Clinical trial NCT04640194
Conditions: Acute Respiratory Distress Syndrome
Sponsor: Boehringer Ingelheim
Start date: 2020-12-16; Completion date: 2022-07-25
Enrollment: 104 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a study in adults with severe breathing problems because of COVID-19. People who are in hospital on breathing support can participate in the study. The purpose of the study is to find out whether a medicine called alteplase helps people get better faster.

The study has 2 parts. In the first part, participants are put into 3 groups by chance. Participants in 2 of the groups get 2 different doses of alteplase, in addition to standard treatment.

Participants in the third group get standard treatment. In the second part of the study, participants are put into 2 groups by chance. One group gets alteplase and standard treatment. The other group gets only standard treatment. Alteplase is given as an infusion into a vein. In both study parts, treatments are given for 5 days. Doctors monitor patients and check whether their breathing problems improve. They compare results between the groups after 1 month.

Participants are in the study for 3 months.

Detailed description:

Arms: arm 1: 0.3 milligram/kilogram (mg/kg) over 2 hours (Day 1) immediately followed by daily infusion of 0.02 mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5), plus Standard of Care (SOC). One optional additional infusion of 0.3 mg/kg over 2 hours could be given once on Days 2 to 5 in case of clinical worsening, per investigator judgement. Part 1 subjects were randomized equally (1:1:1) across the three Part 1 arms. arm 2: 0.6 milligram/kilogram (mg/kg) over 2 hours (Day 1) immediately followed by daily infusion of 0.04 mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5), plus Standard of Care (SOC). One optional additional infusion of 0.6 mg/kg over 2 hours could be given once on Days 2 to 5 in case of clinical worsening, per investigator judgement. Part 1 subjects were randomized equally (1:1:1) across the three Part 1 arms. arm 3: Standard of Care included best possible treatment regimen established locally and was in line with current guidelines for Acute respiratory distress syndrome treatment. Part 1 subjects were randomized equally (1:1:1) across the three Part 1 arms. arm 4: 0.6 milligram/kilogram (mg/kg) over 2 hours (Day 1) immediately followed by daily infusion of 0.04 mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5), plus Standard of Care (SOC). One optional additional infusion of 0.6 mg/kg over 2 hours could be given once on Days 2 to 5 in case of clinical worsening, per investigator judgement. Non-invasive mechanical ventilation (NIV) patients are those with a baseline World Health Organization (WHO) Clinical Progression Scale value of 6. Part 2 subjects were randomized 2 (Alteplase) to 1 (SOC). arm 5: Standard of Care included best possible treatment regimen established locally and was in line with current guidelines for Acute respiratory distress syndrome treatment. Non-invasive mechanical ventilation (NIV) patients are those with a baseline World Health Organization (WHO) Clinical Progression Scale value of 6. Part 2 subjects were randomized 2 (Alteplase) to 1 (SOC). arm 6: 0.6 milligram/kilogram (mg/kg) over 2 hours (Day 1) immediately followed by daily infusion of 0.04 mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5), plus Standard of Care (SOC). One optional additional infusion of 0.6 mg/kg over 2 hours could be given once on Days 2 to 5 in case of clinical worsening, per investigator judgement. Invasive mechanical ventilation (IMV) patients are those with a baseline World Health Organization (WHO) Clinical Progression Scale value of 7, 8 or 9. Part 2 subjects were randomized 2 (Alteplase) to 1 (SOC). arm 7: Standard of Care included best possible treatment regimen established locally and was in line with current guidelines for Acute respiratory distress syndrome treatment. Invasive mechanical ventilation (IMV) patients are those with a baseline World Health Organization (WHO) Clinical Progression Scale value of 7, 8 or 9. Part 2 subjects were randomized 2 (Alteplase) to 1 (SOC).
Interventions: intervention 1: Standard of care intervention 2: Alteplase low dose intervention 3: Alteplase high dose
Intervention descriptions: intervention 1: Standard of Care (SOC) includes any supportive measures applied in hospital, specifically on an intensive care unit (ICU), such as for example the use of non-invasive or invasive ventilation, oxygen masks, haemodynamic support, if needed, sedation, as well as medical therapies commonly used in patients suffering from acute respiratory distress syndrome (ARDS) or its complications. SOC should include best possible treatment regimen established locally and should be in line with current guidelines for ARDS treatment. intervention 2: 0.3 milligram/kilogram (mg/kg) over 2 hours (Day 1) immediately followed by daily infusion of 0.02 mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5). One optional additional infusion of 0.3 mg/kg over 2 hours could be given once on Days 2 to 5 in case of clinical worsening, per investigator judgement. intervention 3: 0.6 milligram/kilogram (mg/kg) over 2 hours (Day 1) immediately followed by daily infusion of 0.04 mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5). One optional additional infusion of 0.6 mg/kg over 2 hours could be given once on Days 2 to 5 in case of clinical worsening, per investigator judgement.

Protocol documents:
Prot_000.pdf: 
001-MCS-40-106_RD-03 (18.0) / Saved on: 10 Jul 2019
Clinical Trial Protocol
TITLE PAGE
Document Number:
c32434345-04
EudraCT No.
EU Trial No.
2020-002913-16
BI Trial No.
0135-0347
BI Investigational
Medicinal Product(s)
Alteplase (recombinant tissue-type plasminogen activator, rt-PA)
Title
The TRISTARDS trial - ThRombolysIS Therapy for ARDS
A Phase IIb/III operationally seamless, open-label, randomised, 
sequential, parallel-group adaptive study to evaluate the efficacy 
and safety of daily intravenous alteplase treatment given up to 5 
days on top of standard of care (SOC) compared with SOC alone, 
in patients with acute respiratory distress syndrome (ARDS) 
triggered by COVID-19.
Lay Title
The TRISTARDS trial - ThRombolysIS Therapy for ARDS
A study to test whether different doses of alteplase help people 
with severe breathing problems because of COVID-19.
Clinical Phase
IIb/III
Clinical Trial Leader
Phone: 
Fax:
Coordinating 
Investigator
Tel. 
Fax.
Version and Date
Version: 4.0 
Date: 17 Feb 2022
Page 1 of 140
Proprietary confidential information.
2022 Boehringer Ingelheim International GmbH or one or more of its affiliated companies. All rights reserved.
This document may not - in full or in Part - be passed on, roduced, published or otherwise used without prior written permission.


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CLINICAL TRIAL PROTOCOL SYNOPSIS
Company name
Boehringer Ingelheim (BI)
Protocol date
21 July 2020
Revision date
17 Feb 2022
BI trial number
0135-0347
Title of trial
The TRISTARDS trial - ThRombolysIS Therapy for ARDS
A Phase IIb/III operationally seamless, open-label, randomised, 
sequential, parallel-group adaptive study to evaluate the efficacy and 
safety of daily intravenous alteplase treatment given up to 5 days in 
addition to standard of care (SOC) compared with SOC alone, in 
patients with acute respiratory distress syndrome (ARDS) triggered 
by COVID-19.
Coordinating 
Investigator
Tel.
Fax
Trial site(s)
Multi-centre trial conducted in approximately 22 countries
Clinical phase
IIb/III
Trial rationale
ARDS is an acute illness of various etiologies (e.g. infections such as 
COVID-19) and is one of the major causes of mortality in COVID-
19. ARDS due to COVID-19 is associated with fibrin wall thickening 
and micro-/macrovascular thrombi formation, while at the same time 
fibrinolysis is impaired. There is accumulating data from animal 
studies, historical trials in ARDS and clinical cases / case series
reported during the COVID-19 pandemic that application of 
thrombolytic drugs are associated with increased oxygenation and 
improved clinical outcomes of ARDS, as a result of clearance of 
fibrin from the alveolar space and lysis of microclots. Alteplase
(recombinant tissue-type plasminogen activator) may therefore have a 
role in the treatment of ARDS associated with COVID-19.   
Trial objective(s)
To evaluate the efficacy and safety of intravenous alteplase in ARDS 
triggered by COVID-19.
Trial endpoints
The same primary endpoint will be evaluated in Part 1 and in Part 2
as follows:

Time to clinical improvement or hospital discharge up to Day 
28, defined as the time from randomisation to either 
an improvement of two points on the 11-point WHO Clinical 
Progression Scale or discharge from the hospital, whichever 


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comes first. 
In Part 2, key secondary endpoints are:

Treatment failure defined as all cause mortality or mechanical 
ventilation at Day 28

All cause mortality at Day 28
Secondary endpoints to evaluate safety and efficacy in Part 2 are as 
follows:

Number of oxygen-free days up to Day 28

Length of hospital stay up to Day 28

Major bleeding events (MBE) (according to International 
Society on Thrombosis and Haemostasis [ISTH] definition) 
until Day 6

PaO2/FiO2 ratio (or inferred PaO2/FiO2 ratio from SpO2) 
change from baseline to Day 6 
Trial design
Open-label, randomised, sequential parallel design comparison of:  
Part 1: two treatment arms and a standard of care (SOC) group (1:1:1 
randomisation)
Part 2: one treatment arm and a standard of care (SOC) group
(2:1 randomisation)    
Total number of patients
randomised
Part 1: approximately 60 patients 
Part 2: approximately 260 patients, thereof 210 non-invasive 
mechanical ventilation (NIV) and up to 50 invasive mechanical 
ventilation (IMV) patients
Randomisation will be stratified by type of ventilation support 
(invasive mechanical / non-invasive)
Additionally in Part 2: 
Randomisation will be stratified by D-Dimer levels (≥ULN to <5-fold 
ULN, versus ≥5-fold ULN)
Randomisation of IMV patients will be stopped when a maximum of 
50 IMV patients have been randomized in Part 2.
Randomisation for the whole trial will be stopped when 
approximately 210 NIV patients have been randomized in Part 2, 
regardless of how many IMV patients have been randomised so far. 
Number of patients on 
each treatment
Part 1: approximately 20 patients in SOC arm and 20 patients in each 
treatment arm
Part 2: NIV patients: approximately 70 patients in SOC arm and 140
in treatment arm 


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IMV patients: up to approximately16 patients in SOC arm and 32 in 
treatment arm
Diagnosis
ARDS due to COVID-19
Main in- and exclusion 
criteria
Patients participating in Part 1 are not eligible for Part 2.
Main criteria for inclusion:
1. Age ≥ 18 years (or above legal age)
2. ARDS with PaO2*/FiO2 ratio >100 and ≤300, either on non-
invasive ventilator support, OR on mechanical ventilation (<48 
hours since intubation), 

with bilateral opacities in chest X-ray or CT scan (not fully 
explained by effusions, lobar/lung collapse, or nodules)

with respiratory failure (not fully explained by cardiac 
failure/fluid overload)
*or estimation of PaO2/FiO2 from pulse oximetry (SpO2/FiO2)
3. SARS-CoV-2 positive (laboratory-confirmed RT-PCR test)
4. Fibrinogen level  ≥ lower limit of normal
5. D-Dimer ≥upper limit of normal (ULN) according to local 
laboratory
6. Signed and dated written informed consent in accordance with 
ICH Good Clinical Practice (GCP) and local legislation prior to 
admission to the trial.
Main criteria for exclusion:
1. Massive confirmed pulmonary embolism (PE) with 
haemodynamic instability at trial entry, or any (suspected or 
confirmed) PE that is expected to require therapeutic dosages of 
anticoagulants during the treatment period
2. Indication for therapeutic dosages of anticoagulants at trial entry
3. Mechanical ventilation for longer than 48 hours
4. Chronic pulmonary disease i.e. with known forced expiratory 
volume in 1 second (FEV1) <50% requiring home oxygen, or oral 
steroid therapy or hospitalisation for exacerbation within 
12 months, or significant chronic pulmonary disease in the 
Investigator’s opinion, or primary pulmonary arterial 
hypertension
5. Has a Do-Not-Intubate (DNI) or Do-Not-Resuscitate (DNR) order
6. In the opinion of the investigator, is not expected to survive for 
> 48 hours after screening.
7.
Planned interventions during the first 5 days after randomisation, 
such as surgery, insertion of central catheter or arterial line, 
drains, etc.
Further criteria apply to exclude patients at higher risk of bleeding.


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Test product(s)
Alteplase, recombinant tissue-plasminogen activator, rt- PA, powder 
and solvent for infusion
dose
Part 1: 
Dosing regimen A: Initial i.v. infusion of alteplase 0.3 mg/kg over 2 
hours (Day 1) immediately followed by daily i.v. infusion of 0.02 
mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5 days at 
maximum)
One optional additional i.v. infusion of 0.3 mg/kg over 2 hours can be 
given once on Days 2 to 5 in case of clinical worsening (as per 
investigator judgement)
OR:
Dosing regimen B: Initial i.v. infusion of alteplase 0.6 mg/kg over 2 
hours (Day 1) immediately followed by daily i.v. infusion of 0.04 
mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5 at 
maximum)
One optional additional i.v. infusion of 0.6 mg/kg over 2 hours can be 
given once on Days 2 to 5 in case of clinical worsening (as per 
investigator judgement)
Part 2: 
Initial i.v. infusion of alteplase 0.6 mg/kg over 2 hours (Day 1) 
immediately followed by daily i.v. infusion of 0.04 mg/kg/hour over 
12 hours (starting on Day 1 and up to Day 5* at maximum)
One optional additional i.v. infusion of 0.6 mg/kg over 2 hours can be 
given once on Days 2 to 5 in case of clinical worsening (as per 
investigator judgement)
* Exception: Treatment period can be exceeded beyond Day 5, in 
case of unavoidable interruptions of the treatment.
           
mode of administration
Intravenous (i.v.)
Comparator product(s)
N/A
dose
N/A
mode of administration
N/A
Duration of treatment
Maximum of 5 days.
In case of unavoidable and urgent reasons, treatment interruptions are 
permitted up to a total of 72 hrs, and the treatment period will be 
extended accordingly.


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Statistical methods
The trial consists of two parts which, for the purpose of statistical 
analyses, will be treated independently. Part 1 will be descriptive, and 
Part 2 will be confirmatory. In both parts, the respective full analysis
set (FAS) will be used and the same primary endpoint for both Parts 1
and 2 will apply. Only patients randomised into Part 1 will be 
included in the analyses of Part 1, and only patients randomised into 
Part 2 will be included in the confirmatory analyses of Part 2. 
The primary endpoint for Part 1 will be analysed in a similar manner 
to Part 2, albeit in Part 1 this will be exploratory in nature.
Following the completion of Part 1, the DMC will review the 
unblinded efficacy and safety data to make a recommendation for 
proceeding into Phase III (Part 2). This recommendation will be with 
respect to selecting one dosing regimen for Phase III.
Part 2 will consist of two cohorts, the NIV patient cohort and the IMV 
patient cohort, and these two cohorts will be analysed separately. 
Only the NIV patient cohort will be part of the main statistical 
objectives of the study, and therefore part of the confirmatory 
hierarchical testing procedure. 
In Part 2, a confirmatory hierarchical testing procedure in the NIV 
patient cohort will be applied for the primary endpoint and the two 
key secondary endpoints. In a first step, the primary endpoint 
hypothesis in NIV patients will be tested. If the null hypothesis is 
rejected, the key secondary endpoint of Treatment failure at Day 28
will then be tested in the NIV patient cohort as a second step. If this 
null hypothesis is rejected, the key secondary endpoint All cause 
mortality at Day 28 will then be tested in the NIV patient cohort as a 
third step.
For Part 2, the primary analysis of the time to clinical improvement or 
hospital discharge up to Day 28 will be analysed using the Cox 
proportional hazard model. This endpoint is defined as the time from 
randomisation to either an improvement of two points on the 11-point 
WHO Clinical Progression Scale or discharge from the hospital, 
whichever comes first. Patients will be censored at Day 28 if they die 
prior to Day 28. If they receive bail-out therapy, they will be censored 
on the day of receiving the bail-out therapy. Patients who have not 
had clinical improvement by Day 28 will be censored on Day 28.
For the primary endpoint the Cox proportional hazard model will be 
used to estimate the hazard ratio, the 95% confidence interval and p-
value. In the NIV cohort, adjustment will be made for the number of 
days under NIV support, baseline D-Dimer level and age. For the 


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IMV cohort, adjustment will be made for the baseline WHO value, 
baseline D-Dimer level and age.
For each of the two key secondary endpoints, treatment failure at Day
28 and all cause mortality at Day 28, the delta method and average 
marginal effect method will be used to calculate the risk difference, 
95% confidence intervals and p-values. Adjustment will be made for 
the same set of covariates as in the primary analysis of the primary 
endpoint. 
Descriptive statistics will be used to analyse all other endpoints and 
for all analyses of the IMV patient cohort.
If the NIV and IMV patient cohorts show homogeneity, then a meta-
analysis will be performed to obtain an overall treatment estimate, 
albeit in an exploratory manner.


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FLOW CHART
Trial Periods
Scree
ning 
Intravenous open-label treatment
Follow-Up
Visit number
1
2a
2b
2c
2d
2e / 
EOT
3
4
5
6 /
EOS
Day
0
1
2
3
4
5
6
7
28
90
Hours
-24 -
0
0-14
26-38
50-62
74-86
98-
110
134
Time windows [hrs, if not 
specified otherwise]
+6
±2
±2
±2
+6
±12
+1 
day
+2 
days
±7 
days
Informed consent 
(separate IC for patients 
participating in PK 
sampling) 
X
Demographics
X
Medical history
X
Physical examination
X
X
X
Weight
X
Height
X
Vital signs 1
X
X
X
X
X
X
X
X
X
12-lead ECG2
X
(X only needed in case of clinical event)2
X
Review of in-/exclusion 
criteria
X
IRT call
X
X
X
Randomisation3
X
Dispense trial drugs
X
Administer trial drug4
X
X
X
X
X
PK Sampling5
X
Laboratory Tests6
X
X
X
X6
X
D-dimer assessment7
X
X
X
X
X
Fibrinogen, aPTT and
antithrombin assessment8
X
X
X
X
X
X
X
X
X
PaO2/FiO2 ratio (or 
inferred PaO2/FiO2 ratio 
from SpO2)9
X
X
X
X
X
X
X
X
X
Trial specific 
examinations: 
SOFA score10
X
X
X
11-point WHO Clinical 
Progression Scale (see 
Section 5.1.1) 11
X
X
X
X
X
X
X
X
X
Pregnancy testing12
X
X
SARS-CoV-2 test13
X
All adverse events/Serious 
adverse events14
X
X
X
X
X
X
X
X
X
Concomitant therapy
X
X
X
X
X
X
X
X
X
Completion of patient 
participation15
X
Vital status collection16
X


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.Footnotes:
1.
Vital signs measurement shall include BP measurement, body temperature, respiratory rate and pulse
rate.
2.
In addition to the scheduled ECGs (electrocardiograms), ECGs should be done and documented at any 
time of clinical event (e.g. acute event of arrhythmia, tachy- or bradycardia, angina, or MI).
3.
Randomisation of patients shall happen not later than 24 hours after the confirmation of the inclusion 
and exclusion criteria. Study drug administration shall start within 6 hours of the randomisation. The 
time-point of obtaining D-Dimer levels can be as long as four calendar days before screening.
4.
In Part 2: Initial i.v. infusion of alteplase 0.6 mg/kg over 2 hours (Day 1) immediately followed by daily 
i.v. infusion of 0.04 mg/kg/hour over 12 hours (starting on Day 1 and up to Day 5 at maximum, plus 
possible interruption time for urgent reasons). Interruptions up to 72 hours in total for urgent reasons are
allowed and accordingly, the time window of the visits may be extended. The very first infusion of 
alteplase should start at least 10 hours after the last injection of a LMWH. Each daily 12-hour infusion 
of alteplase is followed by an interruption of approximately 12 hours. Does not apply for SOC treatment 
arm.
5.
PK samples collection (on a voluntary basis, 1st sample: prior to first drug administration on Day 1 
(Visit 2a pre-dose) (planned time: -1:00 h; time window -2:00 h to just prior drug administration), 2nd 
sample: at steady state of the initial i.v. infusion on Day 1 (Visit 2a post-dose initial i.v. infusion) 
(planned time 1:30 h; time window: between 1:00 h after start of initial i.v. infusion and just prior to end 
of initial i.v. infusion), 3rd sample: at steady state of the long-term i.v. infusion on Day 1 (Visit 2a post-
dose long-term i.v. infusion) (planned time: 6:00 h; time window: between 2 h after start of long-term 
i.v. infusion and just prior to end of long-term i.v. infusion) For further details refer to Section 5.3.1.
Separate informed consent has to be signed before including patient in PK collection.   
6.
On Day 6 only lab samples required for SOFA score and PaO2/FiO2 shall be collected.
7.
D-dimer levels shall be measured at Day 0 (or no more than 4 calendar days before screening), days 3, 
5, 7 and 28
8.
During the treatment period, fibrinogen should be measured daily. The aPTT should be measured at 
least daily, but preferably more often. The aPTT (for all patients on unfractionated heparin [UFH]) or 
anti-Xa (for all patients on low-molecular weight heparin [LMWH]) should be used to check the 
intensity of anticoagulation. In patients with moderate (creatinine clearance 30-50 ml/min) and mild 
(creatinine clearance 50-80 ml/min) renal impairment, careful clinical monitoring of anticoagulation 
intensity according to institutional guidance is advised. (more details see Section 4.2.1)   
9.
PaO2/FiO2 is only measured during hospital stay. The qualifying PaO2/FiO2 ratio should be the worst 
measurement in the screening period. During the treatment period the worst measurement on a given 
day should be documented in the case report form. During the first 6 days and during the stay on ICU 
(whatever is longer), PaO2/FiO2 ratio should be measured at least three times daily in all patients, 
together with a recording of the patient`s position.
10. The SOFA score will be assessed at baseline, Day 6 and Day 28 (or just before hospital discharge 
whichever comes first including vital signs assessment and laboratory samples needed for score 
calculations. (Please refer to Appendix 10.1). If patient is discharged from hospital prematurely, last 
evaluation during hospital stay is expected at day of discharge from hospital.
11. WHO clinical progression scale should be collected daily during hospital stay. The last measurement on
a given day should be recorded in the case report form. On Day 1 (randomisation day) an assessment 
just before randomisation shall be collected as a baseline value. 
12. Only applicable for women of childbearing potential. Urine pregnancy test will be performed at 
screening and at Visit 5. Study drug should only be administered in case of a negative test result. A 
woman is considered of childbearing potential, i.e. fertile, following menarche and until becoming post-
menopausal unless permanently sterile. Permanent sterilisation methods include hysterectomy, bilateral 
salpingectomy and bilateral oophorectomy. Tubal ligation is NOT a method of permanent sterilisation. 
A postmenopausal state is defined as no menses for 12 months without an alternative medical cause.
13. SARS-CoV-2 laboratory confirmation (RT-PCR test) as a pre-requisite before or at screening. The RT-
PCR test needs to be in timely association with the current hospital stay. For eligibility, patients should
be hospitalized with the diagnosis of COVID-19 ARDS. The choice of laboratory tests should follow 
local practice.


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14. The rules for Adverse Event Reporting exemptions still apply, please see Section 5.2.6.2.4.
15. Patients who discontinue trial treatment prematurely should still follow the trial schedule until 
Visit 6/EOS, if possible, please see Section 3.3.4.
16. Patients will be followed-up until Day 90 for vital status collection. No onsite visit is required, any 
locally approved way of obtaining vital status information is accepted.


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TABLE OF CONTENTS
TITLE PAGE ...........................................................................................................................1
CLINICAL TRIAL PROTOCOL SYNOPSIS .....................................................................2
FLOW CHART........................................................................................................................8
TABLE OF CONTENTS ......................................................................................................11
ABBREVIATIONS................................................................................................................15
1.
INTRODUCTION...............................................................................................18
1.1
MEDICAL BACKGROUND.............................................................................18
1.2
DRUG PROFILE................................................................................................21
1.3
RATIONALE FOR PERFORMING THE TRIAL.........................................23
1.4
BENEFIT - RISK ASSESSMENT.....................................................................24
1.4.1
Benefits.................................................................................................................24
1.4.2
Risks .....................................................................................................................26
1.4.3
Discussion.............................................................................................................30
2.
TRIAL OBJECTIVES AND ENDPOINTS......................................................32
2.1
MAIN OBJECTIVES, PRIMARY AND SECONDARY ENDPOINTS........32
2.1.1
Main objectives....................................................................................................32
2.1.2
Primary endpoint................................................................................................32
2.1.3
Secondary endpoints...........................................................................................32
3.
DESCRIPTION OF DESIGN AND TRIAL POPULATION.........................35
3.1
OVERALL TRIAL DESIGN.............................................................................35
3.2
DISCUSSION OF TRIAL DESIGN, INCLUDING THE CHOICE OF 
CONTROL GROUP(S)......................................................................................36
3.3
SELECTION OF TRIAL POPULATION .......................................................37
3.3.1
Main diagnosis for trial entry ............................................................................38
3.3.2
Inclusion criteria .................................................................................................38
3.3.3
Exclusion criteria ................................................................................................39
3.3.4
Withdrawal of patients from treatment or assessments..................................40
3.3.4.1
Discontinuation of trial treatment .........................................................................40
3.3.4.2
Withdrawal of consent to trial participation .........................................................41
3.3.4.3
Discontinuation of the trial by the sponsor...........................................................42
4.
TREATMENTS...................................................................................................43
4.1
INVESTIGATIONAL TREATMENTS ...........................................................43
4.1.1
Identity of the Investigational Medicinal Products..........................................43
4.1.2
Selection of doses in the trial and dose modifications......................................47
4.1.3
Method of assigning patients to treatment arms..............................................47
4.1.4
Drug assignment and administration of doses for each patient......................48


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4.1.5
Blinding and procedures for unblinding...........................................................49
4.1.5.1
Blinding.................................................................................................................49
4.1.5.2
Unblinding and breaking the code ........................................................................50
4.1.6
Packaging, labeling, and re-supply....................................................................50
4.1.7
Storage conditions...............................................................................................50
4.1.8
Drug accountability.............................................................................................50
4.2
OTHER TREATMENTS, EMERGENCY PROCEDURES, 
RESTRICTIONS ................................................................................................51
4.2.1
Other treatments and emergency procedures..................................................51
4.2.2
Restrictions ..........................................................................................................54
4.2.2.1
Restrictions regarding concomitant treatment ......................................................54
4.2.2.2
Restrictions on diet and life style..........................................................................54
4.2.2.3
Contraception requirements..................................................................................55
4.3
TREATMENT COMPLIANCE........................................................................55
5.
ASSESSMENTS..................................................................................................56
5.1
ASSESSMENT OF EFFICACY........................................................................56
5.1.1
WHO Clinical Progression Scale.......................................................................56
5.1.2
Oxygen- free and Ventilator-free days, days in ICU .......................................57
5.1.3
PaO2/FiO2 ratio....................................................................................................57
5.1.4
SOFA Score .........................................................................................................57
5.2
ASSESSMENT OF SAFETY.............................................................................57
5.2.1
Physical examination ..........................................................................................57
5.2.2
Vital signs.............................................................................................................57
5.2.3
Safety laboratory parameters ............................................................................58
5.2.4
Electrocardiogram ..............................................................................................60
5.2.5
Other safety parameters.....................................................................................60
5.2.5.1
Bleeding events.....................................................................................................60
5.2.6
Assessment of adverse events.............................................................................61
5.2.6.1
Definitions of adverse events................................................................................61
5.2.6.2
Adverse event collection and reporting ................................................................64
5.3
DRUG CONCENTRATION MEASUREMENTS AND 
PHARMACOKINETICS...................................................................................65
5.3.1
Assessment of pharmacokinetics .......................................................................65
5.3.2
Methods of sample collection .............................................................................66
5.4
ASSESSMENT OF BIOMARKERS.................................................................66
5.5
BIOBANKING ....................................................................................................66
5.6
OTHER ASSESSMENTS...................................................................................67
5.7
APPROPRIATENESS OF MEASUREMENTS..............................................67
6.
INVESTIGATIONAL PLAN.............................................................................68
6.1
VISIT SCHEDULE.............................................................................................68
6.2
DETAILS OF TRIAL PROCEDURES AT SELECTED VISITS .................68
6.2.1
Screening and run-in periods.............................................................................69
6.2.2
Treatment periods...............................................................................................69


