Study to Elucidate the Association of the Renin-angiotensin System and Right Ventricular Function in Mechanically Ventilated Patients
Phase: NA
Conditions: Acute Lung Injury
Sponsor: GlaxoSmithKline

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TITLE PAGE
Protocol Title: Study to elucidate the association of the Renin-angiotensin system and 
right ventricular function in mechanically ventilated patients
Protocol Number: 205821 – Amendment 3
Short Title: Study of Renin-angiotensin system in mechanically ventilated patients
Compound Number: Non-Compound Specific 
Sponsor Name and Legal Registered Address: 
GlaxoSmithKline Research & Development Limited
980 Great West Road
Brentford
Middlesex, TW8 9GS
UK
Medical Monitor Name and Contact Information can be found in the Study 
Reference Manual.
Approval Date: 27-FEB-2019
Copyright 2019 the GlaxoSmithKline group of companies.  All rights reserved.  
Unauthorised copying or use of this information is prohibited.
 
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PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE
DOCUMENT HISTORY
Document
Date
Protocol Amendment 3
27-Feb-2019
Protocol Amendment 2
27-Nov-2017
Protocol Amendment 1
02-Jun-2017
Original Protocol
26-Apr-2017
The Summary of Changes tables for Protocol Amendment 1 (02-Jun-2017) and Protocol 
Amendment 2 (27-Nov-2017) are in Section 12.6 (Appendix 6).
Amendment 3 (27-FEB-2019)
Overall Rationale for the Amendment:  The primary reason for the protocol 
amendment is to increase the duration that a participant may be mechanically ventilated, 
before they are included in the study – from ≤24 hours to ≤48 hours.  This change has 
been made to assist enrolment of participants into the study, but the sponsor considers 
that it will not impact the objectives of the study.  Additionally, minor clarifications have 
been made.
In the table below, in the column ‘Description of Change’, new text is shown in bold and 
deleted text has been striked through.
Section # and Name
Description of Change
Brief Rationale
Sponsor Signatory
Andy Fowler
Discovery Medicine Physician, 
Respiratory Therapy Area
Aili Lazaar, MD,
Discovery Medicine
Clinical Pharmacology and 
Experimental Medicine
Sponsor signatory change due 
to change in study personnel.
2. Schedule of activities –
Simplified acute physiology 
score (SAPS II)
SAPS II score to be taken 
within approximately 24 hours 
of admission to intensive care. 
Not at time of intubation.
To align the protocol with 
standard of care.
 
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Section # and Name
Description of Change
Brief Rationale
2. Schedule of activities
Echocardiogram
First echocardiogram must be 
completed within 24 48 hours 
of starting mechanical 
ventilation (but ≤24 hours, 
whenever possible)
To align the protocol with the 
revised inclusion criteria – that 
a participant may be 
mechanically ventilated for 
≤48 hours before inclusion in 
the study.
5. Study Design –  
5.1 Overall study design
The initial echocardiogram will 
take place within 24 48 hours 
of mechanical ventilation (but 
≤24 hours, whenever 
possible).
Figure 1 updated to show up 
to 48 hours from mechanical 
ventilation to inclusion.
To align the protocol with the 
revised inclusion criteria – that 
a participant may be 
mechanically ventilated for 
≤48 hours before inclusion in 
the study.
6. Study Population
6.1 Inclusion Criteria
Inclusion 2
2. Participants who are 
receiving invasive mechanical 
ventilation (duration of 
ventilation ≤24 ≤48 hours, but 
≤24 hours, whenever 
possible).
To increase the duration that a 
participant may be mechanically 
ventilated, before they are 
included in the study – from 
≤24 hours to ≤48 hours.  This 
change has been made to 
assist enrolment of participants 
into the study.
6. Study Population
6.2 Exclusion Criteria
Investigator will make every 
effort to ensure that the 
following exclusion criteria 3 to 
10 are met; however, in some 
instances it may not be 
possible to assess all of these 
criteria within the 24 48-hour 
window.  
To align the protocol with the 
revised inclusion criteria – that 
a participant may be 
mechanically ventilated for 
≤48 hours before inclusion in 
the study.
9.2 Pharmacodynamics
9.2.1 Echocardiograms
The first echocardiogram must 
be undertaken within 24
48 hours of the start of 
mechanical ventilation (but 
within 24 hours, whenever 
possible)
To align the protocol with the 
revised inclusion criteria – that 
a participant may be 
mechanically ventilated for 
≤48 hours before inclusion in 
the study.
9.5 Participant Status
9.5.1 SAPS II
The SAPS II score will be 
measured at screening only 
and is comprised of the 
following components:
Removed timing of SAPS II
score, because this is described 
more accurately in the 
Schedule of Activities 
(Section 2).
 
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Section # and Name
Description of Change
Brief Rationale
9.7.1 Serious Adverse 
Events (SAE)
Although no investigational 
product will be given, SAEs 
will be recorded, as follows:  
SAEs relating to study 
procedures; and SAEs 
related to GlaxoSmithKline 
(GSK) medication used as 
co-medication. 
This ensures that any SAEs 
relating to study procedures or 
any GSK medication used as 
co-medication, are captured.
Clarification made in the SAE 
section, but no change in the 
guidance.
 
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TABLE OF CONTENTS
PAGE
PROTOCOL AMENDMENT SUMMARY OF CHANGES TABLE..........................3
1.
SYNOPSIS.....................................................................................................8
2.
SCHEDULE OF ACTIVITIES (SOA).............................................................10
3.
INTRODUCTION..........................................................................................13
3.1.
Study Rationale .................................................................................13
3.2.
Background........................................................................................13
3.3.
Benefit/Risk Assessment ...................................................................14
3.3.1.
Risk Assessment .................................................................14
3.3.2.
Benefit Assessment .............................................................15
3.3.3.
Overall Benefit:Risk Conclusion...........................................15
4.
OBJECTIVES AND ENDPOINTS.................................................................16
5.
STUDY DESIGN...........................................................................................19
5.1.
Overall Design ...................................................................................19
5.2.
Number of Participants ......................................................................19
5.3.
Participant and Study Completion......................................................20
5.4.
Scientific Rationale for Study Design.................................................20
5.5.
Dose Justification...............................................................................20
6.
STUDY POPULATION .................................................................................20
6.1.
Inclusion Criteria................................................................................20
6.2.
Exclusion Criteria...............................................................................21
6.3.
Lifestyle Restrictions..........................................................................22
6.4.
Screen Failures..................................................................................22
7.
TREATMENTS .............................................................................................22
7.1.
Concomitant Therapy ........................................................................22
8.
DISCONTINUATION CRITERIA ..................................................................23
8.1.
Discontinuation of Study Treatment...................................................23
8.2.
Withdrawal from the Study.................................................................23
9.
STUDY ASSESSMENTS AND PROCEDURES...........................................23
9.1.
Demographic/Medical History Assessments......................................24
9.2.
Pharmacodynamics ...........................................................................24
9.2.1.
Echocardiograms.................................................................24
9.2.2.
Electrical Impedance Tomography (EIT)..............................25
9.3.
Ventilator Settings..............................................................................25
9.4.
Lung Function....................................................................................25
9.5.
Participant Status...............................................................................26
9.5.1.
SAPS II ................................................................................26
 
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9.5.2.
Sequential Organ Failure Assessment (SOFA) score..........26
9.5.3.
Disease diagnosis................................................................27
9.5.4.
General management status................................................27
9.6.
Additional Clinical assessments.........................................................28
9.7.
Adverse Events..................................................................................28
9.7.1.
Serious Adverse Events (SAE)............................................28
9.7.2.
Time Period and Frequency for Collecting SAE 
Information...........................................................................28
9.7.3.
Method of Detecting SAEs...................................................29
9.7.4.
Follow-up of SAEs ...............................................................29
9.7.5.
Regulatory Reporting Requirements for SAEs.....................29
9.8.
Safety Assessments ..........................................................................29
9.8.1.
Vital Signs............................................................................29
9.8.2.
Clinical Laboratory Assessments.........................................29
9.9.
Biomarkers.........................................................................................30
10. STATISTICAL CONSIDERATIONS..............................................................30
10.1. Sample Size Determination ...............................................................30
10.2. Populations for Analyses ...................................................................33
10.3. Statistical Analyses............................................................................34
10.3.1.
Pharmacodynamic Analyses................................................34
10.3.2.
Safety Analyses...................................................................35
10.3.3.
Interim Analyses ..................................................................35
11. REFERENCES.............................................................................................40
12. APPENDICES ..............................................................................................42
12.1. Appendix 1: Abbreviations and Trademarks ......................................42
12.2. Appendix 2: Clinical Laboratory Tests ...............................................44
12.3. Appendix 3: Study Governance Considerations ................................45
12.3.1.
Ethical Considerations .........................................................45
12.3.2.
Financial Disclosure.............................................................45
12.3.3.
Informed Consent Process ..................................................45
12.4. Appendix 4: Adverse Events: Definitions and Procedures for 
Recording, Evaluating, Follow-up, and Reporting..............................50
12.5. Appendix 5: Assumptions for sample size calculations......................55
12.5.1.
Distribution of Ang II ............................................................55
12.5.2.
Definition of outcome: an objective measure of right 
ventricular (RV) dysfunction.................................................56
12.5.3.
"Success" and "futility" criteria .............................................58
12.5.4.
Assumptions for simulated datasets ....................................59
12.5.5.
Analysis of simulated data ...................................................60
12.5.6.
Metrics for operating characteristics ....................................61
12.6. Appendix 6: Protocol Amendment History .........................................62
 
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1.
SYNOPSIS
Protocol Title: Study to elucidate the association of the Renin-angiotensin system and 
right ventricular function in mechanically ventilated patients
Short Title: Study of Renin-angiotensin system in mechanically ventilated patients
Rationale:
Right ventricular (RV) dysfunction may result from increased RV afterload secondary to 
increased pulmonary vascular resistance in patients with severe respiratory failure,
including acute respiratory distress syndrome (ARDS). RV dysfunction, particularly 
severe dysfunction [acute cor pulmonale (ACP)], has been associated with worse 
outcomes in critically ill patients, including patients with ARDS. Increased mortality has 
been associated with RV dilatation and increased pulmonary artery pressures [termed 
pulmonary circulatory dysfunction (PCD)]. The Renin-angiotensin system (RAS) is a key 
regulator of vascular tone in the pulmonary vasculature and Angiotensin II (Ang II) in 
particular drives both vasoconstriction and vascular remodelling in human diseases such 
as pulmonary arterial hypertension. RAS peptides such as Ang II and Ang(1-7) may play 
a role in the observed changes in pulmonary vasoconstriction in patients with severe 
respiratory failure with consequent adverse effects on the right ventricle. This study is 
designed to assess whether circulating Ang II and Ang(1-7) levels are associated with RV 
dysfunction in mechanically ventilated patients. It is also designed to further characterise 
the patient population, including severity of RV dysfunction.  
Objectives and Endpoints:
Objectives
Endpoints
Primary

To evaluate the association between 
plasma Ang II levels and RV function 
in mechanically ventilated 
participants. 

Ang II levels 

Echocardiographic measures: 
Ratio of right ventricular to left 
ventricular end-diastolic area
Paradoxical septal motion
Pulmonary arterial systolic pressure 
(PASP) estimated from 
transtricuspid pressure and right 
atrial pressure or inferior vena cava 
(IVC) diameter (whichever is 
available)
Up to and including Day 3 of observation
 
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Objectives
Endpoints
Secondary

To define the incidence of ACP and 
PCD in mechanically ventilated 
participants.

Presence of PCD, ACP (severe PCD) 
and severe ACP 
Up to and including Day 3 of observation

To evaluate the association between 
plasma Ang(1-7) levels, Ang 
II/Ang(1-7) ratio and RV function in 
mechanically ventilated participants.

Ang(1-7) levels 

Ang II/Ang(1-7) ratio 

Echocardiographic measures:
Ratio of right ventricular to left 
ventricular end-diastolic area
Paradoxical septal motion
PASP estimated from transtricuspid 
pressure and right atrial pressure or 
IVC diameter (whichever is 
available)
Up to and including Day 3 of observation
Overall Design:
This study will investigate the association of RAS peptides and markers of RV function,
as measured by echocardiography, in participants requiring acute mechanical ventilation. 
It is a low interventional study without an investigational product. Participants will be 
evaluated over a 3-day period using standard of care investigations, including 
transthoracic echocardiography (TTE) and/or transesophageal echocardiography (TOE). 
Three separate echocardiographs over three consecutive days will be undertaken, and at 
the same time point blood samples will be taken for assessment of RAS peptides to allow 
the association with right ventricular function to be assessed.  
Number of Participants:
A total of approximately 150 participants are planned to be included.
Treatment Groups and Duration:
There are no treatment groups as no investigational product will be administered.
The total duration of this study for participants is 28 days. The study period is 3 days and 
health outcomes will be recorded daily for up to 28 days. Health outcomes will not be 
recorded beyond hospital discharge. 
 
