, observed at 3–4 hours after LPS 
administration (Dillingh, 2014). 
experience with LPS and the in vitro activity of 
the actual batch of LPS to be given. 
During the dose finding phase of the study only 
one participant will be dosed at a time with LPS. 
The participants will be dosed with LPS in a 
MHRA accredited clinical research unit and will 
have regular monitoring of vital signs post dose. 
The frequency of monitoring can be increased if 
there are clinical concerns. The dosed 
participants will be confined to clinical unit for 
approx. 24 hours post dosing with LPS. 
The age range for participants in this study is 
restricted to 18-45 years old to minimise the 
possibility of exaggerated response to LPS.
Participants will receive telemetry for a minimum 
of 6 hours post-dose or until their telemetry 
shows no clinical significant findings for 4 hours 
(whichever is longer).
Participants will be prehydrated with intravenous 
fluids prior to LPS challenge, and intravenous 
fluid hydration will continue following the 
challenge.  Normal saline will be infused at a rate 
of 250ml/hr for 4 hours prior to LPS dosing and 8
hours subsequently.
Risk of severe vagal response 
IV LPS endotoxin challenge has been 
administered in the higher dose range of 2 - 4 
ng/kg (10 EU/ng) to thousands of individuals with 
Participants with previous history of vasovagal 
syncope will be excluded from the study.
Participants will be pre-hydrated with intravenous 

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
no serious or permanent adverse events 
(Fullerton, 2016). However isolated accounts of 
severe vagal reactions have been reported (van 
Eijk, 2004). The aetiology presumably represents 
high resting vagal tone, volume depletion after 
overnight fasting, and catecholamine release 
with the onset of fever, chills and symptoms, 
culminating in an exaggerated Bezold-Jarisch 
reflex.
fluids prior to LPS challenge, and intravenous 
fluid hydration will continue following the 
challenge.  Normal saline will be infused at a rate 
of 250ml/hour for 4 hours prior to LPS dosing 
and 8 hours subsequently.
Endotoxin tolerance 
It has been shown that endotoxin tolerance may 
persist in some individuals for an unknown length 
of time in vivo. (Draisma, 2008, Kox, 2011)
Participants with previous experimental exposure 
to LPS will be excluded.
GM-CSF
Risk of Fluid Retention
Oedema, capillary leak syndrome, pleural and/or 
pericardial effusion have been reported in 
patients after LEUKINE administration. In 156 
patients enrolled in placebo-controlled studies 
using LEUKINE at a dose of 250 mcg/m2/day by 
2-hour IV infusion, the reported incidences of 
fluid retention (LEUKINE vs. placebo) were as 
follows: peripheral oedema, 11% vs. 7%; pleural 
effusion, 1% vs. 0%; and pericardial effusion, 4% 
vs. 1%. Capillary leak syndrome was not 
observed in this limited number of studies; based 
on other uncontrolled studies and reports from 
users of marketed LEUKINE, the incidence is 
estimated to be less than 1%. In patients with 
pre-existing pleural and pericardial effusions, 
administration of LEUKINE may aggravate fluid 
Use of healthy volunteers is a mitigation as these 
findings were observed in patients with cancer in 
receipt of chemotherapy.
A lower, single dose of GM-CSF is planned
which means that the AUC and Cmax will be 
lower than in the patients in which this was 
observed.

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
retention; however, fluid retention associated 
with or worsened by LEUKINE has been 
reversible after interruption or dose reduction of 
LEUKINE with or without diuretic therapy. 
LEUKINE should be used with caution in patients 
with pre-existing fluid retention, pulmonary 
infiltrates or congestive heart failure.
Risk of Respiratory Symptoms
Sequestration of granulocytes in the pulmonary 
circulation has been documented following 
LEUKINE infusion12 and dyspnoea has been 
reported occasionally in patients treated with 
LEUKINE. Special attention should be given to 
respiratory symptoms during or immediately 
following LEUKINE infusion, especially in 
patients with pre-existing lung disease. In 
patients displaying dyspnoea during LEUKINE 
administration, the rate of infusion should be 
reduced by half. If respiratory symptoms worsen 
despite infusion rate reduction, the infusion 
should be discontinued. Subsequent IV infusions 
may be administered following the standard dose 
schedule with careful monitoring. LEUKINE 
should be administered with caution in patients 
with hypoxia.
Patients with known respiratory disease will be 
excluded from the study (as described in the 
Exclusion criteria), as well as patients with 
current or recent (<30 days) infection.
Risk of cardiovascular Symptoms
Occasional transient supraventricular arrhythmia 
has been reported in uncontrolled studies during 
LEUKINE administration, particularly in patients 
with a previous history of cardiac arrhythmia. 
However, these arrhythmias have been 
reversible after discontinuation of LEUKINE. 
Patients with pre-existing cardiac disease will not 
be eligible to participate.  In addition, we will use 
telemetry for a minimum of 6 hours following 
dosing, or until normal for 4 hours (whichever is 
longer).

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
LEUKINE should be used with caution in patients 
with pre-existing cardiac disease.
Risk of renal and Hepatic Dysfunction
In some patients with pre-existing renal or 
hepatic dysfunction enrolled in uncontrolled 
clinical trials, administration of LEUKINE has 
induced elevation of serum creatinine or bilirubin 
and hepatic enzymes. Dose reduction or 
interruption of LEUKINE administration has 
resulted in a decrease to pre-treatment values. 
However, in controlled clinical trials the 
incidences of renal and hepatic dysfunction were 
comparable between LEUKINE (250 
mcg/m2/day by 2-hour IV infusion) and placebo-
treated patients. Monitoring of renal and hepatic 
function in patients displaying renal or hepatic 
dysfunction prior to initiation of treatment is 
recommended at least every other week during 
LEUKINE administration.
Patients with pre-existing renal or hepatic 
dysfunction will be excluded from the study.

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3.4.2.
Benefit Assessment
There will be no benefit to the participant taking part in this study. However, participants 
will be contributing to the process of developing new therapies by taking part in the 
study. 
3.4.3.
Overall Benefit : Risk Conclusion
The measures taken to minimize the potential risks identified in association with 
cantharidin, LPS and GM-CSF are considered sufficient to justify participation by 
healthy volunteer participants. 
4.
OBJECTIVES AND ENDPOINTS
Objective
Endpoint
Primary

To compare and define the time 
course of soluble and cellular 
inflammatory biomarkers (and 
urinary prostaglandins for LPS 
only) following systemic 
challenge with LPS or GM-CSF 
in healthy participants

For LPS only: time course and 
magnitude of upregulation of 
circulating TNF-α and IL-6 as well 
as urinary tetranor PGDM.

For GM-CSF only: time course and 
magnitude of upregulation of 
circulating total leukocyte numbers
Secondary

To compare soluble and cellular 
inflammatory biomarkers in 
cantharidin-induced skin blisters 
at baseline versus systemic 
challenge with LPS or GM-CSF 
in healthy participants

Soluble inflammatory biomarkers 
in skin blisters (may include but 
not limited to IL-1b, IL-2, IL-6, 
IL-8, IFNg, TNF-α, MCP-1, GM-
CSF, CRP). 

Blister volumes and differential 
cell counts (cellular activation 
markers may include, but not 
limited to expression of CD16, 
CD86, CD80, CD163, CD206, 
CD83, CD40, CD209, HLA-DR 
in blister leukocytes)

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Objective
Endpoint

To define the time course of 
circulating soluble and cellular 
inflammatory biomarker 
upregulation following systemic 
challenge with LPS or GM-CSF 
in healthy participants

Time course of regulation of 
circulating soluble inflammatory 
biomarkers (may include but is 
not limited to IL-1b, IL-2, IL-6, 
IL-8, IFNg, TNF-α, MCP-1, GM-
CSF, CRP). 

Time course of regulation of 
circulating leukocyte numbers 
and cellular activation markers 
(may include, but not limited to 
expression of CD16, CD86, 
CD80, CD163, CD206, CD83, 
CD40, CD209, HLA-DR in 
circulating leukocytes)
Exploratory

To compare suitability of blisters 
sampled at 24 and 48 hours after 
induction for maximising observed 
effects of systemic challenge

Primary and Secondary endpoints 
measured in 24 versus 48 hour 
blisters

To assess the safety and tolerability 
profile of the challenge agents and to 
ensure it is not materially different 
from previous experience (as 
described in Section 3.2)

Adverse events, clinical laboratory 
measures, vital signs, pain scale 
assessments

Determining whether 
carboxyesterase-1 (CES-1) 
expression is present in monocytes 
following systemic GM-CSF 
challenge

Number of monocytes which are 
CES-1+ in blood

To compare prostaglandins in urine, 
blister fluid and plasma at baseline 
versus systemic challenge with LPS 
or GM-CSF 

Quantification of prostaglandins
including (but not limited to) 
tetranor -PGDM, tetranor-PGEM, 
PGD2 and PGE2

To assess changes in whole blood 
transcriptome induced by 
systemic LPS- or GM-CSF-
challenge

Analysis of differential gene 
expression in whole blood taken 
pre-challenge and at selected 
times post- challenge

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Objective
Endpoint

For LPS challenge only: to assess
whether the dose of in -vivo LPS 
challenge is sufficient to induce 
innate immune tolerance 

Quantification of inflammatory 
markers including (but not limited 
to) TNF-α and IL-6 in blood drawn 
pre- and 6 hour post-systemic LPS 
challenge following ex vivo 
incubation in ‘LPS-TruCulture’ 
tubes and LPS-null TruCulture’ 
tubes

To assess the effects of systemic 
LPS and GM-CSF challenges on 
blister healing times

Blister healing times (self-reported
in participant diary card)

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5.
STUDY DESIGN
Figure 1
Study design schematic
5.1.
Overall Design
This is an exploratory study comprising of 2 parts. In both parts of the study, participants 
will have 2 sessions. In session 1 (baseline) all participants will have 2 blisters induced 
on each forearm by application of cantharidin solution (based on method described by 
Day et al. (Day, 2001)) followed by blood draws and a blister harvest on each forearm at 
24 and 48 hours post-induction. After a minimum of 14 days blister healing period, 
participants will return for a second session (in vivo challenge session). This open label 
study will randomise participants to have either a LPS or GM-CSF in vivo challenge (4 
completed participants in each group). Participants will then have 2 blisters induced on 
each forearm by application of cantharidin solution, followed by multiple, timed, blood 
draws (15 minutes to 48 hours post dose) and a blister harvest on each forearm at 24 and 
48 hours post-induction. 

