A Prospective, Longitudinal Study to Investigate the Effect of Thermal Injury on Intestinal Permeability and Systemic Inflammation (HESTIA)
Phase: PHASE1
Conditions: Burns
Sponsor: GlaxoSmithKline

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TITLE PAGE
Protocol Title: A Prospective, Longitudinal Study to Investigate the Effect of Thermal 
Injury on Intestinal Permeability and Systemic Inflammation (HESTIA)
Protocol Number: 01
Short Title: Investigation of Thermal Injury and Intestinal Permeability
Compound Number:
NONE
Sponsor Name and Legal Registered Address: 
GlaxoSmithKline Research & Development Limited
980 Great West Road
Brentford
Middlesex, TW8 9GS
UK
Medical Monitor Name and Contact Information: 
 MBBS, MRCP, PhD
Director, Clinical Development
R&D Infectious Disease
Stockley Park West, 1-3 Ironbridge Road, Uxbridge, Middlesex, UB11 1BT
United Kingdom
phone:
Regulatory Agency Identifying Number(s): NA
Approval Date: 02-MAR-2017
Copyright 2017 the GlaxoSmithKline group of companies.  All rights reserved.  
Unauthorised copying or use of this information is prohibited.
PPD
PPD

P P D
P P D

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TABLE OF CONTENTS
PAGE
1.
SYNOPSIS...............................................................................................................6
2.
SCHEDULE OF ACTIVITIES (SOA).........................................................................9
2.1.
Healthy Participants (Group 1)......................................................................9
2.2.
Thermal Injury Participants (Group 2) .........................................................11
3.
INTRODUCTION....................................................................................................14
3.1.
Study Rationale ..........................................................................................14
3.2.
Background ................................................................................................14
3.2.1.
Thermal Injury, Intestinal Permeability and Multi-Organ 
Dysfunction Syndrome.................................................................14
3.2.2.
Intestinal Barrier Function and Its Measurement..........................15
3.2.3.
The Intestinal Microbiome and Thermal Injury .............................16
3.3.
Benefit/Risk Assessment ............................................................................17
3.3.1.
Risk Assessment .........................................................................17
3.3.2.
Benefit Assessment.....................................................................21
3.3.3.
Overall Benefit:Risk Conclusion...................................................21
4.
OBJECTIVES AND ENDPOINTS...........................................................................21
5.
STUDY DESIGN ....................................................................................................23
5.1.
Overall Design ............................................................................................23
5.2.
Number of Participants ...............................................................................23
5.3.
Participant and Study Completion...............................................................24
5.4.
Scientific Rationale for Study Design ..........................................................24
5.4.1.
Co-recruitment to the SIFTI-2 Study ............................................24
5.4.2.
Recruitment and Sampling Schedule...........................................25
5.4.3.
Inclusion of Healthy Participants..................................................27
5.4.4.
Preliminary Data Review..............................................................27
5.4.5.
The Use of the Sugar Test Materials (Lactulose, Mannitol 
and Sucralose) ............................................................................27
6.
STUDY POPULATION...........................................................................................27
6.1.
Recruiting Participants with Differing Mental Capacity ................................28
6.1.1.
Adult Participants Determined to Have Mental Capacity at 
Study Entry and Throughout the HESTIA Study...........................28
6.1.2.
Adult Participants Lacking Mental Capacity for the 
Duration of the HESTIA Study .....................................................28
6.1.3.
Adult Participants Lacking Mental Capacity (either at 
Study Entry or During the Study) Who Later Regain 
Capacity and Are Required to Provide Informed Consent............28
6.1.4.
Adult Participants with Mental Capacity to Provide 
Consent at Study Entry Who are Later Deemed No Longer 
to have Mental Capacity ..............................................................29
6.2.
Inclusion Criteria for Healthy Participants (Group 1) ...................................29
6.3.
Inclusion Criteria for Thermal Injury Participants (Group 2).........................29
6.3.1.
Age..............................................................................................30
6.3.2.
Type of Participant and Disease Characteristics..........................30
6.3.3.
Other Inclusions...........................................................................30

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6.3.4.
Gender ........................................................................................30
6.4.
Exclusion Criteria for Healthy Participants (Group 1) ..................................30
6.5.
Exclusion Criteria for Thermal Injury Participants (Group 2)........................30
6.6.
Lifestyle Restrictions...................................................................................31
6.6.1.
Meals and Dietary Restrictions ....................................................31
6.6.2.
Alcohol/Exercise/Aspirin (Healthy Participants only) ....................32
6.7.
Screen Failures...........................................................................................32
7.
NON-INVESTIGATIONAL MEDICINAL PRODUCT (NIMP) ...................................32
7.1.
STM Administered ......................................................................................33
7.2.
Dose Modification .......................................................................................34
7.3.
Method of STM Administration: Treatment Assignment ..............................34
7.4.
Blinding.......................................................................................................34
7.5.
Preparation/Handling/Storage/Accountability..............................................34
7.6.
STM Compliance ........................................................................................34
7.7.
Concomitant Therapy..................................................................................35
7.8.
Treatment after the End of the Study ..........................................................35
8.
DISCONTINUATION CRITERIA.............................................................................35
8.1.
Discontinuation of STM administration........................................................35
8.2.
Withdrawal from the Study..........................................................................35
8.2.1.
Other Withdrawal Criteria.............................................................36
8.3.
Lost to Follow Up........................................................................................36
9.
STUDY ASSESSMENTS AND PROCEDURES .....................................................37
9.1.
Efficacy Assessments.................................................................................37
9.2.
Adverse Events...........................................................................................37
9.2.1.
Monitoring and reporting responsibilities......................................37
9.2.2.
Time Period and Frequency for Collecting AE and SAE 
Information...................................................................................38
9.2.3.
Method of Detecting AEs and SAEs.............................................38
9.2.4.
Follow-up of AEs and SAEs.........................................................39
9.2.5.
Regulatory Reporting Requirements for SAEs .............................39
9.2.6.
Disease-Related Events and/or Disease-Related 
Outcomes Not Qualifying as SAEs...............................................39
9.3.
Treatment of Overdose...............................................................................40
9.4.
Safety Assessments ...................................................................................40
9.4.1.
Physical Examinations.................................................................41
9.4.2.
Vital Signs....................................................................................41
9.4.3.
Clinical Safety Laboratory Assessment........................................41
9.5.
Study Procedures .......................................................................................41
9.5.1.
Fluid Balance Measurement ........................................................42
9.5.2.
Wound Healing ............................................................................42
9.5.3.
Other Clinical Responses ............................................................42
9.6.
Pharmacokinetics .......................................................................................42
9.7.
Pharmacodynamics ....................................................................................42
9.8.
Intestinal Permeability Assessments...........................................................42
9.9.
Genetics .....................................................................................................43
9.10.
Sample Collection for Biomarker Analysis...................................................43
9.10.1.
Blood Biomarkers ........................................................................43
9.10.2.
Stool Sample Collection...............................................................44
9.10.3.
Urine Sample Collection ..............................................................44

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10. STATISTICAL CONSIDERATIONS........................................................................45
10.1.
Hypotheses.................................................................................................45
10.2.
Sample Size Determination ........................................................................45
10.3.
Data Analyses Consideration......................................................................45
10.4.
Populations for Analyses ............................................................................46
10.5.
Statistical Analyses.....................................................................................46
10.5.1.
Safety Analyses...........................................................................46
10.5.2.
Other Analyses ............................................................................47
10.5.3.
Interim Analyses ..........................................................................47
10.6.
Analyses of lactulose/mannitol ratio............................................................47
11. REFERENCES.......................................................................................................49
12. APPENDICES........................................................................................................51
12.1.
Appendix 1: Abbreviations and Trademarks................................................51
12.2.
Appendix 2: Clinical Laboratory Tests.........................................................53
12.3.
Appendix 3: Study Governance Considerations..........................................54
12.3.1.
Regulatory and Ethical Considerations ........................................54
12.3.2.
Recruiting participants under the Mental Capacity Act 
2005 ............................................................................................54
12.3.3.
Determining Whether a Participant has Capacity Under 
the Mental Capacity Act (2005)....................................................55
12.3.4.
Financial Disclosure.....................................................................58
12.3.5.
Informed Consent Process ..........................................................58
12.3.6.
Data Protection............................................................................58
12.3.7.
Committees Structure ..................................................................58
12.3.8.
Publication Policy.........................................................................59
12.3.9.
Dissemination of Clinical Study Data ...........................................59
12.3.10. Data Quality Assurance ...............................................................59
12.3.11. Source Documents ......................................................................60
12.3.12. Study and Site Closure ................................................................60
12.4.
Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting .......................................61
12.5.
Appendix 5..................................................................................................68

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1.
SYNOPSIS
Protocol Title: A Prospective, Longitudinal Study to Investigate the Effect of Thermal 
Injury on Intestinal Permeability, and Systemic Inflammation (HESTIA)
Short Title: Investigation of Thermal Injury and Intestinal Permeability
Rationale:
Patients with severe thermal injury [>20% total body surface area (TBSA)] are at risk for 
organ dysfunction and may develop multiple organ dysfunction syndrome (MODS). This 
study aims to explore the relationship between thermal injury, changes in intestinal 
permeability and the onset of MODS. The results of this study will be used to inform the
design of future drug studies of a novel medicinal product, which is predicted to reduce 
this increased permeability.
No GSK study treatment will be employed in this study.
One of the central hypotheses over the last two decades to explain the onset of MODS in 
the context of critical illness imputes that an increase in intestinal permeability results in 
the translocation of bacteria and bacterial products to the systemic circulation where they 
drive inflammation and injury to distal organs [Deitch, 2006]. This hypothesis is 
supported by data showing that there is a significant increase in intestinal permeability in 
critically ill patients, including patients with thermal injury, and that the degree of 
intestinal permeability correlates with the onset and severity of MODS [Doig, 1998]. As 
such, therapies directed at minimising these changes in intestinal permeability, thereby 
limiting the translocation of bacteria to the systemic circulation, are hypothesised to have 
an impact on clinical outcome in these patients.
Previous literature demonstrates that patients with severe thermal injury (>20% TBSA) 
display a significant and rapid increase in intestinal permeability that declines gradually 
over a two-week period following the injury [Olquin, 2005]. Additionally, these changes 
in intestinal permeability, correlate with the severity of sepsis observed in these patients 
[Ziegler, 1988].
The central hypothesis of this study, therefore, is that thermal injury alters intestinal 
barrier function allowing the translocation of bacteria and bacterial products to the 
systemic circulation where they contribute to the onset of MODS.
The aims of the study are:
1. To establish the magnitude and time course of changes in intestinal permeability to 
inform timing and duration of future investigational medicinal product administration.
2. To establish the optimal method for assessment of intestinal permeability in patients 
with thermal injury.
3. To describe the patient population most likely to benefit from a new medicinal 
product which could prevent changes in intestinal permeability.

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4. To improve our understanding of the links between intestinal damage, changes in the 
gut microbiome and microbial translocation to the systemic circulation following 
thermal injury.
In order to enter this study thermally injured participants will be required to co-enrol in
this study and an allied study entitled: A Multi-centre, Prospective Study to Examine the 
Relationship between Neutrophil Function and Sepsis in Adults and Children with Severe 
Thermal Injury (SIFTI-2) (reference number IRAS ID: 200366). Clinical data, standard 
of care laboratory data and investigational biomarker data will be shared from the SIFTI-
2 study to this study and the combined data from both studies will be used to address 
exploratory endpoints. Participants of the SIFTI-2 study will be appropriately consented 
for this data sharing.
Objectives and Endpoints:
Objective
Endpoint
Co-Primary
1.
To determine the impact of thermal injury 
on the magnitude of small intestinal 
permeability change as soon as possible 
following injury compared to healthy 
participants

Lactulose/Mannitol (L/M) ratio at entry
2.
To characterise the effect of thermal 
injury on small intestinal permeability over 
time

Changes in L/M ratio over time
Overall Design:
A prospective, longitudinal study will be conducted in adult (≥18 years old) men and 
women admitted to a hospital following thermal injury. Measurements of intestinal 
permeability, inflammation and microbial translocation will be taken over a six month 
period. A cohort of healthy participants will also be recruited in order to determine the 
reference against which post-burn permeability measurements and other biomarkers will 
be compared.
The lactulose-to-mannitol ratio is traditionally used to assess small intestinal permeability 
and sucralose to assess colonic permeability. Lactulose, mannitol and sucralose
[henceforth referred to as sugar test material (STM)] will be intermittently administered 
enterally for the purpose of intestinal permeability measurement to examine permeability 
at different points along the GI tract and is described in Section 7. It is important to note
that the administration of the STM is not therapeutic (lactulose and mannitol can be used 
as laxatives, however the amount in the STM is sub-therapeutic) and as such should be 
regarded as a non-investigational medicinal product (NIMP) [Guidance Documents 
Applying Investigational Medicinal Products (NIMPS), 2011].

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An internal preliminary data review will be conducted. This review is described in 
Section 10.
Number of Participants:
Table 1 in Section 5.2 described the number of participants proposed for the study. 
Sample size is based on feasibility. No formal calculation of power or sample size has 
been performed, but a sample size of 15 healthy participants and 25 thermal injury 
participants (≥15% TBSA) should be sufficient to provide useful estimates of variability 
in lactutose:manitol ratios, and any change in L/M ratio over time.
Treatment Groups and Duration:
Group 1: Healthy Participants
The total duration of this study for healthy participants will be approximately two weeks, 
in addition to the screening window:

Screening: up to 28 days before Day 1

Day 1: study start and assessments will be performed

Day 8 and Day 15
Group 2: Thermally injured participants
Thermal injury participants will be asked to participate for a total of 6 months (plus or 
minus 14 days).

There will be no screening period. Thermal injury participants will be recruited 
within 24 hours of their admission to the burns unit at the study site.

Intense monitoring phase: Assessments will be performed on alternate days for 
the first 14 days following study enrolment. If the participant is discharged prior 
to 14 days, the intense monitoring phase will end, but the participant will remain 
enrolled in the study.

Convalescent monitoring phase: Assessments at 28 days and 6 months will be 
made on an outpatient basis if the participant has already been discharged from 
hospital.
Exception to monitoring periods:

If a discharged participant attends the centre for routine clinical care on any 
of days 8-14, then study assessments will be made and samples will be taken

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2.
SCHEDULE OF ACTIVITIES (SOA)
2.1.
Healthy Participants (Group 1)
Procedure
Screening 
Treatment Period [Out patient days] (1 day)
Notes
D1
D8
D15
Review of inclusion/ 
exclusion criteria and 
informed consent
X
Demography
X
Medical history (includes 
substance and alcohol 
usage at screening) and 
Medication history
X
Substance testing (urine)
X
Substances: [Recreational 
Drugs and Alcohol]
HIV, Hepatitis B and C
screening
X
If test otherwise performed 
within 3 months prior to 
study entry, testing is not 
required
Laboratory assessments
X
Pregnancy test (WOCBP 
only) (urine)
X
(X)
Only performed again on 
Day 1 if patient at risk of 
pregnancy at or since 
screening

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Procedure
Screening 
Treatment Period [Out patient days] (1 day)
Notes
D1
D8
D15
Blood sampling for 
biomarkers
X
20ml of blood will be 
sampled in a single draw 
on one day, preferably Day 
1
Stool sample collection
X
Participants will be given a 
collection container at 
screening to bring with 
them on Day 1.
Brief physical examination 
including measurements 
of height and weight
Vital Signs (systolic and 
diastolic blood pressure 
and heart rate)
X
X
X
X
BMI calculated from height 
and weight at screening 
only
Examinations should be 
conducted the day after 
intestinal permeability 
measurement.
Medical review 
(assessment of health 
status)
X
X
X
Monitor for signs and 
symptoms of gastro-
intestinal infections and 
other emergent issues
STM Training
X
Refresher training may be 
provided as needed
Intestinal permeability 
measurement: STM 
administration followed by 
a 24-hour urine collection
X
X
X
Please refer to SRM for full 
information
Intestinal permeability 
measurement to be 
performed at home the day 
before the study visit.

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Procedure
Screening 
Treatment Period [Out patient days] (1 day)
Notes
D1
D8
D15
AE/SAE and Concomitant 
medication reviews
(X)
=============================
Day 1 will include 
concomitant medication 
review only
2.2.
Thermal Injury Participants (Group 2)
Procedure
D1 
(≤ 24 hours 
of 
admission)
Treatment Period [ICU Days] (+/4 hours)
6
months
(14
days)
Notes
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D28 
(3 
days)
Review of inclusion/ 
exclusion criteria and 
informed consent
X
Participants will be co-
consented to the SIFTI-2 
study
Medical history 
(includes substance and 
alcohol usage) and 
Medication history
X
Substance testing 
(urine)
X
Substances: 
[Recreational Drugs, 
Alcohol]
Pregnancy test 
(WOCBP only) (urine)
X
X
To be repeated at 6 
months prior to final STM 
administration
HIV, Hepatitis B and C 
screening
X
If test otherwise 
performed within 3 
months prior to study 
entry, testing is not 
required

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Procedure
D1 
(≤ 24 hours 
of 
admission)
Treatment Period [ICU Days] (+/4 hours)
6
months
(14
days)
Notes
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D28 
(3 
days)
Initial assessment of 
Burns
X
Calculation of %TBSA
Location of thermal injury 
and depth
Fluid balance (total 
input/output)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X*
Daily (over 24 hr)
*only if participant still 
admitted
Intestinal permeability 
measurement: STM 
administration followed 
by a 24-hour urine 
collection
X
X
X
X
X
X
X
X
X
Measurements every 48 
hours from first measure. 
Preference: D1, 3, 5, 7, 
9, 11, 13. Otherwise: D2, 
4, 6, 8, 10, 12, 14.
Please refer to SRM for 
full method.
Brief Physical 
Examination
X
X
Following final intestinal 
permeability 
measurement. Can be 
omitted if patient is still 
admitted to hospital.
Stool sample collection
=============================
(A sample of the first stool produced following admission)
X
X
X
Time to first stool 
collection; preferably on 
Day 1. Then on Days 14 
and 28 and 6 months
Wound Healing 
Assessments
X
X
X
This is to assess time to 
95% wound healing

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Procedure
D1 
(≤ 24 hours 
of 
admission)
Treatment Period [ICU Days] (+/4 hours)
6
months
(14
days)
Notes
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D28 
(3 
days)
AE/SAE and 
Concomitant medication 
reviews
X
=============================
X
AE/SAE monitoring will 
begin after the first 
administration of STM

The timing and number of planned study assessments may be altered during the course of the study based on newly available 
data or factors outside of the study such as priority medical care to ensure appropriate monitoring.

Any changes in the timing or addition of time points for any planned study assessments must be documented and approved by 
the relevant study team member and then archived in the sponsor and site study files, but will not constitute a protocol 
deviation or require a protocol amendment. The IRB/IEC will be informed of any safety issues that require alteration of the 
safety monitoring scheme or amendment of the ICF.

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3.
INTRODUCTION
3.1.
Study Rationale
The purpose of this study is to describe the kinetics and magnitude of increases in 
intestinal permeability which are observed as a result of thermal injury. The results of this 
study will be used to inform the design of future drug studies of a novel medicinal 
product, which is predicted to reduce this increased permeability.
3.2.
Background
3.2.1.
Thermal Injury, Intestinal Permeability and Multi-Organ 
Dysfunction Syndrome
Patients with severe thermal injury >20% TBSA are at risk for organ dysfunction and 
may develop multiple organ dysfunction syndrome (MODS). This study aims to explore 
the relationship between thermal injury, changes in intestinal permeability and the onset
of MODS.
One of the central hypotheses over the last two decades to explain the onset of MODS in 
the context of critical illness imputes that an increase in intestinal permeability results in 
the translocation of bacteria and bacterial products to the systemic circulation where they 
drive inflammation and injury to distal organs [Deitch, 2006]. This hypothesis is 
supported by data showing that there is a significant increase in intestinal permeability in 
critically ill patients, including patients with thermal injury, and that the degree of 
intestinal permeability correlates with the onset and severity of MODS [Doig, 1998]. As 
such, therapies directed at minimising these changes in intestinal permeability, thereby 
limiting the translocation of bacteria to the systemic circulation, are hypothesised to have 
an impact on clinical outcome in these patients (Figure 1).
Figure 1
Changes in Intestinal Permeability
Critical 
Illness
Intestinal
Permeability
Bacteria/
Bacterial products
MODS
Figure 1 (Left panel) Hypothesis: The gut is the major driver of MODS in critical illness
(Right panel) Correlation between intestinal permeability as measured by 
lactulose/mannitol ratio and the severity of MODS in critically ill patients. Intestinal 
permeability was determined by measuring the differential absorption of lactulose 
(increased in the damaged gut) and mannitol (freely absorbed in the normal and damaged 

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gut) following oral administration and expressed as the ratio of lactulose to mannitol 
(L/M) [Olquin; 2005].
There are some data showing that patients with severe thermal injury (>20% TBSA) 
display a significant and rapid increase in intestinal permeability that declines gradually 
over a two-week period following the injury [Olquin, 2005]. Additionally, these changes 
in intestinal permeability, correlate with the severity of sepsis observed in these patients 
[Ziegler, 1988].
The central hypothesis of this study is that thermal injury alters intestinal barrier function 
allowing the translocation of bacteria and bacterial products to the systemic circulation 
where they contribute to the onset of MODS.
The aims of the study are:
1. To establish the magnitude and time course of changes in intestinal permeability to 
inform timing and duration of future investigational medicinal product administration.
2. To establish the optimal method for assessment of intestinal permeability in patients 
with thermal injury.
3. To describe the patient population most likely to benefit from a new medicinal 
product which could prevent changes in intestinal permeability.
4. To improve our understanding of the links between intestinal damage, changes in the 
gut microbiome and microbial translocation to the systemic circulation following 
thermal injury.
3.2.2.
Intestinal Barrier Function and Its Measurement
The intestinal barrier combines a physical and immunological barrier. Epithelial cells are 
connected by tight junctions and prevent the passage of bacteria, toxins and antigens into 
the systemic circulation [Bjarnason, 1995]. Paneth cells, located in crypts of the small 
intestine, produce anti-microbial substances (e.g. lysozyme and defensins) and other 
immune cells patrol the lamina propria [Ayabe, 2000]. Barrier function can be disturbed 
by diseases such as inflammatory bowel disease; by drugs such as aspirin and alcohol; by 
ischaemia and has been observed following a number of acute injuries such as burns, 
trauma and radiation injury [Bjarnason, 1995; Derikx, 2006]. This disturbance results in 
the translocation of the intestinal flora (pathogenic or commensal) to the systemic 
circulation. Microbes are accompanied by proteins which normally form part of the tight 
intracellular junctions of the intestinal epithelium, such as claudins, and other enterocyte-
derived proteins [Grootjans, 2010]. In this study, intestinal permeability will be measured 
directly using oligosaccharide absorption and indirectly by looking for micro-organisms 
and soluble markers of intestinal barrier dysfunction in the systemic circulation.
Since the 1970s, oligosaccharides have been used as test probes to measure the function 
of the intestinal barrier [Menzies, 1972]. Lactulose, a large polysaccharide, does not 
normally cross the intestinal barrier, but following damage can cross the epithelium and 
enter the systemic circulation. It is not metabolised, so is filtered in the kidney and 
excreted in the urine. The fractional excretion (amount administered / amount recovered 
in urine) of lactulose is therefore a measure of intestinal permeability. The amount of 
lactulose entering the urine is dependent on a number of factors including renal function, 

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gastric emptying, and degradation in the large bowel by commensal bacteria; thus, a 
monosaccharide such as mannitol, which passes freely across the healthy intestinal
barrier, is often co-administered to ‘normalise’ lactulose measurements. Following 
administration of both lactulose and mannitol the fractional excretion of the sugars is 
expressed as a ratio where mannitol is the denominator. Lactulose and mannitol 
absorption occurs mainly in the proximal small intestine and is complete within 
approximately 5 hours of oral administration. This approach has been previously used 
successfully in patients with severe burn injury and intestinal permeability was found to 
correlate with episodes of sepsis [Doig, 1998].
In order to assess the permeability of the large bowel a third oligosaccharide, sucralose, 
will also form part of the sugar absorption test. This synthetic sweetener (marketed by 
Tate and Lyle as ‘Splenda’) is not subject to the same degradation by large bowel 
commensal flora as lactulose and is therefore a better measure of large bowel 
permeability than lactulose. Again, this has formed part of previously described studies 
aiming to measure intestinal permeability [Del Valle-Pinero, 2013].
Lactulose, mannitol and sucralose will be co-administered to both healthy participants
and participants following thermal injury. In order to document accurately the time 
course of change in permeability, thermally injured participants will be asked to undergo 
the test on alternate days for 14 days (the intense monitoring phase) followed by two 
convalescent samples at day 28 and month 6 (thermal injury participants). In order to 
produce an accurate baseline measurement, healthy participants will be asked to undergo 
three measurements of intestinal permeability over approximately a two week period.
3.2.3.
The Intestinal Microbiome and Thermal Injury
During the intense and convalescent monitoring phases of this study, samples of stool 
will be collected. These samples will undergo gene sequence analysis in order to 
determine the composition of the intestinal microbiota. These results will be compared 
with culture results from peripheral whole blood samples. The hypothesis is that raised 
intestinal permeability will correlate with an increased frequency of bacteraemia and that 
the particular bacteria detected in blood will correlate with the composition of the 
intestinal microbiota.
Additionally, it has been demonstrated that thermal injury alters the composition of the 
intestinal microbiome [Hammer, 2015]. The ultimate aim is to be able to block changes 
in intestinal permeability which might affect this change in composition and are therefore 
interested, in the current study, to assess the impact of thermal injury on the microbiome.

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3.3.
Benefit/Risk Assessment
3.3.1.
Risk Assessment
Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Non-investigational Medicinal Product Use
Oral administration of sucralose in solution is 
unlikely to be palatable.
The dose of sucralose to be administered is 2g 
per test. A tolerable sweetness score (e.g. 11.3 
(diet Pepsi)) would require the sucralose to be 
administered in 4 litres of water.
Sucralose is to be administered in capsules 
when given by mouth. If being administered by 
nasogastric or nasojejunal tube the capsules are 
to be emptied into the lactulose and mannitol
solution
Depending on the volume of administration, the 
final STM solution may be hyper-osmolar. 
Administration directly into the jejunum via a 
nasojejunal tube may then result in osmotic 
movement of water into the intestine causing 
distention and discomfort.
This is based on the physiological principle that 
the stomach normally regulates the osmolality of 
its contents passing into the small intestine. 
Administration via nasojejunal tube (but not 
nasogastric tube) bypasses this process.
The lactulose, mannitol and sucralose will be 
delivered nasojejunally in 50ml of water and 
followed by a 50ml water flush. This makes the 
solution iso-osmolar (300 mosmol/Kg in situ). 
following nasojejunal administration 
Lactulose and mannitol can produce an osmotic 
laxative effect following enteral administration.
Both lactulose and mannitol are used clinically as 
osmotic laxatives. The typical dose of lactulose 
(for the treatment of constipation) would be 
approximately 21g daily.
The amount of lactulose being used in this study 
is 5g, 75% below the standard laxative dose. 
The dose of mannitol is equally low compared to 
that contained in laxatives.

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Study Procedures
STM administration for the measurement of 
intestinal permeability requires a hold in feeding 
which may be longer than a standard feeding 
hold.
Intestinal permeability assessment requires the 
administration of STM (see Section 7) and the 
collection of urine samples at defined time points 
(see the SRM). This procedure has previously 
been used in patients with severe thermal injury
[Doig, 1998] but has not been studied 
extensively; participant fasting is required pre and 
post administration of these sugars; and repeated 
administration as per the SoA tables is also 
unexplored.
Where possible, tube feeding targets will be 
volume and not time-based to reduce the 
amount of feed missed on test days. Moreover, 
fasts for the tests will, where possible, be 
aligned with clinically indicated feeding holds
(such as fasts required before theatre, or 
scheduled overnight feed holds).
Other
The degree of injury sustained by some 
participants may be severe.
Participants with severe thermal injury experience 
significant morbidity and high levels of mortality. 
Therefore it is anticipated that these participants 
will be subject to multiple medical complications 
which may impact the study assessments and 
period (See SoA tables) 
Ensure that routine care in the burns unit is not 
compromised by study participation. Prompt 
reporting of any adverse events which are 
related to study procedures and may affect study 
safety.
Omission of lactulose and sennosides which are 
frequently administered as a part of routine burn 
management.
Lactulose is a part of the STM and its 
administration for clinical reasons would 
complicate intestinal permeability measurement 
significantly. Equally, the mechanism of actions of 
sennosides is to cause irritation of the GI tract 
Polyethylene glycol (e.g. Movicol) will be used 
as an alternative osmotic laxative.
If senna is required for clinical purposes, then its 
use must be documented in the CRF.

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
and may result in increased permeability.
The STM will be prepared for administration on 
the assessment day and will not be sterile.
There is a risk that the sugar test material 
formulation may be contaminated with yeast or 
bacteria. The presence of contamination within 
the STM formulation delivered via the feeding 
tube in thermal injury participants could present 
an infection risk.
Microbiology release testing will be conducted 
by Tayside pharmaceuticals. The STM will be 
prepared on the assessment day to minimise 
this risk.
Lactulose/Mannitol will be refrigerated from the 
point of manufacture and until use. The site 
investigators have been consulted on this risk 
and felt that it was low.
Incomplete 24 hour urine collections
Ambulant, uncatheterised patients and healthy 
participants 24-hour urine collections could be 
incomplete.
Ambulant, uncatheterised patients and healthy 
participants will receive careful education and 
written instructions of the importance of 
complete urine collections. In addition, 
documentation of incomplete collection, in 
addition to sampling collection times, will be 
recorded.

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Phlebotomy for biomarkers (healthy participants)
Phlebotomy can be painful and result in bruising, 
bleeding and puncture site infection.
Phlebotomy from thermally injured participants
will not be conducted as a part of this protocol.
Healthy participants will undergo one blood draw 
during screening and a further draw on day one. 
Phlebotomy will be performed by an 
appropriately trained member of the site study 
team with aseptic non-touch technique to avoid 
infection.

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3.3.2.
Benefit Assessment
Study participants will not benefit directly from involvement in this study. However, the 
results of this study may contribute significantly to our understanding of changes in 
intestinal permeability and their relationship to morbidity and mortality in the context of 
thermal injury. This knowledge is paramount to designing future medicinal interventional 
studies, aiming to modulate intestinal permeability and, potentially, to improve outcomes 
for patients following thermal injury.
3.3.3.
Overall Benefit:Risk Conclusion
The primary outcome measure of this study is the determination of intestinal permeability 
in healthy and thermal injury participants. Interventions in this study are the 
administration of STM by mouth or feeding tube (if one is site for routine clinical care) 
and the collection of urine and stool samples.
The risk of adverse events is minimised for the population being investigated in the 
proposed study as no drug intervention will be investigated and study assessments being 
conducted are non-invasive (with the exception of STM administration and phlebotomy 
in healthy participants).
4.
OBJECTIVES AND ENDPOINTS
Objective
Endpoint
Co-Primary
1.
To determine the impact of thermal injury 
on the magnitude of small intestinal 
permeability change as soon as possible 
following injury compared to healthy 
participants

Lactulose/Mannitol (L/M) ratio at entry
2.
To characterise the effect of thermal 
injury on small intestinal permeability over 
time and establish the clinical and 
demographic factors which can influence 
it

Changes in L/M ratio over time
Exploratory
1.
To determine the impact of thermal injury 
on colonic permeability as soon as 
possible following injury compared to 
healthy participants

Fractional excretion of sucralose at entry
2.
To characterise the effect of thermal 
injury on colonic permeability over time

Changes in the fractional excretion of 
sucralose over time

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Objective
Endpoint
3.
To assess the relationship between 
severity of a participant’s condition 
following thermal injury and changes in 
intestinal permeability†

Number of ventilator-free days

Number of vasopressor-free days

Number of hemofiltration-free days

Number of episodes of confirmed infection 
and sepsis

Number of surgical interventions

Total length of hospital stay

Calculate critical care and thermal injury 
severity scores
4.
To assess plasma and urine biomarkers 
of intestinal permeability, bacterial 
translocation and renal tubular 
dysfunction following thermal injury†

Change in markers of intestinal mucosal 
damage samples from blood

Change in urine protein:creatinine and urine 
albumin:creatinine ratios
5.
To assess the impact of thermal injury 
and intestinal permeability on the 
intestinal microbiome compared to 
healthy participants

Changes in microbiome of acute and 
convalescent stool samples
6.
To assess the impact of pre-existing co-
morbid conditions on intestinal 
permeability and clinical outcome 
following thermal injury†

Medical history and drug history at the time of 
admission
7.
To assess wound healing

Time to wound recovery (e.g. 95%)
8.
To characterise parameters that may 
influence drug PK/PD†

Determine fluid input/output balance over time

Changes in serum albumin and plasma 
creatinine
9.
To characterise intestinal microbiota, and 
correlate its composition with both 
intestinal permeability and bacterial 
detection in blood†

Changes in intestinal microbiome

Bacterial markers of translocation
†Clinical data, routine laboratory results or blood/urine biomarker results obtained from 
the SIFTI-2 study will be used in the analysis of this exploratory endpoint see Section 
5.4.1.

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5.
STUDY DESIGN
5.1.
Overall Design
This is a longitudinal, prospective study of healthy participants and participants who have 
sustained a thermal injury. The following schematic summarises study measures and their
frequency for healthy and thermal injury participants.
Figure 2
Study Schematic
5.2.
Number of Participants
Table 1 describes the number of participants proposed for the study. Sample size is based 
on feasibility. No formal calculation of power or sample size has been performed, but a 
sample size of 15 healthy participants and 25 thermal injury participants (≥15% TBSA) 
should be sufficient to provide useful estimates of variability in lactutose:manitol ratios, 
and any change in L/M ratio over time.

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Table 1
Recruitment Stratification
Number of participants
Group 1
Healthy participants
15
Group 2
Thermal Injury participants
Percent Total Burn Surface Area (TBSA)
15%
25
The healthy participants (Group 1) will be recruited with an age range similar to that 
typical in thermal injury participants based on historic hospital admission data from the 
UK and data from the SIFTI1 study [Hampson, 2016].
If participants prematurely discontinue the study, additional replacement participants may 
be recruited at the discretion of the Sponsor in consultation with the investigator.
5.3.
Participant and Study Completion
The full duration of the study for healthy participants is approximately two week and for 
thermal injury participants is approximately 6 months.
Thermal injury participants who withdraw prior to week 4 or healthy participants who 
withdraw prior to week 2 will be considered for analysis, although it is acknowledged 
that any missing data at later stages of the study may be related to outcome (either 
positive or negative). Given this is an exploratory study, the impact of missing data will 
be explored by assessing the sensitivity of results to different missing data approaches 
(for example, analyse all available data, analyse only complete data across time points 
and explore imputation of worst or best case scenarios).
Study withdrawals may also include participants who are consented to the study under 
Section 30 of the Mental Capacity Act 2005. In the event that participants do not re-
confirm consent when they regain capacity, they will be withdrawn from the study.
The end of the study is defined as the date of the last visit of the last participant in the 
study.
5.4.
Scientific Rationale for Study Design
5.4.1.
Co-recruitment to the SIFTI-2 Study
Thermally injured participants who are eligible for this study must also be eligible for,
and enrolled in a partner study named SIFTI-2.
SIFTI-2 is an observational study currently recruiting healthy participants and thermally 
injured participants and follows a successfully delivered predecessor study SIFTI1

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[Hampson, 2016]. The objectives and endpoints of SIFTI-2 are included in the SIFTI-2 
study protocol (reference number IRAS ID: 200366). The design of this study has been 
aligned with the SIFTI-2 study to support the strategy of co-consenting thermally injured 
participants to both studies. This will reduce the overall impact of research in this 
population in the following ways:

There is sufficient residual blood from collection in SIFTI-2 to allow testing of 
blood biomarkers of interest for the HESTIA study. This strategy therefore limits 
impact on participants as no additional blood sampling is required for 
participation in HESTIA (with the exception of HIV, Hepatitis B and C testing at 
baseline). SIFTI-2 participants will be explicitly consented for their samples and
data to be shared in this way.

Sampling time points and study visits in SIFTI-2 and HESTIA have been aligned 
to reduce the impact of co-recruitment on thermally injured participants.
Clinical data from both studies can be generated from the same participant therefore 
allowing biomarker, microflora and intestinal permeability data to be compared. In 
contrast to thermally injured participants, healthy participants enrolling in this study will 
not be required to co-consent for participation in SIFTI-2.
Data will be shared from the SIFTI-2 study with GSK through a secure electronic 
database.
A summary of the origin (HESTIA or SIFTI-2) of data and samples collected for the 
HESTIA study is available in Appendix 5 Section 12.5.
5.4.2.
Recruitment and Sampling Schedule
Severely burned patients (with an injury affecting greater than 20% total body surface 
area), display a significant and rapid increase in intestinal permeability that has been 
shown to decline over time (Figure 3) [Olquin, 2005]. What is less well understood is 
whether a greater severity of thermal injury correlates with greater intestinal 
permeability. Moreover, the time to complete restoration of normal permeability and 
other factors which may influence permeability (other than the initial injury) are also not 
well understood.

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Figure 3
Burn Injury Results in a Rapid Increase in Intestinal Permeability
This study aims to recruit participants as soon as possible following their admission in 
order to capture the initial changes in permeability. Serial measurement of intestinal 
permeability and sampling of the biomarkers of bacterial translocation, intestinal damage 
and inflammation are required during the acute phase (days 1-14) of admission in order to 
correlate them with clinical events (e.g. surgery), severity scores and clinical outcomes.
The convalescent time points (28 days and 6 months) are required to determine if 
intestinal permeability has returned to normal and to correlate observed changes on days 
1-14 with longer-term clinical outcomes (e.g. wound healing).
Gut microbiome assessments will be made less frequently than intestinal permeability 
assessments as changes in the microbiome are predicted to evolve more slowly. Ideally a 
stool sample will be collected from thermal injury participants at study entry (limited, of 
course, by when participants first pass stool following admission). A convalescent sample 
is requested to assess if the gut microbiome is able to restore to a more normal 
composition (and will be compared with that of healthy participants to make that 
assessment).

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Blood samples (with the exception of HIV, Hepatitis B and Hepatitis C testing at 
baseline) will not be taken from thermally injured participants during this study. Instead, 
biomarker data from blood samples taken during the SIFTI-2 study will be used. A single 
blood draw will be required from healthy participants on day 1 of participation.
5.4.3.
Inclusion of Healthy Participants
Patient facing material (i.e. poster) will be used to facilitate recruitment of the healthy 
participants. Healthy participants will be recruited to this study to provide a baseline for 
endpoint measures on intestinal permeability and the gut microbiome.
Three measurements of intestinal permeability are required in order to define an average 
given the variability in healthy participants reported previously. The timing of the 
replicates follows the intense time course of the study to control for day-to-day variation 
over a 15 day period.
5.4.4.
Preliminary Data Review
A preliminary review of the initial healthy participant’s data will be performed to explore 
the practicality of intestinal permeability measurement and laboratory quantification of 
STM in urine and will also explore L/M ratio data at entry and over the time course of the 
study to assess whether the variability in L/M ratio is similar to that upon which the study 
was based.  If the variability is much greater than expected, the number of subjects 
recruited may be increased.
The Reporting and Analysis Plan (RAP) will describe the planned preliminary review in 
greater detail.
5.4.5.
The Use of the Sugar Test Materials (Lactulose, Mannitol and 
Sucralose)
As described in Section 3.2.2, lactulose, mannitol and sucralose will be administered to 
both thermally injured participants and healthy participants to measure permeability of 
the small and large intestine. The amount of each of the sugars to be used is based on 
previous successful clinical studies employing this method and balances having enough 
sugar present for detection in urine with their potential laxative effect (Del Valle-Pinero, 
2013; Doig, 1998; Menzies, 1972).
6.
STUDY POPULATION
The study population will comprise healthy and thermal injury participants presenting at 
enrolling study sites. Please note the following:

Prospective approval of protocol deviations to recruitment and enrolment criteria, 
also known as protocol waivers or exemptions, is not permitted.