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2.
SCHEDULE OF ACTIVITIES (SOA)
Procedure
Study Period
Notes
Screening1
Day 
1
Day 
2
Day 
3
Up to day 
28
Start of mechanical 
ventilation  
X
Inclusion/exclusion 
criteria
X
Reason for intubation (underlying diagnosis 
e.g. sepsis, pneumonia) to be collected 
where possible.
Informed consent 
(ICF)
X
Consent process will be followed as outlined 
in Informed Consent Process (Section 
12.3.3)
Demography
X
If it is not possible to collect at screening can 
be collected at any point during the study.
Height and weight 
X
Collected for the purposes of evaluating BMI. 
Clinical estimates are acceptable if direct 
measurement is not possible.
Medical history 
X
If possible to collect, includes medication and 
cardiovascular risk factors. If it is not possible 
to collect at screening can be collected at any 
point during the study.
Diagnosis of 
pulmonary 
circulatory 
dysfunction (PCD)
and acute cor 
pulmonale (ACP) 
X
X
X
As per echocardiographic output.
Participant status 
X
X
X
X
Intubation status, proned status; diagnosis of 
acute respiratory distress syndrome (ARDS)
including severity at time of diagnosis (if 
available). Post screening status to be 
assessed at the same time point as 
echocardiogram (1 hour).
Participant 
management
X
X
X
X
Management with extracorporeal membrane
oxygenation (ECMO) and extracorporeal CO2
(ECCO2R) removal. Post screening status to 
be assessed at the same time point as 
echocardiogram (1 hour).
Simplified acute 
physiology score 
(SAPS II)
X
To be taken within approximately 24 hours of 
admission into the intensive care unit, as per 
standard of care.
 
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Procedure
Study Period
Notes
Screening1
Day 
1
Day 
2
Day 
3
Up to day 
28
Sequential Organ 
Failure (SOFA)
score
X
X
X
X
Echocardiogram
X
X
X
Transthoracic echocardiogram (TTE) and/or 
transoesophageal echocardiogram (TOE). 
First echocardiogram must be completed 
within 48 hours of starting mechanical 
ventilation (but ≤24 hours, whenever 
possible); the following echocardiograms will 
be undertaken as per standard of care 24 
hours apart (± 2 hours) on subsequent 
consecutive days (Day 2 and 3).
Vital signs
X3
X
X
X
Blood pressure (mean arterial, systolic and 
diastolic), heart rate, body temperature to 
match echocardiogram time points (± 30 
minutes)2.
Laboratory 
assessments
X
X
X
X
Clinical chemistry and haematology.
Ventilator settings 
X3
X
X
X
Includes tidal volume, respiratory rates, static 
respiratory compliance, level of positive end 
expiratory pressure (PEEP), peak and 
plateau ventilator pressures, mean airway 
pressure and driving pressure. 
Measurements to be taken at intubation and 
at the time of each echocardiogram (± 30 
mins)2.
Lung function and 
blood gas measures 
X3
X
X
X
Oxygen saturation(SaO2) via pulse oximetry. 
Arterial Blood Gases (ABGs) measuring 
partial pressure of oxygen and carbon 
dioxide (PaO2 and PaCO2).4 Oxygen 
requirement (FiO2) should be documented at 
the same time point. 
pH, lactate and bicarbonate levels and base 
excess or deficit when available should also 
be documented when ABGs are assessed. 
Measurements to be taken at intubation and 
at the time of each echocardiogram (± 30 
mins)2.
Electrical 
Impendance 
Tomography
X
X
X
X
Measurements to be taken at intubation and 
at the time of each echocardiogram when 
available (±2 hours)2.
 
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Procedure
Study Period
Notes
Screening1
Day 
1
Day 
2
Day 
3
Up to day 
28
Serious Adverse 
Event (SAE) Review
<=======================>
SAEs will only be collected during the 3-day
observation period or until participant 
withdrawal, whichever is sooner.  
Concomitant 
medication review
<=======================>
Only specified prescription medication 
(name, start and end dates required) as 
described in Section 7.1. Vasopressor use to 
be documented for each time point of 
echocardiography.
Intensive Care Unit 
(ICU) length of stay
<=======================>
Will be recorded daily up to 28 days.
Ventilator free days
<=======================>
Will be recorded daily up to 28 days; 
however, follow up is not necessary beyond 
hospital discharge.
In hospital mortality
<=======================>
Will be recorded daily up to 28 days; 
however, follow up is not necessary beyond 
hospital discharge.
Biomarkers 
Blood sample for 
renin-angiotensin 
system (RAS) 
biomarkers
X
X
X
Blood samples to be taken at the same time 
as echocardiography (± 30 mins)2.
Blood sample for 
biomarkers
X
X
X
Blood samples to be taken at the same time 
as echocardiography (± 30 mins)2.
1.
Screening may be considered part of Day 1.
2.
When multiple measurements are to be taken at the same time in relation to echocardiography, blood 
sampling for RAS peptides should be given priority. Other measurements (e.g. ventilator settings) to be
prioritized as per investigator for patient care.
3.
Will be recorded at the time of intubation if available.
4.
Where available, a blood sample is required for arterial blood gases (ABGs) for PaO2/FiO2 and oxygenation 
index. A FiO2 measurement should be assessed at the same time point as ABGs are taken for PaO2.
Any changes in the timing or addition of time points for any planned study assessments 
must be documented and approved by the relevant study team member and then archived 
in the sponsor and site study files, but will not constitute a protocol amendment. The 
Independent Ethics Committees (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
Right ventricular (RV) dysfunction may result from increased RV afterload secondary to 
increased pulmonary vascular resistance in patients with severe respiratory failure 
including acute respiratory distress syndrome (ARDS). RV dysfunction, particularly 
severe dysfunction [acute cor pulmonale (ACP)], has been associated with worse 
outcomes in critically ill patients including patients with ARDS [Boissier 2013; 
Mekontso-Dessap 2016]. Patients with changes in RV size and pulmonary artery 
pressures [termed pulmonary circulatory dysfunction (PCD)] have also been observed to 
have an increased mortality with particularly high mortality in those patients with ACP 
(designated as severe PCD in this study) [Bull 2010, Boissier 2013]. The renin-
angiotensin system (RAS) is a key regulator of vascular tone in the pulmonary 
vasculature and Angiotensin II (Ang II) in particular is thought to play a role in both 
vasoconstriction and vascular remodelling in human diseases such as pulmonary arterial 
hypertension. RAS peptides such as Ang II and Ang(1-7) may play a role in the observed 
changes in pulmonary vasoconstriction in patients with severe respiratory failure. This 
study is designed to assess whether circulating Ang II and Ang(1-7) levels are associated 
with RV dysfunction in mechanically ventilated patients. It is also designed to further 
characterise the patient population including the severity of RV dysfunction.  
3.2.
Background
Increased pulmonary vascular resistance (PVR) is a common finding in patients with 
severe respiratory failure [Zapol 1977; Bull 2010]. There is evidence of worse outcomes
for patients with increased PVR. PVR has been observed to fall in survivors, whilst 
remaining elevated in patients who died [Zapol 1977]. PVR may often lead to pulmonary 
hypertension and increased RV afterload, a frequent complication of ARDS [Ware 2000]. 
The acutely increased after-load of the RV causes RV dilatation, impaired RV function, 
and reduced cardiac output. Worse outcomes, including increased mortality, have been 
observed in patients with ARDS and RV dysfunction [Bull 2010; Boissier 2013]. In a 
study of 752 patients, hospital mortality in ARDS patients with ACP was 48% (compared 
with 42% in non ACP patients), rising to 57% with severe ACP [Mekontso-Dessap
2016]. Patients with ACP exhibit a higher incidence of shock, increased heart rates and 
lower arterial pressures. 
The mechanism for increased RV afterload in patients with respiratory failure is multi-
factorial and may result from pulmonary vasoconstriction, micro-embolism and/or 
endothelial dysfunction in addition to the direct effects of mechanical ventilation. 
Pulmonary vasoconstriction may be mediator induced and the RAS is a key regulator of 
vascular tone in the pulmonary vasculature. Ang II in particular drives both acute 
vasoconstriction and vascular remodelling in human diseases including pulmonary 
arterial hypertension. Ang(1-7) appears to be a counter-regulatory peptide in the RAS, 
associated with vasodilation, anti-proliferative and anti-inflammatory activity, and 
reduced vascular leak. Specifically, the RAS regulates pulmonary vascular tone with an 
increase in the Ang II/Ang(1-7) ratio, driving increased PVR with subsequent deleterious 
effects on RV function. Therefore, whilst other effects e.g. mechanical ventilation may 
 
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affect pulmonary vascular resistance, in some patients with respiratory failure, activation 
of the RAS may contribute to pulmonary vascular and consequently RV dysfunction. 
Attenuation of RAS-mediated vasoconstriction, without compromising the normo-
adaptive pulmonary vascular response, may improve pulmonary blood flow, cardiac 
output, ventilation/perfusion matching and reduce afterload which should ultimately 
improve outcomes in mechanically ventilated patients. RAS mediated vasoconstriction 
might be improved through the dual action of angiotensin converting enzyme (ACE2) by 
simultaneously reducing Ang II and forming Ang(1-7). Thus, increasing the total amount 
of active enzyme may be beneficial in the treatment of RV dysfunction in patients with 
severe respiratory failure [Tom 2001; Idell 1987; Santos 2003; Wenz 2000].
Data exploring the RAS status and its association with acute changes in pulmonary 
hemodynamics and worsening RV function is limited in patients with severe respiratory 
failure, including in those who are mechanically ventilated. This study is designed to test 
the hypothesis that an association exists between RAS peptides, primarily plasma Ang II 
and Ang(1-7) levels, and RV function in mechanically ventilated patients. The hypothesis 
will be tested by assessing the plasma levels of RAS peptides and RV function as 
measured by echocardiography assessing RV size, sepal kinetics and degree of 
pulmonary hypertension as measured by pulmonary arterial systolic pressure (PASP). A 
secondary aim of the study is to further characterise this population to inform future 
interventional studies, by assessing the incidence and severity of RV dysfunction, and 
assessing the variability of key echocardiographic parameters including RV size and 
PASP as well as RAS peptide levels in mechanically ventilated patients.  An exploratory 
aim of the study is to consider regional lung ventilation and perfusion assessed by global 
inhomogeneity index, which may improve understanding of venitilation/perfusion
matching in patients undergoing mechanical ventilation with changes in pulmonary 
vascular resistance.    
3.3.
Benefit/Risk Assessment
3.3.1.
Risk Assessment
There is no investigational product administered in this study. 
This is a low interventional study without administration of an investigational product. 
All procedures with the exception of 3 blood draws are standard of care in the involved 
centres, including TTE/TOE assessments. Therefore, there is a limited extra risk to the 
participants beyond standard of care.
 
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Potential Risk of 
Clinical Significance
Summary of 
Data/Rationale for Risk
Mitigation Strategy
Study Procedures (if applicable)
Phlebotomy for 
biomarkers: Bruising, 
irritation or redness at 
the site of blood draw 
Blood samples will be 
collected for assessment of 
RAS peptides and other 
biomarkers of relevance 
Careful blood draw to 
minimise bruising, irritation 
and redness; samples may be 
collected from central or 
arterial line already in place. 
3.3.2.
Benefit Assessment
There is no benefit to the individual participant from being included as part of the study.  
3.3.3.
Overall Benefit:Risk Conclusion
Although individual participants will not benefit, the information derived from the study 
will inform future interventions in an area of high unmet medical need. Given the limited 
additional procedures beyond standard of care (3 blood draws) there is limited extra risk 
to the participants beyond standard of care. Consequently, given the potential future 
benefit to critically unwell participants, the small additional risk of the blood draws is 
considered justified. 
 
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4.
OBJECTIVES AND ENDPOINTS
Objectives
Endpoints
Primary

To evaluate the association between 
plasma Ang II levels and RV function 
in mechanically ventilated 
participants.

Ang II levels 

Echocardiographic measures: 

Ratio of right ventricular to left 
ventricular end-diastolic area

Paradoxical septal motion

PASP estimated from transtricuspid 
pressure and right atrial pressure or 
IVC diameter (whichever is 
available)
Up to and including Day 3 of observation
Secondary 

To define the incidence of ACP and 
PCD in mechanically ventilated 
participants.