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In part I of the study, a dose exploration of each of the challenge agents (LPS and GM-
CSF) will be required to find a dose that provides a robust inflammatory response. Up to 
6 cohorts of 4 participants will be tested and all cohorts will have 2 sessions, as described 
above. For Part I, an initial cohort of 4 participants (Cohort 1) will proceed with session 
1. After their blister healing period, Cohort 1 will return for their session 2 visit in two 
groups of 2 participants (Group A and Group B) on different days. Group A will be dosed 
on the same day (one with LPS and one with GM-CSF) and Group B will be dosed on a 
different day (one participant with LPS and one participant with GM-CSF) after group A. 
Once all 4 participants have been dosed, a dose escalation meeting will be held to make 
an assessment of tolerability and inflammatory response from the first Cohort (e.g. the 
primary endpoints) to determine the dose for Cohort 2. This dose escalation meeting will 
be held prior to Group A in Cohort 2 proceeding to be dosed with the challenge agents in 
Session 2. This same dose escalation procedure will continue until a well-tolerated dose 
for LPS and GM-CSF showing a robust inflammatory response has been identified. Once 
the well-tolerated dose with robust inflammatory response has been determined, 
additional participants will be administered this same dose until there are 8 evaluable 
participants (4 with LPS at the same dose and 4 with GM-CSF at the same dose) 
completed in Part I.
During this dose exploration phase the response to each challenge agent will be assessed 
by dose escalation meeting after 2 participants have been exposed to that agent. The 
response to LPS will be primarily assessed based on IL-6, TNF-α and urinary tetranor 
PGDM. The response to GM-CSF will primarily be based on leukocyte counts.. The 
decision whether to adjust the dose will be made by the Principal Investigator in 
consultation with the medical monitor based on the response outlined above in the 
context of the safety and tolerability at that dose. During the dose escalation phase, 
samples collected for secondary and exploratory endpoints will only be processed if the 
assay is required to be done immediately. Otherwise, collected samples will be batch 
processed once the optimal dose for each challenge has been established.
For Part II, an additional cohort of up to 8 participants (4 participants with GM-CSF and 
4 participants with LPS challenge) at the same dose as the 8 evaluable participants from
Part I may be added if the interim analysis indicates additional participants would provide 
further precision on estimates of primary endpoints. The same two-session design, as 
described above, would be used. 

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Figure 2
Study Design schematic of session 1 and 2 for each participant
5.2.
Number of Participants
In Part I, up to 8 evaluable participants will be required at the optimal dose (4 of each 
challenge agent). Following an interim analysis, a sample size re-estimation will be 
conducted and up to a further 8 evaluable participants (up to 4 in each arm of challenge 
agent) in Part II may be enrolled and dosed at the same dose as Part I. Accounting for 
dose escalation exploration, an estimated 24-30 (maximum of 40 including replacement 
for drop-outs) participants will be enrolled in total. If participants prematurely 
discontinue the study, additional replacement participants may be recruited and assigned
to the same treatment sequence, if that participant has not completed session 2.
5.3.
Participant and Study Completion
A participant is considered to have completed the study if he/she has completed up to the 
end of session 2 of the study. If a participant withdraws before the end of session 2 they 
will be encouraged to return for a follow-up visit as per the SOA. If a participant 
withdraws before the end of session 2 their data may still be used for analysis, depending 
on how much data is available. If there is insufficient data for analysis, at the discretion 
of the study team, this participant will be replaced.

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The end of the study is defined as the date of the last visit of the last participant in the 
study, e.g. the second follow up session for the last participant.
5.4.
Scientific Rationale for Study Design
These challenges provide a method to demonstrate pharmacodynamic effects of novel 
therapeutics that modulate systemic and local inflammation by measurement of 
inflammatory mediator production, leukocyte numbers, immune cell phenotypes and 
opportunity for ex vivo assays.  The combination of blister induction after GM-CSF or 
LPS challenge provides a paradigm to investigate chemotaxis and the ability to recognise 
phenotype switching in mononuclear myeloid cells as local inflammation is thought to be 
exacerbated by systemic inflammation.
5.5.
Dose Justification
5.5.1.
Cantharidin
In many studies employing this model (Day, 2001, Morris, 2009, Landis, 2010, Dinh,
2011) [GlaxoSmithKline Document Number (GM2008/00294/00)], 25 µL of 0.1% 
cantharidin in acetone has been used to induce blisters. Skin blister induction with 0.1% 
cantharidin solution is generally well tolerated and safe, with side-effects limited to 
transient hyperaesthesia, discomfort upon pressure and occasional transient 
hyperpigmentation (Day, 2001). In a previous GSK study [GlaxoSmithKline Document 
Number (GM2008/00294/00)] there were no significant adverse events attributed to 
application of 25 µL of 0.1% cantharidin in acetone solution, via soaking a 7-8 mm 
diameter filter disc, which was held in place in contact with the skin for up to 40 hours. 
The model was well tolerated in healthy male volunteers. 
In a more recent study [GlaxoSmithKline Document Number: (AM2010/00011/01)] the 
application of cantharidin was refined to produce smaller, more consistently sized 
blisters. This was achieved by omitting the filter disc and directly applying a lower 
volume (5 µL) of 0.2 % solution to an area of participants’ skin delimited by a small ring 
of Vaseline (approximately 5-6 mm in diameter).  Blisters were harvested up to 72 hours
after application of cantharidin. This usually resulted in blisters of up to 10 mm in 
diameter containing between 100 – 500 μL of fluid. In this way the amount of cantharidin 
used per blister was both reduced and standardised, with a large resultant decrease in 
observed variability of blister volume (both within (79% to 31%) and between (94% to 
44%) participants). This dose represents a 2.5-fold reduction in the amount of cantharidin 
used per blister, compared to most previous blister studies. It is important to note that 
these blister studies themselves had used cantharidin at 1/7 of the concentration 
recommended for direct skin application for treatment of warts and Molluscum
contagiosum in children, using multiple simultaneous applications (Noda-Cabrera, Martín 
et al. 2015). The developed methodology thus represents a substantial reduction in 
exposure compared to that recommended for therapeutic use; it is the dose and method of 
application that will be used for all blister induction in this study. This new methodology 
was well tolerated by healthy male volunteers.  

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5.5.2.
LPS
The initial dose level for LPS is proposed to be 0.5 ng/kg (bioactivity of proposed batch
LPS is 6 EU / ng, therefore a dose of 3.0 EU / kg). This dose is associated with a 
measurable systemic cytokine response but is well tolerated based on the literature. 
Participants will be hydrated prior to administration of LPS with normal saline at a rate of 
250 mL/hr for 4 hours prior to dosing and 8 hours after dosing.
If dose escalation is required to achieve a robust (well tolerated) systemic cytokine 
response, then this will proceed in increments not exceeding a doubling of the previous 
dose; the highest permitted dose will be 4 ng/kg. (i.e. 24 EU/kg). Dose escalation will be 
guided by 1) safety and tolerability 2) the systemic cytokine response although other 
secondary and exploratory markers may also be taken into account. The goal is to achieve 
informative biomarker excursions with absent or trivial symptoms experienced by 
participants.
5.5.3.
GM-CSF
Recombinant human GM-CSF (Sargramostim) is FDA approved for the reconstitution of 
myeloid cells after bone marrow transplantation, neutrophil recovery following 
chemotherapy in AML patients or mobilisation of peripheral blood progenitor cells, with 
a recommended dose of 250 μg/m2/day. GM-CSF is well tolerated in healthy (van 
Pelt, 1996) and immuno-compromised participants (Lieschke, 1989, Gianni, 1990, 
Mehta, 2015). A wide range of doses have been administered to oncology patients (0.3 –
30 μg/kg/day) over several consecutive days and whilst are generally well tolerated, with 
adverse effects including bone pain, myalgia and rash at 15 μg / kg / day. Pericarditis 
was a dose-limiting toxicity where GM-CSF doses exceeded 15 μg / kg / day 
(Lieschke, 1989).
A GM-CSF dose of 5 μg / kg, administered subcutaneously, is associated with a 
measurable leukocyte response but is well tolerated based on the literature. 1.5 μg/kg 
dose infused intravenously over 2 hours provides the same exposure (AUC) due to the 
increased bioavailability and higher Cmax concentrations [Cebon, 1990].  For an average 
male body surface area of 1.9m2 this equates to a dose of approximately 60µg/m2, and to 
remain consistent with clinical practice in dosing GM-CSF therapeutically the 60µg/m2
will be the starting dose.
If dose escalation is required to achieve a robust (well tolerated) leukocyte response then 
this will proceed in increments up to a maximum dose of 480 μg/m2 . Dose escalation 
will be guided by 1) safety and tolerability 2) the systemic leukocyte response although 
other secondary and exploratory markers may also be taken into account. The goal is to 
achieve informative biomarker excursions with absent or trivial symptoms experienced 
by participants.  
6.
STUDY POPULATION
Prospective approval of protocol deviations to recruitment and enrolment criteria, also 
known as protocol waivers or exemptions, is not permitted.

