Clinical trial NCT04676763
Conditions: Skin Diseases
Keywords: Substance P Wheal response Flare response Skin challenge
Sponsor: GlaxoSmithKline
Start date: 2021-03-02; Completion date: 2021-07-21
Enrollment: 32 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
The objectives of this enabling study are to characterize the wheal and flare responses over time following skin challenges with ascending concentrations of Substance P. This will be a 2-part study: Part 1 will aid in the understanding of the wheal and flare responses following Substance P. Part 2 will investigate the variability of the responses. Participants may be enrolled into Part 1 or Part 2, not both.

Detailed description:

Arms: arm 1: Eligible participants will receive control challenge with saline and histamine, followed by challenge with ascending concentrations of Substance P. Part 1 will include a single challenge visit. arm 2: Eligible participants will receive control challenge with saline and histamine, followed by challenge with ascending concentrations of Substance P. Part 2 will include two challenge visits.
Interventions: intervention 1: Substance P intervention 2: Normal Saline intervention 3: Histamine
Intervention descriptions: intervention 1: Participants will receive Substance P intervention 2: Participants will receive normal saline as Negative control intervention 3: Participants will receive histamine as Positive control

Protocol documents:
Prot_000.pdf: 
 
 
 
 
Protocol 
 
Study ID:  213224 
 
Official Title of Study: An open label, single centre, enabling study to investigate the 
optimum method for use of intradermal Substance P as a challenge agent in healthy 
participants 
 
NCT ID: NCT04676763 
 
 
Date of Document: 03-NOV-2020 
 
 


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TITLE PAGE
Protocol Title: An open label, single centre, enabling study to investigate the optimum 
method for use of intradermal Substance P as a challenge agent in healthy participants
Protocol Number: 213224
Compound Number
or Name:
None
Study Phase: N/A
Short Title: Intradermal Substance P challenge in healthy participants
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): NA
Approval Date: 03-NOV-2020
Copyright 2020 the GlaxoSmithKline group of companies.  All rights reserved.  
Unauthorised copying or use of this information is prohibited.


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SPONSOR SIGNATORY: 
Nicolas Wisniacki 
VP, Discovery Medicine, CPEM, GSK 
2 
213224 
Date 
PPD
PPD
PPD


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TABLE OF CONTENTS
PAGE
1.
PROTOCOL SUMMARY..........................................................................................6
1.1.
Synopsis.......................................................................................................6
1.2.
Schema ........................................................................................................9
1.3.
Schedule of Activities (SoA)........................................................................10
1.3.1.
Screening ....................................................................................11
1.3.2.
Part 1 & Part 2 Challenge Visits...................................................12
1.3.3.
Each challenge ............................................................................14
2.
INTRODUCTION....................................................................................................15
2.1.
Study Rationale ..........................................................................................15
2.2.
Background ................................................................................................15
2.3.
Benefit/Risk Assessment ............................................................................15
2.3.1.
Risk Assessment .........................................................................16
2.3.2.
Benefit Assessment.....................................................................20
2.3.3.
Overall Benefit: Risk Conclusion..................................................20
3.
OBJECTIVES AND ENDPOINTS...........................................................................20
4.
STUDY DESIGN ....................................................................................................22
4.1.
Overall Design ............................................................................................22
4.2.
Scientific Rationale for Study Design ..........................................................23
4.3.
Justification for Substance P concentrations...............................................24
4.4.
End of Study Definition ...............................................................................24
5.
STUDY POPULATION...........................................................................................25
5.1.
Inclusion Criteria.........................................................................................25
5.2.
Exclusion Criteria........................................................................................26
5.3.
Lifestyle Considerations..............................................................................28
5.3.1.
Caffeine, Alcohol, and Tobacco ...................................................28
5.3.2.
Activity .........................................................................................29
5.4.
Screen Failures...........................................................................................29
6.
STUDY INTERVENTION AND CONCOMITANT THERAPY ..................................29
6.1.
Study Intervention(s) Administered .............................................................29
6.2.
Preparation/Handling/Storage/Accountability..............................................30
6.3.
Measures to Minimize Bias: Randomization and Blinding ...........................31
6.4.
Study Intervention Compliance ...................................................................31
6.5.
Modification of Substance P concentrations................................................31
6.6.
Intervention after the End of the Study........................................................31
6.7.
Treatment of Overdose...............................................................................31
6.8.
Concomitant Therapy..................................................................................32
7.
DISCONTINUATION OF STUDY INTERVENTION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL.....................................................................32
7.1.
Study Stopping criteria................................................................................32
7.2.
Discontinuation of Study Intervention..........................................................33
7.3.
Participant Discontinuation/Withdrawal from the Study ...............................33
7.4.
Lost to Follow Up........................................................................................33


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8.
STUDY ASSESSMENTS AND PROCEDURES .....................................................34
8.1.
Efficacy Assessments.................................................................................34
8.2.
Safety Assessments ...................................................................................34
8.2.1.
Physical Examinations.................................................................34
8.2.2.
Vital Signs....................................................................................35
8.2.3.
Electrocardiograms......................................................................35
8.2.4.
Clinical Safety Laboratory Assessments ......................................35
8.2.5.
Pregnancy Testing.......................................................................36
8.3.
Adverse Events and Serious Adverse Events.............................................36
8.3.1.
Time Period and Frequency for Collecting AE and SAE 
Information...................................................................................36
8.3.2.
Method of Detecting AEs and SAEs.............................................37
8.3.3.
Follow-up of AEs and SAEs.........................................................37
8.3.4.
Regulatory Reporting Requirements for SAEs .............................37
8.3.5.
Pregnancy ...................................................................................37
8.4.
Pharmacokinetics .......................................................................................38
8.5.
Pharmacodynamics ....................................................................................38
8.5.1.
Visual check of planned challenge sites and administration 
of challenge agents......................................................................38
8.5.2.
Wheal and Flare Assessments ....................................................38
8.6.
Genetics .....................................................................................................38
8.7.
Biomarkers .................................................................................................39
8.8.
Immunogenicity Assessments.....................................................................39
8.9.
Health Economics.......................................................................................39
9.
STATISTICAL CONSIDERATIONS........................................................................39
9.1.
Statistical Hypotheses.................................................................................39
9.2.
Sample Size Determination ........................................................................40
9.3.
Populations for Analyses ............................................................................40
9.4.
Statistical Analyses.....................................................................................40
9.4.1.
General Considerations ...............................................................40
9.4.2.
Primary Endpoints .......................................................................41
9.4.3.
Secondary Endpoints...................................................................41
9.4.4.
Tertiary/Exploratory Endpoints.....................................................41
9.5.
Interim Analyses .........................................................................................42
10. SUPPORTING DOCUMENTATION AND OPERATIONAL
CONSIDERATIONS...............................................................................................43
10.1.
Appendix 1: Regulatory, Ethical, and Study Oversight 
Considerations............................................................................................43
10.1.1.
Regulatory and Ethical Considerations ........................................43
10.1.2.
Financial Disclosure.....................................................................43
10.1.3.
Informed Consent Process ..........................................................44
10.1.4.
Data Protection............................................................................44
10.1.5.
Committees Structure ..................................................................45
10.1.6.
Dissemination of Clinical Study Data ...........................................45
10.1.7.
Data Quality Assurance ...............................................................46
10.1.8.
Source Documents ......................................................................46
10.1.9.
Study and Site Start and Closure.................................................47
10.1.10. Publication Policy.........................................................................47
10.2.
Appendix 2: Clinical Laboratory Tests.........................................................49


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10.3.
Appendix 3: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting .......................................51
10.3.1.
Definition of AE............................................................................51
10.3.2.
Definition of SAE..........................................................................52
10.3.3.
Recording and Follow-Up of AE and SAE....................................53
10.3.4.
Reporting of SAE to GSK.............................................................55
10.4.
Appendix 4: Contraceptive Guidance and Collection of Pregnancy 
Information..................................................................................................56
10.4.1.
Definitions:...................................................................................56
10.4.2.
Contraception Guidance: .............................................................57
10.5.
Appendix 5: Abbreviations and Trademarks................................................58
11. REFERENCES.......................................................................................................60


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1.
PROTOCOL SUMMARY
1.1.
Synopsis
Protocol Title: An open label single-centre, enabling study to investigate the optimum 
method for use of intradermal Substance P as a challenge agent in healthy participants
Short Title: Intradermal Substance P challenge in healthy participants
Rationale:
This study aims to establish a Substance P (SP) intradermal challenge model which will 
allow the future clinical evaluation of the pharmacological effects of antagonists to block 
or decrease the induced wheal and flare response.
Objectives and Endpoints:
Part 1
Objectives
Endpoints
Primary

To characterise the wheal response 
over time following skin challenge with 
ascending concentrations of SP on a 
single day

Wheal response during the 2 h post-
challenge period area under the curve 
(AUC) at each concentration of SP
Secondary

To characterise the wheal response 
over time following skin challenge with 
ascending concentrations of SP on a 
single day

Wheal response during the 2 h post-
challenge period at each concentration 
of SP (including maximum observed 
wheal response, time to maximum 
observed wheal response, time of 
complete wheal response 
disappearance)


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Objectives
Endpoints

To characterise the flare response over 
time following skin challenge with 
ascending concentrations of SP on a 
single day

Flare response during the 2 h post-
challenge period at each concentration 
of SP (including AUC, maximum 
observed flare response, time to 
maximum observed flare response, time 
of complete flare response 
disappearance)

To evaluate the safety and tolerability of 
SP

Incidence of Adverse Events (AE) and
Serious Adverse Events (SAE)

Incidence of findings of clinical important 
changes in:
o
vital signs 
o
laboratory safety data 
o
12-lead electrocardiogram 
(ECG)
Part 2
Objectives
Endpoints
Primary

To compare the wheal response over 
time following skin challenge with 
ascending concentrations of SP on 2 
days

Wheal response during the 2 h post-
challenge period (AUC) at each 
concentration of SP across 2 challenge 
days
Secondary

To compare the wheal response over 
time following skin challenge with 
ascending concentrations of SP on 2 
days

Wheal response during the 2 h post-
challenge period at each concentration of 
SP across 2 challenge days (including 
maximum observed wheal response, time 
to maximum observed wheal response, 
time of complete wheal response 
disappearance)


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Objectives
Endpoints

To compare the flare response over 
time following skin challenge with 
ascending concentrations of SP on 2 
days

Flare response during the 2 h post-
challenge period at each concentration of 
SP across 2 challenge days (including 
AUC, maximum observed flare response, 
time to maximum observed flare response, 
time of complete flare response 
disappearance)

To evaluate the safety and tolerability 
of SP

Incidence of Adverse Events (AE) and
Serious Adverse Events (SAE)

Incidence of findings of clinical important 
changes in:
o
vital signs 
o
laboratory safety data 
o
12-lead ECG
Overall Design:
This is an open label single-centre, 2-part, prospective enabling study of SP intradermal 
challenge in healthy participants.  Parts 1 and 2 will run sequentially.  A schematic of the 
overall study design is provided in Figure 1.
Disclosure Statement: This is a 2-part, single arm, sequential study with no masking.
Number of Participants:
Part 1 will enrol a sufficient number of participants to achieve up to 12 participants who 
have completed the challenge visit.  Once Part 1 is complete, SP concentrations to be 
given in Part 2 will be confirmed.  Part 2 will occur only if Part 1 is successful.
Part 2 will enrol a sufficient number of participants to achieve up to 20 participants who 
have completed the 2 challenge visits.  The study may be stopped after review of data 
from about the first 10 participants.  
Participants who withdraw or are withdrawn from the study may be replaced.  In Part 2,
following screening, replacement participants must start at Challenge Visit 1.
Participants may be enrolled into Part 1 or Part 2, not both.  Therefore, the anticipated 
sample size is up to 32 participants (not including replacements).
Intervention Groups and Duration:
All participants will attend the clinical unit for a screening visit (V0) within 28 days 
before their first or only challenge visit.  


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In Part 1, participants will attend the unit as outpatients for 1 challenge visit.  The 
duration of Part 1, including screening, is not expected to exceed 4 weeks for each 
participant.  
In Part 2, participants will attend the unit as outpatients for 2 challenge visits and have 
1 follow--up phone call; the timing of visits will be decided based on emerging data.  
The duration of Part 2, including screening and follow-up is not expected to exceed 7 
weeks for each participant.  
A schematic of each challenge day is provided in Figure 2.
Data Monitoring or Other Committee: No
1.2.
Schema
Figure 1
Overall study design schematic
Abbreviations:  SP = Substance P.


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Figure 2
Schematics of challenge days
Challenge Visit 1
Challenge Visit 2
Part 1
Wheal and flare post SP challenges (Panel 
A) only
Not applicable
Part 2
Wheal and flare post SP challenges (Panel 
A); and
IDM during SP challenges (Panel B)
Wheal and flare post SP challenges (Panel 
A)
IDM during SP challenges (Panel B)
Biopsy post challenges (Panel C)
Before first and after last SP challenge:  
biomarker blood sample^
Panel A:  Wheal and Flare measurement
Panel B:  IDM measurement
Panel C:  Biopsy sample
Abbreviations:  IDM = intradermal microdialysis; SP = Substance P.
^ To coincide with safety laboratory blood sampling.
* Acceptable saline and histamine responses are defined in the Study Reference Manual (SRM).
1.3.
Schedule of Activities (SoA)
Protocol waivers or exemptions are not allowed. Therefore, adherence to the study design 
requirements, including those specified in the SOA, are essential and required for study 
conduct. This section lists the procedures and parameters of each planned study 
assessment. The exact timing of each assessment is listed in the SOA. 


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1.3.1.
Screening
Visit (V)
V0
Notes
Procedure
Screening 
(up to 28 days before Day 1)
Informed consent
X
Inclusion and exclusion criteria
X
Demography
X
Complete physical examination including height and 
weight
X
Medical /medication/drug/alcohol/tobacco history
X
Includes Substance usage and current medical condition 
Drug/alcohol/cotinine test
X
As per standard local practice
HIV, Hepatitis B and C screening
X
If test otherwise performed within 3 months before first challenge day, 
testing at screening is not required.
Serum pregnancy test (WCBP only)
X
FSH and estradiol (in WNCBP)
X
As required, if postmenopausal state in women is in doubt.
12-lead ECG
X
In triplicate.  Mean of 3 measure used to confirm eligibility.
Vital signs
X
Blood pressure and heart rate, in triplicate.  Mean of 3 measure used 
to confirm eligibility.  Single measurement of temperature.
Control challenge (histamine/saline)
X
Clinical chemistry/Haemtology and urinalysis
X
SAE review
X
From signing consent
Abbreviations:  ECG = electrocardiogram; HIV = human immunodeficiency virus; SAE = serious adverse event; WCBP = women of childbearing potential; WNCBP = women of non 
childbearing potential.