Presence of PCD, ACP (severe PCD) 
and severe ACP 
Up to and including Day 3 of observation

To evaluate the association between 
plasma Ang(1-7) levels, 
AngII/Ang(1-7) ratio and RV function 
in mechanically ventilated participants.

Ang(1-7) levels

Ang II/Ang(1-7) ratio 

Echocardiographic measures:

Ratio of right ventricular to left
ventricular end-diastolic area

Paradoxical septal motion 

PASP estimated from transtricuspid 
pressure and right atrial pressure or 
IVC diameter (whichever is 
available)
Up to and including Day 3 of observation
 
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Objectives
Endpoints
Exploratory 

To evaluate the association between 
plasma Ang II and Ang(1-7) levels and 
other measures of right ventricular 
function in mechanically ventilated 
participants. 

Ang II, Ang(1-7) levels  

Echocardiographic measures:

Paradoxical septal motion (systolic 
eccentricity index)

Maximal velocity of s wave (tissue 
Doppler imaging) at the tricuspid 
annulus 

Fractional area contraction (end 
diastolic area minus end systolic 
area normalised to end diastolic 
area)

Acceleration time of RV ejection 
flow

Mean acceleration of RV ejection 
flow (maximum velocity/
acceleration time) at end-expiration 
and end-inspiration

Inferior vena cava diameter at 
end-expiration

Respiratory variations of RV 
velocity time integral (end-
expiration minus end-inspiration
normalised to end-expiration)

Tricuspid annular plane systolic 
excursion (TAPSE) 
Up to and including Day 3 of observation

To evaluate the association between 
plasma Ang II and Ang(1-7) levels and 
other measures of cardiac function and 
pulmonary hemodynamics in 
mechanically ventilated participants. 

Ang II and Ang(1-7)

Central venous pressure (if available)

Mean arterial pressure

Echocardiographic measures:

Respiratory variations of diameter of 
superior vena cava

Cardiac output (pulsed wave 
Doppler, left ventricle (LV) outflow 
track) 

LV ejection fraction 

LV Velocity (E’) tissue doppler 
imaging (TDI) of annulus 

LV early to late ventricular filling 
velocities (E/A)
Up to and including Day 3 of observation
 
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Objectives
Endpoints

To characterise disease severity by 
clinical assessment.

Oxygen saturation (SpO2) via pulse 
oximetry 

pH of arterial blood gases

PaO2/FiO2 ratio

PaCO2

Peak and plateau ventilator pressure

PEEP

Oxygenation index

Static respiratory system compliance

Mean airway pressure

Driving pressure (plateau pressure –
total PEEP)

Clinical Safety labs

Vital Signs 
Up to and including Day 3 of observation

To characterise levels of organ 
dysfunction.
Sequential Organ Failure Assessment 
(SOFA) score
Up to and including Day 3 of observation

To assess the association of cardiac 
and inflammatory biomarkers and RV 
function.

PCD and ACP status

Biomarkers: for example, cardiac 
biomarkers (B-type natriuretic peptide 
(BNP)) or inflammatory biomarkers 
(cytokines such as IL-6, IL-8 and 
sTNFR1) may be analysed as sample 
availability allows
Up to and including Day 3 of observation

To evaluate the association between 
additional markers of the RAS system 
(if samples permit) and 
primary/secondary or exploratory 
endpoints.
Markers could include but are not limited 
to: Ang(1-5) in relation to primary, 
secondary or exploratory endpoints if 
sufficient sample is available
Up to and including Day 3 of observation

To characterize health outcome 
measures.

Duration of intubation

Length of hospital stay 

ICU length of stay 

Ventilator free days 

In ICU and hospital mortality 
Up to 28 days after first echocardiogram

To assess lung ventilation and 
perfusion distribution (in selected site 
only).

Lung regional ventilation and perfusion
with global inhomogeneity (GI) index
Up to and including Day 3 of observation
 
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5.
STUDY DESIGN
5.1.
Overall Design
This study will investigate the association of RAS peptides and markers of RV function 
in participants requiring acute mechanical ventilation. This is a low interventional study 
without an investigational product. Participants will be evaluated over a 3-day period 
using standard of care investigations, including TTE and/or TOE echocardiography. 
Additional investigations will be limited to blood samples for RAS peptides and cardiac
biomarkers (inflammatory markers if samples allow).
Participants will be enrolled in the study following intubation and mechanical ventilation. 
The initial echocardiogram will take place within 48 hours of mechanical ventilation (but 
≤24 hours, whenever possible); this will be considered Day 1. Participants will be 
assessed every 24 hours for a further two days with daily echocardiography to monitor 
right and left ventricular function. Time 0 h for the study will be defined as the start of 
the first echocardiogram. Blood samples for RAS peptides will be collected at the time of 
the echocardiogram on each day. Additional parameters (including ventilator settings and
arterial blood gases) will be collected at the time of intubation and daily as per standard 
of care. Daily assessment of mechanical ventilation and mortality will continue until 
hospital discharge or 28 days whichever is sooner.
Figure 1
Study Schematic
5.2.
Number of Participants
Up to a maximum of 150 participants may be enrolled.  
If participants prematurely discontinue the study, additional replacement participants may 
be recruited at the discretion of the Sponsor in consultation with the investigator.
 
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5.3.
Participant and Study Completion
A participant is considered to have completed the study if he/she has completed Day 3 
assessments as shown in the SoA. If participants are extubated after the first 
echocardiogram and prior to the second/third echocardiogram he/she will remain in the 
study and be followed using echocardiography if conducted as per standard of care. In the 
event that further echocardiographs are undertaken, RAS peptide sampling will continue 
as per SoA. 
Participants enrolled but not undergoing any echocardiograms will be considered 
withdrawn from the study.  
The end of the study is defined as the date of last scheduled assessment of the last 
participant in the trial.
5.4.
Scientific Rationale for Study Design
This study is designed to test the hypothesis that an association exists between RAS 
peptides, primarily plasma Ang II and Ang(1-7) levels, and RV function in mechanically 
ventilated participants. Therefore, this a prospective study to collect blood samples to 
assess RAS peptide levels and RV function as measured by echocardiography.
5.5.
Dose Justification
Not applicable as there is no investigational product. 
6.
STUDY POPULATION
Prospective approval of protocol deviations to recruitment and enrolment criteria, also 
known as protocol waivers or exemptions, is not permitted.
6.1.
Inclusion Criteria
Participants are eligible to be included in the study only if all of the following criteria 
apply:
1. Male or female participant must be 18 to 80 years of age inclusive, at the time of 
enrolment. 
2. Participants who are receiving invasive mechanical ventilation (duration of 
ventilation ≤48 hours, but ≤24 hours, whenever possible).
Weight
3. Body mass index within the range 18.0 – 38.0kg/m2 (inclusive). Clinical estimate of 
height and weight is acceptable.
Informed Consent
4. Capable of giving signed informed consent as described in Section 12.3.3 (Appendix
3) which includes compliance with the requirements and restrictions listed in the ICF 
 
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and in this protocol. If participants are not capable of giving signed informed consent 
an emergency consent procedure will be followed as detailed in Section 12.3.3
(Appendix 3).
In France, a subject will be eligible for inclusion in this study only if either affiliated 
to or a beneficiary of a social security category.
6.2.
Exclusion Criteria
Participants are excluded from the study if any of the following criteria apply:
1. Participants who are moribund or whose clinical condition is deteriorating rapidly or 
any participant for whom the investigator does not consider there is a reasonable 
expectation that they will be able to complete the 3 days of observation in the study.
2. Participants undergoing elective surgery. 
Investigator will make every effort to ensure that the following exclusion criteria 3 to 10
are met; however, in some instances it may not be possible to assess all these criteria 
within the 48-hour window.  In this case, a participant can be included, and investigator 
will obtain the information when available.
3. COPD requiring long term oxygen treatment or home mechanical ventilation.
4. Documented pre-existing chronic pulmonary hypertension.
5. Massive pulmonary embolism (defined by pulmonary embolism with systemic 
hypotension (defined as a systolic arterial pressure <90 mmHg or a drop in systolic 
arterial pressure of at least 40 mmHg for at least 15 min which is not caused by new 
onset arrhythmias) or shock (manifested by evidence of tissue hypoperfusion and 
hypoxia, including an altered level of consciousness, oliguria, or cool, clammy 
extremities)).
6. Pulmonary vasculitis or pulmonary haemorrhage including diffuse alveolar 
haemorrhage.
7. Lung transplantation within last 6 months.
8. Cardiopulmonary arrest during concurrent illness.
Concomitant Therapy
9. Any use of RAS modulators including ACE type 1 inhibitors, Renin inhibitors and 
Angiotensin Receptor Blockers within 4 days or 5.5 half-lives, whichever is longer.
If unable to obtain medication history at time of enrolment, participant is allowed to be 
included. 
Other Exclusions
10. Do not resuscitate status.
 
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6.3.
Lifestyle Restrictions
No restrictions are required.
6.4.
Screen Failures
Screen failures are defined as participants who consent (or are consented on behalf of) to 
participate in the clinical study, but are not subsequently enrolled 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 and
eligibility criteria.
Individuals who do not meet the criteria for participation in this study (screen failures) 
may not be rescreened. 
7.
TREATMENTS
No investigational product will be administered. Treatments will be based on local 
standard of care including fluid resuscitation, use of vasopressor drugs and renal support. 
Vasopressor use and renal replacement therapy will be documented.
Mechanical ventilation will be applied based on local practice: in the volume-assist 
control mode, with a target tidal volume of 6–8 ml/kg (predicted body weight) and a 
target plateau pressure (Pplat) of <30 cmH2O. Respiratory rate can be increased in case of 
high arterial carbon dioxide partial pressure (PaCO2) level, while avoiding intrinsic 
PEEP. Level of PEEP is left to the discretion of the frontline intensivist.
The use of prone position and use of inhaled nitric oxide is left to the discretion of the 
attending physician but should be documented in the Case Report Form (CRF).
7.1.
Concomitant Therapy
Any prescription medication or vaccine (if known) that the participant is receiving at the 
time of enrolment or receives during the study must be recorded. Specifically collect any 
medication that may affect the RAS, cardiovascular or pulmonary homeostasis including 
but not limited to vasopressors, diuretics, beta blockers, steroids, induction agents such as 
Propofol, and inhaled nitric oxide. 
The date and time of inhaled nitric oxide and vasopressor use must be recorded. 
The following do not need to be documented: medication doses, administration of fluids 
or electrolyte infusions and nutrition.  
 
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8.
DISCONTINUATION CRITERIA
8.1.
Discontinuation of Study Treatment
Not applicable as there is no study treatment.
8.2.
Withdrawal from the Study

A participant may withdraw from the study at any time at his/her own request, 
request by their LAR, or may be withdrawn at any time at the discretion of the 
investigator for safety, behavioural, compliance or administrative reasons.

If a participant withdraws from the study or the LAR requests that the 
participant is withdrawn after consenting, no additional blood samples 
will be taken. Samples already taken but not sent to central laboratory 
can be destroyed at patient or LAR’s request. The investigator must 
document this in the site study records. 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.

Participants who are withdrawn may be replaced. 
9.
STUDY ASSESSMENTS AND PROCEDURES
This section lists the parameters of each planned study assessment.  Participants will be
evaluated using standard of care investigations including parameters derived from 
ventilator readings, arterial blood gases, safety laboratory measures, and 
echocardiographic readings (TTE and/or TOE). Additional daily blood samples will be 
collected on Days 1, 2 and 3 to assess RAS peptides and other biomarkers which are not 
part of the standard of care. Health outcomes will also be measured including mortality, 
ICU status and ventilator free days up to a maximum of 28 days.
Detailed procedures for obtaining each assessment are provided in the Study Reference 
Manual (SRM). Whenever vital signs, lung function, ventilator settings, echocardiogram 
and blood draws are scheduled for the same nominal time, the assessments should occur 
in the following order: echocardiogram, blood draws, vital signs, ventilator settings, lung
function. The timing of the assessments should allow the blood draw to occur at the exact 
nominal time. Time 0h will be defined as the start of the first echocardiogram.

Study procedures and their timing are summarized in the schedule of assessments 
(SoA; Section 2).

Protocol waivers or exemptions are not allowed. 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 
 
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log to record details of all participants screened and to confirm eligibility or record 
reasons for screening failure, as applicable. 

Procedures conducted as part of the participant’s routine clinical management (eg, 
blood count) and obtained before signing of ICF may be utilized for screening or 
baseline purposes provided the procedure met the protocol-specified criteria and was 
performed within the time frame defined in the SoA.