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6.1.
Inclusion Criteria
Participants are eligible to be included in the study only if all of the following criteria 
apply:
Age
1.
Participant must be 18 to 45 years of age inclusive, at the time of signing the 
informed consent.
Type of Participant and Disease Characteristics
2.
Participants who are overtly healthy as determined by medical evaluation including: 
medical history, physical examination, laboratory tests, and ECG.
Weight
3.
Body mass index (BMI) within the range 19.0-30.0 kg/m2 (inclusive).
Sex
4.
Male 
All participants must agree to use contraception as detailed in Appendix 5 of this protocol 
during session 2 and refrain from donating sperm from session 2 to end of study (follow-
up 2 visit).
Informed Consent
Capable of giving signed informed consent as described in Appendix 3 which includes 
compliance with the requirements and restrictions listed in the informed consent form 
(ICF) and in this protocol.
6.2.
Exclusion Criteria
A participant will not be eligible for inclusion in this study if any of the following criteria 
apply:
1.
A positive pre-study Hepatitis B surface antigen or positive Hepatitis C antibody 
result within 3 months of screening 
2.
A positive test for HIV antibody.
3.
Persistent abnormal CRP/ WCC levels at screening.
4.
Abnormal liver function tests at screening. For healthy participants: aspartate 
aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase and 
bilirubin ≥1.5xupper limit of normal (ULN) (isolated bilirubin >1.5xULN is 
acceptable if bilirubin is fractionated and direct bilirubin <35%) at screening (Refer 
to Appendix 1). 
5.
A positive pre-study drug/alcohol screen.  
6.
Current, or chronic history of:

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
liver disease or known hepatic or biliary abnormalities (with the exception of 
Gilbert's syndrome or asymptomatic gallstones), anaphylaxis, and /or anaphylactoid 
(resembling anaphylaxis) reactions (Sampson, 2006).

cardiac, respiratory or renal disease (childhood asthma can be included).

Sensitivity or severe allergic responses to any of the challenge agents or cantharidin, 
or components thereof or a history of drug or other allergy that, in the opinion of the 
Investigator or GSK Medical Monitor, contraindicates their participation.

vasovagal syncope

surgery or significant trauma in 3 months leading to study enrolment

relevant skin conditions (e.g. recent h/o eczema or recurrent eczema, keloid, skin 
allergies, psoriasis, atopic dermatitis, and vitiligo) which in the opinion of the 
investigator could pose safety issues or cause interference with study procedures. 

sepsis or known coagulation disorders

peripheral oedema, lymphangitis, lymphoedema, pleural or pericardial effusion.

respiratory conditions including but not limited to asthma, COPD, and bronchiectasis
and any current respiratory infection.
7.
Presence on either forearm of tattoos, naevi, hypertrophic scars, keloids, hyper– or 
hypo- pigmentation. Participants with very fair skin, very dark skin, excessive hair or 
any skin abnormalities that may, in the opinion of the Investigator, interfere with 
study assessments.
8.
Unable to refrain from the use of prescription drugs taken on an intermittent (as 
needed) basis or non-prescription drugs; these include NSAIDs, vitamins, herbal and 
dietary supplements (including St John’s Wort) within 7 days (or 14 days if the drug 
is a potential enzyme inducer) or 5 half-lives (whichever is longer) prior to Day 1 of 
session 1 and continuing until the final follow up visit).
9.
The participant has participated in a clinical trial and has received an investigational 
product within the following time period prior to the first dosing day in the current 
study: 90 days, 5 half-lives or twice the duration of the biological effect of the 
investigational product (whichever is longer) or currently in a study of an 
investigational device. 
10. Previous exposure to LPS in a clinical research setting.

Where participation in the study would result in donation of blood or blood products 
in excess of 500 mL within a 56-day period.

Current smoker or former regular smoker within 6 months before the screening visit.

Unwillingness or inability to follow the procedures outlined in the protocol.

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6.3.
Lifestyle Restrictions
6.3.1.
Caffeine and Alcohol

During each session, participants will abstain from caffeine and alcohol for 24 
hours before the start of procedures until after collection of the final 
pharmacodynamics sample. 
6.3.2.
Activity

Participants will abstain from strenuous exercise for 48 hours before each session. 
Participants may participate in light recreational activities during studies (e.g., 
watching television, reading), but should avoid: 

Strenuous exercise to the upper limbs whilst blister in place 

Getting the blister dressing wet during bathing 

Sunbathing (or sun-bed) on the forearms during the study

Topical application of any creams to the forearms for the duration of the study
6.4.
Screen Failures
Screen failures are defined as participants who consent to participate in the clinical study 
but are not subsequently entered in the study. A minimal set of screen failure information 
is required to ensure transparent reporting of screen failure participants to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes 
demography, screen failure details, eligibility criteria, and any serious adverse events 
(SAEs).
Individuals who do not meet the criteria for participation in this study (screen failure) 
may be rescreened. 
7.
TREATMENTS
Study treatment is defined as any investigational treatment(s), marketed product(s), 
placebo, or medical device(s) intended to be administered to a study participant according 
to the study protocol.

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7.1.
Treatments Administered
Study 
Treatment 
Name:
Cantharone 
(Cantharidi
n)
LPS
GM-CSF
Intravenous 
Hydration with 
saline solution
(for LPS only)
Dosage 
formulation:
Liquid 
(mixture)
Ether (42.8 
% W/V), 
Acetone 
(36.0 % 
V/V), 
Alcohol 
(14.2 % 
W/V),
Camphor 
(1.2 % 
W/V), 
Cantharidin 
(0.7 % 
W/V) 
Balance 
(5.1%W/V) 
mixture of 
pyroxylin 
and castor 
oil
LPS is 
lyophilized 
in a 1 
microgram 
vial, 
formulated 
in 1% 
lactose and 
0.1% 
PEG6000 
The vial of 
lyophilized 
LEUKINE 
contains 250 mcg 
(1.4 × 106
IU/vial) 
sargramostim. 
The reconstituted 
lyophilized 
LEUKINE vial 
also contain 40 
mg/mL mannitol, 
USP; 10 mg/mL 
sucrose, NF; and 
1.2 mg/mL 
tromethamine, 
USP, as 
excipients
0.9% Sodium 
Chloride IV bags
Unit dose 
strength(s)/Dosa
ge level(s):
0.7% 
cantharidin 
liquid which 
will be 
diluted with 
acetone to 
0.2%
0.5 ng/kg (6 
EU/ng) with 
possible 
escalation up 
to 4ng/kg if 
participants
asymptomati
c and no 
cytokine 
response 
observed. 
60µg/m2 to a 
maximum of 480 
µg/m2
1L
Route of 
Administration 
Topical
IV
IV infusion
Intravenous

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Study 
Treatment 
Name:
Cantharone 
(Cantharidi
n)
LPS
GM-CSF
Intravenous 
Hydration with 
saline solution
(for LPS only)
Dosing 
instructions:
Apply 5 μl 
of 0.2 % 
Cantharidin 
solution 
(diluted in 
acetone) 
directly onto 
skin in area 
of ~ 1 cm2
IV injection 
of 0.5 ng/kg 
(6 EU/ng) 
body weight 
formulated 
as 
suspension 
in normal 
saline (or 
other LPS 
dose 
following 
escalation, 
see above).  
LPS will be 
provided in a 
final volume 
of 10 mL 
normal 
saline in a 
10 mL 
syringe, and 
administered 
over 1-2 
minutes.
Dose will be
calculated by 
calculating body 
surface area
using the 
Mosteller 
formula to give 
IV infusion dose
in µg/m2 given
over 
approximately 2 
hours
Administer 
intravenously at a 
rate of 250 mL/hr 
for 4 hours prior 
to dosing with 
LPS and 8 hours 
after dosing with 
LPS
Packaging and 
Labeling
N/A
N/A
N/A
N/A

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Study 
Treatment 
Name:
Cantharone 
(Cantharidi
n)
LPS
GM-CSF
Intravenous 
Hydration with 
saline solution
(for LPS only)
Manufacturer
Dormer 
Laboratories 
Inc. 
ADDRESS: 
91 Kelfield 
St. # 5 
Rexdale
Ontario 
Canada 
M9W 5A3
List 
Biological 
Laboratories, 
INC
540 Division 
Street, 
Campbell
California 
95008-6906
USA
Leukine is a 
registered 
trademark 
licensed to 
Genzyme 
Corporation.
Manufactured by:
sanofi-aventis
U.S. LLC
Bridgewater, NJ 
08807
A SANOFI 
COMPANY
US License No. 
1752
© April 2013 
sanofi-aventis 
U.S. LLC
Phone:
Baxter Inc.
Caxton Way
Thetford
IP24 3SE
7.2.
Method of Treatment Assignment
Participants will each have 4 blisters induced in session 1, and 4 blisters in session 2. All 
blisters will be within the central 60 % of the volar forearm surface. 2 blisters will be 
induced on each forearm in each session. Blisters induced in the second session will be in 
the same area of the forearm, but at least 2 cm away from a previous blister site.
In session 2, participants will be randomised (centrally) to have either a LPS or GM-CSF 
in vivo challenge, in a 1:1 ratio in accordance with a randomisation schedule generated by 
GSK Clinical statistics, prior to the start of the study, using a validated internal
randomisation software.  Randomisation cannot be reassigned.
7.3.
Preparation/Handling/Storage/Accountability
The investigator, institution, or the head of the medical institution (where applicable) is 
responsible for study treatment accountability, reconciliation, and record maintenance.  
PPD
PPD