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1.3.2.
Part 1 & Part 2 Challenge Visits  
Visit (V)
V1, V2 (Part 2 only)
Follow-
up phone 
call
(5-7 days
post last 
challenge 
day)
Early 
withdrawal
Notes
Procedure
Pre-
challenge
Within 1 h before 
first control 
challenge
At least 20 min 
before first SP 
challenge
SP challenges
2 h after last 
SP challenge
Clinical 
chemistry/ 
Haematology 
and urinalysis 
X
X*
X
*In Part 2, 2 h post last SP 
challenge at last challenge visit 
only.
12-lead ECG
X
X
X
Triplicate measurements.
Vital signs
X
X
X
Refer to Section 8.2.2.
Brief PE
X
Urine 
pregnancy test 
(WCBP only)
X
Visual check 
of planned 
challenge 
sites
X
Prior to any other study 
procedures.
Saline and 
Histamine 
challenges
X
Histamine to be administered at 
least 20 cm from any IDM sampling
SP challenge
X
Part 1:  Up to 4 SP injections:  
increasing concentrations of SP at 
a minimum of 20-min intervals.  
Part 2:  Up to 10 SP injections:
up to 4 increasing 
concentrations of SP at a 
minimum of 20-min intervals, in 
duplicate


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Visit (V)
V1, V2 (Part 2 only)
Follow-
up phone 
call
(5-7 days
post last 
challenge 
day)
Early 
withdrawal
Notes
Procedure
Pre-
challenge
Within 1 h before 
first control 
challenge
At least 20 min 
before first SP 
challenge
SP challenges
2 h after last 
SP challenge
a further 2 SP injections at one 
concentration may also be given, 
if ongoing data suggest biopsy 
samples are required
Wheal and 
flare 
assessment
See Section 1.3.3
Blood sample 
for exploratory 
biomarkers
X
X
Part 2, V2 only.
IDM
See Section 1.3.3
Part 2 only
Skin punch 
biopsy
See Section 1.3.3
Part 2, V2 only.  May or may not be 
performed based on ongoing data.
AE review

==========================================================
X
SAE review

==========================================================
X
Concomitant 
medication 
review
X
Review of medications since last 
visit, if applicable.
Follow-up on 
possible 
reactions at 
sites of 
IDM/biopsy
X
Part 2 only.
Abbreviations:  AE = adverse event; ECG = electrocardiogram; IDM = intradermal microdialysis; SAE = serious adverse event; SP = substance P.


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1.3.3.
Each challenge
Procedure
Min in relation to time of each intradermal injection of each SP or saline, or the time 
of skin prick administration of histamine
Notes
Pre-challenge
0
5
10
15
20
30
40
60
90
120
Challenge
X
Wheal and flare 
assessment
X
X
X
X
X
X
X
X
See Section 8.5.2 for methods.
IDM (SP and saline 
challenged sites, as 
indicated in 
Figure 2)
X
=================================================
Part 2 only (see Figure 2).  Tubing inserted at least 2 h 
before baseline sampling.  A 30-min pre-challenge sample 
will be taken within 1 h before challenge.  Post-challenge 
samples will be collected in 10-min interval from 0–120 min 
post-challenge.
Skin punch biopsy 




X


X


Part 2, Challenge Visit 2 only (see Figure 2).  20-min and 
60-min samples to be taken from different SP challenged 
sites; a third biopsy to be taken from a non-challenged area 
of skin to be collected at approximately the same time as the 
20-min SP biopsy.  Biopsy sampling may or may not be 
included in the Part 2 based on ongoing data.
Abbreviations:  IDM = intradermal microdialysis; SP = substance P.

The timing and number of planned study assessments, including the challenge or sampling and assessment time points in Part 2
(safety, wheal and flare, biomarker samples [including IDM and skin punch biopsy]) may be altered during the course of the study 
based on newly available data. 

Any changes to the timing or addition of time points for any planned study assessments in Part 2 as the result of emerging data from 
Part 1 of this study 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 Competent Authority (CA) and ethics committee (EC) will be 
informed of any safety issues that constitute a substantial amendment and require alteration of the safety monitoring scheme or 
amendment of the informed consent form (ICF). The changes will be approved by the EC before implementation.

Acceptable time windows around the nominal time points for specific assessments will be included in the Study Reference Manual 
(SRM) and assessments performed within these time windows will not constitute a protocol deviation.


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2.
INTRODUCTION
2.1.
Study Rationale
This study aims to establish and validate a Substance P (SP) intradermal challenge model 
which will allow the future clinical evaluation of the pharmacological effects of 
antagonists to block or decrease the induced wheal and flare response.
2.2.
Background
SP is a neuropeptide with an important role in neurogenic inflammation [Mashaghi, 
2016].  When it is injected intradermally it causes wheal and flare responses, which 
increase in size with increased concentrations of SP [Borici-Mazi, 1999; Grouzmann, 
2011; Sabroe, 1997; Zirbs, 2013].  While wheal and flare response to SP has been 
previously studied, the methods used to determine effect size differ between published 
reports and variability is difficult to establish.  In addition, previous studies did not 
investigate possible modulation of pro-inflammatory markers and immune cells within 
the skin following SP challenge.  This knowledge will aid the understanding of the 
mechanistic pathways downstream of SP activation.
Techniques such as intradermal microdialysis (IDM) can be used to take samples of 
interstitial fluid [Holmgaard, 2012; Baumann, 2019].  IDM involves insertion of dialysis 
membranes into the dermis, which is then perfused at a low speed with a physiological 
solution (the perfusate).  Endogenous or exogenous molecules soluble in the extracellular 
fluid diffuse into the membrane and are collected in small vials for analysis.  The
technique has not previously been used to take samples from SP challenged skin.
This will be a 2-part study:  Part 1 will aid understanding of the wheal and flare responses 
following intradermal SP Part 2 will investigate the variability of the responses, including 
the release of locally acting mediators and may include immune cells markers that can be 
detected in the skin after challenge..    
2.3.
Benefit/Risk Assessment
More detailed information about the known and expected benefits and risks and 
reasonably expected adverse events of SP can be found in the Participant Information 
Leaflet.


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2.3.1.
Risk Assessment
Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Intradermal Substance P (SP) Challenge Agent
Substance P intradermal challenge
Transient local and limited regional skin 
flushing and local urticarial-like oedema 
has been observed with intradermal 
administration.  One study with total 
doses of 5575 pmol intradermally in a 
single day had no systemic effects noted 
[Zirbs, 2013].  A second study 
administered a total dose of 5555 pmol 
[Grouzmann, 2011] with only minor 
flushing and no blood pressure effects.
Transient flushing, tachycardia, dizziness, 
and decreased blood pressure have been 
observed with intravenous dosing at 
>16 pmol/min for 6 minutes (96 pmol). 
[Newby , 1997].
Only small doses of Substance P will be 
administered intradermally. For Part 1 the 
planned doses will total 220 pmol and 
Part 2 the planned doses will total 740 
pmol resulting in a 25x and 7.5x coverage 
from the previously safe doses 
administered in reported publications 
[Zirbs, 2013; Grouzmann, 2011].
There is a provision to administer up to 500 
pmol should no wheal and flare occur at 
lower doses.  This top allowed dose would 
result in a Part 1 total dose of 715 pmol and 
a Part 2 total dose of 2430 pmol, still 
resulting in a 7.8x and 2.3x coverage from 
the previously safe doses administered in 
reported publications [Zirbs, 2013; 
Grouzmann, 2011].
The participants will be closely monitored 
during administration of SP challenge. The 
clinical site will have appropriate 
precautions and treatments in place during 


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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
administration and post-administration 
observation.
Skin prick test-Histamine Control Agent
Histamine intradermal challenge
Highly sensitised patients may experience 
stronger local reactions.  In extremely rare 
cases generalised side reactions and even 
severe systemic reactions (anaphylactic 
shock) may occur.
Mild systemic adverse reactions include 
rhinitis, conjunctivitis, asthma, generalized 
exanthema, or urticaria.  More severe 
adverse reactions include itching of tongue, 
throat, palms or soles.  The reaction may 
progress to cyanosis, tachycardia, 
dizziness, headache, bronchial constriction, 
urticaria, asthma, marked hypotension or 
hypertension, vomiting, urination or 
defecation.
Histamine challenge is an established 
proinflammation/allergy test.
Only small concentrations will be 
administered via skin prick. One drop (~50 
μL) of 1.7 mg/mL solution) is applied to 
the skin and a prick needle is passed 
through to introduce a small amount 
intradermally. This dose and technique are
commonly used in histamine skin challenge 
test. The participants will be closely 
monitored during administration of 
histamine challenge. The clinical site will 
have appropriate precautions and 
treatments in place, per the product label,
during administration and post-
administration observation.


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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Study Procedures
Intradermal Microdialysis
Mild pain, bruising and irritation at dialysis 
site
Site staff under supervision of experienced 
staff will follow local standard approaches 
to perform the procedure.
Phlebotomy
Fainting, mild pain, bruising, irritation or 
redness at a phlebotomy site may be 
associated with blood draws.
Experienced site staff will follow local 
standard approaches for managing events 
related to blood draws.
Participants are instructed to visually 
inspect phlebotomy sites after clinical visit
are informed what signs of
inflammation/infection to watch for and 
contact the investigator with any concerns.
Skin Biopsy
Complications of punch skin biopsy are 
uncommon but may include bleeding, 
infection, and scarring.
Scarring risk is increased in individuals 
with a history of scarring and in individuals 
with darker skin.
Exclusion criterion #3:
History of risk for or actual experience of 
complications from skin biopsy including 
excess bleeding, infection, or 
scarring/keloid formation (Participants with 
darker skin (Fitzpatrick>2) will be 
excluded).
Observation of the subject while in the 
clinical unit and at follow-up after 
discharge from the unit. 


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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Participants are instructed to visually
inspect skin biopsy sites after clinical visit
and are informed what signs of 
inflammation/infection to watch for and 
contact the investigator with any concerns.


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2.3.2.
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 methodologies to assess future 
medicines by taking part in the study.
2.3.3.
Overall Benefit: Risk Conclusion
The measures taken to minimize the potential risks identified in association with 
intradermal administration of SP are considered sufficient to justify participation by 
healthy participants.
3.
OBJECTIVES AND ENDPOINTS
Part 1
Objectives
Endpoints
Primary

To characterise the wheal response 
over time following skin challenge with
ascending concentrations of SP on a 
single day

Wheal response during the 2 h post-
challenge period area under the curve 
(AUC) at each concentration of SP
Secondary

To characterise the wheal response 
over time following skin challenge with 
ascending concentrations of SP on a 
single day

Wheal response during the 2 h post-
challenge period at each concentration 
of SP (including maximum observed 
wheal response, time to maximum 
observed wheal response, time of 
complete wheal response 
disappearance)


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
To characterise the flare response over 
time following skin challenge with
ascending concentrations of SP on a 
single day

Flare response during the 2 h post-
challenge period at each concentration 
of SP (including AUC, maximum 
observed flare response, time to 
maximum observed flare response, time 
of complete flare response 
disappearance)

To evaluate the safety and tolerability of 
SP

Incidence of Adverse Events (AE) and
Serious Adverse Events (SAE)

Incidence of findings of clinical important 
changes in:
o
vital signs 
o
laboratory safety data 
o
12-lead electrocardiogram 
(ECG)
Part 2
Objectives
Endpoints
Primary

To compare the wheal response over 
time following skin challenge with
ascending concentrations of SP on 
two days

Wheal response during the 2 h post-
challenge period (AUC) at each 
concentration of SP across 2 challenge
days
Secondary

To compare the wheal response over 
time following skin challenge with 
ascending concentrations of SP on 
two days

Wheal response during the 2 h post-
challenge period at each concentration of 
SP across 2 challenge days (including 
maximum observed wheal response, time 
to maximum observed wheal response, 
time of complete wheal response 
disappearance)


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Objectives
Endpoints

To compare the flare response over 
time following skin challenge with
ascending concentrations of SP on 
two days

Flare response during the 2 h post-
challenge period at each concentration of 
SP across 2 challenge days (including 
AUC, maximum observed flare response, 
time to maximum observed flare response, 
time of complete flare response 
disappearance)

To evaluate the safety and tolerability 
of SP

Incidence of Adverse Events (AE) and
Serious Adverse Events (SAE)

Incidence of findings of clinical important 
changes in:
o
vital signs 
o
laboratory safety data 
o
12-lead ECG
Exploratory

To investigate the effect of intradermal 
SP challenges on local exploratory 
biomarkers of mast cell activation.

Change in levels of exploratory 
biomarkers in skin during up to 2hr post 
challenge time at each of the 2 challenge 
days.
4.
STUDY DESIGN
4.1.
Overall Design
This is an open label, single centre, 2-part, prospective enabling study of SP intradermal 
challenge in healthy participants.  Parts 1 and 2 will run sequentially.  Part 1 will include 
1 challenge visit; Part 2 will include two challenge visits.  
All participants will attend the clinical unit for a screening visit within 28 days before 
their first or only challenge visit.  Eligible participants will be enrolled into Part 1 or 
Part 2, based on their availability.

In Part 1, participants will attend the unit as outpatients for 1 challenge visit.  The 
duration of Part 1, including screening, is not expected to exceed 4 weeks for each 
participant. .  

In Part 2, participants will attend the unit as outpatients for 2 challenge visits and 
have 1 follow-up phone call; the timing of visits will be decided based on emerging 
data.  The duration of Part 2, including screening and follow-up is not expected to 
exceed 7 weeks for each participant.


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At each challenge visit, up to 4 concentrations of SP will be assessed, as follows:

In Parts 1 and 2:

Participants will first receive saline, by intradermal injection, and histamine by 
skin prick, as negative and positive controls.  

If the wheal responses within the 20 min after each control challenge do not 
meet the acceptable saline and histamine responses (as defined in the SRM), 
the participant will not receive SP.  

If it happens in Part 1 or on Challenge Visit 1 in Part 2, the participant will 
be withdrawn from the study.  

If it happens on Challenge Visit 2 in Part 2 and if the participant 
marginally fails to meet the control criteria (as defined in the SRM), they 
may have the tests repeated.  If they still fail to meet the control criteria, 
they will be withdrawn from the study.

Following confirmation of acceptable control responses, participants will receive 
up to 4 intradermal injections of SP at different concentrations; injections will be 
given sequentially from lowest to highest concentration; wheal and flare 
responses will be assessed after each challenge.  
In Part 2 only:

Participants will receive up to 6 additional SP intradermal injections for biomarker 
analysis:  

IDM challenges:  4 increasing concentrations of SP (the same concentrations as 
those administered in the wheal and flare challenges); IDM samples will be taken 
at the site of each challenge, before and after challenge. 

Biopsy challenges at Challenge Visit 2:  2 at the same concentration of SP; 3 
biopsies would be taken – one from each challenged site and one from a non-
challenge area of skin. (these challenges and biopsies may or may not be 
performed, based on review of ongoing data.)

Biomarker blood samples will be taken before the first SP challenge and after the last
SP challenge at Challenge Visit 2.
Wheal response data will be reviewed during the study:

After about 3, 6 and 12 participants who have completed the challenge visit (as
applicable) in Part 1 – to confirm SP concentrations to be given in Part 2

After about 10 participants who have completed the two challenge visits in Part 2 – to 
determine whether enough data have been generated to understand the variability of 
the wheal response
4.2.
Scientific Rationale for Study Design
The primary endpoint in this study is the wheal response, which directly links SP to 
inflammation.  The wheal response is caused by accumulation of mast cell proteins in the 
dermis and presents as a raised oedema [Takahashi, 2004].  Flare, a secondary effect of 
degranulation, will also be measured.  Mast cell degranulation stimulates neuropeptide 
release from neurons, which in turn causes superficial vasodilation presenting as a flare 
response, characterised by a reddened area of skin [Schmelz, 2000].   