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6.2.3
Follow-up period and trial completion..............................................................70
7.
STATISTICAL METHODS AND DETERMINATION OF SAMPLE SIZE
...............................................................................................................................71
7.1
NULL AND ALTERNATIVE HYPOTHESES ...............................................71
7.2
PLANNED ANALYSES.....................................................................................72
7.2.1
General considerations .......................................................................................72
7.2.2
Primary endpoint analyses.................................................................................73
7.2.3
Secondary endpoint analyses .............................................................................74
7.2.5
Safety analyses.....................................................................................................75
7.2.6
Interim Analyses .................................................................................................76
7.3
HANDLING OF MISSING DATA ...................................................................76
7.4
RANDOMISATION ...........................................................................................76
7.5
DETERMINATION OF SAMPLE SIZE .........................................................77
8.
INFORMED CONSENT, TRIAL RECORDS, DATA PROTECTION, 
PUBLICATION POLICY, AND ADMINISTRATIVE STRUCTURE ........79
8.1
TRIAL APPROVAL, PATIENT INFORMATION, INFORMED 
CONSENT ...........................................................................................................79
8.2
DATA QUALITY ASSURANCE ......................................................................80
8.3
RECORDS...........................................................................................................80
8.3.1
Source documents ...............................................................................................81
8.3.2
Direct access to source data and documents.....................................................82
8.3.3
Storage period of records ...................................................................................82
8.4
EXPEDITED REPORTING OF ADVERSE EVENTS ..................................82
8.5
STATEMENT OF CONFIDENTIALITY AND PATIENT PRIVACY........82
8.5.1
Collection, storage and future use of biological samples and corresponding 
data.......................................................................................................................83
8.6
TRIAL MILESTONES.......................................................................................83
8.7
ADMINISTRATIVE STRUCTURE OF THE TRIAL ...................................84
9.
REFERENCES....................................................................................................86
9.1
PUBLISHED REFERENCES............................................................................86
9.2
UNPUBLISHED REFERENCES......................................................................93
10.
APPENDICES.....................................................................................................95
10.1
SOFA SCORE AND GLASGOW COMA SCALE .........................................95
10.2
TABLE TO DERIVE PAO2/FIO2 RATIO FROM SPO2................................96
10.3
FIO2 RANGES FOR COMMON OXYGEN DELIVERY DEVICES...........97
10.4
POTENTIAL MODIFICATION OF TRIAL CONDUCT IN CASE OF 
RESTRICTIONS DUE TO COVID-19.............................................................97
10.5
ALTEPLASE DOSING TABLE........................................................................99
10.6
TREATMENT RECOMMENDATIONS FOR COVID-19 ARDS (WITH A 
FOCUS ON NIV PATIENTS) .........................................................................103
11.
DESCRIPTION OF GLOBAL AMENDMENT(S) .......................................111


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11.1
GLOBAL AMENDMENT 1 ............................................................................111
11.2
GLOBAL AMENDMENT 2 ............................................................................114
11.3
GLOBAL AMENDMENT 3 ............................................................................135


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ABBREVIATIONS
ACE
angiotensin-converting enzyme
AE
adverse event
ALCOA
attributable, legible, contemporaneous, original, accurate 
ALT
alanine aminotransferase
AME
Average marginal effect
 
aPTT
activated partial thromboplastin time
ARDS
acute respiratory distress syndrome
ASA
acetylsalicylic acid
AST
aspartate aminotransferase
BI
Boehringer Ingelheim
BP
blood pressure
COVID-19
coronavirus disease 2019
CRA
Clinical Research Associate
CRF
Case report form, paper or electronic (sometimes referred to as “eCRF”)
CRI
Constant rate infusion
CRO
Contract research organisation
CT Leader
Clinical Trial Leader
CTP
clinical trial protocol
CTR
clinical trial report
DBL
database lock
DMC
Data Monitoring Committee
EC
Ethics Committee
ECG
Electrocardiogram
ECMO
extracorporeal membrane oxygenation
eCRF
electronic case report form
EOT
End of treatment
EOS
End of trial
EudraCT
European Clinical Trials Database
FAS
Full analysis set


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FEV1
forced expiratory volume in 1 second
FiO2
fraction of inspired oxygen
FPI
First patient in
GI
Gastrointestinal
GCP
Good Clinical Practice
GPIIb/IIIa
glycoprotein IIb/IIIa
GUSTO
Global Utilization of Streptokinase and Tissue Plasminogen Activator for 
Occluded Coronary Arteries
HIT
Heparin-induced thrombocytopenia
i.v.
Intravenous
IB
Investigator’s Brochure
ICU
intensive care unit
IMP
Investigational medicinal product
IMV
Invasive mechanical ventilation
INR
International normalised ratio
IRB
Institutional Review Board
IRT
Interactive response technology
ISF
Investigator site file
ISTH
International Society on Thrombosis and Haemostasis
kDa
Kilodalton
LMWH
low-molecular weight heparin
LPLT
Last patient last treatment
MBE
Major bleeding events
MedDRA
Medical Dictionary for Drug Regulatory Activities
MI
myocardial infarction
MRT
magnetic resonance tomography
NIV
Non-invasive mechanical ventilation
OE
Outcome event
PAI-1
plasminogen activator inhibitor Type 1
PaO2
partial oxygen pressure in arterial blood
PaO2/FiO2
Oxidation Index (Horowitz Index)
PE
pulmonary embolism


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PK
Pharmacokinetics
PPS
Per Protocol Set
rt-PA
recombinant tissue-type plasminogen activator
RBC
red blood cell
REP
residual effect period
RRT
renal replacement therapy
RS
Randomised Set
SARS-CoV-2
severe acute respiratory syndrome coronavirus 2
SAE
serious adverse event
SC
Steering Committee
SGPT
serum glutamic-pyruvic transaminase
SGOT
serum glutamic-oxaloacetic transaminase
SOC
Standard of Care
SmPC
Summary of Product Characteristics
SOFA 
Sequential (sepsis-related) Organ Failure Assessment
SOP
standard operating procedure
SpO2
Oxygen saturation
SUSAR
Suspected unexpected serious adverse reactions
t1/2
Half life time
T2DM
Type 2 diabetes mellitus
TS
Treated Set
TSAP
Trial Statistical Analysis Plan
UFH
unfractionated heparin
ULN
upper limit of normal
WBC
white blood cell
WHO
World Health Organisation


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1.
INTRODUCTION
1.1
MEDICAL BACKGROUND
The current knowledge of COVID-19 is evolving. In 2019, an outbreak of acute respiratory 
illness emerged. In February 2020, the International Committee on Taxonomy of Viruses 
announced “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2) as the name of 
the new virus, and the WHO designated the disease “coronavirus disease 2019,” or 
COVID-19. The WHO declared COVID-19 a public health emergency in January 2020 and a 
pandemic in March 2020. During this pandemic, cases have grown exponentially around the 
world. 
The infection is a self-limiting illness in most people, but in a significant proportion of the 
population, particularly the elderly, results in ARDS, with severe progressive pneumonia, 
multiorgan failure, and death [R20-1310, R20-1309]. Experience suggests that 5 to 16% of 
patients hospitalised with COVID-19 will undergo prolonged intensive care and 50 to 70% 
thereof require mechanical ventilation [R20-1310, R20-1309]. The mortality rate is 25 to 
60% in severely affected patients with ARDS with the current standard of care [R20-1652].
COVID-19 and other infections are associated with ARDS. The exact mechanism 
contributing to a rapid lung injury in patients with ARDS is not fully understood, but diffuse 
alveolar damage typically marks the onset of ARDS. In early phases, epithelial and 
endothelial injury is characterized by loss of integrity of the barrier with plasma rich in 
albumin and fibrinogen and cellular debris exuding into the alveolar spaces which probably 
accentuates the inflammatory process [R20-1654]. A procoagulant state stems from increased 
tissue factor expression and suppression of fibrinolytic activity due to a rise in plasminogen 
activator inhibitor Type 1 (PAI-1) [P94-80931]. This leads to the formation of microthrombi 
in the lungs, as demonstrated by autopsy, further compromising gas exchange. [R20-1366, 
R20-1643, R20-1952].   
Additionally, fibrin formation within the lungs starts a vicious cycle in which leukocytes 
localized to the clot through integrin binding sites within the fibrinogen molecule exacerbate 
inflammation and fibrinolysis [R20-1653]. Emerging evidence indicates that activation of 
coagulation as demonstrated by elevated D-dimer and fibrin degradation product levels are 
associated with poorer outcomes in novel coronavirus patients [R20-1655]. 
Current treatment standards for COVID-19 are emerging. Current treatment may include 
supportive measures applied in hospital, specifically on an intensive care unit (ICU), such as 
for example the use of non-invasive or invasive ventilation, oxygen masks, haemodynamic 
support, if needed, sedation, as well as medical therapies commonly used in patients suffering 
from ARDS or its complications. Standard of care follows the standard therapies established 
locally at the study site.
Since the outbreak of COVID-19, treatment options for hospitalized patients with COVID-19 
who require oxygen and ventilation support are evolving. Data and guidance to target the 


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hyperinflammatory status of COVID-19 patients are available [R21-2944, R20-4049, R21-
2943, R21-2942, R21-2936]. [R20-1647, R21-2937, R21-2938, R20-1645, R21-2941].
With regard to the need and intensity of concomitant anticoagulation, the evidence rapidly 
evolved during the SARS-CoV-2 pandemic. Guidelines recommend prophylactic 
anticoagulation for all patients with COVID-19 in the ICU and do not recommend therapeutic 
anticoagulation in absence of known thromboembolic events [R20-1852, R21-2851]. The 
status of intermediate dosing of anticoagulants in critically ill patients is still under scientific
debate [P21-01227, R21-2935]. 
Critically ill COVID-19 patients with hypercoagulable state, presenting with a significant 
increase in D-dimer levels, might benefit from a thrombolytic therapy on top of 
anticoagulation with unfractionated or low-molecular weight heparins. As an established 
thrombolytic therapy, alteplase may have a role in targeting the coagulation and fibrinolytic 
systems to improve the treatment and possibly outcome of ARDS, an approach that has been 
proposed for at least the past two decades [R20-1650]. In particular, the use of plasminogen 
activators to limit ARDS progression and reduce ARDS-induced death has received strong 
support from animal models and human clinical trials. A meta-analysis of 22 preclinical 
studies in different species showed a significant reduction in mortality when using 
fibrinolytics via different routes, and conclude that fibrinolytic therapy may be an effective 
pharmaceutic strategy for ARDS in animal models [R20-1656]. 
A case series of five patients with COVID-19-associated ARDS receiving a bolus 
administration of alteplase (50 mg) and three patients with an additional re-bolus and 
concomitant heparin treatment and alteplase appeared to have an improved respiratory status 
following alteplase [P20-04591]. In a study with three severe COVID-19 related ARDS 
patient under mechanical ventilation, the administration of an intravenous bolus of alteplase 
(25 mg) followed by an infusion of 25 mg over 22 hours, led to a temporary improvement in 
their respiratory status (ranging from 38% to a ~100% improvement), with one of them 
having a sustained response, the two others having transient improvements [P20-03126]. A 
further study with four severe COVID-19 related patients with ARDS, under mechanical 
ventilation and a bolus-infusion concept (50 mg over 2 hours) showed a post treatment 
immediate improvement in gas exchange and/or hemodynamics [P20-04704]. The authors 
concluded that thrombolysis improved alveolar ventilation by restoring blood flow to 
previously occluded regions.  
Results of phase II (Part 1)
Efficacy
Observed data from Phase II (Part 1), in line with the dose-finding objective, demonstrated 
the alteplase 0.6/0.04 mg/kg dose in addition to SOC was associated with faster clinical 
improvement or hospital discharge, compared with the alteplase 0.3/0.02 mg/kg + SOC, or 
SOC alone treatments.
Data from patients in the alteplase 0.6/0.04 mg/kg group show numerically more favourable 
outcomes of clinical improvement of ≥2 points on the 11-point WHO Clinical Progression 


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Scale or hospital discharge by Day 28 than those in the SOC group (adjusted HR 2.04 [95% 
CI 0.83 to 5.01], p=0.1183). The alteplase 0.6/0.04 mg/kg group had a median time of 19 
days to clinical improvement or hospital discharge; the median was not achieved in the 
alteplase 0.3/0.02 mg/kg or the SOC groups. No difference on the primary endpoint was 
observed between the alteplase 0.3/0.02 mg/kg and the SOC groups.  
The favorable results on the primary endpoint in the alteplase 0.6/0.04 mg/kg dosing group 
were supported by similar trends for other prespecified analyses, including sensitivity 
analyses of the primary endpoint, as well as secondary efficacy endpoints of lower all-cause 
mortality and lower all-cause mortality or mechanical ventilation up to Day 28 compared 
with the SOC group, greater improvement in median PaO2/FiO2 ratio from baseline to Day 
6 and more ventilator-free days from the start of treatment to Day 28 than in the alteplase 
0.3/0.02 mg/kg and SOC groups. 
A Kaplan-Meier estimate of the unadjusted primary analysis showed a separation of the
alteplase 0.6/0.04 mg/kg group compared with the other two treatment groups, beginning at
approximately Day 11. The risk difference for the alteplase 0.6/0.04 mg/kg group compared
with the SOC group at Day 28 was 19.1% in favour of alteplase (95% CI -10.6%, 48.8%).
Secondary and further endpoint results supported the clinical benefit of alteplase in addition 
to SOC, particularly for the alteplase 0.6/0.04 mg/kg group. Both alteplase groups had 
numerically fewer cases of all-cause mortality than in the SOC group at Day 28. All-cause 
mortality or mechanical ventilation at Day 28 was considered as treatment failure; both 
alteplase groups had fewer cases of all-cause mortality or mechanical ventilation at Day 28 
than the SOC group. The alteplase 0.6/0.04 mg/kg group had more ventilator-free days from 
the start of treatment to Day 28 than the alteplase 0.3/0.02 mg/kg and SOC groups. In 
addition, the alteplase 0.6/0.04 mg/kg group had a greater improvement in median PaO2/FiO2
ratio from baseline to Day 6 compared with the alteplase 0.3/0.02 mg/kg or SOC group.. 
 
 
 
 
 


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Safety 
The overall safety results were generally comparable across the treatment groups. Patients 
treated with alteplase and those receiving only standard of care had similar frequencies of any 
adverse events (AEs), severe AEs, and serious AEs. The majority of reported AEs were of 
mild or moderate intensity. No new safety signals were identified. 
The most common AEs by system organ class were: respiratory, thoracic and mediastinal
disorders (alteplase 0.3/0.02 mg/kg: 7 patients/35.0%, alteplase 0.6/0.04 mg/kg:
12 patients/60.0%, SOC: 9 patients/40.9%), infections and infestations (9 patients/45.0%,
4 patients/20.0%, 11 patients/50.0%), and general disorders and administration site conditions
(7 patients/35.0%, 7 patients/35.0%, 4 patients/18.2%).
Serious AEs reported for >2 patients in a treatment group were respiratory failure (alteplase
0.3/0.02 mg/kg: 2 patients/10.0%, alteplase 0.6/0.04 mg/kg: 0 patients, SOC:
6 patients/27.3%), pulmonary embolism (2 patients/10.0%, 3 patients/15.0%, 1 patient/4.5%),
septic shock (1 patient/5.0%, 0 patients, 4 patients/18.2%), and epistaxis (3 patients/15.0%,
1 patient/5.0%, 0 patients).
Treatment-emergent bleeding events were more frequent in the alteplase treatment groups, 
both for any bleeding events and major bleeding events. Major bleeding events up to Day 6 
were reported only in the alteplase treatment groups (1 patient in the 0.3/0.02 mg/kg dosing 
group and 4 patients in the 0.6/0.04 mg/kg dosing group). Most bleeding events until Day 12 
were non-major (4 patients, alteplase 0.3/0.02 mg/kg; 9 patients, alteplase 0.6/0.04 mg/kg; 3 
patients, SOC group). There were no fatal bleeding events.
The study results of Part 1 support the continuation of the study into Phase III (Part 2), 
implementing a comparison of the alteplase 0.6/0.04 mg/kg dose versus SOC alone.
1.2
DRUG PROFILE
Alteplase is a tissue plasminogen activator produced by recombinant DNA technology. 
Alteplase is a serine protease consisting of 527 amino acid residues with a molecular weight 
of 65 kDa. It shows extensive homology in primary structure with urokinase and has a 
catalytic site homologous with trypsin and other serine proteases. It contains an 
aminoterminal region that has a high degree of sequence homology with the "kringle" 
regions of plasminogen [P96-2358] and prothrombin. A kringle is a characteristic triple 
disulfide structure originally described in the "pro" fragment of prothrombin [R96-2065]. 
The aminoterminal region is responsible for the fibrin-specific activation of alteplase. The 
carboxyterminal end of the molecule contains a domain responsible for the protease activity 


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of alteplase. The primary pharmacological effect of alteplase is activation of plasminogen by 
converting it to plasmin. This occurs extremely efficiently in the presence of fibrin. Fibrin 
has been shown to reduce the apparent Michaelis constant (Km) for plasminogen activation 
by about 8-fold [P90-53645]. When administered into the systemic circulation at therapeutic 
concentrations, alteplase binds to fibrin in a thrombus. Subsequent conversion of 
fibrin-bound plasminogen to plasmin by alteplase results in the dissolution of thrombi.
Mode of action
Simplified, alteplase binds to fibrin clots and activates plasminogen, leading to the generation 
of plasmin and to the degradation of fibrin clots or blood coagulates. For a more detailed 
description of the alteplase profile, please refer to the current Investigator’s Brochure (IB).
Key pharmacokinetic characteristics
The pharmacokinetic profile of alteplase after intravenous administration is well documented: 
alteplase is cleared rapidly from the circulating blood and metabolised mainly by the liver 
(plasma clearance 550 - 680 mL/min). The relevant plasma half-life t1/2α is 4 – 5 minutes. 
This means that after 20 minutes less than 10% of the initial value is present in the plasma 
[U87-0935, U85-0697, U86-0354, U87-0368, P89-49823]. Alteplase w
as shown to have a high clearance rate and a removal from the circulation by specific uptake 
and degradation in the liver. The volume of distribution is confined to the blood volume and 
well-perfused tissues [P85-16126].
Drug interactions
No formal interaction studies with alteplase and medicinal products commonly administered 
in patients with acute myocardial infarction (MI) have been performed.
The risk of haemorrhage is increased if coumarine derivatives, oral anticoagulants, platelet
aggregation inhibitors, unfractionated heparin or LMWH or active substances which interfere 
with coagulation are co-administered (before, during or within the first 24 hours after 
treatment with alteplase). Concomitant use of glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists 
increases the risk of bleeding.
Concomitant treatment with angiotensin-converting enzyme (ACE) inhibitors may enhance 
the risk of suffering a hypersensitivity reaction.
Residual effect period
A single dose of 0.25 mg/kg alteplase (approximately 20 mg) infused over 30 minutes in 
healthy volunteers had no effect on coagulation parameters, indicative that systemic 
fibrinogenolysis was absent. This is consistent with the fibrin specificity and physiologic 
function of native alteplase [P89-49823]. For the residual amount remaining in deep 
compartments, a beta-half-life of about 40 minutes was measured.
The residual effect period (REP) of alteplase for this trial is defined as 7 days. This is the 
period after the last dose where pharmacodynamic effects are likely be present.


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Data from non-clinical studies
For a more detailed description of the alteplase profile, please refer to the current 
Investigator’s Brochure (IB) and to the Summary of Product Characteristics (SmPC)/local 
prescribing information.
Data from clinical studies
Alteplase is a well-established drug, first authorized in Europe in June 1987. It has been 
authorized worldwide in more than 100 countries.
Alteplase is currently indicated for the thrombolytic treatment of acute MI, acute massive PE
with haemodynamic instability and acute ischemic stroke. 
In a study including more than 40,000 patients with an acute MI (GUSTO) the administration 
of alteplase 100 mg over 90 minutes, with concomitant intravenous heparin infusion, led to a 
lower mortality after 30 days (6.3%) compared with the administration of streptokinase
(1.5 million units over 60 minutes) with concomitant use of subcutaneous or intravenous 
heparin (7.3%) [P93-77527].
The results of ECASS III show a positive net-clinical benefit for alteplase in patients with 
acute ischemic stroke in the 3 to 4.5-hour time window [P08-12177]. Treatment 
administration in the ECASS III study was in line with the European SmPC for alteplase in 
its stroke indication. Further, pooled data demonstrating that the net-clinical benefit is no 
longer favorable for alteplase in the time window beyond 4.5 hours.
In patients with acute massive PE with haemodynamic instability thrombolytic treatment with 
alteplase leads to a fast reduction of the thrombus size and a reduction of pulmonary artery 
pressure. The approved alteplase dosage in haemodynamic-relevant pulmonary embolism is
10 mg as bolus followed by 90 mg over 2 hours (i.e. 100 mg in total given over 2 hours).
For a more detailed description of the alteplase profile, please refer to the current IB and for
to the SmPC.
1.3
RATIONALE FOR PERFORMING THE TRIAL
The current study is needed to appropriately clinically evaluate the efficacy and safety of 
adding intravenous alteplase to standard medical care for patients with ARDS associated with 
COVID-19, and to determine the appropriate dose. 
As an established thrombolytic therapy, alteplase may offer benefit for patients with ARDS 
associated with COVID-19 by targeting the fibrinolytic system [R20-1760]. Currently, such 
severely ill patients lack effective treatment besides supportive care. 
The study results of Phase II of the current trial (Part 1) provided proof of concept and 
support the continuation of the study into Phase III (Part 2), the confirmatory part of the 
study, implementing a comparison of the alteplase 0.6/0.04 mg/kg dose versus SOC alone.


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The current study is designed to enroll a study population that is in line with patients with 
ARDS, due to COVID-19, who are presently only receiving standard of care, i.e. supportive 
care. The study eligibility criteria, including age range, is anticipated to enroll a study 
population of severely ill patients with ARDS who have the capacity to benefit from study 
treatment.
In order to be able to address future scientific questions, patients will be asked to voluntarily 
donate samples for pharmacokinetic measurements in both parts of the trial (please see 
Section 5.3.1). 
1.4
BENEFIT - RISK ASSESSMENT
1.4.1
Benefits
Alteplase is a currently approved, established fibrinolytic agent with a well-known safety and 
efficacy profile. Extensive experience with alteplase has been obtained over the past 30 years. 
Alteplase is licensed for the thrombolytic treatment of acute MI, massive PE with 
haemodynamic instability, and acute ischaemic stroke. In the current study, alteplase is 
expected to beneficially address the thrombogenic state and compromised respiratory status 
associated with ARDS due to COVID-19. Alteplase can be administered in a regimen and 
approach that is suitable to address these clinical issues as well as to mitigate risks.
Dissolving extensive microvasculature thrombosis with potent and fast acting fibrinolytic 
substances such as alteplase is likely to be highly beneficial for patients with ARDS due to 
COVID-19, leading to improved oxygen exchange and end organ perfusion after alteplase 
application. Pre-clinical and previous clinical trial data in patients with ARDS suggest a 
positive effect of fibrinolytic compounds by improving oxygen exchange in these patients
[P00-10646, P13-04526, R20-1656, P12-04032, R20-1650, R20-1934]. Current pathology 
findings from deceased COVID-19 patients with ARDS show profound thrombotic 
obliterations of mainly (but not exclusively) end stream vasculature in lungs and other organs
[R20-1649, P20-04704, P20-04176, R20-1851 , R20-1941 , R20-1804, R20-1852].
Additionally, histopathological evidence of widespread fibrin deposits in alveoli have been 
observed in these patients. Microvascular thrombosis in lung capillaries and fibrin deposits in 
the alveoli are considered to play a major role in the deterioration of lung function and 
potentially right ventricular function in patients with ARDS due to COVID-19. Impaired 
oxygen exchange in the alveoli (either affected by capillary thrombosis or fibrin deposition
into alveoli) are key pathological processes, which make high flow oxygen support or even 
mechanical ventilation necessary in severely affected patients with ARDS due to COVID-19. 
An additional complexity in severely affected patients with ARDS due to COVID-19 appears 
to be a prolonged thrombogenic status during active infection, which may lead to re-
thrombosis and/or ongoing microvascular thrombosis contributing to associated organ 
dysfunctions. This prolonged thrombogenic status resulting in thrombosis-related organ 
dysfunction seems to be difficult to address with parenteral anticoagulants such as UFH or 
LMWH alone. UFH and LMWH will prevent or at least slow down the progression of (new) 
thrombus formation by its anticoagulant activity. UFH or LMWH will not directly dissolve 


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existing thrombi. Adding alteplase to UFH or LMWH will additionally directly and 
immediately dissolve newly formed thrombi, which are re-occluding the microvasculature 
despite UFH or LMWH treatment. Since alteplase has a short half-life of maximal 
40 minutes, a bolus application will only be effective and beneficial for a short time point.
Low dose constant rate infusion of alteplase is considered to be a dose regimen which
addresses on a time-as-needed basis the ongoing re-formation of microthrombi during active
SARS-CoV-2 infection [U95-2167, U99-1584]. The combination of an initial 2 hours
infusion of alteplase 0.3 mg/kg over 2 hours or 0.6 mg/kg over 2 hours combined with a 
constant rate infusion (CRI) of low dose alteplase 0.02 to 0.04 mg/kg/h over 12 hours is
believed to dissolve the present thrombotic obstructions and the newly formed microthrombi 
in patients with ARDS due to COVID-19. The results of Phase II of the current trial showed 
that patients receiving a dose of 0.6 mg/kg over 2 hours immediately followed by a constant 
rate infusion of alteplase 0.04 mg/kg/h over 12 hours (up to 5 days) in addition to SOC was 
associated with faster clinical improvement or hospital discharge, compared with patients 
receiving alteplase 0.3/0.02 mg/kg on top of SOC, or SOC alone. The study results of Phase 
II (Part 1), support the continuation of the study into Phase III (Part 2), the confimartory part 
of the study.
Alteplase can be given in combination with UFH or LMWH. This is routinely performed
when applying treatment to acute MI patients or acute PE patients.
During the alteplase treatment period of up to a maximum of 5 days (plus a possible
extension in case of unavoidable interruptions of up to 72 hours) the concomitant use of 
LMWH in Part 2 will be limited to prophylactic doses and the use of UFH will be limited to 
target levels of aPTT between 1.0 to 1.5 fold ULN according to local laboratory.
Treating patients with a moderate initial dose of 0.6 mg/kg over 2 hours (for example, this 
corresponds to 42 mg in a patient with 70 kg of body weight) combined with a constant rate 
infusion (CRI) of low dose alteplase of 0.04 mg/kg/h over 12 hours is considered to reduce 
the risk of alteplase-related bleeding events in comparison to application of high alteplase
doses (up to 100 mg) used in other indications (e.g. massive pulmonary embolism with 
hemodynamic instability, acute ischemic stroke, acute myocardial infarction). Also a regular 
temporary interruption of CRI over days is believed to reduce the bleeding risk by allowing 
the body to regenerate its plasma fibrinogen levels.
The risk of bleeding may be further mitigated because these patients are under close 
surveillance. Blood pressure, glucose levels, plasma fibrinogen levels are being closely
monitored and intervention immediately applied in this setting as standard of care. Because 
alteplase has a very short half-life, interrupting the CRI will have an immediate effect on 
fibrinolysis. Bleeding is addressed rapidly either surgically or medically; e.g. with fresh 
frozen plasma (FFP), fibrinogen concentrate, or including i.v. tranexamic acid may be 
considered. 
While previous historical studies evaluating fibrinolytic therapy for treatment of ARDS used 
urokinase and streptokinase, the more contemporary approach to thrombolytic therapy 
involves the use of alteplase due to higher efficacy of clot lysis with comparable bleeding 
risk. This trial hypothesizes that administration of alteplase as a short-term infusion (0.3 