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The investigator or the head of the medical institution, or designated site staff (e.g., 
storage manager, where applicable) must maintain study treatment accountability records 
throughout the course of the study. 
7.3.1.
Cantharidin
Cantharidin is not expected to pose significant occupational safety risk to site staff under
proposed conditions of use and administration.  Adequate precautions will be taken to 
avoid direct eye or skin contact and the generation of aerosols or mists. Precaution will be 
taken to avoid direct contact with the challenge agent.  A Material Safety Data Sheet 
(MSDS) describing occupational hazards and recommended handling precautions will be 
provided to the investigator.  In the case of unintentional occupational exposure, the 
monitor, medical monitor and/or study manager will be notified.  
One part of Cantharone will be mixed with 2.5 parts of pharmaceutical grade acetone in 
the CUC pharmacy according to standard practice.  This reconstituted challenge agent 
will be used within 4 hours and any residual material will be discarded according to 
standard GSK waste-streams.  
7.3.2.
LPS
LPS is not expected to pose significant occupational safety risk to site staff under the 
proposed conditions of use and administration.  Adequate precautions will be taken to 
avoid direct eye or skin contact and the generation of aerosols or mists. Precaution will be 
taken to avoid direct contact with the challenge agent.  A Material Safety Data Sheet 
(MSDS) describing occupational hazards and recommended handling precautions will be 
provided to the investigator.  In the case of unintentional occupational exposure, the 
monitor, medical monitor and/or study manager will be notified.
The dose of LPS will be calculated according to body weight and infused as a bolus over 
less than 2 minutes. Any residual material will be discarded according to standard GSK 
waste-streams.  
7.3.3.
GM-CSF
GM-CSF is not expected to pose significant occupational safety risk to site staff under the 
proposed conditions of use and administration.  Adequate precautions will be taken to 
avoid direct eye or skin contact and the generation of aerosols or mists. Precaution will be 
taken to avoid direct contact with the challenge agent.  A Material Safety Data Sheet 
(MSDS) describing occupational hazards and recommended handling precautions will be 
provided to the investigator.  In the case of unintentional occupational exposure the 
monitor, medical monitor and/or study manager will be notified.
The dose of GM-CSF will be calculated based on body surface area (Mosteller formula: 
BSA =  0.016667 × Weight (kg)0.5 × Height (cm)0.5) [Mosteller, 1987] and infused over 
approximately a 2 hour period. Any residual material will be discarded according to 
standard GSK waste-streams.

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7.4.
Treatment Compliance

When the individual dose for a participant is prepared from a bulk supply, the 
preparation of the dose will be confirmed by a second member of the study site staff. 

Participants will be dosed at the site and will receive study treatment directly from 
the investigator or designee, under medical supervision.  The date and time of each 
dose administered in the clinic will be recorded in the source documents and eCRF.  
The dose of study treatment and study participant identification will be confirmed at 
the time of dosing by a member of the study site staff other than the person 
administering the study treatment.  
7.5.
Concomitant Therapy
Any medication or vaccine (including over-the-counter or prescription medicines, 
vitamins, and/or herbal supplements) that the participant is receiving at the time of 
enrolment or receives during the study must be recorded along with:

reason for use

dates of administration including start and end dates

dosage information including dose and frequency
The Medical Monitor should be contacted if there are any questions regarding 
concomitant or prior therapy.
Participants must abstain from taking prescription or non-prescription drugs (including
paracetamol, NSAIDs, steroids, vitamins and dietary or herbal supplements) within 7 
days (or 14 days if the drug is a potential enzyme inducer) or 5 half-lives (whichever is 
longer) before the start of study treatment until completion of the follow-up visit, unless, 
in the opinion of the investigator and sponsor, the medication will not interfere with the 
study. 
Other concomitant medication may be considered on a case-by-case basis by the 
investigator in consultation with the Medical Monitor if required.
8.
DISCONTINUATION CRITERIA
8.1.
Withdrawal from the Study

A participant may withdraw from the study at any time at his/her own request, 
or may be withdrawn at any time at the discretion of the investigator for safety, 
behavioural, compliance or administrative reasons.

If the participant withdraws consent for disclosure of future information, the 
sponsor may retain and continue to use any data collected before such a 
withdrawal of consent.

If a participant withdraws from the study, he/she may request destruction of any 
samples taken and not tested, and the investigator must document this in the site 
study records.

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
Refer to the SoA for data to be collected at the time of study discontinuation and 
follow-up and for any further evaluations that need to be completed.

Individual participants may be withdrawn from the study if, in the judgement of 
the investigator, they have exhibited symptoms in response to LPS or GM-CSF 
that are exaggerated or unanticipated.
8.2.
Lost to Follow Up
A participant will be considered lost to follow-up if he or she repeatedly fails to return for 
scheduled visits and is unable to be contacted by the study site. 
The following actions must be taken if a participant fails to return to the clinic for a 
required study visit:

The site must attempt to contact the participant and reschedule the missed visit 
as soon as possible and counsel the participant on the importance of maintaining 
the assigned visit schedule and ascertain whether or not the participant wishes to 
and/or should continue in the study.

Before a participant is deemed lost to follow up, the investigator or designee 
must make every effort to regain contact with the participant (where possible, 3 
telephone calls and, if necessary, a certified letter to the participant’s last known 
mailing address or local equivalent methods). These contact attempts should be 
documented in the participant’s medical record.

Should the participant continue to be unreachable, he/she will be considered to 
have withdrawn from the study with a primary reason of lost to follow-up. 
9.
STUDY ASSESSMENTS AND PROCEDURES

Study procedures and their timing are summarized in the SoA (Section 2).

Protocol waivers or exemptions are not allowed 

Immediate safety concerns should be discussed with the sponsor immediately upon 
occurrence or awareness to determine if the participant should continue or 
discontinue study treatment.

Samples will be stored securely and made accessible only to scientists contributing 
to and supporting this research.  Clear custodianship responsibilities will be in place 
at all times and receipt/use/disposal details will be tracked on databases as per GSK 
internal processes.  The samples will be stored for a maximum of 5 years, after 
which they will be disposed of in accordance with the Human Tissue Authority’s 
Code of Practice.

Adherence to the study design requirements, including those specified in the SoA, is 
essential and required for study conduct.

All screening evaluations must be completed and reviewed to confirm that potential 
participants meet all eligibility criteria. The investigator will maintain a screening 
log to record details of all participants screened and to confirm eligibility or record 
reasons for screening failure, as applicable. 

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
The maximum amount of blood collected from each participant over the 
duration of the study, including any extra assessments that may be required, will 
not exceed 500 mL.

Repeat or unscheduled samples may be taken for safety reasons or for technical 
issues with the samples.
9.1.
Adverse Events
The definitions of an AE or SAE can be found in Appendix 4.
The investigator and any designees are responsible for detecting, documenting, and 
reporting events that meet the definition of an AE or SAE and remain responsible for 
following up AEs that are serious, considered related to the study treatment or the study, 
or that caused the participant to discontinue the study. 
9.1.1.
Time Period and Frequency for Collecting AE and SAE 
Information

All SAEs will be collected from the time when the ICF is signed by the 
participants until the follow-up visit at the time points specified in the SoA 
(Section 2).

All AEs will be collected from the start of session 1 until the follow-up visit at 
the time points specified in the SoA (Section 2).

Medical occurrences that begin before the start of study treatment but after 
obtaining informed consent will be recorded on the Medical History/Current 
Medical Conditions section of the case report form (CRF) not the AE section.

All SAEs will be recorded and reported to the sponsor or designee immediately 
and under no circumstance should this exceed 24 hours, as indicated in 
Appendix 4. The investigator will submit any updated SAE data to the sponsor 
within 24 hours of it being available.

Investigators are not obligated to actively seek AEs or SAEs in former study 
participants. However, if the investigator learns of any SAE, including a death, 
at any time after a participant has been discharged from the study, and he/she 
considers the event to be reasonably related to the study treatment or study 
participation, the investigator must promptly notify the sponsor.

The method of recording, evaluating, and assessing causality of AEs and SAEs
and the procedures for completing and transmitting SAE reports are provided in 
Appendix 4.
9.1.2.
Method of Detecting AEs and SAEs
Care will be taken not to introduce bias when detecting AE and/or SAE. Open-ended and 
non-leading verbal questioning of the participant is the preferred method to inquire about 
AE occurrence. 

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9.1.3.
Follow-up of AEs and SAEs
After the initial AE/SAE report, the investigator is required to proactively follow each 
participant at subsequent visits/contacts. All SAEs will be followed until the event is 
resolved, stabilized, otherwise explained, or the participant is lost to follow-up (as 
defined in Section 8.2). Further information on follow-up procedures is given in 
Appendix 4.
9.1.4.
Regulatory Reporting Requirements for SAEs

Prompt notification by the investigator to the sponsor of a SAE is essential so 
that legal obligations and ethical responsibilities towards the safety of 
participants and the safety of a study treatment under clinical investigation are 
met. 