Where possible, written informed consent will be obtained from each subject prior 
to participation in this study. Recruitment of subjects who lack mental capacity is 
discussed in Section 6.1.

Healthy participants will be consented to the HESTIA study only. 

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
Thermally injured participants are required to be co-consented to the SIFTI-2 and 
HESTIA studies outlined in Section 6.1. A diagrammatic overview of the SIFTI-2 
and HESTIA thermal injury participant recruitment is given in Figure 4. (see 
Appendix 3).
6.1.
Recruiting Participants with Differing Mental Capacity
Following evaluation of capacity (as outlined in the Mental Capacity Act (MCA) 2005), 
thermally injured participants enrolled in this study will fall into the following groups:
6.1.1.
Adult Participants Determined to Have Mental Capacity at Study 
Entry and Throughout the HESTIA Study
Those participants who present with capacity and meet study entry criteria will be 
provided with a Participant Information Leaflet (PIL) outlining the study. If the 
participant agrees to consent to the HESTIA study following a discussion with the 
research team, they will be asked to sign a consent form.
Given the short (24 hour) window for recruitment, those patients who are acutely 
unwell will initially be presented with an abbreviated PIL. Once stable, this will be 
followed by the standard information leaflet and re-confirmation of consent.
6.1.2.
Adult Participants Lacking Mental Capacity for the Duration of 
the HESTIA Study
It is anticipated that some subjects who meet eligibility criteria for this study will not 
be able to give informed consent due to their medical condition or its management 
(e.g. sedation, opioid analgesia, intubation). In such cases, participants may be 
enrolled in the study in accordance with Section 30- Section 34 of the MCA 2005. 
The decision to enrol the participant will be discussed with a legally acceptable 
representative (LAR) (also termed a ‘consultee’). This decision may or may not be 
witnessed by an independent witness according to the decision of the principal 
investigator.
6.1.3.
Adult Participants Lacking Mental Capacity (either at Study Entry 
or During the Study) Who Later Regain Capacity and Are
Required to Provide Informed Consent
As soon as is practically possible following a participant regaining capacity, 
participants will be asked to provide informed consent to remain in the study. If they 
decline, then they will be withdrawn from the study as soon as it is safe to do so 
(likely immediately given the design of this study). Samples and data collected prior 
to study withdrawal may be retained. The participant will be asked about this at the 
point of study withdrawal.

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6.1.4.
Adult Participants with Mental Capacity to Provide Consent at
Study Entry Who are Later Deemed No Longer to have Mental 
Capacity
The decision for the participant to remain in the study will be discussed with a LAR 
and recorded. If the participant subsequently regains capacity again, they will be 
asked to re-consent to study participation.
When considering enrolment of participants who lack the mental capacity to consent, the 
following should be noted:
Section 3.2.1 of the SIFTI-2 protocol describes the consent process for that study
in detail and should be read in conjunction with this protocol. Please note that the 
SIFTI 2 protocol refers to a LAR as the Patient’s Personal Consultee (PC) or 
Nominated Consultee (NC).

A Study Information Leaflet will be provided to the LAR outlining the HESTIA 
trial before being asked to sign a form supporting the participant’s enrolment in 
the study.

The investigator and/or the site’s IEC/IRB have responsibility for acting in 
accordance with the MCA 2005 in the matter of assessing who has the capacity to 
consent and who qualifies as a LAR of a potential subject. The investigator will 
also decide if an independent witness is required.

Further information regarding the assessment of mental capacity and the 
appointing of LARs/PCs/NCs is given in Appendix 3 (Section 12.3.2 and Section 
12.3.3).

If a patient loses mental capacity subsequent to their consent and enrolment to the 
HESTIA study, samples and data collected prior to loss of capacity will be 
retained even if approval of continued study participation by a LAR is declined.
6.2.
Inclusion Criteria for Healthy Participants (Group 1)
1.
Males or Females must be ≥18 years of age at the time of signing informed consent.
2.
Participants who are healthy as determined by the investigator following medical 
evaluation including medical history, physical examination, and laboratory tests
(these are listed in Appendix 2).
3.
Female participants:
A female participant is eligible to participate if she is not pregnant (negative 
pregnancy testing at screening or Day 1 as needed) and not breastfeeding.
4.
Capable of giving signed informed consent as described in Appendix 3 which
includes compliance with the requirements and restrictions listed in the informed 
consent form (ICF) and in this protocol.
6.3.
Inclusion Criteria for Thermal Injury Participants (Group 2)
Thermal injury participants are eligible to be included in the study only if all of the 
following criteria apply:

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6.3.1.
Age
1.
Participant must be ≥18 years of age.
6.3.2.
Type of Participant and Disease Characteristics
2.
Participants who have sustained a burn (thermal injury) with a TBSA ≥ 15%.
6.3.3.
Other Inclusions
3.
Admission to the burn centre (study site) ≤24 hours of injury.
4.
Able to take enteral fluids either orally or via a nasogastric tube (depends on facial 
burn damage).
6.3.4.
Gender
5.
Male and female.
a. Female participants: 
A female participant is eligible to participate if she is not pregnant (negative 
pregnancy testing at study entry) and not breastfeeding.
6.4.
Exclusion Criteria for Healthy Participants (Group 1)
1.
Healthy participants are excluded from this study if they are receiving anti-
coagulation therapy.
2.
Pregnancy or breastfeeding.
3.
A body mass index >34kg/m2
4.
An active history of alcohol dependency
5.
History of sensitivity to any of the STM, or components thereof or a history of drug 
or other allergy that, in the opinion of the Investigator and/or GSK Medical Monitor, 
contraindicates their participation.
6.
A positive pre-study Hepatitis B surface antigen or positive Hepatitis C antibody and 
confirmatory Hepatitis C PCR result within 3 months of screening.
7.
A positive pre-study urine drug/alcohol screen.
8.
A positive test for HIV antibody.
9.
Participants unable to swallow large capsules (the capsules will be shown to 
participants at screening).
10. Galactosaemia or severe lactose intolerance.
11. Use of an antibiotic 2 weeks prior to study start (i.e. administration of the STM).
12. Gastroenteritis in the 2 weeks prior to study start (i.e. administration of the STM).
6.5.
Exclusion Criteria for Thermal Injury Participants (Group 2)
Thermal injury participants are excluded from the study if any of the following criteria 
apply:

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Medical Conditions
1.
Chemical or electrical burn.
2.
Multiple traumatic injuries with an Injury Severity Score (ISS) ≥16 (note: excludes 
burn in score system).
Prior/Concomitant Therapy
3.
Patient received substantial undocumented management prior to arrival at the study 
site (burn centre) e.g. from paramedics or in a local accident and emergency 
department.
4.
Systemic corticoidsteroid use.
5.
Intravenous (IV) mannitol use.
Prior/Concurrent Conditions
6.
Human immunodeficiency virus (HIV) infection.
7.
Gastrointestinal disease (e.g. inflammatory bowel disease) which may affect 
intestinal permeability.
8.
Previous bowel resection (e.g. hemicolectomy, small bowel resection)
9.
Galatosaemia or severe lactose intolerance.
10. Bowel obstruction.
11. Renal dysfunction requiring renal replacement therapy (i.e. end-stage renal failure 
prior to thermal injury).
12. Active autoimmune disease and receiving immunomodulatory therapy e.g.
rheumatoid arthritis anti-TNF.
13. Active chemotherapy for cancers or immunoremittive therapies (prednisolone, 
adalimumab) within 60 days of thermal injury.
14. Premorbid conditions of malignancy currently under treatment.
15. Previous bilateral lower extremity amputation.
Diagnostic assessments
16. Decision not to treat the patient due to futility.
6.6.
Lifestyle Restrictions
6.6.1.
Meals and Dietary Restrictions

Participants will be fasted (or feed stopped) for 3 hours prior to STM administration
and for 3-5 hours afterwards. For thermally injured participants these fasts should be 
aligned with those required for routine clinical care (feed holds, before surgical 
interventions) where possible.

Refrain from consumption of the following for 24 hours before and after the 
administration of STM:

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o
Foods/drinks/medicines and other products which contain sucralose, lactulose or 
mannitol as artificial sweeteners.
N.B. If cannot be avoided, then clear documentation of its administration is required and 
the current test to stop. If urine samples have been collected PRIOR to 
administration of the drug, then these can still be sent for analysis
6.6.2.
Alcohol/Exercise/Aspirin (Healthy Participants only)
Alcohol, aspirin and vigorous exercise [Sequeira, 2014] are all known to cause transient 
increases in intestinal permeability. Healthy participants will therefore be requested to 
avoid alcohol, aspirin and physical exercise for 48 hours before taking the STM and for 
the 24-hour urine collection period.
6.7.
Screen Failures
There will be no screening period for thermal injury participants. Screening will be up to
28 days before Day 1 for healthy participants.
Screen failures are defined as participants who consent to participate in the clinical study 
but are not subsequently entered in the study. A minimal set of screen failure information 
is required to ensure transparent reporting of screen failure participants to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes 
demography, screen failure details, eligibility criteria, and any serious adverse events 
(SAEs).
Individuals who do not meet the criteria for participation in this study (screen failure) 
may be rescreened. Rescreened participants should be assigned the same participant 
number as for the initial screening.
7.
NON-INVESTIGATIONAL MEDICINAL PRODUCT (NIMP)
A study treatment is defined as any investigational treatment(s), marketed product(s), 
placebo, or medical device(s) intended to be administered to a study participant according 
to the study protocol. According to this definition, no GSK study treatment will be 
employed in this study.
The STM (comprising lactulose, mannitol and sucralose) will be intermittently 
administered enterally as a study challenge agent to measure permeability at different 
points along the GI tract. Lactulose and Mannitol assess small intestine permeability and 
sucralose to assess colonic permeability.
It is important to note that the administration of the STM is not therapeutic (lactulose and 
mannitol can be used as laxatives, however the amount in the STM is sub-therapeutic) 
and as such should be regarded as a non-investigational medicinal product (NIMP)
[Guidance Documents Applying Investigational Medicinal Products (NIMPS), 2011].

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7.1.
STM Administered
Study 
Treatment 
Name
Lactulose
(4-o-β-D-
galactopyranosyl-D-
fructofuranose)
Mannitol
(D-mannitol)
GRAS listed
Sucralose 
(1,6-Dichloro-1,6-dideoxy-β-
D-fructofuranosyl-4-chloro-
4-deoxy-α-D-
galactopyranoside)
Dosage 
formulation
oral solution
oral solution
Capsules (powder)
Unit dose 
strength(s) 
Adults
5g
2g
2g (3 capsules to deliver 
total 2g sucralose)
Route of 
Administration
Oral / nasogastric / 
nasojejunal
Oral / nasogastric / 
nasojejunal
Oral / nasogastric / 
nasojejunal (capsules to 
be opened and contents 
added to lactulose and 
mannitol for tube 
administration)
Preparation
and Dosing 
instructions
For oral administration, the lactulose and mannitol will be prepared as a 
100ml drink to be taken with 3 sucralose capsules.
For feeding tube administration, lactulose/mannitol/sucralose will be prepared 
as a 50ml solution and given via a feeding tube followed by an immediate 
50ml drinking water flush
Preparation refer to Study Reference Manual together with SoA tables
(Section 2)
Packaging and 
Labelling
Lactulose and Mannitol will be supplied pre-
mixed in an amber bottle (or equivalent) for 
single use. Each container will be labelled as 
required per country requirement.
Sucralose will be provided 
as capsules in a storage 
container. Each container 
will be labelled as 
required per country 
requirement.
Manufacturer
Tayside Pharmaceuticals, UK
Storage
Lactulose/Mannitol formulation should be stored under refrigerated conditions. 
The sucralose capsules should be stored at room temperature in a dry 
environment away from direct sunlight.
Shelf-life
Lactulose/Mannitol pre-mix formulation and sucralose capsules supplied by 
Tayside Pharmaceuticals will have at least 3 month shelf-life when stored at 
the correct storage conditions.

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The preparation of the STM for oral use and nasogastric/nasojejunal tube administration
can be found in the Study Reference Manual.
7.2.
Dose Modification
Dose modification will not be required. Unit dose is described in Section 7.1.
7.3.
Method of STM Administration: Treatment Assignment
There is no element of randomisation in the study and all study participants will receive 
the STM according to the relevant SoA. The method of administration can be found in 
the Study Reference Manual.
7.4.
Blinding
No GSK study treatment will be employed in this study. All participants will receive the 
same STM and all thermal injury participants will perform the same study procedures.
7.5.
Preparation/Handling/Storage/Accountability
1.
The investigator or designee must confirm appropriate temperature conditions 
have been maintained during transit for all STM received and any discrepancies are 
reported and resolved before use of the STM.
2.
Only participants enrolled in the study may receive STM and only authorized site 
staff may supply or administer STM unless adequate training is provided such as in 
the case of healthy participants. All STM must be stored in a secure, temperature
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 STM accountability, reconciliation, and record 
maintenance, as needed.
4.
Further guidance and information for the final disposition of unused STM are 
provided in the Study Reference Manual.
5. Under normal conditions of handling and administration, STM is not expected to pose 
significant safety risks to site staff.
7.6.
STM Compliance

When participants undergo intestinal permeability testing at the site, they will 
receive STM directly from the investigator or designee, under medical supervision. 
The date and time of each dose administered in the clinic will be recorded in the 
source documents. The dose of STM and study participant identification will be 
confirmed at the time of dosing by a member of the study site staff other than the 
person administering the STM.

If healthy participants need to prepare and administer the STM off-site such as at 
home, STM training will be provided and a record maintained by the investigator or 
designee.

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7.7.
Concomitant Therapy

Refrain from consumption of the following for 24 hours before and after the 
administration of STM:
o
Lactulose or mannitol-containing laxatives. Study sites will be asked to use 
movicol (polyethylene glycol) in place of lactulose.
o
Medicines with mannitol as an excipient (chlorthiazide sodium, some albumin 
preparations, some laxatives, tablets as a bulking agent).
o
Products containing sucralose.

For healthy participants only, refrain from consumption of aspirin for 48 hours
before taking the STM and for the 24-hour urine collection period see Section 6.6.2.

For healthy participants only, antibiotic use 2 weeks prior to STM administration and 
during the study is not permitted.

Sennoside laxatives should be avoided. These can cause gastrointestinal irritation 
and may contribute to raised intestinal permeability.

Additional Glutamine supplementation in excess of that delivered with a standard 
feeding protocol should be avoided during the first 28 days of study participation. If 
supplementation is given inadvertently, then the patient will remain in the study, but 
the total dose and duration of additional glutamine supplementation must be recorded 
in the CRF.

Thermal injury participants that receive Intravenous (IV) mannitol for renal failure or 
raised intracranial pressure (testing to be delayed until 12 hours after last 
administration).
7.8.
Treatment after the End of the Study
There will be no ongoing STM administration following the end of this study.
8.
DISCONTINUATION CRITERIA
8.1.
Discontinuation of STM administration
Discontinuation of STM administration can be considered by the investigator in the event 
that an adverse event to the STM is observed. Withdrawal of further STM administration
does not require withdrawal from the study.
8.2.
Withdrawal from the Study

A participant may withdraw from the study at any time at his/her own request, 
or may be withdrawn at any time at the discretion of the investigator for safety, 
behavioural, compliance or administrative reasons.

If the participant withdraws consent for disclosure of future information, the 
sponsor may retain and continue to use any data collected before such a 
withdrawal of consent.

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
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.
8.2.1.
Other Withdrawal Criteria

A participant will be withdrawn from the study following positive HIV test at 
screening; Serologic evidence of Hepatitis B (HB) infection based on the results 
of testing for HBsAg, and anti-HBc and positive test for Hepatitis C antibody 
confirmed by HCV RNA. If HCV RNA is not available, then the positive test 
for Hepatitis C antibody alone would be exclusionary. Results must be discussed 
with the medical monitor to withdraw the subject from the study and commence 
therapy according to local practice.

Healthy participants that are treated with antibiotics during the duration of the 
study.

Participants that experience signs and symptoms of gastro-intestinal infections
during the duration of the study.

Withdrawals related to mental capacity as described in Section 6 and Appendix 
3.
8.3.
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 study site for a 
required study visit:

The site must attempt to contact the participant and reschedule the missed visit 
as soon as possible and counsel the participant on the importance of maintaining 
the assigned visit schedule and ascertain whether or not the participant wishes to 
and/or should continue in the study.

Before a participant is deemed lost to follow up, the investigator or designee 
must make every effort to regain contact with the participant (where possible, 3 
telephone calls and, if necessary, a certified letter to the participant’s last known 
mailing address or local equivalent methods). These contact attempts should be 
documented in the participant’s medical record.

Should the participant continue to be unreachable, he/she will be considered to 
have withdrawn from the study with a primary reason of lost to follow-up.

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9.
STUDY ASSESSMENTS AND PROCEDURES

Study procedures and their timing are summarized in the SoA.

Protocol waivers or exemptions are not allowed.

Safety concerns related to the STM should must be discussed with the sponsor 
immediately upon occurrence or awareness to determine if the participant should 
continue to be administered the STM.

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 thermally injured participant’s routine clinical 
management (e.g., weight measurement) and obtained before signing of ICF may be 
used for screening or baseline purposes provided the procedure met the protocol-
specified criteria and was performed within the time frame defined in the SoA.
Procedures (the administration of STM) are not part of routine care for either healthy 
or thermal injury participants.

Healthy participants will be asked to donate a single blood sample on Day 1. No 
blood collection is specified in the SoA of this protocol for thermal injury 
participants (with the exception of HIV, Hepatitis B and Hepatitis C testing at 
baseline). The results of clinical laboratory blood tests will be recorded in the SIFTI-
2 study and the data used in this study. Likewise, blood collection for exploratory 
biomarker detection will be included in the SIFTI-2 study and the data used in this 
study.
9.1.
Efficacy Assessments
The administration of the STM is for the purpose of intestinal permeability measurement 
and is not therapeutic, therefore no efficacy will be assessed.
9.2.
Adverse Events
9.2.1.
Monitoring and reporting responsibilities
Healthy participants will be recruited to the HESTIA study alone and all AEs or SAEs 
occurring in this group should be managed according to this protocol.
Thermally injured participants recruited to this study will also be recruited to the SIFTI-2 
study. The following guidance relates only to AEs or SAEs which the investigator 
reasonably believes to be the result of a procedure or requirement unique to this (the 
HESTIA) protocol. All other AEs or SAEs will be reported and managed in accordance 
with the SIFTI-2 protocol.

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Unique procedures and requirements of HESTIA
1. The administration of STM
2. The collection of stool samples
3. Changes to standard of care for thermally injured participants:
a.
The fasts required during the measurement of intestinal permeability
b. The use of alternative laxatives to lactulose and sennosides.
The definitions of an AE or SAE for this study can be found in Appendix 4.
The investigator and any designees are responsible for detecting, documenting, and 
reporting events that meet the definition of an AE or SAE and remain responsible for 
following up AEs that are serious or that caused the participant to discontinue intestinal 
permeability measurement with the STM (see Section 8).
9.2.2.
Time Period and Frequency for Collecting AE and SAE 
Information

All SAEs will be collected from the start of unique HESTIA study procedures
until the final visit at the time points specified in the SoA (Section 2).

All AEs will be collected from the start of unique HESTIA study procedures
until the final visit at the time points specified in the SoA (Section 2).

Medical occurrences that begin before the start of unique HESTIA study 
procedures 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 within 24 
hours, as indicated in Appendix 4. The investigator will submit any updated 
SAE data to the sponsor within 24 hours of it being available.

Investigators are not obligated to actively seek AEs or SAEs in former study 
participants. However, if the investigator learns of any SAE, including a death, 
at any time after a participant has been discharged from the study, and he/she 
considers the event to be reasonably related to the STM administration or study 
participation, the investigator must promptly notify the sponsor.

The method of recording, evaluating, and assessing causality of AEs and SAEs
and the procedures for completing and transmitting SAE reports are provided in 
Appendix 4.
9.2.3.
Method of Detecting AEs and SAEs
Care will be taken not to introduce bias when detecting AEs and/or SAEs. If the 
participants are conscious, open-ended and non-leading verbal questioning of the 
participant is the preferred method to inquire about AE occurrence. For unconscious 
patients or participants not always able to provide valid verbal responses to open-ended 
questions, the investigator or designee will need to identify AEs and/or SAEs through 
relevant clinical signs and/or investigations.

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9.2.4.
Follow-up of AEs and SAEs
After the initial AE/SAE report, the investigator is required to follow proactively each 
participant at subsequent visits/contacts. All SAEs, will be followed until the event is 
resolved, stabilized, otherwise explained, or the participant is lost to follow-up (as 
defined in Section 8.3). Further information on follow-up procedures is given in 
Appendix 4.
9.2.5.
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 are met.

The sponsor has a legal responsibility to notify both the local regulatory 
authority and other regulatory agencies about the safety of the study. The 
sponsor will comply with country-specific regulatory requirements relating to 
safety reporting to the regulatory authority, Institutional Review Boards 
(IRB)/Independent Ethics Committees (IEC), and investigators.

Investigator safety reports must be prepared for suspected unexpected serious 
adverse reactions (SUSAR) according to local regulatory requirements and 
sponsor policy and forwarded to investigators as necessary.

An investigator who receives an investigator safety report describing a SAE or 
other specific safety information e.g., summary or listing of SAE) from the 
sponsor will review and then file it and will notify the IRB/IEC, if appropriate 
according to local requirements.
9.2.6.
Disease-Related Events and/or Disease-Related Outcomes Not 
Qualifying as SAEs
The following broad disease related events (DREs) are common in thermally injured 
participants and can be serious/life threatening:

Deterioration of condition.

Death (may be expected in burns of a large surface area).

Prolongation of hospital stay.

Persistent or significant disability or incapacity.
Because these events are typically associated with the disease under study, they will not 
be reported according to the standard process for expedited reporting of SAEs to GSK 
(even though the event may meet the definition of a SAE). These events will be recorded 
on the DRE page in the participant’s CRF within [the appropriate time frame agreed 
upon by the SRT for completion of DRE CRF pages]. These DREs will be monitored by
clinical study team on a routine basis.

NOTE: However, if the investigator considers that there is a reasonable 
possibility that the event was related to administration of STM or another 

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unique or required element of the study (as defined in Section 9.2.1) then the 
event must be recorded and reported as an SAE (instead of a DRE).

A comprehensive list of further thermal injury related DREs can be found in 
Appendix 4 (Section 12.4).
9.3.
Treatment of Overdose
For this study, an overdose is defined as any dose of STM greater than defined in Section
7.1. No specific treatment is recommended for an overdose and treatment is at the 
discretion of the investigator.  The GSK medical monitor must be notified promptly.
9.4.
Safety Assessments
Planned time points for all safety assessments are provided in the SoA and summarised 
here.
Safety Assessment
When conducted
Healthy Participants
Thermally injured 
participants
Laboratory tests
Screening. Only repeated 
if clinically indicated in 
the opinion of the 
investigator.
Only if clinically 
indicated.
Brief Physical Examination 
including Vital Signs 
Recording
Screening, Day 1, Day 8, 
Day 15
As a part of routine 
clinical care whilst 
admitted (not protocol 
specified). Following day 
28 and 6 month intestinal 
permeability 
measurements (if patient 
not still admitted)
Detection of AEs
Day 1, Day 8, Day 15
Throughout the study
Assessment of health status
Screening, Day 1, Day 8, 
Day 15
Not required

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9.4.1.
Physical Examinations

A brief physical examination will include, at a minimum, assessments of the 
skin, lungs, cardiovascular system, and abdomen (liver and spleen).
9.4.2.
Vital Signs

A single vital sign measurement will be obtained at each time point indicated in 
SoA Table, and will include systolic and diastolic blood pressure and heart rate. 
Any abnormalities and changes in measurements will be communicated to the 
medical monitor.

Blood pressure and pulse measurements will be assessed 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, mobile phones).

Vital signs to be taken before blood collection for laboratory tests.

Repeat or unscheduled measurements may be taken at the discretion of the 
investigator.
9.4.3.
Clinical Safety Laboratory Assessment

All study related laboratory assessments will be performed by a local laboratory.
The laboratory reports must be reviewed by the investigator, this review 
documented and both report and review are to be filed with the source 
documents. 

Refer to Appendix 2 for the list of clinical laboratory tests to be performed and 
to the SoA for the timing and frequency.

All protocol-required laboratory assessments, as defined in Appendix 2, must be 
conducted in accordance with the laboratory manual and the SoA.

If laboratory values from non-protocol specified laboratory assessments 
performed at the institution’s local laboratory require a change in participant 
management or are considered clinically significant by the investigator (e.g., 
SAE or AE), then the investigator must review the laboratory report, document 
this review, and record any clinically relevant in the AE section of the CRF.

Clinically significant abnormal laboratory findings are those which are not 
associated with the underlying disease, unless judged by the investigator to be 
more severe than expected for the participant's condition.
9.5.
Study Procedures
The following procedures will be carried out during the study. 

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9.5.1.
Fluid Balance Measurement
All fluid input and output will be recorded every 24 hours for thermally injured 
participants.
9.5.2.
Wound Healing
Assessment of wound healing will be the time to 95% wound healing. Physical 
parameters of the wound (e.g., rate of healing) will be recorded and collected as a part of 
both the HESTIA and the SIFTI-2 studies.
9.5.3.
Other Clinical Responses
To assess the relationship between severity of a participant’s condition following thermal 
injury and changes in intestinal permeability the following will be recorded and collected 
as a part of the SIFTI-2 study. Details can be found in the SIFTI-2 study protocol.

Number of ventilator-free days (ventilator start/restart/end date/time)

Number of vasopressor-free days (medication chart review)

Number of hemofiltration-free days (notes review)

Number of episodes of confirmed infection and sepsis

Number of surgical interventions

Total length of hospital stay

Calculate critical care and thermal injury severity scores
9.6.
Pharmacokinetics
PK parameters are not evaluated in this study.
9.7.
Pharmacodynamics
PD parameters are not evaluated in this study.
9.8.
Intestinal Permeability Assessments

Intestinal permeability will be determined by measuring the excretion of 
lactulose, mannitol and sucralose in urine following their enteral administration. 
It will be conducted in both healthy participants and thermally injured 
participants at the time points specified in the SoA.

The complete method for administration of STM and measurement of intestinal 
permeability is detailed in the SRM.

Urinary excretion of the orally ingested STM will be quantified using a 
technique such as capillary column gas chromatography.

Urine samples will be collected in plastic bottles for analysis. Urine collection 
will begin immediately following STM administration. Urine samples will be 
collected over 24 hours post-STM administration. Accurate collection of the 
total volume voided during this 24 hour period is critical.

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
Sample Preparation
Details of urine sample collection, processing, storage and shipping procedures are 
provided in the SRM.
9.9.
Genetics
Genetics are not evaluated in this study.
9.10.
Sample Collection for Biomarker Analysis
The following biomarkers will be collected during the study. Details of sample 
processing, storage and shipping are included in the study reference manual.
Blood, stool and urine samples will be collected and stored. Timing of analyses and 
selected biomarkers will be dependent on the results of the intestinal permeability tests 
results.
9.10.1.
Blood Biomarkers
Healthy participants

Blood samples will be taken for healthy participants recruited in this study over 
the time period specified in the SOA. Blood will be taken adhering to standard 
operating procedure from venae puncture. 

The results of blood biomarker analysis will be evaluated in this study and 
compared to measures of intestinal permeability. 
Twenty (20) ml blood will be collected on Day 1 for biomarker analysis, and 
20ml blood will be collected at screening for screening tests. The biomarkers to 
be measured may include, but are not limited to:

Markers of microbial translocation

Markers of intestinal damage

Inflammatory markers: e.g. C-Reactive Protein, Procalcitonin, cytokines 
(including TNF-a, IL-6, IL-8, IFN-g, IL-10, IL-1b, IL-12p70, IL-17, IL-4, 
IL13, IL1Ra, MIP1a, MIP1b, MIP2, GCSF, GMCSF, MCP-1, RANTES, 
HMGB1).
Thermally injured participants

The blood required for this analysis in thermally injured participants will be 
collected as a part of the SIFTI-2 (IRAS 200366) study to which all thermally 
injured participants will be co-recruited. Details of the schedule for blood 
collection and the total volume of blood collected can be found in the SIFTI-2 
study protocol.

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9.10.2.
Stool Sample Collection

Stool samples will be collected from all participants in this study over the time 
period specified in Section 2, Schedule of activities (SOA). Stool samples will 
be collected adhering to standard operating procedure. 

For thermally injured participants, the initial sample will be taken as close to 
time of injury as possible (“first stool sample produced upon admission”) and 
Day 14. Further samples will be taken on day 28 (3 days) and at month 6 (14
days).

For healthy participants, a single sample will be collected at study entry
(participants will be given a collection container at screening).
9.10.3.
Urine Sample Collection

Urine samples will be collected as a part of the measurement of intestinal 
permeability which is described in Section 9.4.

Additional urine samples will be collected from patients as part of the SIFTI-2 
study to which all thermally injured participants will be co-recruited. These will 
be used for, among other tests, the quantification of protein and microbial 
metabolites.

It is standard practice that patients admitted with burns of TBSA ≥15% will 
have a urinary catheter inserted on admission to ensure the accurate maintenance 
of fluid balance. A clean urine sample will be taken from the appropriate port on 
the urinary catheter. In patients who are not catheterised, a mid-stream urine 
(MSU) should be collected in a clean universal container where possible.

N.B. During the 24 hours following STM administration (during intestinal 
permeability measurement) urine samples must only be taken from the 5-hour of 
24-hour urine collections after the aliquots for sugar quantification have been 
taken.
Sample Preparation

Details of urine sample collection, processing, storage and shipping procedures 
are provided in the SRM.

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10.
STATISTICAL CONSIDERATIONS
All statistical analyses will be performed using SAS (SAS Institute Inc., Cary, North 
Carolina, USA), version 9.2 or higher.  Before database lock, a reporting and analysis 
plan (RAP) will be issued as a separate document, providing detailed methods for the 
analyses outlined below.  Any deviations from the planned analyses will be described in a 
RAP addendum and justified in the final integrated clinical study report.
10.1.
Hypotheses
As this is an enabling study designed to better understand the biomarkers of intestinal 
permeability and other biomarkers in participants with moderate to severe burns, the 
statistical analysis for this study will be exploratory in order to better understand the 
parameters to inform future investigational medicinal product studies.
The key factors of interest in this study are to understand (i) the nature of any differences 
at entry in intestinal permeability between healthy participants and thermal injury 
participants (ii) to understand the trajectory of changes in intestinal permeability over 
time.
The key endpoint to be explored is the lactulose:mannitol (L/M) ratio, but other 
permeability biomarkers will also be explored. The analysis approaches to address these 
questions are exploratory, but will initially be conducted as outlined in Section 10.5 and 
Section 10.6.
10.2.
Sample Size Determination
Sample size is based on feasibility. No formal calculation of power or sample size has 
been performed, but a sample size of 15 healthy participants and 25 thermal injury 
participants (>15% TBSA) should be sufficient to provide useful estimates of variability 
in lactutose:manitol ratios, and any change in L/M ratio over time.
Although the key aim is to estimate the variability and L/M ratio and assess the 
difference in L/M ratio between thermal injury and healthy participants, for illustration, a 
trial including 25 thermal injury and 15 healthy participants would have 89% power to 
detect a 3-fold difference in L/M ratio between thermal injury and healthy participants
using a 2-sided significance level of p<0.10.  This calculation uses a (log) b etween-
subject SD of 1.15, as estimated from the literature [Olquin, 2005].
10.3.
Data Analyses Consideration
In general, descriptive summaries will include number of subjects, mean, standard 
deviation, median, minimum, and maximum for continuous variables.  If data are log-
normally distributed data will be presented as number of subjects, geometric mean, 
coefficient of variation, minimum, and maximum; and percent for categorical variables. 
Summaries will present data by dose level and where appropriate, by assessment time.

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10.4.
Populations for Analyses
The Safety Population will consist of all subjects who receive at least 1 dose of STM
and have at least on post-dose safety assessment.
The Evaluable Population will consist of all subjects who are entered into the study and 
have evaluable L/M ratio measurements.
10.5.
Statistical Analyses
10.5.1.
Safety Analyses
Administration of STM is for the measurement of intestinal permeability. The safety of 
this administration is not an endpoint of this study, but will be monitored and reported.
All safety data will be presented in data listings. Subject demographics, medical history, 
and prior and concomitant medications will be summarized using descriptive statistics. 
For continuous variables, these summaries will include number of subjects, mean, 
median, standard deviation, minimum, and maximum.
For categorical variables, the summaries will include frequencies and corresponding 
percentages. No inferential hypothesis testing will be performed on the safety variables.
Adverse events will be coded using the MedDRA classification system.
For healthy participants (who are recruited only to HESTIA), STM-emergent AEs will be 
defined as any AEs, regardless of relationship to STM administration, that occur after the
first dose of STM until the final follow-up visit. The STM-emergent AEs will be 
summarized for the overall number of AEs and the percentage of subjects who 
experience them. The total number of AEs will be summarized overall. The AEs will be 
further summarized by severity and relationship to STM. If relationship information is 
missing, the AE will be considered STM-related. Listings for the subsets of SAEs and 
STM-related SAEs will be provided. The SAEs and number of AEs leading to 
discontinuation of STM administration will be summarized. The incidence of AEs will 
also be summarized by system organ class and preferred term.
For thermally-injured participants, STM-emergent AEs will be defined as any AE 
deemed related to STM administration that occurs after STM administration until the 
follow-up visit. The STM-related emergent AEs will be summarized for the overall 
number of AEs and the percentage of subjects who experience them. Listings for the 
subsets of SAEs and STM-related SAEs will be provided. The SAEs and number of AEs 
leading to discontinuation of STM administration will be summarized. The incidence of 
AEs will also be summarized by system organ class and preferred term.
As laboratory data and vital signs are only collected at screening for healthy participants, 
these data will be listed only. Clinical laboratory values that are outside of the reference 
ranges will be flagged and evaluated for clinical significance by the investigator. Physical 
examination findings will be listed. For thermally-injured participants, physical 

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examination findings and clinical laboratory values will be highly abnormal and as such 
any data collected will only be listed.  Disease-related findings and changes will not be 
reported.
10.5.2.
Other Analyses
Biomarker exploratory analyses will be described in the RAP.
10.5.3.
Interim Analyses
A preliminary review of the initial healthy participant’s data will be performed to explore 
the practicality of intestinal permeability measurement and laboratory quantification of 
STM in urine and will also explore L/M ratio data at entry and over the time course of the 
study to assess whether the variability in L/M ratio is similar to that upon which the study 
was based.  If the variability is much greater than expected, the number of subjects 
recruited may be increased.
The RAP will describe the planned preliminary review in greater detail.
10.6.
Analyses of lactulose/mannitol ratio
In all analyses the variable TBSA will be a categorical variable defined as “Yes” for 
thermally injured participants, and “No” for healthy participants.
Differences in permeability at entry
Intestinal permeability biomarkers will be summarised by TBSA group and overall.  Data 
will summarised by geometric mean, CV, minimum, maximum and N.  A model will be 
fitted, defined as
Log (L/M ratio) = intercept + TBSA
Trajectory of the L/M ratio over time
Intestinal permeability biomarkers will be summarised over time, by TBSA group and 
overall.  Data will summarise geometric mean, CV, minimum, maximum and N.  A 
model will be fitted, defined as
Log (L/M ratio at time X / L/M ratio at entry) = intercept + Time + TBSA + Time*TBSA.
This will be a repeated measurement analysis and will assess the rate of improvement in 
L/M ratio over time, and assess how this changes relative to healthy participants.  If 
required, further modelling assessing more complex relationships between L/M ratio and 
time may be undertaken.  Given this is an exploratory study the most appropriate 
variance-covariance matrix regarding the correlation of data over time will be explored as 
part of the statistical analysis.
Data from this model may also be used to estimate the time to 50% improvement (or 
other degrees of improvement) in L/M ratio in relation to L/M ratio values seen in 

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healthy participants.  This will be used to assess the clinical relevance and sensitivity of 
such measures.
A model fitting log (AUC of L/M ratio) = intercept + TBSA will also be fitted.  AUC will 
be calculated using all measurements taken over time.  This will provide a summary of 
the weighted average L/M ratio value over time.
The use of %TBSA will also be assessed in the above analyses as a continuous covariate.  
The effects of age and Baux score will also be evaluated to understand differences in 
intestinal permeability in these groups [Osler, 2010].