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Exploratory biomarkers of mast cell activation and the resulting immune response may be 
measured, at the site of SP challenge and systemically, to aid understanding of the wheal 
and flare responses.  
Wheal and flare responses to intradermal SP challenge peak and resolve over about 2 h in 
healthy people [Borici-Mazi, 1999].  Therefore, a 2 h sampling window following 
challenge is deemed appropriate.  If review of wheal response data from Part 1 indicate
that a longer or shorter sampling window is more appropriate, the sampling window will 
be changed accordingly; the maximum post challenge- sampling window will be 4 h.
It is unknown whether SP responses could change upon repeated challenge – e.g. because 
of exhaustion of mast cells.  Timing of challenge days in Part 2 of this study will be 
chosen to evaluate anticipated intervals between challenge days in subsequent studies of 
antagonists.  
4.3.
Justification for Substance P concentrations
Published data indicate that the wheal and flare responses to intradermal SP continue to 
increase with increasing concentrations, even up to 100 mol/L [Zirbs, 2013]. However, 
at the cellular level in an in-vitro mast cell line, SP begins to exert effects at ~0.1 mol/L 
and the effect plateaus at ~10 mol/L.  The probable reason for the result in human skin 
is as the concentration of SP increases, it diffuses to a larger area of the dermis, causing 
larger reactions by activating more mast cells.  To avoid concentrations of SP that are 
well above maximum effectiveness, the human mast cell line data were used to determine 
the SP concentrations in this study.  Concentrations of 0.1, 0.3, 1 and 3 mol/L SP are 
expected to give a range up to ~95% of the maximum effect at the cellular level.  Those 
concentrations have been shown to cause wheal and flare responses when given 
intradermally to healthy people [Borici-Mazi, 1999; Grouzmann, 2011; Sabroe, 1997; 
Zirbs, 2013].
To allow for possible differences between published data and our SP challenge model, 
the concentrations of SP may be changed during the study in order to elicit the desired 
wheal response.  For example, if the wheal response following one or more SP 
concentrations was indistinguishable from the wheal response following saline, and/or 
the wheal response was indistinguishable between 2 concentrations of SP, the 
concentrations could be adjusted accordingly.  The maximum amount of SP administered 
in a single injection will not exceed 50 L of 10 mol/L.
The SP concentrations administered in Part 2 will be selected to ensure that the lowest 
concentration gives a wheal response larger than the wheal response following saline, and 
that each increasing concentration gives a wheal response larger than that at the previous 
concentration.
4.4.
End of Study Definition
A participant is considered to have completed the study if he/she has completed all 
phases of the study including the follow up phone call as shown in the Schedule of 
Activities.


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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.
5.
STUDY POPULATION
Prospective approval of protocol deviations to recruitment and enrolment criteria, also 
known as protocol waivers or exemptions, is not permitted.
5.1.
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 50 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, ECGs and vital signs. 
3.
Participants who responded positive to histamine skin prick test and negative to 
saline injection at screening.
4.
Participants with Fitzpatrick skin type I-II (Caucasian).
Weight
5.
Body weight 50 kg and body mass index (BMI) within the range 19–29.9 kg/m2
(inclusive).
Sex
Sex and Contraceptive/Barrier Requirements
6.
Male participants are eligible to participate in the study


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
A female participant is eligible to participate if she is not pregnant or breastfeeding, 
and one of the following conditions applies:
o
Is a woman of non-childbearing potential (WONCBP) as defined in Section 
10.4 : Contraceptive and Barrier Guidance.
OR
o
Is a WOCBP and using a contraceptive method that is highly effective, with a 
failure rate of <1%, as described in Section 10.4  during the study 
intervention period. The investigator should evaluate potential for 
contraceptive method failure (e.g., noncompliance, recently initiated) in 
relationship to the first challenge. 

A sensitive pregnancy test is required to be negative on the day of each challenge.  
This pregnancy test is able to detect a pregnancy near the threshold of implantation, 
correlating with the earliest point of potential exposure.   Due to the short half-life of 
the interventions, a negative highly sensitive pregnancy test as scheduled in the 
protocol mitigates the risk of pregnancy exposure because exposure will be 
completely gone even with a worst case but unlikely scenario of conception occurring 
near the time of dosing. See Section 8.2.5 Pregnancy Testing

If a urine test cannot be confirmed as negative (e.g., an ambiguous result), a serum 
pregnancy test is required. In such cases, the participant must be excluded from 
participation if the serum pregnancy result is positive.

Additional requirements for pregnancy testing during and after study intervention is
located in Section 8.2.5 .

The investigator is responsible for review of medical history, menstrual history, and 
recent sexual activity to decrease the risk for inclusion of a woman with an early 
undetected pregnancy  
Informed Consent
7.
Capable of giving signed informed consent as described in Appendix 1 which 
includes compliance with the requirements and restrictions listed in the informed 
consent form (ICF) and in this protocol.
5.2.
Exclusion Criteria
Participants are excluded from the study if any of the following criteria apply:
Medical Conditions
1.
Significant history of or current, cardiovascular (including hypotension, severe 
hypertension, vasomotor instability), respiratory (including asthma), renal, 
gastrointestinal, endocrine, haematological, infectious or neurological disorders 
constituting a risk when taking part in the study or interfering with the interpretation 
of data.


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2.
History or presence of significant skin disorder (such as but not limited to chronic 
urticaria, atopic dermatitis, severe eczema, psoriasis or skin cancer).
3.
History of risk for or actual experience of complications from skin biopsy including 
excess bleeding, infection, or scarring/keloid formation.
4.
Abnormal blood pressure as determined by the investigator.
5.
Alanine transaminase (ALT) >1.5x upper limit of normal (ULN).
6.
Total bilirubin >1.5xULN (isolated bilirubin >1.5xULN is acceptable if total 
bilirubin is fractionated and direct bilirubin <35%). 
7.
Current or chronic history of liver disease, or known hepatic or biliary abnormalities 
(with the exception of Gilbert's syndrome or asymptomatic gallstones).
8.
QT interval corrected for heart rate according to Fridericia’s formula (QTcF) >450 
msec, based on the mean of triplicate ECGs .
NOTE: The QTc is the QT interval corrected for heart rate according to Fridericia’s 
formula (QTcF).
Prior/Concomitant Therapy
9.
Use of any form of H1 or H2 antihistamine, tricyclic antidepressants, beta2 agonists, 
dopamine, or beta blocking agents within 14 days before the first challenge visit
through final assessments.
10. Use of topical medications such as but not limited to retinoids, steroids, and 
transdermal hormone replacement therapies on or near the intended site of 
application within 8 weeks prior to dosing through treatment follow up.  Use of other 
topical preparations such as those containing vitamins, supplements or herbal within 
2 weeks prior to dosing through treatment follow up.
11. Past or intended use of any other non-topical over-the-counter or prescription 
medication, including herbal medications, within 7 days before the first challenge
visit, unless, in the opinion of the investigator and GSK Medical Monitor, the 
medication will not constitute a risk when taking the study intervention or interfere
with the interpretation of data.
Prior/Concurrent Clinical Study Experience
12. Participation in the study would result in loss of blood or blood products in excess of 
500 mL within 3 months.
13. Current enrolment in any clinical study involving an investigational study 
intervention or any other type of medical research.
14. Current enrolment or past participation in this study.
Diagnostic assessments
15. Presence of Hepatitis B surface antigen (HBsAg) or hepatitis B core antibody 
(HbcAb) at screening or within 3 months before the first challenge day.
16. Positive Hepatitis C antibody test result at screening or within 3 months before the 
first challenge day.


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NOTE:  Subjects with positive Hepatitis C antibody due to prior resolved disease can 
be enrolled, only if a confirmatory negative Hepatitis C RNA test is obtained
17. Positive Hepatitis C RNA test result at screening or within 3 months before the first 
challenge day.
NOTE: Test is optional and subjects with negative Hepatitis C antibody test are not 
required to also undergo Hepatitis C RNA testing
18. Positive pre-study drug/alcohol screen.
19. Positive human immunodeficiency virus (HIV) antibody test at screening or within 
3 months before the first challenge day.
20. Current use of known drugs of abuse.
Other Exclusions
21. Participants who present with damaged skin including sunburn, scar tissue, moles, 
uneven skin tones and dark skin tone (Fitzpatrick>2), tattoos, body piercings, 
branding or other skin disfiguration on or near the intended site of application which 
could interfere with the assessments.
22. Regular alcohol consumption within 6 months before the study defined as an average 
weekly intake of >21 units for males or >14 units for females.  One unit is equivalent 
to 8 g of alcohol: a half-pint (~240 mL) of beer, 1 glass (125 mL) of wine or 1 (25 
mL) measure of spirits. 
23. Smoking test result indicative of smoking, history or regular use of tobacco- or 
nicotine-containing products (e.g. nicotine patches or vaporizing devices) within 
6 months prior to screening.
24. Sensitivity to any of the study interventions, or components thereof, or drug or other 
allergy that, in the opinion of the investigator or medical monitor, contraindicates 
participation in the study.
25. Unable to refrain from the use of topical medications from before the first to after the 
last challenge visit.
5.3.
Lifestyle Considerations
Not applicable.
5.3.1.
Caffeine, Alcohol, and Tobacco

During each visit, participants will abstain from ingesting caffeine- or xanthine-
containing products (e.g., coffee, tea, cola drinks, and chocolate).

Participants will abstain from alcohol for 24 hours before each visit. 

Only non-smokers are allowed to participate in this study, therefore the use of 
tobacco products will not be allowed during the study.


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5.3.2.
Activity

Participants will abstain from strenuous exercise for 48 hours before each blood 
collection for clinical laboratory tests. Participants may participate in light 
recreational activities during studies (e.g., watching television, reading).
5.4.
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, any protocol deviations and any 
serious adverse events (SAEs).
Individuals who do not meet the criteria for participation in this study (screen failure) 
may be rescreened only once after consultation and on approval of the GSK Medical 
Monitor. Rescreened participants should be assigned a new participant number. In the 
event of out -of -range results of safety results, the test maybe repeated once within the 
screening window without this being considered a rescreen. If a retest result is again 
outside the reference range and considered clinically significant by the investigator and 
GSK medical monitor, the participant will be considered a screen failure.  
Individuals who meet the eligibility criteria and are reserve participants who are 
subsequently not required may also be rescreened for a start date later in the study.
6.
STUDY INTERVENTION AND CONCOMITANT THERAPY
Study intervention is defined as any investigational intervention(s), marketed product(s), 
placebo, or medical device(s) intended to be administered to a study participant according 
to the study protocol.
6.1.
Study Intervention(s) Administered
Participants will receive saline and SP by intradermal injection, and histamine by skin 
prick.  
Saline and histamine will be sourced by the site; histamine will be administered in 
accordance with manufacturer’s instructions.
SP drug substance will be supplied by GSK and prepared into drug product 
extemporaneously at the trial site prior to administration.


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Intervention Name
Substance P
Type 
Drug
Formulation
Solution for injection
Unit Strength(s)
3.0, 1.0, 0.3 and 0.1 µmol/L (with possible increase up to 
10 µmol/L, if no wheal and flare response is observed)
Dosage Level(s)
50 L of: 150, 50, 15 and 5 pmol (with possible increase 
up to 500 pmol if no wheal and flare response is observed)
Route of Administration
Intradermal injection
Use
Challenge agent
Sourcing
Drug substance provided centrally by sponsor then 
prepared to drug product locally by the trial site.
Packaging and Labelling
Study Intervention will be provided in vials. Each vial will be 
labelled as required per country requirement.
6.2.
Preparation/Handling/Storage/Accountability
1.
The investigator or designee must confirm appropriate temperature conditions have 
been maintained during transit for all study intervention received and any 
discrepancies are reported and resolved before use of the study intervention.
2.
Only participants enrolled in the study may receive study intervention and only 
authorized site staff may supply or administer study intervention. All study 
interventions must be stored in a secure, environmentally controlled, and monitored 
(manual or automated) area in accordance with the labeled storage conditions with 
access limited to the investigator and authorized site staff.
3.
The investigator, institution, or the head of the medical institution (where applicable) 
is responsible for study intervention accountability, reconciliation, and record 
maintenance (i.e., receipt, reconciliation, and final disposition records).
4.
Further guidance and information for the final disposition of unused study 
intervention are provided in the Pharmacy Manual.
5.
Information on the biomarker sampling device can be found in the Study Reference 
Manual (SRM)

Under normal conditions of handling and administration, study intervention is not 
expected to pose significant safety risks to site staff.  Take adequate precautions to 
avoid direct eye or skin contact and the generation of aerosols or mists. In the case of 
unintentional occupational exposure notify the monitor, Medical Monitor and/or 
GSK study contact.


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
A Material Safety Data Sheet (MSDS)/equivalent document describing occupational 
hazards and recommended handling precautions either will be provided to the 
investigator, where this is required by local laws, or is available upon request from 
GSK.
6.3.
Measures to Minimize Bias: Randomization and Blinding
Not applicable.
6.4.
Study Intervention Compliance

When the individual SP concentration for a participant is prepared from a bulk 
supply, the preparation will be confirmed by a second member of the study site staff.

When participants are administered SP at the site, they will receive study 
intervention directly from the investigator or designee, under medical supervision.  
The date and time of each administration in the clinic will be recorded in the source 
documents.  The concentration of study intervention and study participant
identification will be confirmed at the time of administration by a member of the
study site staff other than the person administering the study intervention.  
6.5.
Modification of Substance P concentrations
Following data reviews at the data review committee (DRC) (see Section 10.1.5 ) in 
Part 1, higher/lower concentrations of SP might be administered to subsequent 
participants, depending on the wheal reactions observed (see Section 4.3).  The maximum 
amount of SP administered in a single injection will not exceed 50 L of 10 mol/L, and 
up to a maximum of 10 injections of SP will be allowed per challenge visit. 
6.6.
Intervention after the End of the Study
There will be no intervention offered at the end of the study.
6.7.
Treatment of Overdose

Intradermal SP doses up to ~7.5-fold higher than those planned for this study and up 
to ~2.3-fold higher than those permitted in this study have been reported to show no 
important side effects [Grouzmann, 2011; Zirbs, 2013], and thus an overdose is not
anticipated given the use and local delivery. In case of any overexposure to SP refer 
to the MSDS. 

Histamine dose selection is based on the product label for histamine injection as a 
positive skin test control.  An overdose is not anticipated given the per-product label 
use and local delivery, refer to the product label.
In case of any overexposure or evidence of systemic adverse reactions to histamine, 
please refer to the product label for appropriate treatment response including, but not 
limited to tourniquet application, epinephrine administration, and antihistamine 
administration.


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In the event of an overdose, the investigator should:
1.
Contact the Medical Monitor immediately.
2.
Closely monitor the participant for AE/SAE and laboratory abnormalities until stable 
or resolved. 
3.
Document the quantity of the excess concentration as well as the duration of the 
overdosing in the CRF.
Decisions regarding dose interruptions or modifications will be made by the investigator 
in consultation with the Medical Monitor based on the clinical evaluation of the 
participant.
6.8.
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 
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 
intervention until completion of the last visit, unless, in the opinion of the investigator 
and sponsor, the medication will not interfere with the study. 
Paracetamol, at doses of 2 grams/day, is permitted for use any time during 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.
7.
DISCONTINUATION OF STUDY INTERVENTION AND 
PARTICIPANT DISCONTINUATION/WITHDRAWAL
7.1.
Study Stopping criteria

If a SAE, considered reasonably attributable to study intervention (SP, histamine) 
in the opinion of the Investigator, occurs in one participant, then dosing will be 
temporarily halted, and no further participants will be enrolled or dosed until a 
full safety review of the data has taken place. Relevant reporting and discussion 
with the Medical Monitor, relevant site personnel, and the IRB/IEC will take 
place before resumption of enrolment or dosing.


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
If the significant clinical or laboratory abnormality, considered reasonably 
attributable to study intervention (SP, histamine) in the opinion of the 
Investigator, occurs in more than 2 participants, then dosing will be temporarily 
halted, and no further participants will be enrolled or dosed until a full safety 
review of the data has taken place. Relevant reporting and discussion with the 
Medical Monitor, relevant site personnel, and the IRB/IEC will take place before 
resumption of enrolment or dosing.
7.2.
Discontinuation of Study Intervention
In rare instances, it may be necessary for a participant to permanently discontinue study 
intervention. If study intervention is permanently discontinued, the participant will 
remain in the study until the end of that challenge visit to be evaluated for safety. See the 
SOA for data to be collected at the time of discontinuation of study intervention and 
follow-up and for any further evaluations that need to be completed.
7.3.
Participant Discontinuation/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. This is expected to be 
uncommon.