The maximum amount of blood collected from each participant over the duration of 
the study for the purpose of meeting the objectives will not exceed 30 mL.  This does 
not include any standard of care assessments.

A single repeat of a sample may be taken for technical reasons as long as they are 
within the timelines defined in the SoA. 
9.1.
Demographic/Medical History Assessments
The following demographic parameters will be captured: year of birth and gender. If it is 
not possible to collect at screening can be collected at any point during the study.
Medical and medication history including cardiovascular risk will be collected. If it is not 
possible to collect at screening can be collected at any point during the study.
Height and weight will be collected at screening. Clinical estimates are acceptable if 
direct measurement is not possible.
9.2.
Pharmacodynamics
9.2.1.
Echocardiograms
As per standard of care, TTE will be conducted on all participants with TOE 
measurements undertaken in those participants in whom adequate windows cannot be 
obtained via the transthoracic route. The first echocardiogram must be undertaken within 
48 hours of the start of mechanical ventilation (but within 24 hours, whenever possible),
with subsequent echocardiograms approximately 24 hours apart on Day 2 and Day 3. As 
previously described [Mekontso-Dessap 2016], echocardiography will be undertaken by 
trained operators in the field of advanced critical care echocardiography and in the case 
of TOE will be performed using a multiplane oesophageal probe. Images will be stored in 
a digital format at site and a computer assisted consensual interpretation will be 
performed off line by at least two trained senior investigators (see SRM for further details 
on echocardiography standards).
Variables that will be recorded from TTE and/or TOE, as data permit, include:

Ratio of right ventricular to left ventricular end-diastolic area

Paradoxical septal motion (systolic eccentricity index) 

PASP (estimated from transtricuspid pressure gradient and right atrial pressure or 
IVC diameter (whichever is available))

Maximal velocity of s wave (tissue Doppler imaging) at the tricuspid annulus
 
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
Fractional area contraction: End Diastolic Area minus End Systolic Area normalised 
to End Diastolic Area 

Acceleration time of RV ejection flow (Tacc)

Mean acceleration of RV ejection flow (maximum velocity/ acceleration time) at 
end-expiration and end-inspiration 

Inferior vena cava diameter at end expiration

Respiratory variations of RV velocity time integral (end-expiration minus             
end-inspiration / end-expiration)

Tricuspid annular plane systolic excursion

Respiratory variations of superior vena cava diameter

Cardiac output (pulsed wave Doppler, LV outflow track) 

LV ejection fraction 

LV Velocity (E’) tissue doppler imaging (TDI) of annulus 

LV Early to late ventricular filling velocities (E/A)
9.2.2.
Electrical Impedance Tomography (EIT)
The Global Inhomogeneity (GI) index will be measured based on electrical impendence 
(Enlight, Timpel, Brazil) to assess lung regional and/or perfusion in centres where 
measurements are taken routinely. Measurements will be taken daily at time of 
echocardiography for up to three days. The GI index will be measured according to the 
methods set out by Zhao 2009.
9.3.
Ventilator Settings
Measurements will include tidal volume, respiratory rates, respiratory static compliance 
of the respiratory system, level of total PEEP, peak and plateau ventilator pressures, mean 
airway pressure and driving pressure.
9.4.
Lung Function
Measurements will include oxygen saturation (SpO2) via pulse oximetry and Partial 
pressure of oxygen and carbon dioxide in arterial blood (PaO2 and PaCO2). The pH, 
lactate and bicarbonate levels of ABGs will also be documented when ABGs are assessed
as well as the base excess/deficit if available. Oxygen requirement (FiO2) will be
collected and documented at the same time as ABG collection.
 
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9.5.
Participant Status
9.5.1.
SAPS II
The SAPS II score is comprised of the following components: 

Age

Heart Rate

Systolic Blood Pressure

Temperature

Mechanical Ventilation or Continuous Positive Airway Pressure

PaO2

FiO2

Urine Output

Blood Urea Nitrogen

Sodium

Potassium

Bicarbonate

Bilirubin

White Blood Cell

Chronic Diseases

Type of Admission

Glasgow Coma Score (GCS)
If the patient is sedated at the time of assessment, an estimated GCS prior to sedation will 
be used instead.
More information is provided in the SRM.
9.5.2.
Sequential Organ Failure Assessment (SOFA) score
The SOFA score is made up of the following components:

PaO2/FiO2 (mmHg)

Mean Arterial Pressure or administration of vasopressors required

Bilirubin

Platelets

Creatinine (or urine output)
 
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Due to the difficulties in accurately scoring Glasgow coma score in sedated patients, the 
neurologic system will not be included.
More information is provided in the SRM.
9.5.3.
Disease diagnosis
Diagnosis of ARDS, PCD and ACP.

ACP is defined as a dilated RV in the mid-esophagus longitudinal view or apical 4-
chamber view [end-diastolic RV / left ventricle (LV) area ratio (0.6)] associated 
with the presence of a septal dyskinesia in the (transgastric) short-axis view of the 
heart.

Severe ACP is defined as severely dilated RV (end-diastolic RV / LV area ratio ≥1) 
with septal dyskinesia. Septal dyskinesia will be particularly sought at end-systole, 
while analysing loops in slow motion.

PCD is defined as moderate dysfunction (PASP [>40 mmHg] or a dilated RV end-
diastolic RV / left ventricle (LV) area ratio [≥0.6] but without septal dyskinesia). In 
case of the inability of measuring transtricuspid pressure gradient, pulmonary 
hypertension will be defined as a Tacc below 100 ms.

ARDS is defined as per the Berlin definition (Ranieri 2012):
Timing 
Within 1 week of a known clinical insult or new or worsening 
respiratory symptoms
Chest imaging
Bilateral opacities—not fully explained by effusions, lobar/lung 
collapse, or nodules
Origin of edema
Respiratory failure not fully explained by cardiac failure or fluid 
overload
Need objective assessment (eg, echocardiography) to exclude 
hydrostatic edema if no risk factor present
Oxygenation
      Mild
200 mm Hg _<  PaO2/FIO2 _ ≤ 300 mm Hg with PEEP or  CPAP 
≥_5 cm H2Oc
      Moderate
100 mm Hg <_ PaO2/FIO2  ≤_ 200 mm Hg with PEEP _5 cm H2O
      Severe
PaO2/FIO2 ≤_ 100 mm Hg with PEEP ≥_5 cm H2O
9.5.4.
General management status
The following measurements of general management status will be recorded at the time 
of echocardiogram for each of the three study days:

Proned status

Intubation status

Management with extracorporeal membrane oxygenation (ECMO) 

Management with extracorporeal CO2 (ECCO2R) removal
 
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9.6.
Additional Clinical assessments
The following data will be collected for up to a maximum of 28 days:

Mortality

ICU status

Ventilator free days
Date and time of any concomitant surgery will also be recorded.
9.7.
Adverse Events
Safety assessments will be conducted as part of standard of care and include serious 
adverse event collection, safety laboratory assessments, and vital signs. More information 
will be provided in the SRM.
9.7.1.
Serious Adverse Events (SAE)
The definitions of an adverse event (AE) or SAE can be found in Appendix 4.
Although no investigational product will be given, SAEs will be recorded, as follows:  
SAEs relating to study procedures; and SAEs related to GlaxoSmithKline (GSK) 
medication used as co-medication. 
This ensures that any SAEs relating to study procedures or any GSK medication used as 
co-medication are captured.
AEs (other than SAEs, as described above) will not be captured in the study.
The investigator is responsible for the detection and documentation of events meeting the
criteria and definition of an SAE, as provided in this protocol. During the study when
there is a safety evaluation, the investigator or site staff will be responsible for detecting,
documenting and reporting SAEs, as detailed in both this section of the protocol and in
the SAE section of the study reference manual.
9.7.2.
Time Period and Frequency for Collecting SAE Information

All SAEs will be collected over the 3 days of study period from the time of 
enrolment.

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 SAEs in former study participants. 
However, if the investigator learns of any SAE, including a death, at any time after 
a participant has been discharged from the study, and he/she considers the event to 
be reasonably related to the study procedure or GSK medication, the investigator 
must promptly notify the sponsor.
 
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
The method of recording, evaluating, and assessing causality of SAEs and the 
procedures for completing and transmitting SAE reports are provided in Appendix 
4.
9.7.3.
Method of Detecting SAEs
SAEs will be detected by the medical team. 
9.7.4.
Follow-up of SAEs
After the initial SAE report, the investigator is required to proactively follow each 
participant at subsequent visits/contacts. All SAEs, will be followed until the event is 
resolved, stabilized, otherwise explained, or the participant is discharged from ICU or up 
to 28 days (whichever is sooner).
9.7.5.
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 any GSK product or study 
procedure. The sponsor will comply with country-specific regulatory requirements 
relating to safety reporting to the regulatory authority, 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.
9.8.
Safety Assessments
Planned time points for all safety assessments are provided in the SoA (Section 2) as per 
standard of care.
9.8.1.
Vital Signs

Systolic and diastolic and mean arterial blood pressure and heart rate will be 
assessed.

Blood pressure and heart measurements will be assessed (supine if possible) with a 
completely automated device. Manual techniques will be used only if an automated 
device is not available.
9.8.2.
Clinical Laboratory Assessments
Refer to Appendix 2 for the list of clinical laboratory tests to be performed and to the 
SoA (Section 2) for the timing and frequency. 
 
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9.9.
Biomarkers
The following samples are required and will be collected from all participants in this 
study as specified in the SoA

The levels of Ang II and Ang(1-7) will be analysed in plasma. Additional 
components of the RAS [e.g. Ang(1-5)] may be evaluated from the same blood 
sample to better understand the correlation of the RAS to RV function if samples 
allow. 

Collection of samples for other biomarker research is also part of this study. For 
example cardiac biomarkers (eg. BNP) and other biomarkers (eg. Inflammatory 
markers or lung injury markers) may be evaluated if sample availability allows.

Samples will be stored to assess future biomarkers of interest in lung injury 
pathophysiology as sample availability allows.
10.
STATISTICAL CONSIDERATIONS
10.1.
Sample Size Determination
Up to 150 participants may be enrolled in the study (as described in Section 5.2). The 
sample size is deemed to be sufficient to evaluate the secondary objectives of the study. 
In particular, the sample size of 150 participants would provide a precision of ±5 
percentage points (defined as the half-width of the 95% Wald confidence interval) around 
a point estimate for ACP incidence (based on an estimated incidence rate of 10%).
The primary analysis (association between Ang II and RV function measured by PASP 
and RV size ratio) will be evaluated after ~50 participants have been enrolled in the 
study. If results are inconclusive, then a second analysis after ~100 participants will be 
carried out. Full details of the assumptions and simulations used to determine the sample 
size are given in Section 12.5.
In the event of a conclusion of "success" or "inconclusive" at an interim analysis, only the 
decision to continue with the study will be communicated with the study sites, to mitigate 
against the possibility of the nature of the interim conclusion influencing the data 
collection process and introducing bias into the later interim and final analysis results. In 
the event of a "futility" decision, recruitment to the study will be halted.
Simulations were carried out to evaluate the operating characteristics of the 
success/futility criteria (described Section 10.3.3) at the primary analysis timepoint (i.e. 
after 50 participants have completed their assessments), under a range of scenarios 
describing the correlation between Ang II and the two endpoints of PASP and RV size 
ratio. The main results from these simulations are as follows:

In the case of there being no relationship between Ang II and either of the 
endpoints, the decision-making criteria will return a conclusion of "futility" 99.1% 
of the time.
 
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
In the case of there being a strong relationship (defined as a correlation of 0.6) 
between Ang II and both of the endpoints, the mean difference in probabilities of 
RV dysfunction between the two reference values will be 33.9%, and the decision-
making criteria will return a conclusion of "success" 74.7% of the time (with 
"inconclusive" being returned the remaining 25.3% of the time).

When the relationship between Ang II and the endpoints is less clear, there is a 
greater likelihood of returning an inconclusive result and continuing the study up to 
the backup interim analysis at 100 participants.

The conclusions returned by the decision-making criteria are more sensitive to Ang 
II's correlation with RV size ratio than to its correlation with PASP. For example, 
when the correlation between Ang II and RV size ratio is reduced from 0.6 to 0.3, 
the decision-making criteria return "success" only 12.6% of the time and the mean 
difference in probabilities of RV dysfunction is 22.5%. Conversely, if the 
correlation between Ang II and PASP is reduced from 0.6 to 0.3, the decision-
making criteria will return "success" 30.1% of the time and the mean difference in 
probabilities of RV dysfunction is only reduced to 26.9%.
A full description of the simulation methods is given in Appendix 5 (Section 12.5) and a 
summary of the results from all the scenarios simulated is given in Table 1.
 