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11.
REFERENCES
Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, Ouellette AJ. Secretion of 
Microbicidal Alpha-Defensins by Intestinal Paneth Cells in Response to Bacteria. Nat 
Immunol 2000; 1: 113-118
Bjarnason I, MacPherson A, Hollander D. Intestinal Permeability: An Overview. 
Gastroenterology 1995; 108: 1566-1581
Deitch EA, XU D, Kaisse VL, Role of the Gut in the Development of Injury and Shock 
Induced SIRS and MODS: the Gut-Lymph Hypothesis, A Review. Front Biosci 2006; 
11:520-528
Deitch EA. Intestinal Permeability Is Increased in Burn Patients Shortly After Injury. 
British Journal of Surgery 1990; 77:587-592. 
Del Valle-Pinero A, Van Deventer H, Fourie N et al. Gastrointestinal Permeability in 
Patients with Irritable Bowel Syndrome Assessed Using a Four Probe Permeability 
Solution. Clin Chim Acta. 2013 March 15; 418: 97–101.
Derikx J, Van Waardenburg D, Granzen B et al: Detection of Chemotherapy-Induced 
Enterocyte Toxicity with Circulating Intestinal Fatty Acid Binding Protein. J Paediatr 
Hematol Oncol 2006;28:267-269
Doig CJ, Sutherland LR, Sandham JD, Fick GH, Verhoef M, Meddings JB: Increased 
Intestinal Permeability is Associated with the Development of Multiple Organ 
Dysfunction Syndrome in Critically Ill ICU Patients.  Am J Respir Crit Care Med 
1998;158:444-451
European Commission, Health and Consumers Directorate-General.  The Rules 
Governing Medicinal Products in the European Union Volume10-Guidance Documents 
Applying Investigational Medicinal Products (NIMPS) (Rev. 1,March 2011) Ref. 
Ares(2011)300458 - 18/03/2011
Grootjans J, Thuijls G, Verdam F et al. World J Gastrointest Surg 2010 March 27; 2(3): 
61-69
Hammer A, Morris N, Earley Z, Choudhry M. The First Line of Defense: The Effects of 
Alcohol on Post-Burn Intestinal Barrier, Immune Cells, and Microbiome. Alcohol Res. 
2015;37(2):209-22
Hampson P et al. Neutrophyl Dysfunction, Immature Granulocytes, and Cell Free DNA 
Are Early Biomarkers of Sepsis in Burn-Injured Patients. Annals of Surgery Ann Surg.
2016 May 26. [Epub ahead of print]
Menzies IS. Intestinal Permeability in Coeliac Disease. Gut 1972;13:847

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Olquin F, Araya M, Hirsch S, Brunser O, Ayala V, Rivera R, Gotteland M:Prebiotic
Ingestion Does Not Improve Gastrointestinal Barrier Function in Burn Patients. Burns 
2005 Jun; 31 (4):482-8
Osler T, Glance LG, Hosmer DW. Simplified estimates of the probability of death after 
burn injuries: extending and updating the baux score. J Trauma. 2010;68(3):690-7
Sequeira IR, Lentle RG, Kruger MC, Hurst RD. Standardising the Lactulose Mannitol 
Test of Gut Permeability to Minimise Error and Promote Comparability. PLoS ONE. 
2014;9(6):e99256
Ziegler TR, Smith RJ, O’Dwyer ST, Demling RH, Wilmore DW: Increased Intestinal 
Permeability Associated with Infection in Burn Patients. Arch Surg 1988 Nov; 123 
(11):1313-9

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12.
APPENDICES
12.1.
Appendix 1: Abbreviations and Trademarks
ACR
Albumin/creatinine ratio
AE
Adverse Event
Anti-HBc
Anti-Hepatitis C
ART
Anti-retrovial treatment
CFR
Code of Federal Regulations
D
Day
G
Grams
eCRF
Electronic Case Report Form
ICU
Intensive Care Unit
GCP
Good Clinical Practice
GCSP
Global Clinical Safety and 
Pharmacovigilance
GI
Gatrointestinal 
HB
Hepatitis B
HBs AG
Hepatitis B Antigen
HCV
Hepatitis C
GSK
GlaxoSmithKline
HIV
Human Immunodeficiency Virus
ICF
Informed Consent Form
ICH
International Conference on 
Harmonization of Technical Requirements 
for Registration of Pharmaceuticals for 
Human Use
ICU
Intensive Care Unit
IEC
Independent Ethics Committee

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IRB
Institutional Review Board
ISS
Investigator Sponsored Study
L/M
Lactulose/mannitol ratio
LAR
Legally Authorised Representative
mL
Milliliter
MODS
Multi-organ dysfunction syndrome
NIMP
Non-investigational medicinal product
NC
Nominated Consultee
PC
Personal Consultee
PCR
Polymerase Chain Reaction
RAP
Reporting and Analysis Plan
SAE
Serious adverse event
SIFTI-2
A Multi-centre, Prospective Study to 
Examine the Relationship between 
Neutrophil Function and Sepsis in Adults 
and Children with Severe Thermal 
Injuries
SoA
Schedule of Activities
SRM
Study Reference Manual
STM
Sugar Test Material
TBSA
Total body surface area
TNF
Tumour Necrosis Factor
WOCBP
Women of Child Bearing Potential
Trademark Information
Trademarks of the GlaxoSmithKline 
group of companies
Trademarks not owned by the 
GlaxoSmithKline group of companies
NONE
SAS

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12.2.
Appendix 2: Clinical Laboratory Tests

All clinical laboratory tests will be performed in the local laboratory.

Protocol-specific requirements for inclusion or exclusion of participants are
detailed in Section 6 of the protocol.

Additional tests may be performed at any time during the study as determined 
necessary by the investigator or required by local regulations.
Table 2
Protocol-Required Safety Laboratory Assessments for Healthy 
Participants
Laboratory 
Assessments
Parameters
Haematology
Platelet Count
WBC count with Differential:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
RBC Count
Hemoglobin
Hematocrit
Clinical 
Chemistry1
Urea
Potassium
Total bilirubin
Creatinine
Sodium
Alanine 
Aminotransferase
(ALT)/ 
Total Protein
Glucose 
nonfasting
Calcium
Alkaline 
phosphatase
Routine 
Urinalysis

pH, glucose, protein, blood, ketones by dipstick

Microscopic examination (if blood or protein is abnormal)
Other 
Screening 
Tests

Urine alcohol and drug screen (to include at minimum: amphetamines, 
barbiturates, cocaine, opiates, cannabinoids and benzodiazepines)]

Urine human chorionic gonadotropin (hCG) pregnancy test (as needed for 
women of childbearing potential)1

Serology (HIV antibody, hepatitis B surface antigen [HBsAg], and hepatitis C 
virus antibody)
NOTES :
1.
Local urine testing will be standard for the protocol unless serum testing is required by local regulation or IRB/IEC.

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12.3.
Appendix 3: Study Governance Considerations
12.3.1.
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 and other relevant documents (e.g., 
advertisements) must be submitted to an IRB/IEC by the investigator and 
reviewed and approved by the IRB/IEC before the study is initiated. 

Any amendments to the protocol will require IEC/IRB approval before 
implementation of changes made to the study design, except for changes 
necessary to eliminate an immediate hazard to study participants. 

Any amendments to the SIFTI-2 protocol which impact on this protocol will be 
reviewed and may result in changes to this protocol being required. Any such 
changes will be subject to IEC/IRB approval before implementation.

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
12.3.2.
Recruiting participants under the Mental Capacity Act 2005
On admission into hospital the patient’s capacity will be assessed. A patient may lack 
capacity due to the severity of their injury, arriving intubated and ventilated or due to a 
pre-existing co-morbidity.
Please note, the same process will also be followed for the SIFTI-2 study to which 
thermally injured participants will be co-recrruited.
If a patient does not have the capacity to make an informed decision, the research team 
will approach a patient’s LAR, also known as a Personal Consultee. Examples of 
personal consultees include:

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
A family member, carer or friend

An attorney acting under a Lasting Power of Attorney

A court appointed deputy, provided that they had a relationship with, or personal 
knowledge of, the person lacking capacity before their appointment as deputy.
There may be circumstances in which a personal consultee is not available, some 
examples of this are:

Where no family member or friend is willing to act as a personal consultee

Where the family member or friends live a long distance away and/or are not in 
frequent contact with the patient who lacks capacity

Where the regular carers of the person who lacks capacity are doing so for 
payment or in a professional capacity (e.g. care home staff or nurses)

Where someone is acting on a professional role (e.g. their GP or solicitor)
In this case, a nominated consultee will be required. A nominated consultee is considered 
to be a medical professional that has no connection to the research trial, but has an 
understanding of the implications of the research trial on the participant.
In these circumstances, examples of nominated consultees are:

An emergency department doctor, preferably Consultant level.

Intensive Care doctor, preferably Consultant level.

Doctor from the burns team, not directly involved in the research study.
Once a personal or nominated consultee has been identified, they will be provided with a 
specific information leaflet about the trial. The personal and nominated consultee will be 
asked if they feel the study would be something the participant would have no objections 
to. If in their opinion the participant would have no objection to being recruited into a 
research trial the consultee will be asked to sign a declaration form.
12.3.3.
Determining Whether a Participant has Capacity Under the 
Mental Capacity Act (2005)
Prior to deciding that a patient does not have the capacity to give informed consent the 
researcher must follow the Mental Capacity Act (2005) to ensure that the participant does 
not hold capacity. The principles of the MCA which we will adhere are as follows:

A person must be assumed to have capacity unless it is established that he/she
lacks capacity.

A person is not to be treated as unable to make a decision unless all practical steps 
to help him/her to do so have been taken without success.

A person is not to be treated as unable to make a decision merely because he/she
makes an unwise decision.

An act done or decision made, under this Act for or on behalf of a person who 
lacks capacity must be done, or made, in his best interests.

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
Before the act is done, or the decision is made, regard must be had to whether the 
purpose for which it is needed can be as effectively achieved in a way that is less 
restrictive of the person's rights and freedom of action.
A decision to appoint a consultee on behalf of a patient will be made if the participant is 
unable to:
1. (a) understand the information relevant to the decision,
(b) retain that information,
(c) use or weigh that information as part of the process of making the decision, or
(d) communicate his/her decision (whether by talking, using sign language or any 
other means).
2. A person is not to be regarded as unable to understand the information relevant to a 
decision if he/she is able to understand an explanation of it given to him in a way that is 
appropriate to his circumstances (using simple language, visual aids or any other means).
3. The fact that a person is able to retain the information relevant to a decision for a short 
period only does not prevent him/her from being regarded as able to make the decision.
4. The information relevant to a decision includes information about the reasonably 
foreseeable consequences of
(a) deciding one way or another, or
(b) failing to make the decision.

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Figure 4
HESTIA Thermal Injury Participant Recruitment

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12.3.4.
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.
12.3.5.
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.

Healthy participants who are rescreened are required to sign a new ICF.
12.3.6.
Data Protection

Participants will be assigned a unique identifier by the sponsor. Any participant 
records or datasets that are transferred to the sponsor will contain the identifier 
only; participant names or any information which would make the participant 
identifiable will not be transferred.

The participant must be informed that his/her personal study-related data will be 
used by the sponsor in accordance with local data protection law. The level of 
disclosure must also be explained to the participant.

The participant must be informed that his/her medical records may be examined 
by Clinical Quality Assurance auditors or other authorized personnel appointed 
by the sponsor, by appropriate IRB/IEC members, and by inspectors from 
regulatory authorities.
12.3.7.
Committees Structure
An Independent Data Monitoring Committee or similar review group will not be used in 
this study, but an internal preliminary data review will be conducted.

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The Data Review team will consist of the GSK medical monitor, clinical and operational 
leads, statistician, early development lead and the safety officer. They will meet at 
intervals specified within the data review charter to review data relevant to the future 
conduct of the study, and will also assess any risk to study participants.
12.3.8.
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 
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.
12.3.9.
Dissemination of Clinical Study Data

Study information from this protocol will be posted on publicly available clinical 
trial registers before enrollment of subjects begins.

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.

The procedures and timing for public disclosure of the results summary and for 
development of a manuscript for publication will be in accordance with GSK 
Policy.

A manuscript will be progressed for publication in the scientific literature if the 
results provide important scientific or medical knowledge.
12.3.10.
Data Quality Assurance

All participant data relating to the study will be recorded on printed or electronic 
CRF unless transmitted to the sponsor or designee electronically (e.g., 
laboratory data). The investigator is responsible for verifying that data entries 
are accurate and correct by physically or electronically signing the CRF.

The investigator must maintain accurate documentation (source data) that 
supports the information entered in the CRF.

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
The investigator must permit study-related monitoring, audits, IRB/IEC review, 
and regulatory agency inspections and provide direct access to source data 
documents.

The sponsor or designee is responsible for the data management of this study 
including quality checking of the data.

Study monitors will perform ongoing source data verification to confirm that 
data entered into the CRF by authorized site personnel are accurate, complete, 
and verifiable from source documents; that the safety and rights of participants 
are being protected; and that the study is being conducted in accordance with the 
currently approved protocol and any other study agreements, ICH GCP, and all 
applicable regulatory requirements.

Records and documents, including signed ICF, pertaining to the conduct of this 
study must be retained by the investigator for 15 years after study completion 
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.
12.3.11.
Source Documents

Source documents provide evidence for the existence of the participant and 
substantiate the integrity of the data collected. Source documents are filed at the 
investigator’s site.

Data reported on the CRF or entered in the eCRF that are transcribed from 
source documents must be consistent with the source documents or the 
discrepancies must be explained. The investigator may need to request previous 
medical records or transfer records, depending on the study. Also, current 
medical records must be available.

Definition of what constitutes source data can be found in the study specific 
Source Data Verification document.
12.3.12.
Study and Site Closure
GSK or its designee reserves the right to close the study site or terminate the study at any 
time for any reason at the sole discretion of GSK. Study sites will be closed upon study 
completion. A study site is considered closed when all required documents and study 
supplies have been collected and a study-site closure visit has been performed.
The investigator may initiate study-site closure at any time, provided there is reasonable 
cause and sufficient notice is given in advance of the intended termination.
Reasons for the early closure of a study site by the sponsor or investigator may include 
but are not limited to:

Failure of the investigator to comply with the protocol, the requirements of the 
IRB/IEC or local health authorities, the sponsor's procedures, or GCP guidelines

Inadequate recruitment of participants by the investigator

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12.4.
Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting
Definition of AE
AE Definition

An AE is any untoward medical occurrence in a clinical study participant, temporally 
associated with study participation, whether or not considered related to the study.

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 study participation.

NOTE: As detailed in Section 9.2.1, only AEs deemed to be related to procedures or 
requirements unique to the HESTIA study will be recorded/reported for thermally 
injured participants. All other AEs will be recorded/reported through the SIFTI-2 
study.
Events Meeting the AE Definition 

Any abnormal laboratory test results (haematology, 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 STM 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. 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.
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. Table 3
provides a list of commonly occurring AEs in participants with severe thermal injury 
which may meet this definition.

The disease/disorder being studied or expected progression, signs, or symptoms of 

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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.
Table 3
Complications of severe thermal injury which can be considered as 
associated with the underlying disease and do not require reporting 
as AEs unless judged to be more severe than expected for the 
participant’s condition or related to HESTIA study procedures.
If any of the following are observed, then an AE will be recorded in the SIFTI2 study 
CRF.
If any of the following are observed and deemed to be related to STM administration or 
other unique requirements of the HESTIA study, then to be recorded in the HESTIA CRF
and reported to GSK as per guidance below.
System Assessment
Complication type
Action
Airway problems
Failed extubation
Record AE
Tracheostomy complication
Breathing Problems
Pneumothorax
Record AE
Pulmonary Oedema
Respiratory Arrest
Pneumonia
VAP
Acute lung injury (ALI)
ARDS
Circulatory Problems
Haemodynamic instability
Record AE
Record ionotrope dose in 
con-meds
Increasing vasoactive drug 
support
Arrhythmia

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System Assessment
Complication type
Action
Endocarditis
Acute LVF/CCF
Cardiac Arrest
MI
Neurological Problems
Reduced GCS (off sedation)
Record AE
Intra-Cranial bleed
CVA
Acute confusion/Delirium
Meningitis-bacterial
Oedema Complications
Abdominal Compartment 
Syndrome (ACS)
Record AE
Acute Limb compartment 
syndrome
Microbiological problems
Sepsis 
Record AE
Record in Microbiology 
form
Chest Infection
Lower Respiratory Tract 
Infection
UTI
Bloodstream Infection (BSI)
Wound infection
Intra-vascular catheter (line) 
infection
Infective diarrhoea
Clostridium difficile 
infection/pseudomembranous 
colitis
Renal/Urology problems
Acute rhabdomyolysis
Record AE

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System Assessment
Complication type
Action
Acute renal failure
Ensure biochemistry and 
CK results recorded n CRF
Acute urinary retention
Renal replacement therapy
Thromboembolic 
complications
Lower limb DVT
Record AE with location of 
thrombus
Upper limb DVT
Pulmonary embolism
Other VTE
Fat embolism
Wound complications
Major graft loss
Record AE with details
Major skin substitute loss
Wound infection
Invasive wound infection
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).
Please note that, as described in Section 9.2.6 a, b, c and d below are considered as 
‘disease related events’ as they occur commonly in patients following thermal injury
unless, in the opinion of the investigator, they are directly related to STM administration 
or other unique requirements of the HESTIA study.
A SAE is defined as any untoward medical occurrence that:
a.
Results in death
b.
Is life-threatening
The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
participant was at risk of death at the time of the event. It does not refer to an event, 
which hypothetically might have caused death, if it were more severe.
c.
Requires inpatient hospitalization or prolongation of existing hospitalization
In general, hospitalization signifies that the participant has been detained (usually 

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involving at least an overnight stay) at the hospital or emergency ward for observation 
and/or treatment that would not have been appropriate in the physician’s office or 
outpatient setting. Complications that occur during hospitalization are AE. If a 
complication prolongs hospitalization or fulfils any other serious criteria, the event is 
serious. When in doubt as to whether “hospitalization” occurred or was necessary, the AE
should be considered serious.
Hospitalization for elective treatment of a pre-existing condition that did not worsen from 
baseline is not considered an AE.
d.
Results in persistent disability/incapacity

The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions.

This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 
and accidental trauma (e.g., sprained ankle) which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption.
e.
Is a congenital anomaly/birth defect
f.
Other situations:

Medical or scientific judgment should be exercised in deciding whether SAE 
reporting is appropriate in other situations such as important medical events that may 
not be immediately life-threatening or result in death or hospitalization but may 
jeopardize the participant or may require medical or surgical intervention to prevent 
one of the other outcomes listed in the above definition. These events should usually 
be considered serious.
Examples of such events include invasive or malignant cancers, intensive treatment 
in an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency 
or drug abuse.
Recording AE and SAE
AE and SAE Recording

When an AE/SAE occurs, it is the responsibility of the investigator to review all 
documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) 
related to the event.

The investigator will then record all relevant AE/SAE information in the CRF.

It is not acceptable for the investigator to send photocopies of the participant’s 
medical records to GSK in lieu of completion of the GSK /AE/SAE CRF page.

There may be instances when copies of medical records for certain cases are 
requested by GSK. In this case, all participant identifiers, with the exception of the 
participant number, will be redacted on the copies of the medical records before 

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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 sufficiently discomfort and interferes with normal 
everyday activities.

Severe: An event that prevents normal everyday activities. An AE that is assessed as 
severe should not be confused with an SAE. Severe is a category utilized for rating 
the intensity of an event; and both AE and SAE can be assessed as severe.
An event is defined as ‘serious’ when it meets at least 1 of the predefined outcomes 
as described in the definition of an SAE, NOT when it is rated as severe.
Assessment of Causality

The investigator is obligated to assess the relationship between administration of the 
NIMP (STM) 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 STM administration 
will be considered and investigated.

The investigator will also consult the Product Information, for marketed products, in 
his/her assessment.

For each AE/SAE, the investigator must document in the medical notes that he/she 
has reviewed the AE/SAE and has provided an assessment of causality.

There may be situations in which an SAE has occurred and the investigator has 
minimal information to include in the initial report to GSK. However, it is very 
important that the investigator always make an assessment of causality for 
every event before the initial transmission of the SAE data to GSK.

The investigator may change his/her opinion of causality in light of follow-up 
information and send an SAE follow-up report with the updated causality 
assessment.

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
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.

The investigator will submit any updated SAE data to GSK within 24 hours of 
receipt of the information.
Reporting of SAE to GSK
SAE Reporting to GSK via 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 for more than 24 hours, then the site will use 
the paper SAE data collection tool (see next section).

The site will enter the SAE data into the electronic system as soon as it becomes 
available.

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 to the medical monitor by telephone.

Contacts for SAE reporting can be found in 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.

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.

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complete and sign the SAE CRF pages within the designated reporting time frames.

Contacts for SAE reporting can be found in SRM.
12.5.
Appendix 5
The following table of study objectives specifies, for each objective, the provenance of 
clinical data and samples which will be used to explore that endpoint
Objective
Endpoint
Origin of Data/Samples
Co-Primary
1.
To determine the impact of 
thermal injury on the magnitude 
of small intestinal permeability 
change as soon as possible 
following injury compared to 
healthy participants

Lactulose/Mannitol 
(L/M) ratio at entry

HESTIA study
2.
To characterise the effect of 
thermal injury on small 
intestinal permeability over time 
and establish the clinical and 
demographic factors which can 
influence it

Changes in L/M 
ratio over time

HESTIA Study
Exploratory
3.
To determine the impact of 
thermal injury on colonic 
permeability as soon as 
possible following injury 
compared to healthy 
participants

Fractional excretion 
of sucralose at entry

HESTIA Study
4.
To characterise the effect of 
thermal injury on colonic 
permeability over time 

Changes in the 
fractional excretion 
of sucralose over 
time

HESTIA Study
5.
To assess the relationship 
between severity of a 
participant’s condition following 
thermal injury and changes in 
intestinal permeability†

Number of 
ventilator-free days

Number of 
vasopressor-free 
days

Number of 

All clinical data will be 
obtained from SIFTI2.

Permeability measurements 
will be obtained from HESTIA

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Objective
Endpoint
Origin of Data/Samples
hemofiltration-free 
days

Number of episodes 
of confirmed 
infection and sepsis

Number of  surgical 
interventions

Total length of 
hospital stay

Calculate critical 
care and thermal 
injury severity 
scores
6.
To assess plasma and urine 
biomarkers of intestinal 
permeability, bacterial 
translocation and renal tubular 
dysfunction following thermal 
injury†

Change in markers 
of intestinal 
mucosal damage 
samples from blood

Change in urine 
protein:creatinine 
and urine 
albumin:creatinine 
ratios

Blood biomarkers obtained 
from SIFTI-2 (and HESTIA 
for healthy participants)

Urine for microbial metabolite 
analysis, claudin 3 and KIM 1
obtained from SIFTI-2 (and 
HESTIA for Healthy 
participants)

Urine albumin:creatinine and 
protein:creatinine ratios 
obtained from SIFTI-2 (and 
HESTIA for healthy 
participants)

Permeability data (STM 
absorption) from HESTIA
7.
To assess the impact of 
thermal injury and intestinal 
permeability on the intestinal 
microbiome compared to 
healthy participants

Changes in
microbiome of acute 
and convalescent 
stool samples

Stool samples collected in 
HESTIA protocol

Permeability data from 
HESTIA
8.
To assess the impact of pre-
existing co-morbid conditions 
on intestinal permeability and 
clinical outcome following 
thermal injury†

Medical history and 
drug history at the 
time of admission

Medical History data from 
HESTIA

Permeability data from 
HESTIA

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Objective
Endpoint
Origin of Data/Samples
9.
To assess wound healing

Time to wound 
recovery (e.g. 95%)

Wound healing assessment 
data from clinical notes will 
be captured in HESTIA at 14 
day, 28 day and 6 month 
visits.
10. To characterise intestinal 
microbiota, and correlate its 
composition with both intestinal 
permeability and bacterial 
detection in blood†

Changes in 
intestinal 
microbiome

Bacterial markers of 
translocation

Microbiome data from 
HESTIA study

Blood Biomarker data from 
SIFTI-2 (thermally injured 
participants) and HESTIA 
(healthy participants.

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TITLE PAGE
Protocol Title: A Prospective, Longitudinal Study to Investigate the Effect of Thermal
Injury on Intestinal Permeability and Systemic Inflammation (HESTIA)
Protocol Number: 206243/01
Short Title: A Prospective, Longitudinal Study to Investigate the Effect of Thermal 
Injury on Intestinal Permeability and Systemic Inflammation (HESTIA)
Compound Number:
NONE
Sponsor Name and Legal Registered Address: 
GlaxoSmithKline Research & Development Limited
980 Great West Road
Brentford
Middlesex, TW8 9GS
UK
Medical Monitor Name and Contact Information will be provided separately.
Regulatory Agency Identifying Number(s): NA
Approval Date: 15-AUG-2017
Copyright 2017 the GlaxoSmithKline group of companies.  All rights reserved.  
Unauthorised copying or use of this information is prohibited.

P P D
P P D
P P D

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PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE
DOCUMENT HISTORY
Document
Date
Amendment 1
15-Aug-2017
Original Protocol
02-Mar-2017
Amendment 1 15-Aug-2017
Overall Rationale for the Amendment: The interim analysis has been removed since 
lactulose and mannitol data in healthy controls was unlikely to change the required 
sample size in thermally injured participants.

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Section # 
and Name
Description of Change
Brief Rationale
10.5.3 Interim 
Analysis
Removed preliminary review of 
lactulose/mannitol ratio data.
Analysis of lactulose and mannitol data in 
healthy controls would be unlikely to 
change the study size, and will no longer 
be performed.
5.2 Number of 
Participants
Included withdrawn participants 
may be replaced.
To ensure the target number of evaluable 
population is maintained.
5.4.4
Preliminary 
Data Review
Modified the preliminary data 
review.
To describe the change in preliminary data 
review.
Synopsis
Updated preliminary data review.
For consistency.
6.5 Exclusion 
Criteria
Revision to eligibility criteria to 
exclude hepatitis infection for 
thermally injured participants.
Addition the personal consultee will 
not be notified if a patient lacking 
mental capacity was found to be 
HIV positive.
Consistency correction. Hepatitis screening 
will be performed.
To provide Ethic Committee reassurance 
and address any ambiguity of this 
information.
8.2.1 Other 
Withdraw 
Criteria
Added the withdraw of participants 
receiving hemodialysis.
Renal replacement therapy (i.e. dialysis) 
may filter out lactulose and mannitol.
9.2.2 Time 
Period and 
Frequency for 
Collecting AE 
and SAE 
Information
Added:
Any SAEs assessed as related to 
study participation will be recorded 
from the time a participant 
consents.
All SAEs will be recorded and 
reported to the sponsor or designee 
immediately and under no 
circumstance should this exceed 24 
hours.
To align with the updates to Clinical 
Protocol Template (Vol 7) effective 7th April 
2017.
9.4 Clinical 
Safety 
Laboratory 
Assessments
Removal of if laboratory values 
from non-protocol specified 
laboratory assessments performed 
at the institution’s local laboratory.
To align with the updates to Clinical 
Protocol Template (Vol 7) effective 7th April 
2017.
Appendix 4: 
Adverse 
Events: 
Reporting SAE to GSK if the 
electronic system is unavailable.
To align with the updates to Clinical 
Protocol Template (Vol 7) effective 7th April 

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Section # 
and Name
Description of Change
Brief Rationale
Definitions 
and 
Procedures 
for Recording, 
Evaluating, 
Follow-up, 
and Reporting
2017.

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TABLE OF CONTENTS
PAGE
PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE.....................................3
1.
SYNOPSIS...............................................................................................................9
2.
SCHEDULE OF ACTIVITIES (SOA).......................................................................12
2.1.
Healthy Participants (Group 1)....................................................................12
2.2.
Thermal Injury Participants (Group 2) .........................................................14
3.
INTRODUCTION....................................................................................................17
3.1.
Study Rationale ..........................................................................................17
3.2.
Background ................................................................................................17
3.2.1.
Thermal Injury, Intestinal Permeability and Multi-Organ 
Dysfunction Syndrome.................................................................17
3.2.2.
Intestinal Barrier Function and Its Measurement..........................18
3.2.3.
The Intestinal Microbiome and Thermal Injury .............................19
3.3.
Benefit/Risk Assessment ............................................................................20
3.3.1.
Risk Assessment .........................................................................20
3.3.2.
Benefit Assessment.....................................................................24
3.3.3.
Overall Benefit:Risk Conclusion...................................................24
4.
OBJECTIVES AND ENDPOINTS...........................................................................24
5.
STUDY DESIGN ....................................................................................................26
5.1.
Overall Design ............................................................................................26
5.2.
Number of Participants ...............................................................................26
5.3.
Participant and Study Completion...............................................................27
5.4.
Scientific Rationale for Study Design ..........................................................27
5.4.1.
Co-recruitment to the SIFTI-2 Study ............................................27
5.4.2.
Recruitment and Sampling Schedule...........................................28
5.4.3.
Inclusion of Healthy Participants..................................................30
5.4.4.
Preliminary Data Review..............................................................30
5.4.5.
The Use of the Sugar Test Materials (Lactulose, Mannitol 
and Sucralose) ............................................................................30
6.
STUDY POPULATION...........................................................................................30
6.1.
Recruiting Participants with Differing Mental Capacity ................................31
6.1.1.
Adult Participants Determined to Have Mental Capacity at 
Study Entry and Throughout the HESTIA Study...........................31
6.1.2.
Adult Participants Lacking Mental Capacity for the 
Duration of the HESTIA Study .....................................................31
6.1.3.
Adult Participants Lacking Mental Capacity (either at 
Study Entry or During the Study) Who Later Regain 
Capacity and Are Required to Provide Informed Consent............31
6.1.4.
Adult Participants with Mental Capacity to Provide 
Consent at Study Entry Who are Later Deemed No Longer 
to have Mental Capacity ..............................................................31
6.2.
Inclusion Criteria for Healthy Participants (Group 1) ...................................32
6.3.
Inclusion Criteria for Thermal Injury Participants (Group 2).........................32
6.3.1.
Age..............................................................................................32

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6.3.2.
Type of Participant and Disease Characteristics..........................32
6.3.3.
Other Inclusions...........................................................................33
6.3.4.
Gender ........................................................................................33
6.4.
Exclusion Criteria for Healthy Participants (Group 1) ..................................33
6.5.
Exclusion Criteria for Thermal Injury Participants (Group 2)........................33
6.6.
Lifestyle Restrictions...................................................................................35
6.6.1.
Meals and Dietary Restrictions ....................................................35
6.6.2.
Alcohol/Exercise/Aspirin (Healthy Participants only) ....................35
6.7.
Screen Failures...........................................................................................35
7.
NON-INVESTIGATIONAL MEDICINAL PRODUCT (NIMP) ...................................35
7.1.
STM Administered ......................................................................................37
7.2.
Dose Modification .......................................................................................38
7.3.
Method of STM Administration: Treatment Assignment ..............................38
7.4.
Blinding.......................................................................................................38
7.5.
Preparation/Handling/Storage/Accountability..............................................38
7.6.
STM Compliance ........................................................................................38
7.7.
Concomitant Therapy..................................................................................39
7.8.
Treatment after the End of the Study ..........................................................39
8.
DISCONTINUATION CRITERIA.............................................................................39
8.1.
Discontinuation of STM administration........................................................39
8.2.
Withdrawal from the Study..........................................................................39
8.2.1.
Other Withdrawal Criteria.............................................................40
8.3.
Lost to Follow Up........................................................................................40
9.
STUDY ASSESSMENTS AND PROCEDURES .....................................................41
9.1.
Efficacy Assessments.................................................................................41
9.2.
Adverse Events...........................................................................................41
9.2.1.
Monitoring and reporting responsibilities......................................41
9.2.2.
Time Period and Frequency for Collecting AE and SAE 
Information...................................................................................42
9.2.3.
Method of Detecting AEs and SAEs.............................................42
9.2.4.
Follow-up of AEs and SAEs.........................................................43
9.2.5.
Regulatory Reporting Requirements for SAEs .............................43
9.2.6.
Disease-Related Events and/or Disease-Related 
Outcomes Not Qualifying as SAEs...............................................43
9.3.
Treatment of Overdose...............................................................................44
9.4.
Safety Assessments ...................................................................................44
9.4.1.
Physical Examinations.................................................................45
9.4.2.
Vital Signs....................................................................................45
9.4.3.
Clinical Safety Laboratory Assessment........................................45
9.5.
Study Procedures .......................................................................................45
9.5.1.
Fluid Balance Measurement ........................................................45
9.5.2.
Wound Healing ............................................................................46
9.5.3.
Other Clinical Responses ............................................................46
9.6.
Pharmacokinetics .......................................................................................46
9.7.
Pharmacodynamics ....................................................................................46
9.8.
Intestinal Permeability Assessments...........................................................46
9.9.
Genetics .....................................................................................................47
9.10.
Sample Collection for Biomarker Analysis...................................................47
9.10.1.
Blood Biomarkers ........................................................................47

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9.10.2.
Stool Sample Collection...............................................................47
9.10.3.
Urine Sample Collection ..............................................................48
10. STATISTICAL CONSIDERATIONS........................................................................49
10.1.
Hypotheses.................................................................................................49
10.2.
Sample Size Determination ........................................................................49
10.3.
Data Analyses Consideration......................................................................49
10.4.
Populations for Analyses ............................................................................50
10.5.
Statistical Analyses.....................................................................................50
10.5.1.
Safety Analyses...........................................................................50
10.5.2.
Other Analyses ............................................................................51
10.5.3.
Interim Analyses ..........................................................................51
10.6.
Analyses of lactulose/mannitol ratio............................................................51
11. REFERENCES.......................................................................................................53
12. APPENDICES........................................................................................................55
12.1.
Appendix 1: Abbreviations and Trademarks................................................55
12.2.
Appendix 2: Clinical Laboratory Tests.........................................................57
12.3.
Appendix 3: Study Governance Considerations..........................................58
12.3.1.
Regulatory and Ethical Considerations ........................................58
12.3.2.
Recruiting participants under the Mental Capacity Act 
2005 ............................................................................................58
12.3.3.
Determining Whether a Participant has Capacity Under 
the Mental Capacity Act (2005)....................................................59
12.3.4.
Financial Disclosure.....................................................................61
12.3.5.
Informed Consent Process ..........................................................62
12.3.6.
Data Protection............................................................................62
12.3.7.
Committees Structure ..................................................................62
12.3.8.
Publication Policy.........................................................................63
12.3.9.
Dissemination of Clinical Study Data ...........................................63
12.3.10. Data Quality Assurance ...............................................................63
12.3.11. Source Documents ......................................................................64
12.3.12. Study and Site Closure ................................................................64
12.4.
Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting .......................................65
12.5.
Appendix 5..................................................................................................73

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1.
SYNOPSIS
Protocol Title: A Prospective, Longitudinal Study to Investigate the Effect of Thermal 
Injury on Intestinal Permeability, and Systemic Inflammation (HESTIA)
Short Title: Investigation of Thermal Injury and Intestinal Permeability
Rationale:
Patients with severe thermal injury [>20% total body surface area (TBSA)] are at risk for 
organ dysfunction and may develop multiple organ dysfunction syndrome (MODS). This 
study aims to explore the relationship between thermal injury, changes in intestinal 
permeability and the onset of MODS. The results of this study will be used to inform the
design of future drug studies of a novel medicinal product, which is predicted to reduce 
this increased permeability.
No GSK study treatment will be employed in this study.
One of the central hypotheses over the last two decades to explain the onset of MODS in 
the context of critical illness imputes that an increase in intestinal permeability results in 
the translocation of bacteria and bacterial products to the systemic circulation where they 
drive inflammation and injury to distal organs [Deitch, 2006]. This hypothesis is 
supported by data showing that there is a significant increase in intestinal permeability in 
critically ill patients, including patients with thermal injury, and that the degree of 
intestinal permeability correlates with the onset and severity of MODS [Doig, 1998]. As 
such, therapies directed at minimising these changes in intestinal permeability, thereby 
limiting the translocation of bacteria to the systemic circulation, are hypothesised to have 
an impact on clinical outcome in these patients.
Previous literature demonstrates that patients with severe thermal injury (>20% TBSA) 
display a significant and rapid increase in intestinal permeability that declines gradually 
over a two-week period following the injury [Olquin, 2005]. Additionally, these changes 
in intestinal permeability, correlate with the severity of sepsis observed in these patients 
[Ziegler, 1988].
The central hypothesis of this study, therefore, is that thermal injury alters intestinal 
barrier function allowing the translocation of bacteria and bacterial products to the 
systemic circulation where they contribute to the onset of MODS.
The aims of the study are:
1. To establish the magnitude and time course of changes in intestinal permeability to 
inform timing and duration of future investigational medicinal product administration.
2. To establish the optimal method for assessment of intestinal permeability in patients 
with thermal injury.
3. To describe the patient population most likely to benefit from a new medicinal 
product which could prevent changes in intestinal permeability.

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4. To improve our understanding of the links between intestinal damage, changes in the 
gut microbiome and microbial translocation to the systemic circulation following 
thermal injury.
In order to enter this study thermally injured participants will be required to co-enrol in 
this study and an allied study entitled: A Multi-centre, Prospective Study to Examine the 
Relationship between Neutrophil Function and Sepsis in Adults and Children with Severe 
Thermal Injury (SIFTI-2) (reference number IRAS ID: 200366). Clinical data, standard 
of care laboratory data and investigational biomarker data will be shared from the SIFTI-
2 study to this study and the combined data from both studies will be used to address 
exploratory endpoints. Participants of the SIFTI-2 study will be appropriately consented 
for this data sharing.
Objectives and Endpoints:
Objective
Endpoint
Co-Primary

To determine the impact of thermal injury 
on the magnitude of small intestinal 
permeability change as soon as possible 
following injury compared to healthy 
participants

Lactulose/Mannitol (L/M) ratio at entry

To characterise the effect of thermal 
injury on small intestinal permeability over 
time

Changes in L/M ratio over time
Overall Design:
A prospective, longitudinal study will be conducted in adult (≥18 years old) men and 
women admitted to a hospital following thermal injury. Measurements of intestinal 
permeability, inflammation and microbial translocation will be taken over a six month 
period. A cohort of healthy participants will also be recruited in order to determine the 
reference against which post-burn permeability measurements and other biomarkers will 
be compared.
The lactulose-to-mannitol ratio is traditionally used to assess small intestinal permeability 
and sucralose to assess colonic permeability. Lactulose, mannitol and sucralose 
[henceforth referred to as sugar test material (STM)] will be intermittently administered 
enterally for the purpose of intestinal permeability measurement to examine permeability 
at different points along the GI tract and is described in Section 7. It is important to note 
that the administration of the STM is not therapeutic (lactulose and mannitol can be used 
as laxatives, however the amount in the STM is sub-therapeutic) and as such should be 
regarded as a non-investigational medicinal product (NIMP) [Guidance Documents 
Applying Investigational Medicinal Products (NIMPS), 2011].

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An internal preliminary data review will be conducted. This review is described in 
Section 5.4.4.
Number of Participants:
Table 1 in Section 5.2 described the number of participants proposed for the study. 
Sample size is based on feasibility. No formal calculation of power or sample size has 
been performed, but a sample size of 15 healthy participants and 25 thermal injury 
participants (≥15% TBSA) should be sufficient to provide useful estimates of variability 
in lactutose:manitol ratios, and any change in L/M ratio over time.
Treatment Groups and Duration:
Group 1: Healthy Participants
The total duration of this study for healthy participants will be approximately two weeks, 
in addition to the screening window:

Screening: up to 28 days before Day 1

Day 1: study start and assessments will be performed

Day 8 and Day 15
Group 2: Thermally injured participants
Thermal injury participants will be asked to participate for a total of 6 months (plus or 
minus 14 days).

There will be no screening period. Thermal injury participants will be recruited 
within 24 hours of their admission to the burns unit at the study site.

Intense monitoring phase: Assessments will be performed on alternate days for 
the first 14 days following study enrolment. If the participant is discharged prior 
to 14 days, the intense monitoring phase will end, but the participant will remain 
enrolled in the study.

Convalescent monitoring phase: Assessments at 28 days and 6 months will be 
made on an outpatient basis if the participant has already been discharged from 
hospital.
Exception to monitoring periods:

If a discharged participant attends the centre for routine clinical care on any 
of days 8-14, then study assessments will be made and samples will be taken

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2.
SCHEDULE OF ACTIVITIES (SOA)
2.1.
Healthy Participants (Group 1)
Procedure
Screening 
Treatment Period [Out patient days] (1 day)
Notes
D1
D8
D15
Review of inclusion/ 
exclusion criteria and 
informed consent
X
Demography
X
Medical history (includes 
substance and alcohol 
usage at screening) and 
Medication history
X
Substance testing (urine)
X
Substances: [Recreational 
Drugs and Alcohol]
HIV, Hepatitis B and C
screening
X
If test otherwise performed 
within 3 months prior to 
study entry, testing is not 
required
Laboratory assessments
X
Pregnancy test (WOCBP 
only) (urine)
X
(X)
Only performed again on 
Day 1 if patient at risk of 
pregnancy at or since 
screening

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Procedure
Screening 
Treatment Period [Out patient days] (1 day)
Notes
D1
D8
D15
Blood sampling for 
biomarkers
X
20ml of blood will be 
sampled in a single draw 
on one day, preferably Day 
1
Stool sample collection
X
Participants will be given a 
collection container at 
screening to bring with 
them on Day 1.
Brief physical examination 
including measurements 
of height and weight
Vital Signs (systolic and 
diastolic blood pressure 
and heart rate)
X
X
X
X
BMI calculated from height 
and weight at screening 
only
Examinations should be 
conducted the day after 
intestinal permeability 
measurement.
Medical review 
(assessment of health 
status)
X
X
X
Monitor for signs and 
symptoms of gastro-
intestinal infections and 
other emergent issues
STM Training
X
Refresher training may be 
provided as needed
Intestinal permeability 
measurement: STM 
administration followed by 
a 24-hour urine collection
X
X
X
Please refer to SRM for full 
information
Intestinal permeability 
measurement to be 
performed at home the day 
before the study visit.