At the time of study discontinuation, the participant should undergo the 
assessments for early withdrawal shown in the SoA.

The participant will be permanently discontinued both from the study 
intervention and from the study at that time.

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.
7.4.
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.


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
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. 
8.
STUDY ASSESSMENTS AND PROCEDURES

Study procedures and their timing are summarized in the SoA. 

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 intervention.

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. 

Procedures conducted as part of the participant’s routine clinical management (e.g., 
blood count) and obtained before signing of ICF may be utilized for screening or 
baseline purposes provided the procedure met the protocol-specified criteria and was 
performed within the time frame defined in the SoA.

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.
8.1.
Efficacy Assessments
Not applicable.
8.2.
Safety Assessments
Planned time points for all safety assessments are provided in the SoA.
8.2.1.
Physical Examinations

A complete physical examination will include, at a minimum, assessments of the 
Skin, Cardiovascular, Respiratory, Gastrointestinal and Neurological systems. 
Height and weight will also be measured and recorded.


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
A brief physical examination will include, at a minimum, assessments of the 
skin, lungs, cardiovascular system, and abdomen (liver and spleen).
8.2.2.
Vital Signs

Tympanic body temperature, pulse rate and blood pressure will be assessed as a 
single or triplicate measurement(s), as described below.

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 without distractions (e.g., 
television, cell phones).

At screening, vital signs will consist of 1 temperature, and 3 pulse and blood 
pressure measurements.  On Challenge days, vital signs will consist of 1 pulse 
and 3 blood pressure measurements.  Vital signs will to be taken before blood 
collection for laboratory tests.  Triplicate readings will consist of 3 consecutive 
pulse and/or blood pressure readings recorded at intervals of at least 1 minute.
8.2.3.
Electrocardiograms

Triplicate 12-lead ECG will be obtained as outlined in the SoA (see Section 1.3) 
using an ECG machine that automatically calculates the heart rate and measures 
PR, QRS, QT, and QTc intervals. Refer to Section 7 for QTc withdrawal criteria 
and additional QTc readings that may be necessary.

At Screening where a triplicate ECG are required, 3 individual ECG tracings 
should be obtained as closely as possible in succession, but no more than 
2 minutes apart. The full set of triplicates should be completed in less than 
4 minutes.
8.2.4.
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. 

All laboratory tests with values considered clinically significantly abnormal 
during participation in the study or within one day after the last administration
of study intervention should be repeated until the values return to normal or 
baseline or are no longer considered significantly abnormal by the investigator 
or medical monitor. 


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
If such values do not return to normal/baseline within a period of time judged 
reasonable by the investigator, the etiology 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.
8.2.5.
Pregnancy Testing

Refer to Section 5.1 Inclusion Criteria for pregnancy testing entry criteria.

Pregnancy testing (urine or serum as required by local regulations) should be 
conducted prior to each challenge during the study intervention period.

Pregnancy testing (urine or serum as required by local regulations) should be 
conducted at the end of relevant systemic exposure.

Additional serum or urine pregnancy tests may be performed, as determined 
necessary by the investigator or required by local regulation, to establish the 
absence of pregnancy at any time during the participant’s participation in the 
study.
8.3.
Adverse Events and Serious Adverse Events
The definitions of an AE or SAE can be found in Appendix 3.
AEs will be reported by the participant (or, when appropriate, by a caregiver, surrogate, 
or the participant's legally authorized representative).
The investigator and any qualified 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 intervention or the 
study, or that caused the participant to discontinue the study (see Section 7).
8.3.1.
Time Period and Frequency for Collecting AE and SAE 
Information

All SAEs will be collected from the signing of the informed consent form until 
2 hrs after the last SP challenge, as specified in the SoA (Section 1.3).

All AEs will be collected from the first challenge-related procedure until 2 hrs 
after the last SP challenge, as specified in the SoA (Section 1.3).

Medical occurrences that begin before the start of study intervention 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 3. The investigator will submit any updated SAE data to the sponsor 
within 24 hours of it being available.


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
Investigators are not obligated to actively seek AEs or SAEs after the conclusion 
of the study participation. 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 
intervention or study participation, the investigator must promptly notify the 
sponsor.
8.3.2.
Method of Detecting AEs and SAEs

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 3.

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. 
8.3.3.
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 7.4). Further information on follow-up procedures is given in 
Appendix 3.
8.3.4.
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 intervention 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 intervention
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.

An investigator who receives an investigator safety report describing a SAE or 
other specific safety information (e.g., summary or listing of SAEs) from the 
sponsor will review and then file it along with other relevant documents and will 
notify the IRB/IEC, if appropriate according to local requirements.
8.3.5.
Pregnancy

Details of all pregnancies in female participants will be collected after the start of 
study intervention and until end of last challenge visit. If a pregnancy is reported, the 
investigator will record pregnancy information on the appropriate form and submit it 
to GSK within 24 hours of learning of the female participant pregnancy. While
pregnancy itself is not considered to be an AE or SAE, any pregnancy complication 


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or elective termination of a pregnancy for medical reasons will be reported as an AE 
or SAE.

Abnormal pregnancy outcomes (e.g., spontaneous abortion, fetal death, stillbirth, 
congenital anomalies, ectopic pregnancy) are considered SAEs and will be reported 
as such.

The participant will be followed to determine the outcome of the pregnancy. The 
investigator will collect follow-up information on the participant and the neonate and 
the information will be forwarded to the sponsor.

Any post-study pregnancy-related SAE considered reasonably related to the study 
intervention by the investigator will be reported to the sponsor as described in Section
8.3.4 . While the investigator is not obligated to actively seek this information in 
former study participants she may learn of an SAE through spontaneous reporting.

Any female participant who becomes pregnant while participating in the study will 
discontinue study intervention or be withdrawn from the study.
8.4.
Pharmacokinetics
Not applicable to this study.
8.5.
Pharmacodynamics
8.5.1.
Visual check of planned challenge sites and administration of 
challenge agents
At each challenge visit, before any challenge related procedures are performed, the 
investigator (or delegate) will visually inspect the planned challenge sites for any 
markings or reactions that could interfere with the interpretation of data (e.g. carryover
effects from a previous challenge day).
Saline and SP will be delivered by intradermal injection and histamine will be delivered 
by skin prick, as described in the SRM.
8.5.2.
Wheal and Flare Assessments
Callipers will be used to measure the area of the responses at the sites of saline, histamine 
and SP challenge.  Responses will also be digitally photographed for confirmation of 
results.  In addition, perfusion of each challenge site will be measured using laser speckle
contrast imaging.
Further details are provided in the SRM.  
8.6.
Genetics
Not applicable to this study.


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8.7.
Biomarkers
At challenge visits defined in the SoA, the following samples will be collected to 
measure exploratory biomarkers:

IDM probes will be inserted intradermally in the skin of the forearm – in total, 
approximately 450 l of dialysate will be collected from each probe

3 punch biopsy samples of up to 3mm in diameter each will be collected

Up to 56mL of blood will be collected via venipuncture or cannula 
Details of the processing, storage and shipping procedures for all samples are provided in 
the SRM.
IDM samples will be collected to investigate the effect of intradermal SP challenge on 
local exploratory biomarkers of mast cell activation and their variability. Biomarkers may
include but not be limited to histamine. 
Skin biopsies may be collected to investigate the effect of intradermal SP challenge on 
local exploratory biomarkers of mast cell activation and their variability. Biomarkers will
include tryptase and/or markers of cells including mast cells, eosinophils, basophils, T 
cells, macrophages and neutrophils. 
Blood samples will be stored and analysis may be performed on biomarkers thought to 
play a role in mast cell activation or SP challenge response.
GSK may store samples for up to 15 years after the end of the study to achieve study 
objectives. Additionally, with participants consent, samples may be used for further 
research by GSK or others such as universities or other companies to contribute to the 
understanding of mast cell related or other diseases, the development of related or new 
treatments or research methods.
Exploratory biomarker results may be entered into the clinical study database or reported 
separately.
8.8.
Immunogenicity Assessments
Not applicable to this study.
8.9.
Health Economics
Not applicable to this study.
9.
STATISTICAL CONSIDERATIONS
9.1.
Statistical Hypotheses
The objectives of this enabling study are to characterise the wheal and flare responses 
over time following skin challenges with ascending concentrations of SP. No formal 
hypotheses will be tested.


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9.2.
Sample Size Determination
A sufficient number of participants will be screened in order to enrol approximatively 32 
participants in the study (up to 12 in part 1 and up to 20 in part 2).
The sample size for part 1 and part 2 is not based on statistical considerations and is 
based on previous published data.
9.3.
Populations for Analyses
The following populations are defined:
Population
Description
Screened
All participants who signed the ICF and were screened for 
eligibility.
Enrolled
All participants in the Screened population who entered the study  
Safety
All participants in the Enrolled population who received at least 
one challenge-related study procedure on their (first) challenge
day
9.4.
Statistical Analyses
The reporting and analysis plan (RAP) will include a more technical and detailed 
description of the statistical analyses described in this section. This section is a summary 
of the planned statistical analyses of the most important endpoints including primary and 
key secondary endpoints.
9.4.1.
General Considerations
This is an exploratory enabling study which is primarily designed to define the 
relationship between SP concentrations and wheal and flare response, within a range of 
intradermal SP concentrations. There are no formal hypotheses to be tested.
All the statistical analyses will be carried out in the Bayesian framework. Models will be 
fitted by Markov chain Monte Carlo simulations. The following summaries of the 
posterior distribution will be reported: posterior mean, posterior standard deviation (SD), 
posterior median, 95% equal tailed credible interval, posterior probabilities of the true 
effect being greater than pre-specified thresholds. Vague prior distributions will be used.
For all endpoints, the baseline value will be the negative control value. No missing 
baseline will be allowed (as part of the inclusion/exclusion criteria).


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9.4.2.
Primary Endpoints
For parts 1 and 2, the primary endpoint is the wheal response-time area under the curve 
(AUC) over the 2h post-challenge period following skin challenge with ascending 
concentrations of SP on a single day and 2 days, respectively. The wheal response
variable will be area in millimetres squared. Other methods may be investigated as 
outlined in Section 8.5.2. These variables may be adjusted for baseline response.
The primary analysis on the Safety population and on participants who have completed 
the challenge visits (complete case analysis) will assess the difference in wheal response-
time area under the curve (AUC) over the 2h post-challenge period between the different 
challenges (saline, SP). The following statistical models may be used:
-
Part 1: Analysis of covariance (ANCOVA) 
-
Part 2: Mixed effect model for repeat measurements (MMRM), including 
predictors and random effects
Alternative approaches may be investigated. Further details will be provided in the RAP.
9.4.3.
Secondary Endpoints
For part 1 and 2, the following parameters related to the wheal response will be 
calculated (not limited to) and summarised:

Maximum observed wheal response

Time to maximum observed wheal response

Time of complete wheal response disappearance 
For parts 1 and 2, the secondary endpoints are the flare response over time (including but 
not limited to AUC, maximum observed flare response, time to maximum observed flare 
response, time of complete flare response disappearance) following skin challenge with
ascending concentrations of SP on a single day and 2 days, respectively. The proposed 
statistical analyses planned for the primary endpoint (Section 9.4.2) will be repeated for
the flare response-time area under the curve (AUC) over the 2h post-challenge period.
All safety analyses will be performed on the Safety population. Safety data will be 
presented in tabular and/or graphical format and summarized descriptively according to 
GSK’s Integrated Data Standards Library (IDSL) standards. No formal statistical testing 
will be performed on safety data.
Further details will be provided in the RAP.
9.4.4.
Tertiary/Exploratory Endpoints
Full details will be described in the RAP.


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9.5.
Interim Analyses
No formal interim analysis will be performed.  The following data monitoring will be 
performed based on data collected during the study.  At a minimum, all reviews will 
include available wheal response data after each injection of SP.

Part 1 data for about 3 participants who have completed the challenge visit will be
reviewed prior to enrolment of subsequent participants, to allow any concentration 
adjustment.

Part 1 data for about 6 participants who have completed the challenge visit will be
reviewed prior to enrolment of subsequent participants, to allow any concentration 
adjustment and/or to confirm if sufficient data is already available to progress to Part 
2.

Part 1 data will be reviewed prior to participants being enrolled into Part 2.

Part 2 data for about 10 participants who have completed the 2 challenge visits will
be reviewed; enrolment into Part 2 may continue during the review.


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10.
SUPPORTING DOCUMENTATION AND OPERATIONAL
CONSIDERATIONS
10.1.
Appendix 1: Regulatory, Ethical, and Study Oversight 
Considerations
10.1.1.
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 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
10.1.2.
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.


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10.1.3.
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. 

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 center. 

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. 
A participant who is rescreened is not required to sign another ICF if the rescreening 
occurs within (28) days from the previous ICF signature date.
GSK (alone or working with others) may use participant’s coded study data and samples 
and other information to carry out this study; understand the results of this study; learn 
more about SP challenge or about the disease related to mast cells; publish the results of 
these research efforts; work with government agencies or insurers to have study 
interventions approved for medical use or approved for payment coverage.
The ICF contains a separate section that addresses the use of participant data and 
remaining samples for optional further research. The investigator or authorised designee 
will inform each participant of the possibility of further research not related to the 
study/disease. 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 tick box will be required to document a participant's agreement to allow any 
participant data and/or remaining leftover samples to be used for further research not 
related to the study/disease. Participants who decline further research will tick the 
corresponding “No” box.
10.1.4.
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 


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disclosure must also be explained to the participant who will be required to give 
consent for their data to be used as described in the informed consent. 

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.
10.1.5.
Committees Structure
A data review committee (DRC) will review data during the study, at time points 
described in Section 9.5.  At a minimum, available wheal response data after each SP 
challenge will be reviewed.  Other data, such flare response, safety and biomarker data 
may also be reviewed, if applicable.  The DRC will consist of the following people (or 
their delegate):  GSK Medical Monitor, GSK Clinical Science Lead, GSK Clinical 
Delivery Lead, GSK Clinical Pharmacology Modelling and Simulation (CPMS) 
representative, a GSK Global Clinical Safety and Pharmacovigilance (GCSP) 
representative and GSK Statistician representative.  Other study team members may 
attend, as needed.  The DRC may decide to change the concentrations of SP to be 
administered to subsequent participants, as described in Section 6.5.
10.1.6.
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 subjects, as appropriate.

GSK will provide the investigator with the randomization codes for their site 
only after completion of the full statistical analysis.

The procedures and timing for public disclosure of the protocol and results 
summary and for development of a manuscript for publication for this study will 
be in accordance with GSK Policy.

GSK intends to make anonymized patient-level data from this trial available to 
external researchers for scientific analyses or to conduct further research that 
can help advance medical science or improve patient care. This helps ensure the 
data provided by trial participants are used to maximum effect in the creation of 
knowledge and understanding

A manuscript will be progressed for publication in the scientific literature if the 
results provide important scientific or medical knowledge. Specifications about 
the data and publication are incorporated into the clinical trial agreement.


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10.1.7.
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. 

Guidance on completion of CRFs will be provided in the InForm portal

Quality tolerance limits (QTLs) will be pre-defined in the QTL plan to identify 
systematic issues that can impact participant safety and/or reliability of study 
results. These pre-defined parameters will be monitored during and at the end of 
the study and all deviations from the QTLs and remedial actions taken will be 
summarized in the clinical study report.