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mg/kg over 2 hours or 0.6 mg/kg over 2 hours) plus low-dose long-term infusion for patients 
with COVID-19-related ARDS will improve pulmonary gas exchange and oxygenation via a 
decrease in pulmonary vascular microthrombi and lysis of fibrin in alveoli.
There is emerging evidence that patients with COVID-19-related ARDS have pulmonary 
thrombi in segmental and sub segmental vessels. Alteplase is proven to have a higher efficacy 
of clot lysis in arterial and venous thrombosis and might improve ventilation capacity 
(monitored by PaO2/FiO2 ratio) of COVID-19 related ARDS.
COVID-19 ARDS patients appear to have a strong prothrombotic tendency and have a 
relative resistance to the action of anticoagulants. This condition is worsened due to an 
impairment of fibrinolysis which is addressed by the use of alteplase. While alteplase does 
not prevent recurrence of thrombosis, the concomitant use of anticoagulation is intended to 
further prevent formation of new micro- or macroclots.
1.4.2
Risks
Bleeding events
Fibrinolytic treatment always harbours the risk of bleeding events. Depending on the 
anatomical location and severity of bleeding, the bleeding can be life threatening or even turn 
out fatal. The bleeding risk is potentially augmented when combining alteplase with 
anticoagulants or platelet inhibitor due to the combination of fibrinolysis, anticoagulation 
and/or platelet inhibition. The bleeding risk of alteplase also differs in relation to dose and 
rate. High doses of alteplase given at high rates increases bleeding risk. Low levels of 
fibrinogen (<100 mg/dL) have also been identified to increase bleeding risk in relation to 
alteplase. 
The bleeding risk can be partially estimated from the plasma level of fibrinogen. Due to its 
relative fibrin-specificity, alteplase results in a modest decrease of the circulating plasma 
fibrinogen levels to about 60% at 4 hours, which is generally reverted to more than 80% after 
24 hours. Plasminogen and alpha-2-antiplasmin decrease to about 20% and 35% respectively 
after 4 hours and increase again to more than 80% at 24 hours [P86-5233]. A marked and 
prolonged decrease of the circulating fibrinogen level is only seen in few patients [P89-
46753]. A plasma level of fibrinogen below 150 mg/dL has been associated with an elevated 
risk of bleeding [P13-13102]. The bleeding risk can be minimized by intensive monitoring of 
plasma fibrinogen levels and establishing stopping criteria or by dose reduction. 
Vasculature health of the patient seem to play a major role in the bleeding risk. Confounding 
conditions affecting the vasculature health include high blood pressure, T2DM and advanced 
age. These conditions are considered patient-related risk factors for bleeding. 
Alteplase administered intravenously carries a risk of bleeding and the extent of this risk is 
multifactorial (e.g. dose, weight, age, gender, blood pressure, diabetes) [P15-13160]. The 
highest number of bleeding events were seen in trials involving acute ischemic stroke
patients, where the reported complications with alteplase total doses (up to 90 mg) given over 


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60 minutes were symptomatic intracerebral haemorrhage (6%) and other major haemorrhage 
(2%). Minor haemorrhage (e.g. bleeding at site of venepuncture) accounting for 20-25%. 
Similarly, in patients with acute MI where alteplase doses (up to 100 mg) given over 90 min
were applied, the risk of intracranial haemorrhage is <1%. In multivariable models the main 
risk factors were older age, female sex, black ethnicity, systolic blood pressure (BP) of 140 
mm Hg or above, diastolic blood pressure of 100 mm Hg or above, history of stroke, 
alteplase dose of more than 1.5 mg/kg, and lower body weight [P98-10462]. 
In the Phase II part of the current trial, no new safety signals were identified. Major bleeding 
events until Day 6 were overall infrequent (1 patient in the 0.3/0.02 mg/kg dosing group and 
3 patients in the 0.6/0.04 mg/kg dosing group), and a trend towards a dose-dependent 
increase was mainly observed for non-major bleeding events.
Mitigation strategies to prevent bleeding events in Phase III (Part 2) of the current study are 
implemented in Section 3.3.4.1.   
Hypersensitivity
Further known risks of alteplase are hypersensitivity reactions. These can occur at any time
and are not predictable. If detected at an early stage, any form of hypersensitivity reaction 
(anaphylaxis, anaphylactic reactions like angioedema) can be effectively treated with 
medication such as adrenaline/epinephrine, glucocorticoids, antihistamines, complement 
inhibitor, bradykinin antagonists and fluids. 
Effect of Alteplase on D-Dimer and fibrinogen
In patients with acute myocardial infarction (AMI) or deep vein thrombosis (DVT), D-dimer 
and fibrin degradation products significantly increased during administration of alteplase, 
while fibrinogen and plasminogen concentrations significantly decreased compared to 
baseline [P90-52475, P91-62566]. The increase in D-dimer levels was correlated with 
thrombus mass and can be considered as indicator for efficient thrombolytic treatment in
AMI and DVT patients [P90-52475, P92-71704].
Overview of trial-related risks
An overview of trial-related risks is provided in Table 1.4.2:1.


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Table 1.4.2:1
Overview of trial-related risks
Possible or known risks of 
clinical relevance for this 
trial
Summary of data, rationale 
for the risk
Mitigation strategy
Investigational Medicinal Product / alteplase
Cardiovascular: 
hypotension, bradycardia
  GI: nausea, vomiting,
GI tract bleeding
Haematologic: spontaneous 
bleeding, bone marrow 
depression
Musculoskeletal: 
musculoskeletal pain
Respiratory: Pulmonary 
edema
Skin: bruising, flushing
Other: fever, edema, 
phlebitis  
More detailed description 
see IB, Section 7.6
Timely detection,
evaluation, and follow-up of 
laboratory alterations in 
selected laboratory 
parameters to ensure 
patients´ safety. ECG 
monitoring in ICU, repeated 
ECGs. Monitoring for 
bleeding including 
neurological assessment to 
rule out intracranial 
haemorrhage. Monitor 
hypersensitivity reactions 
such as angioedema, 
profound hypotension, rash,
urticaria. Supportive 
treatment for bleeding 
including infusion therapy, 
red blood cell (RBC) and 
FFP transfusion. In case of 
hypersensitivity 
glucocorticoids, 
antihistamines, and 
adrenaline (epinephrine)
address acute 
hypersensitivity. Intubation 
readiness


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Table 1.4.2:1
Overview of trial-related risks (cont)
Clinical events
Minor bleeding particularly 
traumatic –at site of 
venepuncture
Supportive treatment, and 
use caution during suction,
measurement of plasma 
fibrinogen levels
Major bleeding including 
intracerebral haemorrhage
or bleeding from a critical 
organ
Common
Measurement of plasma 
fibrinogen levels, 
recommendation to stop
alteplase infusion, and 
administer fibrinogen 
concentrate or 
cryoprecipitate, if 
fibrinogen is <150 mg/dL
and other supportive 
treatment including i.v.
tranexamic acid should be 
considered  
Hypersensitivity reactions 
(e.g. rash, urticaria, 
bronchospasm, angio-
oedema, hypotension, 
shock).
Rare 
Anti-allergic medications  
Serious anaphylaxis
Very rare
Treat per local protocols 
and stop further 
administration of 
medication
Other risks
Additional blood samples
Bleeding related to 
venipuncture
Compression
Transfusion of blood 
products, fibrinogen or 
supportive treatment like 
i.v. tranexamic acid.
Related to bleeding event.
Anaphylaxis due to 
transfusion.
Treatment as per local 
protocols and product 
information


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Risk evaluation in relation to COVID-19:
Patients participating in this trial are at higher risk for severe consequences resulting from 
COVID-19. In the event of restriction to visit the investigative site (for patients after 
discharge) and in case a visit to the hospital is not possible for an individual patient, certain 
procedures can be done remotely, and local labs obtained from a laboratory located outside 
the hospital can be used instead of the hospital laboratory. These changes are meant to keep 
the integrity of the trial and they will not affect the benefit-risk for the individual patient.
1.4.3
Discussion
Consideration of therapies that are not recognized for this indication but of potential benefit, 
such as fibrinolytic agents, is warranted in this unprecedented public health emergency of 
COVID-19. 
The bleeding risk described in the literature typically include high dosages of alteplase (up to 
90 to 100 mg given as infusion over 1-3 hours) and the concurrent use of therapeutic 
antithrombotics. However, the alteplase short-term infusion dosages being used in this study 
(0.3 mg/kg/2h or 0.6 mg/kg/2h) are lower than those used in studies for MI, massive PE and 
stroke. Additionally, by the low dose during long-term infusion over 12 hours, the rapid 
inactivation of alteplase in the circulation is projected to be associated with plasma fibrinogen 
levels well above the threshold of 150 mg/dL, which are known to be linked to the risk 
bleeding c32479735-01; P13-13102 . The safety results from Phase II (part 1) of the current 
trial were generally comparable across the treatment groups, except for bleeding events,
which are a known side effect of alteplase. In Phase II, mainly non-major bleeding events 
were observed, and some of them were related to interventions. Additional risk minimization 
strategies to avoid intervention-related bleeding events have accordingly been implemented 
for Part 2, phase III of the study.
The bleeding risk is predicted from the plasma fibrinogen level which provides a measure of 
the alteplase absorbed. This will be monitored and if the level falls below 150 mg/dL,
alteplase has to be temporarily interrupted. Beside the extent of fibrinogen breakdown, D-
Dimer levels, thrombocytopenia, the prolongation of the activated partial thromboplastin time 
(aPTT) will be monitored continuously. Furthermore, elevated blood pressure as a risk of 
intracranial hemorrhage and the co-administration of ACE inhibitors related to the risk of 
angioedema will be monitored.
Taken together, the concept of combining a moderate initial dose of alteplase applied over 
2 hours followed by long-term low dose infusion to COVID-19 related patients with ARDS is
considered to have a favorable benefit-risk ratio. The expected benefit is an improved 
pulmonary oxygenation via lysis of pulmonary vascular microthrombi and fibrin deposition 
in the alveoli. The risk of bleeding is considered acceptable in this severely ill population and 
will be monitored by measurements of fibrinogen and coagulation parameters. The study
results of Phase II (Part 1), support the continuation of the study into Phase III (Part 2), the 
confirmatory part of the study. The alteplase 0.6/0.04 mg/kg dose in addition to SOC was 
associated with faster clinical improvement 
, compared with the 


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alteplase 0.3/0.02 mg/kg + SOC, or SOC alone treatments.
 
. The safety results were 
generally comparable across the treatment groups. No new safety signals were identified.
Major bleeding events were overall infrequent, and a trend towards a dose-dependent 
increase was mainly observed for non-major bleeding events.
Precautionary recommendations for temporary interruption of alteplase infusion (up to 72
hours in total for urgent reasons, bleeding or drop in fibrinogen), a limitation of concomitant 
administration of heparins to prophylactic doses and recommendations to avoid intervention-
related bleeding events are implemented in Part 2 to mitigate the risk of bleeding. 
Taken together, the overall benefit-risk ratio of alteplase at the studied dosing regimen is 
considered favourable.


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2.
TRIAL OBJECTIVES AND ENDPOINTS
2.1
MAIN OBJECTIVES, PRIMARY AND SECONDARY ENDPOINTS
2.1.1
Main objectives
Main objective is to evaluate the efficacy and safety of two (Part 1) different dosing regimen
and of one dosing regimen (Part 2) of intravenous alteplase given for up to 5 days on top of 
standard of care (SOC) compared with SOC alone in ARDS associated with COVID-19.
SOC includes supportive measures, such as the use of non-invasive or invasive ventilation, 
haemodynamic support, if needed, sedation, as well as medical therapies commonly used in 
patients suffering from ARDS or its complications. SOC follows the standard therapies
established locally.
2.1.2
Primary endpoint
The same primary endpoint will be evaluated in Part 1 and in Part 2, as follows: 

Time to clinical improvement or hospital discharge up to Day 28, defined as the 
time from randomisation to either an improvement of two points on the 11-point 
WHO Clinical Progression Scale or discharge from the hospital, whichever comes 
first. See Section 5.1.1.
2.1.3
Secondary endpoints
The following secondary endpoints to assess safety and efficacy in this setting have been 
evaluated in Part 1: 

All cause mortality at Day 28

Number of ventilator-free days from start of treatment to Day 28

Improvement of Sequential (sepsis-related) Organ Failure Assessment (SOFA) score 
by ≥2 points from baseline to end of Day 6

Major bleeding events (MBE) (according to International Society on Thrombosis and 
Haemostasis [ISTH] definitionR05-0344until Day 6, see Section 5.2.5.1.

Daily average PaO2/FiO2 ratio (or inferred PaO2/FiO2 ratio from SpO2) change from 
baseline to Day 6

All-cause mortality or on mechanical ventilation at Day 28
In Part 2, the following key secondary endpoints will be evaluated:

Treatment failure defined as all cause mortality or mechanical ventilation at Day 28

All cause mortality at Day 28
In Part 2, the following secondary endpoints will be evaluated:


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
Number of oxygen-free days up to Day 28

Length of hospital stay up to Day 28

Major bleeding events (MBE) (according to International Society on Thrombosis and 
Haemostasis [ISTH] definition R05-0344) until Day 6, see Section 5.2.5.1.

PaO2/FiO2 ratio (or inferred PaO2/FiO2 ratio from SpO2) change from baseline to 
Day 6 


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3.
DESCRIPTION OF DESIGN AND TRIAL POPULATION
3.1
OVERALL TRIAL DESIGN 
This is an open-label operationally seamless
Phase
IIb/III randomised, sequential,
parallel-group adaptive clinical trial in patients experiencing ARDS triggered by COVID-19, 
comparing daily intravenous infusion of alteplase, up to a maximum treatment duration of 5 
days on top of SOC, versus SOC alone. This trial will investigate the efficacy and safety of 
different dosing regimens in hospitalized patients with ARDS of mild to moderate severity.
The study comprises two parts in an operationally seamless sequential adaptive design: Part 1 
(dose-finding, “proof of concept”, Phase IIb) with approximately 60 patients using a 1:1:1 
randomisation scheme to two dose regimens of alteplase treatment on top of SOC, or SOC 
alone; and Part 2 (confirmatory, Phase III) with approximately 260 patients carrying one 
dosing regimen forward and using a 2:1 randomisation to alteplase treatment on top of SOC, 
or SOC alone. 
The two parts include the same patient population and have same primary endpoint. Visit 
schedules are the same in both parts. There will be one interim assessment after Part 1.
One dosing regimen of alteplase (0.6/0.04 mg/kg dosing, see Section 4.1.1) has been selected 
for Part 2, based on the benefit-risk profile observed in Part 1.
To minimize the bias, eligible patients will be randomised using Interactive Response 
Technology (IRT). The randomisation list will be generated using a validated system, which 
involves a pseudo-random number generator so that the resulting treatment will be 
reproducible and non-predictable. The block size will be documented in the CTR. Access to 
the codes will be controlled and documented.
Randomisation in Part 1 will be 1:1:1 to one of the three arms:

initial i.v. infusion of alteplase 0.3 mg/kg/2h (Day 1) followed by daily i.v. long-term 
infusion of 0.02 mg/kg over 12 hours (Dosing regimen A), 

initial i.v. infusion of alteplase 0.6mg/kg/2h (Day 1) followed by daily i.v. long-term 
infusion of 0.04 mg/kg/h over 12 hours (Dosing regimen B) 

or SOC.
Randomisation will be stratified by type of ventilation (non-invasive or invasive mechanical).
Randomisation in Part 2 will be 2:1 to dosing regimen B of alteplase (initial i.v. infusion of 
alteplase 0.6mg/kg/2h (Day 1)) followed by daily i.v. long-term infusion of 0.04 mg/kg/h 
over 12 hours or to standard of care, and will be stratified by type of ventilation (non-invasive 
or invasive mechanical) and by D-dimer levels (≥ULN to <5-fold ULN, versus ≥5-fold 
ULN). 
Recruitment of patients on mechanical ventilation will be stopped when a maximum of 50 
patients have been randomized in Part 2. Randomisation for the whole trial will be stopped 
when approximately 210 NIV patients have been randomized in Part 2, regardless of how 
many IMV patients have been randomised so far. 


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This is an open-label study; therefore no blinding will apply for patients, investigators and 
personnel at the site involved in trial conduct. More details please see Section 4.1.5.1.
A schematic overview of trial design is shown in (Figure 3.1:1):
Figure 3.1:1 Study design
3.2
DISCUSSION OF TRIAL DESIGN, INCLUDING THE CHOICE OF 
CONTROL GROUP(S)
Operationally seamless trial design, with separate proof-of-concept part (Part 1, Phase IIb) 
and confirmation of efficacy and safety part (Part 2, Phase III). The trial will include pre-
defined decision criteria for selecting one alteplase dosing regimen for Part 2, with the intent 
of ensuring that a positive proof-of-concept will quickly be followed by the confirmation of 
efficacy and safety of alteplase in patients with ARDS. As Part 1 results indicated the need 
for some design modifications in Part 2 beyond those pre-specified in the protocol, these
required modifications have been implemented via the current global CTP amendment. 
An additional advantage of the operationally seamless adaptive design for the trial sites is 
mainly to gain experience with the protocol measures. The anticipated short duration between 
the end of Part 1 and the start of Part 2 will reduce the risk of potential measurement errors 
and protocol deviations in Part 2, the confirmatory part of the trial. The overall expectation is 
an increase in site efficiency and study quality. In addition, the Part 1 experience with 
protocol measures is expected to lead to a reduction in the variability associated with efficacy 
measurements thereby increasing the precision of treatment estimates particularly in the 
confirmatory part (Part 2) and to ensure that safety assessments are as objective as possible.


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Finally, the accelerated recruitment compared with two separate trials may reduce time to 
availability of an effective treatment filling unmet medical needs in patients with ARDS. 
A parallel group, sequential, randomized, open label trial was considered most appropriate to 
demonstrate proof of concept of alteplase in Part 1 in patients with COVID-19-induced 
ARDS. The SOC control group in Parts 1 and 2 is thus required to compare both efficacy and 
safety with the alteplase treatment arm. Sites are expected to ensure that patients are provided 
with best possible standard of care for the treatment of ARDS according to local guidelines.
As an established thrombolytic therapy, alteplase may offer benefit for patients with ARDS, 
associated with COVID-19, by targeting the coagulation and fibrinolytic systems [R20-1760]. 
Currently, such severely ill patients lack effective treatment besides supportive care.
In order to ensure the patient’s safety during the trial, a fully external data monitoring 
committee (DMC), independent of the trial and project teams, will be set-up to review all 
available un-blinded safety data as well as selected efficacy data at regular intervals 
following first patient in (FPI) for Part 1 and Part 2, respectively. The role of DMC is given 
below, further details including pre-specified rules for decision-making, will be provided in a
DMC charter.
The DMC`s role is to monitor patients` safety continuously and to put it in context with 
observed treatment benefits. The DMC may provide recommendations to the Sponsor and the 
Expert Board on the following: (1) Changes in dosing (e.g. change in dose or timing of 
infusion schedule, changing criteria for treatment interruption, concomitant or restricted 
medication, potential closure of one dose group in Part 1, additional safety measures to be 
implemented), (2) Changes in population selection (e.g. exclude select patients based on data 
generated from the trial, modify in-/exclusion criteria), (3) Termination of the trial in case of 
serious safety concerns, (4) Stop the trial for futility, (5) Any other measure that affects
patients` safety. After Part 1, the DMC has provided a recommendation to continue into Part 
2, together with a recommendation to select dosing regimen B for Part 2. For further details,
see the DMC Charter and the DMC recommendation after completion of Part 1.
3.3
SELECTION OF TRIAL POPULATION
Approximately 60 patients are planned to be randomised in Part 1 at approximately 30 to 
35 study sites; each site is expected to randomise approximately 2 patients. In Part 2, there 
are approximately 260 patients planned to be randomised at approximately 110-130 sites and 
approximately 2-3 patients per site are planned to be randomised per site in Part 2. The 
majority of sites in Part 1 are located in 10 countries in Western Europe, but if recruitment 
numbers are not achieved or the incidence of new COVID-19 cases shifts to other regions of 
the world, the trial may expand to Eastern Europe, Russia, China, India, Africa, South 
America and other countries to focus on places where the incidence of COVID-19 is high and 
vaccination rate is slow. Approximately 22 countries are planned for Part 2. Given the 
expected regional fluctuation in SARS-CoV-2 infection rates, it may become necessary to 
open new countries and sites and to potentially suspend existing sites, or countries.


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Screening of patients for this trial is competitive, i.e. screening for the trial will stop at all 
sites at the same time once a sufficient number of patients has been screened. Investigators 
will be notified about screening completion and will then not be allowed to screen additional 
patients for this trial. This applies to both parts of the study.
In part 2 of the trial, a randomisation cap will be applied for patients on mechanical 
ventilation at baseline: when 50 patients on mechanical ventilation have been randomized. 
Recruitment of further patients on mechanical ventilation will be stopped via IRT.
A log of all patients enrolled into the trial (i.e. who have signed informed consent) will be 
maintained in the Investigator Site File (ISF) irrespective of whether they have been treated 
with investigational drug or not.
If a patient is enrolled in error (does not meet all inclusion criteria or meets one or more 
exclusion criteria on the day of enrolment), the sponsor should be contacted immediately.
Patients will be selected for the trial according to pre-defined eligibility criteria and written 
informed consent has to be obtained prior to initiation of any trial related procedures.
Randomisation of patients shall happen no later than 24 hours after the confirmation of the 
eligibility criteria, and study drug administration will be started within 6 hours of the 
randomisation. The time-point of obtaining D-Dimer levels can be as long as four calendar 
days before screening.
The respiratory status of the enrolled patients is monitored by PaO2/FiO2 ratio (or inferred 
PaO2/FiO2 ratio from SpO2) and should be measured at least three times per day in all 
patients together with a recording of the patient`s position, and all available data points will 
be collected until discharge, death, or Day 28, whichever occurs first. The qualifying  
PaO2/FiO2 ratio should be the worst measurement in the screening period. During the 
treatment period the worst measurement on a given day should be documented in the case 
report form.
The end of trial for an enrolled patient is the date of death or Day 28, whichever occurs first. 
3.3.1
Main diagnosis for trial entry
Please refer to Section_8.3.1 for the documentation requirements pertaining to the eligibility
criteria.
In both parts, Part 1 (proof of concept) and Part 2 (confirmatory), the trial will include 
patients with ARDS due to COVID-19 infection.
3.3.2
Inclusion criteria
Patients participating in Part 1 are not eligible for Part 2.


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1. Age ≥ 18 years (or above legal age)
2. ARDS with PaO2*/FiO2 ratio >100 and ≤300, either on non-invasive ventilator support, 
OR on mechanical ventilation (<48 hours since intubation), 
-
with bilateral opacities in chest X-ray or CT scan (not fully explained by effusions, 
lobar/lung collapse, or nodules)
-
with respiratory failure (not fully explained by cardiac failure/fluid overload)
*or estimation of PaO2/FiO2 from pulse oximetry (SpO2/FiO2) (see Appendix 10.2 for 
SpO2/FiO2 estimation)
3. SARS-CoV-2 positive (laboratory-confirmed RT PCR test)
4. Fibrinogen level ≥ lower limit of normal (according to local laboratory)
5. D-Dimer ≥ upper limit of normal (ULN) according to local laboratory
6. Signed and dated written informed consent in accordance with ICH Good Clinical 
Practice (GCP) and local legislation prior to admission to the trial.
3.3.3
Exclusion criteria
1. Massive, confirmed PE with haemodynamic instability at trial entry, or any (suspected or 
confirmed) PE that is expected to require therapeutic dosages of anticoagulants during the 
treatment period
2. Indication for therapeutic dosages of anticoagulants at trial entry
3. Mechanical ventilation for longer than 48 hours
4. Chronic pulmonary disease i.e. with known FEV1 <50%, requiring home oxygen, or oral 
steroid therapy or hospitalisation for exacerbation within 12 months, or significant
chronic pulmonary disease in the Investigator’s opinion, or primary pulmonary arterial 
hypertension
5. Has a Do-Not-Intubate (DNI) or Do-Not-Resuscitate (DNR) order
6. In the opinion of the investigator not expected to survive for > 48 hours after screening.
7. Planned interventions during the first 5 days after randomisation, such as surgery, 
insertion of central catheter or arterial line, drains, etc.
8. Known hypersensitivity to the active substance alteplase, gentamicin (a trace residue from 
the manufacturing process) or to any of the excipients
9. Significant bleeding disorder at present or within the past 6 months, known haemorrhagic 
diathesis
10. Patients receiving effective oral anticoagulant treatment, e.g. vitamin K antagonists with 
INR >1.3, or any direct oral anticoagulant within the past 48 hours
11. Any history of central nervous system damage (i.e. neoplasm, aneurysm, intracranial or 
spinal surgery)
12. History or evidence or suspicion of intracranial haemorrhage including sub-arachnoid 
haemorrhage
13. Severe uncontrolled arterial hypertension (according to the investigator`s judgement)
14. Major surgery or significant trauma in the past 10 days, recent trauma to head or cranium
15. Cardiac arrest and/or cardiopulmonary resuscitation during the current hospital stay
16. Obstetrical delivery within the past 10 days
17. Severe hepatic dysfunction i.e. Child-Pugh B and C, including biopsy confirmed hepatic 
cirrhosis, portal hypertension, hepatic encephalopathy, or active hepatitis
18. Bacterial endocarditis, pericarditis


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19. Acute pancreatitis
20. Documented ulcerative gastro-intestinal disease during the last 3 months
21. Severe heart failure (New York Heart Association Class IV)
22. Arterial aneurysms, arterial/venous malformations
23. Malignancy (Stage IV) with increased bleeding risk
24. Haemorrhagic stroke or stroke of unknown origin at any time
25. Ischaemic stroke or transient ischaemic attack (TIA) in the preceding 6 months
26. Patients who must or wish to continue the intake of restricted medications (see Section 
4.2.2.1) or any drug considered likely to interfere with the safe conduct of the trial. Re-
screening is allowed once within the same hospital stay.
27. Women who are pregnant. (Note that women of childbearing potential are not excluded as 
eligible patients are severely ill during the observation period of the trial.)
28. End stage renal disease (Stage ≥ 4) or need for renal replacement therapy at baseline
29. Need for ECMO at baseline 
30. Platelet count <100 x 109/L or history of Heparin-Induced Thrombocytopenia 
31. Lumbar puncture with in past 3 days
32. Known abdominal or thoracic aneurysm
33. Active tuberculosis
3.3.4
Withdrawal of patients from treatment or assessments
Patients may discontinue trial treatment or withdraw consent to trial participation as a whole
(“withdrawal of consent”) with very different implications; please see Sections 3.3.4.1 and 
3.3.4.2 below.
Every effort should be made to keep the patients in the trial: if possible on treatment, or at 
least to collect important trial data (e.g. data required for primary and secondary outcome 
measures or at least vital status at planned EOS for the patient). 
Measures to control the withdrawal rate include careful patient selection, appropriate 
explanation of the trial requirements and procedures prior to trial enrolment, as well as the
explanation of the consequences of withdrawal. 
The decision to discontinue trial treatment or withdraw consent to trial participation and the 
reason must be documented in the patient files and eCRF. If applicable, consider the 
requirements for Adverse Event collection reporting (please see Sections 5.2.6.2.1 and 
5.2.6.2).
3.3.4.1
Discontinuation of trial treatment
An individual patient will discontinue trial treatment if:

The patient wants to discontinue trial treatment, without the need to justify the 
decision.