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Procedure
Screening 
Treatment Period [Out patient days] (1 day)
Notes
D1
D8
D15
AE/SAE and Concomitant 
medication reviews
(X)
=============================
Day 1 will include 
concomitant medication 
review only
2.2.
Thermal Injury Participants (Group 2)
Procedure
D1 
(≤ 24 hours 
of 
admission)
Treatment Period [ICU Days] (+/4 hours)
6 
months
(14 
days)
Notes
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D28 
(3 
days)
Review of inclusion/ 
exclusion criteria and 
informed consent
X
Participants will be co-
consented to the SIFTI-2 
study
Medical history 
(includes substance and 
alcohol usage) and 
Medication history
X
Substance testing 
(urine)
X
Substances: 
[Recreational Drugs, 
Alcohol]
Pregnancy test 
(WOCBP only) (urine)
X
X
To be repeated at 6 
months prior to final STM 
administration

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Procedure
D1 
(≤ 24 hours 
of 
admission)
Treatment Period [ICU Days] (+/4 hours)
6 
months
(14 
days)
Notes
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D28 
(3 
days)
HIV, Hepatitis B and C 
screening
X
If test otherwise 
performed within 3 
months prior to study 
entry, testing is not 
required
Initial assessment of 
Burns
X
Calculation of %TBSA
Location of thermal injury 
and depth
Fluid balance (total 
input/output)
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X*
Daily (over 24 hr)
*only if participant still 
admitted
Intestinal permeability 
measurement: STM 
administration followed 
by a 24-hour urine 
collection
X
X
X
X
X
X
X
X
X
Measurements every 48 
hours from first measure. 
Preference: D1, 3, 5, 7, 
9, 11, 13. Otherwise: D2, 
4, 6, 8, 10, 12, 14.
Please refer to SRM for 
full method.
Brief Physical 
Examination
X
X
Following final intestinal 
permeability 
measurement. Can be 
omitted if patient is still 
admitted to hospital.

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Procedure
D1 
(≤ 24 hours 
of 
admission)
Treatment Period [ICU Days] (+/4 hours)
6 
months
(14 
days)
Notes
D2
D3
D4
D5
D6
D7
D8
D9
D10
D11
D12
D13
D14
D28 
(3 
days)
Stool sample collection
=============================
(A sample of the first stool produced following admission)
X
X
X
Time to first stool 
collection; preferably on 
Day 1. Then on Days 14 
and 28 and 6 months
Wound Healing 
Assessments
X
X
X
This is to assess time to 
95% wound healing
AE/SAE and 
Concomitant medication 
reviews
X
=============================
X
AE/SAE monitoring will 
begin after the first 
administration of STM

The timing and number of planned study assessments may be altered during the course of the study based on newly available 
data or factors outside of the study such as priority medical care to ensure appropriate monitoring.

Any changes in the timing or addition of time points for any planned study assessments must be documented and approved by 
the relevant study team member and then archived in the sponsor and site study files, but will not constitute a protocol 
deviation or require a protocol amendment. The IRB/IEC will be informed of any safety issues that require alteration of the 
safety monitoring scheme or amendment of the ICF.

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3.
INTRODUCTION
3.1.
Study Rationale
The purpose of this study is to describe the kinetics and magnitude of increases in 
intestinal permeability which are observed as a result of thermal injury. The results of this 
study will be used to inform the design of future drug studies of a novel medicinal 
product, which is predicted to reduce this increased permeability.
3.2.
Background
3.2.1.
Thermal Injury, Intestinal Permeability and Multi-Organ 
Dysfunction Syndrome
Patients with severe thermal injury >20% TBSA are at risk for organ dysfunction and 
may develop multiple organ dysfunction syndrome (MODS). This study aims to explore 
the relationship between thermal injury, changes in intestinal permeability and the onset 
of MODS.
One of the central hypotheses over the last two decades to explain the onset of MODS in 
the context of critical illness imputes that an increase in intestinal permeability results in 
the translocation of bacteria and bacterial products to the systemic circulation where they 
drive inflammation and injury to distal organs [Deitch, 2006]. This hypothesis is 
supported by data showing that there is a significant increase in intestinal permeability in 
critically ill patients, including patients with thermal injury, and that the degree of 
intestinal permeability correlates with the onset and severity of MODS [Doig, 1998]. As 
such, therapies directed at minimising these changes in intestinal permeability, thereby 
limiting the translocation of bacteria to the systemic circulation, are hypothesised to have 
an impact on clinical outcome in these patients (Figure 1).
Figure 1
Changes in Intestinal Permeability
Critical 
Illness
Intestinal
Permeability
Bacteria/
Bacterial products
MODS
Figure 1 (Left panel) Hypothesis: The gut is the major driver of MODS in critical illness
(Right panel) Correlation between intestinal permeability as measured by 
lactulose/mannitol ratio and the severity of MODS in critically ill patients. Intestinal 
permeability was determined by measuring the differential absorption of lactulose 
(increased in the damaged gut) and mannitol (freely absorbed in the normal and damaged 

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gut) following oral administration and expressed as the ratio of lactulose to mannitol 
(L/M) [Olquin; 2005].
There are some data showing that patients with severe thermal injury (>20% TBSA) 
display a significant and rapid increase in intestinal permeability that declines gradually 
over a two-week period following the injury [Olquin, 2005]. Additionally, these changes 
in intestinal permeability, correlate with the severity of sepsis observed in these patients 
[Ziegler, 1988].
The central hypothesis of this study is that thermal injury alters intestinal barrier function 
allowing the translocation of bacteria and bacterial products to the systemic circulation 
where they contribute to the onset of MODS.
The aims of the study are:
1. To establish the magnitude and time course of changes in intestinal permeability to 
inform timing and duration of future investigational medicinal product administration.
2. To establish the optimal method for assessment of intestinal permeability in patients 
with thermal injury.
3. To describe the patient population most likely to benefit from a new medicinal 
product which could prevent changes in intestinal permeability.
4. To improve our understanding of the links between intestinal damage, changes in the 
gut microbiome and microbial translocation to the systemic circulation following 
thermal injury.
3.2.2.
Intestinal Barrier Function and Its Measurement
The intestinal barrier combines a physical and immunological barrier. Epithelial cells are 
connected by tight junctions and prevent the passage of bacteria, toxins and antigens into 
the systemic circulation [Bjarnason, 1995]. Paneth cells, located in crypts of the small 
intestine, produce anti-microbial substances (e.g. lysozyme and defensins) and other 
immune cells patrol the lamina propria [Ayabe, 2000]. Barrier function can be disturbed 
by diseases such as inflammatory bowel disease; by drugs such as aspirin and alcohol; by 
ischaemia and has been observed following a number of acute injuries such as burns, 
trauma and radiation injury [Bjarnason, 1995; Derikx, 2006]. This disturbance results in 
the translocation of the intestinal flora (pathogenic or commensal) to the systemic 
circulation. Microbes are accompanied by proteins which normally form part of the tight 
intracellular junctions of the intestinal epithelium, such as claudins, and other enterocyte-
derived proteins [Grootjans, 2010]. In this study, intestinal permeability will be measured 
directly using oligosaccharide absorption and indirectly by looking for micro-organisms 
and soluble markers of intestinal barrier dysfunction in the systemic circulation.
Since the 1970s, oligosaccharides have been used as test probes to measure the function 
of the intestinal barrier [Menzies, 1972]. Lactulose, a large polysaccharide, does not 
normally cross the intestinal barrier, but following damage can cross the epithelium and 
enter the systemic circulation. It is not metabolised, so is filtered in the kidney and 
excreted in the urine. The fractional excretion (amount administered / amount recovered 
in urine) of lactulose is therefore a measure of intestinal permeability. The amount of 
lactulose entering the urine is dependent on a number of factors including renal function, 

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gastric emptying, and degradation in the large bowel by commensal bacteria; thus, a 
monosaccharide such as mannitol, which passes freely across the healthy intestinal 
barrier, is often co-administered to ‘normalise’ lactulose measurements. Following 
administration of both lactulose and mannitol the fractional excretion of the sugars is 
expressed as a ratio where mannitol is the denominator. Lactulose and mannitol 
absorption occurs mainly in the proximal small intestine and is complete within 
approximately 5 hours of oral administration. This approach has been previously used 
successfully in patients with severe burn injury and intestinal permeability was found to 
correlate with episodes of sepsis [Doig, 1998].
In order to assess the permeability of the large bowel a third oligosaccharide, sucralose, 
will also form part of the sugar absorption test. This synthetic sweetener (marketed by 
Tate and Lyle as ‘Splenda’) is not subject to the same degradation by large bowel 
commensal flora as lactulose and is therefore a better measure of large bowel 
permeability than lactulose. Again, this has formed part of previously described studies 
aiming to measure intestinal permeability [Del Valle-Pinero, 2013].
Lactulose, mannitol and sucralose will be co-administered to both healthy participants 
and participants following thermal injury. In order to document accurately the time 
course of change in permeability, thermally injured participants will be asked to undergo 
the test on alternate days for 14 days (the intense monitoring phase) followed by two 
convalescent samples at day 28 and month 6 (thermal injury participants). In order to 
produce an accurate baseline measurement, healthy participants will be asked to undergo 
three measurements of intestinal permeability over approximately a two week period.
3.2.3.
The Intestinal Microbiome and Thermal Injury
During the intense and convalescent monitoring phases of this study, samples of stool 
will be collected. These samples will undergo gene sequence analysis in order to 
determine the composition of the intestinal microbiota. These results will be compared 
with culture results from peripheral whole blood samples. The hypothesis is that raised 
intestinal permeability will correlate with an increased frequency of bacteraemia and that 
the particular bacteria detected in blood will correlate with the composition of the 
intestinal microbiota.
Additionally, it has been demonstrated that thermal injury alters the composition of the 
intestinal microbiome [Hammer, 2015]. The ultimate aim is to be able to block changes 
in intestinal permeability which might affect this change in composition and are therefore 
interested, in the current study, to assess the impact of thermal injury on the microbiome.

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3.3.
Benefit/Risk Assessment
3.3.1.
Risk Assessment
Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Non-investigational Medicinal Product Use
Oral administration of sucralose in solution is 
unlikely to be palatable.
The dose of sucralose to be administered is 2g 
per test. A tolerable sweetness score (e.g. 11.3 
(diet Pepsi)) would require the sucralose to be 
administered in 4 litres of water.
Sucralose is to be administered in capsules 
when given by mouth. If being administered by 
nasogastric or nasojejunal tube the capsules are 
to be emptied into the lactulose and mannitol 
solution
Depending on the volume of administration, the 
final STM solution may be hyper-osmolar. 
Administration directly into the jejunum via a 
nasojejunal tube may then result in osmotic 
movement of water into the intestine causing 
distention and discomfort.
This is based on the physiological principle that 
the stomach normally regulates the osmolality of 
its contents passing into the small intestine. 
Administration via nasojejunal tube (but not 
nasogastric tube) bypasses this process.
The lactulose, mannitol and sucralose will be 
delivered nasojejunally in 50ml of water and 
followed by a 50ml water flush. This makes the 
solution iso-osmolar (300 mosmol/Kg in situ). 
following nasojejunal administration 
Lactulose and mannitol can produce an osmotic 
laxative effect following enteral administration.
Both lactulose and mannitol are used clinically as 
osmotic laxatives. The typical dose of lactulose 
(for the treatment of constipation) would be 
approximately 21g daily.
The amount of lactulose being used in this study 
is 5g, 75% below the standard laxative dose. 
The dose of mannitol is equally low compared to 
that contained in laxatives.

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Study Procedures
STM administration for the measurement of 
intestinal permeability requires a hold in feeding 
which may be longer than a standard feeding 
hold.
Intestinal permeability assessment requires the 
administration of STM (see Section 7) and the 
collection of urine samples at defined time points 
(see the SRM). This procedure has previously 
been used in patients with severe thermal injury 
[Doig, 1998] but has not been studied 
extensively; participant fasting is required pre and 
post administration of these sugars; and repeated 
administration as per the SoA tables is also 
unexplored.
Where possible, tube feeding targets will be 
volume and not time-based to reduce the 
amount of feed missed on test days. Moreover, 
fasts for the tests will, where possible, be 
aligned with clinically indicated feeding holds 
(such as fasts required before theatre, or 
scheduled overnight feed holds).
Other
The degree of injury sustained by some 
participants may be severe.
Participants with severe thermal injury experience 
significant morbidity and high levels of mortality. 
Therefore it is anticipated that these participants 
will be subject to multiple medical complications 
which may impact the study assessments and 
period (See SoA tables) 
Ensure that routine care in the burns unit is not 
compromised by study participation. Prompt 
reporting of any adverse events which are 
related to study procedures and may affect study 
safety.
Omission of lactulose and sennosides which are 
frequently administered as a part of routine burn 
management. 
Lactulose is a part of the STM and its 
administration for clinical reasons would 
complicate intestinal permeability measurement 
significantly. Equally, the mechanism of actions of 
sennosides is to cause irritation of the GI tract 
Polyethylene glycol (e.g. Movicol) will be used 
as an alternative osmotic laxative.
If senna is required for clinical purposes, then its 
use must be documented in the CRF.

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
and may result in increased permeability.
The STM will be prepared for administration on 
the assessment day and will not be sterile.
There is a risk that the sugar test material 
formulation may be contaminated with yeast or 
bacteria. The presence of contamination within 
the STM formulation delivered via the feeding 
tube in thermal injury participants could present 
an infection risk.
Microbiology release testing will be conducted 
by Tayside pharmaceuticals. The STM will be 
prepared on the assessment day to minimise 
this risk.
Lactulose/Mannitol will be refrigerated from the 
point of manufacture and until use. The site 
investigators have been consulted on this risk 
and felt that it was low.
Incomplete 24 hour urine collections
Ambulant, uncatheterised patients and healthy 
participants 24-hour urine collections could be 
incomplete.
Ambulant, uncatheterised patients and healthy 
participants will receive careful education and 
written instructions of the importance of 
complete urine collections. In addition, 
documentation of incomplete collection, in 
addition to sampling collection times, will be 
recorded.

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Potential Risk of Clinical Significance
Summary of Data/Rationale for Risk
Mitigation Strategy
Phlebotomy for biomarkers (healthy participants)
Phlebotomy can be painful and result in bruising, 
bleeding and puncture site infection.
Phlebotomy from thermally injured participants 
will not be conducted as a part of this protocol.
Healthy participants will undergo one blood draw 
during screening and a further draw on day one. 
Phlebotomy will be performed by an 
appropriately trained member of the site study 
team with aseptic non-touch technique to avoid 
infection.

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3.3.2.
Benefit Assessment
Study participants will not benefit directly from involvement in this study. However, the 
results of this study may contribute significantly to our understanding of changes in 
intestinal permeability and their relationship to morbidity and mortality in the context of 
thermal injury. This knowledge is paramount to designing future medicinal interventional 
studies, aiming to modulate intestinal permeability and, potentially, to improve outcomes 
for patients following thermal injury.
3.3.3.
Overall Benefit:Risk Conclusion
The primary outcome measure of this study is the determination of intestinal permeability 
in healthy and thermal injury participants. Interventions in this study are the 
administration of STM by mouth or feeding tube (if one is site for routine clinical care) 
and the collection of urine and stool samples.
The risk of adverse events is minimised for the population being investigated in the 
proposed study as no drug intervention will be investigated and study assessments being 
conducted are non-invasive (with the exception of STM administration and phlebotomy 
in healthy participants).
4.
OBJECTIVES AND ENDPOINTS
Objective
Endpoint
Co-Primary
1.
To determine the impact of thermal injury 
on the magnitude of small intestinal 
permeability change as soon as possible 
following injury compared to healthy 
participants

Lactulose/Mannitol (L/M) ratio at entry
2.
To characterise the effect of thermal 
injury on small intestinal permeability over 
time and establish the clinical and 
demographic factors which can influence 
it

Changes in L/M ratio over time
Exploratory
1.
To determine the impact of thermal injury 
on colonic permeability as soon as 
possible following injury compared to 
healthy participants

Fractional excretion of sucralose at entry
2.
To characterise the effect of thermal 
injury on colonic permeability over time

Changes in the fractional excretion of 
sucralose over time

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Objective
Endpoint
3.
To assess the relationship between 
severity of a participant’s condition 
following thermal injury and changes in 
intestinal permeability†

Number of ventilator-free days

Number of vasopressor-free days

Number of hemofiltration-free days

Number of episodes of confirmed infection 
and sepsis

Number of surgical interventions

Total length of hospital stay

Calculate critical care and thermal injury 
severity scores
4.
To assess plasma and urine biomarkers 
of intestinal permeability, bacterial 
translocation and renal tubular 
dysfunction following thermal injury†

Change in markers of intestinal mucosal
damage samples from blood

Change in urine protein:creatinine and urine 
albumin:creatinine ratios
5.
To assess the impact of thermal injury 
and intestinal permeability on the 
intestinal microbiome compared to 
healthy participants

Changes in microbiome of acute and 
convalescent stool samples
6.
To assess the impact of pre-existing co-
morbid conditions on intestinal 
permeability and clinical outcome 
following thermal injury†

Medical history and drug history at the time of 
admission
7.
To assess wound healing

Time to wound recovery (e.g. 95%)
8.
To characterise parameters that may 
influence drug PK/PD†

Determine fluid input/output balance over time

Changes in serum albumin and plasma 
creatinine
9.
To characterise intestinal microbiota, and 
correlate its composition with both 
intestinal permeability and bacterial 
detection in blood†

Changes in intestinal microbiome

Bacterial markers of translocation
†Clinical data, routine laboratory results or blood/urine biomarker results obtained from 
the SIFTI-2 study will be used in the analysis of this exploratory endpoint see Section 
5.4.1.

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5.
STUDY DESIGN
5.1.
Overall Design
This is a longitudinal, prospective study of healthy participants and participants who have 
sustained a thermal injury. The following schematic summarises study measures and their 
frequency for healthy and thermal injury participants.
Figure 2
Study Schematic
5.2.
Number of Participants
Table 1 describes the number of participants proposed for the study. Sample size is based 
on feasibility. No formal calculation of power or sample size has been performed, but a 
sample size of 15 healthy participants and 25 thermal injury participants (≥15% TBSA) 
should be sufficient to provide useful estimates of variability in lactutose:manitol ratios, 
and any change in L/M ratio over time.

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Table 1
Recruitment Stratification
Number of participants
Group 1
Healthy participants
15
Group 2
Thermal Injury participants
Percent Total Burn Surface Area (TBSA)
15%
25
The healthy participants (Group 1) will be recruited with an age range similar to that 
typical in thermal injury participants based on historic hospital admission data from the 
UK and data from the SIFTI1 study [Hampson, 2016].
If participants prematurely discontinue or are withdrawn from the study, additional 
replacement participants may be recruited at the discretion of the Sponsor in consultation 
with the investigator see Section 8.2.1.
5.3.
Participant and Study Completion
The full duration of the study for healthy participants is approximately two weeks and for 
thermal injury participants is approximately 6 months.
Thermal injury participants who withdraw prior to week 4 or healthy participants who 
withdraw prior to week 2 will be considered for analysis, although it is acknowledged 
that any missing data at later stages of the study may be related to outcome (either 
positive or negative). Given this is an exploratory study, the impact of missing data will 
be explored by assessing the sensitivity of results to different missing data approaches 
(for example, analyse all available data, analyse only complete data across time points 
and explore imputation of worst or best case scenarios).
Study withdrawals may also include participants who are consented to the study under 
Section 30 of the Mental Capacity Act 2005. In the event that participants do not re-
confirm consent when they regain capacity, they will be withdrawn from the study.
The end of the study is defined as the date of the last visit of the last participant in the 
study.
5.4.
Scientific Rationale for Study Design
5.4.1.
Co-recruitment to the SIFTI-2 Study
Thermally injured participants who are eligible for this study must also be eligible for, 
and enrolled in a partner study named SIFTI-2.

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SIFTI-2 is an observational study currently recruiting healthy participants and thermally 
injured participants and follows a successfully delivered predecessor study SIFTI1
[Hampson, 2016]. The objectives and endpoints of SIFTI-2 are included in the SIFTI-2 
study protocol (reference number IRAS ID: 200366). The design of this study has been 
aligned with the SIFTI-2 study to support the strategy of co-consenting thermally injured 
participants to both studies. This will reduce the overall impact of research in this 
population in the following ways:

There is sufficient residual blood from collection in SIFTI-2 to allow testing of 
blood biomarkers of interest for the HESTIA study. This strategy therefore limits 
impact on participants as no additional blood sampling is required for 
participation in HESTIA (with the exception of HIV, Hepatitis B and C testing at 
baseline). SIFTI-2 participants will be explicitly consented for their samples and
data to be shared in this way.

Sampling time points and study visits in SIFTI-2 and HESTIA have been aligned 
to reduce the impact of co-recruitment on thermally injured participants.
Clinical data from both studies can be generated from the same participant therefore 
allowing biomarker, microflora and intestinal permeability data to be compared. In 
contrast to thermally injured participants, healthy participants enrolling in this study will 
not be required to co-consent for participation in SIFTI-2.
Data will be shared from the SIFTI-2 study with GSK through a secure electronic 
database.
A summary of the origin (HESTIA or SIFTI-2) of data and samples collected for the 
HESTIA study is available in Appendix 5 Section 12.5.
5.4.2.
Recruitment and Sampling Schedule
Severely burned patients (with an injury affecting greater than 20% total body surface 
area), display a significant and rapid increase in intestinal permeability that has been 
shown to decline over time (Figure 3) [Olquin, 2005]. What is less well understood is 
whether a greater severity of thermal injury correlates with greater intestinal 
permeability. Moreover, the time to complete restoration of normal permeability and 
other factors which may influence permeability (other than the initial injury) are also not 
well understood.

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Figure 3
Burn Injury Results in a Rapid Increase in Intestinal Permeability
This study aims to recruit participants as soon as possible following their admission in 
order to capture the initial changes in permeability. Serial measurement of intestinal 
permeability and sampling of the biomarkers of bacterial translocation, intestinal damage 
and inflammation are required during the acute phase (days 1-14) of admission in order to 
correlate them with clinical events (e.g. surgery), severity scores and clinical outcomes.
The convalescent time points (28 days and 6 months) are required to determine if 
intestinal permeability has returned to normal and to correlate observed changes on days 
1-14 with longer-term clinical outcomes (e.g. wound healing).
Gut microbiome assessments will be made less frequently than intestinal permeability 
assessments as changes in the microbiome are predicted to evolve more slowly. Ideally a 
stool sample will be collected from thermal injury participants at study entry (limited, of 
course, by when participants first pass stool following admission). A convalescent sample 
is requested to assess if the gut microbiome is able to restore to a more normal 
composition (and will be compared with that of healthy participants to make that 
assessment).

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Blood samples (with the exception of HIV, Hepatitis B and Hepatitis C testing at 
baseline) will not be taken from thermally injured participants during this study. Instead, 
biomarker data from blood samples taken during the SIFTI-2 study will be used. A single 
blood draw will be required from healthy participants on day 1 of participation.
5.4.3.
Inclusion of Healthy Participants
Patient facing material (i.e. poster) will be used to facilitate recruitment of the healthy 
participants. Healthy participants will be recruited to this study to provide a baseline for 
endpoint measures on intestinal permeability and the gut microbiome.
Three measurements of intestinal permeability are required in order to define an average 
given the variability in healthy participants reported previously. The timing of the 
replicates follows the intense time course of the study to control for day-to-day variation 
over a 15 day period.
5.4.4.
Preliminary Data Review
A safety review based on raw data will be conducted when approximately 10 participants 
have completed the study. This review would include data from any participant, either 
healthy or thermally injured, that has been collected at the time the review is conducted.
5.4.5.
The Use of the Sugar Test Materials (Lactulose, Mannitol and 
Sucralose)
As described in Section 3.2.2, lactulose, mannitol and sucralose will be administered to 
both thermally injured participants and healthy participants to measure permeability of 
the small and large intestine. The amount of each of the sugars to be used is based on 
previous successful clinical studies employing this method and balances having enough 
sugar present for detection in urine with their potential laxative effect (Del Valle-Pinero, 
2013; Doig, 1998; Menzies, 1972).
6.
STUDY POPULATION
The study population will comprise healthy and thermal injury participants presenting at 
enrolling study sites. Please note the following:

Prospective approval of protocol deviations to recruitment and enrolment criteria, 
also known as protocol waivers or exemptions, is not permitted.

Where possible, written informed consent will be obtained from each subject prior 
to participation in this study. Recruitment of subjects who lack mental capacity is 
discussed in Section 6.1.

Healthy participants will be consented to the HESTIA study only. 

Thermally injured participants are required to be co-consented to the SIFTI-2 and 
HESTIA studies outlined in Section 6.1. A diagrammatic overview of the SIFTI-2 
and HESTIA thermal injury participant recruitment is given in Figure 4. (see 
Appendix 3).

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6.1.
Recruiting Participants with Differing Mental Capacity
Following evaluation of capacity (as outlined in the Mental Capacity Act (MCA) 2005), 
thermally injured participants enrolled in this study will fall into the following groups:
6.1.1.
Adult Participants Determined to Have Mental Capacity at Study 
Entry and Throughout the HESTIA Study
Those participants who present with capacity and meet study entry criteria will be 
provided with a Participant Information Leaflet (PIL) outlining the study. If the 
participant agrees to consent to the HESTIA study following a discussion with the 
research team, they will be asked to sign a consent form.
Given the short (24 hour) window for recruitment, those patients who are acutely 
unwell will initially be presented with an abbreviated PIL. Once stable, this will be 
followed by the standard information leaflet and re-confirmation of consent.
6.1.2.
Adult Participants Lacking Mental Capacity for the Duration of 
the HESTIA Study
It is anticipated that some subjects who meet eligibility criteria for this study will not 
be able to give informed consent due to their medical condition or its management 
(e.g. sedation, opioid analgesia, intubation). In such cases, participants may be 
enrolled in the study in accordance with Section 30- Section 34 of the MCA 2005. 
The decision to enrol the participant will be discussed with a legally acceptable 
representative (LAR) (also termed a ‘consultee’). This decision may or may not be 
witnessed by an independent witness according to the decision of the principal 
investigator.
6.1.3.
Adult Participants Lacking Mental Capacity (either at Study Entry 
or During the Study) Who Later Regain Capacity and Are
Required to Provide Informed Consent
As soon as is practically possible following a participant regaining capacity, 
participants will be asked to provide informed consent to remain in the study. If they 
decline, then they will be withdrawn from the study as soon as it is safe to do so 
(likely immediately given the design of this study). Samples and data collected prior 
to study withdrawal may be retained. The participant will be asked about this at the 
point of study withdrawal.
6.1.4.
Adult Participants with Mental Capacity to Provide Consent at
Study Entry Who are Later Deemed No Longer to have Mental 
Capacity
The decision for the participant to remain in the study will be discussed with a LAR 
and recorded. If the participant subsequently regains capacity again, they will be 
asked to re-consent to study participation.

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When considering enrolment of participants who lack the mental capacity to consent, the 
following should be noted:
Section 3.2.1 of the SIFTI-2 protocol describes the consent process for that study 
in detail and should be read in conjunction with this protocol. Please note that the 
SIFTI 2 protocol refers to a LAR as the Patient’s Personal Consultee (PC) or 
Nominated Consultee (NC).

A Study Information Leaflet will be provided to the LAR outlining the HESTIA 
trial before being asked to sign a form supporting the participant’s enrolment in 
the study.

The investigator and/or the site’s IEC/IRB have responsibility for acting in 
accordance with the MCA 2005 in the matter of assessing who has the capacity to 
consent and who qualifies as a LAR of a potential subject. The investigator will 
also decide if an independent witness is required.

Further information regarding the assessment of mental capacity and the 
appointing of LARs/PCs/NCs is given in Appendix 3 (Section 12.3.2 and Section 
12.3.3).

If a patient loses mental capacity subsequent to their consent and enrolment to the 
HESTIA study, samples and data collected prior to loss of capacity will be 
retained even if approval of continued study participation by a LAR is declined.
6.2.
Inclusion Criteria for Healthy Participants (Group 1)
1.
Males or Females must be ≥18 years of age at the time of signing informed consent.
2.
Participants who are healthy as determined by the investigator following medical 
evaluation including medical history, physical examination, and laboratory tests
(these are listed in Appendix 2).
3.
Female participants:
A female participant is eligible to participate if she is not pregnant (negative 
pregnancy testing at screening or Day 1 as needed) and not breastfeeding.
4.
Capable of giving signed informed consent as described in Appendix 3 which
includes compliance with the requirements and restrictions listed in the informed 
consent form (ICF) and in this protocol.
6.3.
Inclusion Criteria for Thermal Injury Participants (Group 2)
Thermal injury participants are eligible to be included in the study only if all of the 
following criteria apply:
6.3.1.
Age
1.
Participant must be ≥18 years of age.
6.3.2.
Type of Participant and Disease Characteristics
2.
Participants who have sustained a burn (thermal injury) with a TBSA ≥ 15%.

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6.3.3.
Other Inclusions
3.
Admission to the burn centre (study site) ≤24 hours of injury.
4.
Able to take enteral fluids either orally or via a nasogastric tube (depends on facial 
burn damage).
6.3.4.
Gender
5.
Male and female.
a. Female participants: 
A female participant is eligible to participate if she is not pregnant (negative 
pregnancy testing at study entry) and not breastfeeding.
6.4.
Exclusion Criteria for Healthy Participants (Group 1)
1.
Healthy participants are excluded from this study if they are receiving anti-
coagulation therapy.
2.
Pregnancy or breastfeeding.
3.
A body mass index >34kg/m2
4.
An active history of alcohol dependency
5.
History of sensitivity to any of the STM, or components thereof or a history of drug 
or other allergy that, in the opinion of the Investigator and/or GSK Medical Monitor, 
contraindicates their participation.
6.
A positive pre-study Hepatitis B surface antigen or positive Hepatitis C antibody and 
confirmatory Hepatitis C PCR result within 3 months of screening.
7.
A positive pre-study urine drug/alcohol screen.
8.
A positive test for HIV antibody.
9.
Participants unable to swallow large capsules (the capsules will be shown to 
participants at screening).
10. Galactosaemia or severe lactose intolerance.
11. Use of an antibiotic 2 weeks prior to study start (i.e. administration of the STM).
12. Gastroenteritis in the 2 weeks prior to study start (i.e. administration of the STM).
6.5.
Exclusion Criteria for Thermal Injury Participants (Group 2)
Thermal injury participants are excluded from the study if any of the following criteria 
apply:
Medical Conditions
1.
Chemical or electrical burn.
2.
Multiple traumatic injuries with an Injury Severity Score (ISS) ≥16 (note: excludes 
burn in score system).

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Prior/Concomitant Therapy
3.
Patient received substantial undocumented management prior to arrival at the study 
site (burn centre) e.g. from paramedics or in a local accident and emergency
department.
4.
Systemic corticoidsteroid use.
5.
Intravenous (IV) mannitol use.
Prior/Concurrent Conditions
6.
Human immunodeficiency virus (HIV) infection.
7.
Viral Hepatitis B or C infection.
8.
Gastrointestinal disease (e.g. inflammatory bowel disease) which may affect 
intestinal permeability.
9.
Previous bowel resection (e.g. hemicolectomy, small bowel resection)
10. Galatosaemia or severe lactose intolerance.
11. Bowel obstruction.
12. Renal dysfunction requiring renal replacement therapy (i.e. end-stage renal failure 
prior to thermal injury).
13. Active autoimmune disease and receiving immunomodulatory therapy e.g. 
rheumatoid arthritis anti-TNF.
14. Active chemotherapy for cancers or immunoremittive therapies (prednisolone, 
adalimumab) within 60 days of thermal injury.
15. Premorbid conditions of malignancy currently under treatment.
16. Previous bilateral lower extremity amputation.
NOTE: Due to the rapid recruitment period (within 24 hours of admission) for thermally 
injured participants, HIV and viral Hepatitis test results may not be available at the time 
of enrolment. In the event these tests return positive, the participant will be informed of 
the result(s) and withdrawn from the study (see Section 8.2.1). In the event that the 
participant has been recruited who lacked mental capacity to consent, the reason for their 
withdrawal (the positive HIV and/or viral Hepatitis test results) must not be shared with a 
personal consultee. According to UK law and the guidance of the British HIV 
association, if the participant’s physician believes there is an overriding public interest to 
disclose the participant’s results to a personal consultee who is a current or former sexual 
partner, then the result(s) may be disclosed without the participant’s consent. This must 
be as a last resort. If the participant regains capacity, he/she will be informed of the 
positive test result(s).
Diagnostic assessments
17. Decision not to treat the patient due to futility.

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6.6.
Lifestyle Restrictions
6.6.1.
Meals and Dietary Restrictions

Participants will be fasted (or feed stopped) for 3 hours prior to STM administration 
and for 3-5 hours afterwards. For thermally injured participants these fasts should be 
aligned with those required for routine clinical care (feed holds, before surgical 
interventions) where possible.

Refrain from consumption of the following for 24 hours before and after the 
administration of STM:
o
Foods/drinks/medicines and other products which contain sucralose, lactulose or 
mannitol as artificial sweeteners.
N.B. If cannot be avoided, then clear documentation of its administration is required and 
the current test to stop. If urine samples have been collected PRIOR to 
administration of the drug, then these can still be sent for analysis
6.6.2.
Alcohol/Exercise/Aspirin (Healthy Participants only)
Alcohol, aspirin and vigorous exercise [Sequeira, 2014] are all known to cause transient 
increases in intestinal permeability. Healthy participants will therefore be requested to 
avoid alcohol, aspirin and physical exercise for 48 hours before taking the STM and for 
the 24-hour urine collection period.
6.7.
Screen Failures
There will be no screening period for thermal injury participants. Screening will be up to
28 days before Day 1 for healthy participants.
Screen failures are defined as participants who consent to participate in the clinical study 
but are not subsequently entered in the study. A minimal set of screen failure information 
is required to ensure transparent reporting of screen failure participants to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes 
demography, screen failure details, eligibility criteria, and any serious adverse events 
(SAEs).
Individuals who do not meet the criteria for participation in this study (screen failure) 
may be rescreened. Rescreened participants should be assigned the same participant 
number as for the initial screening.
7.
NON-INVESTIGATIONAL MEDICINAL PRODUCT (NIMP)
A study treatment is defined as any investigational treatment(s), marketed product(s), 
placebo, or medical device(s) intended to be administered to a study participant according 
to the study protocol. According to this definition, no GSK study treatment will be 
employed in this study.

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The STM (comprising lactulose, mannitol and sucralose) will be intermittently 
administered enterally as a study challenge agent to measure permeability at different 
points along the GI tract. Lactulose and Mannitol assess small intestine permeability and 
sucralose to assess colonic permeability.
It is important to note that the administration of the STM is not therapeutic (lactulose and 
mannitol can be used as laxatives, however the amount in the STM is sub-therapeutic) 
and as such should be regarded as a non-investigational medicinal product (NIMP) 
[Guidance Documents Applying Investigational Medicinal Products (NIMPS), 2011].

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7.1.
STM Administered
Study 
Treatment 
Name
Lactulose
(4-o-β-D-
galactopyranosyl-D-
fructofuranose)
Mannitol
(D-mannitol)
GRAS listed
Sucralose 
(1,6-Dichloro-1,6-dideoxy-β-
D-fructofuranosyl-4-chloro-
4-deoxy-α-D-
galactopyranoside)
Dosage 
formulation
oral solution
oral solution
Capsules (powder)
Unit dose 
strength(s) 
Adults
5g
2g
2g (3 capsules to deliver 
total 2g sucralose)
Route of 
Administration
Oral / nasogastric / 
nasojejunal
Oral / nasogastric / 
nasojejunal
Oral / nasogastric / 
nasojejunal (capsules to 
be opened and contents 
added to lactulose and 
mannitol for tube 
administration)
Preparation 
and Dosing 
instructions
For oral administration, the lactulose and mannitol will be prepared as a 
100ml drink to be taken with 3 sucralose capsules.
For feeding tube administration, lactulose/mannitol/sucralose will be prepared 
as a 50ml solution and given via a feeding tube followed by an immediate 
50ml drinking water flush
Preparation refer to Study Reference Manual together with SoA tables 
(Section 2)
Packaging and 
Labelling
Lactulose and Mannitol will be supplied pre-
mixed in an amber bottle (or equivalent) for 
single use. Each container will be labelled as 
required per country requirement.
Sucralose will be provided 
as capsules in a storage 
container. Each container 
will be labelled as 
required per country 
requirement.
Manufacturer
Tayside Pharmaceuticals, UK
Storage
Lactulose/Mannitol formulation should be stored under refrigerated conditions. 
The sucralose capsules should be stored at room temperature in a dry 
environment away from direct sunlight.
Shelf-life
Lactulose/Mannitol pre-mix formulation and sucralose capsules supplied by 
Tayside Pharmaceuticals will have at least 3 month shelf-life when stored at 
the correct storage conditions.

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The preparation of the STM for oral use and nasogastric/nasojejunal tube administration 
can be found in the Study Reference Manual.
7.2.
Dose Modification
Dose modification will not be required. Unit dose is described in Section 7.1.
7.3.
Method of STM Administration: Treatment Assignment
There is no element of randomisation in the study and all study participants will receive 
the STM according to the relevant SoA. The method of administration can be found in 
the Study Reference Manual.
7.4.
Blinding
No GSK study treatment will be employed in this study. All participants will receive the 
same STM and all thermal injury participants will perform the same study procedures.
7.5.
Preparation/Handling/Storage/Accountability
1.
The investigator or designee must confirm appropriate temperature conditions 
have been maintained during transit for all STM received and any discrepancies are 
reported and resolved before use of the STM.
2.
Only participants enrolled in the study may receive STM and only authorized site 
staff may supply or administer STM unless adequate training is provided such as in 
the case of healthy participants. All STM must be stored in a secure, temperature
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 STM accountability, reconciliation, and record 
maintenance, as needed.
4.
Further guidance and information for the final disposition of unused STM are 
provided in the Study Reference Manual.
5. Under normal conditions of handling and administration, STM is not expected to pose 
significant safety risks to site staff.
7.6.
STM Compliance

When participants undergo intestinal permeability testing at the site, they will 
receive STM directly from the investigator or designee, under medical supervision. 
The date and time of each dose administered in the clinic will be recorded in the 
source documents. The dose of STM and study participant identification will be 
confirmed at the time of dosing by a member of the study site staff other than the 
person administering the STM.

If healthy participants need to prepare and administer the STM off-site such as at 
home, STM training will be provided and a record maintained by the investigator or 
designee.

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7.7.
Concomitant Therapy

Refrain from consumption of the following for 24 hours before and after the 
administration of STM:
o
Lactulose or mannitol-containing laxatives. Study sites will be asked to use 
movicol (polyethylene glycol) in place of lactulose.
o
Medicines with mannitol as an excipient (chlorthiazide sodium, some albumin 
preparations, some laxatives, tablets as a bulking agent).
o
Products containing sucralose.

For healthy participants only, refrain from consumption of aspirin for 48 hours
before taking the STM and for the 24-hour urine collection period see Section 6.6.2.

For healthy participants only, antibiotic use 2 weeks prior to STM administration and 
during the study is not permitted.

Sennoside laxatives should be avoided. These can cause gastrointestinal irritation 
and may contribute to raised intestinal permeability.