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Table 1
Operating characteristics of decision-making criteria at the primary 
interim analysis (N = 50) under a range of scenarios regarding the 
relationship between Ang II and the two endpoints PASP and RV 
size ratio
a) Probabilities of returning conclusions of success, futility and inconclusive, by scenario
Correlations between Ang II and
Probability of reaching a conclusion of
(%)
PASP
RV size ratio
Success
Futility
Inconclusive
Extreme null scenario: no correlation between Ang II and either component of RV 
dysfunction
0.0
0.0
0.0
99.1
0.9
Extreme positive scenario: strong correlation between Ang II and both components of RV 
dysfunction
0.6
0.6
74.7
0.0
25.3
"Middling" scenario: weak correlation between Ang II and both components of RV 
dysfunction
0.3
0.3
0.6
49.3
50.1
Mixture scenarios (I): Ang II correlated more strongly with PASP than with RV size ratio
0.3
0.0
0.0
92.6
7.4
0.6
0.0
0.6
60.9
38.5
0.6
0.3
12.6
10.9
76.5
Mixture scenarios (II): Ang II correlated more strongly with RV size ratio than with 
PASP
0.0
0.3
0.2
81.3
18.5
0.0
0.6
12.6
12.5
74.9
0.3
0.6
30.1
2.1
67.8
Other assumptions in the simulation: N = 50 participants; correlations between PASP and RV size = 0.5.
For each scenario, results are based on 1000 simulated study datasets, each analysed using a MCMC 
simulation with 3 chains of 1000 iterations (after a burn-in of 1000 iterations) each.
b) Mean difference in predicted probability of RV dysfunction, PASP and RV size ratio 
between estimated pre-dose and post-dose Ang II reference values, by scenario
Correlations between Ang II and
Mean difference between predicted values
PASP
RV size ratio
P (RV 
dysfunction) 
%
PASP 
(mmHg)
RV size 
ratio
Extreme null scenario: no correlation between Ang II and either component of RV 
dysfunction
0.0
0.0
0.3
0.0
0.00
Extreme positive scenario: strong correlation between Ang II and both components of RV 
dysfunction
 
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Correlations between Ang II and
Mean difference between predicted values
PASP
RV size ratio
P (RV 
dysfunction) 
%
PASP 
(mmHg)
RV size 
ratio
0.6
0.6
33.9
5.7
0.11
"Middling" scenario: weak correlation between Ang II and both components of RV 
dysfunction
0.3
0.3
14.9
2.8
0.05
Mixture scenarios (I): Ang II correlated more strongly with PASP than with RV size ratio
0.3
0.0
6.1
2.9
0.00
0.6
0.0
13.2
5.6
0.00
0.6
0.3
22.5
5.6
0.05
Mixture scenarios (II): Ang II correlated more strongly with RV size ratio than with 
PASP
0.0
0.3
9.1
0.0
0.05
0.0
0.6
22.5
0.0
0.11
0.3
0.6
26.9
2.9
0.11
Other assumptions in the simulation: N = 50 participants; correlations between PASP and RV size = 0.5.
For each scenario, results are based on 1000 simulated study datasets, each analysed using a MCMC 
simulation with 3 chains of 1000 iterations (after a burn-in of 1000 iterations) each.
10.2.
Populations for Analyses
For purposes of analysis, the following populations are defined:
Population
Description
Completed
All participants for whom PASP, RV size ratio, Ang II and Ang(1-7)
data have been recorded for all three study days. For the purpose of 
evaluating ACP incidence, participants who have ACP either on entry or 
occurring during the study period will also be included in this 
population, even if they subsequently do not have three days’ worth of 
study assessments, provided consent is not withdrawn.
Evaluable
All participants for whom PASP, RV size ratio, Ang II and Ang(1-7)
data have been recorded for at least one study time point.
Safety
All participants for whom at least one echocardiograph and/or blood 
sample has been taken.
 
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10.3.
Statistical Analyses
10.3.1.
Pharmacodynamic Analyses
Endpoint
Statistical Analysis Methods
Primary
The association of Ang II with RV function (measured by PASP and RV 
size ratio) will be modelled using a Bayesian repeated measures mixed 
effects linear regression model, where PASP and RV size ratio are joint
response variables, and including subject as a random effect and 
log(Ang II), age and sex as fixed effects. The model will be used to 
make a probabilistic prediction of PASP and RV size ratio for two 
reference values of Ang II, and hence an estimate for the probability of 
RV dysfunction (defined as PASP > 40 mmHg or RV size ratio > 0.6, 
following Boissier 2013) for the two given Ang II levels will be 
obtained using mean values for the other continuous covariates and 
averaging over all levels of class effects for categorical covariates.
The above modelling may use a modified Ang II value (modification 
based on the corresponding Ang(1-7) result to account for potential 
inhibitory interactions); details to be provided in the reporting and 
analysis plan (RAP).
The primary analysis will use the Evaluable population.
Secondary
Association of Ang(1-7) with RV function (measured by PASP and RV 
size ratio) and of Ang II/Ang(1-7) with RV function (measured by 
PASP and RV size ratio) will both be modelled as per the primary 
analysis described above. These analyses will be done using the 
Evaluable population.
A model including both Ang II and Ang(1-7) as co-predictors of RV 
function may also be built, to explore the relationship between RV 
function and the envisaged dual action of angiotensin converting 
enzyme (ACE)2 effecting a reduction in Ang II together with a 
simultaneous increase in Ang(1-7).
Incidence of PCD and ACP during the three days of the study period 
and prevalence of pre-existing PCD/ ACP on entry to the study will be 
reported with 95% confidence intervals. This analysis will be done using 
the Completed population (which includes a clause to include 
participants who have an onset of PCD/ ACP but are subsequently 
withdrawn from the study before all three days' worth of data are 
collected without withdrawing consent).
The distribution of PASP, RV size ratio and other measurements of RV 
function in mechanically ventilated participants will be assessed via a 
combination of descriptive summary tables and outputs from the 
statistical modelling.
Exploratory
Will be described in the reporting and analysis plan
 
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Given that the hypothesis to this study is that RAS-mediated pulmonary vasoconstriction 
may be reduced through the dual action of ACE2 by simultaneously reducing Ang II and 
forming Ang(1-7) (see Section 3.2), an important secondary analysis will be to explore 
the joint effect of both Ang II and Ang(1-7) as related to RV dysfunction. That is to say, 
in addition to exploring the individual hypotheses that Ang II is deleterious to RV 
function and that Ang(1-7) is beneficial, a model will be developed that integrates the 
combined effects of these two mediators, since the expected effect of treatment with 
ACE2 is that both will change. Several models may be analysed in order to explore the 
exact nature of the relationship between the dual action of changes in Ang II and Ang(1-
7) and the probability of RV dysfunction as measured by PASP and RV size ratio.
10.3.2.
Safety Analyses
All safety analyses will be performed on the Safety Population. Safety data will be
presented in tabular and/or graphical format and summarised descriptively according to
GSK’s Integrated Data Standards Library (IDSL) standards.
10.3.3.
Interim Analyses
Two interim analyses are planned during the course of this study, both of which will 
provide an opportunity to stop the study for futility (close study to future recruitment but 
allow existing participants to complete the study as planned). A third interim analysis 
may optionally be performed if the second is inconclusive. The primary analysis of the 
association between Ang II and RV function (measured by PASP and RV size ratio) will 
be performed at each interim analysis, and will be used for the purposes of decision-
making. The results of these analyses are to be considered "rules of thumb" and may be 
overridden by other considerations such as an observed association between the 
secondary RAS biomarkers and measurements of RV (dys)function that is strong enough 
to warrant further investigation. The interim analysis may be postponed if there is 
insufficient coverage in the first 25 participants of Ang II values to make an interpolative 
prediction of RV (dys)function for the two pre-specified reference values (see below).
RV function will be measured using PASP and RV size ratio. The general principle 
underlying all the analyses of RV function and RAS peptide biomarkers will be:

Model the joint distribution of PASP and RV size ratio as a function of the RAS 
peptide(s) in question (together with confounding variables also included as 
covariates)

Choose reference values of the RAS peptide(s) in question and estimate a 
posterior distribution of PASP and RV size ratio for those given values, and

Use the posterior distribution to determine the probability of RV dysfunction
(for a future individual participant), defined (following the definition of 
moderate/severe pulmonary circulatory disease given in Figure 2 of Boissier
2013) as:
o
PASP > 40 mmHg
o
RV size ratio > 0.6
 
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The advantage of this method is that all the information on the continuous scales of PASP 
and RV size ratio is maintained throughout the modelling process, and that dichotomising 
participants into binary categories takes place at the end after the modelling has been 
done.
In order to evaluate the estimated effect of administering rhACE2 on RV outcomes (via 
its effect on the RAS peptides), predictions for the probability of RV dysfunction will be 
calculated for specific, pre-determined values of RAS peptide measurements that 
represent reasonable estimates for each RAS peptide both before and after dosing with 
rhACE2 in this patient population. These values will be hereafter referred to as reference 
values. Reference values for the RAS peptides will be defined based on available 
observed data from an investigator sponsored study (NCT01884051) (where RAS 
peptides were observed both before and after dosing with rhACE2) and will be calculated 
using the geometric mean of the observed pre-dose values (for the first reference value, 
hereafter referred to as the pre-dose reference) and the geometric mean of the observed 2 
hours post-dose values (for the second reference value, hereafter referred to as the post-
dose reference). (Note: the terminology "pre-dose" and "post-dose" should not be taken 
to imply that investigational product is used in this study; these values should be taken as 
being borrowed from that interventional study. Furthermore, the term "reference values" 
in this context should not be interpreted as upper or lower limits to the normal range. 
They represent the best guess available for a patient's typical experience before and after 
rhACE2 administration that may reasonably relate to a change in pulmonary 
haemodynamics.)
In the case that an assessment of inconclusive or futile conclusions based on Ang II at an 
interim analysis Ang 1-7 and the ratio of Ang II:Ang1-7 (and/or other peptides of the 
RAS as possible) will be evaluated and may lead to continuation of the study. In all the 
results from the simulation exercises described in this section, Ang II was taken to be the 
analyte in question for the purpose of evaluating the operating characteristics of the 
decision-making criteria, had they been strictly applied, with no fallback on the analyses 
of other RAS peptides. Additionally, for the sake of simplicity, only one measurement 
per participant was assumed (rather than three repeated measurements). This represents 
the worst case scenario (in terms of independence of samples) where all measurements 
within the same participant are perfectly correlated with each other.
At the time of writing, the reference values for Ang II were as follows (based on the two 
available observations of sufficient quality from the aforementioned investigator 
sponsored study (NCT01884051).

Pre-dose reference value: 55.4 pg/ml

Post-dose reference value 7.1 pg/ml
For the purposes of evaluating sample size considerations, these were the reference 
values for Ang II that were used in the assumptions underlying the simulations. An initial 
set of reference values for all RAS peptides measured in this study will be agreed before 
the first subject enters the study, and will be detailed in the RAP.
 
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The first interim analysis will take place after approximately 25 evaluable participants 
have completed the 3 day observational part of the study. The primary analysis of the 
association between Ang II and RV function will be performed and a probabilistic 
prediction for the difference in probability of RV dysfunction associated with a change in 
Ang II from the pre-dose reference value to the post-dose reference value will be 
obtained. If the median estimate of this difference is positive (i.e. probability of RV 
dysfunction at the pre-dose reference value is lower than that at the post-dose reference 
value) then additional follow-on analyses of the association of the other RAS biomarkers 
with RV (dys)function will be conducted. If similar conclusions are drawn from these 
analyses, then the study may be stopped for futility. If an association between another 
RAS biomarker and RV (dys)function is observed in the absence of an association 
between Ang II and RV (dys)function, then this biomarker may be substituted in place of 
Ang II as the primary endpoint for subsequent analyses.
As part of the sample size simulations (see Section 10.1), the operating characteristics of 
the first interim analysis were assessed to evaluate the probability of falsely declaring 
futility after approximately 25 participants when the study would have gone on to show 
success after 150 participants. None of the simulations, in any of the scenarios returned a 
futility result after 25 participants that would have gone on to be a success after 150 
participants. Full results are given in Table 2.
 