The patient needs to take a concomitant medication that interferes with the 
investigational medicinal product (IMP) or other trial treatment. Please refer to 
Section 4.2.2 for restricted medications during this trial.

The patient can no longer receive trial treatment for medical reasons, such as surgery, 
adverse events, etc. including:


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o
safety reasons, such as active bleeding, or any other condition that is 
considered a safety risk at the discretion of the investigator
o
in case of clinical improvement of respiratory situation with sustained (as per 
investigator`s judgement) PaO2/FiO2 ratio above 300.
o
Need for ECMO or RRT. No additional dose of alteplase 0.3 mg/kg/2 h or 0.6 
mg/kg/2 h (Part 1) or 0.6 mg/kg/2 h in Part 2 should be given under these 
circumstances. 
Recommendation for temporary interruption of alteplase daily infusions:

Interventions (e.g. insertion of lines, catheters, drains and tubes) during the treatment 
period with alteplase should be avoided, whenever possible. If absolutely necessary, 
they need to be performed in the alteplase infusion-free interval (i.e. in the 12-hours
break between two infusions) and ideally not on the first day. 

Alteplase infusion can be resumed at least 6 hours after the intervention. This duration 
can be extended in case of complications during the intervention.

If the fibrinogen level decreases to levels <150 mg/dL. The infusion should be 
restarted when fibrinogen level is 150 mg/dL or higherc32479735-01; P13-13102 

Interruptions for unavoidable interventions, drop in fibrinogen level or bleeding 
(beyond the 12 hours alteplase infusion-free interval per protocol) are allowed for up
to a maximum of 72 hours. The time window of the subsequent visits and infusions 
may be extended accordingly. It is preferable to achieve normal haemostasis 
parameters before the next infusion of alteplase.
If new efficacy/safety information becomes available, BI will review the benefit-risk
assessment and, if needed, pause or discontinue the trial treatment for all patients or take any 
other appropriate action to guarantee the safety of the trial patients. 
Even if the trial treatment is discontinued, the patients remain in the trial and, given their
agreement, will undergo the procedures for early treatment discontinuation and follow-up
until Visit 6/EOS as outlined in the Flow chart and Section 6.2.3.
3.3.4.2
Withdrawal of consent to trial participation
Patients may withdraw their consent to trial participation at any time without the need to 
justify the decision.
If a patient wants to withdraw consent, the investigator should be involved in the discussion 
with the patient and explain the difference between trial treatment discontinuation and 
withdrawal of consent to trial participation, as well as explain the options for continued 
follow-up after trial treatment discontinuation, please see Section 3.3.4.1 above.
For patients who discontinue treatment prematurely, vital status shall be collected at planned 
completion date of each individual patient.


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3.3.4.3
Discontinuation of the trial by the sponsor
Boehringer Ingelheim reserves the right to discontinue the trial overall or at a particular trial
site at any time for the following reasons:
1. Failure to meet expected enrolment goals overall or at a particular trial site.
2. New efficacy or safety information invalidating the earlier positive benefit-risk-
assessment, please see Section 3.3.4.1.
3. Deviations from GCP, the trial protocol, or the contract impairing the appropriate conduct 
of the trial.
Further follow up of patients affected will occur as described in Section 3.3.4.1.
The investigator / the trial site will be reimbursed for reasonable expenses incurred in case of 
trial termination (except in case of the third reason).


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4.
TREATMENTS
4.1
INVESTIGATIONAL TREATMENTS
4.1.1
Identity of the Investigational Medicinal Products
Table 4.1.1:1
Test product 
Substance:
Alteplase (recombinant tissue plasminogen activator)
Pharmaceutical formulation:
Powder and solvent for solution for injection/infusion
Source:
Boehringer Ingelheim Pharma GmbH & Co. KG 
Unit strength:
50 mg/vial powder and 50 mL solvent 
Posology:
Initial i.v. infusion (Day 1 only):
Part 1:

Dosing regimen A: 0.3 mg/kg given over 2 hours

Dosing regimen B: 0.6 mg/kg given over 2 hours
Alteplase 50 mg powder for solution for injection/ 
infusion will be reconstituted in 50 mL water for 
injections to yield a concentration of 1 mg/mL (= stock 
solution).
A Perfusor Syringe of 50 mL should be used.
Dosing regimen A, Initial i.v. infusion (0.3 mg/kg to be
infused over 2 h)
Dosing regimen B, Initial i.v. infusion (0.6 mg/kg to be
infused over 2 h)
Part 2: One dosing regimen is carried forward based on 
results from Part 1 and recommendation by the DMC for 
Part 2.
Dosing regimen B, Initial i.v. infusion (0.6 mg/kg to be
infused over 2 h).


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Table 4.1.1:1
Test product (cont)
Substance:
Alteplase (recombinant tissue plasminogen activator)
Pharmaceutical formulation:
Powder and solvent for solution for injection/infusion
Source:
Boehringer Ingelheim Pharma GmbH & Co. KG 
Unit strength:
50 mg/vial powder and 50 mL solvent 
Posology:
Long-Term i.v. infusion for 12 hours per day, from Day
1 onwards starting right after the initial 2h i.v. infusion, 
followed by an approximately 12 hour break. This 
infusion scheme (12 hour infusion, followed by an 
approximately 12 hour break) is repeated daily up to at 
maximum Day 5). Preferably, the infusion will be started 
at approximately the same time at each day.
Please note: On Day 1, the total infusion time is 2 hours 
plus 12 hours = 14 hours. 
Please note: 
If the 12-hour infusion has to be temporarily interrupted
due to an urgent intervention, a drop of fibrinogen level
< 150 mg/dL and / or bleeding, it can be restarted within 
the next 72 hours. If these interruptions extend
beyond the protocol-recommended 12-hour alteplase-
infusion free interval, the time-windows for subsequent 
long-term infusions move accordingly, and the last long-
term infusion may be given later than day 5 in the study. 
Alteplase 50 mg powder for solution for injection/ 
infusion will be reconstituted in 50 mL water for 
injections to yield a concentration of 1 mg/mL (=stock 
solution).
A Perfusor Syringe of 50 mL should be used. 
A new vial of alteplase should be taken every day.


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Table 4.1.1:1
Test product (cont)
Substance:
Alteplase (recombinant tissue plasminogen activator)
Pharmaceutical formulation:
Powder and solvent for solution for injection/infusion
Source:
Boehringer Ingelheim Pharma GmbH & Co. KG
Unit strength:
50 mg/vial powder and 50 mL solvent
Posology:
Dose of Long-Term i.v. infusion in Part 1:
Dosing regimen A: 0.02 mg/kg/hour given over 12 hours
Dosing regimen B: 0.04 mg/kg /hour given over 12
hours
Dose of Long-Term i.v. infusion in Part 2:
One dosing regimen is carried forward based on results 
from Part 1 and recommendation by the DMC.
Dosing regimen B: 0.04 mg/kg /hour given over 12
hours
Optional additional i.v. infusion (allowed one time, 
during Days 2 to 5):
Part 1: One optional additional i.v. infusion:
Dosing regimen A: 0.3 mg/kg given over 2 hours
Dosing regimen B:  0.6 mg/kg given over 2 hours
This additional i.v. infusion can be given one time
during Days 2 to 5 in case of clinical worsening (as per 
investigator judgement)
Please note: This optional single additional infusion of 
alteplase may only be given one time. This additional 
dose may be administered on either Day 2, 3, 4, or 5. 
The duration of the long-term infusion (0.02mg/kg/h 
over 12 hours in Dosing regimen A; 0.04mg/kg/h over 
12 hours in Dosing regimen B) on the day of optional 


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Table 4.1.1:1
Test product (cont)
Substance:
Alteplase (recombinant tissue plasminogen activator)
Pharmaceutical formulation:
Powder and solvent for solution for injection/infusion
Source:
Boehringer Ingelheim Pharma GmbH & Co. KG 
Unit strength:
50 mg/vial powder and 50 mL solvent
additional infusion should sum up to 12 hours. The long-
term-infusion should not be given in parallel to the 
optional additional dose, but should be interrupted for 
approximately 2 hours, when the optional additional 
infusion of 0.3mg/kg/2h or 0.6mg/kg/2h is given. 
On the day of optional additional infusion, the total 
infusion time is 2 hours plus 12 hours = 14 hours.
For the optional additional i.v. infusion application, it is 
recommended that fibrinogen concentrations are ≥ 
300mg/dL.
Part 2: One dosing regimen is carried forward based on 
results from Part 1 and recommendation by the DMC.
One optional additional i.v. infusion:
Dosing regimen B:  0.6 mg/kg given over 2 hours 
This additional i.v. infusion can be given once during the 
treatment period in case of clinical worsening (as per 
investigator judgement)
For Instructions for Preparation and Administration of 
Trial Medication, please see ISF. 
For dose calculation and infusion rates, please see 
Appendix 10.5
Mode of administration:
Intravenous infusion


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4.1.2
Selection of doses in the trial and dose modifications
Selection of doses in the study took into consideration the established safety profile of 
alteplase and was in addition based on pharmacokinetic and pharmacodynamics (PK-PD)
modelling results c32479735-01. Doses for Part 1 were chosen to be lower than in the 
current indication of alteplase for massive, haemodynamic relevant PE, because COVID-19 
is associated with a constant pro-inflammatory trigger of coagulation activation, leading 
primarily to fibrin deposition, micro-clots and non-massive PE. Dose selection was based on 
the fact that the fibrinogen level should not fall bellow 4.4 µM (150 mg/dL), which has been 
reported as threshold for increased risk of bleeding P13-13102. With both dosing regimens 
(A and B) the fibrinogen level is likely to remain above this threshold. Both dosing regimens
are considered to deliver benefits for the treatment of the disease with an acceptable safety 
profile. The dose -response relationship and benefit-risk ratio of each dosing regimen will be 
evaluated in Part 1, and a decision will be taken after Part 1 to carry one dosing regimen into 
Part 2. 
For dosing regimen A and B see Table 4.1.1:1 above.
In Part 2 only dosing regimen B is applicable. 
Dose modifications are not foreseen unless recommended by the DMC. One optional 
additional i.v. infusion of 0.3 mg/kg infused over 2 hours (Dosing regimen A) or 0.6 mg/kg
infused over 2 hours (Dosing regimen B) can be given once on Days 2 to 5 in case of clinical 
worsening (as per investigator judgement). In Part 2, only dosing regimen B can be given as 
an additional i.v. infusion, as per investigator judgement.
4.1.3
Method of assigning patients to treatment arms
After the assessment of all eligibility criteria, each eligible patient will be randomised to 
treatment arms according to a randomisation plan in a 1:1:1 ratio at Visit 2a (same day as 
screening or day afterwards) in Part 1, and in a 2:1 randomisation rate in Part 2 via Interactive 
Response Technology (IRT). The appropriate medication number will be assigned and 
documented in the eCRF.
In Part 1 the randomisation will be stratified by the type of ventilation support (invasive 
mechanical / non-invasive) and in Part 2 by both type of ventilation support and by baseline 
D-dimer level (≥ULN to <5-fold ULN, versus ≥5-fold ULN), and access to the 
randomisation code will be controlled and documented. 
Randomisation of IMV patients will be stopped when a maximum of 50 IMV patients have 
been randomized in Part 2.
The block sizes of the randomisation will be documented in the clinical trial report (CTR)
and patients will be randomized sequentially.


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Note that the medication number is different from the patient number (the latter is generated 
during screening via the IRT System).
4.1.4
Drug assignment and administration of doses for each patient
At Day 1, patients will be randomized to one of three treatment arms (1:1:1) in Part 1 or to 
one of two treatment arms (2:1) in Part 2 by the IRT.
Part 1:
9 vials will be allocated per each patient in higher dose arm and 7 vials will be allocated per 
each patient in lower dose arm. In case additional kits are needed reflecting calculated 
amount needed per body weight, site has to call IRT and register a replacement call to 
allocate the additionally needed kits.
Part 2:
Number of vials will be allocated per each patient depending on patient`s weight in treatment 
arm by IRT. 
Each patient kit contains:
1 vial alteplase (TPA-05) Powder for solution for injection/infusion (50 mg/vial) with 50 mL 
solvent for alteplase (TPA-05) powder in a box. All components are labelled in an open label 
fashion. Single panel labels are placed on vials and box.
The pharmacist or his/her qualified delegate will prepare the trial medication at the dose level 
requested by the investigator. For dose calculation and infusion rates please refer to Alteplase 
dosing table in Appendix 10.5.
Handling and preparation of the medicinal product should be performed according to routine 
site procedures using aseptic techniques. Prior to the reconstitution, preparation and the 
infusion, the vials should be checked for any changes (e.g. color) and for the absence of 
particles. The lyo cake should be a white to pale yellow cake, the solution should be clear and 
colorless to slightly yellow. If you have any questions or concerns regarding appearance of 
the solution, please contact your CRA or the local Clinical Monitor. 
The reconstitution should be done immediately before use.
Reconstitution can be performed with a transfer cannula (if available in the kit) or with 
syringe and cannula. More details on preparation, administration and in-use stability of
alteplase solution will be provided in Instructions for Preparation and Administration of 
Alteplase 50 mg for solution for injection/infusion in ISF.


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Table 4.1.4:1 Dosage and treatment schedule (Part 1)
Initial i.v. infusion (Day 1):

Dosing regimen A: 0.3 mg/kg infused over 2 hours

Dosing regimen B: 0.6 mg/kg infused over 2 hours
Daily long-term i.v. infusion over 12 hours from Day 1 onwards, up to at maximum Day
5:
Dosing regimen A
Dosing regimen B
Target Dose 
0.02 mg/kg/h
0.04 mg/kg/h
Optional additional i.v. infusion of 0.3 mg/kg infused over 2 hours (Dosing Regimen A) 
or 0.6 mg/kg infused over 2 hours (Dosing regimen B) is allowed once, on Days 2 to 5.
Part 2: One dosing regimen is carried forward based on results from Part 1 and 
recommendation by the DMC.
Initial i.v. infusion (Day 1):

Dosing regimen B: 0.6 mg/kg infused over 2 hours
Daily long-term i.v. infusion over 12 hours from Day 1 onwards, up to at maximum Day 
5 (plus up to 72 hours in case of unexpected interruptions):
Dosing regimen B
Target Dose 
0.04 mg/kg/h
Optional additional i.v. infusion of 0.6 mg/kg infused over 2 hours (Dosing regimen B) is 
allowed once during the treatment period.
4.1.5
Blinding and procedures for unblinding
4.1.5.1
Blinding
This is an open-label study, therefore no blinding will apply for patients, investigators and 
personnel at the site involved in trial conduct
The access to the randomisation code list will be kept restricted until it is released for 
analysis.
The randomisation codes will be provided to bioanalytics prior to last patient completed in 
each part of the trial to allow for a timely analysis of pharmacokinetic (PK) samples. 


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4.1.5.2
Unblinding and breaking the code
As this is an open-label trial, unblinding does not apply for the investigator and study site 
personnel.
Due to the requirements to report Suspected Unexpected Serious Adverse Reactions 
(SUSARs), it may be necessary for a representative from BI’s Pharmacovigilance (PV) group 
to access the randomisation code for individual patients during trial conduct. The access to 
the code will only be given to authorised PV representatives for processing in the PV 
database system and not be shared further. 
4.1.6
Packaging, labeling, and re-supply
The IMPs will be provided by BI or a designated Contract Research Organisation (CRO). 
They will be packaged and labelled in accordance with the principles of Good Manufacturing 
Practice. Re-supply to the sites will be managed via an IRT system, which will also monitor 
expiry dates of supplies available at the sites.
For details of packaging and the description of the label, refer to the ISF.
4.1.7
Storage conditions
Drug supplies will be kept in their original packaging and in a secure limited access storage 
area according to the recommended storage conditions on the medication label. A
temperature log must be maintained for documentation.
If the storage conditions are found to be outside the specified range, the Clinical Research 
Associate (CRA), as provided in the list of contacts, must be contacted immediately.
4.1.8
Drug accountability
The investigator or designee will receive the investigational drugs delivered by the sponsor
when the following requirements are fulfilled:

Approval of the clinical trial protocol (CTP) by the Institutional Review Board (IRB)
/ Ethics Committee (EC),

Availability of a signed and dated clinical trial contract between the sponsor and the 
investigational site,

Approval/notification of the regulatory authority, e.g. Competent Authority (CA),

Availability of the curriculum vitae of the Principal Investigator,

Availability of a signed and dated CTP,

Availability of the proof of a medical license for the Principal Investigator,
Investigational drugs are not allowed to be used outside the context of this protocol. They 
must not be forwarded to other investigators or clinics. 
The investigator or designee must maintain records of the product’s delivery to the trial site, 
the inventory at the site, the use by each patient, and the return to the sponsor or warehouse / 
drug distribution centre or alternative disposal of unused products. If applicable, the sponsor 
or warehouse / drug distribution centre will maintain records of the disposal. 


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These records will include dates, quantities, batch / serial numbers, expiry (‘use- by’) dates, 
and the unique code numbers assigned to the IMP and trial patients. The investigator or 
designee will maintain records that document adequately that the patients were provided the 
doses specified by this CTP and reconcile all IMPs received from the sponsor. At the time of 
return to the sponsor or appointed CRO, the investigator or designee must verify that no 
remaining supplies are in the investigator’s possession.
4.2
OTHER TREATMENTS, EMERGENCY PROCEDURES, 
RESTRICTIONS
4.2.1
Other treatments and emergency procedures
Anticoagulant medications should be given to prevent the formation of new clots, as follows:
Alteplase arm, Day 1 to 5 (treatment period):
The investigator may choose between Option A or B:
Option A: Option A can only be applied in patients with an eGFR ≥30ml/min/1.73m2.
Low-dose LMWH in prophylactic dosages given every day at the end of long-term i.v. 
alteplase infusion
Recommendations for LMWH dosages (these represent examples, and other LMWHs in 
prophylactic dosages are also allowed):
Dalteparin 5000 U, subcutaneously (SC) once 
daily (OD)
Enoxaparin 40 mg (4000 U), SC OD
Fondaparinux 2.5 mg, SC OD
Nadroparin 5700 U, SC OD (patients >70 kg)
Nadroparin 3800 U, SC OD (patients ≤70 kg)
Tinzaparin 4500 U, SC OD
In patients with moderate (creatinine clearance 30-50 ml/min/1.73m2) and mild (creatinine 
clearance 50-80 ml/min/1.73m2) renal impairment, careful clinical monitoring is advised.
or:
Option B: Option B can be chosen in all patients, irrrespective of their eGFR.
Unfractionated heparin (UFH, e.g. 10 IU/ kg body weight/hour) concomitantly to long-term 
alteplase i.v. infusion, to be started immediately after initial 2 hour alteplase i.v. infusion, 
with a target aPTT of 1.0 to 1.5 fold of ULN according to local laboratory
[U99-0193, P99-02520].
UFH in a dosage of up to 5000 IU, SC BID (twice daily) can be given alternatively. 


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It is recommended to administer antithrombin III to patients where no further anticoagulant
effects with dose increases of heparin are seen.
Alteplase arm, post treatment period (Day 5 and onwards), and SOC arm:
Option A: LMWH with target anti-Xa level according to local practice
or
Option B: Unfractionated heparin with target aPTT level according to local practice
Heparin should not be given simultaneously with the initial 2h i.v. infusion of alteplase, or at 
time of an optional additional 2h i.v. infusion of alteplase, or when an intervention needs to 
be performed. The additional 2h i.v. infusion of alteplase should preferably be given after 
UFH has been stopped for 2 hours, or ideally 12 hours after the last LMWH injection, 
however, these interruption intervals may not apply in an emergency situation (according to 
the investigator`s judgement). 
The interruption intervals for concomitant heparin treatment before an intervention should 
follow local practice. Please see product information of respective heparins for appropriate 
interruption intervals before intervention (for temporary interruption of alteplase, please see 
Section 3.3.4.1).
The anti Xa level should be monitored regularly, in patients with mild to moderate renal 
impairment. The aPTT level should be monitored at least daily and preferably more 
frequently, while given concomitantly with alteplase. 
Occurrence of heparin-induced thrombocytopenia (HIT) must result in cessation of UFH or 
LMWH without recommencement regardless of treatment assignment. Use of an acceptable 
alternative agent is required in this instance as clinically indicated. Occurrence of HIT has to 
be reported as adverse event (AE).
Glucocorticoid treatment
Current or scheduled use of systemic (intravenous or oral) corticosteroid therapy should be 
included in the SOC regimen (e.g. 6 mg of dexamethasone orally) for up to 10 days or until 
hospital discharge. As patients in the alteplase treatment arm should also receive best possible 
SOC, this recommendation applies in the vs. non-invasive
alteplase arm as well.
Total daily dose equivalencies to dexamethasone 6 mg are provided in Appendix 10.6.
Standard of Care (SOC) arm
SOC includes any supportive measures applied in hospital, specifically on an ICU, such as 
for example the use of non-invasive or invasive ventilation, oxygen masks, haemodynamic 
support, if needed, sedation, as well as medical therapies commonly used in patients suffering 
from ARDS or its complications. SOC should include best possible treatment regimen
established locally and should be in line with current guidelines for COVID-19 ARDS 
treatment.


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For additional recommended SOC therapies please refer to Appendix 10.6.
Recommendations before the start of alteplase:

Arterial lines and central venous catheters, tubes (e.g. for intubation and feeding) and 
drains (e.g. pleural and abdominal) should be placed at least 6 hours before starting 
the first treatment with alteplase.

A delay of the alteplase treatment of approximately 6 hours after the insertion of the 
last line/catheter is recommended to allow for complete hemostasis (this time-period 
can be extended to up to 24 hours after complicated interventions)

Before the start of the very first alteplase infusion, there should have been a time 
period of at least 10 hours since the last LMWH (S.C.) administration to ensure no 
significant overlap of the LMWH and alteplase.
Emergency procedures
Management of Bleeding during Alteplase Treatment:

In case of bleeding, the alteplase infusion can be interrupted up to a maximum of 72
hours.

A restart is possible, if complete haemostasis is achieved, and if lab results support a 
restart (fibrinogen ≥150mg/dL, haemoglobin is stable, anti-Xa 4 hours after s.c. 
administration is < 0.5, or aPTT ratio is < 1.5-fold of upper limit of normal).

In case of interruptions of more than 12 hours, the time-windows for subsequent long-
term infusions move accordingly, and the last long-term infusion may be given later 
than day 5 in the study. In total, up to 5x long-term infusions (of 12 hours each) can 
be given.
Management of serious bleeding events:
In case of serious bleeding, in particular cerebral or pulmonary haemorrhage, the fibrinolytic 
therapy must be discontinued, and concomitant heparin administration should be terminated 
immediately. Administration of protamine should be considered if unfractionated heparin has 
been administered. In patients who fail to respond to these conservative measures, judicious 
use of transfusion products may be indicated. Transfusion of cryoprecipitate, fresh frozen 
plasma, and platelets should be considered with clinical and laboratory reassessment after 
each administration. A target fibrinogen level of 1g/L (100 mg/dL) is desirable with 
cryoprecipitate infusion. Antifibrinolytic agents should also be considered.
Beyond patient-specific risk factors for bleeding, the bleeding risk can be estimated by the 
plasma fibrinogen level. Fibrinogen will be monitored daily during the treatment period with 
alteplase, and if the level falls below 150 mg/dL, the investigator should interrupt the 
alteplase infusion.
The alteplase infusion can be restarted, if fibrinogen recovers to ≥150 mg/dL during the 
following 72 hours.


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In case of bleeding, intervention or surgery, the treatment with alteplase may require a 
temporary interruption or permanent discontinuation of alteplase, according to the 
investigator`s discretion and the severity of the bleed. While a major bleeding may in general 
require a temporary interruption or premature discontinuation of alteplase, minor bleeds may 
not lead to changes in study drug application. For details on temporary interruption of 
alteplase, please see Section 3.3.4.1.
4.2.2
Restrictions
4.2.2.1
Restrictions regarding concomitant treatment
The following medications are not allowed to be used concomitantly during the treatment 
period in the alteplase arm:

Vitamin K antagonists*

Direct oral anticoagulants**

Antiplatelet agents (with the exception of acetyl salicylic acid, ASA, up to 100
mg/day)

GPIIb/IIIa antagonists

Other fibrinolytic drugs (for ultima ratio/bail-out situation, see below).

LMWH/unfractionated heparins in therapeutic dosages

Any medication that may increase the risk of bleeding, except 
LMWH/unfractionated heparin as described in Section 4.2.1
The above mentioned restrictions do not apply after the completion of the treatment period in 
the Alteplase arm.
*Patients who previously received a vitamin K antagonist can only be randomised, if INR is <1.3. 
**Patients who previously received a direct oral anticoagulant, can only be included, if the direct 
oral anticoagulant has been stopped for at least 48 hours prior to randomisation.
Except for the use of fibrinolytic drugs, these restrictions do not apply for patients in the SOC
arm, these patients should be treated according to institutional guidelines. 
Ultima ratio situation - Bail-out: 
Preferably, patients should stay on initially assigned treatment for the first 5 days, and 
marketed Actilyse® should not be given during the treatment period. However, if a patient 
fulfills an indication according to the local label for Actilyse® (e.g. ischemic stroke, massive 
haemodynamically unstable PE, MI), marketed Actilyse® (non-study drug) is allowed in 
study treatment arms and SOC arms as ultima ratio, at the discretion of investigator at any 
time during the trial.
4.2.2.2
Restrictions on diet and life style
Not applicable.


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4.2.2.3
Contraception requirements
No contraception requirements are applicable, as patients are hospitalized and in general are 
severely ill during the whole observational period.
4.3
TREATMENT COMPLIANCE
As all study treatments will be given intravenously in-hospital, no compliance problems are 
foreseen. The only reasons for deviations from the treatment schedule in the protocol would 
be due to the rare events of dosing mistakes or damaged treatment kits. Information on dosing 
will be collected in the eCRF.


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5.
ASSESSMENTS
5.1
ASSESSMENT OF EFFICACY
5.1.1
WHO Clinical Progression Scale
The following 11-point WHO Clinical Progression Scale will be used for the assessment of 
clinical improvement [R20-2002] (Table 5.1.1: 1). On Day 1 (randomisation day) an 
assessment just before randomisation shall be collected as baseline value. The last assessment 
per day should be recorded in the case report form.
Table 5.1.1:1
WHO Clinical Progression Scale
Patient State
Descriptor
Score
Uninfected
Uninfected ; no viral RNA detected
0
Ambulatory mild disease
Asymptomatic ; viral RNA detected
1
Symptomatic ; independent
2
Symptomatic ; assistance needed
3
Hospitalised : moderate 
disease
Hospitalised ; no oxygen therapy*
4
Hospitalised ; oxygen by mask or nasal prongs
5
Hospitalised : severe 
diseases
Hospitalised ; oxygen by NIV or high flow
6
Intubation and mechanical ventilation, PaO2/FiO2
≥150 or SpO2/FiO2 ≥200
7
Mechanical ventilation PaO2/FiO2<150 
(SpO2/FiO2<200) or vasopressors
8
Mechanical ventilation PaO2/FiO2 <150 and 
vasopressors, dialysis, or ECMO
9
Dead
Dead
10
Source: Lancet Infect Dis. 2020, published online June12, 2020 R20-2002




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5.1.2
Oxygen- free and Ventilator-free days, days in ICU
Oxygen-free and ventilator-free days until Day 28 will be captured from source documents. 
Time-points of intubation and extubation will be recorded in the eCRF. Days on ICU, 
intermediate care, step-down unit (normal ward) will be counted until Day 28 based on 
source documents. Date of discharge from hospital and/or death will be recorded in the 
eCRF. 
5.1.3
PaO2/FiO2 ratio
The respiratory status of the enrolled patients is monitored by the PaO2/FiO2 ratio (or inferred 
PaO2/FiO2 ratio from SpO2, see Appendix 10.2) regularly and all available data points will be 
collected until discharge, death, or Day 28, whatever occurs first. Fraction of Inspired 
Oxygen (FiO2) for common oxygen delivery systems are provided in Appendix 10.3.
During the first 6 days and during stay on ICU (whatever is longer) PaO2/FiO2 ratio should be 
measured three times per day in all patients, together with a recording of the patient`s 
position. The qualifying PaO2/FiO2 ratio should be the worst measurement in the screening 
period. The worst measurement on a given day should be documented in the case report form.
Thereafter, less frequent measurements are acceptable and should be obtained according to 
local clinical practice. The average daily measurements for each patient will be calculated 
and later analysed.
5.1.4
SOFA Score
Change in Sequential (sepsis-related) Organ Failure Assessment (SOFA) score is assessed 
during hospital stay from baseline to end of Day 28. If patient is discharged from hospital 
prematurely, the last evaluation needs to be performed at the day of discharge from hospital.
(Appendix 10.1)
5.2
ASSESSMENT OF SAFETY
5.2.1
Physical examination
A physical examination will be performed at the time points specified in the Flow chart. It 
includes at minimum the assessment of general appearance, neck, lungs, cardiovascular 
system, abdomen, extremities, and skin. 
Measurement of body weight and height will be performed at the time points specified in the 
Flow chart. The results must be included in the source documents available at the site. 
5.2.2
Vital signs
Vital signs will be evaluated at the time points specified in the Flow chart.