Additional Glutamine supplementation in excess of that delivered with a standard 
feeding protocol should be avoided during the first 28 days of study participation. If 
supplementation is given inadvertently, then the patient will remain in the study, but 
the total dose and duration of additional glutamine supplementation must be recorded 
in the CRF.

Thermal injury participants that receive Intravenous (IV) mannitol for renal failure or 
raised intracranial pressure (testing to be delayed until 12 hours after last 
administration).
7.8.
Treatment after the End of the Study
There will be no ongoing STM administration following the end of this study.
8.
DISCONTINUATION CRITERIA
8.1.
Discontinuation of STM administration
Discontinuation of STM administration can be considered by the investigator in the event 
that an adverse event to the STM is observed. Withdrawal of further STM administration 
does not require withdrawal from the study.
8.2.
Withdrawal from the Study

A participant may withdraw from the study at any time at his/her own request, 
or may be withdrawn at any time at the discretion of the investigator for safety, 
behavioural, compliance or administrative reasons.

If the participant withdraws consent for disclosure of future information, the 
sponsor may retain and continue to use any data collected before such a 
withdrawal of consent.

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
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.
8.2.1.
Other Withdrawal Criteria

A participant will be withdrawn from the study following positive (and
confirmed) HIV test at screening; Serologic evidence of active Hepatitis B (HB) 
infection based on the results of testing for HBsAg, and anti-HBc or a positive 
test for Hepatitis C antibody confirmed by Hepatitis C RNA or antigen testing. 
If HCV RNA is not available, then the positive test for Hepatitis C antibody 
alone would be exclusionary. Results must be discussed with the medical 
monitor to withdraw the participant from the study and commence therapy 
according to local practice.

Healthy participants that are treated with antibiotics during the duration of the 
study.

Participants that experience signs and symptoms of gastro-intestinal infections 
during the duration of the study.

Withdrawals related to mental capacity as described in Section 6 and Appendix 
3.

Participants that received haemodialysis during the first 48 hours of the study 
(i.e. during the first measurement of intestinal permeability) will be excluded 
from the evaluable population and a replacement will be recruited.

Participants that received haemodialysis during later time points will not be 
excluded, but consideration will be given to recruiting an additional 
participant if 3 or more intestinal permeability measurements occur 
concurrently with haemodialysis.
8.3.
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 study site for a 
required study visit:

The site must attempt to contact the participant and reschedule the missed visit 
as soon as possible and counsel the participant on the importance of maintaining 
the assigned visit schedule and ascertain whether or not the participant wishes to 
and/or should continue in the study.

Before a participant is deemed lost to follow up, the investigator or designee 
must make every effort to regain contact with the participant (where possible, 3 
telephone calls and, if necessary, a certified letter to the participant’s last known 
mailing address or local equivalent methods). These contact attempts should be 
documented in the participant’s medical record.

Should the participant continue to be unreachable, he/she will be considered to 
have withdrawn from the study with a primary reason of lost to follow-up.

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9.
STUDY ASSESSMENTS AND PROCEDURES

Study procedures and their timing are summarized in the SoA.

Protocol waivers or exemptions are not allowed.

Safety concerns related to the STM should must be discussed with the sponsor 
immediately upon occurrence or awareness to determine if the participant should 
continue to be administered the STM.

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 thermally injured participant’s routine clinical 
management (e.g., weight measurement) and obtained before signing of ICF may be 
used for screening or baseline purposes provided the procedure met the protocol-
specified criteria and was performed within the time frame defined in the SoA. 
Procedures (the administration of STM) are not part of routine care for either healthy 
or thermal injury participants.

Healthy participants will be asked to donate a single blood sample on Day 1. No 
blood collection is specified in the SoA of this protocol for thermal injury 
participants (with the exception of HIV, Hepatitis B and Hepatitis C testing at 
baseline). The results of clinical laboratory blood tests will be recorded in the SIFTI-
2 study and the data used in this study. Likewise, blood collection for exploratory 
biomarker detection will be included in the SIFTI-2 study and the data used in this 
study.
9.1.
Efficacy Assessments
The administration of the STM is for the purpose of intestinal permeability measurement 
and is not therapeutic, therefore no efficacy will be assessed.
9.2.
Adverse Events
9.2.1.
Monitoring and reporting responsibilities
Healthy participants will be recruited to the HESTIA study alone and all AEs or SAEs 
occurring in this group should be managed according to this protocol.
Thermally injured participants recruited to this study will also be recruited to the SIFTI-2 
study. The following guidance relates only to AEs or SAEs which the investigator 
reasonably believes to be the result of a procedure or requirement unique to this (the 
HESTIA) protocol. All other AEs or SAEs will be reported and managed in accordance 
with the SIFTI-2 protocol.

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Unique procedures and requirements of HESTIA
1. The administration of STM
2. The collection of stool samples
3. Changes to standard of care for thermally injured participants:
a.
The fasts required during the measurement of intestinal permeability
b. The use of alternative laxatives to lactulose and sennosides.
The definitions of an AE or SAE for this study can be found in Appendix 4.
The investigator and any designees are responsible for detecting, documenting, and 
reporting events that meet the definition of an AE or SAE and remain responsible for 
following up AEs that are serious or that caused the participant to discontinue intestinal 
permeability measurement with the STM (see Section 8).
9.2.2.
Time Period and Frequency for Collecting AE and SAE 
Information

All SAEs will be collected from the start of unique HESTIA study procedures
until the final visit at the time points specified in the SoA (Section 2). However, 
any SAEs assessed as related to study participation (e.g., STM, protocol-
mandated procedures, invasive tests, or change in existing therapy) will be 
recorded from the time a participant consents to participate in the study.

All AEs will be collected from the start of unique HESTIA study procedures 
until the final visit at the time points specified in the SoA (Section 2).

Medical occurrences that begin before the start of unique HESTIA study 
procedures but after obtaining informed consent will be recorded on the Medical 
History/Current Medical Conditions section of the case report form (CRF) not 
the AE section.

All SAEs will be recorded and reported to the sponsor or designee immediately 
and under no circumstance should this exceed 24 hours, as indicated in 
Appendix 4. The investigator will submit any updated SAE data to the sponsor 
within 24 hours of it being available.

Investigators are not obligated to actively seek AEs or SAEs in former study 
participants. However, if the investigator learns of any SAE, including a death, 
at any time after a participant has been discharged from the study, and he/she 
considers the event to be reasonably related to the STM administration or study 
participation, the investigator must promptly notify the sponsor.

The method of recording, evaluating, and assessing causality of AEs and SAEs 
and the procedures for completing and transmitting SAE reports are provided in 
Appendix 4.
9.2.3.
Method of Detecting AEs and SAEs
Care will be taken not to introduce bias when detecting AEs and/or SAEs. If the 
participants are conscious, open-ended and non-leading verbal questioning of the 
participant is the preferred method to inquire about AE occurrence. For unconscious 

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patients or participants not always able to provide valid verbal responses to open-ended 
questions, the investigator or designee will need to identify AEs and/or SAEs through 
relevant clinical signs and/or investigations.
9.2.4.
Follow-up of AEs and SAEs
After the initial AE/SAE report, the investigator is required to follow proactively each 
participant at subsequent visits/contacts. All SAEs, will be followed until the event is 
resolved, stabilized, otherwise explained, or the participant is lost to follow-up (as 
defined in Section 8.3). Further information on follow-up procedures is given in 
Appendix 4.
9.2.5.
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 are met.

The sponsor has a legal responsibility to notify both the local regulatory 
authority and other regulatory agencies about the safety of the study. The 
sponsor will comply with country-specific regulatory requirements relating to 
safety reporting to the regulatory authority, Institutional Review Boards 
(IRB)/Independent Ethics Committees (IEC), and investigators.

Investigator safety reports must be prepared for suspected unexpected serious 
adverse reactions (SUSAR) according to local regulatory requirements and 
sponsor policy and forwarded to investigators as necessary.

An investigator who receives an investigator safety report describing a SAE or 
other specific safety information e.g., summary or listing of SAE) from the 
sponsor will review and then file it and will notify the IRB/IEC, if appropriate 
according to local requirements.
9.2.6.
Disease-Related Events and/or Disease-Related Outcomes Not 
Qualifying as SAEs
The following broad disease related events (DREs) are common in thermally injured 
participants and can be serious/life threatening:

Deterioration of condition.

Death (may be expected in burns of a large surface area).

Prolongation of hospital stay.

Persistent or significant disability or incapacity.
Because these events are typically associated with the disease under study, they will not 
be reported according to the standard process for expedited reporting of SAEs to GSK 
(even though the event may meet the definition of a SAE). These events will be recorded 
on the DRE page in the participant’s CRF within [the appropriate time frame agreed 
upon by the SRT for completion of DRE CRF pages]. These DREs will be monitored by
clinical study team on a routine basis.

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
NOTE: However, if the investigator considers that there is a reasonable 
possibility that the event was related to administration of STM or another 
unique or required element of the study (as defined in Section 9.2.1) then the 
event must be recorded and reported as an SAE (instead of a DRE).

A comprehensive list of further thermal injury related DREs can be found in 
Appendix 4 (Section 12.4).
9.3.
Treatment of Overdose
For this study, an overdose is defined as any dose of STM greater than defined in Section
7.1. No specific treatment is recommended for an overdose and treatment is at the 
discretion of the investigator.  The GSK medical monitor must be notified promptly.
9.4.
Safety Assessments
Planned time points for all safety assessments are provided in the SoA and summarised 
here.
Safety Assessment
When conducted
Healthy Participants
Thermally injured 
participants
Laboratory tests
Screening. Only repeated 
if clinically indicated in 
the opinion of the 
investigator.
Only if clinically 
indicated.
Brief Physical Examination 
including Vital Signs 
Recording
Screening, Day 1, Day 8, 
Day 15
As a part of routine 
clinical care whilst 
admitted (not protocol 
specified). Following day 
28 and 6 month intestinal 
permeability 
measurements (if patient 
not still admitted)
Detection of AEs
Day 1, Day 8, Day 15
Throughout the study
Assessment of health status
Screening, Day 1, Day 8, 
Day 15
Not required

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9.4.1.
Physical Examinations

A brief physical examination will include, at a minimum, assessments of the 
skin, lungs, cardiovascular system, and abdomen (liver and spleen).
9.4.2.
Vital Signs

A single vital sign measurement will be obtained at each time point indicated in 
SoA Table, and will include systolic and diastolic blood pressure and heart rate. 
Any abnormalities and changes in measurements will be communicated to the 
medical monitor.

Blood pressure and pulse measurements will be assessed 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, mobile phones).

Vital signs to be taken before blood collection for laboratory tests.

Repeat or unscheduled measurements may be taken at the discretion of the 
investigator.
9.4.3.
Clinical Safety Laboratory Assessment

All study related laboratory assessments will be performed by a local laboratory.
The laboratory reports must be reviewed by the investigator, this review 
documented and both report and review are to be filed with the source 
documents. 

Refer to Appendix 2 for the list of clinical laboratory tests to be performed and 
to the SoA for the timing and frequency.

All protocol-required laboratory assessments, as defined in Appendix 2, must be 
conducted in accordance with the laboratory manual and the SoA.

Clinically significant abnormal laboratory findings are those which are not 
associated with the underlying disease, unless judged by the investigator to be 
more severe than expected for the participant's condition.
9.5.
Study Procedures
The following procedures will be carried out during the study. 
9.5.1.
Fluid Balance Measurement
All fluid input and output will be recorded every 24 hours for thermally injured
participants.

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9.5.2.
Wound Healing
Assessment of wound healing will be the time to 95% wound healing. Physical 
parameters of the wound (e.g., rate of healing) will be recorded and collected as a part of 
both the HESTIA and the SIFTI-2 studies.
9.5.3.
Other Clinical Responses
To assess the relationship between severity of a participant’s condition following thermal 
injury and changes in intestinal permeability the following will be recorded and collected 
as a part of the SIFTI-2 study. Details can be found in the SIFTI-2 study protocol.

Number of ventilator-free days (ventilator start/restart/end date/time)

Number of vasopressor-free days (medication chart review)

Number of hemofiltration-free days (notes review)

Number of episodes of confirmed infection and sepsis

Number of surgical interventions

Total length of hospital stay

Calculate critical care and thermal injury severity scores
9.6.
Pharmacokinetics
PK parameters are not evaluated in this study.
9.7.
Pharmacodynamics
PD parameters are not evaluated in this study.
9.8.
Intestinal Permeability Assessments

Intestinal permeability will be determined by measuring the excretion of 
lactulose, mannitol and sucralose in urine following their enteral administration. 
It will be conducted in both healthy participants and thermally injured 
participants at the time points specified in the SoA.

The complete method for administration of STM and measurement of intestinal 
permeability is detailed in the SRM.

Urinary excretion of the orally ingested STM will be quantified using a 
technique such as capillary column gas chromatography.

Urine samples will be collected in plastic bottles for analysis. Urine collection 
will begin immediately following STM administration. Urine samples will be 
collected over 24 hours post-STM administration. Accurate collection of the 
total volume voided during this 24 hour period is critical.

Sample Preparation
Details of urine sample collection, processing, storage and shipping procedures are 
provided in the SRM.

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9.9.
Genetics
Genetics are not evaluated in this study.
9.10.
Sample Collection for Biomarker Analysis
The following biomarkers will be collected during the study. Details of sample 
processing, storage and shipping are included in the study reference manual.
Blood, stool and urine samples will be collected and stored. Timing of analyses and 
selected biomarkers will be dependent on the results of the intestinal permeability tests 
results.
9.10.1.
Blood Biomarkers
Healthy participants

Blood samples will be taken for healthy participants recruited in this study over 
the time period specified in the SOA. Blood will be taken adhering to standard 
operating procedure from venae puncture. 

The results of blood biomarker analysis will be evaluated in this study and 
compared to measures of intestinal permeability. 
Twenty (20) ml blood will be collected on Day 1 for biomarker analysis, and 
20ml blood will be collected at screening for screening tests. The biomarkers to 
be measured may include, but are not limited to:

Markers of microbial translocation

Markers of intestinal damage

Inflammatory markers: e.g. C-Reactive Protein, Procalcitonin, cytokines 
(including TNF-a, IL-6, IL-8, IFN-g, IL-10, IL-1b, IL-12p70, IL-17, IL-4, 
IL13, IL1Ra, MIP1a, MIP1b, MIP2, GCSF, GMCSF, MCP-1, RANTES, 
HMGB1).
Thermally injured participants

The blood required for this analysis in thermally injured participants will be 
collected as a part of the SIFTI-2 (IRAS 200366) study to which all thermally 
injured participants will be co-recruited. Details of the schedule for blood 
collection and the total volume of blood collected can be found in the SIFTI-2 
study protocol.
9.10.2.
Stool Sample Collection

Stool samples will be collected from all participants in this study over the time 
period specified in Section 2, Schedule of activities (SOA). Stool samples will 
be collected adhering to standard operating procedure. 

For thermally injured participants, the initial sample will be taken as close to 
time of injury as possible (“first stool sample produced upon admission”) and 

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Day 14. Further samples will be taken on day 28 (3 days) and at month 6 (14
days).

For healthy participants, a single sample will be collected at study entry 
(participants will be given a collection container at screening).
9.10.3.
Urine Sample Collection

Urine samples will be collected as a part of the measurement of intestinal 
permeability which is described in Section 9.4.

Additional urine samples will be collected from patients as part of the SIFTI-2 
study to which all thermally injured participants will be co-recruited. These will 
be used for, among other tests, the quantification of protein and microbial 
metabolites.

It is standard practice that patients admitted with burns of TBSA ≥15% will 
have a urinary catheter inserted on admission to ensure the accurate maintenance 
of fluid balance. A clean urine sample will be taken from the appropriate port on 
the urinary catheter. In patients who are not catheterised, a mid-stream urine 
(MSU) should be collected in a clean universal container where possible.

N.B. During the 24 hours following STM administration (during intestinal 
permeability measurement) urine samples must only be taken from the 5-hour of 
24-hour urine collections after the aliquots for sugar quantification have been 
taken.
Sample Preparation

Details of urine sample collection, processing, storage and shipping procedures 
are provided in the SRM.

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10.
STATISTICAL CONSIDERATIONS
All statistical analyses will be performed using SAS (SAS Institute Inc., Cary, North 
Carolina, USA), version 9.2 or higher.  Before database lock, a reporting and analysis 
plan (RAP) will be issued as a separate document, providing detailed methods for the 
analyses outlined below.  Any deviations from the planned analyses will be described in a 
RAP addendum and justified in the final integrated clinical study report.
10.1.
Hypotheses
As this is an enabling study designed to better understand the biomarkers of intestinal 
permeability and other biomarkers in participants with moderate to severe burns, the 
statistical analysis for this study will be exploratory in order to better understand the 
parameters to inform future investigational medicinal product studies.
The key factors of interest in this study are to understand (i) the nature of any differences 
at entry in intestinal permeability between healthy participants and thermal injury 
participants (ii) to understand the trajectory of changes in intestinal permeability over 
time.
The key endpoint to be explored is the lactulose:mannitol (L/M) ratio, but other 
permeability biomarkers will also be explored. The analysis approaches to address these 
questions are exploratory, but will initially be conducted as outlined in Section 10.5 and 
Section 10.6.
10.2.
Sample Size Determination
Sample size is based on feasibility. No formal calculation of power or sample size has 
been performed, but a sample size of 15 healthy participants and 25 thermal injury 
participants (>15% TBSA) should be sufficient to provide useful estimates of variability 
in lactutose:manitol ratios, and any change in L/M ratio over time.
Although the key aim is to estimate the variability and L/M ratio and assess the 
difference in L/M ratio between thermal injury and healthy participants, for illustration, a 
trial including 25 thermal injury and 15 healthy participants would have 89% power to 
detect a 3-fold difference in L/M ratio between thermal injury and healthy participants 
using a 2-sided significance level of p<0.10.  This calculation uses a (log) b etween-
subject SD of 1.15, as estimated from the literature [Olquin, 2005].
10.3.
Data Analyses Consideration
In general, descriptive summaries will include number of subjects, mean, standard 
deviation, median, minimum, and maximum for continuous variables.  If data are log-
normally distributed data will be presented as number of subjects, geometric mean, 
coefficient of variation, minimum, and maximum; and percent for categorical variables. 
Summaries will present data by dose level and where appropriate, by assessment time.

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10.4.
Populations for Analyses
The Safety Population will consist of all subjects who receive at least 1 dose of STM 
and have at least on post-dose safety assessment.
The Evaluable Population will consist of all subjects who are entered into the study and 
have evaluable L/M ratio measurements.
10.5.
Statistical Analyses
10.5.1.
Safety Analyses
Administration of STM is for the measurement of intestinal permeability. The safety of 
this administration is not an endpoint of this study, but will be monitored and reported.
All safety data will be presented in data listings. Subject demographics, medical history, 
and prior and concomitant medications will be summarized using descriptive statistics. 
For continuous variables, these summaries will include number of subjects, mean, 
median, standard deviation, minimum, and maximum.
For categorical variables, the summaries will include frequencies and corresponding 
percentages. No inferential hypothesis testing will be performed on the safety variables.
Adverse events will be coded using the MedDRA classification system.
For healthy participants (who are recruited only to HESTIA), STM-emergent AEs will be 
defined as any AEs, regardless of relationship to STM administration, that occur after the
first dose of STM until the final follow-up visit. The STM-emergent AEs will be 
summarized for the overall number of AEs and the percentage of subjects who 
experience them. The total number of AEs will be summarized overall. The AEs will be 
further summarized by severity and relationship to STM. If relationship information is 
missing, the AE will be considered STM-related. Listings for the subsets of SAEs and 
STM-related SAEs will be provided. The SAEs and number of AEs leading to 
discontinuation of STM administration will be summarized. The incidence of AEs will 
also be summarized by system organ class and preferred term.
For thermally-injured participants, STM-emergent AEs will be defined as any AE 
deemed related to STM administration that occurs after STM administration until the 
follow-up visit. The STM-related emergent AEs will be summarized for the overall 
number of AEs and the percentage of subjects who experience them. Listings for the 
subsets of SAEs and STM-related SAEs will be provided. The SAEs and number of AEs 
leading to discontinuation of STM administration will be summarized. The incidence of 
AEs will also be summarized by system organ class and preferred term.
As laboratory data and vital signs are only collected at screening for healthy participants, 
these data will be listed only. Clinical laboratory values that are outside of the reference 
ranges will be flagged and evaluated for clinical significance by the investigator. Physical 
examination findings will be listed. For thermally-injured participants, physical 

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examination findings and clinical laboratory values will be highly abnormal and as such 
any data collected will only be listed.  Disease-related findings and changes will not be 
reported.
10.5.2.
Other Analyses
Biomarker exploratory analyses will be described in the RAP.
10.5.3.
Interim Analyses
No formal interim analysis will be performed.
10.6.
Analyses of lactulose/mannitol ratio
In all analyses the variable TBSA will be a categorical variable defined as “Yes” for 
thermally injured participants, and “No” for healthy participants.
Differences in permeability at entry
Intestinal permeability biomarkers will be summarised by TBSA group and overall.  Data 
will summarised by geometric mean, CV, minimum, maximum and N.  A model will be 
fitted, defined as
Log (L/M ratio) = intercept + TBSA
Trajectory of the L/M ratio over time
Intestinal permeability biomarkers will be summarised over time, by TBSA group and 
overall.  Data will summarise geometric mean, CV, minimum, maximum and N.  A 
model will be fitted, defined as
Log (L/M ratio at time X / L/M ratio at entry) = intercept + Time + TBSA + Time*TBSA.
This will be a repeated measurement analysis and will assess the rate of improvement in 
L/M ratio over time, and assess how this changes relative to healthy participants.  If 
required, further modelling assessing more complex relationships between L/M ratio and 
time may be undertaken.  Given this is an exploratory study the most appropriate 
variance-covariance matrix regarding the correlation of data over time will be explored as 
part of the statistical analysis.
Data from this model may also be used to estimate the time to 50% improvement (or 
other degrees of improvement) in L/M ratio in relation to L/M ratio values seen in 
healthy participants.  This will be used to assess the clinical relevance and sensitivity of 
such measures.
A model fitting log (AUC of L/M ratio) = intercept + TBSA will also be fitted.  AUC will 
be calculated using all measurements taken over time.  This will provide a summary of 
the weighted average L/M ratio value over time.

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The use of %TBSA will also be assessed in the above analyses as a continuous covariate.  
The effects of age and Baux score will also be evaluated to understand differences in 
intestinal permeability in these groups [Osler, 2010].

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11.
REFERENCES
Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, Ouellette AJ. Secretion of 
Microbicidal Alpha-Defensins by Intestinal Paneth Cells in Response to Bacteria. Nat 
Immunol 2000; 1: 113-118
Bjarnason I, MacPherson A, Hollander D. Intestinal Permeability: An Overview. 
Gastroenterology 1995; 108: 1566-1581
Deitch EA, XU D, Kaisse VL, Role of the Gut in the Development of Injury and Shock 
Induced SIRS and MODS: the Gut-Lymph Hypothesis, A Review. Front Biosci 2006; 
11:520-528
Deitch EA. Intestinal Permeability Is Increased in Burn Patients Shortly After Injury. 
British Journal of Surgery 1990; 77:587-592. 
Del Valle-Pinero A, Van Deventer H, Fourie N et al. Gastrointestinal Permeability in 
Patients with Irritable Bowel Syndrome Assessed Using a Four Probe Permeability 
Solution. Clin Chim Acta. 2013 March 15; 418: 97–101.
Derikx J, Van Waardenburg D, Granzen B et al: Detection of Chemotherapy-Induced 
Enterocyte Toxicity with Circulating Intestinal Fatty Acid Binding Protein. J Paediatr 
Hematol Oncol 2006;28:267-269
Doig CJ, Sutherland LR, Sandham JD, Fick GH, Verhoef M, Meddings JB: Increased 
Intestinal Permeability is Associated with the Development of Multiple Organ 
Dysfunction Syndrome in Critically Ill ICU Patients.  Am J Respir Crit Care Med 
1998;158:444-451
European Commission, Health and Consumers Directorate-General.  The Rules 
Governing Medicinal Products in the European Union Volume10-Guidance Documents 
Applying Investigational Medicinal Products (NIMPS) (Rev. 1,March 2011) Ref. 
Ares(2011)300458 - 18/03/2011
Grootjans J, Thuijls G, Verdam F et al. World J Gastrointest Surg 2010 March 27; 2(3):
61-69
Hammer A, Morris N, Earley Z, Choudhry M. The First Line of Defense: The Effects of 
Alcohol on Post-Burn Intestinal Barrier, Immune Cells, and Microbiome. Alcohol Res. 
2015;37(2):209-22
Hampson P et al. Neutrophyl Dysfunction, Immature Granulocytes, and Cell Free DNA 
Are Early Biomarkers of Sepsis in Burn-Injured Patients. Annals of Surgery Ann Surg.
2016 May 26. [Epub ahead of print]
Menzies IS. Intestinal Permeability in Coeliac Disease. Gut 1972;13:847

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Olquin F, Araya M, Hirsch S, Brunser O, Ayala V, Rivera R, Gotteland M:Prebiotic 
Ingestion Does Not Improve Gastrointestinal Barrier Function in Burn Patients. Burns 
2005 Jun; 31 (4):482-8
Osler T, Glance LG, Hosmer DW. Simplified estimates of the probability of death after 
burn injuries: extending and updating the baux score. J Trauma. 2010;68(3):690-7
Sequeira IR, Lentle RG, Kruger MC, Hurst RD. Standardising the Lactulose Mannitol 
Test of Gut Permeability to Minimise Error and Promote Comparability. PLoS ONE. 
2014;9(6):e99256
Ziegler TR, Smith RJ, O’Dwyer ST, Demling RH, Wilmore DW: Increased Intestinal 
Permeability Associated with Infection in Burn Patients. Arch Surg 1988 Nov; 123 
(11):1313-9

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12.
APPENDICES
12.1.
Appendix 1: Abbreviations and Trademarks
ACR
Albumin/creatinine ratio
AE
Adverse Event
Anti-HBc
Anti-Hepatitis C
ART
Anti-retrovial treatment
CFR
Code of Federal Regulations
D
Day
G
Grams
eCRF
Electronic Case Report Form
ICU
Intensive Care Unit
GCP
Good Clinical Practice
GCSP
Global Clinical Safety and 
Pharmacovigilance
GI
Gatrointestinal
HB
Hepatitis B
HBs AG
Hepatitis B Antigen
HCV
Hepatitis C
GSK
GlaxoSmithKline
HIV
Human Immunodeficiency Virus
ICF
Informed Consent Form
ICH
International Conference on Harmonization 
of Technical Requirements for Registration 
of Pharmaceuticals for Human Use
ICU
Intensive Care Unit
IEC
Independent Ethics Committee
IRB
Institutional Review Board
ISS
Investigator Sponsored Study
L/M
Lactulose/mannitol ratio
LAR
Legally Authorised Representative
mL
Milliliter
MODS
Multi-organ dysfunction syndrome
NIMP
Non-investigational medicinal product
NC
Nominated Consultee
PC
Personal Consultee
PCR
Polymerase Chain Reaction
RAP
Reporting and Analysis Plan
SAE
Serious adverse event
SIFTI-2
A Multi-centre, Prospective Study to 
Examine the Relationship between 
Neutrophil Function and Sepsis in Adults 
and Children with Severe Thermal Injuries
SoA
Schedule of Activities
SRM
Study Reference Manual

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STM
Sugar Test Material
TBSA
Total body surface area
TNF
Tumour Necrosis Factor
WOCBP
Women of Child Bearing Potential
Trademark Information
Trademarks of the GlaxoSmithKline 
group of companies
Trademarks not owned by the 
GlaxoSmithKline group of companies
NONE
SAS

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12.2.
Appendix 2: Clinical Laboratory Tests

All clinical laboratory tests will be performed in the local laboratory.

Protocol-specific requirements for inclusion or exclusion of participants are
detailed in Section 6 of the protocol.

Additional tests may be performed at any time during the study as determined 
necessary by the investigator or required by local regulations.
Table 2
Protocol-Required Safety Laboratory Assessments for Healthy 
Participants
Laboratory 
Assessments
Parameters
Haematology
Platelet Count
WBC count with Differential:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
RBC Count
Hemoglobin
Hematocrit
Clinical 
Chemistry1
Urea
Potassium
Total bilirubin
Creatinine
Sodium
Alanine 
Aminotransferase
(ALT)/ 
Total Protein
Glucose 
nonfasting
Calcium
Alkaline 
phosphatase
Routine 
Urinalysis

pH, glucose, protein, blood, ketones by dipstick

Microscopic examination (if blood or protein is abnormal)
Other 
Screening 
Tests

Urine alcohol and drug screen (to include at minimum: amphetamines, 
barbiturates, cocaine, opiates, cannabinoids and benzodiazepines)]

Urine human chorionic gonadotropin (hCG) pregnancy test (as needed for 
women of childbearing potential)1

Serology (HIV antibody, hepatitis B surface antigen [HBsAg], and hepatitis C 
virus antibody)
NOTES :
1.
Local urine testing will be standard for the protocol unless serum testing is required by local regulation or IRB/IEC.

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12.3.
Appendix 3: Study Governance Considerations
12.3.1.
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 and other relevant documents (e.g., 
advertisements) must be submitted to an IRB/IEC by the investigator and 
reviewed and approved by the IRB/IEC before the study is initiated. 

Any amendments to the protocol will require IEC/IRB approval before 
implementation of changes made to the study design, except for changes 
necessary to eliminate an immediate hazard to study participants. 

Any amendments to the SIFTI-2 protocol which impact on this protocol will be 
reviewed and may result in changes to this protocol being required. Any such 
changes will be subject to IEC/IRB approval before implementation.

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
12.3.2.
Recruiting participants under the Mental Capacity Act 2005
On admission into hospital the patient’s capacity will be assessed. A patient may lack 
capacity due to the severity of their injury, arriving intubated and ventilated or due to a 
pre-existing co-morbidity.
Please note, the same process will also be followed for the SIFTI-2 study to which 
thermally injured participants will be co-recrruited.
If a patient does not have the capacity to make an informed decision, the research team 
will approach a patient’s LAR, also known as a Personal Consultee. Examples of 
personal consultees include:

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
A family member, carer or friend

An attorney acting under a Lasting Power of Attorney

A court appointed deputy, provided that they had a relationship with, or personal 
knowledge of, the person lacking capacity before their appointment as deputy.
There may be circumstances in which a personal consultee is not available, some 
examples of this are:

Where no family member or friend is willing to act as a personal consultee

Where the family member or friends live a long distance away and/or are not in 
frequent contact with the patient who lacks capacity

Where the regular carers of the person who lacks capacity are doing so for 
payment or in a professional capacity (e.g. care home staff or nurses)

Where someone is acting on a professional role (e.g. their GP or solicitor)
In this case, a nominated consultee will be required. A nominated consultee is considered 
to be a medical professional that has no connection to the research trial, but has an 
understanding of the implications of the research trial on the participant.
In these circumstances, examples of nominated consultees are:

An emergency department doctor, preferably Consultant level.

Intensive Care doctor, preferably Consultant level.

Doctor from the burns team, not directly involved in the research study.
Once a personal or nominated consultee has been identified, they will be provided with a 
specific information leaflet about the trial. The personal and nominated consultee will be 
asked if they feel the study would be something the participant would have no objections 
to. If in their opinion the participant would have no objection to being recruited into a 
research trial the consultee will be asked to sign a declaration form.
12.3.3.
Determining Whether a Participant has Capacity Under the 
Mental Capacity Act (2005)
Prior to deciding that a patient does not have the capacity to give informed consent the 
researcher must follow the Mental Capacity Act (2005) to ensure that the participant does 
not hold capacity. The principles of the MCA which we will adhere are as follows:

A person must be assumed to have capacity unless it is established that he/she
lacks capacity.

A person is not to be treated as unable to make a decision unless all practical steps 
to help him/her to do so have been taken without success.

A person is not to be treated as unable to make a decision merely because he/she
makes an unwise decision.

An act done or decision made, under this Act for or on behalf of a person who 
lacks capacity must be done, or made, in his best interests.

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
Before the act is done, or the decision is made, regard must be had to whether the 
purpose for which it is needed can be as effectively achieved in a way that is less 
restrictive of the person's rights and freedom of action.
A decision to appoint a consultee on behalf of a patient will be made if the participant is 
unable to:
1. (a) understand the information relevant to the decision,
(b) retain that information,
(c) use or weigh that information as part of the process of making the decision, or
(d) communicate his/her decision (whether by talking, using sign language or any 
other means).
2. A person is not to be regarded as unable to understand the information relevant to a 
decision if he/she is able to understand an explanation of it given to him in a way that is 
appropriate to his circumstances (using simple language, visual aids or any other means).
3. The fact that a person is able to retain the information relevant to a decision for a short 
period only does not prevent him/her from being regarded as able to make the decision.
4. The information relevant to a decision includes information about the reasonably 
foreseeable consequences of
(a) deciding one way or another, or
(b) failing to make the decision.

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Figure 4
HESTIA Thermal Injury Participant Recruitment
12.3.4.
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. 

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Investigators are responsible for providing information on financial interests during the 
course of the study and for 1 year after completion of the study.
12.3.5.
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.

Healthy participants who are rescreened are required to sign a new ICF.
12.3.6.
Data Protection

Participants will be assigned a unique identifier by the sponsor. Any participant 
records or datasets that are transferred to the sponsor will contain the identifier 
only; participant names or any information which would make the participant 
identifiable will not be transferred.

The participant must be informed that his/her personal study-related data will be 
used by the sponsor in accordance with local data protection law. The level of 
disclosure must also be explained to the participant.

The participant must be informed that his/her medical records may be examined 
by Clinical Quality Assurance auditors or other authorized personnel appointed 
by the sponsor, by appropriate IRB/IEC members, and by inspectors from 
regulatory authorities.
12.3.7.
Committees Structure
An Independent Data Monitoring Committee or similar review group will not be used in 
this study, but an internal preliminary data review will be conducted.
The Data Review team will consist of the GSK medical monitor, clinical and operational 
leads, statistician, early development lead and the safety officer. They will meet at 

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intervals specified within the data review charter to review data relevant to the future 
conduct of the study, and will also assess any risk to study participants.
12.3.8.
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 
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.
12.3.9.
Dissemination of Clinical Study Data

Study information from this protocol will be posted on publicly available clinical 
trial registers before enrollment of subjects begins.

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.

The procedures and timing for public disclosure of the results summary and for 
development of a manuscript for publication will be in accordance with GSK 
Policy.

A manuscript will be progressed for publication in the scientific literature if the 
results provide important scientific or medical knowledge.
12.3.10.
Data Quality Assurance

All participant data relating to the study will be recorded on printed or electronic 
CRF unless transmitted to the sponsor or designee electronically (e.g., 
laboratory data). The investigator is responsible for verifying that data entries 
are accurate and correct by physically or electronically signing the CRF.

The investigator must maintain accurate documentation (source data) that 
supports the information entered in the CRF.

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
The investigator must permit study-related monitoring, audits, IRB/IEC review, 
and regulatory agency inspections and provide direct access to source data 
documents.

The sponsor or designee is responsible for the data management of this study 
including quality checking of the data.

Study monitors will perform ongoing source data verification to confirm that 
data entered into the CRF by authorized site personnel are accurate, complete, 
and verifiable from source documents; that the safety and rights of participants 
are being protected; and that the study is being conducted in accordance with the 
currently approved protocol and any other study agreements, ICH GCP, and all 
applicable regulatory requirements.

Records and documents, including signed ICF, pertaining to the conduct of this 
study must be retained by the investigator for 15 years after study completion 
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.
12.3.11.
Source Documents

Source documents provide evidence for the existence of the participant and 
substantiate the integrity of the data collected. Source documents are filed at the 
investigator’s site.

Data reported on the CRF or entered in the eCRF that are transcribed from 
source documents must be consistent with the source documents or the 
discrepancies must be explained. The investigator may need to request previous 
medical records or transfer records, depending on the study. Also, current 
medical records must be available.

Definition of what constitutes source data can be found in the study specific 
Source Data Verification document.
12.3.12.
Study and Site Closure
GSK or its designee reserves the right to close the study site or terminate the study at any 
time for any reason at the sole discretion of GSK. Study sites will be closed upon study 
completion. A study site is considered closed when all required documents and study 
supplies have been collected and a study-site closure visit has been performed.
The investigator may initiate study-site closure at any time, provided there is reasonable 
cause and sufficient notice is given in advance of the intended termination.
Reasons for the early closure of a study site by the sponsor or investigator may include 
but are not limited to:

Failure of the investigator to comply with the protocol, the requirements of the 
IRB/IEC or local health authorities, the sponsor's procedures, or GCP guidelines

Inadequate recruitment of participants by the investigator

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12.4.
Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting
Definition of AE
AE Definition

An AE is any untoward medical occurrence in a clinical study participant, temporally 
associated with study participation, whether or not considered related to the study.

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 study participation.

NOTE: As detailed in Section 9.2.1, only AEs deemed to be related to procedures or 
requirements unique to the HESTIA study will be recorded/reported for thermally 
injured participants. All other AEs will be recorded/reported through the SIFTI-2 
study.
Events Meeting the AE Definition 

Any abnormal laboratory test results (haematology, 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 STM 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. 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.
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. Table 3
provides a list of commonly occurring AEs in participants with severe thermal injury 
which may meet this definition.

The disease/disorder being studied or expected progression, signs, or symptoms of 

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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.
Table 3
Complications of severe thermal injury which can be considered as 
associated with the underlying disease and do not require reporting 
as AEs unless judged to be more severe than expected for the 
participant’s condition or related to HESTIA study procedures.
If any of the following are observed, then an AE will be recorded in the SIFTI2 study 
CRF.
If any of the following are observed and deemed to be related to STM administration or 
other unique requirements of the HESTIA study, then to be recorded in the HESTIA CRF 
and reported to GSK as per guidance below.
System Assessment
Complication type
Action
Airway problems
Failed extubation
Record AE
Tracheostomy complication
Breathing Problems
Pneumothorax
Record AE
Pulmonary Oedema
Respiratory Arrest
Pneumonia
VAP
Acute lung injury (ALI)
ARDS
Circulatory Problems
Haemodynamic instability
Record AE
Record ionotrope dose in 
con-meds
Increasing vasoactive drug 
support
Arrhythmia

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System Assessment
Complication type
Action
Endocarditis
Acute LVF/CCF
Cardiac Arrest
MI
Neurological Problems
Reduced GCS (off sedation)
Record AE
Intra-Cranial bleed
CVA
Acute confusion/Delirium
Meningitis-bacterial
Oedema Complications
Abdominal Compartment 
Syndrome (ACS)
Record AE
Acute Limb compartment 
syndrome
Microbiological problems
Sepsis 
Record AE
Record in Microbiology 
form
Chest Infection
Lower Respiratory Tract 
Infection
UTI
Bloodstream Infection (BSI)
Wound infection
Intra-vascular catheter (line) 
infection
Infective diarrhoea
Clostridium difficile 
infection/pseudomembranous 
colitis
Renal/Urology problems
Acute rhabdomyolysis
Record AE

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System Assessment
Complication type
Action
Acute renal failure
Ensure biochemistry and 
CK results recorded n CRF
Acute urinary retention
Renal replacement therapy
Thromboembolic 
complications
Lower limb DVT
Record AE with location of 
thrombus
Upper limb DVT
Pulmonary embolism
Other VTE
Fat embolism
Wound complications
Major graft loss
Record AE with details
Major skin substitute loss
Wound infection
Invasive wound infection
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).
Please note that, as described in Section 9.2.6 a, b, c and d below are considered as 
‘disease related events’ as they occur commonly in patients following thermal injury 
unless, in the opinion of the investigator, they are directly related to STM administration 
or other unique requirements of the HESTIA study.
A SAE is defined as any untoward medical occurrence that:
a.
Results in death
b.
Is life-threatening
The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
participant was at risk of death at the time of the event. It does not refer to an event, 
which hypothetically might have caused death, if it were more severe.
c.
Requires inpatient hospitalization or prolongation of existing hospitalization
In general, hospitalization signifies that the participant has been detained (usually 

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involving at least an overnight stay) at the hospital or emergency ward for observation 
and/or treatment that would not have been appropriate in the physician’s office or 
outpatient setting. Complications that occur during hospitalization are AE. If a 
complication prolongs hospitalization or fulfils any other serious criteria, the event is 
serious. When in doubt as to whether “hospitalization” occurred or was necessary, the AE 
should be considered serious.
Hospitalization for elective treatment of a pre-existing condition that did not worsen from 
baseline is not considered an AE.
d.
Results in persistent disability/incapacity

The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions.