The sponsor has a legal responsibility to notify both the local regulatory 
authority and other regulatory agencies about the safety of a study treatment 
under clinical investigation. The sponsor will comply with country-specific 
regulatory requirements relating to safety reporting to the regulatory authority, 
Institutional Review Boards (IRB)/Independent Ethics Committees (IEC), and 
investigators.

Investigator safety reports must be prepared for suspected unexpected serious 
adverse reactions (SUSAR) according to local regulatory requirements and 
sponsor policy and forwarded to investigators as necessary.

An investigator who receives an investigator safety report describing a SAE or 
other specific safety information e.g. summary or listing of SAE) from the 
sponsor will review and then file it along with the Investigator’s Brochure and 
will notify the IRB/IEC, if appropriate according to local requirements.
9.2.
Treatment of Overdose
Cantharidin: Treatment of Challenge Agent Overdose

Participants will not have access to cantharidin and overdose is therefore 
extremely unlikely. In the event of overdose, the clinical management will be 
based on symptomatic treatment and supportive measures as indicated and 
required according to current UK guidelines.

In case of any overexposure to Cantharone refer to Material Safety Data Sheet 
(MSDS).

Inhalation: Remove victim to fresh air. Give oxygen or artificial respiration if 
necessary.

Skin Contact: Immediately flood affected skin with water while removing and 
isolating all contaminated clothing. Gently wash all affected skin areas thoroughly 
with soap and water. Seek medical attention if warranted.

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
Eye Contact: First check the victim for contact lenses and remove if present. 
Flush victim's eyes with water or normal saline solution for 20 to 30 minutes 
while simultaneously calling a hospital or poison control centre. Do not put any 
ointments, oils, or medication in the victim's eyes without specific instructions 
from a physician. Immediately transport the victim after flushing eyes to a 
hospital even if no symptoms (such as redness or irritation) develop.

Ingestion: Do not induce vomiting. If the victim is conscious and not convulsing, 
give 1 or 2 glasses of water to dilute the chemical and immediately call a hospital 
or poison control centre. Immediately transport the victim to a hospital. If the 
victim is convulsing or unconscious, do not give anything by mouth, ensure that 
the victim's airway is open, and lay the victim on his/her side with the head lower 
than the body. Transport the victim immediately to a hospital.
LPS: Treatment of Challenge Agent Overdose

LPS dose selection has been made on the basis of a lower dose and potency than 
is usually reported in the literature, and thus overdose is not anticipated.

Intravenous LPS has the potential to be extremely harmful in overdose and induce 
symptoms and organ dysfunction in keeping with septic shock.  Participants who 
receive an overdose of intravenous LPS will be hydrated with rapid intravenous 
fluid boluses.  Clinical staff may need to expedite immediate transfer to hospital 
in the event that volunteers exhibit clinical signs of septic shock.  Mild pyrexia 
and malaise may be observed.
GM-CSF: Treatment of Challenge Agent Overdose

In case of overdose, participant will be carefully monitored for WBC increase and 
respiratory symptoms. Symptomatic management will be carried out according to 
existing guidelines.
9.3.
Safety Assessments
Planned time points for all safety assessments are provided in the SoA.
9.3.1.
Height and body weight
Height and body weight will be measured and recorded in PIMS. Body weight at session 
2 Day -1 will be used to calculate the dose of LPS and GM-CSF to be used. 
9.3.2.
Physical Examinations

A complete physical examination will include, at a minimum, assessments of the 
skin, cardiovascular, respiratory, gastrointestinal and neurological systems. 

A brief physical examination will include, at a minimum, assessments of the 
skin, lungs, cardiovascular system and abdomen (liver and spleen).

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9.3.3.
Assessment of blister sites

Visual assessment of blister site will be carried out at selected visits.
9.3.4.
Vital Signs

Vital sign measurements will include systolic and diastolic blood pressure, 
respiratory rate, SpO2 and pulse rate.

Blood pressure and pulse measurements will be assessed in a semi-supine 
position with a completely automated device. Manual techniques will be used 
only if an automated device is not available.

Blood pressure and pulse measurements should be preceded by at least 5 
minutes of rest for the participant in a quiet setting in a semi supine position
without distractions (e.g., television, cell phones).

Vital signs (to be taken before blood collection for laboratory tests at times 
detailed in SoA) will consist of 1 pulse and 1 blood pressure measurement. 

Temperature will also be taken
9.3.5.
Electrocardiograms

Triplicate 12-lead ECG will be obtained at screening and baseline as outlined in 
the SoA using an ECG machine that automatically calculates the heart rate and 
measures PR, QRS, QT, and QTc intervals. At all other time points a single 
ECG measurement will be taken. 

All scheduled time points for ECG measurements should be preceded by at least 
5 minutes of rest for the participant in a quiet setting in a semi supine position 
without distractions (e.g., television, cell phones).

At each time point at which triplicate ECG are required, 3 individual ECG 
tracings will be obtained as closely as possible in succession.

Participants dosed with LPS, will receive telemetry for a minimum of 6 hours 
post-dose or until their telemetry shows no clinical significant findings for 4 
hours (whichever is longer).

Participants undergoing the GM-CSF challenge may be connected to continuous 
cardiac telemetry during their stay in the unit at the discretion of the investigator 
if they are symptomatic.
9.3.6.
Participant Diary Card
Participants will be given a diary card for them to record the healing time of the blisters
as well as to record any adverse events or medications taken whilst outside of the unit. 

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9.3.7.
Pain scale
After the final blister sample in each session is taken, participants will be asked to rate 
their level of pain from blister induction on an 11-point scale (0-10), with 0 being “no 
pain” and 10 being “worst pain imaginable” (Farrar, 2001).
9.3.8.
Clinical Safety Laboratory Assessments

Refer to Appendix 2 for the list of clinical laboratory tests to be performed and 
to the SoA for the timing and frequency. 

The investigator must review the laboratory report, document this review, and 
record any clinically relevant changes occurring during the study in the AE 
section of the CRF. The laboratory reports must be filed with the source 
documents. Clinically significant abnormal laboratory findings are those which 
are not associated with the underlying disease, unless judged by the investigator 
to be more severe than expected for the participant's condition.

All laboratory tests with values considered clinically significantly abnormal 
during participation in the study or within 7 days after the last dose of study 
treatment should be repeated until the values return to normal or baseline or are 
no longer considered significantly abnormal by the investigator or medical 
monitor. 

If such values do not return to normal/baseline within a period of time judged 
reasonable by the investigator, the aetiology should be identified and the sponsor 
notified.

All protocol-required laboratory assessments, as defined in Appendix 2, must be 
conducted in accordance with the laboratory manual and the SoA.
9.4.
Genetics
Genetics are not evaluated in this study.  Transcriptome analysis will utilise Next 
Generation Sequencing (NGS) methods, which are described in Section 9.7.1.4.  
9.5.
Pharmacokinetics
Pharmacokinetics are not evaluated in this study.  
9.6.
Pharmacodynamics
Some of the pharmacodynamic endpoints may be reported separately from the main 
clinical study report.
9.7.
Biomarkers
There will be several different types of biomarker samples collected during the study, as 
outlined in the SOA, including biomarkers from blood, urine and blister fluid. Some of 
the exploratory biomarkers may be reported separately from the main clinical study 
report.

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9.7.1.
Biomarkers in blood
9.7.1.1.
Mediators blood sample
Approximately 6 mL of blood will be collected into SST or Na Heparin tubes according 
to the timings on the SoA. This will allow for analysis of serum (or plasma) to measure 
inflammatory mediators. Methods of analysis which may be used on these samples are 
detailed in the Study Reference Manual. Measurement of soluble inflammatory mediators 
will be carried out in batch analysis. 
9.7.1.2.
Flow cytometry blood sample
Blood samples of approximately 2 mL will be collected into SST or Na Heparin tubes for 
measurement of leukocyte number and activation markers by flow cytometry (see 
indicative list in endpoints), at times specified in the SoA.
9.7.1.3.
Ex vivo stimulation blood sample
For the LPS challenge participants only, 2x 1 mL whole blood samples will be collected 
at pre-dose and 6 hours post-LPS dose. These samples will be drawn into TruCulture 
tubes containing LPS and the other in a TruCulture null tube, and incubated for 24 hours. 
Cellular and soluble contents will be separated and inflammatory mediators analysed in 
the soluble fraction.
9.7.1.4.
Transcriptome blood sample
In session 2, whole blood will be collected into PAXgene tubes for transcriptome 
analysis.  Blood (2.5 ml) will be collected prior and several time points post LPS or GM-
CSF challenge as detailed in SoA. Samples will be used to extract RNA and prepare 
cDNA which will subsequently be used for transcriptome analysis by Next Generation 
Sequencing (NGS) methods.  NGS data will be aligned and mapped to human genome 
reference sequences and analysed for differential expression for each challenge type. 
9.7.2.
Biomarkers in urine
The pre-challenge urine sample will be collected during session 1. The post-challenge
urine samples will be collected during session 2 after LPS challenge. In session 2, 
participants will be encouraged to pass urine immediately before LPS challenge dose and 
urine voids will be collected from after LPS until 12 hours post-LPS and the time of the 
urine collection will be recorded. These samples will be collected for measurement of 
tetranor-PGDM and other inflammatory mediators.  Tetranor-PGDM is the stable urinary 
metabolite of the prostaglandin PGD2. While tetranor-PGDM has very low natural 
abundance in normal healthy individuals, it has been shown to be elevated in the urine of 
patients with Duchenne muscular dystrophy.  Elevation of urinary tetranor-PGDM in 
normal healthy volunteers through LPS challenge would allow for evaluation of target 
engagement in that population Tetranor-PGDM and related metabolite will be measure 
by mass spectroscopy as samples are collected.