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This includes systolic and diastolic blood pressure, body temperature, respiratory rate and 
pulse rate (electronically or by palpation count for 1 minute) after 5 minutes of rest. The 
results must be included in the source documents available at the site.
5.2.3
Safety laboratory parameters
Safety laboratory parameters to be assessed are listed in Table 5.2.3:1. For the sampling time 
points please see the Flow chart. Local laboratory results will be used throughout the trial.
All analyses will be performed by the local laboratory, the respective reference ranges will be 
provided in the ISF. More details on collection of safety laboratory parameters are 
summarized in Table 5.2.3:1
Patients do not have to be fasted for the blood sampling for the safety laboratory. 


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Table 5.2.3:1 
Safety laboratory tests
Haematology

Haematocrit

Haemoglobin

RBCs / Erythrocytes

White Blood Cells (WBC) / Leukocytes

Platelet Count / Thrombocytes
Clinical chemistry

Albumin*

Alkaline phosphatase*

ALT (alanine transaminase, serum glutamic-pyruvic transaminase [SGPT])

AST (aspartate transaminase, serum glutamic-oxaloacetic transaminase [SGOT])

Bilirubin total*

Creatinine (estimated GFR to be calculated according to local practice)

Creatine kinase (CK) *

Troponin I or T*

Lactate dehydrogenase

Glucose*

Potassium

Sodium

Urea (BUN) *

Type B natriuretic peptide (BNP), or NT-proBNP*
Coagulation and Inflammation

aPTT (Activated partial thromboplastin time; in patients receiving UFH)

Prothrombin time, (or alternatively INR) 

D-Dimer

Fibrinogen

Antithrombin*

Anti-Xa (in patients receiving LMWH)

C-reactive Protein

Procalcitonin*

Interleukin-6*

Plasminogen activator inhibitor 1*

Thrombomodulin*

Ferritin*
*If available locally


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Clinically relevant abnormal findings as judged by the investigator will be reported as 
adverse events (please refer to Section 5.2.6.1).
5.2.4
Electrocardiogram
The 12-lead ECGs must be administered by a qualified personnel and results will be recorded 
as scheduled in the Flow chart. The investigator or a designee will evaluate whether the ECG 
is normal or abnormal and assess clinical relevance. ECGs may be repeated for quality 
reasons and a repeated recording may be used for evaluation. In addition to the scheduled 
ECGs, ECGs should be done and documented at any time of clinical event (e.g. acute event 
of arrhythmia, tachy- or bradycardia, angina, or MI).
Dated and signed printouts of ECG with findings should be documented in the patient’s 
medical records.
Clinically relevant abnormal findings will be reported either as a baseline condition (if 
identified at the Screening Visit) or otherwise as adverse events and will be followed up 
and/or treated as medically appropriate.
5.2.5
Other safety parameters
5.2.5.1
Bleeding events
Patients should be carefully assessed for signs and symptoms of bleeding. Bleeding
definitions are provided below. The location of the bleeding including the specific critical 
area or organ into which the bleeding occurred (e.g. GI, intracranial, pulmonary bleed) and 
whether or not it prolongs hospitalization will be recorded.
Definition of any bleed
This is the sum of all major and minor bleeds. 
Definition of a major bleed
Major bleeds will be defined according to the ISTH definition of a major bleed, as follows 
(R05-0344):

Symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, 
intraocular, retroperitoneal, intra-articular or pericardial, or intramuscular with 
compartment syndrome,
and/or

Bleeding associated with a reduction in hemoglobin of at least 2g/dL (1.24 mmol/L), 
or leading to transfusion of two or more units of blood or packed cells1
and/or

Fatal bleed
1Bleeding should be overt and the hemoglobin drop should be considered to be due to and temporally related to the bleeding 
event.


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Definition of a fatal bleed
Fatal bleeding is defined as a bleeding event that the investigator determines is the primary 
cause of death or contributes directly to death.
Definition of intracranial hemorrhage
Intracranial hemorrhage comprises the subtypes of intracerebral bleeds, subdural bleeds, 
epidural bleeds and subarachnoid bleed and will be recorded in eCRF.
Definition of a minor bleed
Minor bleeds are clinical bleeds that do not fulfill the criteria for major bleeds. 
5.2.6
Assessment of adverse events
5.2.6.1
Definitions of adverse events
5.2.6.1.1
Adverse event
An adverse event is defined as any untoward medical occurrence in a patient or clinical 
investigation patient administered a medicinal product and which does not necessarily have to 
have a causal relationship with this treatment.
An adverse event can therefore be any unfavourable and unintended sign (including an 
abnormal laboratory finding), symptom, or disease temporally associated with the use of a 
medicinal product, whether or not considered related to the medicinal product.
The following should also be recorded as an adverse event in the eCRF and BI SAE form (as
applicable):
Worsening of the underlying disease or of other pre-existing conditions 
Changes in vital signs, ECG, physical examination and laboratory test results, if they 
are judged clinically relevant by the investigator.
If such abnormalities already exist prior to trial inclusion, they will be considered as baseline 
conditions and should be collected in the eCRF only.
5.2.6.1.2
Serious adverse event
A serious adverse event (SAE) is defined as any adverse event, which fulfills at least one of 
the following criteria:
-
results in death,
-
is life-threatening, which refers to an event in which the patient was at risk of death at 
the time of the event; it does not refer to an event that hypothetically might have 
caused death if more severe,
-
requires inpatient hospitalisation or prolongation of existing hospitalisation
-
results in persistent or significant disability or incapacity, 
-
is a congenital anomaly / birth defect,


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-
is deemed serious for any other reason if it is an important medical event when based 
on appropriate medical judgement which may jeopardise the patient and may require 
medical or surgical intervention to prevent one of the other outcomes listed in the 
above definitions. Examples of such events are intensive treatment in an emergency 
room or at home for allergic bronchospasm, blood dyscrasias or convulsions that do 
not result in hospitalisation or development of dependency or abuse. 
5.2.6.1.3
Adverse events considered “Always Serious”
In accordance with the European Medicines Agency initiative on Important Medical Events, 
BI has set up a list of adverse events, which by their nature, can always be considered to be 
“serious” even though they may not have met the criteria of an SAE as defined above.
The latest list of “Always Serious Adverse Events” can be found in the Electronic Data 
Capture System and there is an algorithm in clinical database installed which detects non 
serious events which are per list definition serious. A copy of the latest list of “Always 
Serious Adverse Events” will be provided upon request. For patients on Alteplase treatment,
these events should always be reported as serious adverse events, as described in Section 
5.2.6.2. For patients on SOC, adverse events should be considered serious if they meet any of 
the seriousness criteria, outlined in Section 5.2.6.1.2.
Efficacy and safety endpoints are considered to be disease-related or are well-known side 
effects of fibrinolytics. These events are considered Outcome Events (OEs) and will need to 
be reported as serious adverse events on the SAE form if they meet criteria of an SAE and 
consequently will be reported in an expedited manner to the Competent Authorities. Please 
refer to Section 5.2.6.2.4 for Exemptions to SAE reporting.
Outcome Events are defined as follows:
Any bleeding including intracranial hemorrhage
All deaths 
All myocardial infarctions
All strokes
All systemic embolisms
All transient ischemic attacks
All deep vein thromboses
All pulmonary embolisms 
A standard narrative ( if requested ) and any required supporting documentation (including 
e.g. transesophageal echocardiogram [TEE], transthoracic echocardiogram [TTE], magnetic 
resonance tomography [MRT], computerized tomography, ECG examinations, or other) must 
also be provided for assessment of the DMC. 
A key objective of the independent DMC is to protect patient safety by monitoring the 
incidence and clinical relevance of safety data including OEs, collected throughout the 
conduct of this study.


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Cancers of new histology and exacerbations of existing cancer must be classified as a serious 
event regardless of the time since discontinuation of the drug and must be reported as 
described in Section 5.2.6.2, subSections “AE Collection” and “AE reporting to sponsor 
and timelines”.
5.2.6.1.4
Adverse events of special interest
No adverse events of special interest have been defined and no DILI procedures are required 
for this trial as alteplase is a marketed product with well-known safety profile.
5.2.6.1.5
Intensity (severity) of Adverse events
The intensity (severity) of the AE should be judged based on the following:
Mild:
Awareness of sign(s) or symptom(s) that is/are easily tolerated.
Moderate:
Sufficient discomfort to cause interference with usual activity.
Severe:
Incapacitating or causing inability to work or to perform usual activities.
5.2.6.1.6
Causal relationship of adverse events
Medical judgement should be used to determine whether there is a reasonable possibility of a 
causal relationship between the adverse event and the given study treatment, considering all 
relevant factors, including pattern of reaction, temporal relationship, de-challenge or 
re-challenge, confounding factors such as concomitant medication, concomitant diseases and 
relevant history. 
Arguments that may suggest that there is a reasonable possibility of a causal relationship 
could be:

The event is consistent with the known pharmacology of the drug.

The event is known to be caused by or attributed to the drug class.

A plausible time to onset of the event relative to the time of drug exposure.

Evidence that the event is reproducible when the drug is re-introduced.

No medically sound alternative aetiologies that could explain the event (e.g. 
pre-existing or concomitant diseases, or co-medications).

The event is typically drug-related and infrequent in the general population not 
exposed to drugs (e.g. Stevens-Johnson syndrome).

An indication of dose-response (i.e. greater effect size if the dose is increased, smaller 
effect size if dose is reduced).
Arguments that may suggest that there is no reasonable possibility of a causal relationship 
could be:

No plausible time to onset of the event relative to the time of drug exposure is evident 
(e.g. pre-treatment cases, diagnosis of cancer or chronic disease within days/weeks of 
drug administration; an allergic reaction weeks after discontinuation of the drug 
concerned).


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
Continuation of the event despite the withdrawal of the medication, taking into 
account the pharmacological properties of the compound (e.g. after 5 half-lives).
Of note, this criterion may not be applicable to events whose time course is prolonged 
despite removing the original trigger.

Additional arguments amongst those stated before, like alternative explanation (e.g. 
situations where other drugs or underlying diseases appear to provide a more likely 
explanation for the observed event than the drug concerned).

Disappearance of the event even though the trial drug treatment continues or remains 
unchanged.
5.2.6.2
Adverse event collection and reporting
5.2.6.2.1
Adverse event collection
The investigator shall maintain and keep detailed records of all adverse events in the patient
files. The following must be collected and documented on the appropriate eCRF(s) by the 
investigator:
•
From signing the informed consent onwards until the individual patient’s end of trial (the 
End of Study (EOS) Visit): 
all adverse events (serious and non-serious).
•
After the individual patient’s end of trial: 
the investigator does not need to actively monitor the patient for new adverse events but 
should only report any occurrence of cancer and trial treatment related serious adverse
events of which the investigator may become aware of by any means of communication, 
e.g. phone call. Those adverse events should be reported on the BI SAE form (see 
Section 5.2.6.2.2), but not on the eCRF.
The rules for Adverse Event Reporting exemptions still apply, please see Section 5.2.6.2.4.
5.2.6.2.2
Adverse event reporting to the sponsor and timelines
For the patients on Alteplase treatment arm, the investigator must report serious adverse 
events, and non-serious adverse events which are relevant for the reported SAE on the BI 
SAE form via fax (except of Russia where SAE form is to be send via Clinergize)
immediately (within 24 hours) to the sponsor’s unique entry point (country-specific contact 
details will be provided in the ISF). The same timeline applies if follow-up information 
becomes available. In specific occasions, the investigator could inform the sponsor upfront 
via telephone. This does not replace the requirement to complete and fax the BI SAE form.
With receipt of any further information to these events, a follow-up SAE form has to be 
provided. For follow-up information the same rules and timeline apply as for initial 
information. All serious and adverse events, including those persisting after individual 
patient’s end of trial must be followed up until they have resolved, have been assessed as 
“chronic” or “stable”, or no further information can be obtained.
For patients not exposed to Alteplase treatment, but to SOC only: No expedited reporting 
via SAE form is required. All safety data (AEs and SAEs) should be included in the eCRF.


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Consequently, the investigator is encouraged to report all adverse events for SOC group 
drugs in accordance to the local regulatory requirements for spontaneous AE reporting at the 
investigator’s discretion by using their locally established routes and AE report forms.
5.2.6.2.3
Pregnancy
Not applicable for this trial. This trial will exclude pregnant women, and patients are severely 
ill during the observation period of the trial. To rule-out potential pregnancy urine pregnancy 
test is performed during screening and at Day 28. A woman is considered of childbearing 
potential, i.e. fertile, following menarche and until becoming post-menopausal unless 
permanently sterile. Permanent sterilisation methods include hysterectomy, bilateral 
salpingectomy and bilateral oophorectomy. Tubal ligation is NOT a method of permanent 
sterilisation.
A postmenopausal state is defined as no menses for 12 months without an alternative medical 
cause.
5.2.6.2.4
Exemptions to SAE reporting
Disease progression of the underlying ARDS reflects the natural course of the disease and is 
exempted from reporting as an (S)AE. Progression of the subject's underlying ARDS will be 
recorded on the appropriate pages of the (e)CRF only and will not be reported as an SAE.
The specific preferred terms (PTs) that are exempt are:

Acute respiratory distress syndrome

Respiratory distress

Acute respiratory failure

Respiratory failure
Death due to disease progression is also to be recorded on the appropriate eCRF page and not 
on the SAE form. Disease progression and death due to disease progression will therefore not 
be entered in the safety database and hence not get expeditiously reported.
However, if there is evidence suggesting a causal relationship between the study drugs and
the progression of the underlying ARDS, the event must be reported as SAE on the
eCRF, as well as on the SAE form.
Exempted events will be monitored at appropriate intervals by the external DMC. 
5.3
DRUG CONCENTRATION MEASUREMENTS AND 
PHARMACOKINETICS
5.3.1
Assessment of pharmacokinetics
Participation in PK sampling is voluntary and not a prerequisite for participation in the trial. 
PK sampling will only occur after a separate PK informed consent has been given in 
accordance with local ethical and regulatory requirements. It is intended to obtain PK 
samples from at least 10 patients per dose group in Part 1. As PK samples collection included
less than 10 patients per treatment arm in Part 1, collection of PK samples will continue in 
Part 2. It is intended to obtain PK samples from at least 17 patients receiving Alteplase in Part 
2.


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Three samples per patient will be collected:

1st sample: prior to first drug administration on Day 1 (Visit 2a pre-dose) (planned 
time: -1:00 h; time window -2:00 h to just prior drug administration)

2nd sample: at steady state of the initial i.v. infusion on Day 1 (Visit 2a post-dose 
initial i.v. infusion) (planned time 1:30 h; time window: between 1 h after start of 
initial i.v. infusion and just prior to end of initial i.v. infusion)

3rd sample: at steady state of the long-term i.v. infusion on Day 1 (Visit 2a post-dose 
long-term i.v. infusion) (planned time: 6:00 h; time window: between 2 h after start of 
long--term i.v. infusion and just prior to end of long-term i.v. infusion)
The actual sampling date and time for blood samples will be reported in the eCRF. To allow a 
valid PK analysis, it is of utmost importance to document the exact clock time of start/end of 
infusion and blood sample collection. Every attempt should be made to adhere to a constant 
infusion rate of approximately 1 hour prior to drawing of sample 2 and approximately 2 hours
prior to drawing of sample 3.
5.3.2
Methods of sample collection
For quantification of analyte plasma concentrations, blood will be taken from an antecubital 
or forearm vein, at the times indicated in the Flow chart. Blood will be withdrawn by means 
of single-time venipuncture.
Details on sample characteristics, collection, processing, handling and shipment are provided 
in the Laboratory Manual in the ISF.  
Samples will be stored in a freezer at the analytical laboratory until finalization of the clinical
trial report (CTR). The plasma samples may be used for further methodological 
investigations (e.g. for stability testing). However, only data related to the analyte will be 
generated by these additional investigations. The study samples will be discarded after 
completion of the additional investigations according to local legislation, but not later than at 
the time of completion of the CTR. 
5.4
ASSESSMENT OF BIOMARKERS
No exploratory biomarkers are planned. Established biomarkers of efficacy and safety are 
described and discussed in Section 5.1 and Section 5.2.
5.5
BIOBANKING
Not applicable.


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5.6
OTHER ASSESSMENTS
Not applicable.
5.7
APPROPRIATENESS OF MEASUREMENTS
The primary endpoint measurements will be consistent with the recognized standard for 
ARDS in the setting of COVID-19 to assess treatment effects, including the clinical 
functional status.
All secondary 
 measurements performed during this trial are standard
measurements and will be performed in order to determine the efficacy and safety in an
appropriate way. The planned measurements are appropriate to see drug-induced changes in
vital signs, functional parameters, standard laboratory values and coagulation parameters
specific to efficacy and safety of the study drug. The endpoints are widely used and accepted
for evaluation of efficacy and safety on an intravenously applied drug in a hospital setting.
Therefore, the appropriateness of all measurements applied in this trial is given.


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6.
INVESTIGATIONAL PLAN
The trial consists of three periods, a screening period, an intravenous open-label treatment 
period and a follow-up period. 
During the treatment period, patients will receive an open-label infusion with alteplase on top 
of SOC, or SOC alone.
Instructions on alteplase infusion dosage and treatment schedule is provided in Table 4.1.4:1.
All required procedures are described in the Flow chart and Section 5.
After completion of the treatment period patients will be followed-up on Day 6, Day 7, Day
28 and Day 90/EOS.
If patient is discharged from hospital before completion of Day 28, the patient has to be 
invited to complete subsequent visits as per Flow chart until Day 90 including vital status 
collection.
6.1
VISIT SCHEDULE
No study procedures may be initiated prior to the patient or patient’s legally acceptable
representative signing the informed consent. A patient who could not provide informed 
consent at the start of the trial due to the severity of the disease, may have their legally 
acceptable representative to sign the consent on their behalf, in accordance with applicable 
laws and regulations.  Once patient is recovered, they would need to provide their own
consent to continue in the study.
An independent witness may be required in the event that the patient is able to give verbal 
consent, but unable to sign the consent form. Please refer to Section 8.1 for more details on 
consenting process.
During the Screening Visit at any time of the day or night, eligible patients who are 
exhibiting signs and symptoms of COVID-19 induced ARDS may be asked to participate in 
the study.
Patients fulfilling all eligibility criteria will be randomised to one of three treatment arms in 
Part 1, and to one of two treatment arms in Part 2. 
For potential modification of trial conduct in case of restrictions due to COVID-19, please 
refer to Appendix 10.4.
6.2
DETAILS OF TRIAL PROCEDURES AT SELECTED VISITS
Study procedures to be performed at each visit are listed in the Flow chart and the respective 
protocol Sections.


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6.2.1
Screening and run-in periods
Screening Period
Once the patient has consented, the patient is considered to be enrolled in the trial. The 
patient should be recorded on the enrollment log and be registered in the IRT.
Each patient will be assigned a unique patient number and enrollment will be recorded in 
eCRF.
For the schedule of assessments and procedures during the screening period refer to the Flow
Chart.
Laboratory samples shall be collected as per Table 5.2.3:1 for haematology, clinical 
chemistry, renal function and coagulation including urine pregnancy testing (only applicable
for women of childbearing potential). Local laboratories are intended to be used for all 
assessments.
The qualifying PaO2/FiO2 ratio should be the worst measurement in the screening period.
For eligibility, patients should be hospitalized with the diagnosis of COVID-19 ARDS.
SARS-CoV-2 laboratory confirmation (RT-PCR test) is a pre-requisite at screening. The RT-
PCR test needs to be in timely association with the current hospital stay. 
Re-screening 
A patient may be re-screened once within same hospital stay if the patient does not fulfill
eligibility criteria. In case of re-screening patient will obtain new patient number per IRT.
Run-in Period
Not applicable.
6.2.2
Treatment periods
Randomisation of patients (Day 1, Visit 2a) shall happen not later than 24 hours after the 
confirmation of the inclusion and exclusion criteria. Study drug dispensing and 
administration shall start within 6 hours of randomisation. The time-point of obtaining D-
Dimer levels can be as long as four calendar days before screening. 
Treatment period duration is 5 days.
Interruptions for urgent reasons for a total of up to 72 hours are allowed and accordingly, the 
time window of the visits may be extended.
Patients who discontinue trial treatment prematurely should still follow the trial schedule 
until Visit 6/EOS, if possible, please see Section 3.3.4, including vital status collection.
For the schedule of assessments and procedures during the treatment period refer to the Flow
Chart.


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Please refer to Table 4.1.4:1 for Dosing and Treatment schedule and Instructions for Dose 
calculation and Infusion rates of Alteplase (Appendix 10.5)
For details on PK sampling collection please refer to Section 5.3.1.
During the treatment period the worst measurement on a given day should be documented in 
the case report form. During the first 6 days and during the stay on ICU (whatever is longer),
PaO2/FiO2 ratio should be measured at least three times daily in all patients, together with a 
recording of the patient`s position.
Assessment of time-point of intubation and extubation, or start and end of non-invasive 
ventilation and oxygen support is collected. 
In addition to the scheduled ECGs, ECGs should be done and documented at any time of 
clinical event (e.g. acute event of arrhythmia, tachy- or bradycardia, angina, or MI).
6.2.3
Follow-up period and trial completion
Patients will be followed after treatment period up to Day 90. Follow-up period consists of 
4 visits:  Day 6, Day 7, Day 28 and Day 90/vital status collection.
For the schedule of assessments and procedures during the follow-up period refer to the Flow
Chart.
Vital status shall be collected for all patients at V6/EOS (see Flow Chart ). Vital status can be 
collected by any means approved and accepted by local legislation in each individual country
and does not require an on-site visit of the patient at the study site.


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7.
STATISTICAL METHODS AND DETERMINATION OF
SAMPLE SIZE
The eligible patients for this trial will be randomised in Part 1 to one of three treatment arms 
-
alteplase 0.3mg/kg over 2 hours, followed by daily long-term (12h) infusion of 0.02 
mg/kg/h over 5 days, “alteplase dosing regimen A”
-
alteplase 0.6mg/kg over 2 hours, followed by daily long-term (12h) infusion of 0.04 
mg/kg/h over 5 days, “alteplase dosing regimen B”
-
standard of care
in a 1:1:1 ratio, stratified according to ventilation (invasive mechanical vs. non-invasive).
In Part 2, patients will be randomised to 
-
alteplase (one dosing regimen), depending on results of Part 1. Accordingly, the 
following dosing regimen will be carried forward in Part 2: alteplase 0.6mg/kg over 2 
hours, followed by daily long-term (12h) infusion of 0.04 mg/kg/h over 5 days, 
“alteplase dosing regimen B“
-
standard of care
in a 2:1 ratio, stratified according to baseline ventilation status (invasive mechanical vs. non-
invasive) and baseline D-Dimer level (≥ULN to <5 vs ≥5-fold ULN).
Throughout this whole Section, the NIV (non-invasive ventilation) patient cohort are those 
with a baseline WHO value of 6, and the IMV (invasive mechanical ventilation) patient
cohort are those with a value 7, 8 or 9 at baseline.
7.1
NULL AND ALTERNATIVE HYPOTHESES
Throughout this Section, alteplase refers to the randomised treatment group, which is in 
addition to SOC. With regards to Part 1, reference will be made to alteplase dosing regimen 
A and alteplase dosing regimen B and in Part 2, reference will be made to alteplase.
The primary endpoint related to Part 1, secondary endpoints, 
 will be 
evaluated in an exploratory manner.
For Part 2:
The primary objective of the statistical analysis is to determine whether alteplase significantly 
increases the chances of clinical improvement or hospital discharge up to Day 28, in 
comparison to SOC alone. The key secondary objectives of the statistical analysis are to 
determine whether alteplase significantly prevents treatment failure (defined as WHO value 
≥7) at Day 28, or all cause mortality at Day 28, in comparison to SOC alone.
For the primary analysis of the primary endpoint a Cox proportional hazards model will be 
used. In the NIV patient cohort, adjustment will be made for the number of days under NIV 
support, baseline D-Dimer level and age. For the IMV patient cohort, adjustment will be 
made for the baseline WHO value, baseline D-Dimer level and age.