This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 
and accidental trauma (e.g., sprained ankle) which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption.
e.
Is a congenital anomaly/birth defect
f.
Other situations:

Medical or scientific judgment should be exercised in deciding whether SAE 
reporting is appropriate in other situations such as important medical events that may 
not be immediately life-threatening or result in death or hospitalization but may 
jeopardize the participant or may require medical or surgical intervention to prevent 
one of the other outcomes listed in the above definition. These events should usually 
be considered serious.
Examples of such events include invasive or malignant cancers, intensive treatment 
in an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency 
or drug abuse.
Recording AE and SAE
AE and SAE Recording

When an AE/SAE occurs, it is the responsibility of the investigator to review all 
documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) 
related to the event.

The investigator will then record all relevant AE/SAE information in the CRF.

It is not acceptable for the investigator to send photocopies of the participant’s 
medical records to GSK in lieu of completion of the GSK /AE/SAE CRF page.

There may be instances when copies of medical records for certain cases are 
requested by GSK. In this case, all participant identifiers, with the exception of the 
participant number, will be redacted on the copies of the medical records before 

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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 sufficiently discomfort and interferes with normal 
everyday activities.

Severe: An event that prevents normal everyday activities. An AE that is assessed as 
severe should not be confused with an SAE. Severe is a category utilized for rating 
the intensity of an event; and both AE and SAE can be assessed as severe.
An event is defined as ‘serious’ when it meets at least 1 of the predefined outcomes 
as described in the definition of an SAE, NOT when it is rated as severe.
Assessment of Causality

The investigator is obligated to assess the relationship between administration of the 
NIMP (STM) 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 STM administration 
will be considered and investigated.

The investigator will also consult the Product Information, for marketed products, in 
his/her assessment.

For each AE/SAE, the investigator must document in the medical notes that he/she 
has reviewed the AE/SAE and has provided an assessment of causality.

There may be situations in which an SAE has occurred and the investigator has 
minimal information to include in the initial report to GSK. However, it is very 
important that the investigator always make an assessment of causality for 
every event before the initial transmission of the SAE data to GSK.

The investigator may change his/her opinion of causality in light of follow-up 
information and send an SAE follow-up report with the updated causality 
assessment.

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
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.

The investigator will submit any updated SAE data to GSK within 24 hours of 
receipt of the information.
Reporting of SAE to GSK
SAE Reporting to GSK via 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.

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 to the medical monitor by telephone.

Contacts for SAE reporting can be found in 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.

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 

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complete and sign the SAE CRF pages within the designated reporting time frames.

Contacts for SAE reporting can be found in SRM.

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12.5.
Appendix 5
The following table of study objectives specifies, for each objective, the provenance of 
clinical data and samples which will be used to explore that endpoint
Objective
Endpoint
Origin of Data/Samples
Co-Primary
1.
To determine the impact of 
thermal injury on the magnitude 
of small intestinal permeability 
change as soon as possible 
following injury compared to 
healthy participants

Lactulose/Mannitol 
(L/M) ratio at entry

HESTIA study
2.
To characterise the effect of 
thermal injury on small 
intestinal permeability over time 
and establish the clinical and 
demographic factors which can 
influence it

Changes in L/M 
ratio over time

HESTIA Study
Exploratory
3.
To determine the impact of 
thermal injury on colonic 
permeability as soon as 
possible following injury 
compared to healthy 
participants

Fractional excretion 
of sucralose at entry

HESTIA Study
4.
To characterise the effect of 
thermal injury on colonic 
permeability over time 

Changes in the 
fractional excretion 
of sucralose over 
time

HESTIA Study
5.
To assess the relationship 
between severity of a 
participant’s condition following 
thermal injury and changes in 
intestinal permeability†

Number of 
ventilator-free days

Number of 
vasopressor-free 
days

Number of 
hemofiltration-free 
days

Number of episodes 

All clinical data will be 
obtained from SIFTI2.

Permeability measurements 
will be obtained from HESTIA

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Objective
Endpoint
Origin of Data/Samples
of confirmed 
infection and sepsis

Number of  surgical 
interventions

Total length of 
hospital stay

Calculate critical 
care and thermal 
injury severity 
scores
6.
To assess plasma and urine 
biomarkers of intestinal 
permeability, bacterial 
translocation and renal tubular 
dysfunction following thermal 
injury†

Change in markers 
of intestinal 
mucosal damage 
samples from blood

Change in urine 
protein:creatinine 
and urine 
albumin:creatinine 
ratios

Blood biomarkers obtained 
from SIFTI-2 (and HESTIA 
for healthy participants)

Urine for microbial metabolite 
analysis, claudin 3 and KIM 1 
obtained from SIFTI-2 (and 
HESTIA for Healthy 
participants)

Urine albumin:creatinine and 
protein:creatinine ratios 
obtained from SIFTI-2 (and 
HESTIA for healthy 
participants)

Permeability data (STM 
absorption) from HESTIA
7.
To assess the impact of 
thermal injury and intestinal 
permeability on the intestinal 
microbiome compared to 
healthy participants

Changes in
microbiome of acute 
and convalescent 
stool samples

Stool samples collected in 
HESTIA protocol

Permeability data from 
HESTIA
8.
To assess the impact of pre-
existing co-morbid conditions 
on intestinal permeability and 
clinical outcome following 
thermal injury†

Medical history and 
drug history at the 
time of admission

Medical History data from 
HESTIA

Permeability data from 
HESTIA
9.
To assess wound healing

Time to wound 
recovery (e.g. 95%)

Wound healing assessment 
data from clinical notes will 
be captured in HESTIA at 14 

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Objective
Endpoint
Origin of Data/Samples
day, 28 day and 6 month 
visits.
10. To characterise intestinal 
microbiota, and correlate its 
composition with both intestinal 
permeability and bacterial 
detection in blood†

Changes in 
intestinal 
microbiome

Bacterial markers of 
translocation

Microbiome data from 
HESTIA study

Blood Biomarker data from 
SIFTI-2 (thermally injured 
participants) and HESTIA 
(healthy participants.

2016N289648_02 
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GlaxoSmithKline group of companies 
 
206243 
 
1 
 
TITLE PAGE 
Protocol Title: A Prospective, Longitudinal Study to Investigate the Effect of Thermal 
Injury on Intestinal Permeability and Systemic Inflammation (HESTIA) 
Protocol Number: 206243/02 
Short Title: A Prospective, Longitudinal Study to Investigate the Effect of Thermal 
Injury on Intestinal Permeability and Systemic Inflammation (HESTIA) 
Compound Number: NONE 
 
Sponsor Name and Legal Registered Address:  
GlaxoSmithKline Research & Development Limited 
980 Great West Road 
Brentford 
Middlesex, TW8 9GS 
UK 
Medical Monitor Name and Contact Information will be provided separately. 
Regulatory Agency Identifying Number(s): NA 
Approval Date: 26-SEP-2017 
 
 
 
 
 
 
 
 
 
 
Copyright 2017 the GlaxoSmithKline group of companies.  All rights reserved.  
Unauthorised copying or use of this information is prohibited. 

PPD
PPD

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PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE 
DOCUMENT HISTORY 
Document 
Date 
Amendment 2 
26-Sep-2017 
Amendment 1 
15-Aug-2017 
Original Protocol 
02-Mar-2017 
 
Amendment 2: 26-Sep-2017 
Overall Rationale for the Amendment: The Ethics Committee requested the legally 
acceptable representative be updated with personal or nominated consultee in the 
protocol. 
 
Section # 
and Name 
Description of Change 
Brief Rationale 
6.1.2 
Legally acceptable representative 
was replaced with personal or 
nominated consultee. 
At the request of the Ethics Committee. 
6.1.4 
Legally acceptable representative 
was replaced with personal or 
nominated consultee. 
For consistency. 
12 
Removed LAR abbreviation 
For consistency. 
12.3.2, 12.3 
and 12.5 
Removed LAR 
For consistency. 
6.7 
Screen failure will be assigned a 
new participant number. 
Previous language was in conflict with FDA 
guidelines on subject numbering for re-
screened subjects as defined in the Study 
Data Technical Conformance Guide v2 
(issued in December, 2014). 
Appendix 6 
Protocol Amendment History was 
added. 
Per template instructions. 

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TABLE OF CONTENTS 
PAGE 
PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE ..................................... 3 
1. 
SYNOPSIS ................................................................................................................. 7 
2. 
SCHEDULE OF ACTIVITIES (SOA) ........................................................................ 10 
2.1. 
Healthy Participants (Group 1) ..................................................................... 10 
2.2. 
Thermal Injury Participants (Group 2) .......................................................... 12 
3. 
INTRODUCTION ..................................................................................................... 15 
3.1. 
Study Rationale ............................................................................................ 15 
3.2. 
Background .................................................................................................. 15 
3.2.1. 
Thermal Injury, Intestinal Permeability and Multi-Organ 
Dysfunction Syndrome .................................................................. 15 
3.2.2. 
Intestinal Barrier Function and Its Measurement .......................... 16 
3.2.3. 
The Intestinal Microbiome and Thermal Injury .............................. 17 
3.3. 
Benefit/Risk Assessment .............................................................................. 18 
3.3.1. 
Risk Assessment .......................................................................... 18 
3.3.2. 
Benefit Assessment ...................................................................... 22 
3.3.3. 
Overall Benefit:Risk Conclusion .................................................... 22 
4. 
OBJECTIVES AND ENDPOINTS ............................................................................ 22 
5. 
STUDY DESIGN ...................................................................................................... 24 
5.1. 
Overall Design .............................................................................................. 24 
5.2. 
Number of Participants ................................................................................. 24 
5.3. 
Participant and Study Completion ................................................................ 25 
5.4. 
Scientific Rationale for Study Design ........................................................... 25 
5.4.1. 
Co-recruitment to the SIFTI-2 Study ............................................. 25 
5.4.2. 
Recruitment and Sampling Schedule ............................................ 26 
5.4.3. 
Inclusion of Healthy Participants ................................................... 28 
5.4.4. 
Preliminary Data Review ............................................................... 28 
5.4.5. 
The Use of the Sugar Test Materials (Lactulose, Mannitol 
and Sucralose) .............................................................................. 28 
6. 
STUDY POPULATION ............................................................................................. 28 
6.1. 
Recruiting Participants with Differing Mental Capacity ................................. 29 
6.1.1. 
Adult Participants Determined to Have Mental Capacity at 
Study Entry and Throughout the HESTIA Study ........................... 29 
6.1.2. 
Adult Participants Lacking Mental Capacity for the 
Duration of the HESTIA Study ...................................................... 29 
6.1.3. 
Adult Participants Lacking Mental Capacity (either at 
Study Entry or During the Study) Who Later Regain 
Capacity and Are Required to Provide Informed Consent ............ 29 
6.1.4. 
Adult Participants with Mental Capacity to Provide 
Consent at Study Entry Who are Later Deemed No Longer 
to have Mental Capacity ............................................................... 29 
6.2. 
Inclusion Criteria for Healthy Participants (Group 1) .................................... 30 
6.3. 
Inclusion Criteria for Thermal Injury Participants (Group 2) ......................... 30 
6.3.1. 
Age ................................................................................................ 30 

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6.3.2. 
Type of Participant and Disease Characteristics .......................... 30 
6.3.3. 
Other Inclusions ............................................................................ 30 
6.3.4. 
Gender .......................................................................................... 31 
6.4. 
Exclusion Criteria for Healthy Participants (Group 1) ................................... 31 
6.5. 
Exclusion Criteria for Thermal Injury Participants (Group 2) ........................ 31 
6.6. 
Lifestyle Restrictions .................................................................................... 33 
6.6.1. 
Meals and Dietary Restrictions ..................................................... 33 
6.6.2. 
Alcohol/Exercise/Aspirin (Healthy Participants only) .................... 33 
6.7. 
Screen Failures ............................................................................................ 33 
7. 
NON-INVESTIGATIONAL MEDICINAL PRODUCT (NIMP) .................................... 33 
7.1. 
STM Administered ........................................................................................ 35 
7.2. 
Dose Modification ......................................................................................... 36 
7.3. 
Method of STM Administration: Treatment Assignment ............................... 36 
7.4. 
Blinding ........................................................................................................ 36 
7.5. 
Preparation/Handling/Storage/Accountability ............................................... 36 
7.6. 
STM Compliance .......................................................................................... 36 
7.7. 
Concomitant Therapy ................................................................................... 37 
7.8. 
Treatment after the End of the Study ........................................................... 37 
8. 
DISCONTINUATION CRITERIA .............................................................................. 37 
8.1. 
Discontinuation of STM administration ......................................................... 37 
8.2. 
Withdrawal from the Study ........................................................................... 38 
8.2.1. 
Other Withdrawal Criteria .............................................................. 38 
8.3. 
Lost to Follow Up ......................................................................................... 38 
9. 
STUDY ASSESSMENTS AND PROCEDURES ...................................................... 40 
9.1. 
Efficacy Assessments .................................................................................. 40 
9.2. 
Adverse Events ............................................................................................ 40 
9.2.1. 
Monitoring and reporting responsibilities ...................................... 40 
9.2.2. 
Time Period and Frequency for Collecting AE and SAE 
Information .................................................................................... 41 
9.2.3. 
Method of Detecting AEs and SAEs ............................................. 41 
9.2.4. 
Follow-up of AEs and SAEs .......................................................... 42 
9.2.5. 
Regulatory Reporting Requirements for SAEs ............................. 42 
9.2.6. 
Disease-Related Events and/or Disease-Related 
Outcomes Not Qualifying as SAEs ............................................... 42 
9.3. 
Treatment of Overdose ................................................................................ 43 
9.4. 
Safety Assessments ..................................................................................... 43 
9.4.1. 
Physical Examinations .................................................................. 44 
9.4.2. 
Vital Signs ..................................................................................... 44 
9.4.3. 
Clinical Safety Laboratory Assessment ........................................ 44 
9.5. 
Study Procedures ......................................................................................... 45 
9.5.1. 
Fluid Balance Measurement ......................................................... 45 
9.5.2. 
Wound Healing ............................................................................. 45 
9.5.3. 
Other Clinical Responses ............................................................. 45 
9.6. 
Pharmacokinetics ......................................................................................... 45 
9.7. 
Pharmacodynamics ...................................................................................... 45 
9.8. 
Intestinal Permeability Assessments ............................................................ 45 
9.9. 
Genetics ....................................................................................................... 46 
9.10. 
Sample Collection for Biomarker Analysis ................................................... 46 
9.10.1. 
Blood Biomarkers .......................................................................... 46 

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9.10.2. 
Stool Sample Collection ................................................................ 47 
9.10.3. 
Urine Sample Collection ............................................................... 47 
10. STATISTICAL CONSIDERATIONS ......................................................................... 48 
10.1. 
Hypotheses .................................................................................................. 48 
10.2. 
Sample Size Determination .......................................................................... 48 
10.3. 
Data Analyses Consideration ....................................................................... 48 
10.4. 
Populations for Analyses .............................................................................. 49 
10.5. 
Statistical Analyses ...................................................................................... 49 
10.5.1. 
Safety Analyses ............................................................................ 49 
10.5.2. 
Other Analyses ............................................................................. 50 
10.5.3. 
Interim Analyses ........................................................................... 50 
10.6. 
Analyses of lactulose/mannitol ratio ............................................................. 50 
11. REFERENCES ........................................................................................................ 52 
12. APPENDICES .......................................................................................................... 54 
12.1. 
Appendix 1: Abbreviations and Trademarks ................................................ 54 
12.2. 
Appendix 2: Clinical Laboratory Tests .......................................................... 56 
12.3. 
Appendix 3: Study Governance Considerations ........................................... 57 
12.3.1. 
Regulatory and Ethical Considerations ......................................... 57 
12.3.2. 
Recruiting participants under the Mental Capacity Act 
2005 .............................................................................................. 57 
12.3.3. 
Determining Whether a Participant has Capacity Under 
the Mental Capacity Act (2005) ..................................................... 58 
12.3.4. 
Financial Disclosure ...................................................................... 60 
12.3.5. 
Informed Consent Process ........................................................... 60 
12.3.6. 
Data Protection ............................................................................. 61 
12.3.7. 
Committees Structure ................................................................... 61 
12.3.8. 
Publication Policy .......................................................................... 61 
12.3.9. 
Dissemination of Clinical Study Data ............................................ 62 
12.3.10. Data Quality Assurance ................................................................ 62 
12.3.11. Source Documents ....................................................................... 63 
12.3.12. Study and Site Closure ................................................................. 63 
12.4. 
Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting ........................................ 64 
12.5. 
Appendix 5: Origin of Data/Samples ............................................................ 72 
12.6. 
Appendix 6: Protocol Amendment History .................................................... 75 
 

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1. 
SYNOPSIS 
Protocol Title: A Prospective, Longitudinal Study to Investigate the Effect of Thermal 
Injury on Intestinal Permeability, and Systemic Inflammation (HESTIA) 
Short Title: Investigation of Thermal Injury and Intestinal Permeability 
Rationale: 
Patients with severe thermal injury [>20% total body surface area (TBSA)] are at risk for 
organ dysfunction and may develop multiple organ dysfunction syndrome (MODS). This 
study aims to explore the relationship between thermal injury, changes in intestinal 
permeability and the onset of MODS. The results of this study will be used to inform the 
design of future drug studies of a novel medicinal product, which is predicted to reduce 
this increased permeability. 
 
No GSK study treatment will be employed in this study. 
 
One of the central hypotheses over the last two decades to explain the onset of MODS in 
the context of critical illness imputes that an increase in intestinal permeability results in 
the translocation of bacteria and bacterial products to the systemic circulation where they 
drive inflammation and injury to distal organs [Deitch, 2006]. This hypothesis is 
supported by data showing that there is a significant increase in intestinal permeability in 
critically ill patients, including patients with thermal injury, and that the degree of 
intestinal permeability correlates with the onset and severity of MODS [Doig, 1998]. As 
such, therapies directed at minimising these changes in intestinal permeability, thereby 
limiting the translocation of bacteria to the systemic circulation, are hypothesised to have 
an impact on clinical outcome in these patients. 
 
Previous literature demonstrates that patients with severe thermal injury (>20% TBSA) 
display a significant and rapid increase in intestinal permeability that declines gradually 
over a two-week period following the injury [Olquin, 2005]. Additionally, these changes 
in intestinal permeability, correlate with the severity of sepsis observed in these patients 
[Ziegler, 1988]. 
 
The central hypothesis of this study, therefore, is that thermal injury alters intestinal 
barrier function allowing the translocation of bacteria and bacterial products to the 
systemic circulation where they contribute to the onset of MODS. 
 
The aims of the study are: 
 
1. To establish the magnitude and time course of changes in intestinal permeability to 
inform timing and duration of future investigational medicinal product administration. 
2. To establish the optimal method for assessment of intestinal permeability in patients 
with thermal injury. 
3. To describe the patient population most likely to benefit from a new medicinal 
product which could prevent changes in intestinal permeability. 

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4. To improve our understanding of the links between intestinal damage, changes in the 
gut microbiome and microbial translocation to the systemic circulation following 
thermal injury. 
 
In order to enter this study thermally injured participants will be required to co-enrol in 
this study and an allied study entitled: A Multi-centre, Prospective Study to Examine the 
Relationship between Neutrophil Function and Sepsis in Adults and Children with Severe 
Thermal Injury (SIFTI-2) (reference number IRAS ID: 200366). Clinical data, standard 
of care laboratory data and investigational biomarker data will be shared from the SIFTI-
2 study to this study and the combined data from both studies will be used to address 
exploratory endpoints. Participants of the SIFTI-2 study will be appropriately consented 
for this data sharing. 
 
Objectives and Endpoints: 
Objective 
Endpoint 
Co-Primary 
• To determine the impact of thermal injury 
on the magnitude of small intestinal 
permeability change as soon as possible 
following injury compared to healthy 
participants 
• Lactulose/Mannitol (L/M) ratio at entry 
• To characterise the effect of thermal 
injury on small intestinal permeability over 
time 
• Changes in L/M ratio over time 
 
Overall Design: 
A prospective, longitudinal study will be conducted in adult (≥18 years old) men and 
women admitted to a hospital following thermal injury. Measurements of intestinal 
permeability, inflammation and microbial translocation will be taken over a six month 
period. A cohort of healthy participants will also be recruited in order to determine the 
reference against which post-burn permeability measurements and other biomarkers will 
be compared. 
The lactulose-to-mannitol ratio is traditionally used to assess small intestinal permeability 
and sucralose to assess colonic permeability. Lactulose, mannitol and sucralose 
[henceforth referred to as sugar test material (STM)] will be intermittently administered 
enterally for the purpose of intestinal permeability measurement to examine permeability 
at different points along the GI tract and is described in Section 7. It is important to note 
that the administration of the STM is not therapeutic (lactulose and mannitol can be used 
as laxatives, however the amount in the STM is sub-therapeutic) and as such should be 
regarded as a non-investigational medicinal product (NIMP) [Guidance Documents 
Applying Investigational Medicinal Products (NIMPS), 2011]. 

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An internal preliminary data review will be conducted. This review is described in 
Section 5.4.4. 
Number of Participants: 
Table 1 in Section 5.2 described the number of participants proposed for the study. 
Sample size is based on feasibility. No formal calculation of power or sample size has 
been performed, but a sample size of 15 healthy participants and 25 thermal injury 
participants (≥15% TBSA) should be sufficient to provide useful estimates of variability 
in lactutose:manitol ratios, and any change in L/M ratio over time. 
Treatment Groups and Duration: 
Group 1: Healthy Participants 
The total duration of this study for healthy participants will be approximately two weeks, 
in addition to the screening window: 
• 
Screening: up to 28 days before Day 1 
• 
Day 1: study start and assessments will be performed 
• 
Day 8 and Day 15 
Group 2: Thermally injured participants 
Thermal injury participants will be asked to participate for a total of 6 months (plus or 
minus 14 days). 
• There will be no screening period. Thermal injury participants will be recruited 
within 24 hours of their admission to the burns unit at the study site. 
• Intense monitoring phase: Assessments will be performed on alternate days for 
the first 14 days following study enrolment. If the participant is discharged prior 
to 14 days, the intense monitoring phase will end, but the participant will remain 
enrolled in the study. 
• Convalescent monitoring phase: Assessments at 28 days and 6 months will be 
made on an outpatient basis if the participant has already been discharged from 
hospital. 
Exception to monitoring periods: 
• 
If a discharged participant attends the centre for routine clinical care on any 
of days 8-14, then study assessments will be made and samples will be taken 
 

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2. 
SCHEDULE OF ACTIVITIES (SOA) 
2.1. 
Healthy Participants (Group 1) 
Procedure 
Screening  
Treatment Period [Out patient days] (± 1 day) 
Notes 
D1 
D8 
D15 
Review of inclusion/ 
exclusion criteria and 
informed consent 
X 
 
 
 
 
Demography 
X 
 
 
 
 
Medical history (includes 
substance and alcohol 
usage at screening) and 
Medication history 
X 
 
 
 
 
Substance testing (urine) 
X 
 
 
 
Substances: [Recreational 
Drugs and Alcohol] 
HIV, Hepatitis B and C 
screening 
X 
 
 
 
If test otherwise performed 
within 3 months prior to 
study entry, testing is not 
required 
Laboratory assessments 
X 
 
 
 
 
Pregnancy test (WOCBP 
only) (urine) 
X 
(X) 
 
 
Only performed again on 
Day 1 if patient at risk of 
pregnancy at or since 
screening 

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Procedure 
Screening  
Treatment Period [Out patient days] (± 1 day) 
Notes 
D1 
D8 
D15 
Blood sampling for 
biomarkers 
 
X 
 
 
20ml of blood will be 
sampled in a single draw 
on one day, preferably Day 
1 
Stool sample collection 
 
X 
 
 
Participants will be given a 
collection container at 
screening to bring with 
them on Day 1. 
Brief physical examination 
including measurements 
of height and weight 
Vital Signs (systolic and 
diastolic blood pressure 
and heart rate) 
X 
X 
X 
X 
BMI calculated from height 
and weight at screening 
only 
Examinations should be 
conducted the day after 
intestinal permeability 
measurement. 
Medical review 
(assessment of health 
status) 
 
X 
X 
X 
Monitor for signs and 
symptoms of gastro-
intestinal infections and 
other emergent issues 
STM Training 
X 
 
 
 
Refresher training may be 
provided as needed 
Intestinal permeability 
measurement: STM 
administration followed by 
a 24-hour urine collection 
 
X 
X 
X 
Please refer to SRM for full 
information 
Intestinal permeability 
measurement to be 
performed at home the day 
before the study visit. 

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Procedure 
Screening  
Treatment Period [Out patient days] (± 1 day) 
Notes 
D1 
D8 
D15 
AE/SAE and Concomitant 
medication reviews 
(X) 
============================= 
Day 1 will include 
concomitant medication 
review only 
2.2. 
Thermal Injury Participants (Group 2) 
Procedure 
D1  
(≤ 24 hours 
of 
admission) 
Treatment Period [ICU Days] (+/4 hours) 
6 
months 
(± 14 
days) 
Notes 
D2 
D3 
D4 
D5 
D6 
D7 
D8 
D9 
D10 
D11 
D12 
D13 
D14 
D28 
(± 3 
days) 
Review of inclusion/ 
exclusion criteria and 
informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Participants will be co-
consented to the SIFTI-2 
study 
Medical history 
(includes substance and 
alcohol usage) and 
Medication history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Substance testing 
(urine) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Substances: 
[Recreational Drugs, 
Alcohol] 
Pregnancy test 
(WOCBP only) (urine) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
To be repeated at 6 
months prior to final STM 
administration 

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Procedure 
D1  
(≤ 24 hours 
of 
admission) 
Treatment Period [ICU Days] (+/4 hours) 
6 
months 
(± 14 
days) 
Notes 
D2 
D3 
D4 
D5 
D6 
D7 
D8 
D9 
D10 
D11 
D12 
D13 
D14 
D28 
(± 3 
days) 
HIV, Hepatitis B and C 
screening 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
If test otherwise 
performed within 3 
months prior to study 
entry, testing is not 
required 
Initial assessment of 
Burns 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Calculation of %TBSA 
Location of thermal injury 
and depth 
Fluid balance (total 
input/output) 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X* 
 
Daily (over 24 hr) 
*only if participant still 
admitted 
Intestinal permeability 
measurement: STM 
administration followed 
by a 24-hour urine 
collection 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
Measurements every 48 
hours from first measure. 
Preference: D1, 3, 5, 7, 
9, 11, 13. Otherwise: D2, 
4, 6, 8, 10, 12, 14. 
Please refer to SRM for 
full method. 
Brief Physical 
Examination 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
Following final intestinal 
permeability 
measurement. Can be 
omitted if patient is still 
admitted to hospital. 

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Procedure 
D1  
(≤ 24 hours 
of 
admission) 
Treatment Period [ICU Days] (+/4 hours) 
6 
months 
(± 14 
days) 
Notes 
D2 
D3 
D4 
D5 
D6 
D7 
D8 
D9 
D10 
D11 
D12 
D13 
D14 
D28 
(± 3 
days) 
Stool sample collection 
============================= 
(A sample of the first stool produced following admission) 
X 
X 
X 
Time to first stool 
collection; preferably on 
Day 1. Then on Days 14 
and 28 and 6 months 
Wound Healing 
Assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
This is to assess time to 
95% wound healing 
AE/SAE and 
Concomitant medication 
reviews 
X 
============================= 
X 
AE/SAE monitoring will 
begin after the first 
administration of STM 
 
• The timing and number of planned study assessments may be altered during the course of the study based on newly available 
data or factors outside of the study such as priority medical care to ensure appropriate monitoring. 
• Any changes in the timing or addition of time points for any planned study assessments must be documented and approved by 
the relevant study team member and then archived in the sponsor and site study files, but will not constitute a protocol 
deviation or require a protocol amendment. The IRB/IEC will be informed of any safety issues that require alteration of the 
safety monitoring scheme or amendment of the ICF. 
 

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3. 
INTRODUCTION 
3.1. 
Study Rationale 
The purpose of this study is to describe the kinetics and magnitude of increases in 
intestinal permeability which are observed as a result of thermal injury. The results of this 
study will be used to inform the design of future drug studies of a novel medicinal 
product, which is predicted to reduce this increased permeability. 
3.2. 
Background 
3.2.1. 
Thermal Injury, Intestinal Permeability and Multi-Organ 
Dysfunction Syndrome 
Patients with severe thermal injury >20% TBSA are at risk for organ dysfunction and 
may develop multiple organ dysfunction syndrome (MODS). This study aims to explore 
the relationship between thermal injury, changes in intestinal permeability and the onset 
of MODS. 
One of the central hypotheses over the last two decades to explain the onset of MODS in 
the context of critical illness imputes that an increase in intestinal permeability results in 
the translocation of bacteria and bacterial products to the systemic circulation where they 
drive inflammation and injury to distal organs [Deitch, 2006]. This hypothesis is 
supported by data showing that there is a significant increase in intestinal permeability in 
critically ill patients, including patients with thermal injury, and that the degree of 
intestinal permeability correlates with the onset and severity of MODS [Doig, 1998]. As 
such, therapies directed at minimising these changes in intestinal permeability, thereby 
limiting the translocation of bacteria to the systemic circulation, are hypothesised to have 
an impact on clinical outcome in these patients (Figure 1). 
Figure 1 
Changes in Intestinal Permeability 
Critical 
Illness
Intestinal
Permeability
Bacteria/
Bacterial products
MODS
 
Figure 1 (Left panel) Hypothesis: The gut is the major driver of MODS in critical illness 
(Right panel) Correlation between intestinal permeability as measured by 
lactulose/mannitol ratio and the severity of MODS in critically ill patients. Intestinal 
permeability was determined by measuring the differential absorption of lactulose 
(increased in the damaged gut) and mannitol (freely absorbed in the normal and damaged 

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gut) following oral administration and expressed as the ratio of lactulose to mannitol 
(L/M) [Olquin, 2005]. 
There are some data showing that patients with severe thermal injury (>20% TBSA) 
display a significant and rapid increase in intestinal permeability that declines gradually 
over a two-week period following the injury [Olquin, 2005]. Additionally, these changes 
in intestinal permeability, correlate with the severity of sepsis observed in these patients 
[Ziegler, 1988]. 
The central hypothesis of this study is that thermal injury alters intestinal barrier function 
allowing the translocation of bacteria and bacterial products to the systemic circulation 
where they contribute to the onset of MODS. 
The aims of the study are: 
 
1. To establish the magnitude and time course of changes in intestinal permeability to 
inform timing and duration of future investigational medicinal product administration. 
2. To establish the optimal method for assessment of intestinal permeability in patients 
with thermal injury. 
3. To describe the patient population most likely to benefit from a new medicinal 
product which could prevent changes in intestinal permeability. 
4. To improve our understanding of the links between intestinal damage, changes in the 
gut microbiome and microbial translocation to the systemic circulation following 
thermal injury. 
3.2.2. 
Intestinal Barrier Function and Its Measurement 
The intestinal barrier combines a physical and immunological barrier. Epithelial cells are 
connected by tight junctions and prevent the passage of bacteria, toxins and antigens into 
the systemic circulation [Bjarnason, 1995]. Paneth cells, located in crypts of the small 
intestine, produce anti-microbial substances (e.g. lysozyme and defensins) and other 
immune cells patrol the lamina propria [Ayabe, 2000]. Barrier function can be disturbed 
by diseases such as inflammatory bowel disease; by drugs such as aspirin and alcohol; by 
ischaemia and has been observed following a number of acute injuries such as burns, 
trauma and radiation injury [Bjarnason, 1995; Derikx, 2006]. This disturbance results in 
the translocation of the intestinal flora (pathogenic or commensal) to the systemic 
circulation. Microbes are accompanied by proteins which normally form part of the tight 
intracellular junctions of the intestinal epithelium, such as claudins, and other enterocyte-
derived proteins [Grootjans, 2010]. In this study, intestinal permeability will be measured 
directly using oligosaccharide absorption and indirectly by looking for micro-organisms 
and soluble markers of intestinal barrier dysfunction in the systemic circulation. 
Since the 1970s, oligosaccharides have been used as test probes to measure the function 
of the intestinal barrier [Menzies, 1972]. Lactulose, a large polysaccharide, does not 
normally cross the intestinal barrier, but following damage can cross the epithelium and 
enter the systemic circulation. It is not metabolised, so is filtered in the kidney and 
excreted in the urine. The fractional excretion (amount administered / amount recovered 
in urine) of lactulose is therefore a measure of intestinal permeability. The amount of 
lactulose entering the urine is dependent on a number of factors including renal function, 

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gastric emptying, and degradation in the large bowel by commensal bacteria; thus, a 
monosaccharide such as mannitol, which passes freely across the healthy intestinal 
barrier, is often co-administered to ‘normalise’ lactulose measurements. Following 
administration of both lactulose and mannitol the fractional excretion of the sugars is 
expressed as a ratio where mannitol is the denominator. Lactulose and mannitol 
absorption occurs mainly in the proximal small intestine and is complete within 
approximately 5 hours of oral administration. This approach has been previously used 
successfully in patients with severe burn injury and intestinal permeability was found to 
correlate with episodes of sepsis [Doig, 1998]. 
In order to assess the permeability of the large bowel a third oligosaccharide, sucralose, 
will also form part of the sugar absorption test. This synthetic sweetener (marketed by 
Tate and Lyle as ‘Splenda’) is not subject to the same degradation by large bowel 
commensal flora as lactulose and is therefore a better measure of large bowel 
permeability than lactulose. Again, this has formed part of previously described studies 
aiming to measure intestinal permeability [Del Valle-Pinero, 2013]. 
Lactulose, mannitol and sucralose will be co-administered to both healthy participants 
and participants following thermal injury. In order to document accurately the time 
course of change in permeability, thermally injured participants will be asked to undergo 
the test on alternate days for 14 days (the intense monitoring phase) followed by two 
convalescent samples at day 28 and month 6 (thermal injury participants). In order to 
produce an accurate baseline measurement, healthy participants will be asked to undergo 
three measurements of intestinal permeability over approximately a two week period. 
3.2.3. 
The Intestinal Microbiome and Thermal Injury 
During the intense and convalescent monitoring phases of this study, samples of stool 
will be collected. These samples will undergo gene sequence analysis in order to 
determine the composition of the intestinal microbiota. These results will be compared 
with culture results from peripheral whole blood samples. The hypothesis is that raised 
intestinal permeability will correlate with an increased frequency of bacteraemia and that 
the particular bacteria detected in blood will correlate with the composition of the 
intestinal microbiota. 
Additionally, it has been demonstrated that thermal injury alters the composition of the 
intestinal microbiome [Hammer, 2015]. The ultimate aim is to be able to block changes 
in intestinal permeability which might affect this change in composition and are therefore 
interested, in the current study, to assess the impact of thermal injury on the microbiome 
.

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3.3. 
Benefit/Risk Assessment 
3.3.1. 
Risk Assessment 
Potential Risk of Clinical Significance 
Summary of Data/Rationale for Risk 
Mitigation Strategy 
Non-investigational Medicinal Product Use 
Oral administration of sucralose in solution is 
unlikely to be palatable. 
The dose of sucralose to be administered is 2g 
per test. A tolerable sweetness score (e.g. 11.3 
(diet Pepsi)) would require the sucralose to be 
administered in 4 litres of water. 
Sucralose is to be administered in capsules 
when given by mouth. If being administered by 
nasogastric or nasojejunal tube the capsules are 
to be emptied into the lactulose and mannitol 
solution 
Depending on the volume of administration, the 
final STM solution may be hyper-osmolar. 
Administration directly into the jejunum via a 
nasojejunal tube may then result in osmotic 
movement of water into the intestine causing 
distention and discomfort. 
This is based on the physiological principle that 
the stomach normally regulates the osmolality of 
its contents passing into the small intestine. 
Administration via nasojejunal tube (but not 
nasogastric tube) bypasses this process. 
The lactulose, mannitol and sucralose will be 
delivered nasojejunally in 50ml of water and 
followed by a 50ml water flush. This makes the 
solution iso-osmolar (300 mosmol/Kg in situ). 
following nasojejunal administration  
Lactulose and mannitol can produce an osmotic 
laxative effect following enteral administration. 
Both lactulose and mannitol are used clinically as 
osmotic laxatives. The typical dose of lactulose 
(for the treatment of constipation) would be 
approximately 21g daily. 
The amount of lactulose being used in this study 
is 5g, 75% below the standard laxative dose. 
The dose of mannitol is equally low compared to 
that contained in laxatives. 

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Potential Risk of Clinical Significance 
Summary of Data/Rationale for Risk 
Mitigation Strategy 
Study Procedures 
STM administration for the measurement of 
intestinal permeability requires a hold in feeding 
which may be longer than a standard feeding 
hold. 
Intestinal permeability assessment requires the 
administration of STM (see Section 7) and the 
collection of urine samples at defined time points 
(see the SRM). This procedure has previously 
been used in patients with severe thermal injury 
[Doig, 1998] but has not been studied 
extensively; participant fasting is required pre and 
post administration of these sugars; and repeated 
administration as per the SoA tables is also 
unexplored. 
Where possible, tube feeding targets will be 
volume and not time-based to reduce the 
amount of feed missed on test days. Moreover, 
fasts for the tests will, where possible, be 
aligned with clinically indicated feeding holds 
(such as fasts required before theatre, or 
scheduled overnight feed holds). 
Other 
The degree of injury sustained by some 
participants may be severe. 
Participants with severe thermal injury experience 
significant morbidity and high levels of mortality. 
Therefore it is anticipated that these participants 
will be subject to multiple medical complications 
which may impact the study assessments and 
period (See SoA tables)  
Ensure that routine care in the burns unit is not 
compromised by study participation. Prompt 
reporting of any adverse events which are 
related to study procedures and may affect study 
safety. 
Omission of lactulose and sennosides which are 
frequently administered as a part of routine burn 
management.  
Lactulose is a part of the STM and its 
administration for clinical reasons would 
complicate intestinal permeability measurement 
significantly. Equally, the mechanism of actions of 
sennosides is to cause irritation of the GI tract 
Polyethylene glycol (e.g. Movicol) will be used 
as an alternative osmotic laxative. 
If senna is required for clinical purposes, then its 
use must be documented in the CRF. 