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9.7.3.
Biomarkers in blisters
Biomarkers in blisters will be sampled at 24 and 48 hours post blister induction and 
collected in polypropylene micro-centrifuge tubes. Cellular and soluble contents will be 
separated. The volume of the blister sample will be calculated and recorded in the eCRF.
9.7.3.1.
Flow cytometry blister sample
Blister sample will be used to measure leukocyte number and activation markers by flow 
cytometry (see indicative list in endpoints), at times specified in the SoA.
9.7.3.2.
Mediators blister sample
The soluble fraction from the blister sample will be used to measure inflammatory 
mediators. Methods of analysis which may be used on these samples are detailed in the 
Study Reference Manual. 
9.8.
Health Economics OR Medical Resource Utilization and 
Health Economics
Health Economics/Medical Resource Utilization and Health Economics parameters are 
not evaluated in this study.
10.
STATISTICAL CONSIDERATIONS
This is an exploratory enabling study which is primarily designed to estimate the effect of 
systemic exposure to LPS or GM-CSF challenge on soluble and cellular inflammatory 
biomarkers in cantharidin-induced skin blister model. There are no formal hypotheses to 
be tested. 
10.1.
Sample Size Determination
Participants will be randomised into the study, in a 1:1 randomisation ratio to either LPS 
or GM-CSF challenge. It is estimated that up to 16 participants may be evaluated at the 
optimal dose (8 participants for each challenge agent). 

Part I (up to 28 participants): Up to 6 cohorts of 4 participants (2 for each challenge) 
may be enrolled. A further 4 participants (2 for each challenge) will be enrolled into 
the cohort at the optimal dose selected.  

Part II (up to 8 participants): Based on the decisions from an interim analysis 
following Part I of the study, an additional cohort of up to 8 participants (4 for each 
challenge) may be enrolled into the cohort at  the optimal dose selected.  

For Part I and II (up to 4 participants), if participants prematurely discontinue the 
study, additional replacement participants may be recruited. A 10% drop-out rate has 
been assumed. 
These numbers have been determined partly through feasibility but considerations has 
also been given for this study to generate data that can provide the foundation for future 

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studies testing the effect of compounds that target key pathways in inflammatory and 
autoimmunity. The 2-stage design provides an efficient way to adapt the sample-size, 
based on emerging data from Part I, so that estimates of response and variability of 
primary endpoints can be generated.     
Following assessment of past studies from literature and GSK, no direct estimates of 
primary endpoints based on LPS and GM-CSF challenges within a cantharidin-induced 
skin blister model were reported. However, estimates of inflammatory response for 
circulating TNF-α and IL-6 which may be translatable to those observed in this study at 
the optimal dose selected have been reported, albeit with some degree of uncertainty : 

Estimates for circulating urinary tetranor PGDM and total leukocyte numbers are
very limited and Part I of the study aims to generate in-house estimates to inform
sample size. 

It is assumed for the purposes of sample size calculation, that the between subject 
standard deviation on the loge scale for TNF-α and IL-6 are 0.217 and 0.394 
respectively (GSK study EMI114416). These estimates reflect the variability of 
cytokines within a cantharidin-induced skin blister model, albeit in the absence of 
challenge agent. 

Based on published data (Suffredini, 1999, Dillingh, 2014, Ferguson, 2014, Kiers,
2017) fold increases from baseline were observed in the range of 42 to 200 for TNF-α 
and 100 to 300 for IL-6 using an IV 1 ng/kg (10EU/ng) LPS challenge. Variability 
was reported with limited estimates to be directly used for sample size calculations.
Using an  estimate of SD the loge scale = 0.217 (EM114416) for TNF-α and a sample size 
of 4 and 8 participants, the probability that the true ratio between systemic challenge and 
baseline being greater than 1 can be calculated for a range of unknown true differences. . 
3 below shows the probability of success for the stated assumptions, for a range of true 
treatment ratios.

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Figure 3
Probability of Success for Cytokines
For example, the probability of achieving study success (i.e. based on observed fold 
increases from baseline in literature and therefore representing robust inflammatory 
cytokine responses with the challenge) assuming the true increase of cytokines with 
challenge agent over baseline is 40% (ratio of 1.4) is  greater than 90% (for n=4 and 8 
participants). If the truth is that there is no increase over baseline (ratio=1) then there is a 
10% chance of incorrectly declaring success.
Sample Size Sensitivity
Table 1, provides the probability of achieving study success assuming the true increase of 
cytokines with challenge agent over baseline is 40% (ratio of 1.4) and assuming larger 
variability is observed.  
Table 1
Probability of Success for Cytokines Assuming Higher Variability 
Observed
Assumed SD 
(Loge scale)
True Ratio 
(Challenge/Baseline)
Probability of Success (%)
n = 4
n = 8
0.394 [1]
1.4
66%
87%
0.434 [2]
1.4
61%
82%
0.473 [3]
1.4
56%
77%
[1] Higher variability observed in study EMI114416; [2] Increase of 10% for SD=0.394; [3] Increase of 20% for 
SD=0.3984

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Sample Size Re-estimation
Sample size re-estimation will be conducted following Part I of the study. If there is an 
increase in the precision of estimates for inflammatory responses, then additional 
participants will be enrolled.  
10.2.
Populations for Analyses
For purposes of analysis, the following populations are defined:
Population
Description
Screened
All participants who were screened for eligibility
Enrolled 
All participants who sign the ICF
Randomized
All participants who are randomized to receive and are given the 
treatment (LPS or GM-CSF challenge) 
Evaluable
All participants who complete the study
As defined in Section 5.3, a participant is considered to have completed the study if
he/she has completed up to the end of session 2 of the study. The end of the study is 
defined as the date of the last visit of the last participant in the study, e.g. the second 
follow up session for the last participant.
10.3.
Statistical Analyses
All data will be summarised using descriptive statistics and/or graphical displays and listed.
Endpoint
Statistical Analysis Methods
Primary
For the optimal dose selected, and if sufficient data in available, primary biomarker 
endpoints will be analysed using a Bayesian repeated measures random effects 
model. Data maybe transformed (e.g. log) prior to the analysis if warranted. Terms in 
the model may include: challenge, challenge by time interaction, blister site and 
forearm. Additional covariates maybe included if deemed appropriate. Adjusted means 
and 95% credible intervals will be constructed for each of the challenges at each time 
point in addition to the comparison of systemic challenge with LPS/baseline and GM-
CSF/baseline. Specific probabilities that the true difference (i.e. challenge to baseline) 
is greater than specified quantities may also be produced. 
Full details of the statistical analysis will be documented in the Reporting and Analysis 
Plan (RAP).
Secondary
The proposed statistical analyses planned for the primary biomarker endpoints will be 
repeated for secondary endpoints.

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Endpoint
Statistical Analysis Methods
Exploratory
Full details will be described in the RAP. 
10.3.1.
Interim Analyses
There will be ongoing data reviews conducted by the study team of any available data 
through the study progression. For Part I, data reviews will be performed by the study 
team to support whether to dose escalate to the next cohort for each challenge agent. At 
the end of Part I, an interim analysis will be conducted to decide whether to enrol 
additional participants in Part II of the study, for the optimum dose selected in Part I. 
Full details of the interim analysis will be pre-specified in the RAP.

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inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot 
of Escherichia coli O:113 endotoxin." J Infect Dis 179(5): 1278-1282.
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(1990). "Experimental endotoxemia in humans: analysis of cytokine release and 
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(2000). "Noninflammatory phagocytosis of monosodium urate monohydrate crystals by 
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12.
APPENDICES
12.1.
Appendix 1: Abbreviations and Trademarks
Abbreviations
AE 
Adverse Event
ALT
Alanine Aminotransferase
AST
Aspartate Aminotransferase
AUC 
Area Under the Curve
BMI
Body Mass Index
CD
Cluster of Differentiation
cDNA
Complementary DNA
CIOMS
Council for International Organizations of Medical Sciences
Cmax
Maximum Concentration
CONSORT
Consolidated Standards of Reporting Trials
COPD
Chronic Obstructive Pulmonary Disease
(e)CRF
(electronic) Case Report Form
CRP
C-reactive protein
CSR
Clinical Study Report
CUC
Clinical Unit Cambridge
ECG
Electrocardiogram
GCP
Good Clinical Practice
GM-CSF
Granulocyte-Macrophage Colony-Stimulating Factor
GSK
GlaxoSmithKline
HIPAA
Health Insurance Portability and Accountability Act
HIV
Human Immunodeficiency Virus
HLA-DR
Human Leukocyte Antigen - antigen D Related

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ICF
Informed Consent Form
ICH
International Conference on Harmonisation
IL 
Interleukin
IV
Intravenous 
IRB/IEC
Institutional Review Boards /Independent Ethics Committees
LPS
Lipopolysaccharide
MCH
Mean Corpuscular Hemoglobin
MCP
Monocyte chemoattractant protein
MCV
Mean Corpuscular volume
mRNA
Messenger RNA
MSDS
Material Safety Data Sheet
NF-kB 
Nuclear Factor kB
NGS
Next Generation Sequencing
NSAIDS
Nonsteroidal anti-inflammatory drugs
PBPC
Peripheral Blood Progenitor Cells
PD
Pharmacodynamics
PGD2
Prostaglandin D2
PGE2
Prostaglandin E2
PR
Pulse rate
PRR
Pattern Recognition Receptors
RAP
Reporting Analysis Plan
RBC
Red Blood Cell
SAE
Serious Adverse Event
SD
Standard Deviation
SoA 
Schedule of Activities

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SRM
Study Reference Manual
SST
Serum-Separating Tube
SUSAR
Suspected Unexpected Serious Adverse Reactions
TNF
Tumour Necrosis Factor
ULN
Upper limit of normal
Trademark Information
Trademarks of the GlaxoSmithKline 
group of companies
Trademarks not owned by the 
GlaxoSmithKline group of companies
NONE
Cantharone
Leukine

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12.2.
Appendix 2: Clinical Laboratory Tests

The tests detailed in 2 will be performed by the local laboratory. 