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For the primary analysis of the key secondary endpoints, the delta method and average 
marginal effect method will be used to calculate the risk differences, the 95% confidence 
intervals and p-values. Adjustment will be made for the same set of default covariates as in 
the primary analysis of the primary endpoint.
In line with the primary and key secondary objectives of the statistical analysis, a
confirmatory hierarchical testing procedure will be applied for the following set of 
hypotheses, in the NIV patient cohort, in the following order:-
H0_1: 
The null hypothesis to be tested for the primary endpoint in the NIV patient cohort is that the 
hazard ratio between alteplase and SOC alone equals 1 for all t>0. The alternative hypothesis 
is that the hazard ratio between alteplase and SOC alone is not equal to 1 for all t>0. 
Superiority will be declared if the hazard ratio between alteplase and SOC alone is 
significantly greater than 1 at the two-sided type I error level alpha=5%.
H0_2:
The null hypothesis to be tested for the key secondary endpoint ‘Treatment failure at Day 28’ 
in the NIV patient cohort is that the risk difference between alteplase and SOC alone equals 
0. The alternative hypothesis is that the risk difference between alteplase and SOC alone is 
not equal to 0. Superiority will be declared if the risk difference between alteplase and SOC 
alone is significantly less than 0 at the two-sided type I error level alpha=5%.
H0_3:
The null hypothesis to be tested for the key secondary endpoint ‘All cause mortality at Day 
28’ in the NIV patient cohort is that the risk difference between alteplase and SOC alone
equals 0. The alternative hypothesis is that the risk difference between alteplase and SOC 
alone is not equal to 0. Superiority will be declared if the risk difference between alteplase
and SOC alone is significantly less than 0 at the two-sided type I error level alpha=5%.
In the first step, the primary endpoint hypothesis (H0_1) will be tested at the 2-sided type I 
error level of alpha=5%. If the null hypothesis (H0_1) is rejected, the key secondary endpoint 
hypothesis (H0_2) based on the endpoint Treatment failure at Day 28 will then be tested in a 
second step, again at alpha=5%. If the null hypothesis (H0_2) is rejected, the remaining key 
secondary endpoint hypothesis (H0_3) based on the endpoint all cause mortality at Day 28
will then be tested in a third step, again at alpha=5%.
7.2
PLANNED ANALYSES
7.2.1
General considerations
With regards to each efficacy and safety endpoint, the term "baseline" refers to the last 
observed measurement prior to randomisation for all patients.
The statistical analysis will be based on the following populations:


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The Randomised Set (RS) will consist of all randomised patients.
The Full Analysis Set (FAS) will consist of all randomised patients with at least one baseline 
and one post baseline assessment relating to the primary endpoint of interest - refer to 
Section 2.1.2 for primary endpoints related to Part 1 and Part 2.
The Treated Set (TS) will consist of all patients who were randomised and, for patients in the 
alteplase groups, treated with at least one dose of trial drug.
Per Protocol Set (PPS) will consist of all patients in FAS who were without important 
protocol deviations (IPDs) for efficacy. The definition of IPDs will be specified in the Trial 
Statistical Analysis Plan (TSAP) but this PPS will only be determined for Part 2.
All endpoints will be presented for Parts 1 and Part 2 separately, and at the end of Part 2 most 
endpoints will additionally be pooled for descriptive purposes.
All analyses for all endpoints, both inferential and descriptive, including safety, will be 
performed on the NIV patient cohort, and the IMV patient cohort separately. This is in line 
with the statistical objectives of the study.
For selected endpoints, if homogeneity between the two patient cohorts is observed, 
combined treatment estimates will be determined using suitable meta-analysis techniques. 
These will be outlined in more detail in the Trial Statistical Analysis Plan.
7.2.2
Primary endpoint analyses
For primary endpoints related to Parts 1 and 2, refer to Section 2.1.2. The primary analyses 
will be performed on the FAS with treatment assignments as randomised.
Part 1
The primary endpoint for Part 1 will be analysed in a similar manner to Part 2, albeit in Part 1 
this will be exploratory in nature, comparing alteplase dosing regimen A to SOC alone, and 
alteplase dosing regimen B to SOC alone. 
Part 2
For the analysis of the primary endpoint of Part 2, patients that have not met the endpoint will 
be censored at Day 28 if they die prior to Day 28. Patients receiving bail-out therapy (as 
defined in Section 4.2.2) without having first met the endpoint, will be censored on the day of 
bail-out. This addresses a hypothetical estimand for the scenario of what would have 
happened had the bail-out not been administered. Since patients requiring bail-out would be 
expected to be those already deteriorating, this non-informative censoring could be 
considered a conservative approach to assessing this estimand. Patients who have not had 
clinical improvement by Day 28 will be censored on Day 28.


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A cox proportional hazards model will be used to estimate the hazard ratio comparing 
alteplase to SOC alone. From this model, the 95% confidence interval and corresponding 
Wald p-value for this HR will be produced. In the NIV cohort, adjustment will be made for 
the number of days under NIV support, baseline D-Dimer level and age. For the IMV cohort, 
adjustment will be made for the baseline WHO value, baseline D-Dimer level and age.
The Cox proportional hazard model assumptions will be checked. 
Kaplan Meier estimates will be presented for this endpoint up to Day 28.
In anticipation of the rare situation where bail-out therapy may be used, as defined in Section 
4.2.2, two non-confirmatory supplementary analyses will be conducted, addressing the 
treatment policy estimand and a composite estimand whereby bail-out represents treatment 
failure and thereby, by definition, precludes any response after bail-out. 
In the first of these supplementary analyses, addressing the treatment policy estimand, there 
will be no consideration for the intake of bail-out therapy. 
In the second of these supplementary analyses, addressing the composite estimand, patients 
receiving bail-out therapy will be censored at Day 28, rather than on the day of bail-out.
A sensitivity analysis of the primary endpoint (main estimand) will be carried out on the PPS, 
in line with the primary analysis. 
Following the amendment to the inclusion criteria (CTP Version 4.0), whereby the original 
D-Dimer level changed from ≥3-ULN to ≥ULN, some additional analyses will be performed. 
Firstly, a sensitivity analysis will be added, similar to the primary analysis, just replacing the 
baseline D-Dimer status on the two levels with that on the three levels (≥ULN to <3, ≥3 to 
<5, ≥5-ULN). Secondly, a supplementary analysis will be added, similar to the primary 
analysis, but removing all patients with baseline D-dimer status ≥ULN to <3-ULN, which is 
according to the expanded inclusion criteria.
7.2.3
Secondary endpoint analyses
For the list of key secondary and secondary endpoints, refer to Section 2.1.3. Summary 
statistics will be presented for each endpoint, and at each visit if appropriate. 
The key secondary endpoints are both binary in nature and will be analysed using frequency 
tables, risk differences, 95% confidence intervals and p-values. The delta method and average 
marginal effect (AME) method will be used. In the NIV cohort, adjustment will be made for 
the number of days under NIV support, baseline D-Dimer level and age. For the IMV cohort, 
adjustment will be made for the baseline WHO value, baseline D-Dimer level and age.


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For the safety endpoint (MBE to Day 6) which is binary, the Chang and Zhang exact method 
will be used, without adjustment for covariates.
The continuous endpoints will be analysed descriptively and inferentially, albeit in a non-
confirmatory manner. A restricted maximum likelihood (REML) based ANCOVA will be 
used. In the NIV cohort, adjustment will be made for the number of days under NIV support, 
baseline D-Dimer level and age. For the IMV cohort, adjustment will be made for the 
baseline WHO value, baseline D-Dimer level and age. The treatment difference with 95% 
confidence interval will be presented.
More detailed descriptions will be provided in the TSAP.
7.2.5
Safety analyses
Adverse events will be coded using the Medical Dictionary for Drug Regulatory Activities
(MedDRA). Standard BI summary tables and listings will be produced. All adverse events
with an onset between start of treatment and end of the REP, as defined in Section 1.2,, will
be assigned to the on-treatment period for evaluation.
All treated patients will be included in the safety analysis. In general, safety analyses will be 
descriptive in nature and will be based on BI standards. No hypothesis testing is planned.
Statistical analysis and reporting of adverse events will concentrate on treatment-emergent 
adverse events, i.e. all adverse events occurring between start of treatment and end of the 
REP. Adverse events that start before first drug administration and deteriorate under 
treatment will also be considered as ‘treatment-emergent’.
Frequency, severity, and causal relationship of adverse events will be tabulated by system 
organ class and preferred term after coding according to the current version of MedDRA at 
DBL.


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Laboratory data will be analysed quantitatively and qualitatively. The latter will be done via 
comparison of laboratory data to their reference ranges. Values outside the reference range as 
well as values defined as clinically relevant will be summarised. Treatment arms will be 
compared descriptively with regard to distribution parameters as well as with regard to 
frequency and percentage of patients with abnormal values or clinically relevant abnormal 
values.
Vital signs, physical examinations, or other safety-relevant data observed at screening, 
baseline, during the course of the trial and at the end-of-trial evaluation will be assessed with 
regard to possible changes compared with findings before start of treatment.
Further details on the safety analysis will be specified in the TSAP.
7.2.6
Interim Analyses
One interim analysis is planned after the first (approximately) 60 patients (Part 1). An 
independent DMC will review the efficacy and safety data and provide a recommendation 
regarding the continuation of the trial into Part 2 and the corresponding dose for the alteplase
treatment arm. The DMC charter will lay out the general principles of decision-making of the 
DMC. Further details on the DMC are described in Section 8.7.
7.3
HANDLING OF MISSING DATA
For the analysis of the primary endpoint, time to clinical improvement or hospital discharge 
up to Day 28, patients will be censored at Day 28 if they die prior to Day 28, at the time they 
received bail-out therapy, or have not had clinical improvement by Day 28.
With respect to safety evaluations, it is not planned to impute missing values.
7.4
RANDOMISATION
BI will arrange for the randomisation and the packaging and labelling of trial medication. The 
randomisation list will be generated using a validated system, which involves a 
pseudo-random number generator so that the resulting treatment will be reproducible and 
non-predictable. The block size will be documented in the CTR. Access to the codes will be 
controlled and documented.
Randomisation in Part 1 will be to the three treatment arms of:

alteplase 0.3mg/kg over 2 hours followed by 0.02 mg/kg/h over 12 hours “dosing
regimen A”

alteplase 0.6 mg/kg over 2 hours followed by 0.04 mg/kg/h over12 hours “dosing 
regimen B”

SOC


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With an allocation ratio of 1:1:1, with stratification by the following factor:

Type of ventilation (non-invasive or invasive mechanical)
Randomisation in Part 2 will be to alteplase and to standard of care, with an allocation ratio 
of 2:1, with stratification by the following factors: 

Type of ventilation (non-invasive or invasive mechanical)

D-Dimer levels (≥ULN to <5-fold ULN, versus ≥5-fold ULN)
7.5
DETERMINATION OF SAMPLE SIZE
Part 1 of the study plans to randomise approximately 60 patients in total, with a view to 
exploring the data in a descriptive manner.
Sample size considerations are limited to Part 2 and the NIV patient cohort.
Part 2 has a planned confirmatory analysis at the 2-sided type I error level of alpha=5% with 
a power of 85% to 90%, applicable to the first two steps within the pre-planned hierarchial 
testing procedure. The trial uses a randomisation allocation ratio of 2:1, which leads to 
approximately 140 NIV patients on the treatment arm and approximately 70 NIV patients on 
the SOC alone. 
With regard to the primary analysis of the primary endpoint, H0_1 in the hierarchy testing 
procedure, in the NIV patient cohort, a hazard ratio of 2.0 is assumed, with event rates in the 
SOC alone group and alteplase group of approximately 30% and 50%. In the alteplase arm, it 
is expected to have a median time to event of 20 to 30 days.
With regard to the primary analysis of the key secondary endpoint “Treatment failure at Day
28 ”, H0_2 in the hierarchy testing procedure, in the NIV patient cohort, a risk difference of -
20% is assumed, with event rates in the SOC alone group and alteplase group of
approximately 40% and 20%. 
With regard to the primary analysis of the key secondary endpoint “All cause mortality at 
Day 28”, H0_3 in the hierarchy testing procedure, in the NIV patient cohort, there would be a
power of 85% under the assumptions of a risk difference of -16%. This would be with event 
rates in the SOC alone group and alteplase group of approximately 25% and 9%. These are 
very similar event rates as observed in Part 1.
The sample size determination or justification is in line with the null hypotheses and 
hierarchial testing approach as defined in Section 7.1, whereby it is planned to test for 
superiority of alteplase vs SOC alone in the NIV patient cohort. Whilst the assumptions are 
still rather approximate, the magnitude of these assumptions is similar to the available results 


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in other COVID-19 patients [R20-2001, R20-2246] and to the results observed in Part 1 of 
this study. In this emerging disease and during this pandemic, standard of care is evolving, 
and other factors are having an influence, for example, the introduction of vaccines.
Calculations were performed using the software R Version 3.6.1 and nQuery Version 4.0.
Only patients randomised into Part 1 will be included in the analyses of Part 1, and only 
patients randomised into Part 2 will be included in the confirmatory analyses of Part 2.


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8.
INFORMED CONSENT, TRIAL RECORDS, DATA 
PROTECTION, PUBLICATION POLICY, AND
ADMINISTRATIVE STRUCTURE
The trial will be carried out in compliance with the protocol, the ethical principles laid down 
in the Declaration of Helsinki, in accordance with the ICH Harmonized Guideline for GCP,
relevant BI SOPs, the EU directive 2001/20/EC / EU regulation 536/2014 and other relevant 
regulations. Investigators and site staff must adhere to these principles. Deviation from the 
protocol, the principles of ICH-GCP or applicable regulations as will be treated as “protocol 
deviation”.
Standard medical care (prophylactic, diagnostic and therapeutic procedures) remains the 
responsibility of the treating physician of the patient.
The investigator should inform the sponsor immediately of any urgent safety measures taken 
to protect the trial patients against any immediate hazard, as well as of any serious breaches 
of the protocol or of ICH-GCP.
The BI transparency and publication policy can be found on the following web page: 
https://trials.boehringer-ingelheim.com/. The rights of the investigator and of the sponsor
with regard to publication of the results of this trial are described in the investigator contract. 
As a rule, no trial results should be published prior to finalisation of the CTR.
The certificate of insurance cover is made available to the investigator and the patients and is
stored in the ISF.
8.1
TRIAL APPROVAL, PATIENT INFORMATION, INFORMED CONSENT
This trial will be initiated only after all required legal documentation has been reviewed and 
approved by the respective IRB / EC and Competent Authority (CA) according to national 
and international regulations. The same applies for the implementation of changes introduced 
by amendments.
Prior to patient participation in the study, each prospective subject or their legally acceptable 
representative (in accordance with national legislation and ICH GCP) will be provided with 
all information relating to the study and a copy of the patient information and consent form. 
Once the essential study information has been provided and the investigator is assured that 
each subject, or their legally acceptable representative, understands the implications of
participating in the study, the subject, or their legally acceptable representative, will be asked
to give consent to participate in the study by signing the informed consent form.
An independent impartial witness may be required in the event that the patient is able to give 
verbal consent, but unable to sign the consent form. In this case, the independent impartial 
witness will sign a form to indicate that the information was provided to the patient, the 


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patient appeared to understand the information and gave consent to participate in the study. 
The consent from the subject may be given verbally or by means of an unequivocal sign such 
as nodding or raising their hand in response to a question. The method used must be approved 
by the ethics committee and documented in the medical records.
If a short written informed consent document is initially used, once the patient recovers the 
investigator or delegate must discuss the study and sign the full patient consent and 
information sheet with the patient in order for patient to continue in the study.  A note of the 
type of written informed consent obtained will be made in the subject’s medical
history/patient notes and recorded in the CRF. 
An optional assessment of pharmacokinetics also is planned to be performed in at least 10 
patients per dose group in Part 1 and in approximately 17 patients in Part 2. This 
pharmacokinetic sampling is voluntary and is not a prerequisite for study participation.  
Sampling will only occur after a separate pharmacokinetic informed consent has been given 
in accordance with the local ethics and regulatory requirements. Please refer to Section 5.3.1
for more details on pharmacokinetic sampling process.
All signatures must be dated by each signatory and all completed consent forms will need to 
be retained by the Investigator and copies provided to the subject or the subject’s legally 
acceptable representative.
Re-consenting may become necessary when new relevant information becomes available and 
should be conducted according to the sponsor’s instructions.  The consent and re-consenting 
process should be properly documented in the source documentation.
8.2
DATA QUALITY ASSURANCE
A risk-based approach is used for trial quality management. It is initiated by the assessment 
of critical data and processes for trial patient protection and reliability of the results as well as 
identification and assessment of associated risks. An Integrated Quality and Risk 
Management Plan documents the rationale and strategies for risk management during trial 
conduct including monitoring approaches, vendor management and other processes focusing 
on areas of greatest risk. 
Continuous risk review and assessment may lead to adjustments in trial conduct, trial design 
or monitoring approaches. 
A quality assurance audit/inspection of this trial may be conducted by the sponsor, sponsor’s 
designees, or by IRB / EC or by regulatory authorities. The quality assurance auditor will 
have access to all medical records, the investigator’s trial-related files and correspondence, 
and the informed consent documentation of this clinical trial.
8.3
RECORDS
Electronic CRFs for individual patients will be provided by the sponsor. See Section 4.1.5.2
for rules about emergency code breaks. For drug accountability, refer to Section 4.1.8.


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8.3.1
Source documents
In accordance with regulatory requirements, the investigator should prepare and maintain 
adequate and accurate source documents and trial records that include all observations and 
other data pertinent to the investigation on each trial patient. Source data as well as reported 
data should be attributable, legible, contemporaneous, original and accurate (ALCOA). 
Changes to the data should be traceable (audit trail).
Data reported on the eCRF must be consistent with the source data or the discrepancies must 
be explained.
The current medical history of the patient may not be sufficient to confirm eligibility for the 
trial and the investigator may need to request previous medical histories and evidence of any 
diagnostic tests. In this case, the investigator must make at least one documented attempt to 
retrieve previous medical records. If this fails, a verbal history from the patient or his legal 
representative, documented in their medical records, would be acceptable.
The investigator must ensure that all patient identifiers (e.g. patient’s name, initials, address, 
phone number, social security number) have properly been removed or redacted from any 
copy of the patients’ source documents before sending them to the sponsor.
If the patient is not compliant with the protocol, any corrective action (e.g. re-training) must 
be documented in the patient file.
Due to Covid-19 restrictions study conduct, including site monitoring and access to source 
documents, may need to be adjusted accordingly.  See Appendix 10.4 for further details.
For the eCRF, data must be derived from source documents, for example:

Patient identification: gender, year of birth (in accordance with local laws and 
regulations)

Patient participation in the trial (substance, trial number, patient number, date patient
was informed)

Dates of patient’s visits, including dispensing of trial medication

Medical history (including trial indication and concomitant diseases, if applicable)

Medication history

Adverse events and OEs onset date (mandatory), and end date (if available)

Serious adverse events onset date (mandatory), and end date (if available)

Concomitant therapy (start date, changes)

Originals or copies of laboratory results and other imaging or testing results, with 
proper documented medical evaluation (in validated electronic format, if available)

Completion of patient’s participation in the trial” (end date; in case of premature 
discontinuation document the reason for it).

Prior to allocation of a patient to a treatment into a clinical trial, there must be 
documented evidence in the source data (e.g. medical records) that the trial participant 
meets all inclusion criteria and does not meet any exclusion criteria. The absence of 


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records (either medical records, verbal documented feedback of the patient or testing 
conducted specific for a protocol) to support inclusion/exclusion criteria does not 
make the patient eligible for the clinical trial.
8.3.2
Direct access to source data and documents
During the conduct of the study, it may not be possible for the investigator / institution to 
allow site trial-related monitoring, audits, IRB/EC review or regular inspections due to 
COVID-19 restrictions. However, once site visits are allowed again, the site will need to 
provide a direct access to the eCRF and all source documents/data, including progress notes, 
copies of laboratory and medical test results, which must be available at all times for review 
by the CRA, auditor and regulatory inspector (e.g. FDA). They may review all eCRFs and 
informed consents. The accuracy of the data will be verified by direct comparison with the 
source documents described in Section 8.3.1. The sponsor will also monitor compliance with 
the protocol and GCP.
8.3.3
Storage period of records
Trial sites:
The trial sites must retain the source and essential documents (including ISF) according to 
contract or the local requirements valid at the time of the end of the trial (whatever is longer).
Sponsor:
The sponsor must retain the essential documents according to the sponsor’s SOPs.
8.4
EXPEDITED REPORTING OF ADVERSE EVENTS
BI is responsible to fulfil their legal and regulatory reporting obligation in accordance with
regulatory requirements. The rules for Adverse Event Reporting exemptions still apply, 
please see Section 5.2.6.2.4.
8.5
STATEMENT OF CONFIDENTIALITY AND PATIENT PRIVACY
Data protection and data security measures are implemented for the collection, storage and 
processing of patient data in accordance with the Principles 7 and 12 of the WHO GCP 
handbook.
Individual patient data obtained as a result of this trial is considered confidential and 
disclosure to third parties is prohibited with the following exceptions:

Personalised treatment data may be given to the patient’s personal physician or to 
other appropriate medical personnel responsible for the patient’s welfare. 

Data generated at the site as a result of the trial need to be available for inspection on 
request by the participating physicians, the sponsor’s representatives, by the IRB / EC 
and the regulatory authorities.


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8.5.1
Collection, storage and future use of biological samples and corresponding 
data 
Measures are in place to comply with the applicable rules for the collection, storage and 
future use of biological samples and clinical data, in particular:

The BI-internal facilities storing biological samples from clinical trial participants as 
well as the laboratorie`s activities for clinical trials sponsored by Boehringer 
Ingelheim are regularly audited and qualified for the storage of biological samples 
collected in clinical trials.

An appropriate sample and data management system, including audit trail for clinical 
data and samples to identify and destroy such samples according to ICF is in place.

A fit for the purpose documentation (analysis plan and report) ensures compliant 
usage.

Samples and data are used only if an appropriate informed consent is available.
8.6
TRIAL MILESTONES
The start of the trial is defined as the date when the FPI, in the whole trial, signs informed 
consent.
The end of the trial is defined as the date of the last visit of the last patient in the whole trial
(“Last Patient Completed”).
The “Last Patient Last Treatment” (LPLT) date is defined as the date on which the last 
patient in the whole trial is administered the last dose of trial treatment (as scheduled per 
protocol or prematurely). Individual investigators will be notified of SUSARs occurring with 
the trial medication until 30 days after LPLT at their site.
Early termination of the trial is defined as the premature termination of the trial due to any 
reason before the end of the trial as specified in this protocol.
Temporary halt of the trial is defined as any unplanned interruption of the trial by the 
sponsor with the intention to resume it.
Suspension of the trial is defined as an interruption of the trial based on a Health Authority
request.
The EC / Competent Authority in each participating EU member state will be notified about 
the trial milestones according to the respective laws.
A final report of the clinical trial data will be written only after all patients have completed 
the trial in all countries (EU or non-EU) to incorporate and consider all data in the report.
The sponsor will submit to the EU database a summary of the final trial results within one 
year from the end of a clinical trial as a whole, regardless of the country of the last patient
(EU or non-EU).


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8.7
ADMINISTRATIVE STRUCTURE OF THE TRIAL
The trial is sponsored by BI.
A Coordinating Investigator is responsible to coordinate investigators at the different sites 
participating in this trial. Tasks and responsibilities are defined in a contract. 
A Steering Committee (SC) consisting of independent experts and sponsor representatives 
will be established to support the Coordinating Investigator who will be the chair of the SC. 
The composition of the SC will be documented in the Trial Master File. The tasks and 
responsibilities will be agreed in contracts between the SC members and the sponsor and also 
summarised in a SC Charter.
A DMC will be established. Members of the DMC are independent of BI, they are physicians 
experienced in the treatment of the disease under investigation and/or thrombolytic 
treatments and a statistician. 
The DMC will evaluate safety and efficacy data as well as the results of the interim analysis.
The DMC will receive urgent significant safety events, including cases of intracranial 
haemorrhage and fatal bleeds for immediate evaluation. Regular DMC meetings will be held 
at specified intervals. The DMC will also review at specified intervals the exempted events to 
SAE reporting as specified in Section 5.2.6.2.4. The DMC will recommend continuation, 
modification or termination of the trial as detailed in the DMC charter. DMC 
recommendations as well as the final BI decision will be reported to the appropriate 
Regulatory Authorities (RAs)/Health Authorities (HAs), IRBs/ECs, and to investigators as 
requested by local law. The tasks and responsibilities of the DMC are specified in a charter.
Relevant documentation on the participating (Principal) Investigators (e.g. their curricula 
vitae) will be filed in the ISF.
The investigators will have access to the BI web portal Clinergize to access documents 
provided by the sponsor.
BI has appointed a Clinical Trial Leader (CT Leader), responsible for coordinating all 
required activities, in order to 
-
manage the trial in accordance with applicable regulations and internal SOPs,
-
direct the clinical trial team in the preparation, conduct, and reporting of the trial,
-
ensure appropriate training and information of Clinical Trial Managers, CRAs, and 
investigators of participating countries.
The organisation of the trial in the participating countries will be performed by the respective 
local or regional BI-organisation (Operating Unit, OPU) in accordance with applicable 
regulations and BI SOPs, or by a CRO with which the responsibilities and tasks will have 
been agreed and a written contract filed before initiation of the clinical trial.
Data Management and Statistical Evaluation will be done by BI according to BI SOPs.


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Tasks and functions assigned in order to organise, manage, and evaluate the trial are defined 
according to BI SOPs. A list of responsible persons and relevant local information can be 
found in the ISF.
An IRT vendor will be used in this trial. Details will be provided in the IRT Manual and, 
available in the ISF.


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9.
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9.1
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anticoagulation with heparin in critically ill patients with Covid-19. N Engl 
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Nonintubated Patients With COVID-19 Hypoxemic Respiratory Failure. 
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Concentration Is Dependent on the Oxygen Mask System and Sampling 
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9.2
UNPUBLISHED REFERENCES
c32479735-01
Simulations in support of dose and schedule selection for alteplase in 
patients with ARDS applying a mathematical pharmacokinetic/
pharmacodynamic model for fibrinolytic proteins.
c32361824
Investigator brochure (current version)
U85-0697
. Report on a pharmacokinetic study of
recombination human tissue type plasminogen activator (RT-PA)
(G11021). 1985


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U86-0354
 Pharmacokinetics of recombinant tissue type plasminogen
activator (0.25 mg/kg) after a10 minute intravenous infusion of 2 different
formulations in healthy volunteers. 1986
U87-0368
 Pharmacokinetics, pharmacodynamics and tolerance of a 30
minute infusion of 0.25 mg/kg bodyweight and 0.50 mg/kg bodyweight of
rt-PA in healthy volunteers. 1987
U87-0935
A pharmacokinetic and safety
study of recombinant human tissue type plasminogen activator in human
volunteers. 1985
U95-2167 
 Efficacy of fibrinolytic therapy with rt-PA in 
patients with deep vein thrombosis of lower and upper extremities. 1995
U99-0193 
 A Phase III, randomized, double-blind, parallel-group,
international trial of single bolus of TNK-tissue plasminogen activator
(TNK-tPA) VS accelerated infusion of rt-PA (Alteplase: Activase, Actilyse)
in acute myocardial infarction: Assent II (Assessment of the safety and
efficacy of a new thrombolytic agent). 19 June 1999.
U99-1584 
Comparison of the efficacy and
safety of intravenous locoregional administration of 20 mg rt-PA, 40 mg rt-
PA, compared intravenous, systemic administration of 9 million units
Streptokinase given over 6 hours given up to 5 days in patients with deep
vein thrombosis of the lower extremities. 30 June 1999.
c35578199-01
Interim report of the TRISTARDS 
trial – ThRombolysIS Therapy for ARDS. A Phase IIb/III operationally 
seamless, open-label, randomised, sequential, parallel-group adaptive study 
to evaluate the efficacy and safety of daily intravenous alteplase treatment
given up to 5 days on top of standard of care (SOC) compared with SOC 
alone, in patients with acute respiratory distress syndrome (ARDS) 
triggered by COVID-19. 14 September 2021


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10.
APPENDICES
10.1
SOFA SCORE AND GLASGOW COMA SCALE
The SOFA score R20-1695] is calculated as follows:
GLASGOW COMA SCALE
The GCS score [R20-1697, R20-1710is calculated as follows:


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10.2
TABLE TO DERIVE PAO2/FIO2 RATIO FROM SPO2
The PaO2/FiO2 ratio will be inferred from SpO2, if arterial blood gases (ABG) are
unavailable, based on the following table [R20-1709]:
Measured SpO2 (%)
Imputed PaO2 (mmHg)
100*
167*
99*
134*
98*
104*
97*
91*
96
82
95
76
94
71
93
67
92
64
91
61
90
59
89
57
88
55
87
53
86
51
85
50
84
49
83
47
82
46
81
45
80
44
79
43


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10.2
TABLE TO DERIVE PAO2/FIO2 RATIO FROM SPO2 (cont)
Measured SpO2 (%)
Imputed PaO2 (mmHg)
78
42
77
42
76
41
75
40
74
39
73
39
72
38
71
37
70
37
*Generally considered unreliable on the basis of the 
sigmoidal shape of the hemoglobin-oxygen 
dissociation curve.
§Based on SpO2 99.5%.
10.3
FIO2 RANGES FOR COMMON OXYGEN DELIVERY DEVICES
Fraction of Inspired Oxygen (FiO2) for common oxygen delivery systems are given in the 
tables below [R22-0456]:
10.4
POTENTIAL MODIFICATION OF TRIAL CONDUCT IN CASE OF 
RESTRICTIONS DUE TO COVID-19
Due to restrictions during the COVID-19 pandemic, study conduct may need to be adjusted. 