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Potential Risk of Clinical Significance 
Summary of Data/Rationale for Risk 
Mitigation Strategy 
and may result in increased permeability. 
The STM will be prepared for administration on 
the assessment day and will not be sterile. 
There is a risk that the sugar test material 
formulation may be contaminated with yeast or 
bacteria. The presence of contamination within 
the STM formulation delivered via the feeding 
tube in thermal injury participants could present 
an infection risk. 
Microbiology release testing will be conducted 
by Tayside pharmaceuticals. The STM will be 
prepared on the assessment day to minimise 
this risk. 
Lactulose/Mannitol will be refrigerated from the 
point of manufacture and until use. The site 
investigators have been consulted on this risk 
and felt that it was low. 
Incomplete 24 hour urine collections 
Ambulant, uncatheterised patients and healthy 
participants 24-hour urine collections could be 
incomplete. 
Ambulant, uncatheterised patients and healthy 
participants will receive careful education and 
written instructions of the importance of 
complete urine collections. In addition, 
documentation of incomplete collection, in 
addition to sampling collection times, will be 
recorded. 

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Potential Risk of Clinical Significance 
Summary of Data/Rationale for Risk 
Mitigation Strategy 
Phlebotomy for biomarkers (healthy participants) 
Phlebotomy can be painful and result in bruising, 
bleeding and puncture site infection. 
Phlebotomy from thermally injured participants 
will not be conducted as a part of this protocol. 
Healthy participants will undergo one blood draw 
during screening and a further draw on day one. 
Phlebotomy will be performed by an 
appropriately trained member of the site study 
team with aseptic non-touch technique to avoid 
infection. 
 

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3.3.2. 
Benefit Assessment 
Study participants will not benefit directly from involvement in this study. However, the 
results of this study may contribute significantly to our understanding of changes in 
intestinal permeability and their relationship to morbidity and mortality in the context of 
thermal injury. This knowledge is paramount to designing future medicinal interventional 
studies, aiming to modulate intestinal permeability and, potentially, to improve outcomes 
for patients following thermal injury. 
3.3.3. 
Overall Benefit:Risk Conclusion 
The primary outcome measure of this study is the determination of intestinal permeability 
in healthy and thermal injury participants. Interventions in this study are the 
administration of STM by mouth or feeding tube (if one is site for routine clinical care) 
and the collection of urine and stool samples. 
The risk of adverse events is minimised for the population being investigated in the 
proposed study as no drug intervention will be investigated and study assessments being 
conducted are non-invasive (with the exception of STM administration and phlebotomy 
in healthy participants). 
4. 
OBJECTIVES AND ENDPOINTS 
Objective 
Endpoint 
Co-Primary 
1. To determine the impact of thermal injury 
on the magnitude of small intestinal 
permeability change as soon as possible 
following injury compared to healthy 
participants 
• Lactulose/Mannitol (L/M) ratio at entry 
2. To characterise the effect of thermal 
injury on small intestinal permeability over 
time and establish the clinical and 
demographic factors which can influence 
it 
• Changes in L/M ratio over time 
Exploratory 
1. To determine the impact of thermal injury 
on colonic permeability as soon as 
possible following injury compared to 
healthy participants 
• Fractional excretion of sucralose at entry 
2. To characterise the effect of thermal 
injury on colonic permeability over time 
• Changes in the fractional excretion of 
sucralose over time 

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Objective 
Endpoint 
3. To assess the relationship between 
severity of a participant’s condition 
following thermal injury and changes in 
intestinal permeability† 
• Number of ventilator-free days 
• Number of vasopressor-free days 
• Number of hemofiltration-free days 
• Number of episodes of confirmed infection 
and sepsis 
• Number of surgical interventions 
• Total length of hospital stay 
• Calculate critical care and thermal injury 
severity scores 
4. To assess plasma and urine biomarkers 
of intestinal permeability, bacterial 
translocation and renal tubular 
dysfunction following thermal injury† 
• Change in markers of intestinal mucosal 
damage samples from blood 
• Change in urine protein:creatinine and urine 
albumin:creatinine ratios 
5. To assess the impact of thermal injury 
and intestinal permeability on the 
intestinal microbiome compared to 
healthy participants 
• Changes in microbiome of acute and 
convalescent stool samples 
6. To assess the impact of pre-existing co-
morbid conditions on intestinal 
permeability and clinical outcome 
following thermal injury† 
• Medical history and drug history at the time of 
admission 
7. To assess wound healing 
• Time to wound recovery (e.g. 95%) 
8. To characterise parameters that may 
influence drug PK/PD† 
• Determine fluid input/output balance over time 
• Changes in serum albumin and plasma 
creatinine 
9. To characterise intestinal microbiota, and 
correlate its composition with both 
intestinal permeability and bacterial 
detection in blood† 
• Changes in intestinal microbiome 
• Bacterial markers of translocation 
†Clinical data, routine laboratory results or blood/urine biomarker results obtained from 
the SIFTI-2 study will be used in the analysis of this exploratory endpoint see Section 
5.4.1. 

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5. 
STUDY DESIGN 
5.1. 
Overall Design 
This is a longitudinal, prospective study of healthy participants and participants who have 
sustained a thermal injury. The following schematic summarises study measures and their 
frequency for healthy and thermal injury participants. 
Figure 2 
Study Schematic 
 
5.2. 
Number of Participants 
Table 1 describes the number of participants proposed for the study. Sample size is based 
on feasibility. No formal calculation of power or sample size has been performed, but a 
sample size of 15 healthy participants and 25 thermal injury participants (≥15% TBSA) 
should be sufficient to provide useful estimates of variability in lactutose:manitol ratios, 
and any change in L/M ratio over time. 
 

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Table 1 
Recruitment Stratification 
 
Number of participants 
Group 1 
Healthy participants 
15 
Group 2 
Thermal Injury participants 
Percent Total Burn Surface Area (TBSA) 
≥15% 
25 
 
The healthy participants (Group 1) will be recruited with an age range similar to that 
typical in thermal injury participants based on historic hospital admission data from the 
UK and data from the SIFTI1 study [Hampson, 2016]. 
If participants prematurely discontinue or are withdrawn from the study, additional 
replacement participants may be recruited at the discretion of the Sponsor in consultation 
with the investigator see Section 8.2.1. 
5.3. 
Participant and Study Completion 
The full duration of the study for healthy participants is approximately two weeks and for 
thermal injury participants is approximately 6 months. 
Thermal injury participants who withdraw prior to week 4 or healthy participants who 
withdraw prior to week 2 will be considered for analysis, although it is acknowledged 
that any missing data at later stages of the study may be related to outcome (either 
positive or negative). Given this is an exploratory study, the impact of missing data will 
be explored by assessing the sensitivity of results to different missing data approaches 
(for example, analyse all available data, analyse only complete data across time points 
and explore imputation of worst or best case scenarios). 
Study withdrawals may also include participants who are consented to the study under 
Section 30 of the Mental Capacity Act 2005. In the event that participants do not re-
confirm consent when they regain capacity, they will be withdrawn from the study. 
The end of the study is defined as the date of the last visit of the last participant in the 
study. 
5.4. 
Scientific Rationale for Study Design 
5.4.1. 
Co-recruitment to the SIFTI-2 Study 
Thermally injured participants who are eligible for this study must also be eligible for, 
and enrolled in a partner study named SIFTI-2. 

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SIFTI-2 is an observational study currently recruiting healthy participants and thermally 
injured participants and follows a successfully delivered predecessor study SIFTI1 
[Hampson, 2016]. The objectives and endpoints of SIFTI-2 are included in the SIFTI-2 
study protocol (reference number IRAS ID: 200366). The design of this study has been 
aligned with the SIFTI-2 study to support the strategy of co-consenting thermally injured 
participants to both studies. This will reduce the overall impact of research in this 
population in the following ways: 
• There is sufficient residual blood from collection in SIFTI-2 to allow testing of 
blood biomarkers of interest for the HESTIA study. This strategy therefore limits 
impact on participants as no additional blood sampling is required for 
participation in HESTIA (with the exception of HIV, Hepatitis B and C testing at 
baseline). SIFTI-2 participants will be explicitly consented for their samples and 
data to be shared in this way. 
• Sampling time points and study visits in SIFTI-2 and HESTIA have been aligned 
to reduce the impact of co-recruitment on thermally injured participants. 
Clinical data from both studies can be generated from the same participant therefore 
allowing biomarker, microflora and intestinal permeability data to be compared. In 
contrast to thermally injured participants, healthy participants enrolling in this study will 
not be required to co-consent for participation in SIFTI-2. 
Data will be shared from the SIFTI-2 study with GSK through a secure electronic 
database. 
A summary of the origin (HESTIA or SIFTI-2) of data and samples collected for the 
HESTIA study is available in Appendix 5 Section 12.5. 
5.4.2. 
Recruitment and Sampling Schedule 
Severely burned patients (with an injury affecting greater than 20% total body surface 
area), display a significant and rapid increase in intestinal permeability that has been 
shown to decline over time (Figure 3) [Olquin, 2005]. What is less well understood is 
whether a greater severity of thermal injury correlates with greater intestinal 
permeability. Moreover, the time to complete restoration of normal permeability and 
other factors which may influence permeability (other than the initial injury) are also not 
well understood. 

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Figure 3 
Burn Injury Results in a Rapid Increase in Intestinal Permeability 
 
 
 
This study aims to recruit participants as soon as possible following their admission in 
order to capture the initial changes in permeability. Serial measurement of intestinal 
permeability and sampling of the biomarkers of bacterial translocation, intestinal damage 
and inflammation are required during the acute phase (days 1-14) of admission in order to 
correlate them with clinical events (e.g. surgery), severity scores and clinical outcomes. 
The convalescent time points (28 days and 6 months) are required to determine if 
intestinal permeability has returned to normal and to correlate observed changes on days 
1-14 with longer-term clinical outcomes (e.g. wound healing). 
Gut microbiome assessments will be made less frequently than intestinal permeability 
assessments as changes in the microbiome are predicted to evolve more slowly. Ideally a 
stool sample will be collected from thermal injury participants at study entry (limited, of 
course, by when participants first pass stool following admission). A convalescent sample 
is requested to assess if the gut microbiome is able to restore to a more normal 
composition (and will be compared with that of healthy participants to make that 
assessment). 

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Blood samples (with the exception of HIV, Hepatitis B and Hepatitis C testing at 
baseline) will not be taken from thermally injured participants during this study. Instead, 
biomarker data from blood samples taken during the SIFTI-2 study will be used. A single 
blood draw will be required from healthy participants on day 1 of participation. 
5.4.3. 
Inclusion of Healthy Participants 
Patient facing material (i.e. poster) will be used to facilitate recruitment of the healthy 
participants. Healthy participants will be recruited to this study to provide a baseline for 
endpoint measures on intestinal permeability and the gut microbiome. 
Three measurements of intestinal permeability are required in order to define an average 
given the variability in healthy participants reported previously. The timing of the 
replicates follows the intense time course of the study to control for day-to-day variation 
over a 15 day period. 
5.4.4. 
Preliminary Data Review 
A safety review based on raw data will be conducted when approximately 10 participants 
have completed the study. This review would include data from any participant, either 
healthy or thermally injured, that has been collected at the time the review is conducted. 
5.4.5. 
The Use of the Sugar Test Materials (Lactulose, Mannitol and 
Sucralose) 
As described in Section 3.2.2, lactulose, mannitol and sucralose will be administered to 
both thermally injured participants and healthy participants to measure permeability of 
the small and large intestine. The amount of each of the sugars to be used is based on 
previous successful clinical studies employing this method and balances having enough 
sugar present for detection in urine with their potential laxative effect (Del Valle-Pinero, 
2013; Doig, 1998; Menzies, 1972). 
6. 
STUDY POPULATION 
The study population will comprise healthy and thermal injury participants presenting at 
enrolling study sites. Please note the following: 
• Prospective approval of protocol deviations to recruitment and enrolment criteria, 
also known as protocol waivers or exemptions, is not permitted. 
• Where possible, written informed consent will be obtained from each subject prior 
to participation in this study. Recruitment of subjects who lack mental capacity is 
discussed in Section 6.1. 
• Healthy participants will be consented to the HESTIA study only.  
• Thermally injured participants are required to be co-consented to the SIFTI-2 and 
HESTIA studies outlined in Section 6.1. A diagrammatic overview of the SIFTI-2 
and HESTIA thermal injury participant recruitment is given in Figure 4. (see 
Appendix 3). 

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6.1. 
Recruiting Participants with Differing Mental Capacity 
Following evaluation of capacity (as outlined in the Mental Capacity Act (MCA) 2005), 
thermally injured participants enrolled in this study will fall into the following groups: 
6.1.1. 
Adult Participants Determined to Have Mental Capacity at Study 
Entry and Throughout the HESTIA Study 
Those participants who present with capacity and meet study entry criteria will be 
provided with a Participant Information Leaflet (PIL) outlining the study. If the 
participant agrees to consent to the HESTIA study following a discussion with the 
research team, they will be asked to sign a consent form. 
Given the short (24 hour) window for recruitment, those patients who are acutely unwell 
will initially be presented with an abbreviated PIL. Once stable, this will be followed by 
the standard information leaflet and re-confirmation of consent. 
6.1.2. 
Adult Participants Lacking Mental Capacity for the Duration of 
the HESTIA Study 
It is anticipated that some subjects who meet eligibility criteria for this study will not be 
able to give informed consent due to their medical condition or its management (e.g. 
sedation, opioid analgesia, intubation). In such cases, participants may be enrolled in the 
study in accordance with Section 30- Section 34 of the MCA 2005. The decision to enroll 
the participant will be discussed with a personal or nominated consultee (as defined in 
Section 12.3.2). This decision may or may not be witnessed by an independent witness 
according to the decision of the principal investigator. 
6.1.3. 
Adult Participants Lacking Mental Capacity (either at Study Entry 
or During the Study) Who Later Regain Capacity and Are 
Required to Provide Informed Consent 
As soon as is practically possible following a participant regaining capacity, participants 
will be asked to provide informed consent to remain in the study. If they decline, then 
they will be withdrawn from the study as soon as it is safe to do so (likely immediately 
given the design of this study). Samples and data collected prior to study withdrawal may 
be retained. The participant will be asked about this at the point of study withdrawal. 
6.1.4. 
Adult Participants with Mental Capacity to Provide Consent at 
Study Entry Who are Later Deemed No Longer to have Mental 
Capacity 
The decision for the participant to remain in the study will be discussed with a personal 
or nominated consultee and recorded. If the participant subsequently regains capacity 
again, they will be asked to re-consent to study participation. 
When considering enrolment of participants who lack the mental capacity to consent, the 
following should be noted: 

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• Section 3.2.1 of the SIFTI-2 protocol describes the consent process for that study 
in detail and should be read in conjunction with this protocol. 
• A Study Information Leaflet will be provided to the personal or nominated 
consultee outlining the HESTIA trial before being asked to sign a form supporting 
the participant’s enrolment in the study. 
• The investigator and/or the site’s IEC/IRB have responsibility for acting in 
accordance with the MCA 2005 in the matter of assessing who has the capacity to 
consent and who qualifies as a personal or nominated consultee of a potential 
subject. The investigator will also decide if an independent witness is required. 
• Further information regarding the assessment of mental capacity and the 
appointing of personal or nominated consultee is given in Appendix 3 (Section 
12.3.2 and Section 12.3.3). 
• If a patient loses mental capacity subsequent to their consent and enrolment to the 
HESTIA study, samples and data collected prior to loss of capacity will be 
retained even if approval of continued study participation by a personal or 
nominated consultee is declined. 
6.2. 
Inclusion Criteria for Healthy Participants (Group 1) 
1. Males or Females must be ≥18 years of age at the time of signing informed consent. 
2. Participants who are healthy as determined by the investigator following medical 
evaluation including medical history, physical examination, and laboratory tests 
(these are listed in Appendix 2). 
3. Female participants: 
A female participant is eligible to participate if she is not pregnant (negative 
pregnancy testing at screening or Day 1 as needed) and not breastfeeding. 
4. Capable of giving signed informed consent as described in Appendix 3 which 
includes compliance with the requirements and restrictions listed in the informed 
consent form (ICF) and in this protocol. 
6.3. 
Inclusion Criteria for Thermal Injury Participants (Group 2) 
Thermal injury participants are eligible to be included in the study only if all of the 
following criteria apply: 
6.3.1. 
Age 
1. Participant must be ≥18 years of age. 
6.3.2. 
Type of Participant and Disease Characteristics 
2. Participants who have sustained a burn (thermal injury) with a TBSA ≥ 15%. 
6.3.3. 
Other Inclusions 
3. Admission to the burn centre (study site) ≤24 hours of injury. 

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4. Able to take enteral fluids either orally or via a nasogastric tube (depends on facial 
burn damage). 
6.3.4. 
Gender 
5. Male and female. 
a. Female participants:  
A female participant is eligible to participate if she is not pregnant (negative 
pregnancy testing at study entry) and not breastfeeding. 
6.4. 
Exclusion Criteria for Healthy Participants (Group 1) 
1. Healthy participants are excluded from this study if they are receiving anti-
coagulation therapy. 
2. Pregnancy or breastfeeding. 
3. A body mass index >34kg/m2 
4. An active history of alcohol dependency 
5. History of sensitivity to any of the STM, or components thereof or a history of drug 
or other allergy that, in the opinion of the Investigator and/or GSK Medical Monitor, 
contraindicates their participation. 
6. A positive pre-study Hepatitis B surface antigen or positive Hepatitis C antibody and 
confirmatory Hepatitis C PCR result within 3 months of screening. 
7. A positive pre-study urine drug/alcohol screen. 
8. A positive test for HIV antibody. 
9. Participants unable to swallow large capsules (the capsules will be shown to 
participants at screening). 
10. Galactosaemia or severe lactose intolerance. 
11. Use of an antibiotic 2 weeks prior to study start (i.e. administration of the STM). 
12. Gastroenteritis in the 2 weeks prior to study start (i.e. administration of the STM). 
6.5. 
Exclusion Criteria for Thermal Injury Participants (Group 2) 
Thermal injury participants are excluded from the study if any of the following criteria 
apply: 
Medical Conditions 
1. Chemical or electrical burn. 
2. Multiple traumatic injuries with an Injury Severity Score (ISS) ≥16 (note: excludes 
burn in score system). 
 
 

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Prior/Concomitant Therapy 
3. Patient received substantial undocumented management prior to arrival at the study 
site (burn centre) e.g. from paramedics or in a local accident and emergency 
department. 
4. Systemic corticoidsteroid use. 
5. Intravenous (IV) mannitol use. 
Prior/Concurrent Conditions 
6. Human immunodeficiency virus (HIV) infection. 
7. Viral Hepatitis B or C infection. 
8. Gastrointestinal disease (e.g. inflammatory bowel disease) which may affect 
intestinal permeability. 
9. Previous bowel resection (e.g. hemicolectomy, small bowel resection) 
10. Galatosaemia or severe lactose intolerance. 
11. Bowel obstruction. 
12. Renal dysfunction requiring renal replacement therapy (i.e. end-stage renal failure 
prior to thermal injury). 
13. Active autoimmune disease and receiving immunomodulatory therapy e.g. 
rheumatoid arthritis anti-TNF. 
14. Active chemotherapy for cancers or immunoremittive therapies (prednisolone, 
adalimumab) within 60 days of thermal injury. 
15. Premorbid conditions of malignancy currently under treatment. 
16. Previous bilateral lower extremity amputation. 
NOTE: Due to the rapid recruitment period (within 24 hours of admission) for thermally 
injured participants, HIV and viral Hepatitis test results may not be available at the time 
of enrolment. In the event these tests return positive, the participant will be informed of 
the result(s) and withdrawn from the study (see Section 8.2.1). In the event that the 
participant has been recruited who lacked mental capacity to consent, the reason for their 
withdrawal (the positive HIV and/or viral Hepatitis test results) must not be shared with a 
personal consultee. According to UK law and the guidance of the British HIV 
association, if the participant’s physician believes there is an overriding public interest to 
disclose the participant’s results to a personal consultee who is a current or former sexual 
partner, then the result(s) may be disclosed without the participant’s consent. This must 
be as a last resort. If the participant regains capacity, he/she will be informed of the 
positive test result(s). 
Diagnostic assessments 
17. Decision not to treat the patient due to futility. 

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6.6. 
Lifestyle Restrictions 
6.6.1. 
Meals and Dietary Restrictions 
• 
Participants will be fasted (or feed stopped) for 3 hours prior to STM administration 
and for 3-5 hours afterwards. For thermally injured participants these fasts should be 
aligned with those required for routine clinical care (feed holds, before surgical 
interventions) where possible. 
• 
Refrain from consumption of the following for 24 hours before and after the 
administration of STM: 
o Foods/drinks/medicines and other products which contain sucralose, lactulose or 
mannitol as artificial sweeteners. 
N.B. If cannot be avoided, then clear documentation of its administration is required and 
the current test to stop. If urine samples have been collected PRIOR to 
administration of the drug, then these can still be sent for analysis 
6.6.2. 
Alcohol/Exercise/Aspirin (Healthy Participants only) 
Alcohol, aspirin and vigorous exercise [Sequeira, 2014] are all known to cause transient 
increases in intestinal permeability. Healthy participants will therefore be requested to 
avoid alcohol, aspirin and physical exercise for 48 hours before taking the STM and for 
the 24-hour urine collection period. 
6.7. 
Screen Failures 
There will be no screening period for thermal injury participants. Screening will be up to 
28 days before Day 1 for healthy participants. 
Screen failures are defined as participants who consent to participate in the clinical study 
but are not subsequently entered in the study. A minimal set of screen failure information 
is required to ensure transparent reporting of screen failure participants to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to 
respond to queries from regulatory authorities. Minimal information includes 
demography, screen failure details, eligibility criteria, and any serious adverse events 
(SAEs). 
Individuals who do not meet the criteria for participation in this study (screen failure) 
may be rescreened. Rescreened participants should be assigned a new participant number. 
7. 
NON-INVESTIGATIONAL MEDICINAL PRODUCT (NIMP) 
A study treatment is defined as any investigational treatment(s), marketed product(s), 
placebo, or medical device(s) intended to be administered to a study participant according 
to the study protocol. According to this definition, no GSK study treatment will be 
employed in this study. 
 

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The STM (comprising lactulose, mannitol and sucralose) will be intermittently 
administered enterally as a study challenge agent to measure permeability at different 
points along the GI tract. Lactulose and Mannitol assess small intestine permeability and 
sucralose to assess colonic permeability. 
It is important to note that the administration of the STM is not therapeutic (lactulose and 
mannitol can be used as laxatives, however the amount in the STM is sub-therapeutic) 
and as such should be regarded as a non-investigational medicinal product (NIMP) 
[Guidance Documents Applying Investigational Medicinal Products (NIMPS), 2011]. 

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7.1. 
STM Administered 
Study 
Treatment 
Name 
Lactulose 
(4-o-β-D-
galactopyranosyl-D-
fructofuranose) 
Mannitol 
(D-mannitol) 
GRAS listed  
Sucralose  
(1,6-Dichloro-1,6-dideoxy-β-
D-fructofuranosyl-4-chloro-
4-deoxy-α-D-
galactopyranoside) 
Dosage 
formulation 
oral solution 
oral solution 
Capsules (powder) 
Unit dose 
strength(s) 
Adults 
5g 
2g 
2g (3 capsules to deliver 
total 2g sucralose) 
Route of 
Administration 
Oral / nasogastric / 
nasojejunal 
Oral / nasogastric / 
nasojejunal 
Oral / nasogastric / 
nasojejunal (capsules to 
be opened and contents 
added to lactulose and 
mannitol for tube 
administration) 
Preparation 
and Dosing 
instructions 
For oral administration, the lactulose and mannitol will be prepared as a 
100ml drink to be taken with 3 sucralose capsules. 
For feeding tube administration, lactulose/mannitol/sucralose will be prepared 
as a 50ml solution and given via a feeding tube followed by an immediate 
50ml drinking water flush 
Preparation refer to Study Reference Manual together with SoA tables 
(Section 2) 
Packaging and 
Labelling 
Lactulose and Mannitol will be supplied pre-
mixed in an amber bottle (or equivalent) for 
single use. Each container will be labelled as 
required per country requirement. 
Sucralose will be provided 
as capsules in a storage 
container. Each container 
will be labelled as 
required per country 
requirement. 
Manufacturer 
Tayside Pharmaceuticals, UK 
Storage 
Lactulose/Mannitol formulation should be stored under refrigerated conditions. 
The sucralose capsules should be stored at room temperature in a dry 
environment away from direct sunlight. 
Shelf-life 
Lactulose/Mannitol pre-mix formulation and sucralose capsules supplied by 
Tayside Pharmaceuticals will have at least 3 month shelf-life when stored at 
the correct storage conditions. 
 

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The preparation of the STM for oral use and nasogastric/nasojejunal tube administration 
can be found in the Study Reference Manual. 
7.2. 
Dose Modification 
Dose modification will not be required. Unit dose is described in Section 7.1. 
7.3. 
Method of STM Administration: Treatment Assignment 
There is no element of randomisation in the study and all study participants will receive 
the STM according to the relevant SoA. The method of administration can be found in 
the Study Reference Manual. 
7.4. 
Blinding 
No GSK study treatment will be employed in this study. All participants will receive the 
same STM and all thermal injury participants will perform the same study procedures. 
7.5. 
Preparation/Handling/Storage/Accountability 
1. 
The investigator or designee must confirm appropriate temperature conditions 
have been maintained during transit for all STM received and any discrepancies are 
reported and resolved before use of the STM. 
2. 
Only participants enrolled in the study may receive STM and only authorized site 
staff may supply or administer STM unless adequate training is provided such as in 
the case of healthy participants. All STM must be stored in a secure, temperature 
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 STM accountability, reconciliation, and record 
maintenance, as needed. 
4. 
Further guidance and information for the final disposition of unused STM are 
provided in the Study Reference Manual. 
5. Under normal conditions of handling and administration, STM is not expected to pose 
significant safety risks to site staff. 
7.6. 
STM Compliance 
• 
When participants undergo intestinal permeability testing at the site, they will 
receive STM directly from the investigator or designee, under medical supervision. 
The date and time of each dose administered in the clinic will be recorded in the 
source documents. The dose of STM and study participant identification will be 
confirmed at the time of dosing by a member of the study site staff other than the 
person administering the STM. 

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• 
If healthy participants need to prepare and administer the STM off-site such as at 
home, STM training will be provided and a record maintained by the investigator or 
designee. 
7.7. 
Concomitant Therapy 
• 
Refrain from consumption of the following for 24 hours before and after the 
administration of STM: 
o Lactulose or mannitol-containing laxatives. Study sites will be asked to use 
movicol (polyethylene glycol) in place of lactulose. 
o Medicines with mannitol as an excipient (chlorthiazide sodium, some albumin 
preparations, some laxatives, tablets as a bulking agent). 
o Products containing sucralose. 
• For healthy participants only, refrain from consumption of aspirin for 48 hours 
before taking the STM and for the 24-hour urine collection period see Section 6.6.2. 
• 
For healthy participants only, antibiotic use 2 weeks prior to STM administration and 
during the study is not permitted. 
• 
Sennoside laxatives should be avoided. These can cause gastrointestinal irritation 
and may contribute to raised intestinal permeability. 
• 
Additional Glutamine supplementation in excess of that delivered with a standard 
feeding protocol should be avoided during the first 28 days of study participation. If 
supplementation is given inadvertently, then the patient will remain in the study, but 
the total dose and duration of additional glutamine supplementation must be recorded 
in the CRF. 
• 
Thermal injury participants that receive Intravenous (IV) mannitol for renal failure or 
raised intracranial pressure (testing to be delayed until 12 hours after last 
administration). 
7.8. 
Treatment after the End of the Study 
There will be no ongoing STM administration following the end of this study. 
8. 
DISCONTINUATION CRITERIA 
8.1. 
Discontinuation of STM administration 
Discontinuation of STM administration can be considered by the investigator in the event 
that an adverse event to the STM is observed. Withdrawal of further STM administration 
does not require withdrawal from the study. 

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8.2. 
Withdrawal from the Study 
• 
A participant may withdraw from the study at any time at his/her own request, 
or may be withdrawn at any time at the discretion of the investigator for safety, 
behavioural, compliance or administrative reasons. 
• 
If the participant withdraws consent for disclosure of future information, the 
sponsor may retain and continue to use any data collected before such a 
withdrawal of consent. 
• 
If a participant withdraws from the study, he/she may request destruction of any 
samples taken and not tested, and the investigator must document this in the site 
study records. 
8.2.1. 
Other Withdrawal Criteria 
• 
A participant will be withdrawn from the study following positive (and 
confirmed) HIV test at screening; Serologic evidence of active Hepatitis B (HB) 
infection based on the results of testing for HBsAg, and anti-HBc or a positive 
test for Hepatitis C antibody confirmed by Hepatitis C RNA or antigen testing. 
If HCV RNA is not available, then the positive test for Hepatitis C antibody 
alone would be exclusionary. Results must be discussed with the medical 
monitor to withdraw the participant from the study and commence therapy 
according to local practice. 
• 
Healthy participants that are treated with antibiotics during the duration of the 
study. 
• 
Participants that experience signs and symptoms of gastro-intestinal infections 
during the duration of the study. 
• 
Withdrawals related to mental capacity as described in Section 6 and Appendix 
3. 
• 
Participants that received haemodialysis during the first 48 hours of the study 
(i.e. during the first measurement of intestinal permeability) will be excluded 
from the evaluable population and a replacement will be recruited. 
• 
Participants that received haemodialysis during later time points will not be 
excluded, but consideration will be given to recruiting an additional 
participant if 3 or more intestinal permeability measurements occur 
concurrently with haemodialysis. 
8.3. 
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 study site 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 

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the assigned visit schedule and ascertain whether or not the participant wishes to 
and/or should continue in the study. 
• 
Before a participant is deemed lost to follow up, the investigator or designee 
must make every effort to regain contact with the participant (where possible, 3 
telephone calls and, if necessary, a certified letter to the participant’s last known 
mailing address or local equivalent methods). These contact attempts should be 
documented in the participant’s medical record. 
• 
Should the participant continue to be unreachable, he/she will be considered to 
have withdrawn from the study with a primary reason of lost to follow-up. 

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9. 
STUDY ASSESSMENTS AND PROCEDURES 
• 
Study procedures and their timing are summarized in the SoA. 
• 
Protocol waivers or exemptions are not allowed. 
• 
Safety concerns related to the STM should must be discussed with the sponsor 
immediately upon occurrence or awareness to determine if the participant should 
continue to be administered the STM. 
• 
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 thermally injured participant’s routine clinical 
management (e.g., weight measurement) and obtained before signing of ICF may be 
used for screening or baseline purposes provided the procedure met the protocol-
specified criteria and was performed within the time frame defined in the SoA. 
Procedures (the administration of STM) are not part of routine care for either healthy 
or thermal injury participants. 
• 
Healthy participants will be asked to donate a single blood sample on Day 1. No 
blood collection is specified in the SoA of this protocol for thermal injury 
participants (with the exception of HIV, Hepatitis B and Hepatitis C testing at 
baseline). The results of clinical laboratory blood tests will be recorded in the SIFTI-
2 study and the data used in this study. Likewise, blood collection for exploratory 
biomarker detection will be included in the SIFTI-2 study and the data used in this 
study. 
9.1. 
Efficacy Assessments 
The administration of the STM is for the purpose of intestinal permeability measurement 
and is not therapeutic, therefore no efficacy will be assessed. 
9.2. 
Adverse Events 
9.2.1. 
Monitoring and reporting responsibilities 
Healthy participants will be recruited to the HESTIA study alone and all AEs or SAEs 
occurring in this group should be managed according to this protocol. 
Thermally injured participants recruited to this study will also be recruited to the SIFTI-2 
study. The following guidance relates only to AEs or SAEs which the investigator 
reasonably believes to be the result of a procedure or requirement unique to this (the 
HESTIA) protocol. All other AEs or SAEs will be reported and managed in accordance 
with the SIFTI-2 protocol. 

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Unique procedures and requirements of HESTIA 
1. The administration of STM 
2. The collection of stool samples 
3. Changes to standard of care for thermally injured participants: 
a. The fasts required during the measurement of intestinal permeability 
b. The use of alternative laxatives to lactulose and sennosides. 
The definitions of an AE or SAE for this study can be found in Appendix 4. 
The investigator and any designees are responsible for detecting, documenting, and 
reporting events that meet the definition of an AE or SAE and remain responsible for 
following up AEs that are serious or that caused the participant to discontinue intestinal 
permeability measurement with the STM (see Section 8). 
9.2.2. 
Time Period and Frequency for Collecting AE and SAE 
Information 
• 
All SAEs will be collected from the start of unique HESTIA study procedures 
until the final visit at the time points specified in the SoA (Section 2). However, 
any SAEs assessed as related to study participation (e.g., STM, protocol-
mandated procedures, invasive tests, or change in existing therapy) will be 
recorded from the time a participant consents to participate in the study. 
• 
All AEs will be collected from the start of unique HESTIA study procedures 
until the final visit at the time points specified in the SoA (Section 2). 
• 
Medical occurrences that begin before the start of unique HESTIA study 
procedures but after obtaining informed consent will be recorded on the Medical 
History/Current Medical Conditions section of the case report form (CRF) not 
the AE section. 
• 
All SAEs will be recorded and reported to the sponsor or designee immediately 
and under no circumstance should this exceed 24 hours, as indicated in 
Appendix 4. The investigator will submit any updated SAE data to the sponsor 
within 24 hours of it being available. 
• 
Investigators are not obligated to actively seek AEs or SAEs in former study 
participants. However, if the investigator learns of any SAE, including a death, 
at any time after a participant has been discharged from the study, and he/she 
considers the event to be reasonably related to the STM administration or study 
participation, the investigator must promptly notify the sponsor. 
• 
The method of recording, evaluating, and assessing causality of AEs and SAEs 
and the procedures for completing and transmitting SAE reports are provided in 
Appendix 4. 
9.2.3. 
Method of Detecting AEs and SAEs 
Care will be taken not to introduce bias when detecting AEs and/or SAEs. If the 
participants are conscious, open-ended and non-leading verbal questioning of the 

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participant is the preferred method to inquire about AE occurrence. For unconscious 
patients or participants not always able to provide valid verbal responses to open-ended 
questions, the investigator or designee will need to identify AEs and/or SAEs through 
relevant clinical signs and/or investigations. 
9.2.4. 
Follow-up of AEs and SAEs 
After the initial AE/SAE report, the investigator is required to follow proactively each 
participant at subsequent visits/contacts. All SAEs, will be followed until the event is 
resolved, stabilized, otherwise explained, or the participant is lost to follow-up (as 
defined in Section 8.3). Further information on follow-up procedures is given in 
Appendix 4. 
9.2.5. 
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 are met. 
• 
The sponsor has a legal responsibility to notify both the local regulatory 
authority and other regulatory agencies about the safety of the study. The 
sponsor will comply with country-specific regulatory requirements relating to 
safety reporting to the regulatory authority, Institutional Review Boards 
(IRB)/Independent Ethics Committees (IEC), and investigators. 
• 
Investigator safety reports must be prepared for suspected unexpected serious 
adverse reactions (SUSAR) according to local regulatory requirements and 
sponsor policy and forwarded to investigators as necessary. 
• 
An investigator who receives an investigator safety report describing a SAE or 
other specific safety information e.g., summary or listing of SAE) from the 
sponsor will review and then file it and will notify the IRB/IEC, if appropriate 
according to local requirements. 
9.2.6. 
Disease-Related Events and/or Disease-Related Outcomes Not 
Qualifying as SAEs 
The following broad disease related events (DREs) are common in thermally injured 
participants and can be serious/life threatening: 
• 
Deterioration of condition. 
• 
Death (may be expected in burns of a large surface area). 
• 
Prolongation of hospital stay. 
• 
Persistent or significant disability or incapacity. 
Because these events are typically associated with the disease under study, they will not 
be reported according to the standard process for expedited reporting of SAEs to GSK 
(even though the event may meet the definition of a SAE). These events will be recorded 
on the DRE page in the participant’s CRF within [the appropriate time frame agreed 

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upon by the SRT for completion of DRE CRF pages]. These DREs will be monitored by 
clinical study team on a routine basis. 
• 
NOTE: However, if the investigator considers that there is a reasonable 
possibility that the event was related to administration of STM or another 
unique or required element of the study (as defined in Section 9.2.1) then the 
event must be recorded and reported as an SAE (instead of a DRE). 
• 
A comprehensive list of further thermal injury related DREs can be found in 
Appendix 4 (Section 12.4). 
9.3. 
Treatment of Overdose 
For this study, an overdose is defined as any dose of STM greater than defined in Section 
7.1. No specific treatment is recommended for an overdose and treatment is at the 
discretion of the investigator.  The GSK medical monitor must be notified promptly. 
9.4. 
Safety Assessments 
Planned time points for all safety assessments are provided in the SoA and summarised 
here. 
Safety Assessment 
When conducted 
 
Healthy Participants 
Thermally injured 
participants 
Laboratory tests 
Screening. Only repeated 
if clinically indicated in 
the opinion of the 
investigator. 
Only if clinically 
indicated. 
Brief Physical Examination 
including Vital Signs 
Recording 
Screening, Day 1, Day 8, 
Day 15 
As a part of routine 
clinical care whilst 
admitted (not protocol 
specified). Following day 
28 and 6 month intestinal 
permeability 
measurements (if patient 
not still admitted) 
Detection of AEs 
Day 1, Day 8, Day 15 
Throughout the study 

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Safety Assessment 
When conducted 
 
Healthy Participants 
Thermally injured 
participants 
Assessment of health status 
Screening, Day 1, Day 8, 
Day 15 
Not required 
9.4.1. 
Physical Examinations 
• 
A brief physical examination will include, at a minimum, assessments of the 
skin, lungs, cardiovascular system, and abdomen (liver and spleen). 
9.4.2. 
Vital Signs 
• 
A single vital sign measurement will be obtained at each time point indicated in 
SoA Table, and will include systolic and diastolic blood pressure and heart rate. 
Any abnormalities and changes in measurements will be communicated to the 
medical monitor. 
• 
Blood pressure and pulse measurements will be assessed 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, mobile phones). 
• 
Vital signs to be taken before blood collection for laboratory tests. 
• 
Repeat or unscheduled measurements may be taken at the discretion of the 
investigator. 
9.4.3. 
Clinical Safety Laboratory Assessment 
• 
All study related laboratory assessments will be performed by a local laboratory. 
The laboratory reports must be reviewed by the investigator, this review 
documented and both report and review are to be filed with the source 
documents.  
• 
Refer to Appendix 2 for the list of clinical laboratory tests to be performed and 
to the SoA for the timing and frequency. 
• 
All protocol-required laboratory assessments, as defined in Appendix 2, must be 
conducted in accordance with the laboratory manual and the SoA.  
• 
Clinically significant abnormal laboratory findings are those which are not 
associated with the underlying disease, unless judged by the investigator to be 
more severe than expected for the participant's condition. 