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Table 2
Proportion of simulated studies returning conclusion of futility at N
under a range of scenarios regarding the relationship between Ang 
II and the two endpoints PASP and RV size ratio
Correlations between Ang II and
Probability 
(%) of 
futility at 
N=25
Probability 
(%) of 
futility at 
N=25 
followed by 
success at 
N=150
Probability 
(%) of 
futility at 
N=25 
followed by 
inconclusive
at N=150
PASP
RV size ratio
Extreme null scenario: no correlation between Ang II and either component of RV 
dysfunction
0.0
0.0
49.7
0.0
0.0
Extreme positive scenario: strong correlation between Ang II and both components of RV 
dysfunction
0.6
0.6
0.0
0.0
0.0
"Middling" scenario: weak correlation between Ang II and both components of RV 
dysfunction
0.3
0.3
4.6
0.0
1.4
Mixture scenarios (I): Ang II correlated more strongly with PASP than with RV size ratio
0.3
0.0
23.3
0.0
0.1
0.6
0.0
8.3
0.0
1.0
0.6
0.3
0.4
0.0
0.4
Mixture scenarios (II): Ang II correlated more strongly with RV size ratio than with 
PASP
0.0
0.3
13.4
0.0
0.0
0.0
0.6
1.2
0.0
1.2
0.3
0.6
0.1
0.0
0.1
Other assumptions in the simulation: N = 50 participants; correlations between PASP and RV size = 0.5.
For each scenario, results are based on 1000 simulated study datasets, each analysed using a MCMC 
simulation with 3 chains of 1000 iterations (after a burn-in of 1000 iterations) each.
The second interim analysis will take place after approximately 50 evaluable participants 
have completed the 3-day observational part of the. This interim analysis will be used as 
the main go/no-go decision-making point as to whether to continue gathering data on 
biomarkers and other background information and to start internal GSK preparations for 
future interventional studies. The primary analysis of the association between RV 
(dys)function and Ang II will be performed and a probabilistic prediction for the 
difference in the probability of RV dysfunction associated with a change in Ang II from 
the pre-dose reference value to the post-dose reference value will be obtained (as at the 
first interim analysis). The absolute difference between these probabilities will then be 
calculated as and conclusions made as follows:
 
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
If the absolute difference between P(RV dysfunction at the pre-dose reference 
value and P(RV dysfunction at the post-dose reference value) ≤ 15%, then the 
study may be stopped for futility.

If the absolute difference between P(RV dysfunction at the pre-dose reference 
value and P(RV dysfunction at the post-dose reference value) ≥ 30%, then the 
study may continue, collecting biomarker data up to a maximum of 150 
participants, and GSK may begin enabling activities to prepare for a future 
interventional study.

If the absolute difference lies somewhere between these values (i.e. between 15 
and 30%), then a decision will be made by the Sponsor in conjunction with the 
investigator as to whether or not to continue based on observed associations 
between the secondary RAS biomarkers and RV (dys)function in addition to the 
primary analysis. A further interim analysis after 100 participants may 
additionally be performed in this case.
Recruitment will continue whilst the interim analyses are being performed.
The Reporting and Analysis Plan will describe the planned interim analyses in greater 
detail.
 
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11.
REFERENCES
Boissier F, Katsahian S, Razazi K, Thille AW, Roche-Campo F, Leon R, et al. 
Prevalence and prognosis of cor pulmonale during protective ventilation for acute 
respiratory distress syndrome. Intensive Care Med. 2013 Oct;39(10):1725-1733. 
Bull TM, Clark B, McFann K, Moss M; National Institutes of Health/National Heart, 
Lung, and Blood Institute ARDS Network. Pulmonary vascular dysfunction is associated 
with poor outcomes in patients with acute lung injury. Am J Respir Crit Care Med. 2010 
Nov 1;182(9):1123-1128 
Idell S, Kueppers, F, Lippmann, M, Rosen, H, Niederman, M, Fein, A. ACE elevated in 
ARDS BAL. Angiotensin converting enzyme in bronchoalveolar lavage in ARDS. Chest, 
1987; 91:52-56.
Marshall RP, Webb S, Bellingan GJ, Montgomery HE, Chaudhari B, McAnulty RJ, 
Humphries SE, Hill MR, Laurent GJ. Angiotensin converting enzyme insertion/deletion 
polymorphism is associated with susceptibility and outcome in acute respiratory distress 
syndrome. Am J Respir Crit Care Med. 2002 Sep 1; 166(5):646-650.
Mekontso-Dessap A, Boissier F, Charron C, Bégot E, Repessé X, Legras A, Brun-
Buisson C, Vignon P, Vieillard-Baron A. Acute cor pulmonale during protective 
ventilation for acute respiratory distress syndrome: prevalence, predictors, and clinical 
impact. Intensive Care Med. 2016 May; 42(5):862-870. 
Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, 
Camporota L, Slutsky AS (ARDS Definition Task Force). Acute respiratory distress 
syndrome: the Berlin Definition. JAMA. 2012; 307(23):2526-33. 
Santos RAS, Simoes e Silva, AC, Maric, C, Silva, DMR, machado, RP, de Buhr, 
I,Heringer-Walther, S, Pinheiro, SVB, Lopes, MT, Bader, M, Mendes, EP, Soares 
Lemos,V, Campagnole-Santos, MJ, Schultheiss, H-P, Speth, R and Walther, T. ANG (1-
7) as the agonist of mas. ‘Angiotensin (1-7) is an endogenous ligand for the G-protein 
coupled receptor Mas’ Proc. Natl. Acad. Sci. USA. 2003; 100:8258-8263.
Tom B, de Vries R, Saxena PR, Danser AH. Bradykinin potentiation by angiotensin-(1-7) 
and ACE inhibitors correlates with ACE C- and N-domain blockade. Hypertension. 2001 
Jul; 38(1):95-99.
Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000 
May 4; 342(18):1334-49.
Wenz M, Hoffmann, B, Bohlender, J, Kaczmarczyk, G. ANG II elevated in 
ARDS.Anfgiotensin II formation and endothelin clearance in ARDS participants in 
supine and prone positions. Intensive Care Med. 2000; 26:292-298.
Zapol WM, Snider MT: Pulmonary hypertension in severe acute respiratory failure. N 
Engl J Med 1977, 296: 476–480.
 
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Zhao Z1, Möller K, Steinmann D, Frerichs I, Guttmann J. Evaluation of an electrical 
impedance tomography-based Global Inhomogeneity Index for pulmonary ventilation 
distribution.Intensive Care Med. 2009 Nov;35(11):1900-6
 
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12.
APPENDICES
12.1.
Appendix 1: Abbreviations and Trademarks
ABG
Arterial Blood Gases 
ACP
Acute Cor Pulmonale
ACE
Angiotensin Converting Enzyme
rhACE2
Recombinant Human Angiotensin 
Converting Enzyme 2
AE
Adverse Event 
Ang
Angiotensin
ARDS
Acute Respiratory Distress Syndrome
CRF
Case Report Form
CO2
Carbon Dioxide 
CONSORT
Consolidated Standards of Reporting Trials
cmH2O
Centimeter of Water
E’
LV Velocity
FiO2
Oxygen Requirement
GCS
Glasgow Coma Score
GI
Global Inhomogeneity
GSK
GlaxoSmithKline
h
Hour
ICF
Informed Consent Form 
ICU
Intensive Care Unit
IDSL
Integrated Data Standards Library 
IEC
Independent Ethics Committee 
IRB
Institutional Review Board
IVC
Inferior Vena Cava
Kg/m2
Kilogram/Square Meter
LAR
Legally Acceptable Representative 
LV
Left Ventricle 
Mins
Minutes
mL
Milliliter
mmHg
Millimeter of Mercury
ms
Millisecond
PaCO2
Partial Pressure of Carbon Dioxide
PaO2
Partial Pressure of Oxygen 
PASP
Pulmonary Arterial Systolic Pressure 
PCD
Pulmonary Circulatory Dysfunction
PEEP
Positive End Expiratory Pressure
Pg/ml
Picograms per millilitre
Pplat
Target Plateau Pressure 
PI
Principle Investigator 
PVR
Pulmonary Vascular Resistance
RAP
Reporting and Analysis Plan
RAS
Renin Angiotensin System
 
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RV
Right Ventriculum
SAE
Serious Adverse Event
SAPS 
Simplified Acute Physiology Score
SaO2
Oxygen Saturation
SoA
Schedule of Assessments 
SOFA
Sequential Organ Failure Assessment
SRM
Study Reference Manual 
SUSAR
Suspected Unexpected Serious Adverse 
Reactions
TAPSE
Tricuspid Annular Plane Systolic 
Excursion
TDI
Tissue Doppler Imaging 
TOE
Transesophageal echocardiography
TTE
Transthoracic echocardiography
SpO2
Oxygen Saturation
Trademark Information
Trademarks of the GlaxoSmithKline group 
of companies
Trademarks not owned by the 
GlaxoSmithKline group of companies
NONE
None
 
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12.2.
Appendix 2: Clinical Laboratory Tests

The tests detailed in Table 3 will be performed by the local laboratory.

Additional tests may be performed at any time during the study as determined 
necessary by the investigator or required by local regulations.
Table 3
Protocol-Required Safety Laboratory Assessments
Collected as per standard of care  
Laboratory 
Assessments
Parameters
Haematology
Platelet Count
WBC count with Differential:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
RBC Count
WBC (absolute)
Haemoglobin
Haematocrit
Clinical 
Chemistry
BUN
Potassium
Total bilirubin
Creatinine
Sodium
Troponin I or T
Chloride
Total CO2
 
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12.3.
Appendix 3: Study Governance Considerations
12.3.1.
Ethical Considerations
The study will also be conducted in accordance with ICH Good Clinical Practice (GCP), 
all applicable subject privacy requirements, and the guiding principles of the current 
version of the Declaration of Helsinki

This study will be conducted in accordance with the protocol and with:
Consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical 
Sciences (CIOMS) International Ethical Guidelines
Applicable ICH Good Clinical Practice (GCP) Guidelines
Applicable laws and regulations

The protocol, protocol amendments, ICF, Investigator Brochure, and other relevant 
documents (eg, advertisements) must be submitted to an IEC by the investigator and 
reviewed and approved by the IEC before the study is initiated. 

Any amendments to the protocol will require IEC approval before implementation of 
changes made to the study design, except for changes necessary to eliminate an 
immediate hazard to study participants. 

The investigator will be responsible for the following:
Providing written summaries of the status of the study to the IEC annually or 
more frequently in accordance with the requirements, policies, and procedures 
established by the IEC
Notifying the IEC of SAE or other significant safety findings as required by IEC 
procedures
Providing oversight of the conduct of the study at the site and adherence to 
requirements of 21 CFR, ICH guidelines, the IEC, European regulation 
536/2014 for clinical studies (if applicable), and all other applicable local 
regulations
12.3.2.
Financial Disclosure
Investigators and sub-investigators will provide the sponsor with sufficient, accurate 
financial information as requested to allow the sponsor to submit complete and accurate 
financial certification or disclosure statements to the appropriate regulatory authorities. 
Investigators are responsible for providing information on financial interests during the 
course of the study and for 1 year after completion of the study.
12.3.3.
Informed Consent Process
Given participants may be mechanically ventilated upon enrolment, obtaining informed 
consent directly from the participants may not always be feasible. Therefore, participants
may be enrolled in three ways:
 
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1.
Participant is enrolled by signing the informed consent
If the participant is capable of understanding and signing the informed consent prior to 
enrolment, the below points will apply:

The investigator or his/her representative will explain the nature of the study to 
the participant and answer all questions regarding the study. 

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.
2.
Participant is enrolled by his/her LAR 
If the participant is unable to provide informed consent, best efforts will be undertaken by 
the investigator or delegate to obtain consent from a LAR prior to enrolment.  If consent 
is obtained from a LAR, the following points will apply:

Best efforts will be undertaken to obtain written consent from a LAR; however,
if only oral LAR consent is feasible, participants can be enrolled on LAR’s oral 
consent and a written LAR consent will be sought as soon as possible thereafter.

The investigator has responsibility for applying local laws in the matter of who 
has the capacity to consent and who qualifies as a LAR of a potential subject.

A LAR is defined as an individual or juridical or other body authorized under 
applicable law to consent, on behalf of a prospective subject, to the subject’s 
participation in the clinical trial.

LAR can be a member of the participant’s family, next-of-kin or trusted person 
previously designated by the participant.

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. The subjects will 
decide if they want to allow samples already collected to be used or request for 
them to be discarded if not yet analysed.  
3.
Participant is enrolled upon emergency consent process
If the participant in unable to provide informed consent and the LAR cannot be reached 
when the study selection criteria are fulfilled, the investigator will be able to enrol the 
participant according to the emergency consent process, and the following points will 
apply.