The investigator must permit study-related monitoring, audits, IRB/IEC review, 
and regulatory agency inspections and provide direct access to source data 
documents. 

Monitoring details describing strategy including definition of study critical data 
items and processes (e.g., risk-based initiatives in operations and quality such as 
Risk Management and Mitigation Strategies and Analytical Risk-Based 
Monitoring), methods, responsibilities and requirements, including handling of 
noncompliance issues and monitoring techniques (central, remote, or on-site 
monitoring) are provided in the monitoring plan.

The sponsor or designee is responsible for the data management of this study 
including quality checking of the data. 

The sponsor assumes accountability for actions delegated to other individuals 
(e.g., Contract Research Organizations).

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. 
10.1.8.
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.


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
Definition of what constitutes source data can be found in a study specific 
source document agreement.

The investigator must maintain accurate documentation (source data) that 
supports the information entered in the CRF.

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 
currently approved protocol and any other study agreements, ICH GCP, and all 
applicable regulatory requirements. 
10.1.9.
Study and Site Start and Closure
The study start date is the date on which the clinical study will be open for recruitment of 
participants.
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 intervention development
If the study is prematurely terminated or suspended, the sponsor shall promptly inform 
the Investigators, the IECs/IRBs, the regulatory authorities, and any contract research 
organization(s) used in the study of the reason for termination or suspension, as specified 
by the applicable regulatory requirements. The Investigator shall promptly inform the 
subject and should assure appropriate subject therapy and/or follow-up
10.1.10.
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 multicenter studies only in their entirety and not 


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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.


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10.2.
Appendix 2: Clinical Laboratory Tests

The tests detailed in Table 1 will be performed by the local laboratory.

Protocol-specific requirements for inclusion or exclusion of participants are 
detailed in Section 5 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.

Pregnancy Testing 

Refer to Section 5.1 Inclusion Criteria for screening pregnancy criteria and 
Section 1.3 Schedule of Activities (SoA).

Pregnancy testing (urine or serum as required by local regulations) should be 
conducted prior to each SP challenge visit.

Pregnancy testing (urine or serum as required by local regulations) should be 
conducted at the end of relevant systemic exposure. 

Additional serum or urine pregnancy tests may be performed, as determined 
necessary by the investigator or required by local regulation, to establish the 
absence of pregnancy at any time during the subject's participation in the study.


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Table 1
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
Clinical 
Chemistry1
BUN
Potassium
Aspartate 
Aminotransferase
(AST)/ Serum Glutamic-
Oxaloacetic 
Transaminase (SGOT)
Total and direct 
bilirubin
Creatinine
Sodium
Alanine Aminotransferase
(ALT)/ Serum Glutamic-
Pyruvic Transaminase
(SGPT)
Total Protein
Glucose [non-
fasting]
Calcium
Alkaline phosphatase
Routine 
Urinalysis

Specific gravity

pH, glucose, protein, blood, ketones, by dipstick

Microscopic examination (if blood or protein is abnormal)
Other Screening 
Tests

Follicle-stimulating hormone and estradiol (as needed in women of non-
childbearing potential only)

Breath alcohol test (as per standard local practice)

Drug screen to include at a minimum: amphetamines, barbiturates, 
cocaine, opiates, cannabinoids and benzodiazepines)

Smoking test – breath test or urine cotinine, according to local 
procedures.

Highly sensitive serum or urine human chorionic gonadotropin (hCG) 
pregnancy test (as needed for women of childbearing potential)2

Serology (HIV antibody, hepatitis B surface antigen [HBsAg], and 
hepatitis C virus antibody) 
NOTES :
1.
All events of ALT 3 × upper limit of normal (ULN) and bilirubin 2 × ULN (>35% direct bilirubin) or ALT 3 × 
ULN and international normalized ratio (INR) >1.5, if INR measured, which may indicate severe liver injury 
(possible Hy’s Law), must be reported as an SAE (excluding studies of hepatic impairment or cirrhosis).
2.
Serum pregnancy testing at screening, at other visits urine testing.


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10.3.
Appendix 3: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting
10.3.1.
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 intervention, whether or not 
considered related to the study intervention.

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 intervention.
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 intervention 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 
intervention 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. 
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.


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
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.
10.3.2.
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:
o
Results in death
o
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.
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 AEs. 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.
Results in persistent or significant 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, 


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influenza, and accidental trauma (e.g. sprained ankle) which may interfere with or 
prevent everyday life functions but do not constitute a substantial disruption.
Is a congenital anomaly/birth defect
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.
10.3.3.
Recording and Follow-Up of 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.
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 sufficient discomfort and interferes with normal 
everyday activities.


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
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 intervention 
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 intervention 
administration will be considered and investigated.

The investigator will also consult the MSDS and Product label.

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.
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.


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
The investigator will submit any updated SAE data to GSK within 24 hours of 
receipt of the information.
10.3.4.
Reporting of SAE to GSK
SAE Reporting to GSK via Electronic Data Collection Tool

The primary mechanism for reporting SAE to GSK will be the electronic data 
collection tool.

If the electronic system is unavailable, then the site will use the paper SAE data 
collection tool (see next section) in order to report the event within 24 hours.

The site will enter the SAE data into the electronic system as soon as it becomes 
available.

The investigator or medically-qualified sub-investigator must show evidence 
within the eCRF (e.g., check review box, signature, etc.) of review and verification 
of the relationship of each SAE to IP/study participation (causality) within 
72 hours of SAE entry into the eCRF.

After the study is completed at a given site, the electronic data collection tool will 
be taken off-line to prevent the entry of new data or changes to existing data.

If a site receives a report of a new SAE from a study participant or receives 
updated data on a previously reported SAE after the electronic data collection tool 
has been taken off-line, then the site can report this information on a paper SAE 
form (see next section) or contacts the medical monitor/SAE coordinator by 
telephone.

Contacts for SAE reporting can be found in the SRM.
SAE Reporting to GSK via Paper CRF

Facsimile transmission of the SAE paper CRF is the preferred method to transmit 
this information to the medical monitor or the SAE coordinator.

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 SRM.


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10.4.
Appendix 4: Contraceptive Guidance and Collection of 
Pregnancy Information
10.4.1.
Definitions:
Woman of Childbearing Potential (WOCBP)
A woman is considered fertile following menarche and until becoming post-menopausal 
unless permanently sterile (see below).
If fertility is unclear (e.g., amenorrhea in adolescents or athletes) and a menstrual cycle 
cannot be confirmed before first administration of study intervention, additional 
evaluation should be considered.
Women in the following categories are not considered WOCBP
1.
Premenarchal
2.
Premenopausal female with 1 of the following:

Documented hysterectomy

Documented bilateral salpingectomy

Documented bilateral oophorectomy
For individuals with permanent infertility due to an alternate medical cause other 
than the above, (e.g., mullerian agenesis, androgen insensitivity), investigator 
discretion should be applied to determining study entry.
Note: Documentation can come from the site personnel’s: review of the 
participant’s medical records, medical examination, or medical history interview.
3.
Postmenopausal female

A postmenopausal state is defined as no menses for 12 months without an 
alternative medical cause. 

A high follicle stimulating hormone (FSH) level in the postmenopausal 
range may be used to confirm a postmenopausal state in women not 
using hormonal contraception or hormonal replacement therapy (HRT). 
However, in the absence of 12 months of amenorrhea, confirmation with 
more than one FSH measurement is required. 

Females on HRT and whose menopausal status is in doubt will be required to 
use one of the non-estrogen hormonal highly effective contraception methods 
if they wish to continue their HRT during the study. Otherwise, they must 
discontinue HRT to allow confirmation of postmenopausal status before 
study enrolment.


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10.4.2.
Contraception Guidance:

CONTRACEPTIVESa ALLOWED DURING THE STUDY INCLUDE:

Highly Effective Methodsb That Have Low User Dependency Failure rate of <1% per 
year when used consistently and correctly.

Implantable progestogen-only hormone contraception associated with inhibition of 
ovulationc

Intrauterine device (IUD)

Intrauterine hormone-releasing system (IUS)c

Bilateral tubal occlusion

Azoospermic partner (vasectomized or due to a medical cause)
Azoospermia is a highly effective contraceptive method provided that the partner is the 
sole sexual partner of the woman of childbearing potential and the absence of sperm has 
been confirmed. If not, an additional highly effective method of contraception should be 
used. Spermatogenesis cycle is approximately 90 days.
Note: documentation of azoospermia for a male participant can come from the site 
personnel’s review of the participant’s medical records, medical examination, or medical 
history interview.

Highly Effective Methodsb That Are User Dependent Failure rate of <1% per year when 
used consistently and correctly.

Combined (estrogen- and progestogen-containing) hormonal contraception associated with 
inhibition of ovulationc

oral

intravaginal

transdermal

injectable

Progestogen-only hormone contraception associated with inhibition of ovulationc

oral

injectable

Sexual abstinence

Sexual abstinence is considered a highly effective method only if defined as refraining from 
heterosexual intercourse during the entire period of risk associated with the study 
intervention. The reliability of sexual abstinence needs to be evaluated in relation to the 
duration of the study and the preferred and usual lifestyle of the participant.
a.
Contraceptive use by men or women should be consistent with local regulations regarding the use of 
contraceptive methods for those participating in clinical studies.
b.
Failure rate of <1% per year when used consistently and correctly. Typical use failure rates differ from those 
when used consistently and correctly.
c.
[Male condoms must be used in addition to hormonal contraception]. If locally required, in accordance with 
Clinical Trial Facilitation Group (CTFG) guidelines, acceptable contraceptive methods are limited to those 
which inhibit ovulation as the primary mode of action.
Note: Periodic abstinence (calendar, sympto-thermal, post-ovulation methods), withdrawal (coitus interruptus), 
spermicides only, and lactational amenorrhea method (LAM) are not acceptable methods of contraception. Male 
condom and female condom should not be used together (due to risk of failure from friction)


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10.5.
Appendix 5: Abbreviations and Trademarks
LIST OF ABBREVIATIONS
AE
Adverse event
ALT
Alanine Aminotransferase
ANCOVA
Analysis of covariance 
AUC
Area under the curve 
BMI
Body mass index 
CA
Competent Authority  
CIOMS
Council for International Organizations of Medical Sciences 
CONSORT
Consolidated Standards of Reporting Trials 
CPMS
Clinical Pharmacology Modelling and Simulation 
CSR
Clinical Study Report 
DRC
Data review committee 
EC
Ethics committee 
ECG
Electrocardiogram
GCP
Good Clinical Practice
GCSP
Global Clinical Safety and Pharmacovigilance
GSK
GlaxoSmithKline
HBsAg
Hepatitis B surface antigen 
hCG
Human chorionic gonadotropin 
HIV
Human immunodeficiency virus 
ICF
Informed consent form 
IDM
Intradermal microdialysis 
IDSL
Integrated Data Standards Library 
IEC
Independent Ethics Committees 
IRB
Institutional Review Boards 
MMRM
Mixed effect model for repeat measurements 
QTcF
QT interval corrected for heart rate according to Fridericia’s 
formula 
RAP
Reporting and analysis plan 
SAE
Serious adverse event
SD
Standard deviation (SD
SGOT
Serum Glutamic-Oxaloacetic Transaminase 
SGPT
Serum Glutamic-Pyruvic Transaminase 
SoA
Schedule of Activities
SP
Substance P
SRM
Study Reference Manual
ULN
Upper limit of normal
WCBP
Women of childbearing potential
WNCBP
Women of non-childbearing potential.


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Trademark Information
Trademarks of the GlaxoSmithKline 
group of companies
Trademarks not owned by the 
GlaxoSmithKline group of companies
None
None


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11.
REFERENCES
Borici-Mazi R, Kouridakis S, Kontou-Fili K.  Cutaneous responses to substance P and 
calcitonin gene-related peptide in chronic urticaria: the effect of cetirizine and 
dimethindene.  Allergy.  1999;54:46-56.
Baumann KY, Church MK, Clough GF, Quist SR, Schmelz M, Skov PS et al. Skin 
microdialysis: methods, applications and future opportunities-an EAACI position paper.  
Clin Transl Allergy.  2019;9:24.
Grouzmann E, Bigliardi P, Appenzeller M, Pannatier A, Buclin T.  Substance P-induced 
skin inflammation is not modulated by a single dose of sitagliptin in human volunteers.  
Biol Chem.  2011;392:217-21.
Holmgaard R, Benfeldt E, Nielsen JB, Gatschelhofer C, Sorensen JA, Höfferer C et al.  
Comparison of open-flow microperfusion and microdialysis methodologies when 
sampling topically applied fentanyl and benzoic acid in human dermis ex vivo.  Pharm 
Res.  2012;29:1808-20.
Mashaghi A, Marmalidou A, Tehrani M, Grace PM, Pothoulakis C, Dana R.  
Neuropeptide Substance P and the Immune Response.  Cell Mol Life Sci. 2016;73:4249–
64.
Newby DE, Sciberras DG, Mendel CM, Gertz BJ, Boon NA, Webb DJ. Intra-arterial 
substance P mediated vasodilatation in the human forearm: pharmacology, 
reproducibility and tolerability.  Br J Clin Pharmacol. 1997; 43: 493–9.
Sabroe RA, Kennedy CT, Archer CB.  The effects of topical doxepin on responses to 
histamine, substance P and prostaglandin E2 in human skin.  Br J Dermatol.  
1997;137:386-90.
Schmelz M, Michael K, Weidner C, Schmidt R, Torebjork HE, Handwerker HO.  Which 
nerve fibers mediate the axon reflex flare in human skin?  NeuroReport. 2000;11:645–8.
Takahashi H, Ishida-Yamamoto A, Iizuka H.  Effects of bepotastine, cetirizine, 
exofenadine, and olopatadine on histamine-induced wheal-and flare-response, sedation, 
and psychomotor performance.  Clinical and Experimental Dermatology.  2004;29:526–
32.
Zirbs M, Reindel U, Hermann K, Church MK, Behrendt H, Ring J, et al.  Reduced skin 
reactivity to vasoconstrictor and vasodilator substances in atopic eczema.  Eur J 
Dermatol.  2013;23:812-9.

SAP_001.pdf: 
 
 
 
 
Statistical Analysis Plan 
 
Study ID:  213224 
 
Official Title of Study: An open label, single center, enabling study to investigate the 
optimum method for use of intradermal Substance P as a challenge agent in healthy 
participants. 
 
NCT ID: NCT04676763  
 
 
Date of Document: 01-JUL-2021 
 
 


CONFIDENTIAL 
The GlaxoSmithKline group of companies 
 
213224 
 
 
1 
 
 
TITLE PAGE 
Protocol Title: 
An open label, single center, enabling study to investigate the 
optimum method for use of intradermal Substance P as a 
challenge agent in healthy participants 
Study Number: 
213224 
Compound 
Number: 
None 
Abbreviated Title: 
Intradermal Substance P challenge in healthy participants 
Sponsor Name: GlaxoSmithKline Research & Development Limited  
Regulatory Agency Identifier Number(s):  
 
Registry                              ID 
Clinicaltrials.gov          NCT04676763 
 
 
 
 
 
 
 
 
 
 
 
Copyright 2021 of the GlaxoSmithKline group of companies. All rights reserved. 
Unauthorised copying or use of this information is prohibited. 
 