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In exceptional cases, when it is impossible to conduct the visits for discharged patients at the 
trial site, visits may be performed at the patient’s home or remotely (via telephone and/or 
internet based means of communication). Based on the benefit-risk assessment (see Section 
1.4.1), the visit procedures may be adjusted for the purpose of particular visits, whereby 
critical safety measures will remain in place. All home/remote visits need to be discussed 
with and approved by the sponsor`s trial team. Local regulatory and legal requirements of the 
participating country need to be respected for all modifications.
Under these circumstances, the following modifications can be considered: 
Remote visit
If a patient is not able to come to the site for an outpatient visit, a remote visit (by phone) 
should be performed instead and all assessments that can be done by phone should be 
performed.
Assessments that can be performed during a remote visit are:
Collection of adverse events, concomitant therapy, local laboratory. If home visits by trial 
staff members are possible, further assessments can be done. 
Safety lab, other laboratory tests
Blood analysis for safety laboratory can be done in a local laboratory outside the hospital.
The results of the laboratory tests are to be reported and transferred to the investigator, who 
has to ensure medical review and proper documentation in the eCRF. 
Monitoring
Due to the nature of this trial and the risks associated with Covid-19, site monitoring visits as 
physical on-site interaction may not always be possible. The focus will be on centralized 
oversight/monitoring. If not restricted by local Covid-19 regulations, we recommend 
combination of remote and on-site visits, mainly for initiation and close-out visits. Regular 
interim monitoring visits will occur mainly remotely unless the trial team requires a visit to 
be done on-site due to an identified risk where the required mitigation may need to be 
performed on-site.
Trial monitoring intervals may be adjusted depending on enrolment rates, data quality, 
recruitment and conduct phases, timelines for CRF completion and/or site quality. 


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10.5
ALTEPLASE DOSING TABLE
Dosing regimen A (lower dose) dosing table (only applicable in Part 1 of the trial)
TRISTARDS trial DOSING TABLE for Alteplase (Dosing regimen A - lower dose)
(1mg/mL concentration)
Weight
(kg)*
Short-term infusion over 2 hours
(Day 1 only / or optional re-application)
Long-term infusion over 12 hours
(Daily from Day 1 up to day 5)
Infusion Dose
over 2 hours (mg)
(0.3 mg/kg)
Infusion Rate
(mL/hour)
Infusion Dose
over 12 hours (mg)
(0.02 mg/kg)
Infusion Rate
(mL/hour)
40
12.0
6.0
9.6
0.8
42
12.6
6.3
10.1
0.8
44
13.2
6.6
10.6
0.9
46
13.8
6.9
11.0
0.9
48
14.4
7.2
11.5
1.0
50
15.0
7.5
12.0
1.0
52
15.6
7.8
12.5
1.0
54
16.2
8.1
13.0
1.1
56
16.8
8.4
13.4
1.1
58
17.4
8.7
13.9
1.2
60
18.0
9.0
14.4
1.2
62
18.6
9.3
14.9
1.2
64
19.2
9.6
15.4
1.3
66
19.8
9.9
15.8
1.3
68
20.4
10.2
16.3
1.4
70
21.0
10.5
16.8
1.4
72
21.6
10.8
17.3
1.4
74
22.2
11.1
17.8
1.5
76
22.8
11.4
18.2
1.5
78
23.4
11.7
18.7
1.6
80
24.0
12.0
19.2
1.6
82
24.6
12.3
19.7
1.6
84
25.2
12.6
20.2
1.7
86
25.8
12.9
20.6
1.7
88
26.4
13.2
21.1
1.8
90
27.0
13.5
21.6
1.8
92
27.6
13.8
22.1
1.8
94
28.2
14.1
22.6
1.9
96
28.8
14.4
23.0
1.9


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10.5 
ALTEPLASE DOSING TABLE (cont)
TRISTARDS trial DOSING TABLE for Alteplase (Dosing regimen A - lower dose)
(1mg/mL concentration)
Weight
(kg)*
Short-term infusion over 2 hours
(Day 1 only / or optional re-application)
Long-term infusion over 12 hours
(Daily from Day 1 up to day 5)
Infusion Dose
over 2 hours (mg)
(0.3 mg/kg)
Infusion Rate
(mL/hour)
Infusion Dose
over 12 hours (mg)
(0.02 mg/kg)
Infusion Rate
(mL/hour)
98
29.4
14.7
23.5
2.0
100
30.0
15.0
24.0
2.0
102
30.6
15.3
24.5
2.0
104
31.2
15.6
25.0
2.1
106
31.8
15.9
25.4
2.1
108
32.4
16.2
25.9
2.2
110
33.0
16.5
26.4
2.2
112
33.6
16.8
26.9
2.2
114
34.2
17.1
27.4
2.3
116
34.8
17.4
27.8
2.3
118
35.4
17.7
28.3
2.4
≥120
36.0
18.0
28.8
2.4
*actual weight is preferred 
(estimated weight is acceptable, if actual weight is not available).
Patients which body weight is in between two categories (e.g. 51 kg) should be 
administered the dosage of the next higher dosing category (e.g. 52 kg).


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10.5 
ALTEPLASE DOSING TABLE (cont)
TRISTARDS trial DOSING TABLE for Alteplase (Dosing regimen B - higher dose)
(1mg/mL concentration)
Weight
(kg)*
Short-term infusion over 2 hours
(Day 1 only / or optional re-application)
Long-term infusion over 12 hours
(Daily from Day 1 up to day 5)
Infusion Dose
over 2 hours (mg)
(0.6 mg/kg)
Infusion Rate
(mL/hour)
Infusion Dose
over 12 hours (mg)
(0.04 mg/kg)
Infusion Rate
(mL/hour)
40
24.0
12.0
19.2
1.6
42
25.2
12.6
20.2
1.7
44
26.4
13.2
21.1
1.8
46
27.6
13.8
22.1
1.8
48
28.8
14.4
23.0
1.9
50
30.0
15.0
24.0
2.0
52
31.2
15.6
25.0
2.1
54
32.4
16.2
25.9
2.2
56
33.6
16.8
26.9
2.2
58
34.8
17.4
27.8
2.3
60
36.0
18.0
28.8
2.4
62
37.2
18.6
29.8
2.5
64
38.4
19.2
30.7
2.6
66
39.6
19.8
31.7
2.6
68
40.8
20.4
32.6
2.7
70
42.0
21.0
33.6
2.8
72
43.2
21.6
34.6
2.9
74
44.4
22.2
35.5
3.0
76
45.6
22.8
36.5
3.0
78
46.8
23.4
37.4
3.1
80
48.0
24.0
38.4
3.2
82
49.2
24.6
39.4
3.3
84
50.4
25.2
40.3
3.4
86
51.6
25.8
41.3
3.4
88
52.8
26.4
42.2
3.5
90
54.0
27.0
43.2
3.6
92
55.2
27.6
44.2
3.7
94
56.4
28.2
45.1
3.8
96
57.6
28.8
46.1
3.8
98
58.8
29.4
47.0
3.9
100
60.0
30.0
48.0
4.0


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10.5
ALTEPLASE DOSING TABLE (cont)
TRISTARDS trial DOSING TABLE for Alteplase (Dosing regimen B - higher dose)
(1mg/mL concentration)
Weight
(kg)*
Short-term infusion over 2 hours
(Day 1 only / or optional re-application)
Long-term infusion over 12 hours
(Daily from Day 1 up to day 5)
Infusion Dose
over 2 hours (mg)
(0.6 mg/kg)
Infusion Rate
(mL/hour)
Infusion Dose
over 12 hours (mg)
(0.04 mg/kg)
Infusion Rate
(mL/hour)
102
61.2
30.6
49.0
4.1
104
62.4
31.2
49.9
4.2
106
63.6
31.8
50.9
4.2
108
64.8
32.4
51.8
4.3
110
66.0
33.0
52.8
4.4
112
67.2
33.6
53.8
4.5
114
68.4
34.2
54.7
4.6
116
69.6
34.8
55.7
4.6
118
70.8
35.4
56.6
4.7
≥120
72.0
36.0
57.6
4.8
*actual weight is preferred 
(estimated weight is acceptable, if actual weight is not available).
Patients which body weight is in between two categories (e.g. 51 kg) should be administered the 
dosage of the next higher dosing category (e.g. 52 kg).


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10.6
TREATMENT RECOMMENDATIONS FOR COVID-19 ARDS (WITH A 
FOCUS ON NIV PATIENTS)
Standard of Care should be harmonized as much as possible in each center and across centers. 
Changes in the Standard of Care during the study period are acceptable, if new evidence 
arises and/or new recommendations are published during this period.
Recommendations given below are based on international treatment recommendation as of 
September 2021. The Table below is intended to give the investigator an updated status of 
therapeutics used in COVID 19 that are recommended for local implementation.
Rating of Recommendations: A = Strong; B = Moderate; C = Optional. 
Rating of Evidence: I = One or more randomized trials without major limitations; IIa = 
Other randomized trials or subgroup analyses of randomized trials; IIb = Nonrandomized 
trials or observational cohort studies; III = Expert opinion
Table 10.6:1
Treatment Recommendations for COVID-19 ARDS (with a focus on 
NIV Patients)
SYSTEMIC CORTICOSTEROIDS
RECOMMEN-
DATION

The WHO-Guideline recommends systemic corticosteroids 
rather than no systemic corticosteroids for the treatment of 
patients with severe and critical COVID-19 (strong 
recommendation, based on moderate certainty evidence).

The NIH- Guideline recommends one of the following options 
for patients who require delivery of oxygen through a high-flow 
device or noninvasive ventilation but not invasive mechanical 
ventilation or extracorporeal membrane oxygenation
o
Dexamethasone (AI); or
o
Dexamethasone plus remdesivir (BIII).
The combination of remdesivir plus dexamethasone has 
not been rigorously studied in clinical trials; therefore, 
the safety and efficacy of this combination are 
unknown.


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Table 10.6:1
Treatment Recommendations for COVID-19 ARDS (with a focus on 
NIV Patients) (cont)
SYSTEMIC CORTICOSTEROIDS
ADDITIONAL 
CONSIDERATIONS

Immunosuppressive therapy (e.g., dexamethasone with 
or without baricitinib or tocilizumab) may increase the 
risk of opportunistic infections or reactivation of latent 
infections; however, randomized trials to date have not 
demonstrated an increase in the frequency of 
infections.

Cases of severe and disseminated strongyloidiasis have 
been reported in patients with COVID-19 during 
treatment with tocilizumab and corticosteroids. Many 
clinicians would initiate empiric treatment for 
strongyloidiasis (e.g., with ivermectin) with or without 
serologic testing in patients from areas where 
Strongyloides is endemic (i.e., tropical, subtropical, or 
warm temperate areas).
For contraindications, special warnings and precautions for use 
please refere to the label and further references.
DOSING
Dexamethasone 6 mg IV or PO once daily for up to 10 days or 
until hospital discharge.
If dexamethasone is not available, alternative glucocorticoids 
(e.g., prednisone, methylprednisolone, hydrocortisone) can be 
used.
For these drugs, the total daily dose equivalencies to 
dexamethasone 6 mg (oral or intravenous) are:

Prednisone 40 mg

Methylprednisolone 32 mg

Hydrocortisone 160 mg
REFERENCES
R20-4049, R21-2944, P22-01013, R22-0477


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Table 10.6:1
Treatment Recommendations for COVID-19 ARDS (with a focus on 
NIV Patients) (cont)
IL-6 INHIBITORS (E.G. TOCILIZUMAB OR SARILUMAB)
RECOMMENDATION

The WHO-Guideline recommends treatment with IL-6 
receptor blockers (tocilizumab or sarilumab) for 
patients with severe or critical COVID-19 infection. 
The WHO recommends patients meeting severity 
criteria should receive both corticosteroids and IL-6 
receptor blockers.

The NIH- Guideline recommends one of the following 
options for patients who require delivery of oxygen 
through a high-flow device or noninvasive ventilation 
but not invasive mechanical ventilation or 
extracorporeal membrane oxygenation
o
Dexamethasone (AI); or
o
Dexamethasone plus remdesivir (BIII).

For patients who have rapidly increasing oxygen needs 
and have increased markers of inflammation, add 
either baricitinib (BIIa) or tocilizumab (BIIa) (drugs 
are listed alphabetically) to 1 of the 2 options above.

The Panel recommends against the use of baricitinib in 
combination with tocilizumab for the treatment of 
COVID-19, except in a clinical trial (AIII). Because 
both baricitinib and tocilizumab are potent 
immunosuppressants, there is the potential for an 
additive risk of infection.
ADDITIONAL 
CONSIDERATIONS

Immunosuppressive therapy (e.g., dexamethasone 
with or without baricitinib or tocilizumab) may 
increase the risk of opportunistic infections or 
reactivation of latent infections; however, randomized 
trials to date have not demonstrated an increase in the 
frequency of infections.

Cases of severe and disseminated strongyloidiasis have 
been reported in patients with COVID-19 during 
treatment with tocilizumab and corticosteroids. Many 
clinicians would initiate empiric treatment for 
strongyloidiasis (e.g., with ivermectin) with or without 
serologic testing in patients from areas where 
Strongyloides is endemic (i.e., tropical, subtropical, or 
warm temperate areas).


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Table 10.6:1
Treatment Recommendations for COVID-19 ARDS (with a focus on 
NIV Patients) (cont)
IL-6 INHIBITORS (E.G. TOCILIZUMAB OR SARILUMAB)
ADDITIONAL 
CONSIDERATIONS

Baricitinib or tocilizumab should only be given in 
combination with dexamethasone or another 
corticosteroid. Some clinicians may assess a patient’s 
clinical response to dexamethasone before deciding 
whether adding baricitinib or tocilizumab as a second 
immunomodulatory drug is necessary.

Studies that directly compare baricitinib to tocilizumab 
as treatments for COVID-19 are not available. 
Therefore, the Panel has insufficient evidence to 
recommend one drug over the other. Treatment 
decisions should be made based on local guidance, 
drug availability, and patient comorbidities.

Even though the REMAP-CAP trial supports that 
sarilumab and tocilizumab have similar efficacy in the 
treatment of hospitalized patients with COVID-19, the 
Panel recommends sarilumab only when tocilizumab is 
not available or is not feasible to use (BIIa)
For contraindications, special warnings and precautions for 
use please refere to the label and further references.
DOSING
Tocilizumab is dosed at 8 mg per kilogram of actual body 
weight, up to a maximum of 800 mg. Sarilumab is most 
commonly dosed at 400 mg. 
REFERENCES
R21-2936, R21-2943, P22-01013, R22-0477


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Table 10.6:1
Treatment Recommendations for COVID-19 ARDS (with a focus on 
NIV Patients) (cont)
BARICITINIB (JAK INHIBITOR)
RECOMMENDATION
Please see recommendations for IL6-Inhibitors above.
ADDITIONAL 
CONSIDERATIONS
Please see additional considerations for IL6-Inhibitors above.

The Panel recommends against the use of the 
combination of baricitinib and tocilizumab for the 
treatment of COVID-19 except in a clinical trial (AIII), 
because there is insufficient evidence for the use of this 
combination. Given that both baricitinib and tocilizumab 
are potent immunosuppressants, there is the potential for 
an additive risk of infection.

If baricitinib and IV tocilizumab are not available or not 
feasible to use, tofacitinib can be used instead of 
baricitinib (BIIa) and IV sarilumab can be used instead 
of IV tocilizumab (BIIa). For contraindications, special 
warnings and precautions for use please refere to the 
label and further references.
DOSING

Baricitinib dose is dependent on eGFR; duration of 
therapy is up to 14 days or until hospital discharge.
eGFR ≥60 mL/min/1.73 m2: Baricitinib 4 mg PO once 
daily
eGFR 30 to <60 mL/min/1.73 m2: Baricitinib 2 mg PO 
once daily
eGFR 15 to <30 mL/min/1.73 m2: Baricitinib 1 mg PO 
once daily
eGFR <15 mL/min/1.73 m2: Baricitinib is not 
recommended.
REFERENCES
R21-3171, R21-3178, P22-01013, R22-0477
PROPHYLACTIC ANTICOAGULATION FOR PREVENTION OF DVT/VTE 
RECOMMENDATION

The results of three international randomized controlled 
trials (REMAP-CAP, ACTIV-4a, and ATTACC)
demonstrated that therapeutic-dose of anticoagulation 
with heparin in patients hospitalized with COVID-19 did 
not prevent progression of disease or death in patients 
with critical COVID-19 who are in the ICU, and that this 
dosing regimen might be associated with increased rates 
of significant bleeding [R21-2851].
REFERENCES
R21-2851


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Table 10.6:1
Treatment Recommendations for COVID-19 ARDS (with a focus on 
NIV Patients) (cont)
OXYGENATION, VENTILATION AND AWAKE PRONE POSITIONING
RECOMMENDATION
The COVID-19 Treatment Guidelines Panel (NIH Panel) and 
the Surviving Sepsis Campaign (SSC) recommend:

For adults with COVID-19 and acute hypoxemic 
respiratory failure despite conventional oxygen therapy, 
the Panel recommends HFNC oxygen over NIV (BIIa).

For adults with COVID-19 and acute hypoxemic 
respiratory failure who do not have an indication

for endotracheal intubation and for whom HFNC 
oxygen is not available, the Panel recommends
performing a closely monitored trial of NIV (BIIa).

For patients with persistent hypoxemia who require 
HFNC oxygen and for whom endotracheal intubation is 
not indicated, the Panel recommends a trial of awake 
prone positioning (BIIa). The Panel recommends 
against using awake prone positioning as a rescue 
therapy for refractory hypoxemia to avoid intubation in 
patients who otherwise meet the indications for 
intubation and mechanical ventilation (AIII). If 
intubation becomes necessary, the procedure should be 
performed by an experienced practitioner in a controlled 
setting due to the enhanced risk of exposing health care 
practitioners to SARS-CoV-2 during intubation (AIII).
REFERENCES
R21-2938, R21-3192, P22-01013, R21-3191, R22-0473


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Table 10.6:1
Treatment Recommendations for COVID-19 ARDS (with a focus on 
NIV Patients) (cont)
HEMODYNAMICS
RECOMMENDATION
The COVID-19 Treatment Guidelines Panel (NIH Panel) and 
the Surviving Sepsis Campaign (SSC) recommend:

For adults with COVID-19 and shock, the Panel 
recommends using dynamic parameters, skin 
temperature, capillary refilling time, and/or lactate 
levels over static parameters to assess fluid 
responsiveness (BIIa).

For the acute resuscitation of adults with COVID-19 
and shock, the Panel recommends using 
buffered/balanced crystalloids over unbalanced 
crystalloids (BIIa).

For the acute resuscitation of adults with COVID-19 
and shock, the Panel recommends against the initial 
use of albumin for resuscitation (BI).

For adults with COVID-19 and shock, the Panel 
recommends norepinephrine as the first-choice 
vasopressor (AI).

For adults with COVID-19 and shock, the Panel 
recommends titrating vasoactive agents to target a 
mean arterial pressure (MAP) of 60 to 65 mm Hg over 
higher MAP targets (BI).

The Panel recommends against using hydroxyethyl 
starches for intravascular volume replacement in 
patients with sepsis or septic shock (AI).

When norepinephrine is available, the Panel 
recommends against using dopamine for patients with 
COVID-19 and shock (AI).

As a second line vasopressor, the Panel recommends 
adding either vasopressin (up to 0.03 units/min) (BIIa) 
or epinephrine (BIIb) to norepinephrine to raise MAP 
to target or adding vasopressin (up to 0.03 units/min) 
(BIIa) to decrease norepinephrine dosage.

The Panel recommends against using low-dose 
dopamine for renal protection (AI).

The Panel recommends using dobutamine in patients 
who show evidence of cardiac dysfunction and 
persistent hypoperfusion despite adequate fluid loading 
and the use of vasopressor agents (BIII).

The Panel recommends that all patients who require 
vasopressors have an arterial catheter placed as soon as 
practical, if resources are available (BIII).


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Table 10.6:1
Treatment Recommendations for COVID-19 ARDS (with a focus on 
NIV Patients) (cont)

For adults with refractory septic shock who have 
completed a course of corticosteroids to treat their 
COVID-19, the Panel recommends using low-dose 
corticosteroid therapy (“shock-reversal”) over no 
corticosteroid therapy (BIIa).
REFERENCES
P22-01013, R21-3191, R22-0473
EMPIRIC BROAD-SPECTRUM ANTIMICROBIAL THERAPY
RECOMMENDATION
NIH guideline recommendations:

In patients with COVID-19 and severe or critical 
illness, there is insufficient evidence for the Panel to 
recommend either for or against empiric broad-
spectrum antimicrobial therapy in the absence of 
another indication.

If antimicrobials are initiated, the Panel recommends 
that their use should be reassessed daily to minimize 
the adverse consequences of unnecessary antimicrobial 
therapy (AIII).
REFERENCES
P22-01013
ACUTE KIDNEY INJURY AND RENAL REPLACEMENT THERAPY
RECOMMENDATION
NIH guideline recommendations:

For critically ill patients with COVID-19 who have 
acute kidney injury and who develop indications for 
renal replacement therapy, the Panel recommends 
continuous renal replacement therapy (CRRT), if 
available (BIII).

If CRRT is not available or not possible due to limited 
resources, the Panel recommends prolonged 
intermittent renal replacement therapy rather than 
intermittent hemodialysis (BIII).
REFERENCES
P22-01013
EXTRACORPOREAL MEMBRANE OXYGENATION
RECOMMENDATION
NIH guideline recommendations:

There is insufficient evidence for the Panel to 
recommend either for or against the use of 
extracorporeal membrane oxygenation for patients 
with COVID-19 and refractory hypoxemia.
REFERENCES
P22-01013


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11.
DESCRIPTION OF GLOBAL AMENDMENT(S)
11.1
GLOBAL AMENDMENT 1
Date of amendment
22 Oct 2020
EudraCT number
EU number
2020-002913-16
BI Trial number
0135-0347
BI Investigational Product(s)
Alteplase (recombinant tissue-type plasminogen 
activator, rt-PA)
Title of protocol
The TRISTARDS trial - ThRombolysIS Therapy 
for ARDS
A Phase IIb/III operationally seamless, open-
label, randomised, sequential, parallel-group 
adaptive study to evaluate the efficacy and safety 
of daily intravenous alteplase treatment given up 
to 5 days on top of standard of care (SOC) 
compared with SOC alone, in patients with acute 
respiratory distress syndrome (ARDS) triggered 
by COVID-19.
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
x
To be implemented immediately in order to eliminate hazard –
IRB / IEC / Competent Authority to be notified of change with request for 
approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
Flowchart
Description of change 1
Vital signs and laboratory tests added at Day 6
IRT call and drug dispensation at Day 2,3 and 4 
changed to:
removed
Rationale for change
Mistakenly entered for IRT call; vital signs and 
laboratory tests added at Day 6 because these are 
required for SOFA 
 score 


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calculation.
Section to be changed
Flowchart , Footnote 6
Description of change 2
Laboratory samples - haematology, clinical 
chemistry, renal function and coagulation 
parameters including D-dimer and fibrinogen are 
collected at screening, on Days 3, 5, 7 and 28. 
changed to:
On Day 6 only lab samples required for 
 
, SOFA score and PaO2/FiO2 shall be collected. 
Rationale for change
Wording in Footnote 6 was splitted and separated 
for lab tests supporting scores calculation and D-
dimer and other coagulation parameters 
assessments for more clarity
Section to be changed
Flowchart, Footnote 10
Description of change 3
 
and SOFA will be assessed until Day 6
changed to:
1.
 
nd SOFA will be assessed until 
Day 6 including vital signs assessment and 
laboratory samples needed for score 
calculations. If patient is discharged from 
hospital prematurely, last evaluation during 
hospital stay is expected at day of 
discharge from hospital
Rationale for change
Details on procedures needed to support score 
calculation  
Section to be changed
Section 1.4.2
Description of change 4
Added: Effect of Alteplase on D-Dimer and 
fibrinogen
Rationale for change
Increased safety, guidance for physicians for 
changes in coagulation system
Section to be changed
Section 3.3.1 Inclusion criteria #2
Description of change 5
ARDS with PaO2*/FiO2 ratio >100 and ≤300, 
either on non-invasive ventilator support, OR 
on mechanical ventilation (<48 hours since 
intubation), 


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-
with bilateral opacities in chest X-ray and 
CT scan (not fully explained by effusions, 
lobar/lung collapse, or nodules)
-
changed to:
-
with bilateral opacities in chest X-ray or 
CT scan (not fully explained by effusions, 
lobar/lung collapse, or nodules)
Rationale for change
Typo correction
Section to be changed
Section 5.2.6.2.2
Description of change 6
The investigator must report serious adverse 
events, and non-serious adverse events which are 
relevant for the reported SAE on the BI SAE form 
via fax
Changed to:
The investigator must report serious adverse 
events, and non-serious adverse events which are 
relevant for the reported SAE on the BI SAE form 
via fax (except of Russia and UK where SAE 
form is to be send via Clinergize)
Rationale for change
Reflecting current SAE reporting process flow
Section to be changed
Section 5.3.2
Description of change 7
Blood for quantification of analyte plasma 
concentrations will be withdrawn either by means 
of indwelling venous catheter or venipuncture
Changed to:
Blood for quantification of analyte plasma 
concentrations will be withdrawn by single-time 
venipuncture
Rationale for change
Refinement of pre-analytical phase
Section to be changed
Appendix 10.2
Description of change 8
SOFA score table was replaced with new one and 
reference to calculator for SOFA score removed
Rationale for change
Ensure unique score calculation as referenced
Section to be changed
Section 8.3.1
Description of change 9
Covid-10
Changed to:
Covid-19
Rationale for change
Typo correction


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11.2
GLOBAL AMENDMENT 2
Date of amendment
01 Oct 2021
EudraCT number
EU number
2020-002913-16
BI Trial number
0135-0347
BI Investigational Product(s)
Alteplase (recombinant tissue-type plasminogen 
activator, rt-PA)
Title of protocol
The TRISTARDS trial - ThRombolysIS Therapy 
for ARDS
A Phase IIb/III operationally seamless, open-label, 
randomised, sequential, parallel-group adaptive 
study to evaluate the efficacy and safety of daily 
intravenous alteplase treatment given up to 5 days 
on top of standard of care (SOC) compared with 
SOC alone, in patients with acute respiratory 
distress syndrome (ARDS) triggered by COVID-19.
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
x
To be implemented immediately in order to eliminate hazard –
IRB / IEC / Competent Authority to be notified of change with request for 
approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
Protocol Synopsis
Description of change 1
Added and updated:
o
Key-secondary endpoints
o
Secondary endpoints 
o
Number of patients in Part 2 updated /
planned number of patients on NIV/ IMV 
updated
o
Randomisation additionally stratified by D-
Dimer status
o
Alteplase labelled to alteplase dosing 
regimen B
o
Updated Inclusion/Exclusion criteria


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o
Treatment duration update
Statistical methods :
Part 2 will consist of two cohorts, the NIV patient 
cohort and the IMV patient cohort, and these two 
cohorts will be analysed separately. Only the NIV 
patient cohort will be part of the main statistical 
objectives of the study, and therefore part of the 
confirmatory hierarchical testing procedure. 
In Part 2, a confirmatory hierarchical testing 
procedure in the NIV patient cohort will be applied
for the primary endpoint and the two key secondary 
endpoints. In a first step, the primary endpoint 
hypothesis in NIV patients will be tested. If the null 
hypothesis is rejected, the key secondary endpoint of 
Treatment failure at Day 28 will then be tested in the 
NIV patient cohort as a second step. If this null 
hypothesis is rejected, the key secondary endpoint 
All cause mortality at Day 28 will then be tested in 
the NIV patient cohort as a third step.
In the NIV cohort, adjustment will be made for the 
number of days under NIV support, baseline D-
Dimer level and age. For the IMV cohort, 
adjustment will be made for the baseline WHO 
value, baseline D-Dimer level and age.
For each of the two key secondary endpoints, 
treatment failure at Day 28 and all cause mortality at 
Day 28, the delta method and average marginal 
effect method will be used to calculate the risk 
difference, 95% confidence intervals and p-values. 
Adjustment will be made for the same set of 
covariates as in the primary analysis of the primary 
endpoint. 
Rationale for change
Reflecting results of part 1 and DMC 
recommendations
Section to be changed
Flowchart, Footnote 3
Description of change 2
The time-point of obtaining D-Dimer levels can be 
as long as two calendar days before randomisation
added
Rationale for change
Increase flexibility for D-dimer level assessment to 
confirm eligibility of patients


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Section to be changed
Flowchart, Footnote 4
Description of change 3
Updated dose of Alteplase used in Part 2, update on 
treatment duration as well as duration of treatment 
interruptions
Rationale for change
Update based on results of Phase III for dose 
regimen and additional explanation for sites in case 
treatment interruption for safety reasons is needed
Section to be changed
Flowchart, Footnote 8
Description of change 4
Updated wording for anti-Xa measurements
requirements and timing, frequency and 
requirements for aPTT measurement.
Rationale for change
Concomitant medication with LMWH is limited to 
prophylactic dosages only. Based on labelling 
information for LMWHs, monitoring of anti-Xa 
levels is not required in general. Monitoring of anti-
Xa levels is adviced for patients with mild to
moderate renal impairment according to the 
prescribing information of LMWHs.
Section to be changed
Flowchart, Footnote 9
Description of change 5
Added instructions for PaO2/FiO2 measurement on 
during screening and treatment period:
o
The qualifying PaO2/FiO2 ratio should be the 
worst measurement in the screening period. 
During the treatment period the worst 
measurement on a given day should be 
documented in the case report form. During 
the first 6 days and during the stay on ICU 
(whatever is longer), PaO2/FiO2 ratio should 
be measured at least three times daily in all 
patients. 
Rationale for change
To ensure consistency of data collection across the 
sites
Section to be changed
Flowchart, Footnote 10
Description of change 6
; SOFA 
score assessment only at baseline, Day 6 and Day 
28. Additional instructions on SOFA score 
collection added
Rationale for change
Streamline operational feasibility.
 