The emergency consent process is indicated in last resort (article L1122-1-2 of 
French Public Health code) as per the following arguments: the urgency and 
gravity of the situation has mandated invasive mechanical ventilation and 
intensive care admission; the use of echocardiography for hemodynamic 
monitoring is routine in the participating centres in this situation.
 
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
As soon as it 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. The participants will 
decide if they want to allow samples already collected to be used or request for 
them to be discarded if not yet analysed.  
Documentation of Informed consent process
The management of the informed consent process will be documented in the medical 
source document.  This will include which process was used to obtain consent. 
The following should be noted in regard to the informed consent process: 

If consent is provided by LAR, name, time and date of LAR and name of the 
personnel at site that collected this information 
If consent is not obtained the time and date of attempt(s) must be collected along with the 
name(s) of LAR and personnel at site that attempted contact. Participants/LAR must be 
informed that their participation is voluntary. Participants and/or LAR 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 IEC or study centre. 
A copy of the signed ICF(s) must be provided to the participant or the participant’s LAR.
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 IEC members, and by inspectors from regulatory 
authorities.
Publication Policy

The results of this study may be published or presented at scientific meetings. If 
this is foreseen, the investigator agrees to submit all manuscripts or abstracts to the 
sponsor before submission. This allows the sponsor to protect proprietary 
information and to provide comments.

The sponsor will comply with the requirements for publication of study results. In 
accordance with standard editorial and ethical practice, the sponsor will generally 
support publication of multicentre studies only in their entirety and not as 
 
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individual site data. In this case, a coordinating investigator will be designated by 
mutual agreement.

Authorship will be determined by mutual agreement and in line with International 
Committee of Medical Journal Editors authorship requirements.
Dissemination of Clinical Study Data

Study information from this protocol will be posted on publicly available clinical 
trial registers before enrolment 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.
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 (eg, 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, 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 25 years after study completion 
 
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unless local regulations or institutional policies require a longer retention period. 
No records may be destroyed during the retention period without the written 
approval of the sponsor. No records may be transferred to another location or party 
without written notification to the sponsor. 
Source Documents 

Source documents provide evidence for the existence of the participant and 
substantiate the integrity of the data collected. Source documents are filed at the 
investigator’s site.

Data reported on the CRF or entered in the eCRF that are transcribed from source 
documents must be consistent with the source documents or the discrepancies must 
be explained. The investigator may need to request previous medical records or 
transfer records, depending on the study. Also, current medical records must be 
available.
Study and Site Closure 
GSK or its designee reserves the right to close the study site(s) 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 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 the use of a study treatment*, whether or not considered related to 
the study treatment.

NOTE: An AE can therefore be any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally 
associated with the use of a study treatment.
*Although there is no “study treatment” in this study, AE/SAE definitions still apply to 
reports of serious adverse events explicitly attributed to GSK products or to study 
procedures.
Events Meeting the AE Definition 

Any abnormal laboratory test results (hematology, clinical chemistry, or urinalysis) 
or other safety assessments (eg, ECG, radiological scans, vital signs measurements), 
including those that worsen from baseline, considered clinically significant in the 
medical and scientific judgment of the investigator (ie, not related to progression of 
underlying disease).

Exacerbation of a chronic or intermittent pre-existing condition including either an 
increase in frequency and/or intensity of the condition.

New conditions detected or diagnosed after study treatment administration even 
though it may have been present before the start of the study.

Signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction.

Signs, symptoms, or the clinical sequelae of a suspected overdose of concomitant 
GSK product. 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.

The disease/disorder being studied or expected progression, signs, or symptoms of 
the disease/disorder being studied, unless more severe than expected for the 
 
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Events NOT Meeting the AE Definition 
participant’s condition.

Medical or surgical procedure (eg, endoscopy, appendectomy): the condition that 
leads to the procedure is the AE.

Situations in which an untoward medical occurrence did not occur (social and/or 
convenience admission to a hospital).

Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present 
or detected at the start of the study that do not worsen.
Definition of SAE
If an event is not an AE per definition above, then it cannot be an SAE even if serious 
conditions are met (eg, hospitalization for signs/symptoms of the disease under study, 
death due to progression of disease).
A SAE is defined as any untoward medical occurrence that:
a.
Results in death
b.
Is life-threatening
The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
participant was at risk of death at the time of the event. It does not refer to an event, 
which hypothetically might have caused death, if it were more severe.
c.
Requires inpatient hospitalization or prolongation of existing hospitalization
In general, hospitalization signifies that the participant has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for observation 
and/or treatment that would not have been appropriate in the physician’s office or 
outpatient setting. Complications that occur during hospitalization are AE. If a 
complication prolongs hospitalization or fulfils any other serious criteria, the event is 
serious. When in doubt as to whether “hospitalization” occurred or was necessary, the AE 
should be considered serious.
Hospitalization for elective treatment of a pre-existing condition that did not worsen from 
baseline is not considered an AE.
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 (eg, sprained ankle) which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption.
 
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A SAE is defined as any untoward medical occurrence that:
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 SAE
SAE Recording

When a SAE occurs, it is the responsibility of the investigator to review all 
documentation (eg, hospital progress notes, laboratory, and diagnostics reports) 
related to the event.

The investigator will then record all relevant SAE information in the CRF.

It is not acceptable for the investigator to send photocopies of the participant’s 
medical records to GSK in lieu of completion of the GSK /AE/SAE CRF page.

There may be instances when copies of medical records for certain cases are 
requested by GSK. In this case, all participant identifiers, with the exception of the 
participant number, will be redacted on the copies of the medical records before 
submission to GSK.

The investigator will attempt to establish a diagnosis of the event based on signs, 
symptoms, and/or other clinical information. Whenever possible, the diagnosis (not 
the individual signs/symptoms) will be documented as the SAE.
Assessment of Causality

The investigator is obligated to assess the relationship between study procedure, 
GSK product and each occurrence of each 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.
 
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Assessment of Causality

Alternative causes, such as underlying disease(s), concomitant therapy, and other 
risk factors, as well as the temporal relationship of the event to study treatment 
administration will be considered and investigated.

The investigator will also consult the Investigator’s Brochure (IB) and/or Product 
Information, for marketed products, in his/her assessment.

For each SAE, the investigator must document in the medical notes that he/she has 
reviewed the SAE and has provided an assessment of causality.

There may be situations in which an SAE has occurred, and the investigator has 
minimal information to include in the initial report to GSK. However, it is very 
important that the investigator always make an assessment of causality for 
every event before the initial transmission of the SAE data to GSK.

The investigator may change his/her opinion of causality in light of follow-up 
information and send an SAE follow-up report with the updated causality 
assessment.

The causality assessment is one of the criteria used when determining regulatory 
reporting requirements.
Follow-up of 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 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).
 
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SAE Reporting to GSK via Electronic Data Collection Tool

The site will enter the SAE data into the electronic system as soon as it becomes 
available.

The investigator or medically-qualified sub-investigator must show evidence within 
the eCRF (e.g., check review box, signature, etc.) of review and verification of the 
relationship of each SAE to study procedure or GSK product (causality) within 72 
hours of SAE entry into the eCRF.

After the study is completed at a given site, the electronic data collection tool will be 
taken off-line to prevent the entry of new data or changes to existing data.

If a site receives a report of a new SAE from a study participant or receives updated 
data on a previously reported SAE after the electronic data collection tool has been 
taken off-line, then the site can report this information on a paper SAE form (see 
next Section) or to the medical monitor/SAE coordinator by telephone.

Contacts for SAE reporting can be found in the SRM.
SAE Reporting to GSK via Paper CRF

Facsimile transmission of the SAE paper CRF is the preferred method to transmit 
this information to the medical monitor or the SAE coordinator.

In rare circumstances and in the absence of facsimile equipment, notification by 
telephone is acceptable with a copy of the SAE data collection tool sent by overnight 
mail or courier service.

Initial notification via telephone does not replace the need for the investigator to 
complete and sign the SAE CRF pages within the designated reporting time frames.

Contacts for SAE reporting can be found in the SRM.
 
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12.5.
Appendix 5: Assumptions for sample size calculations
12.5.1.
Distribution of Ang II
For the purposes of these sample size calculations, Ang II was chosen as the primary 
explanatory variable. This choice is based on the a priori belief that lowering Ang II is 
associated with improvements in RV outcomes. Similar modelling approaches can be 
applied to other RAS biomarkers, in particular Ang(1-7) (the other component of the dual 
action of Angiotensin Converting Enzyme (ACE)2); indeed these will be applied in the 
final analysis. However, for the purposes of defining sensible decision criteria, and 
assessing whether the proposed sample size of 50 participants (for the primary interim 
analysis) is suitable for purpose, only Ang II is considered in these simulations.
The assumptions underlying the distribution of Ang II in the participant population are as 
follows:

Ang II follows log-normal distribution with mean and standard deviation as observed 
in 42 participants of the previous ARDS study (ACE114622; baseline 
measurements)

Geometric mean of 16.1 pg/ml.

Standard deviation of log(Ang II) is 1.74; equivalent to 5.7-fold change in 
Ang II.

Implies that ~⅔ of population will be in the range (2.8, 92.1) (one SD 
below mean to one SD above mean)
Figure 2
Distribution of observed Ang II in study ACE116422 with super-
imposed normal distribution curve
 
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12.5.2.
Definition of outcome: an objective measure of right ventricular 
(RV) dysfunction
Following the definition of moderate pulmonary circulatory dysfunction (PCD) presented 
by Boissier 2013, quantitative criteria for an objective measurement of right ventricular 
(RV) dysfunction were set out as follows:

A participant is defined as having right ventricular (RV) dysfunction if one of the 
following two criteria is true:

pulmonary arterial systolic pressure (PASP) > 40 mmHg

ratio of the right ventricular to the left ventricular end-diastolic areas 
(hereafter referred to as RV size ratio) > 0.6
These two criteria can be represented on a 2-dimensional plane divided into two regions 
representing "no RV dysfunction" and "RV dysfunction" (Figure 3a).
However, rather than dichotomising participants into those with and those without RV 
dysfunction based on individual measurements (Figure 3b), the two component 
variables, PASP and RV size ratio, will be modelled as bivariate continuous outcomes, 
with Angiotensin II (Ang-II) level as a continuous predictor variable. The joint posterior 
distribution of PASP and RV size ratio for given reference levels of Ang-II will be 
calculated based on the observed data, and a posterior probability of the mean PASP and 
mean RV jointly lying in the "RV dysfunction" region of the plane calculated from these 
posterior distributions (Figure 3c).
 
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Figure 3
Illustration of the process of modelling the joint distribution of PASP 
and RV size ratio and obtaining a predicted probability of RV 
dysfunction for given Ang II levels
a) The criteria used for defining RV dysfunction 
are represented as a plane divided into two 
regions. Points (representing participant data) 
lying in the green region are categorised as not 
having RV dysfunction ("normal"), points lying 
in the grey region are categorised as having RV 
dysfunction ("dysfunctional").
b) Illustrative mock data showing simulated 
values of the two component variables RV 
dysfunction (PASP and RV size ratio, represented 
on the x and y axes) and their relationship to  Ang 
II values (represented by colour). It is possible to 
dichotomise participants into those with RV 
dysfunction and those with normal RV function at 
this stage; this is not the approach proposed here.
 