Information 
Type: 
Statistical Analysis Plan (SAP) 
 


CONFIDENTIAL 
 
 
213224 
2 
 
TABLE OF CONTENTS 
PAGE 
TITLE PAGE ................................................................................................................... 1 
TABLE OF CONTENTS .................................................................................................. 2 
1. 
INTRODUCTION ...................................................................................................... 5 
1.1. 
Objectives and Endpoints ............................................................................. 5 
1.2. 
Study Design ................................................................................................ 7 
2. 
STATISTICAL HYPOTHESES ................................................................................. 9 
2.1. 
Multiplicity Adjustment .................................................................................. 9 
3. 
ANALYSIS SETS ................................................................................................... 10 
4. 
STATISTICAL ANALYSES ..................................................................................... 10 
4.1. 
General Considerations .............................................................................. 10 
4.1.1. 
General Methodology .................................................................. 10 
4.1.2. 
Baseline Definition ....................................................................... 11 
4.2. 
Primary Pharmacodynamic Analyses .......................................................... 12 
4.2.1. 
Definition of endpoint ................................................................... 12 
4.2.2. 
Main analytical approach ............................................................. 12 
4.2.3. 
Sensitivity analyses ..................................................................... 14 
4.3. 
Secondary Pharmacodynamic Analyses ..................................................... 14 
4.3.1. 
Definition of endpoint ................................................................... 14 
4.3.2. 
Main Analytic approach ................................................................ 15 
4.4. 
Exploratory Biomarker Analyses ................................................................. 16 
4.5. 
Safety Analyses .......................................................................................... 16 
4.5.1. 
Extent of Exposure ...................................................................... 16 
4.5.2. 
Adverse Events............................................................................ 17 
4.5.3. 
Additional Safety Assessments .................................................... 18 
4.6. 
Other Analyses ........................................................................................... 19 
4.6.1. 
Subgroup analyses ...................................................................... 19 
4.6.2. 
Other variables and/or parameters............................................... 19 
4.7. 
Data reviews ............................................................................................... 19 
4.8. 
Changes to Protocol Defined Analyses ....................................................... 21 
5. 
SAMPLE SIZE DETERMINATION ......................................................................... 21 
6. 
SUPPORTING DOCUMENTATION ....................................................................... 22 
6.1. 
Appendix 1 Study Population Analyses ....................................................... 22 
6.1.1. 
Participant Disposition ................................................................. 22 
6.1.2. 
Demographic and Baseline Characteristics .................................. 22 
6.1.3. 
Protocol Deviations ...................................................................... 22 
6.1.4. 
Prior and Concomitant Medications ............................................. 22 
6.1.5. 
Study Intervention Compliance .................................................... 23 
6.1.6. 
Additional Analyses Due to the COVID-19 Pandemic .................. 23 
6.2. 
Appendix 2 Data Derivations Rule .............................................................. 24 
6.2.1. 
Criteria for Potential Clinical Importance ...................................... 24 
6.2.2. 
Study Phase ................................................................................ 25 


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213224 
3 
 
6.2.3. 
Study Day and Reference Dates .................................................. 26 
6.2.4. 
Assessment Window ................................................................... 26 
6.2.5. 
Multiple measurements at One Analysis Time Point .................... 26 
6.2.6. 
Premature Withdrawals ................................................................ 26 
6.2.7. 
Handling of Missing data .............................................................. 27 
7. 
TRADEMARKS & ABBREVIATIONS ...................................................................... 31 
8. 
REFERENCES ....................................................................................................... 32 
 


CONFIDENTIAL 
 
213224 
4 
 
Version history 
SAP 
Version 
Approval Date 
Protocol Version 
(Date) on which 
SAP is Based 
Change 
Rationale 
SAP 
01 Jul 2021 
03-NOV-2020 
Not 
Applicable 
Original version 


CONFIDENTIAL 
 
213224 
5 
 
1. 
INTRODUCTION 
• The purpose of this SAP is to describe the planned analyses and output to be included 
in the Clinical Study Report for study 213224 (Protocol 2019N419175_00). 
1.1. 
Objectives and Endpoints 
Part 1 
Objectives 
Endpoints 
Primary 
 
• To characterise the wheal 
response over time following 
skin challenge with ascending 
concentrations of SP on a 
single day 
• Wheal response during the 2 h post-
challenge period area under the curve 
(AUC) at each concentration of SP  
Secondary 
 
• To characterise the wheal 
response over time following 
skin challenge with ascending 
concentrations of SP on a 
single day 
• Wheal response during the 2 h post-
challenge period at each concentration of 
SP (including maximum observed wheal 
response, time to maximum observed 
wheal response, time of complete wheal 
response disappearance) 
• To characterise the flare 
response over time following 
skin challenge with ascending 
concentrations of SP on a 
single day 
• Flare response during the 2 h post-
challenge period at each concentration of 
SP (including AUC, maximum observed 
flare response, time to maximum 
observed flare response, time of 
complete flare response disappearance) 
• To evaluate the safety and 
tolerability of SP 
• Incidence of Adverse Events (AE) and 
Serious Adverse Events (SAE) 
• Incidence of findings of clinical important 
changes in: 
o vital signs 
o laboratory safety data 
o 12-lead electrocardiogram (ECG) 
 
 


CONFIDENTIAL 
 
213224 
6 
 
Part 2 
Objectives 
Endpoints 
Primary 
 
• To compare the wheal 
response over time 
following skin challenge 
with ascending 
concentrations of SP on 
two days  
• Wheal response during the 2 h post-challenge 
period (AUC) at each concentration of SP across 
2 challenge days  
Secondary 
 
• To characterise the wheal 
response over time 
following skin challenge 
with ascending 
concentrations of SP on 
two days 
• Wheal response during the 2 h post-challenge 
period at each concentration of SP across 
2 challenge days (including maximum observed 
wheal response, time to maximum observed 
wheal response, time of complete wheal response 
disappearance) 
• To characterise the flare 
response over time 
following skin challenge 
with 
ascending concentrations 
of SP on two days 
• Flare response during the 2 h post-challenge 
period at each concentration of SP across 
2 challenge days (including AUC, maximum 
observed flare response, time to maximum 
observed flare response, time of complete flare 
response disappearance) 
• To evaluate the safety and 
tolerability of SP 
• Incidence of Adverse Events (AE) and Serious 
Adverse Events (SAE) 
• Incidence of findings of clinical important 
changes in: 
o vital signs 
o laboratory safety data 
o 12-lead electrocardiogram (ECG) 
Exploratory 
 
• To investigate the effect of 
intradermal SP challenges 
on local exploratory 
biomarkers of mast cell 
activation. 
• Change in levels of exploratory biomarkers in 
skin during up to 2hr post challenge time at each 
of the 2 challenge days.  


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1.2. 
Study Design 
Overview of Study Design and Key Features 
 
Challenge Visit 1 
Challenge Visit 2 
Part 
1 
Wheal and flare post SP challenges 
(Panel A) only 
Not applicable 
Part 
2 
Wheal and flare post SP challenges 
(Panel A); and 
IDM during SP challenges (Panel B) 
Wheal and flare post SP challenges 
(Panel A) 
IDM during SP challenges (Panel B) 
Biopsy post challenges (Panel C) 
Before first and after last SP 
challenge: biomarker blood sample^ 
Panel A: Wheal and Flare measurement 


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Overview of Study Design and Key Features
Panel B: IDM measurement 
 
Panel C: Biopsy sample 
 
 
Design 
Features 
• 
This is an open label, single centre, 2-part, prospective enabling study of 
SP intradermal challenge in healthy participants.  
• 
Parts 1 and 2 will run sequentially.  
• 
Part 1 will include 1 challenge visit; Part 2 will include two challenge 
visits. 
• 
All participants will attend the clinical unit for a screening visit within 
28 days before their first or only challenge visit. Eligible participants 
will be enrolled into Part 1 or Part 2, based on their availability. 
• 
In Part 1, participants will attend the unit as outpatients for 1 challenge 
visit. The duration of Part 1, including screening, is not expected to 
exceed 4 weeks for each participant. 
• 
In Part 2, participants will attend the unit as outpatients for 2 challenge 
visits and have 1 follow-up phone call; the timing of visits will be 
decided based on emerging data. The duration of Part 2, including 
screening and follow-up is not expected to exceed 7 weeks for each 
participant. 
• 
At each challenge visit, up to 4 concentrations of SP will be assessed. 
• 
Wheal and flare responses will be assessed after each challenge (parts 1 
and 2) 
• 
In part 2, participants will receive up to 6 additional SP intradermal 
injections for biomarker analysis:  
• 
IDM challenges: 4 increasing concentrations of SP (the same 
concentrations as those administered in the wheal and flare 
challenges); IDM samples will be taken at the site of each challenge, 
before and after challenge.  
• 
Biopsy challenges at Challenge Visit 2: 2 at the same concentration 
of SP; 3 biopsies would be taken – one from each challenged site and 
one from a non-challenge area of skin. (these challenges and biopsies 
may or may not be performed, based on review of ongoing data.) 
• 
Biomarker blood samples will be taken before the first SP challenge 
and after the last SP challenge at Challenge Visit 2. 


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Overview of Study Design and Key Features 
Study 
intervention 
At each challenge visit, up to 4 concentrations of SP will be assessed, as 
follows: 
In Parts 1 and 2: 
• 
Participants will first receive saline, by intradermal injection, and 
histamine by skin prick, as negative and positive controls.  
o If the wheal responses immediately after the  20 min post 
challenge period do not meet the acceptable saline and 
histamine responses (as defined in the SRM), the participant 
will not receive SP.  
o If it happens in Part 1 or on Challenge Visit 1 in Part 2, the 
participant will be withdrawn from the study.  
o If it happens on Challenge Visit 2 in Part 2 and if the 
participant marginally fails to meet the control criteria (as 
defined in the SRM), they may have the tests repeated. If 
they still fail to meet the control criteria, they will be 
withdrawn from the study. 
• 
Following confirmation of acceptable control responses, participants 
will receive up to 4 intradermal injections of SP at different 
concentrations; injections will be given sequentially from lowest to 
highest concentration; wheal and flare responses will be assessed 
after each challenge.  
In Part 2 only: 
• 
Participants will receive up to 6 additional SP intradermal injections 
for biomarker analysis 
Study 
intervention 
Assignment 
• 
No randomization is planned 
• 
Participants will receive up to 4 ascending SP concentrations 
Data reviews 
• 
See Table 1  
 
2. 
STATISTICAL HYPOTHESES  
The objectives of this enabling study are to characterise the wheal and flare responses 
over time following skin challenges with ascending concentrations of SP. No formal 
hypotheses will be tested. 
2.1. 
Multiplicity Adjustment 
No multiplicity adjustment is planned here. 


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3. 
ANALYSIS SETS 
 Analysis Set 
Definition / Criteria 
Analyses 
Evaluated 
Screened  
• 
All participants who signed the ICF and 
were screened for eligibility. 
• Screen 
Failure 
Enrolled 
• 
All participants in the Screened analysis set 
who entered the study.  
Note: screening failures (who never passed screening 
even if rescreened) and participants screened but 
never enrolled into the study (Met eligibility but not 
needed) are excluded from the Enrolled analysis set 
as they did not enter the study. 
• Study 
Population 
Safety 
• 
All participants in the Enrolled analysis set 
who received at least one challenge-related 
study procedure on their (first) challenge day 
• Safety 
• PD 
 
4. 
STATISTICAL ANALYSES 
The objectives of this enabling study are to characterise the wheal and flare responses 
over time following skin challenges with ascending concentrations of SP. 
4.1. 
General Considerations 
4.1.1. 
General Methodology 
Unless otherwise specified, continuous data will be summarized using descriptive 
statistics: n, mean, standard deviation (SD), median, interquartile range, minimum and 
maximum. Categorical data will be summarized as the number and percentage of 
participants in each category.  
4.1.1.1. 
Bayesian Inference 
All the main statistical analyses will be carried out in the Bayesian framework. Models 
will be fitted by MCMC simulations. Vague priors will be used for modelling parameters 
and will be specified in the following sections. Sensitivity analyses on priors may be 
performed. Covariance structures may also be investigated.. 
The following summaries of the posterior distribution will be reported: posterior median, 
posterior standard deviation (SD), 95% equal-tailed credible interval (95% CrI), posterior 
probabilities of the true difference being greater than pre-specified thresholds. 
The inference will be carried out as follows: 
- 
2 chains will be run, with different (over-dispersed) initial values in order to assess 
convergence. The posterior summaries will use only 1 chain. 
- 
A minimum burn-in period of 10,000 MCMC samples (for each chain) will be used.  


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- 
A minimum of 10,000 MCMC samples (for each chain) will be run to generate 
samples of the posterior distribution.  
- 
A thinning sample may be used to improve convergence. If k is the thinning ratio, 
the number of MCMC samples will be minimum k*10,000. 
To assess convergence (MCMC), the following will be used: 
- 
Ratio Monte Carlo error /posterior SD should be as small as to ensure that only a 
fraction of the posterior variability is due to simulation error.  
The final MCMC samples will be such that this ratio for all the parameters in the 
model is ≤ 0.01. 
- 
Gelman & Rubin diagnostic is based on the ratio of the pooled variance across the 
chains over the overall within-chain variance. Values close to 1 indicate that each 
of the subsets of MCMC samples is close to the target posterior distribution. 
A cut-off of 1.1 will be used. 
- 
Diagnostic plots and visual inspection 
o Trace plots (the distribution of points should not change as the chain 
progresses and the posterior mean and variance are relatively constant) 
o Autocorrelation plots (provide information on how slow or fast the Markov 
chain converges) 
To assess the model adequacy, diagnostic plots of the median predicted value vs median 
residual (should not show evidence of any patterns) will be used. 
All the diagnostic outputs and alternative models fitted (where applicable) will be stored 
in the refdata folder in HARP (production) in the relevant reporting efforts. 
4.1.2. 
Baseline Definition 
For part 1, there will be 1 baseline value (baseline visit 1). For part 2, there will be 2 
baseline values, one for each visit (baseline visit 1 and visit 2).  
For the PD endpoints (wheal and flare responses), the baseline value will be the saline 
control non-missing value pre-SP challenge at challenge visit 1 and challenge visit 2 
(parts 1 and 2). No missing baseline values will be allowed as part of the 
inclusion/exclusion criteria. 
For the other endpoints collected at challenge visits 1 and 2, the baseline value will be the 
latest non-missing pre-1st control challenge assessment value at challenge visit 1 and 
challenge visit 2 (parts 1 and 2), including those from unscheduled visits. 
For Part 2, the Baseline for Visit 2 will be the latest assessment performed after Visit 1 
visits (such as Unscheduled) but before the first Visit 2 challenge.  
Participants with a missing baseline value will be excluded from the change from 
baseline analyses. 
The mean of replicate assessments at any given time point will be used as the value for 
that time point. 


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If baseline data are missing no derivation will be performed and baseline will be set to 
missing. 
4.2. 
Primary Pharmacodynamic Analyses 
4.2.1. 
Definition of endpoint 
For parts 1 and 2, the primary endpoint is the wheal response-time area under the curve 
(AUC) over the 2h post-challenge period following skin challenge with ascending 
concentrations of SP on a single day (challenge visit 1) and 2 days (challenge visits 1 and 
2), respectively, expressed as mm2 /min (see OPS). 
The wheal response variable will be the area in millimetres squared (mm2) and will be 
calculated using the following formula for area of an ellipse (see OPS), using the longest 
and orthogonal diameters measured by the calliper method.  
The wheal response variable will be calculated for each ascending SP concentration and 
each control challenge (saline and histamine) and at each timepoint during the 2h post-
challenge period at challenge visit 1 (parts 1 and 2) and challenge visit 2 (part 2). 
The same as above will be repeated for flare.  
4.2.2. 
 Main analytical approach 
4.2.2.1. 
Summary Measure 
The summary measure is the difference in wheal response AUC between 2 consecutive 
SP concentrations at challenge visit 1 (parts 1 and 2) and at challenge visit 2 (part 2). 
4.2.2.2. 
Population of interest 
The primary pharmacodynamics analyses will be based on the Safety population and on 
participants who have completed the challenge visits (complete case analysis). 
Statistical analyses 
Unless otherwise specified, endpoints / variables defined in Section 4.2.1 will be 
summarised using descriptive statistics, graphically presented and listed (where 
appropriate). 