Protocol-specific requirements for inclusion or exclusion of participants are 
detailed in Section 6 of the protocol.

Additional tests may be performed at any time during the study as determined 
necessary by the investigator or required by local regulations.
Table 2
Protocol-Required Safety Laboratory Assessments
Laboratory 
Assessments
Parameters
Hematology
Platelet Count
RBC Indices:
MCV
MCH
%Reticulocytes
WBC count with 
Differential:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
RBC Count
Hemoglobin
Hematocrit
PT and APTT (at 
screening only)
Clinical 
Chemistry1
BUN
Potassium
Aspartate 
Aminotransferase
(AST)
Total and direct 
bilirubin
Creatinine
Sodium
Alanine 
Aminotransferase
(ALT)
Total Protein
Glucose (fasted 
glucose at 
screening only) 
Calcium
Alkaline 
phosphatase
CRP
Albumin
Urea
Corrected calcium
Routine 
Urinalysis

Urine cotinine

Specific gravity

pH, glucose, protein, blood, ketones by dipstick

Microscopic examination (at investigator discretion if blood or protein 
urine dipstix is abnormal.)
Other tests

HIV

Hepatitis B (HBsAg and HBcAb)

Hepatitis C (Hep C antibody)

Alcohol and drug screen (to include at minimum: amphetamines, 
barbiturates, cocaine, opiates, cannabinoids and benzodiazepines)
NOTES :
1.
Local urine testing will be standard for the protocol unless serum testing is required by local regulation or 
IRB/IEC.

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12.3.
Appendix 3: Study Governance Considerations
Regulatory and Ethical Considerations

This study will be conducted in accordance with the protocol and with:

Consensus ethical principles derived from international guidelines 
including the Declaration of Helsinki and Council for International 
Organizations of Medical Sciences (CIOMS) International Ethical 
Guidelines

Applicable ICH Good Clinical Practice (GCP) Guidelines

Applicable laws and regulations

The protocol, protocol amendments, ICF, Investigator Brochure, and other 
relevant documents (e.g. advertisements) must be submitted to an IRB/IEC by 
the investigator and reviewed and approved by the IRB/IEC before the study is 
initiated. 

Any amendments to the protocol will require IEC/IRB approval before 
implementation of changes made to the study design, except for changes 
necessary to eliminate an immediate hazard to study participants. 

The investigator will be responsible for the following:

Providing written summaries of the status of the study to the IRB/IEC 
annually or more frequently in accordance with the requirements, policies, 
and procedures established by the IRB/EC

Notifying the IRB/IEC of SAE or other significant safety findings as 
required by IRB/IEC procedures

Providing oversight of the conduct of the study at the site and adherence to 
requirements of 21 CFR, ICH guidelines, the IRB/IEC, European 
regulation 536/2014 for clinical studies (if applicable), and all other 
applicable local regulations
Financial Disclosure
Investigators and sub-investigators will provide the sponsor with sufficient, accurate 
financial information as requested to allow the sponsor to submit complete and accurate 
financial certification or disclosure statements to the appropriate regulatory authorities. 
Investigators are responsible for providing information on financial interests during the 
course of the study and for 1 year after completion of the study.
Informed Consent Process

The investigator or his/her representative will explain the nature of the study to 
the participant or his/her legally authorized representative and answer all 
questions regarding the study. 

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
Participants must be informed that their participation is voluntary. Participants 
or their legally authorized representative will be required to sign a statement of 
informed consent that meets the requirements of 21 CFR 50, local regulations, 
ICH guidelines, Health Insurance Portability and Accountability Act (HIPAA) 
requirements, where applicable, and the IRB/IEC or study centre. 

The medical record must include a statement that written informed consent was 
obtained before the participant was enrolled in the study and the date the written 
consent was obtained. The authorized person obtaining the informed consent 
must also sign the ICF.

Participants must be re-consented to the most current version of the ICF(s) 
during their participation in the study. 

A copy of the ICF(s) must be provided to the participant or the participant’s 
legally authorized representative. 

Participants who are rescreened are required to sign a new ICF.
The ICF may contain a separate section that addresses the use of remaining mandatory 
samples for optional exploratory research in accordance with SOP-GSKF-410. The 
investigator or authorized designee will explain to each participant the objectives of the 
exploratory research. Participants will be told that they are free to refuse to participate 
and may withdraw their consent at any time and for any reason during the storage period. 
A separate signature will be required to document a participant's agreement to allow any 
remaining specimens to be used for exploratory research. Participants who decline to 
participate will not provide this separate signature.
Data Protection

Participants will be assigned a unique identifier by the sponsor. Any participant 
records or datasets that are transferred to the sponsor will contain the identifier 
only; participant names or any information which would make the participant 
identifiable will not be transferred. 

The participant must be informed that his/her personal study-related data will be 
used by the sponsor in accordance with local data protection law. The level of 
disclosure must also be explained to the participant. 

The participant must be informed that his/her medical records may be examined 
by Clinical Quality Assurance auditors or other authorized personnel appointed 
by the sponsor, by appropriate IRB/IEC members, and by inspectors from 
regulatory authorities.
Committees Structure
A dose escalation committee will review data after each Group (2 participants) following 
analysis of key primary endpoints to determine a dose which gives a robust inflammatory 
response. 

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Publication Policy

The results of this study may be published or presented at scientific meetings. If 
this is foreseen, the investigator agrees to submit all manuscripts or abstracts to 
the sponsor before submission. This allows the sponsor to protect proprietary 
information and to provide comments.

The sponsor will comply with the requirements for publication of study results. 
In accordance with standard editorial and ethical practice, the sponsor will 
generally support publication of multicentre studies only in their entirety and not 
as individual site data. In this case, a coordinating investigator will be 
designated by mutual agreement.

Authorship will be determined by mutual agreement and in line with 
International Committee of Medical Journal Editors authorship requirements.
Dissemination of Clinical Study Data
Where required by applicable regulatory requirements, an investigator signatory 
will be identified for the approval of the clinical study report.  The investigator 
will be provided reasonable access to statistical tables, figures, and relevant 
reports and will have the opportunity to review the complete study results at a 
GSK site or other mutually-agreeable location.
GSK will also provide the investigator with the full summary of the study results.  
The investigator is encouraged to share the summary results with the study 
participants, as appropriate.
A manuscript will be progressed for publication in the scientific literature if the 
results provide important scientific or medical knowledge.
Data Quality Assurance

All participant data relating to the study will be recorded on printed or electronic 
CRF unless transmitted to the sponsor or designee electronically (e.g. laboratory 
data). The investigator is responsible for verifying that data entries are accurate 
and correct by physically or electronically signing the CRF. 

The investigator must maintain accurate documentation (source data) that 
supports the information entered in the CRF. 

The investigator must permit study-related monitoring, audits, IRB/IEC review, 
and regulatory agency inspections and provide direct access to source data 
documents. 

The sponsor or designee is responsible for the data management of this study 
including quality checking of the data. 

Study monitors will perform ongoing source data verification to confirm that 
data entered into the CRF by authorized site personnel are accurate, complete, 
and verifiable from source documents; that the safety and rights of participants 
are being protected; and that the study is being conducted in accordance with the 

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currently approved protocol and any other study agreements, ICH GCP, and all 
applicable regulatory requirements. 

Records and documents, including signed ICF, pertaining to the conduct of this 
study must be retained by the investigator for 25 years from the issue of the final 
Clinical Study Report (CSR) / equivalent summary unless local regulations or 
institutional policies require a longer retention period. No records may be 
destroyed during the retention period without the written approval of the 
sponsor. No records may be transferred to another location or party without 
written notification to the sponsor. 
Source Documents

Source documents provide evidence for the existence of the participant and 
substantiate the integrity of the data collected. Source documents are filed at the 
investigator’s site.

Data reported on the CRF or entered in the eCRF that are transcribed from 
source documents must be consistent with the source documents or the 
discrepancies must be explained. The investigator may need to request previous 
medical records or transfer records, depending on the study. Also, current 
medical records must be available.

Definition of what constitutes source data can be found in the Source Document 
Agreement.
Study and Site Closure
GSK or its designee reserves the right to close the study site or terminate the study at any 
time for any reason at the sole discretion of GSK. Study sites will be closed upon study 
completion. A study site is considered closed when all required documents and study 
supplies have been collected and a study-site closure visit has been performed.
The investigator may initiate study-site closure at any time, provided there is reasonable 
cause and sufficient notice is given in advance of the intended termination.
Reasons for the early closure of a study site by the sponsor or investigator may include 
but are not limited to:

Failure of the investigator to comply with the protocol, the requirements of the 
IRB/IEC or local health authorities, the sponsor's procedures, or GCP guidelines

Inadequate recruitment of participants by the investigator

Discontinuation of further study treatment development

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12.4.
Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting
Definition of AE
AE Definition

An AE is any untoward medical occurrence in a clinical study participant, temporally 
associated with the use of a study treatment, whether or not considered related to the 
study treatment.