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c) Illustration of posterior prediction regions for 
the two component variables of RV dysfunction 
for two reference values of Ang II.  Instead of 
modelling the association of Ang II with the 
dichotomised RV dysfunction response variable 
(and losing the information from the continuous 
variables), a Bayesian linear regression model is 
fitted with PASP and RV size ratio as bivariate 
outcome variables and Ang II as the explanatory 
variable. Prediction regions and the probability of 
RV dysfunction (defined as the weighted 
proportion of the prediction region being within 
the grey region of the plane) are calculated for 
two reference Ang II values. The absolute 
difference between these two probabilities is then 
calculated. If this difference is ≥30%, then the 
relationship between Ang II and RV dysfunction 
is considered strong enough for there to be 
potential for the action of ACE2 on Ang II to be 
of clinical benefit and further study to be 
conducted ("success"). If the difference is ≤15%, 
then the relationship is considered too weak to 
warrant further inquiry ("futile"). The reference 
values are derived as the geometric mean of the 
observed Ang II values at pre-dose and at 2 hours 
post rhACE2 dose in the investigator sponsored 
study (NCT01884051).
12.5.3.
"Success" and "futility" criteria
Criteria for "success" (that is to say, the strength of association between Ang II and RV 
dysfunction required to confirm potential for the effect of rhACE2 on Ang II to yield 
clinical benefits, warranting further study) and "futility" (an association too weak to 
justify continuing development) are based on posterior predictive regions for PASP and 
RV size ratio for two given Ang II values. These reference values are the geometric mean 
of observed pre-dose and 2 hours’ post-dose Ang II values from an investigator-
sponsored study (NCT01884051), and represent the best estimate for a typical "before 
rhACE2" and "after rhACE2" measurement. The reference values used in this simulation 
are based on the two participants in that study for which, at the time of writing, data of 
sufficient reliability were available, and were derived as follows:

Pre-dose Ang II: 55.4 pg/ml (geometric mean of 121.3 and 25.3 pg/ml)

Post-dose Ang II: 7.1 pg/ml (geometric mean of 16.3 and 3.1 pg/ml)
"Success" and "futility" criteria are derived using the predicted probabilities of RV 
dysfunction for a hypothetical future participant with the reference values listed above. 
The predicted probability of RV dysfunction for a given Ang II value is defined as the 
proportion of the posterior joint predicted region of PASP and RV size ratio (weighted by 
density) that satisfies the criteria for RV dysfunction (i.e. what portion of the prediction 
region lies in the grey zone in Figure 3). The absolute difference between the two 
 
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probabilities is then calculated and compared with the following criteria for success or 
futility (for N = 50, 100 and 150):

"Success": P(RV dysfunction when Ang II = 55.4) - P(RV dysfunction 
when Ang II = 7.1) ≥ 30%

"Futile": P(RV dysfunction when Ang II = 55.4) - P(RV dysfunction when 
Ang II = 7.1) ≤ 15%
The results are deemed "inconclusive" otherwise.
For N = 25, a conclusion of futile was returned if the difference between P (RV 
dysfunction when Ang II = 55.4) and P (RV dysfunction when Ang II = 7.1) was 
negative. A result of inconclusive was returned otherwise.
12.5.4.
Assumptions for simulated datasets
A simulation was carried out to assess the operating characteristics of the above success 
and futility criteria, given reasonable assumptions for the distributions of PASP, RV size 
and Ang II, and the three pairwise correlations between them.
The assumptions for the distributions of these variables are as follows:

Ang II is log-normally distributed, with mean(log(Ang II)) = 2.78 and 
SD(log(Ang II)) = 1.74 (as described above)

PASP is normally distributed, with mean(PASP) = 38 mmHg and 
SD(PASP) = 8 mmHg

RV size ratio is normally distributed, with mean(RV size ratio) = 0.61 and 
SD(RV size ratio) = 0.15
The assumed pairwise correlations between these three variables were as follows:

The correlation between PASP and RV size ratio (the two outcome 
variables) was assumed to be 0.5

Three different scenarios each for the correlation between PASP and 
log(Ang II), and between RV size ratio and log(Ang II) were considered 
(for a total of nine scenarios in all): zero, 0.3 and 0.6. This was to test the 
operating characteristics for a wide variety of scenarios ranging from "no 
association whatsoever" (both correlations zero) to "Ang II strongly 
correlated with both components of RV dysfunction" (both correlations 
0.6).
A single simulated study dataset consisted of 50 values for Ang II, PASP and RV size, 
drawn from a multi-variate normal distribution with the above characteristics.
For each of the nine scenarios, a total of 1000 study datasets were generated and 
analysed, which was the maximum that was feasible given computational constraints.
 
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12.5.5.
Analysis of simulated data
For each individual study dataset that was generated, the relationship between Ang II (as 
the explanatory variable) and the joint bivariate distribution of PASP and RV size ratio 
(the outcome variables) was analysed using a Bayesian multivariate linear regression 
model. Estimates for the posterior distributions of coefficients in the model were obtained 
using an MCMC simulation (using Gibbs sampling) running three chains of 2000 
iterations each. The first 1000 iterations of each chain were discarded (the "burn-in") and 
the posterior distributions estimated from the 3000 remaining iterations (1000 from each 
chain).
The multivariate normal model was specified as follows:



















,
~
2
1
2
1
i
i
i
i
MVNormal
y
y


where yij represents the observed value for the ith subject and the jth outcome (i.e. j=1
representing PASP and j=2 representing RV size ratio), µij represents the expected value 
for the ith subject and the jth outcome (j=1 and j=2 as above), and Σ represents a 2×2 
variance-covariance matrix for the two outcome variables. The expected values, µij, are 
modelled as follows:
i
j
j
ij
x





where αj is the intercept term for the jth outcome (i.e. PASP or RV size), βj represents the 
slope coefficient for log(Ang II) in the model, and xi represents the observed log(Ang II) 
value for the ith subject. For simplicity the simulations have no additional covariates (e.g. 
age, sex), and do not take into account multiple observations in the same participant (i.e. 
they represent the extreme worst case scenario where there is perfect within-participant 
correlation of all variables).
Uninformative prior distributions are assigned for α, β, and σ as follows:

α is assigned a bivariate normal distribution with mean zero (for both 
outcomes), large variances (for both outcomes) and zero covariance 
(between the two outcomes),

β is assigned a bivariate normal distribution with the same specifications 
as for α,

Τ, defined as the inverse of Σ, is assigned an uninformative Wishart prior 
distribution: T ~ Wishart(R, df) where:







001
.0
0
0
001
.0
R
and df=3.
 
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12.5.6.
Metrics for operating characteristics
For each scenario, 1000 simulated datasets of 150 participants each were generated and 
analysed according to the above modelling process, and the following metrics were 
calculated for the first 25, 50, 100 and the whole 150-participant dataset using the 1000 
results so generated:

the proportion of studies returning "success" results

the proportion of studies returning "futile" results

the proportion of studies returning “inconclusive” results

the mean absolute difference in predicted P(RV dysfunction) between the 
two reference Ang II values

the mean difference in predicted PASP between the two reference Ang II 
values

the mean difference in predicted RV size ratio between the two reference 
Ang II values
Results are summarised for the nine scenarios and presented in the main body of the 
protocol (Section 10.1 and Section 10.3.3).
 
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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.
Protocol Amendment 2 (27-Nov-2017)
Overall Rationale for the Amendment: In this amendment, an exploratory outcome and 
emergency consent have been added. In addition, clarification was added throughout the 
protocol. 
Section # and Name
Description of Change
Brief Rationale
2. Schedule of Assessments
Added Sequential Organ 
Failure (SOFA) score
Clarification that SOFA will be 
calculated on a daily basis. All 
components needed to 
calculate the score were 
already being collected. 
2. Schedule of Assessments
The following language was 
added in Footnote 2 of 
Schedule of Assessments:
Other measurements (e.g. 
ventilator settings) to be 
prioritized as per investigator 
for patient care. 
The timing of exploratory 
outcomes can be prioritized by 
investigators in the best interest 
of patients as part of standard 
of care. 
2. Schedule of Assessments
A note was added that SAPS 
II will only be measured at 
screening.
The timing of the assessment 
was clarified.
2. Schedule of Assessments
4. Objectives and Endpoints
9.5.5 General management 
status
Added Participant 
Management which includes 
use of ECMO and ECCO2R. 
Sites have recently included 
use of ECMO and ECCO2R as 
part of patient’s standard 
management. The use needs to 
be documented in case of
interaction with outcomes.
2. Schedule of Assessments
4. Objectives and Endpoints 
9.3 Ventilator settings
Static compliance was added 
as a measurement that will be 
collected with other ventilator 
settings. 
It had previously been missed; 
static compliance is a standard 
measure read from the 
ventilator.
2. Schedule of assessments
4. Objectives and Endpoints
9.4 Lung Function
pH and base excess were 
added as measurements that 
will be collected with other 
lung function measurements.  
These measurements had 
previously been missed.

pH is a standard measure 
automatically calculated 
from blood gas analysis. 

Base excess is a standard 
blood gas analysis. 
Depending on the base 
status of the patient a base 
 
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Section # and Name
Description of Change
Brief Rationale
excess result may be 
collected rather than base 
deficit.
2. Schedule of Assessments
4. Objectives and Endpoints
9.9 Biomarkers
12.2 Appendix 2: Clinical 
Laboratory Tests
Troponin I or T was removed 
from the schedule of 
assessments, objectives and 
endpoints and biomarker 
sections and added to Table 
3. Protocol required safety 
laboratory assessments.
Troponin I or T was removed as 
a test performed by GSK and 
will be performed as a safety 
lab by sites when taken as 
standard of care.
4. Objectives and Endpoints 
Systolic eccentricity index was 
moved from Primary to 
exploratory endpoint. 
The primary analysis will use 
the binary outcome of 
paradoxical septal motion. 
Systolic eccentricity index is a 
continuous variable and was 
included in the primary endpoint 
in error. This will now be 
analysed separately as an 
exploratory outcome.
4. Objectives and Endpoints
9.2.2 Electrical Impedance 
Tomography (EIT) 
New exploratory objective was 
added. 
EIT assesses lung ventilation 
and perfusion. Understanding 
regional ventilation/perfusion 
may be of importance in 
understanding the role of the 
RAS in ventilation/perfusion 
matching in severe respiratory 
failure.
4. Objectives and Endpoints
9.2.1 Echocardiograms 
It was added that the inferior 
vena cava diameter will be 
taken at end-expiration.
The formula for calculating 
Respiratory variations of RV 
velocity time integral was 
added for completeness. The 
formula is (end-expiration 
minus end-inspiration / end-
expiration).
Values that will be recorded 
from the echocardiograms were 
further defined for clarity.
9.1 Demographic/Medical 
History Assessments
Clarification was added that 
demographic and medical 
history assessments could be 
collected at any point in the 
study if it is not possible at 
screening. 
Whilst important to collect this 
data where possible, 
sometimes it is extremely
challenging to collect in the 
acute care setting.
 
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Section # and Name
Description of Change
Brief Rationale
9.5.2 SAPS II
It was clarified that an 
estimated GCS can be used if 
a patient is sedated at the 
time of assessment 
When a patient is sedated the 
GCS cannot be calculated, 
therefore an estimate prior to 
sedation must be used (where 
available).
9.5.3 Disease Diagnosis
ARDS Definition was added.
The definition was missing from 
previous version.  
9.5.3 Disease Diagnosis
PCD definition clarified
Definition was previously 
missing > and ≥ for PASP and 
RV size respectively.  
9.6 Additional clinical 
assessments
It was defined that date and 
time of only concomitant 
surgery will be recorded.
To clarify that only concomitant 
surgery needs to be recorded. 
9.9 Biomarkers
Troponin I or T was removed 
as a test performed by GSK 
and will be performed by sites 
when taken as standard of 
care.
Clarified that samples will be 
stored, and other biomarkers 
may be assessed eg. BNP 
and eg. Inflammatory 
markers/lung injury markers.
Troponin I or T cannot be 
measured at GSK.
To clarify other biomarkers may 
be tested and samples will be 
stored. 
10.3.1 Pharmacodynamics 
Analyses
Statistical analysis (PD) 
wording updated to clarify the 
rationale for additional RAS 
peptide analyses.
The biological effects of the 
RAS are complex and effects of 
peptides beyond Ang II (peptide 
to be assessed in primary 
analysis) may be observed e.g. 
Ang 1-7.
12.2 Appendix 2: Clinical 
Laboratory Tests
It was clarified that the safety 
assessments in Table 3 will be 
as per standard of care.
To ensure assessments are 
only completed as per standard 
of care 
12.3.3 Informed consent 
process
The option to enrol a 
participant with emergency 
consent was added.
To ensure the consenting 
process is appropriate for the 
participant population.
Throughout 
Deleted mention of 
Institutional Review Board 
(IRB) throughout.
This protocol does not require 
submission to an IRB due to 
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Protocol Amendment 1 (02-Jun-2017)
Overall Rationale for the Amendment: Protocol was amended to remove the option for 
participants to be included without informed consent and to change errors in the previous 
version.
Section # and Name
Description of Change
Brief Rationale
2. Schedule of 
Assessments 
And
9.1 Demography and 
Medical History 
Assessments
Height & Weight assessment 
were added as they were 
missed from the prior version. 
Prior version mentioned these parameters 
only under the inclusion criteria, but 
missed them in Section 2 and Section 9.1.
2. Schedule of 
Assessments
Assessments of Diagnosis of 
PCD & ACP were removed 
from Participant status and 
added as a separate line. 
To provide more clarity on the time points 
of these assessments. 
9.5.1 SAPS II
Glasgow Coma scale was 
removed from the 
components defined for the 
SAPS II score.
It was incorrectly included in the prior 
version. 
12.3.3 
Informed consent 
process and 6.1 
inclusion criteria 
Removed the option for 
participants to be included 
without informed consent.  
It was decided participants must have a 
signed informed consent by their legally 
acceptable representative prior to 
enrolment. 
 
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