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9.5. 
Study Procedures 
The following procedures will be carried out during the study.  
9.5.1. 
Fluid Balance Measurement 
All fluid input and output will be recorded every 24 hours for thermally injured 
participants. 
9.5.2. 
Wound Healing 
Assessment of wound healing will be the time to 95% wound healing. Physical 
parameters of the wound (e.g., rate of healing) will be recorded and collected as a part of 
both the HESTIA and the SIFTI-2 studies. 
9.5.3. 
Other Clinical Responses 
To assess the relationship between severity of a participant’s condition following thermal 
injury and changes in intestinal permeability the following will be recorded and collected 
as a part of the SIFTI-2 study. Details can be found in the SIFTI-2 study protocol. 
• 
Number of ventilator-free days (ventilator start/restart/end date/time) 
• 
Number of vasopressor-free days (medication chart review) 
• 
Number of hemofiltration-free days (notes review) 
• 
Number of episodes of confirmed infection and sepsis 
• 
Number of surgical interventions 
• 
Total length of hospital stay 
• 
Calculate critical care and thermal injury severity scores 
9.6. 
Pharmacokinetics 
PK parameters are not evaluated in this study. 
9.7. 
Pharmacodynamics 
PD parameters are not evaluated in this study. 
9.8. 
Intestinal Permeability Assessments 
• 
Intestinal permeability will be determined by measuring the excretion of 
lactulose, mannitol and sucralose in urine following their enteral administration. 
It will be conducted in both healthy participants and thermally injured 
participants at the time points specified in the SoA. 
• 
The complete method for administration of STM and measurement of intestinal 
permeability is detailed in the SRM. 
• 
Urinary excretion of the orally ingested STM will be quantified using a 
technique such as capillary column gas chromatography. 

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• 
Urine samples will be collected in plastic bottles for analysis. Urine collection 
will begin immediately following STM administration. Urine samples will be 
collected over 24 hours post-STM administration. Accurate collection of the 
total volume voided during this 24 hour period is critical. 
• 
Sample Preparation 
Details of urine sample collection, processing, storage and shipping procedures are 
provided in the SRM. 
9.9. 
Genetics 
Genetics are not evaluated in this study. 
9.10. 
Sample Collection for Biomarker Analysis 
The following biomarkers will be collected during the study. Details of sample 
processing, storage and shipping are included in the study reference manual. 
Blood, stool and urine samples will be collected and stored. Timing of analyses and 
selected biomarkers will be dependent on the results of the intestinal permeability tests 
results. 
9.10.1. 
Blood Biomarkers 
Healthy participants 
• 
Blood samples will be taken for healthy participants recruited in this study over 
the time period specified in the SOA. Blood will be taken adhering to standard 
operating procedure from venae puncture.  
• 
The results of blood biomarker analysis will be evaluated in this study and 
compared to measures of intestinal permeability.  
• Twenty (20) ml blood will be collected on Day 1 for biomarker analysis, and 
20ml blood will be collected at screening for screening tests. The biomarkers to 
be measured may include, but are not limited to: 
• 
Markers of microbial translocation 
• 
Markers of intestinal damage 
• 
Inflammatory markers: e.g. C-Reactive Protein, Procalcitonin, cytokines 
(including TNF-a, IL-6, IL-8, IFN-g, IL-10, IL-1b, IL-12p70, IL-17, IL-4, 
IL13, IL1Ra, MIP1a, MIP1b, MIP2, GCSF, GMCSF, MCP-1, RANTES, 
HMGB1). 
Thermally injured participants 
• 
The blood required for this analysis in thermally injured participants will be 
collected as a part of the SIFTI-2 (IRAS 200366) study to which all thermally 
injured participants will be co-recruited. Details of the schedule for blood 

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collection and the total volume of blood collected can be found in the SIFTI-2 
study protocol. 
9.10.2. 
Stool Sample Collection 
• 
Stool samples will be collected from all participants in this study over the time 
period specified in Section 2, Schedule of activities (SOA). Stool samples will 
be collected adhering to standard operating procedure.  
• 
For thermally injured participants, the initial sample will be taken as close to 
time of injury as possible (“first stool sample produced upon admission”) and 
Day 14. Further samples will be taken on day 28 (± 3 days) and at month 6 (± 14 
days). 
• 
For healthy participants, a single sample will be collected at study entry 
(participants will be given a collection container at screening). 
9.10.3. 
Urine Sample Collection 
• 
Urine samples will be collected as a part of the measurement of intestinal 
permeability which is described in Section 9.4. 
• 
Additional urine samples will be collected from patients as part of the SIFTI-2 
study to which all thermally injured participants will be co-recruited. These will 
be used for, among other tests, the quantification of protein and microbial 
metabolites. 
• 
It is standard practice that patients admitted with burns of TBSA ≥15% will 
have a urinary catheter inserted on admission to ensure the accurate maintenance 
of fluid balance. A clean urine sample will be taken from the appropriate port on 
the urinary catheter. In patients who are not catheterised, a mid-stream urine 
(MSU) should be collected in a clean universal container where possible. 
• 
N.B. During the 24 hours following STM administration (during intestinal 
permeability measurement) urine samples must only be taken from the 5-hour of 
24-hour urine collections after the aliquots for sugar quantification have been 
taken. 
Sample Preparation 
• 
Details of urine sample collection, processing, storage and shipping procedures 
are provided in the SRM. 

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10. 
STATISTICAL CONSIDERATIONS 
All statistical analyses will be performed using SAS (SAS Institute Inc., Cary, North 
Carolina, USA), version 9.2 or higher.  Before database lock, a reporting and analysis 
plan (RAP) will be issued as a separate document, providing detailed methods for the 
analyses outlined below.  Any deviations from the planned analyses will be described in a 
RAP addendum and justified in the final integrated clinical study report. 
10.1. 
Hypotheses 
As this is an enabling study designed to better understand the biomarkers of intestinal 
permeability and other biomarkers in participants with moderate to severe burns, the 
statistical analysis for this study will be exploratory in order to better understand the 
parameters to inform future investigational medicinal product studies. 
The key factors of interest in this study are to understand (i) the nature of any differences 
at entry in intestinal permeability between healthy participants and thermal injury 
participants (ii) to understand the trajectory of changes in intestinal permeability over 
time. 
The key endpoint to be explored is the lactulose:mannitol (L/M) ratio, but other 
permeability biomarkers will also be explored. The analysis approaches to address these 
questions are exploratory, but will initially be conducted as outlined in Section 10.5 and 
Section 10.6. 
10.2. 
Sample Size Determination 
Sample size is based on feasibility. No formal calculation of power or sample size has 
been performed, but a sample size of 15 healthy participants and 25 thermal injury 
participants (>15% TBSA) should be sufficient to provide useful estimates of variability 
in lactutose:manitol ratios, and any change in L/M ratio over time. 
 
Although the key aim is to estimate the variability and L/M ratio and assess the 
difference in L/M ratio between thermal injury and healthy participants, for illustration, a 
trial including 25 thermal injury and 15 healthy participants would have 89% power to 
detect a 3-fold difference in L/M ratio between thermal injury and healthy participants 
using a 2-sided significance level of p<0.10.  This calculation uses a (log) b etween-
subject SD of 1.15, as estimated from the literature [Olquin, 2005]. 
10.3. 
Data Analyses Consideration 
In general, descriptive summaries will include number of subjects, mean, standard 
deviation, median, minimum, and maximum for continuous variables.  If data are log-
normally distributed data will be presented as number of subjects, geometric mean, 
coefficient of variation, minimum, and maximum; and percent for categorical variables. 
Summaries will present data by dose level and where appropriate, by assessment time. 

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10.4. 
Populations for Analyses 
The Safety Population will consist of all subjects who receive at least 1 dose of STM 
and have at least on post-dose safety assessment. 
 
The Evaluable Population will consist of all subjects who are entered into the study and 
have evaluable L/M ratio measurements. 
10.5. 
Statistical Analyses 
10.5.1. 
Safety Analyses 
Administration of STM is for the measurement of intestinal permeability. The safety of 
this administration is not an endpoint of this study, but will be monitored and reported. 
 
All safety data will be presented in data listings. Subject demographics, medical history, 
and prior and concomitant medications will be summarized using descriptive statistics. 
For continuous variables, these summaries will include number of subjects, mean, 
median, standard deviation, minimum, and maximum. 
 
For categorical variables, the summaries will include frequencies and corresponding 
percentages. No inferential hypothesis testing will be performed on the safety variables. 
 
Adverse events will be coded using the MedDRA classification system. 
 
For healthy participants (who are recruited only to HESTIA), STM-emergent AEs will be 
defined as any AEs, regardless of relationship to STM administration, that occur after the 
first dose of STM until the final follow-up visit. The STM-emergent AEs will be 
summarized for the overall number of AEs and the percentage of subjects who 
experience them. The total number of AEs will be summarized overall. The AEs will be 
further summarized by severity and relationship to STM. If relationship information is 
missing, the AE will be considered STM-related. Listings for the subsets of SAEs and 
STM-related SAEs will be provided. The SAEs and number of AEs leading to 
discontinuation of STM administration will be summarized. The incidence of AEs will 
also be summarized by system organ class and preferred term. 
 
For thermally-injured participants, STM-emergent AEs will be defined as any AE 
deemed related to STM administration that occurs after STM administration until the 
follow-up visit. The STM-related emergent AEs will be summarized for the overall 
number of AEs and the percentage of subjects who experience them. Listings for the 
subsets of SAEs and STM-related SAEs will be provided. The SAEs and number of AEs 
leading to discontinuation of STM administration will be summarized. The incidence of 
AEs will also be summarized by system organ class and preferred term. 
 
As laboratory data and vital signs are only collected at screening for healthy participants, 
these data will be listed only. Clinical laboratory values that are outside of the reference 
ranges will be flagged and evaluated for clinical significance by the investigator. Physical 
examination findings will be listed. For thermally-injured participants, physical 

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examination findings and clinical laboratory values will be highly abnormal and as such 
any data collected will only be listed.  Disease-related findings and changes will not be 
reported. 
10.5.2. 
Other Analyses 
Biomarker exploratory analyses will be described in the RAP. 
10.5.3. 
Interim Analyses 
No formal interim analysis will be performed. 
10.6. 
Analyses of lactulose/mannitol ratio 
In all analyses the variable TBSA will be a categorical variable defined as “Yes” for 
thermally injured participants, and “No” for healthy participants. 
Differences in permeability at entry 
Intestinal permeability biomarkers will be summarised by TBSA group and overall.  Data 
will summarised by geometric mean, CV, minimum, maximum and N.  A model will be 
fitted, defined as 
Log (L/M ratio) = intercept + TBSA 
Trajectory of the L/M ratio over time 
Intestinal permeability biomarkers will be summarised over time, by TBSA group and 
overall.  Data will summarise geometric mean, CV, minimum, maximum and N.  A 
model will be fitted, defined as 
Log (L/M ratio at time X / L/M ratio at entry) = intercept + Time + TBSA + Time*TBSA. 
This will be a repeated measurement analysis and will assess the rate of improvement in 
L/M ratio over time, and assess how this changes relative to healthy participants.  If 
required, further modelling assessing more complex relationships between L/M ratio and 
time may be undertaken.  Given this is an exploratory study the most appropriate 
variance-covariance matrix regarding the correlation of data over time will be explored as 
part of the statistical analysis. 
Data from this model may also be used to estimate the time to 50% improvement (or 
other degrees of improvement) in L/M ratio in relation to L/M ratio values seen in 
healthy participants.  This will be used to assess the clinical relevance and sensitivity of 
such measures. 
A model fitting log (AUC of L/M ratio) = intercept + TBSA will also be fitted.  AUC will 
be calculated using all measurements taken over time.  This will provide a summary of 
the weighted average L/M ratio value over time. 

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The use of %TBSA will also be assessed in the above analyses as a continuous covariate.  
The effects of age and Baux score will also be evaluated to understand differences in 
intestinal permeability in these groups [Osler, 2010]. 

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11. 
REFERENCES 
Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, Ouellette AJ. Secretion of 
Microbicidal Alpha-Defensins by Intestinal Paneth Cells in Response to Bacteria. Nat 
Immunol 2000; 1: 113-118 
Bjarnason I, MacPherson A, Hollander D. Intestinal Permeability: An Overview. 
Gastroenterology 1995; 108: 1566-1581 
Deitch EA, XU D, Kaisse VL, Role of the Gut in the Development of Injury and Shock 
Induced SIRS and MODS: the Gut-Lymph Hypothesis, A Review. Front Biosci 2006; 
11:520-528 
Deitch EA. Intestinal Permeability Is Increased in Burn Patients Shortly After Injury. 
British Journal of Surgery 1990; 77:587-592.  
Del Valle-Pinero A, Van Deventer H, Fourie N et al. Gastrointestinal Permeability in 
Patients with Irritable Bowel Syndrome Assessed Using a Four Probe Permeability 
Solution. Clin Chim Acta. 2013 March 15; 418: 97–101. 
Derikx J, Van Waardenburg D, Granzen B et al: Detection of Chemotherapy-Induced 
Enterocyte Toxicity with Circulating Intestinal Fatty Acid Binding Protein. J Paediatr 
Hematol Oncol 2006;28:267-269 
Doig CJ, Sutherland LR, Sandham JD, Fick GH, Verhoef M, Meddings JB: Increased 
Intestinal Permeability is Associated with the Development of Multiple Organ 
Dysfunction Syndrome in Critically Ill ICU Patients.  Am J Respir Crit Care Med 
1998;158:444-451 
European Commission, Health and Consumers Directorate-General.  The Rules 
Governing Medicinal Products in the European Union Volume10-Guidance Documents 
Applying Investigational Medicinal Products (NIMPS) (Rev. 1,March 2011) Ref. 
Ares(2011)300458 - 18/03/2011 
Grootjans J, Thuijls G, Verdam F et al. World J Gastrointest Surg 2010 March 27; 2(3): 
61-69 
Hammer A, Morris N, Earley Z, Choudhry M. The First Line of Defense: The Effects of 
Alcohol on Post-Burn Intestinal Barrier, Immune Cells, and Microbiome. Alcohol Res. 
2015;37(2):209-22 
Hampson P et al. Neutrophyl Dysfunction, Immature Granulocytes, and Cell Free DNA 
Are Early Biomarkers of Sepsis in Burn-Injured Patients. Annals of Surgery Ann Surg. 
2016 May 26. [Epub ahead of print] 
Menzies IS. Intestinal Permeability in Coeliac Disease. Gut 1972;13:847 

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Olquin F, Araya M, Hirsch S, Brunser O, Ayala V, Rivera R, Gotteland M:Prebiotic 
Ingestion Does Not Improve Gastrointestinal Barrier Function in Burn Patients. Burns 
2005 Jun; 31 (4):482-8 
Osler T, Glance LG, Hosmer DW. Simplified estimates of the probability of death after 
burn injuries: extending and updating the baux score. J Trauma. 2010;68(3):690-7 
Sequeira IR, Lentle RG, Kruger MC, Hurst RD. Standardising the Lactulose Mannitol 
Test of Gut Permeability to Minimise Error and Promote Comparability. PLoS ONE. 
2014;9(6):e99256 
Ziegler TR, Smith RJ, O’Dwyer ST, Demling RH, Wilmore DW: Increased Intestinal 
Permeability Associated with Infection in Burn Patients. Arch Surg 1988 Nov; 123 
(11):1313-9 

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12. 
APPENDICES 
12.1. 
Appendix 1: Abbreviations and Trademarks 
ACR 
Albumin/creatinine ratio 
AE 
Adverse Event 
Anti-HBc 
Anti-Hepatitis C 
ART 
Anti-retrovial treatment 
CFR 
Code of Federal Regulations 
D 
Day 
G 
Grams 
eCRF 
Electronic Case Report Form 
ICU 
Intensive Care Unit 
GCP 
Good Clinical Practice 
GCSP 
Global Clinical Safety and 
Pharmacovigilance 
GI 
Gatrointestinal  
HB 
Hepatitis B 
HBs AG 
Hepatitis B Antigen 
HCV 
Hepatitis C 
GSK 
GlaxoSmithKline 
HIV 
Human Immunodeficiency Virus 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
of Technical Requirements for Registration 
of Pharmaceuticals for Human Use 
ICU 
Intensive Care Unit 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
ISS 
Investigator Sponsored Study 
L/M 
Lactulose/mannitol ratio 
mL 
Milliliter 
MODS 
Multi-organ dysfunction syndrome 
NIMP 
Non-investigational medicinal product 
NC 
Nominated Consultee 
PC 
Personal Consultee 
PCR 
Polymerase Chain Reaction 
RAP 
Reporting and Analysis Plan 
SAE 
Serious adverse event 
SIFTI-2 
A Multi-centre, Prospective Study to 
Examine the Relationship between 
Neutrophil Function and Sepsis in Adults 
and Children with Severe Thermal Injuries 
SoA 
Schedule of Activities 
SRM 
Study Reference Manual 
STM 
Sugar Test Material 

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TBSA 
Total body surface area 
TNF 
Tumour Necrosis Factor 
WOCBP 
Women of Child Bearing Potential 
 
Trademark Information 
Trademarks of the GlaxoSmithKline 
group of companies 
 
Trademarks not owned by the 
GlaxoSmithKline group of companies 
NONE 
 
SAS 
 

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12.2. 
Appendix 2: Clinical Laboratory Tests 
• 
All clinical laboratory tests will be performed in the local laboratory. 
• 
Protocol-specific requirements for inclusion or exclusion of participants are 
detailed in Section 6 of the protocol. 
• 
Additional tests may be performed at any time during the study as determined 
necessary by the investigator or required by local regulations. 
Table 2 
Protocol-Required Safety Laboratory Assessments for Healthy 
Participants 
Laboratory 
Assessments 
Parameters 
Haematology 
Platelet Count  
 
WBC count with Differential: 
Neutrophils 
Lymphocytes 
Monocytes 
Eosinophils 
Basophils 
RBC Count 
Hemoglobin 
Hematocrit 
Clinical 
Chemistry1 
Urea 
Potassium 
 
Total bilirubin 
 
Creatinine 
Sodium 
Alanine 
Aminotransferase 
 (ALT)/  
Total Protein 
 
Glucose 
nonfasting 
Calcium 
Alkaline 
phosphatase 
 
Routine 
Urinalysis 
• pH, glucose, protein, blood, ketones by dipstick 
• Microscopic examination (if blood or protein is abnormal) 
Other 
Screening 
Tests 
• Urine alcohol and drug screen (to include at minimum: amphetamines, 
barbiturates, cocaine, opiates, cannabinoids and benzodiazepines)] 
• Urine human chorionic gonadotropin (hCG) pregnancy test (as needed for 
women of childbearing potential)1 
• Serology (HIV antibody, hepatitis B surface antigen [HBsAg], and hepatitis C 
virus antibody) 
NOTES : 
1. Local urine testing will be standard for the protocol unless serum testing is required by local regulation or IRB/IEC. 
 

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12.3. 
Appendix 3: Study Governance Considerations 
12.3.1. 
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 and other relevant documents (e.g., 
advertisements) must be submitted to an IRB/IEC by the investigator and 
reviewed and approved by the IRB/IEC before the study is initiated.  
• 
Any amendments to the protocol will require IEC/IRB approval before 
implementation of changes made to the study design, except for changes 
necessary to eliminate an immediate hazard to study participants.  
• 
Any amendments to the SIFTI-2 protocol which impact on this protocol will be 
reviewed and may result in changes to this protocol being required. Any such 
changes will be subject to IEC/IRB approval before implementation. 
• 
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 
12.3.2. 
Recruiting participants under the Mental Capacity Act 2005 
On admission into hospital the patient’s capacity will be assessed. A patient may lack 
capacity due to the severity of their injury, arriving intubated and ventilated or due to a 
pre-existing co-morbidity. 
Please note, the same process will also be followed for the SIFTI-2 study to which 
thermally injured participants will be co-recruited. 
If a patient does not have the capacity to make an informed decision, the research team 
will approach a patient’s personal consultee. Examples of personal consultees include: 

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• A family member, carer or friend 
• An attorney acting under a Lasting Power of Attorney 
• A court appointed deputy, provided that they had a relationship with, or personal 
knowledge of, the person lacking capacity before their appointment as deputy. 
There may be circumstances in which a personal consultee is not available, some 
examples of this are: 
• Where no family member or friend is willing to act as a personal consultee 
• Where the family member or friends live a long distance away and/or are not in 
frequent contact with the patient who lacks capacity 
• Where the regular carers of the person who lacks capacity are doing so for 
payment or in a professional capacity (e.g. care home staff or nurses) 
• Where someone is acting on a professional role (e.g. their GP or solicitor) 
In this case, a nominated consultee will be required. A nominated consultee is considered 
to be a medical professional that has no connection to the research trial, but has an 
understanding of the implications of the research trial on the participant. 
In these circumstances, examples of nominated consultees are: 
• An emergency department doctor, preferably Consultant level. 
• Intensive Care doctor, preferably Consultant level. 
• Doctor from the burns team, not directly involved in the research study. 
Once a personal or nominated consultee has been identified, they will be provided with a 
specific information leaflet about the trial. The personal and nominated consultee will be 
asked if they feel the study would be something the participant would have no objections 
to. If in their opinion the participant would have no objection to being recruited into a 
research trial the consultee will be asked to sign a declaration form. 
12.3.3. 
Determining Whether a Participant has Capacity Under the 
Mental Capacity Act (2005) 
Prior to deciding that a patient does not have the capacity to give informed consent the 
researcher must follow the Mental Capacity Act (2005) to ensure that the participant does 
not hold capacity. The principles of the MCA which we will adhere are as follows: 
• A person must be assumed to have capacity unless it is established that he/she 
lacks capacity. 
• A person is not to be treated as unable to make a decision unless all practical steps 
to help him/her to do so have been taken without success. 
• A person is not to be treated as unable to make a decision merely because he/she 
makes an unwise decision. 
• An act done or decision made, under this Act for or on behalf of a person who 
lacks capacity must be done, or made, in his best interests. 

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• Before the act is done, or the decision is made, regard must be had to whether the 
purpose for which it is needed can be as effectively achieved in a way that is less 
restrictive of the person's rights and freedom of action. 
A decision to appoint a consultee on behalf of a patient will be made if the participant is 
unable to: 
1. (a) understand the information relevant to the decision, 
(b) retain that information, 
(c) use or weigh that information as part of the process of making the decision, or 
(d) communicate his/her decision (whether by talking, using sign language or any 
other means). 
2. A person is not to be regarded as unable to understand the information relevant to a 
decision if he/she is able to understand an explanation of it given to him in a way that is 
appropriate to his circumstances (using simple language, visual aids or any other means). 
3. The fact that a person is able to retain the information relevant to a decision for a short 
period only does not prevent him/her from being regarded as able to make the decision. 
4. The information relevant to a decision includes information about the reasonably 
foreseeable consequences of 
(a) deciding one way or another, or 
(b) failing to make the decision. 

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Figure 4 
HESTIA Thermal Injury Participant Recruitment 
 
12.3.4. 
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. 
12.3.5. 
Informed Consent Process 
• 
The investigator or his/her representative will explain the nature of the study to 
the participant or his/her personal or nominated consultee and answer all 
questions regarding the study. 

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• 
Participants must be informed that their participation is voluntary. Participants 
or their personal or nominated consultee 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 
personal or nominated consultee. 
• 
Healthy participants who are rescreened are required to sign a new ICF. 
12.3.6. 
Data Protection 
• 
Participants will be assigned a unique identifier by the sponsor. Any participant 
records or datasets that are transferred to the sponsor will contain the identifier 
only; participant names or any information which would make the participant 
identifiable will not be transferred. 
• 
The participant must be informed that his/her personal study-related data will be 
used by the sponsor in accordance with local data protection law. The level of 
disclosure must also be explained to the participant. 
• 
The participant must be informed that his/her medical records may be examined 
by Clinical Quality Assurance auditors or other authorized personnel appointed 
by the sponsor, by appropriate IRB/IEC members, and by inspectors from 
regulatory authorities. 
12.3.7. 
Committees Structure 
An Independent Data Monitoring Committee or similar review group will not be used in 
this study, but an internal preliminary data review will be conducted. 
The Data Review team will consist of the GSK medical monitor, clinical and operational 
leads, statistician, early development lead and the safety officer. They will meet at 
intervals specified within the data review charter to review data relevant to the future 
conduct of the study, and will also assess any risk to study participants. 
12.3.8. 
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. 

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• 
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 
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. 
12.3.9. 
Dissemination of Clinical Study Data 
• Study information from this protocol will be posted on publicly available clinical 
trial registers before enrollment of subjects begins. 
• 
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. 
• 
The procedures and timing for public disclosure of the results summary and for 
development of a manuscript for publication will be in accordance with GSK 
Policy. 
• 
A manuscript will be progressed for publication in the scientific literature if the 
results provide important scientific or medical knowledge. 
12.3.10. 
Data Quality Assurance 
• 
All participant data relating to the study will be recorded on printed or electronic 
CRF unless transmitted to the sponsor or designee electronically (e.g., 
laboratory data). The investigator is responsible for verifying that data entries 
are accurate and correct by physically or electronically signing the CRF. 
• 
The investigator must maintain accurate documentation (source data) that 
supports the information entered in the CRF. 
• 
The investigator must permit study-related monitoring, audits, IRB/IEC review, 
and regulatory agency inspections and provide direct access to source data 
documents. 
• 
The sponsor or designee is responsible for the data management of this study 
including quality checking of the data. 
• 
Study monitors will perform ongoing source data verification to confirm that 
data entered into the CRF by authorized site personnel are accurate, complete, 
and verifiable from source documents; that the safety and rights of participants 
are being protected; and that the study is being conducted in accordance with the 

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currently approved protocol and any other study agreements, ICH GCP, and all 
applicable regulatory requirements. 
• 
Records and documents, including signed ICF, pertaining to the conduct of this 
study must be retained by the investigator for 15 years after study completion 
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. 
12.3.11. 
Source Documents 
• 
Source documents provide evidence for the existence of the participant and 
substantiate the integrity of the data collected. Source documents are filed at the 
investigator’s site. 
• 
Data reported on the CRF or entered in the eCRF that are transcribed from 
source documents must be consistent with the source documents or the 
discrepancies must be explained. The investigator may need to request previous 
medical records or transfer records, depending on the study. Also, current 
medical records must be available. 
• 
Definition of what constitutes source data can be found in the study specific 
Source Data Verification document. 
12.3.12. 
Study and Site Closure 
GSK or its designee reserves the right to close the study site or terminate the study at any 
time for any reason at the sole discretion of GSK. Study sites will be closed upon study 
completion. A study site is considered closed when all required documents and study 
supplies have been collected and a study-site closure visit has been performed. 
The investigator may initiate study-site closure at any time, provided there is reasonable 
cause and sufficient notice is given in advance of the intended termination. 
Reasons for the early closure of a study site by the sponsor or investigator may include 
but are not limited to: 
• 
Failure of the investigator to comply with the protocol, the requirements of the 
IRB/IEC or local health authorities, the sponsor's procedures, or GCP guidelines 
• 
Inadequate recruitment of participants by the investigator 

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12.4. 
Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting 
Definition of AE 
AE Definition 
• 
An AE is any untoward medical occurrence in a clinical study participant, temporally 
associated with study participation, whether or not considered related to the study. 
• 
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 study participation. 
• 
NOTE: As detailed in Section 9.2.1, only AEs deemed to be related to procedures or 
requirements unique to the HESTIA study will be recorded/reported for thermally 
injured participants. All other AEs will be recorded/reported through the SIFTI-2 
study. 
 
Events Meeting the AE Definition  
• 
Any abnormal laboratory test results (haematology, 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 STM 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. 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. 
 
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. Table 3 
provides a list of commonly occurring AEs in participants with severe thermal injury 
which may meet this definition. 
• 
The disease/disorder being studied or expected progression, signs, or symptoms of 

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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. 
Table 3 
Complications of severe thermal injury which can be considered as 
associated with the underlying disease and do not require reporting 
as AEs unless judged to be more severe than expected for the 
participant’s condition or related to HESTIA study procedures. 
System Assessment 
Complication type 
Action 
Airway problems 
Failed extubation 
Record AE 
Tracheostomy complication 
Breathing Problems 
Pneumothorax 
Record AE 
Pulmonary Oedema 
Respiratory Arrest 
Pneumonia 
VAP 
Acute lung injury (ALI) 
ARDS 
Circulatory Problems 
Haemodynamic instability 
Record AE 
Record ionotrope dose in 
con-meds 
Increasing vasoactive drug 
support 
Arrhythmia 
Endocarditis 
Acute LVF/CCF 
Cardiac Arrest 

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System Assessment 
Complication type 
Action 
MI 
Neurological Problems 
Reduced GCS (off sedation) 
Record AE 
Intra-Cranial bleed 
CVA 
Acute confusion/Delirium 
Meningitis-bacterial 
Oedema Complications 
Abdominal Compartment 
Syndrome (ACS) 
Record AE 
Acute Limb compartment 
syndrome 
Microbiological problems 
Sepsis  
Record AE 
Record in Microbiology 
form 
Chest Infection 
Lower Respiratory Tract 
Infection 
UTI 
Bloodstream Infection (BSI) 
Wound infection 
Intra-vascular catheter (line) 
infection 
Infective diarrhoea 
Clostridium difficile 
infection/pseudomembranous 
colitis 
Renal/Urology problems 
Acute rhabdomyolysis 
Record AE 
Ensure biochemistry and 
CK results recorded n CRF 
Acute renal failure 
Acute urinary retention 
Renal replacement therapy 

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System Assessment 
Complication type 
Action 
Thromboembolic 
complications 
Lower limb DVT 
Record AE with location of 
thrombus 
Upper limb DVT 
Pulmonary embolism 
Other VTE 
Fat embolism 
Wound complications 
Major graft loss 
Record AE with details 
Major skin substitute loss 
Wound infection 
Invasive wound infection 
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). 
Please note that, as described in Section 9.2.6 a, b, c and d below are considered as 
‘disease related events’ as they occur commonly in patients following thermal injury 
unless, in the opinion of the investigator, they are directly related to STM administration 
or other unique requirements of the HESTIA study. 
A SAE is defined as any untoward medical occurrence that: 
a. Results in death 
b. Is life-threatening 
The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
participant was at risk of death at the time of the event. It does not refer to an event, 
which hypothetically might have caused death, if it were more severe. 
c. 
Requires inpatient hospitalization or prolongation of existing hospitalization 
In general, hospitalization signifies that the participant has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for observation 
and/or treatment that would not have been appropriate in the physician’s office or 
outpatient setting. Complications that occur during hospitalization are AE. If a 
complication prolongs hospitalization or fulfils any other serious criteria, the event is 
serious. When in doubt as to whether “hospitalization” occurred or was necessary, the AE 

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should be considered serious. 
Hospitalization for elective treatment of a pre-existing condition that did not worsen from 
baseline is not considered an AE. 
d. Results in persistent disability/incapacity 
• 
The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions. 
• 
This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 
and accidental trauma (e.g., sprained ankle) which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption. 
e. 
Is a congenital anomaly/birth defect 
f. 
Other situations: 
• 
Medical or scientific judgment should be exercised in deciding whether SAE 
reporting is appropriate in other situations such as important medical events that may 
not be immediately life-threatening or result in death or hospitalization but may 
jeopardize the participant or may require medical or surgical intervention to prevent 
one of the other outcomes listed in the above definition. These events should usually 
be considered serious. 
Examples of such events include invasive or malignant cancers, intensive treatment 
in an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency 
or drug abuse. 
 
If any of the above are observed, then an AE will be recorded in the SIFTI2 study CRF. 
If any of the above are observed and deemed to be related to STM administration or other 
unique requirements of the HESTIA study, then to be recorded in the HESTIA CRF and 
reported to GSK as per guidance below. 
 
Recording AE and SAE 
AE and SAE Recording 
• 
When an AE/SAE occurs, it is the responsibility of the investigator to review all 
documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) 
related to the event. 
• 
The investigator will then record all relevant AE/SAE information in the CRF. 
• 
It is not acceptable for the investigator to send photocopies of the participant’s 
medical records to GSK in lieu of completion of the GSK /AE/SAE CRF page. 
• 
There may be instances when copies of medical records for certain cases are 
requested by GSK. In this case, all participant identifiers, with the exception of the 

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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 sufficiently discomfort and interferes with normal 
everyday activities. 
• 
Severe: An event that prevents normal everyday activities. An AE that is assessed as 
severe should not be confused with an SAE. Severe is a category utilized for rating 
the intensity of an event; and both AE and SAE can be assessed as severe. 
An event is defined as ‘serious’ when it meets at least 1 of the predefined outcomes 
as described in the definition of an SAE, NOT when it is rated as severe. 
 
Assessment of Causality 
• 
The investigator is obligated to assess the relationship between administration of the 
NIMP (STM) 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 STM administration 
will be considered and investigated. 
• 
The investigator will also consult the Product Information, for marketed products, in 
his/her assessment. 
• 
For each AE/SAE, the investigator must document in the medical notes that he/she 
has reviewed the AE/SAE and has provided an assessment of causality. 
• 
There may be situations in which an SAE has occurred and the investigator has 
minimal information to include in the initial report to GSK. However, it is very 
important that the investigator always make an assessment of causality for 
every event before the initial transmission of the SAE data to GSK. 
• 
The investigator may change his/her opinion of causality in light of follow-up 
information and send an SAE follow-up report with the updated causality 

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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. 
• 
The investigator will submit any updated SAE data to GSK within 24 hours of 
receipt of the information. 
 
Reporting of SAE to GSK 
SAE Reporting to GSK via 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. 
• 
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 to the medical monitor by telephone. 
• 
Contacts for SAE reporting can be found in 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. 
• 
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 

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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 SRM. 
 

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12.5. 
Appendix 5: Origin of Data/Samples 
The following table of study objectives specifies, for each objective, the provenance of 
clinical data and samples which will be used to explore that endpoint 
Objective 
Endpoint 
Origin of Data/Samples 
Co-Primary 
 
1. To determine the impact of 
thermal injury on the magnitude 
of small intestinal permeability 
change as soon as possible 
following injury compared to 
healthy participants 
• Lactulose/Mannitol 
(L/M) ratio at entry 
• HESTIA study 
2. To characterise the effect of 
thermal injury on small 
intestinal permeability over time 
and establish the clinical and 
demographic factors which can 
influence it 
• Changes in L/M 
ratio over time 
• HESTIA Study 
Exploratory 
 
3. To determine the impact of 
thermal injury on colonic 
permeability as soon as 
possible following injury 
compared to healthy 
participants 
• Fractional excretion 
of sucralose at entry 
• HESTIA Study 
4. To characterise the effect of 
thermal injury on colonic 
permeability over time  
• Changes in the 
fractional excretion 
of sucralose over 
time 
• HESTIA Study 
5. To assess the relationship 
between severity of a 
participant’s condition following 
thermal injury and changes in 
intestinal permeability† 
• Number of 
ventilator-free days 
• Number of 
vasopressor-free 
days 
• Number of 
hemofiltration-free 
days 
• Number of episodes 
• All clinical data will be 
obtained from SIFTI2. 
• Permeability measurements 
will be obtained from HESTIA 

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Objective 
Endpoint 
Origin of Data/Samples 
of confirmed 
infection and sepsis 
• Number of surgical 
interventions 
• Total length of 
hospital stay 
• Calculate critical 
care and thermal 
injury severity 
scores 
6. To assess plasma and urine 
biomarkers of intestinal 
permeability, bacterial 
translocation and renal tubular 
dysfunction following thermal 
injury† 
• Change in markers 
of intestinal 
mucosal damage 
samples from blood 
• Change in urine 
protein:creatinine 
and urine 
albumin:creatinine 
ratios 
• Blood biomarkers obtained 
from SIFTI-2 (and HESTIA 
for healthy participants) 
• Urine for microbial metabolite 
analysis, claudin 3 and KIM 1 
obtained from SIFTI-2 (and 
HESTIA for Healthy 
participants) 
• Urine albumin:creatinine and 
protein:creatinine ratios 
obtained from SIFTI-2 (and 
HESTIA for healthy 
participants) 
• Permeability data (STM 
absorption) from HESTIA 
7. To assess the impact of 
thermal injury and intestinal 
permeability on the intestinal 
microbiome compared to 
healthy participants 
• Changes in 
microbiome of acute 
and convalescent 
stool samples 
• Stool samples collected in 
HESTIA protocol 
• Permeability data from 
HESTIA 
8. To assess the impact of pre-
existing co-morbid conditions 
on intestinal permeability and 
clinical outcome following 
thermal injury† 
• Medical history and 
drug history at the 
time of admission 
• Medical History data from 
HESTIA 
• Permeability data from 
HESTIA 
9. To assess wound healing 
• Time to wound 
recovery (e.g. 95%) 
• Wound healing assessment 
data from clinical notes will 
be captured in HESTIA at 14 

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Objective 
Endpoint 
Origin of Data/Samples 
day, 28 day and 6 month 
visits. 
10. To characterise intestinal 
microbiota, and correlate its 
composition with both intestinal 
permeability and bacterial 
detection in blood† 
• Changes in 
intestinal 
microbiome 
• Bacterial markers of 
translocation 
• Microbiome data from 
HESTIA study 
• Blood Biomarker data from 
SIFTI-2 (thermally injured 
participants) and HESTIA 
(healthy participants. 
 

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12.6. 
Appendix 6: Protocol Amendment History 
The Protocol Amendment Summary of Changes Table for the current amendment is 
located directly before the Table of Contents (TOC). 
Amendment 01 (15-Aug-2017) 
Overall Rationale for the Amendment: The interim analysis has been removes since 
lactuose and mannitol data in healthy controls was unlikely to change the required sample 
size in thermally injured participants. 
Section # 
and Name 
Description of Change 
Brief Rationale 
10.5.3 Interim 
Analysis 
Removed preliminary review of 
lactulose/mannitol ratio data. 
Analysis of lactulose and mannitol data in 
healthy controls would be unlikely to 
change the study size, and will no longer 
be performed. 
5.2 Number of 
Participants 
Included withdrawn participants 
may be replaced. 
To ensure the target number of evaluable 
population is maintained. 
5.4.4 
Preliminary 
Data Review 
Modified the preliminary data 
review. 
To describe the change in preliminary data 
review. 
Synopsis 
Updated preliminary data review. 
For consistency. 
6.5 Exclusion 
Criteria 
Revision to eligibility criteria to 
exclude hepatitis infection for 
thermally injured participants. 
Addition the personal consultee will 
not be notified if a patient lacking 
mental capacity was found to be 
HIV positive. 
Consistency correction. Hepatitis screening 
will be performed. 
 
To provide Ethic Committee reassurance 
and address any ambiguity of this 
information. 
8.2.1 Other 
Withdraw 
Criteria 
Added the withdraw of participants 
receiving hemodialysis. 
Renal replacement therapy (i.e. dialysis) 
may filter out lactulose and mannitol. 
9.2.2 Time 
Period and 
Frequency for 
Collecting AE 
and SAE 
Information 
Added: 
Any SAEs assessed as related to 
study participation will be recorded 
from the time a participant 
consents. 
All SAEs will be recorded and 
reported to the sponsor or designee 
immediately and under no 
To align with the updates to Clinical 
Protocol Template (Vol 7) effective 7th April 
2017. 

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Section # 
and Name 
Description of Change 
Brief Rationale 
circumstance should this exceed 24 
hours. 
9.4 Clinical 
Safety 
Laboratory 
Assessments 
Removal of if laboratory values 
from non-protocol specified 
laboratory assessments performed 
at the institution’s local laboratory. 
To align with the updates to Clinical 
Protocol Template (Vol 7) effective 7th April 
2017. 
Appendix 4: 
Adverse 
Events: 
Definitions 
and 
Procedures 
for Recording, 
Evaluating, 
Follow-up, 
and Reporting 
Reporting SAE to GSK if the 
electronic system is unavailable. 
To align with the updates to Clinical 
Protocol Template (Vol 7) effective 7th April 
2017. 
 
DOCUMENT HISTORY 
Document 
Date 
Amendment 2 
26-Sep-2017 
Amendment 1 
15-Aug-2017 
Original Protocol 
02-Mar-2017