NOTE: An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally 
associated with the use of a study treatment.
Events Meeting the AE Definition 

Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) 
or other safety assessments (e.g. ECG, radiological scans, vital signs measurements), 
including those that worsen from baseline, considered clinically significant in the 
medical and scientific judgment of the investigator (i.e. not related to progression of 
underlying disease).

Exacerbation of a chronic or intermittent pre-existing condition including either an 
increase in frequency and/or intensity of the condition.

New conditions detected or diagnosed after study treatment administration even 
though it may have been present before the start of the study.

Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction.

Signs, symptoms, or the clinical sequelae of a suspected overdose of either study 
treatment or a concomitant medication. Overdose per se will not be reported as an 
AE/SAE unless it is an intentional overdose taken with possible suicidal/self-
harming intent. Such overdoses should be reported regardless of sequelae.

"Lack of efficacy" or "failure of expected pharmacological action" per se will not be 
reported as an AE or SAE. Such instances will be captured in the efficacy 
assessments. However, the signs, symptoms, and/or clinical sequelae resulting from 
lack of efficacy will be reported as AE or SAE if they fulfil the definition of an AE 
or SAE. 

The signs, symptoms, and/or clinical sequelae resulting from lack of efficacy will be 
reported as AE or SAE if they fulfil the definition of an AE or SAE. Also, "lack of 
efficacy" or "failure of expected pharmacological action" constitutes an AE or SAE.

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Events NOT Meeting the AE Definition 

Any clinically significant abnormal laboratory findings or other abnormal safety 
assessments which are associated with the underlying disease, unless judged by the 
investigator to be more severe than expected for the participant’s condition.

The disease/disorder being studied or expected progression, signs, or symptoms of 
the disease/disorder being studied, unless more severe than expected for the 
participant’s condition.

Medical or surgical procedure (e.g. endoscopy, appendectomy): the condition that 
leads to the procedure is the AE.

Situations in which an untoward medical occurrence did not occur (social and/or 
convenience admission to a hospital).

Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present 
or detected at the start of the study that do not worsen.
Definition of SAE
If an event is not an AE per definition above, then it cannot be an SAE even if serious 
conditions are met (e.g. hospitalization for signs/symptoms of the disease under study, 
death due to progression of disease).
A SAE is defined as any untoward medical occurrence that, at any dose:
a.
Results in death
b.
Is life-threatening
The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
participant was at risk of death at the time of the event. It does not refer to an event, 
which hypothetically might have caused death, if it were more severe.
c.
Requires inpatient hospitalization or prolongation of existing hospitalization
In general, hospitalization signifies that the participant has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for observation 
and/or treatment that would not have been appropriate in the physician’s office or 
outpatient setting. Complications that occur during hospitalization are AE. If a 
complication prolongs hospitalization or fulfils any other serious criteria, the event is 
serious. When in doubt as to whether “hospitalization” occurred or was necessary, the AE 
should be considered serious.
Hospitalization for elective treatment of a pre-existing condition that did not worsen from 
baseline is not considered an AE.

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d.
Results in persistent disability/incapacity

The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions.

This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhoea, influenza, 
and accidental trauma (e.g. sprained ankle) which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption.
e.
Is a congenital anomaly/birth defect
f.
Other situations:

Medical or scientific judgment should be exercised in deciding whether SAE 
reporting is appropriate in other situations such as important medical events that may 
not be immediately life-threatening or result in death or hospitalization but may 
jeopardize the participant or may require medical or surgical intervention to prevent 
one of the other outcomes listed in the above definition. These events should usually 
be considered serious.
Examples of such events include invasive or malignant cancers, intensive treatment 
in an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency 
or drug abuse.
Recording AE and SAE
AE and SAE Recording

When an AE/SAE occurs, it is the responsibility of the investigator to review all 
documentation (e.g. hospital progress notes, laboratory, and diagnostics reports) 
related to the event.

The investigator will then record all relevant AE/SAE information in the CRF.

It is not acceptable for the investigator to send photocopies of the participant’s 
medical records to GSK in lieu of completion of the GSK /AE/SAE CRF page.

There may be instances when copies of medical records for certain cases are 
requested by GSK. In this case, all participant identifiers, with the exception of the 
participant number, will be redacted on the copies of the medical records before 
submission to GSK.

The investigator will attempt to establish a diagnosis of the event based on signs, 
symptoms, and/or other clinical information. Whenever possible, the diagnosis (not 
the individual signs/symptoms) will be documented as the AE/SAE.

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Assessment of Intensity
The investigator will make an assessment of intensity for each AE and SAE reported 
during the study and assign it to 1 of the following categories: 

Mild: An event that is easily tolerated by the participant, causing minimal discomfort 
and not interfering with everyday activities.

Moderate: An event that causes sufficiently discomfort and interferes with normal 
everyday activities.

Severe: An event that prevents normal everyday activities. An AE that is assessed as 
severe should not be confused with an SAE. Severe is a category utilized for rating 
the intensity of an event; and both AE and SAE can be assessed as severe.
An event is defined as ‘serious’ when it meets at least 1 of the predefined outcomes 
as described in the definition of an SAE, NOT when it is rated as severe.
Assessment of Causality

The investigator is obligated to assess the relationship between study treatment and 
each occurrence of each AE/SAE.

A "reasonable possibility" of a relationship conveys that there are facts, evidence, 
and/or arguments to suggest a causal relationship, rather than a relationship cannot 
be ruled out.

The investigator will use clinical judgment to determine the relationship.

Alternative causes, such as underlying disease(s), concomitant therapy, and other 
risk factors, as well as the temporal relationship of the event to study treatment 
administration will be considered and investigated.

The investigator will also consult the Investigator’s Brochure (IB) and/or Product 
Information, for marketed products, in his/her assessment.

For each AE/SAE, the investigator must document in the medical notes that he/she 
has reviewed the AE/SAE and has provided an assessment of causality.

There may be situations in which an SAE has occurred and the investigator has 
minimal information to include in the initial report to GSK. However, it is very 
important that the investigator always make an assessment of causality for 
every event before the initial transmission of the SAE data to GSK.

The investigator may change his/her opinion of causality in light of follow-up 
information and send an SAE follow-up report with the updated causality 
assessment.

The causality assessment is one of the criteria used when determining regulatory 
reporting requirements.

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Follow-up of AE and SAE

The investigator is obligated to perform or arrange for the conduct of supplemental 
measurements and/or evaluations as medically indicated or as requested by GSK to 
elucidate the nature and/or causality of the AE or SAE as fully as possible. This may 
include additional laboratory tests or investigations, histopathological examinations, 
or consultation with other health care professionals.

If a participant dies during participation in the study or during a recognized follow-
up period, the investigator will provide GSK with a copy of any post-mortem 
findings including histopathology.

New or updated information will be recorded in the originally completed CRF.

The investigator will submit any updated SAE data to GSK within 24 hours of 
receipt of the information.
Reporting of SAE to GSK
SAE Reporting to GSK via Paper CRF

Facsimile transmission of the SAE paper CRF is the preferred method to transmit 
this information to the Primary Medical monitor.

In rare circumstances and in the absence of facsimile equipment, notification by 
telephone is acceptable with a copy of the SAE data collection tool sent by overnight 
mail or courier service.

Initial notification via telephone does not replace the need for the investigator to 
complete and sign the SAE CRF pages within the designated reporting time frames.

Contacts for SAE reporting can be found in the study reference manual (SRM).

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12.5.
Appendix 5: Contraceptive Guidance and Collection of 
Pregnancy Information
Contraception Guidance
Male participants

Male participants with female partners of child-bearing potential are eligible to 
participate if they agree to ONE of the following during the protocol-defined 
time frame in Section 6.1: 

Are abstinent from penile-vaginal intercourse as their usual and preferred 
lifestyle (abstinent on a long term and persistent basis) and agree to remain 
abstinent 

Agree to use a male condom plus an additional method of contraception 
with a failure rate of <1% per year when having penile-vaginal intercourse 
with a woman of childbearing potential 

Men with a pregnant or breastfeeding partner must agree to remain abstinent 
from penile-vaginal intercourse or use a male condom during each episode of 
penile penetration during the protocol-defined time frame.

In addition, male participants must refrain from donating sperm from session 2 
to end of final study follow-up.
Collection of Pregnancy Information
Male participants with partners who become pregnant

Investigator will attempt to collect pregnancy information on any male participant’s 
female partner of a male study participant who becomes pregnant while participating 
in this study. This applies only to participants who receive study treatment.

After obtaining the necessary signed informed consent from the pregnant female 
partner directly, the investigator will record pregnancy information on the 
appropriate form and submit it to GSK within 24 hours of learning of the partner’s 
pregnancy. 

Partner will also be followed to determine the outcome of the pregnancy. 
Information on the status of the mother and child will be forwarded to GSK

Generally, follow-up will be no longer than 6 to 8 weeks following the estimated 
delivery date. Any termination of the pregnancy will be reported regardless of foetal
status (presence or absence of anomalies) or indication for procedure.