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Statistical Methodology Specification for Part 1 
Endpoint / Variables 
• SP-induced wheal response-time AUC at challenge visit 1 
Model Specification 
• Bayesian analysis of covariance (ANCOVA) 
Model 
𝑦𝑖 ~ 𝑁𝑜𝑟𝑚𝑎𝑙(𝜇𝑖,  𝜎) 
𝜇𝑖= 𝑿𝑖 𝜷 
Response 
𝑦𝑖= AUC for participant i       
Prior for 𝜎 
σ = between-participant SD ~ 𝐺𝑎𝑚𝑚𝑎(shape = 2 , rate =
1
𝐴)  
with A reasonably large e.g. A=10 (Chung et al, 2013) 
Population-level 
coefficients (β) 
𝛃 = parameters associated to predictors 
βk ~ 𝑁𝑜𝑟𝑚𝑎𝑙(0, 𝑠𝑑= 1e6) 
Predictors (X) 
• 
SP = SP concentration (categorical) 
• 
Saline-induced (Baseline) wheal response-time AUC (continuous) 
Model Checking & Diagnostics 
See Section 4.1.1.1 
Model Results Presentation 
• Summaries of the posterior distributions of true AUC for each SP challenge and the true 
difference in AUC  using the mean baseline to calculate the marginal estimates 
• Probabilities that the true difference in AUC is greater than 0 
 
Statistical Methodology Specification for Part 2 
Endpoint / Variables 
• SP-induced wheal response-time AUC at challenge visits 1 and 2 (using only data when no 
IDM intervention was done) 
Model Specification 
• Bayesian analysis of covariance (ANCOVA) with visit effects  
Model 
Y ~ MVN(μ, Σ) 
μ = Xβ + Zγ 
Outcome 
𝑦𝑖𝑗= SP-induced AUC for participant i at visit j 


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Prior for Σ 
(covariance matrix) 
Σ𝑖𝑗= 𝐶𝑜𝑣[𝑌𝑖, 𝑌𝑗] assumed to be unstructured 
Σ ~ Inverse Wishart (J, R), with J = 2 (visits), R = empirical covariance 
matrix 
Population-level 
coefficients (β) 
𝛃 = parameters associated to predictors 
βk ~ 𝑁𝑜𝑟𝑚𝑎𝑙(0, 𝑠𝑑= 1e6) 
β0 = intercept, Vague: 𝑁𝑜𝑟𝑚𝑎𝑙(0, 𝑠𝑑= 1e6) 
Predictors (X) 
• 
SP = SP concentration (categorical) 
• 
Visit = challenge visit (categorical) 
• 
SP*visit = SP by-visit interactions (categorical) 
• 
Saline_AUC_base = average baseline defined as the mean of the 2 
saline-induced AUCs for each participant (continuous) 
• 
Saline_AUC_visit = Deviation of each visit saline-induced baseline 
from average baseline defined as saline-induced AUC - 
Saline_AUC_base 
• 
Saline_AUC_visit*visit = Saline_AUC_visit by-visit interactions 
Subject-level 
effects (γ) 
• 
𝒁𝑖 = design matrix for the subject-level effects 
• 
γ~ Normal (0, σ2I) 
Model Checking & Diagnostics 
See Section 4.1.1.1 
Model Results Presentation 
• Summaries of the posterior distributions of true AUC for each SP challenge and the true 
difference in AUC using the mean baseline to calculate the marginal estimates 
• 
Probabilities that the true difference in AUC is greater than 0 
4.2.3. 
Sensitivity analyses 
Sensitivity analyses on the priors (in particular the prior for the between-participant and 
within pa SD) will be carried out.  
4.3. 
Secondary Pharmacodynamic Analyses 
4.3.1. 
Definition of endpoint 
Wheal response related endpoints (parts 1 and 2): 
The following endpoints will be calculated at challenge visit 1 (parts 1 and 2) and 
challenge visit 2 (part 2) using the wheal area (mm2) determined by the calliper method 
(see OPS) and for each of the skin challenges: 
- 
Wheal response-time area under the curve (AUC) over the 2h post-challenge period 
- 
Maximum observed wheal area (mm2) during the 2h post-challenge period. 
- 
Time (min) to maximum observed wheal area during the 2h post-challenge period 


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- 
Time (min) of complete wheal area disappearance during the 2h post-challenge 
period. The complete disappearance is defined as a wheal area of 0. If the complete 
disappearance does not occur within the 2h post-challenge period, the time will be set 
to >120min. 
The longest and orthogonal diameters (mm) of the wheal, measured by the calliper 
method, will also be summarised by challenge visit and skin challenge in the same way: 
- 
 Maximum observed wheal longest/orthogonal diameter (mm) during the 2h post-
challenge period. 
- 
Time (min) to maximum observed wheal longest/orthogonal diameter during the 2h 
post-challenge period  
- 
Time (min) of complete wheal longest/orthogonal diameter disappearance during the 
2h post-challenge period. The complete disappearance is defined as a wheal 
longest/orthogonal diameter of 0. If the complete disappearance does not occur within 
the 2h post-challenge period, the time will be set to >120min. 
Flare response related endpoints (part 1 and 2) 
The following endpoints will be calculated at challenge visit 1 (parts 1 and 2) and 
challenge visit 2 (part 2) using the flare area (mm2) as determined by the calliper method 
(see OPS). 
- 
Flare response-time area under the curve (AUC) over the 2h post-challenge period 
- 
Maximum observed flare area during the 2h post-challenge period. 
- 
Time (min) to maximum observed flare area during the 2h post-challenge period  
- 
Time (min) of complete flare area disappearance during the 2h post-challenge period. 
The complete disappearance is defined as a flare area of 0. If the complete 
disappearance does not occur within the 2h post-challenge, the time will be set to 
>120min. 
The longest and orthogonal diameters (mm) of the flare, measured by the calliper 
method, will also be summarised by challenge visit and skin challenge in the same way: 
- 
 Maximum observed flare longest/orthogonal diameter (mm) during the 2h post-
challenge period. 
- 
Time (min) to maximum observed flare longest/orthogonal diameter during the 2h 
post-challenge period  
- 
Time (min) of complete flare longest/orthogonal diameter disappearance during the 
2h post-challenge period. The complete disappearance is defined as a flare 
longest/orthogonal diameter of 0. If the complete disappearance does not occur within 
the 2h post-challenge period, the time will be set to >120min. 
4.3.2. 
Main Analytic approach 
4.3.2.1. 
Summary Measure 
The summary measure is the difference in flare response AUC between 2 consecutive SP 
concentrations at challenge visit 1 (parts 1 and 2) and at challenge visit 2 (part 2). 


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4.3.2.2. 
Population of interest 
The secondary pharmacodynamics analyses will be based on the Safety population, 
unless otherwise specified. 
4.3.2.3. 
Statistical Analyses 
Endpoints / variables defined in Section 4.3.1 will be summarised using descriptive 
statistics, and graphically presented (where appropriate). 
The proposed statistical analyses planned for the primary PD endpoint (Section 0) will be 
repeated for the flare response-time area under the curve (AUC) over the 2h post-
challenge period for parts 1 and 2. 
For the other endpoints, no statistical analyses will be carried out. 
4.4. 
 Exploratory Biomarker Analyses 
The exploratory biomarker analyses will be based on the Safety population and will be 
reported for part 2 only. 
No formal statistical testing will be performed on biomarkers data. 
Histamine concentrations (ng/mL) from intradermal microdialysis during the 2h post-
challenge will be summarised using descriptive statistics and graphically presented 
(where appropriate) at challenge visits 1 and 2 of part 2 in a similar way as the wheal and 
flare responses. 
Additional biomarker analyses of the skin punch biopsies and blood samples (visit 2 
only) may be conducted. These results will be the subject of a post hoc analysis. 
4.5. 
 Safety Analyses 
The safety analyses will be based on the Safety population. No formal statistical testing 
will be performed on safety data. 
Displays will be generated for parts 1 and 2, separately (but in the same file) and for the 
study intervention group “Skin Challenges”. 
4.5.1. 
Extent of Exposure 
A listing of exposure to skin challenges (intervention) will be produced for each subject 
based on location, laterality and directionality for the skin challenges, concentration 
(μmol/L), volume (μL) and dose (pmol) 
For the SP challenges, the dose (pmol) will be derived from the concentration and 
volume (see OPS). 


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4.5.2. 
Adverse Events 
An adverse event (AE) is defined as a study intervention emergent AE if it is an AE that 
started on or after the date/time of first study intervention 
An AE is not a study intervention emergent AE if it started and ended before the 
date/time of first study intervention. 
All AE summaries will be based on study intervention emergent events unless otherwise 
specified. SAE summaries will be based on all SAEs reported regardless of whether they 
meet the definition of study intervention emergent or not. All AE and SAE summaries 
will be by PT only unless otherwise specified. 
Adverse events will be coded using the standard Medical Dictionary for Regulatory 
Affairs (MedDRA dictionary). 
Summary tables will include the number of participants with the AE, the percentage of 
participants with the AE. The following AE categories will be provided: 
• All adverse events by SOC, PT and maximum intensity 
• Common non-serious AEs by SOC and PT (Number of Subjects and 
Occurrences): 
o For disclosure purposes, common is defined as AEs occurring in at 
least 5% of any intervention arm. Due to the small sample size of this 
study this table will include all non-serious AEs. 
• AEs related to study intervention by SOC, PT and maximum intensity: 
o A study intervention-related AE is defined as an AE for which the 
investigator classifies the possible relationship to study interventions 
as “Yes”. A worst-case scenario approach will be taken to handle 
missing data, i.e. the summary table will include events with the 
relationship to study intervention as ‘Yes’ or missing. 
o The summary will be generated for the relationship to SP only, 
histamine only, and both SP and histamine, separately. 
• SAE by SOC, PT and maximum intensity 
• SAE by SOC and PT (Number of Subjects and Occurrences) 
• Serious fatal and non-fatal study intervention related AEs by overall 
frequency 
4.5.2.1. 
Adverse Events of Special Interest 
Not applicable. 
4.5.2.2. 
COVID-19 Assessment and COVID-19 AEs 
It is the investigator who decides whether an AE is a COVID related Adverse event or 
not. 
Listings will be produced for subjects with COVID-19 Adverse Events. 


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4.5.2.3. 
Impact of COVID-19 Pandemic on Safety Results 
The impact of the COVID-19 pandemic on the safety results will be assessed, if any. 
4.5.3. 
Additional Safety Assessments  
4.5.3.1. 
Laboratory Data 
Summary tables for haematology and chemistry laboratory tests as well as urine 
concentration parameters will be produced: 
• Change from baseline in laboratory parameters (clinical chemistry and hematology) 
will be presented for continuous variables, with a summary of baseline values 
included in the tables.  
• Summaries of worst-case results post-baseline relative to baseline will be provided 
for the following tests: 
• 
For lab tests with associated Potential Clinical Importance (PCI) criteria: the 
summary will include baseline data and worst-case post-baseline. The baseline 
categories are: Low, W/in Range and High. The change categories are: To 
Low, To w/in Range or No Change, To High. If a subject has a decrease to 
low and an increase to high during the same time interval, then the subject is 
counted in both the “to Low” and the “to High” categories. 
• 
For categorical urinalysis parameters 
For part 2, change from baseline will be calculated for each visit, (using the visit 1 
baseline for visit 1, and visit 2 baseline for visit 2). 
Baseline and post-baseline data will be included in these tables. If a participant moves to 
both a low and a high clinical concern range during the study intervention period, then 
the participant is counted in both categories.  
Summaries of hepatobiliary laboratory events including possible Hy’s law cases will be 
provided in addition to what has been described above. Possible Hy’s law cases are 
defined as any elevated alanine aminotransferase (ALT)>3×upper limit of normal (ULN), 
total bilirubin≥2×ULN and alkaline phosphatase (ALP)<3×ULN/missing. Total 
bilirubin≥2×ULN can be within 28 days following the ALT elevation and if direct 
bilirubin is available on the same day, it must be ≥ 35% of total bilirubin. 
ALP<3×ULN/missing means it is satisfied unless the ALP is ≥3xULN at the time of 
bilirubin elevation. The summary will be produced for worst case post baseline only. 
4.5.3.2. 
Vital Signs 
A summary of change from baseline vital sign values will be provided by visit and 
intervention group, including systolic blood pressure, diastolic blood pressure, heart rate, 
respiratory rate, height, weight and BMI, with a summary of baseline values included in 
the table. 
The number and percentage of participants with clinical important changes for vital signs 
(systoloic blood pressure, diastolic blood pressure, heart rate) will be generated using the 
PCI criteria stated in Section 6.2.1.3.  


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The mean of replicate assessments will be used as baseline and as value at each timepoint 
for blood pressure and heart rate values collected in triplicates. 
4.5.3.3. 
ECG 
The QTc data analysis will use the collected values based on Fridericia formula. The 
number and percentage of participants with clinical important changes for 12-lead ECG 
will be generated using the PCI criteria stated in Section 6.2.1.2. 
For all ECG analyses, baseline is taken to be the mean of the triplicate values collected 
pre-1st skin challenge.  
The following summaries will be provided by visit: 
• Summary of ECG findings (the clinical significance and interpretation of each 
planned ECG). In the case of triplicate ECG measurements, results will be 
presented separately by measurement number. 
• Summary of change from baseline in ECG values. In the case of triplicate ECG 
measurements, summaries will use the average of the triplicate, listings will 
present all individual measurements (and the average). 
The absolute and change from baseline ECG values will be rounded to the nearest integer 
before being categorized according to the clinical concern ranges. The following 
summaries of participants will be produced: 
• A summary of QTcF absolute values will display the number and percentage of 
participants with a post-baseline increase to a value within each clinical concern 
range for the worst-case post-baseline only.  
• A summary of change from baseline QTcF values will display the number and 
percentage of participants with a change within each range for the worst-case 
post-baseline. Participants with missing baseline values will be excluded from this 
summary. 
4.6. 
Other Analyses 
Not applicable. 
4.6.1. 
Subgroup analyses 
Not applicable 
4.6.2. 
Other variables and/or parameters 
Not applicable 
4.7. 
Data reviews 
All data reviews will be performed by the data review committee (DRC) (see Section 
10.1.5 of the protocol) in accordance to the Dose Escalation Plan and are summarised in 


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Table 1. Study population and safety data will be generated by the site, wheal and flare 
data will be generated by S&P. 
Table 1 
Timing, purpose, outcomes and data to be reviewed at each data 
review 
Data 
review 
number 
Timing 
Purpose 
Potential 
outcomes 
Data to be 
reviewed 
1.1 
~3 participants 
who have 
completed the part 
1 challenge visit 
Review 
safety data 
 
Review PD 
data 
Stop/pause study 
or recommend 
alterations to 
study design or 
conduct for safety 
Modify SP 
concentrations for 
the next 
participants to be 
enrolled in part 1 
Continue 
recruitment to 
part 1 at the same 
SP concentrations 
Study population 
Safety 
Wheal response 
(plots) 
 
1.2 
~6 participants 
who have 
completed the part 
1 challenge visit 
Review 
safety data 
 
Review PD 
data 
Stop/pause study 
or recommend 
alterations to 
study design or 
conduct for safety 
Modify SP 
concentrations for 
the next 
participants to be 
enrolled in part 1 
Stop recruitment 
to part 1 and open 
up part 2 
Continue 
recruitment to 
Study population 
Safety  
Wheal response 
(plots) 
 


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Data 
review 
number 
Timing 
Purpose 
Potential 
outcomes 
Data to be 
reviewed 
part 1 at the same 
SP concentrations 
1.3 
~12 participants 
who have 
completed the part 
1 challenge visit 
Review 
safety data 
 
Review PD 
data 
Stop/pause study 
or recommend 
alterations to 
study design or 
conduct for safety 
Confirm SP 
concentrations for 
part 2 
Stop the study 
Study population 
Safety 
Wheal response 
(plots, summary 
tables) 
Flare response 
(plots, summary 
tables) 
2 
~10 participants 
who have 
completed the part 
2 challenge visit 2 
Review 
safety data 
 
Review PD 
data 
Stop/pause study 
or recommend 
alterations to 
study design or 
conduct for safety 
Continue 
recruitment to 
part 2  
Stop the study 
Study population 
Safety 
Wheal response 
(plots, summary 
tables, stats 
analyses) 
Histamine 
concentrations 
(plots, summary 
tables) if 
available 
4.8. 
Changes to Protocol Defined Analyses 
There were no changes or deviations to the originally planned statistical analysis 
specified in the protocol 2019N419175_00 (Dated: 03-NOV-2020). 
 
5. 
SAMPLE SIZE DETERMINATION 
A sufficient number of participants will be screened in order to enroll approximatively 32 
participants in the study (up to 12 in part 1 and up to 20 in part 2). The sample size for 
part 1 and part 2 is not based on statistical considerations and is based on previous 
published data. 


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6. 
SUPPORTING DOCUMENTATION 
6.1. 
Appendix 1 Study Population Analyses 
The study population analyses will be based on the Screened (Section 6.1.1) and Enrolled 
(Section 6.1.1, Section 6.1.2, Section 6.1.3, Section 6.1.4) populations. 
Study population analyses including analyses of participant’s disposition, protocol 
deviations, demographic and baseline characteristics, prior and concomitant medications, 
and study intervention will be provided. 
6.1.1. 
Participant Disposition 
A summary of the number and percentage of participants who completed the study as 
well as those who prematurely withdrew from the study will be provided. Primary 
reasons for study withdrawal will be summarized.  
A summary of study intervention status will be provided. This display will show the 
number and percentage of participants who have completed the scheduled study 
intervention, or have discontinued study intervention prematurely, as well as primary 
reasons for discontinuation of study intervention. 
6.1.2. 
Demographic and Baseline Characteristics 
The demographic characteristics including age, race, ethnicity, sex, height, screening 
body weight and Body Mass Index (BMI) will be summarized with descriptive statistics.  
In addition, the following age categories will be summarized: 18-64, 65-84 and >=85. 
Summary of race and racial combinations will also be summarised. 
Medical conditions collected at screening will be summarized by past and current.  
6.1.3. 
Protocol Deviations 
Important protocol deviations (including deviations related to study inclusion/exclusion 
criteria, conduct of the trial, participant management or participant assessment) will be 
summarized. 
Protocol deviations will be tracked by the study team throughout the conduct of the 
study. These protocol deviations will be reviewed to identify those considered as 
important as follows: 
o Data will be reviewed prior to freezing the database to ensure all important 
deviations are captured and categorised on the protocol deviations dataset.  
o This dataset will be the basis for the summaries of protocol deviations. 
6.1.4. 
Prior and Concomitant Medications 
Concomitant medications will be coded and summarized using the GSK Drug dictionary.   


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A summary of the number and percentage of subjects with concomitant medications will 
be displayed using the Generic Term without regard to ATC classifications. This 
summary will present single ingredient medications and multi-ingredient medications 
according to the combination of the ingredients. Multi-ingredient medications will be 
labelled according to the sum of their ingredients, i.e., Generic Term, e.g., “Tylenol Cold 
and Flu” would appear as “CHLORPHENAMINE MALEATE + 
DEXTROMETHORPHAN HYDROBROMIDE + PARACETAMOL + 
PSEUDOEPHEDRINE HYDROCHLORIDE”. 
Concomitant medications include any medication that was taken at some point during the 
on-intervention period as defined in Section 6.2.2.1. 
6.1.5. 
Study Intervention Compliance 
Not Applicable. 
6.1.6. 
Additional Analyses Due to the COVID-19 Pandemic 
Not Applicable. 


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6.2. 
Appendix 2 Data Derivations Rule 
6.2.1. 
Criteria for Potential Clinical Importance 
The following criteria will be used to flag potential clinical importance:  
6.2.1.1. 
Laboratory Values 
Haematology 
Laboratory Parameter 
Units 
Category 
Clinical Concern Range 
Low Flag (< x) 
High Flag (>x) 
Haematocrit  
Ratio of 
1 
Male 
 
0.54 
Female 
 
0.54 
∆ from BL 
↓0.075 
 
Haemoglobin  
g/L 
Male 
 
180 
Female 
 
180 
∆ from BL 
↓25 
 
Platelet Count  
x109/ L 
 
100 
550 
While Blood Cell (WBC) 
 
 
 
 
    Absolute Count 
x109/ L 
 
3 
20 
    Neutrophil Count 
x109/ L 
 
1.5 
 
    Lymphocytes 
x109/ L 
 
0.8 
 
 
Clinical Chemistry 
Laboratory Parameter 
Units 
Category 
Clinical Concern Range 
Low Flag (< x) 
High Flag (>x) 
Albumin  
mmol/L 
 
30 
 
Calcium 
mmol/L 
 
2 
2.75 
Creatinine 
mmol/L 
∆ from BL 
 
↑ 44.2 
Glucose (Fasting) 
mmol/L 
 
3 
9 
Potassium 
mmol/L 
 
3 
5.5 
Sodium 
mmol/L 
 
130 
150 
Total CO2 
mmol/L 
 
18 
32 
 
Liver Function 
Test Analyte 
Units 
Category 
Clinical Concern Range 
ALT/SGPT  
U/L 
High 
≥ 2x ULN 
AST/SGOT  
U/L 
High 
≥ 2x ULN 
AlkPhos  
U/L 
High 
≥ 2x ULN 
T Bilirubin  
µmol/L 
High 
≥ 1.5xULN 
T. Bilirubin + ALT 
µmol/L 
High 
1.5xULN T. Bilirubin 
 
+ 
U/L 
≥ 2x ULN ALT 


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6.2.1.2. 
ECG 
ECG Parameter 
Units 
Clinical Concern Range 
Lower 
Upper 
Absolute 
Absolute QTc Interval 
Msec 
 
> 450 
Absolute PR Interval 
Msec 
< 110 
> 220 
Absolute QRS Interval 
Msec 
< 75 
> 110 
Change from Baseline 
Increase from Baseline 
QTc 
Msec 
 
> 60 
6.2.1.3. 
Vital Signs 
Vital Sign Parameter 
(Absolute) 
Units 
Clinical Concern Range 
Lower 
Upper 
Systolic Blood Pressure 
mmHg 
< 85 
> 160 
Diastolic Blood Pressure 
mmHg 
< 45 
> 100 
Heart Rate 
bpm 
< 40 
> 110 
 
Vital Sign Parameter 
(Change from Baseline) 
Units 
Clinical Concern Range 
Decrease  
Increase  
Systolic Blood Pressure 
mmHg 
≥ 20 
≥ 20 
Diastolic Blood Pressure 
mmHg 
≥ 10 
≥ 10 
Heart Rate 
bpm 
≥ 15 
≥ 15 
6.2.2. 
Study Phase 
6.2.2.1. 
Study phases for concomitant medication 
Study Phase 
Definition 
Prior 
If medication end date is not missing and is before 28 days prior to the most 
recent screening visit assessment 
Concomitant 
Any medication that is not a prior 
Notes: 
Please refer to Appendix Section 6.2.7.1: Handling of partial dates for concomitant 
medication. Use the rules in this table if concomitant medication date is completely 
missing. 


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6.2.3. 
Study Day and Reference Dates 
Study day is calculated as the number of days from Date of First skin challenge done at 
Visit 1: 
• Ref Date = Missing             → Study Day = Missing  
• Ref Date < First skin challenge Date → Study Day = Ref Date – Date of first 
Visit 1 Skin Challenge 
• Ref Date ≥ First skin challenge Date → Study Day = Ref Date – (Date of First 
Skin Challenge) + 1 
6.2.4. 
Assessment Window  
No Assessment Windows will be defined for analysis, and all (except Safety) summaries 
and analyses will be based on nominal visits. Safety summaries and analyses will use the 
remapped visits. Please see OPS for further details. 
Unscheduled and withdrawal visits will not be slotted to an analysis timepoint, but they 
will be considered for worse case and maximum displays. If multiple assessments qualify 
for worse case or maximum, the earlier one will be used. 
Acceptable windows are included in the SRM and assessments performed within these 
windows are not protocol deviations. 
6.2.5. 
Multiple measurements at One Analysis Time Point 
When triplicate ECG/Vital Signs assessments are taken, mean of the measurement will be 
calculated first and summary statistics will be based on the calculated mean. This will 
apply to both baseline and post baseline assessments.  
For lab tests on a study day, if more than one assessment is taken on the same day from 
the same type of local lab, the worst case will be used. 
6.2.6. 
Premature Withdrawals 
Element 
Reporting Detail 
General 
• 
Participant study completion (i.e. as specified in the protocol) was defined as 
having completed phases of the study including the follow up phone call as 
shown in the Schedule of Activities (see Protocol) 
• 
All available data from participants who were withdrawn from the study will 
be listed and all available planned data will be included in summary tables and 
figures, unless otherwise specified.  


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6.2.7. 
Handling of Missing data 
Element 
Reporting Detail 
General 
• 
Missing data occurs when any requested data is not provided, leading to 
blank fields on the collection instrument: 
o These data will be indicated by the use of a ‘blank’ in participant 
listing displays. Unless all data for a specific visit are missing in 
which case the data is excluded from the table. 
o Answers such as ‘Not applicable’ and ‘Not evaluable’ are not 
considered to be missing data and should be displayed as such. 
Wheal and 
flare 
• 
No imputation methods will be used for missing value of the longest and 
orthogonal diameters 
AUC 
• 
If there are single non-consecutive missing time points, or 2 consecutive 
missing time points, AUC will be calculated using trapezoidal rule (using 
non missing time points) 
• 
If 3 or more consecutive time points are missing, AUC will be set to missing 
• 
If 50% or more time points are missing, AUC will be set to missing 
• 
If at least 50% points are non-missing, and the last 2 timepoints are missing, 
the AUC will be calculated using non-missing timepoints up to N-2 
Biomarkers 
(IDM) 
• 
No imputation methods will be used for missing value. 
AE 
• 
A worst-case scenario approach will be taken to handle missing data: if the 
relationship to study intervention is missing, the relationship will be “yes”, if 
severity is missing the severity will be set to ‘severe’, if seriousness is 
missing the seriousness will be set to ‘yes’ 
6.2.7.1. 
Handling of Missing & Partial dates 
Element 
Reporting Detail 
General 
• 
Partial dates will be displayed as captured in participant listing 
displays.   
• 
However, where necessary, display macros may impute dates as 
temporary variables for sorting data in listings only. In addition, 
partial dates may be imputed for ‘slotting’ data to study phases or 
for specific analysis purposes as outlined below. 
• 
Imputed partial dates will not be used to derive study day, time to 
onset or duration (e.g., time to onset or duration of adverse events), 
or elapsed time variables (e.g., time since diagnosis). In addition, 
imputed dates are not used for deriving the last contact date in 
overall survival analysis dataset. 


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Element 
Reporting Detail 
Adverse Events 
• 
Imputations in the adverse events dataset are used for slotting 
events to the appropriate study time periods and for sorting in data 
listings 
 
• 
Partial dates for AE recorded in the CRF will be imputed using the 
following conventions: 
Missing start 
day 
If study intervention start date is missing (i.e. 
participant did not start study intervention), then 
set start date = 1st of month. 
Else if study intervention start date is not missing: 
o If month and year of start date = month 
and year of study intervention start date, 
then 
▪ 
If stop date contains a full date and 
stop date is earlier than study 
intervention start date, then set start 
date= 1st of month. 
▪ 
Else set start date = study 
intervention start date. 
Else set start date = 1st of month. 
Missing start 
day and month 
If study intervention start date is missing (i.e. 
participant did not start study intervention), then 
set start date = January 1. 
Else if study intervention start date is not missing: 
o If year of start date = year of study 
intervention start date, then 
▪ 
If stop date contains a full date 
and stop date is earlier than 
study intervention start date, 
then set start date = January 1. 
 
▪ 
Else set start date = study 
intervention start date.       
Else set start date = January 1. 
Missing stop 
day  
A '28/29/30/31' will be used for the day (dependent 
on the month and year) 
Missing stop 
day and month 
No Imputation 


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Element 
Reporting Detail 
Completely 
missing 
start/stop date 
No imputation 
Concomitant 
Medications/Medical 
History 
• 
Partial dates for any concomitant medications recorded in the CRF 
will be imputed using the following convention: 
 
Missing start 
day 
If study intervention start date is missing (i.e. 
participant did not start study Intervention), then 
set start date = 1st of month. 
Else if study intervention start date is not missing: 
o If month and year of start date = month 
and year of study intervention start date, 
then 
▪ 
If stop date contains a full date and 
stop date is earlier than study 
intervention start date, then set start 
date= 1st of month. 
▪ 
Else set start date = study intervention 
start date. 
Else set start date = 1st of month. 
Missing start 
day and month 
If study intervention start date is missing (i.e. 
participant did not start study Intervention), then 
set start date = January 1. 
Else if study intervention start date is not missing: 
o If year of start date = year of study 
intervention start date, then 
▪ If stop date contains a full date and 
stop date is earlier than study 
intervention start date, then set start 
date = January 1. 
▪ Else set start date = study 
intervention start date.       
Else set start date = January 1. 
Missing end 
day 
A '28/29/30/31' will be used for the day (dependent 
on the month and year).  


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Element 
Reporting Detail 
Missing end 
day and month 
A '31' will be used for the day and 'Dec' will be 
used for the month. 
Completely 
missing 
start/end date 
No imputation 
• 
The recorded partial date will be displayed in listings. 
Age 
GSK standard IDSL algorithms will be used for calculating Age. 
• 
Birth date will be presented in listings as ‘YYYY’. 
• 
Birth date and month will be imputed as ‘30th June’ for calculating 
age since only birth year is collected. 
The reference day for age calculation will be screening visit date 
(Screening date should be used to derive age as per programming 
note in Demo table). 


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7. 
TRADEMARKS & ABBREVIATIONS 
Trademarks of the GlaxoSmithKline 
Group of Companies 
 
Trademarks not owned by the 
GlaxoSmithKline Group of Companies 
None 
 
SAS 
 
Abbreviation 
Description 
AE 
Adverse Event 
GSK 
GlaxoSmithKline 
IDM 
Intradermal Micro dialysis 
IDSL 
Integrated Data Standards Library (GSK Standards Library) 
PCI 
Potential Clinical Importance  
SOC 
System Organ Class 
BMI 
Body Mass Index 
DRC 
Data Review Committee 
PT 
Preferred Term 
OPS 
Output &Programming Specification 
PD 
Pharmacodynamic 
QTcF 
Frederica’s QT Interval Corrected for Heart Rate 
SD 
Standard Deviation 
SP 
Substance P 
ECG 
Electrocardiogram 
MCMC 
Monte Carlo Markov Chain 


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8. 
REFERENCES 
Chung, Y., Rabe-Hesketh, S., Dorie, V., Gelman, A., Liu, J. (2013). A nondegenerate 
penalized likelihood estimator for variance parameters in multilevel models. 
Psychometrika, 78, 685–709.