 
 


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Assessment of SOFA was prolonged until Day 
28.
Section to be changed
Flowchart, Footnote 11
Description of change 7
Instructions on timing of baseline and the last 
measurement of WHO score on a given day added 
Rationale for change
To ensure consistency of WHO score assessment
and recording across the sites
Section to be changed
Flowchart, Footnote 12
Description of change 8
Added: A woman is considered of childbearing 
potential, i.e. fertile, following menarche and until 
becoming post-menopausal unless permanently 
sterile. Permanent sterilisation methods include 
hysterectomy, bilateral salpingectomy and bilateral 
oophorectomy. Tubal ligation is NOT a method of 
permanent sterilisation. A postmenopausal state is 
defined as no menses for 12 months without an 
alternative medical cause.
Rationale for change
More detailed definition of ‘women of childbearing 
potential’ requested by regulatory authorities.
Section to be changed
Flowchart, Footnote 13
Description of change 9
The RT-PCR test needs to be in timely association 
with the current hospital stay. For eligibility, 
patients should be hospitalized with the diagnosis of 
COVID-19 ARDS.
Removed COVID-19 RT-PCR test on Day 28
Rationale for change
More precise definition of RT-PCR test timing 
before screening of potential patients. 
Removal of COVID-19 test to reduce complexity 
and operational burden for sites
Section to be changed
Flowchart, Footnote 14
Description of change 10
Added: The rules for Adverse Event Reporting 
exemptions still apply, please see Section 5.2.6.2.4.
Rationale for change
Exemptions for AE reporting in SOC group as no 
investigational medicinal product is administered
Section to be changed
Section 1.1


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Description of change 11
Added:
o
Discussion on evolving treatment options 
since Covid-19 outbreak
o
Critically ill COVID-19 patients with 
hypercoagulable state, presenting with a 
significant increase in D-dimer levels, might 
benefit from a thrombolytic therapy on top of 
anticoagulation with unfractionated or low-
molecular weight heparins
o
Discussion of Part 1 / Phase III. The study 
results of Part 1 support the continuation of 
the study into Phase III (Part 2), 
implementing a comparison of the alteplase 
0.6/0.04 mg/kg dose versus SOC alone.
Rationale for change
Positioning of the use of Alteplase treatment in the 
context of evolving treatment options in COVID-19, 
also including the results of Part 1 of the trial
Section to be changed
Section 1.2
Description of change 12
Added: Alteplase is currently indicated for the 
thrombolytic treatment of acute MI, acute massive 
PE with haemodynamic instability and acute 
ischemic stroke.
The results of ECASS III show a positive net-
clinical benefit for alteplase in patients with acute 
ischemic stroke.
Rationale for change
More precise wording
Section to be changed
Section 1.3
Description of change 13
The results of Part 1 of the current study added: 
were intended to provide proof of concept and 
establish the dose for Part 2, the confirmatory part of 
the study. 
The study results of Phase III of the current trial 
(Part 1) provided proof of concept and support the 
continuation of the study into Phase III (Part 2), the 
confirmatory part of the study, implementing a 
comparison of the alteplase 0.6/0.04 mg/kg dose 
versus SOC alone.
Pharmacokinetic measurements updated: in both 
parts of the trial
Rationale for change
Provided reasoning of continuation of study from 


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proof of concept to confirmatory part of the trial.
Continuation of pharmacokinetic measurements in 
Part 2 due to too sparse samples collected in Part 1 
of the study.
Section to be changed
Section 1.4.1 Benefits
Description of change 14
Added:
o
The results of Phase III of the current trial 
showed that patients receiving a dose of 0.6 
mg/kg over 2 hours inmediately followed  by
a constant rate infusion of alteplase 0.04 
mg/kg/h over 12 hours (up to 5 days) in 
addition to SOC was associated with faster 
clinical improvement or hospital discharge, 
compared with patients receiving alteplase 
0.3/0.02 mg/kg on top of SOC, or SOC alone 
. The study results of Phase III (Part 1), 
support the continuation of the study into 
Phase III (Part 2), the confimartory part of 
the study.
o
Alteplase can be given in combination with 
UFH or LMWH.
o
During the alteplase treatment period of up 
to a maximum of 5 days (plus a possible 
extension in case of unavoidable 
interruptions of up to 72 hours) the 
concomitant use of LMWH in Part 2 is 
limited to prophylactic doses and the use of 
UFH is limited to target levels of aPTT 
between 1.0 to 1.5 fold ULN according to 
local laboratory.
o
Treating patients with a moderate initial dose 
of 0.6 mg/kg over 2 hours (for example, this 
corresponds to 42 mg in a patient with 70 kg 
of body weight) combined with a constant 
rate infusion (CRI) of low dose alteplase of
0.04 mg/kg/h over 12 hours is considered to 
reduce the risk of alteplase-related bleeding 
events in comparison to  application of high 
alteplase doses (up to 100 mg) used in other 
indications (e.g.  massive pulmonary 
embolism with hemodynamic instability, 
acute ischemic stroke, acute myocardial 
infarction).
o
These patients replaced by COVID-19 


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ARDS 
Rationale for change
Summarizing results of Part 1 supporting benefit of 
treatment with Alteplase 0.6 mg/kg over 2 hours 
inmediately followed by a constant rate infusion of 
alteplase 0.04 mg/kg/h over 12 hours (up to 5 days) 
in addition to SOC versus SOC alone.
Section to be changed
Section 1.4.2
Description of change 15
Added: 
The highest number of bleeding events were seen in 
trials involving acute ischemic stroke patients, 
where the reported complications with alteplase total 
doses (up to 90 mg) given over 60 minutes were 
symptomatic intracerebral haemorrhage (6%) and 
other major haemorrhage (2%). Minor haemorrhage 
(e.g. bleeding at site of venepuncture) accounting for 
20-25%. 
Similarly in patients with acute MI where alteplase
doses (up to 100 mg) given over 90 min were
applied, the risk of intracranial haemorrhage is 
<1%.In the Phase III part of the current trial, no new 
safety signals were identified at the chosen alteplase 
dose levels that are in general considerably lower 
than in approved indications (e.g. stroke, MI, PE). 
Major bleeding events until Day 6 were overall 
infrequent (1 patient in the 0.3/0.02 mg/kg dosing 
group and 3 patients in the 0.6/0.04 mg/kg dosing 
group), and a trend towards a dose-dependent 
increase was mainly observed for non-major 
bleeding events.
Mitigation strategies to prevent bleeding events in 
Phase III (Part 2) of the current study are 
implemented in Section 3.3.4.1.    
Rationale for change
No new safety signals observed in Part 1 of the trial, 
an increased risk of bleeding was observed in Part 1 
and accordingly mitigation strategies are now 
adopted to prevent bleeding events that may occur in 
Part 2.
Section to be changed
Table 1.4.2:1
Description of change 16
Added possible or known risks: 
o
Major bleeding including intracerebral 


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haemorrhage or bleeding from a critical 
organ
Mitigation strategy added: 
Rationale for change
Safety information from CCDS added
Section to be changed
Section 1.4.3
Description of change 17
Added:
o
The safety results from Phase III (part 1) of 
the current trial were generally comparable 
across the treatment groups, except for 
bleeding events, which are a known side 
effect of alteplase. In Phase III, mainly non-
major bleeding events were observed, and
some of them were related to interventions. 
Additional risk minimization strategies to 
avoid intervention-related bleeding events 
have accordingly be implemented for Part 2 
of the study. No new safety signals were 
identified. Major bleeding events in Part
1were overall infrequent.
o
If the level of fibrinogen falls alteplase has to 
be temporarily interrupted .
o
The study results of Phase III (Part 1), 
support the continuation of the study into 
Phase III (Part 2), the confimartory part of 
the study. The alteplase 0.6/0.04 mg/kg dose 
in addition to SOC was associated with faster 
clinical improvement or hospital discharge, 
compared with the alteplase 0.3/0.02 mg/kg 
+ SOC, or SOC alone treatments. Patients 
receiving NIV support appeared to derive 
greater benefits than those receiving IMV 
support. The safety results were generally 
comparable across the treatment groups. No 
new safety signals were identified. Major 
bleeding events were overall infrequent, and 
a trend towards a dose-dependent increase 
was mainly observed for non-major bleeding 
events. 
o
Precautionary recommendations for 
temporary interruption of alteplase infusion 
(up to 72 hours in total for urgent reasons, 
bleeding or drop in fibrinogen), a limitation 
of concomitant administration of heparins to 
prophylactic doses and recommendations to 


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avoid intervention-related bleeding eventsare 
implemented in Part 2 to mitigate the risk of 
bleeding. 
o
Taken together, the overall benefit-risk ratio 
of alteplase at the studied dosing regimen is 
considered favourable.
Rationale for change
Discussion of benefits and risks and summary of 
study results to support Part 2 study design. 
Justification of the dose of alteplase that was chosen 
for Part 2 of this trial.
Section to be changed
Section 2.1.3
Description of change 18
Added: In Part 2, the following key secondary 
endpoints will be evaluated:

Treatment failure defined as all cause 
mortality or mechanical ventilation at Day 
28 

All cause mortality at Day 28
In Part 2, the following secondary endpoints will be 
evaluated:

Number of oxygen-free days up to Day 28

Length of hospital stay up to Day 28
Major bleeding events (MBE) (according to 
International Society on Thrombosis and 
Haemostasis [ISTH] definition R05-0344) 
until Day 6, see Section 5.2.5.1.

PaO2/FiO2 ratio (or inferred PaO2/FiO2 ratio 
from SpO2) change from baseline up to 
Day 6 
Rationale for change
Reflecting interim analysis and results of Part 1. 
Implementation of harder endpoints introduced as 
key secondary endpoints and implementation of a 
hierarchical testing approach to increase strength of 
the Phase III results.
Section to be changed
Description of change 19


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Rationale for change
Section to be changed
Section 3.1
Description of change 20
Added characteristics of patient population in Part 2 
and treatment regimen to be used . Stratification 
used in Part 2 updated and recruitment cap for 
patients on IMV and NIV added .
Rationale for change
Reflecting results of Part 1 and DMC 
recommendation
Section to be changed
Figure 3.1.1
Description of change 21
A schematic overview of trial design adapted for 
part 2 – selected dose for alteplase added and 
planned number of patients adapted
Rationale for change
Reflecting results of Part 1 and DMC 
recommendation, reflecting sample size in Part 2
Section to be changed
Section 3.2
Description of change 22
Added reasoning of trial Protocol modifications: As
Part 1 results indicated the need for some design 
modifications in Part 2 beyond those pre-specified in 
the protocol, these required modifications have been
implemented via current global CTP amendment. 
DMC recommendation for Alteplase dosing regimen 
B added
Rationale for change
Reflecting results of Part 1 and DMC 
recommendation
Section to be changed
Section 3.3
Description of change 23
Updated number of participating sites in part 2 to 
110-130.
Added information on randomisation cap for IMV 
patients:
In Part 2 of the trial: 
In part 2 of the trial, a randomisation cap will be 
applied for patients on mechanical ventilation at 
baseline: when 50 patients on mechanical ventilation 
have been randomized. Recruitment of further 
patients on mechanical ventilation will be stopped 


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via IRT.
Randomisation for the whole trial will be stopped 
when approximately 210 NIV patients have been 
randomized in Part 2, regardless of how many IMV 
patients have been randomised so far. 
Rationale for change
Feasibility results show need to increase number of 
participating sites from regions with higher 
predicted incidence rate of Covid-19 infection.
Randomisation cap was set-up reflecting results of 
Part 1 and recommendation of expert board to focus 
the main analyses on NIV patients.
Section to be changed
Section 3.3.2
Section to be changed
Inclusion criteria
Description of change 24
Removed:
-Eligibility criteria for Parts 1 and 2 are the 
identical
Inclusion criteria #1 updated: above > 18 (or above 
legal age)
Rationale for change
UK is no longer participating in the trial, but legal 
age above 16 may be applicable for other 
participating countries
Section to be changed
Exclusion criteria 2 and 7
Description of change 25
Added following exclusion criteria:
o
Indication for therapeutic dosages of 
anticoagulants at trial entry
o
Planned interventions during the first 5 
days after randomisation, such as 
surgery, insertion of central catheter or 
arterial line, drains, etc.
Rationale for change
To avoid potential safety risk to patients
Section to be changed
Exclusion criterion #17
Description of change 26
Severe hepatic dysfunction - added i.e. Child-Pugh 
B and C
Rationale for change
To be in line with CCDS
Section to be changed
Exclusion criterion #23
Description of change 27
Malignancy (Stage IV) added: “with increased 


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bleeding risk”
Rationale for change
To be in line with CCDS
Section to be changed
Section 3.3.4.1
Description of change 28
Need for ECMO or RRT. No additional dose of 
alteplase 0.3 mg/kg/2 h or 0.6 mg/kg/2 h (Part 1) or 
0.6 mg/kg/2 h in Part 2 should be given under these 
circumstances. 
Rationale for change
Clarification how to proceed with treatment in case 
patient requires ECMO or RRT: no additional 2h 
infusion of alteplase should be given
Section to be changed
Section 3.3.4.1 
Description of change 29
Recommendations for temporary interruption of 
alteplase daily infusions updated.
Rationale for change
Provide sites with more detailed instructions for 
temporary interruption of treatment in case of 
interventions needed to be performed during 
treatment with alteplase
Section to be changed
Section 4.1.1 / Tab. 4.1.1:1
Description of change 30
Part 2 added:
o
Dosing regimen B, Initial i.v. infusion (0.6 
mg/kg to be infused over 2 h).
o
Instructions for interruption of infusion 
adapted
o
Instructions for additional infusion added:
i.v. infusion can be given once during the 
treatment period in case of clinical 
worsening (as per investigator judgement)
Rationale for change
Selected dosing regimen based on results of Part 1 
of the trial and operational instructions in case of 
urgent interventions adapted to allow more 
flexibility and prevent treatment discontinuation.
Section to be changed
Section 4.1.2
Description of change 31
Dosing regimen B added as well as instructions that 
in Part 2, only dosing regimen B can be given as an 
additional i.v. infusion, as per investigator 
judgement
Rationale for change
Added selected dose for Part 2; clarification on dose 


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to be used in Part 2
Section to be changed
Section 4.1.3
Description of change 32
Randomisation cap for patients on IMV has been
added.
Rationale for change
Reflecting trial results from Part 1 and 
recommendation of expert board
Section to be changed 33
Section 4.1.4
Description of change
Added wording for Part 2: Number of vials will be 
allocated per each patient depending on patient`s 
weight in treatment arm by IRT. 
Rationale for change
Inform sites on IMP allocation planned in Part 2
based on results of Part 1
Section to be changed
Section 4.2.1
Description of change 34
Alteplase arm, Day 1 to 5 (treatment period):
The investigator may choose between Option A or 
B:
Added wording:
Option A: Option A can only be applied in patients 
with an eGFR ≥ 30ml/min/1.73m2.
Low-dose LMWH: added: in prophylactic dosages
given every day at the end of long-term i.v. alteplase 
infusion
Recommendations for LMWH dosages (these 
represent examples, and other LMWHs in 
prophylactic dosages are also allowed) provided
Recommendation added:
Before the start of the very first alteplase infusion,
there should have been a time period of at least 10 
hours since the last LMWH (S.C.) administration to 
ensure no significant overlap of the LMWH and
alteplase.
In patients with moderate (creatinine clearance 30-
50 ml/min/1.73m2) and mild (creatinine clearance 
50-80 ml/min/1.73m2) renal impairment, careful 
clinical monitoring is advised.


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Option B: Option B can be chosen in all patients, 
irrrespective of their eGFR.
Alteplase arm, post treatment period (Day 5 and 
onwards), and SOC arm:
Recommendation for anti-Xa monitoring:
The anti Xa level should be monitored regularly, in 
patients with mild to moderate renal impairment.
aPTT should be measured at least daily or more 
often
Rationale for change
Implementation of risk mitigation strategies to 
prevent bleeding events by the limitation to 
prophylactic dosages of LMWH only. Advice to 
monitor dosage of LMWH by anti-Xa levels in 
patients with mild to moderate renal impairment 
according to LMWH labelling information.
Implementation of a time gap between LMWH 
administration and first administration of alteplase 
to avoid bleeding events on the first day of alteplase 
treatment with a combined short and long-term 
infusion.
Section to be changed
Section 4.2.1
Description of change 35
Added: 
Current or scheduled use of systemic (intravenous or 
oral) corticosteroid therapy should be included in 
SOC regimen (e.g. 6 mg of dexamethasone orally) 
for up to 10 days or until hospital discharge. This 
applies to both treatment arms.
Total daily dose equivalencies to dexamethasone 6 
mg are provided in Appendix 10.5.
o
Appendix 10.5 with proposed SOC therapies
added
o
Recommendations before start of Alteplase 
treatment regarding insertion of arterial lines 
and central venous catheters, tubes, 
interventional procedures.
o
Management of bleeding and serious 
bleeding events recommendations during 
alteplase treatment period


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Rationale for change
Prevention of bleeding events during alteplase 
treatment period; standardization of SOC therapy by 
providing guideline recommended treatment 
recommendations in Appendix 10.5
Section to be changed
Section 4.2.2.1 Restrictions regarding concomitant 
treatment
Description of change 36
Added: LMWH/unfractionated heparins in 
therapeutic dosages
Patients who previously received a direct oral 
anticoagulant, can only be included, if the direct oral 
anticoagulant has been stopped. Added: for at least 
48 hours prior to randomisation.
Rationale for change
Only prophylactic dosages of heparins allowed in 
combination with alteplase treatment for safety 
reasons.
A time window of 48h after last DOAC dose is 
recommended to prevent overlap of therapies and 
with that risk of bleeding.
Section to be changed
Section 5.1.1
Description of change 37
Added: On Day 1 (randomisation day) an 
assessment just before randomisation shall be 
collected as a baseline value. The last assessment 
per day should be recorded in the case report form.
Rationale for change
Ensure standardization of data collection 
Section to be changed
Table 5.1.1:1
Description of change 38
Mechanical ventilation PaO2/FiO2; corrected  from 
<200 to <150; for Score 9
Rationale for change
Typo correction
Section to be changed
Section 5.1.2
Description of change 39
Added: oxygen free days
Rationale for change
To be aligned with endpoints collection
Section to be changed
Section 5.1.3
Description of change 40
Added wording: The qualifying PaO2/FiO2 ratio 
should be the worst measurement in a screening 
period. The worst measurement on a given day 
should be documented in the case report form.
Rationale for change
Ensure consistency of data capture between sites


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Section to be changed
Section 5.1.4 
Description of change 41
SOFA score evaluation timing added: If patient is 
discharged from hospital prematurely, the last 
evaluation needs to be performed at the day of 
discharge from hospital.
Rationale for change
Provide clear instructions to sites on expectations of 
last SOFA score evaluation
Section to be changed
Section 5.2.3
Description of change 42
Removed wording: 
Mandatory local laboratory parameters are: 
PaO2, haematology, ALT, AST, creatinine, 
potassium, sodium, LDH, CRP, fibrinogen, D-dimer 
and aPTT or anti-Xa.
Added wording:
More details on collection of safety laboratory 
parameters are summarized in Tab. 5.2.3:1
Rationale for change
Text abbreviation
Section to be changed
Section 5.2.6.1.3
Description of change 43
Updated wording for reporting of Always Serious 
AEs: For patients on the Alteplase treatment, these 
events should always be reported as serious adverse 
events, as described in Section 5.2.6.2. For patients 
on SOC, adverse events should be considered 
serious if they meet any of the seriousness criteria, 
outlined in Section 5.2.6.1.2
Rationale for change
The Always serious AE list is not applicable for 
patients on SOC as there is no requirement of 
expedited reporting of SAEs for the patients on 
SOC, as there is no IMP administered. 
Section to be changed
Section 5.2.6.1.3
Description of change 44
For outcome events reporting added wording: Please 
refer to Section 5.2.6.2.4 for Exemptions to SAE 
reporting.
Rationale for change
Decision taken to exempt expedited SAE reporting 
of specific preferred terms (PTs), which are 
considered as progression of underlying disease.
Section to be changed
Section 5.2.6.2.1
Description of change 45
Added wording:


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The rules for Adverse Event Reporting exemptions 
still apply, please see Section 5.2.6.2.4.
Rationale for change
Decision taken to exempt expedited SAE reporting 
of specific preferred terms (PTs), which are 
considered as progression of underlying disease.
Section to be changed
Section 5.2.6.2.2.
Description of change 46
AFor patients not exposed to Alteplase treatment, 
but to SOC only: No expedited reporting 
via SAE form is required. All safety data 
(AEs and SAEs) should be included in the 
eCRF.Consequently, the investigator is 
encouraged to report all adverse events for 
SOC group drugs in accordance to the local 
regulatory requirements for spontaneous AE 
reporting at the investigator’s discretion by 
using their locally established routes and AE 
report forms.
o
For the patients on Alteplase treatment arm,
the investigator must report serious adverse 
events, and non-serious adverse events
which are relevant for the reported SAE on 
the BI SAE form
Rationale for change
There is no requirement of expedited SAE reporting 
for patients on SOC, as there is no BI IMP 
administered
Section to be changed
Section 5.2.6.2.3
Description of change 47
Added: A woman is considered of childbearing 
potential, i.e. fertile, following menarche and until 
becoming post-menopausal unless permanently 
sterile. Permanent sterilisation methods include 
hysterectomy, bilateral salpingectomy and bilateral 
oophorectomy. Tubal ligation is NOT a method of 
permanent sterilisation.
A postmenopausal state is defined as no menses for 
12 months without an alternative medical cause.
Rationale for change
Reflcting definition of women of childbearing 
potential and postmenopausal state
Section to be changed
Section 5.2.6.2.4
Description of change 48
Exemptions to SAE reporting:
Disease progression of the underlying ARDS 
reflects the natural course of the disease, and is 


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exempted from reporting as an (S)AE. Progression 
of the subject's underlying ARDS will be recorded 
on the appropriate pages of the (e)CRF only and will 
not be reported as an SAE.
The specific (preferred terms) PTs that are exempt 
are:

Acute respiratory distress syndrome

Respiratory distress

Acute respiratory failure

Respiratory failure
Death due to disease progression is also to be 
recorded on the appropriate eCRF page and not on 
the SAE form. Disease progression and death due to 
disease progression will therefore not be entered in 
the safety database and hence not get expeditiously 
reported.
However, if there is evidence suggesting a causal 
relationship between the study drugs and
the progression of the underlying ARDS, the event 
must be reported as SAE on the
eCRF, as well as on the SAE form.
Exempted events will be monitored at appropriate 
intervals by the external DMC. 
Rationale for change
Decision taken to exempt expedited SAE reporting
of specific preferred terms (PTs), which are
considered as progression of underlying disease.
Section to be changed
Section 5.3.1
Description of change 49
Updated wording: As PK samples collection 
included less than 10 patients per treatment arm in 
Part 1, collection of PK samples will continue in 
Part 2. It is intended to obtain PK samples from at 
least 17 patients receiving Alteplase in Part 2.
Rationale for change
PK samples collection will continue in part 2 as 
there were not enough PK samples collected in part 
1 of the trial.
Section to be changed
Section 6.2.1
Description of change 50
Removed wording: 
SARS-CoV-2 positive test result has to be available 
before treatment administration.
Updated wording : For eligibility, patients should 
be hospitalized with the diagnosis of COVID-19 
ARDS.
SARS-CoV-2 laboratory confirmation (RT-PCR 


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test) is a pre-requisite at screening. The RT-PCR test 
needs to be in timely association with the current 
hospital stay. 
Rationale for change
More precise definition of eligibility and timely 
association of ARDS diagnosis and COVID-19 
confirmed by RT-PCR. 
Section to be changed
Section 6.2.2
Description of change 51
Updated wording: Randomisation of patients (Day 
1, Visit 2a) shall happen not later than 24 hours after 
the confirmation of the inclusion and exclusion 
criteria. Study drug dispensing and administration 
shall start within 6 hours of randomisation. The time
point of obtaining D-Dimer levels can be as long as 
two calendar days before randomisation. 
Rationale for change
More clear wording on sequence of procedures and 
time window between screening and randomisation. 
Prolonged time point for D-dimer level collection 
for operational feasibility.
Section to be changed
Section 6.2.2
Description of change 52
Added:
o
Interruptions for urgent reasons for a total of 
72 hours are allowed and accordingly, the 
time window of the visits may be extended.
o
wording for PaO2/FiO2 measurement: The 
worst measurement on a given day should be 
recorded in the CRF.
o
oxygen support is collected
Rationale for change
Added instructions on allowed treatment 
interruptions due to urgent safety reasons and timing 
of subsequent visits.
Clear instructions for sites regarding which 
PaO2/FiO2 measurement shall be recorded in CRF.
Oxygen support collection added for secondary 
endpoints assessment
Section to be changed
Section 7 Statistical methods and determination of 
sample size
Description of change 53
o
Randomisation additionally stratified by D-
Dimer status
o
Key secondary statistical objectives added
o
Confirmatory hierarchical testing procedure 
added to include testing of H0_1, Ho_2 and 
H0_3


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o
Analyses will be on the NIV patient cohort, 
and on the IMV patient cohort separately, as 
well as a potential a meta-analysis 
combining the NIV and IMV patient cohorts
o
Model adjustment covariates for the NIV 
patient cohort are the number of days under 
NIV support, baseline D-Dimer level and 
age.
o
Model adjustment covariates for the IMV
patient cohort are the baseline WHO value, 
baseline D-Dimer level and age   
o
Sensitivity analysis for primary endpoint on 
PPS.
o
Addition of ANCOVA for continuous 
secondary endpoints.
Sample size rationale based on Part 1 results and 
changes to design including the introduction of the 
hierarchical testing.
Rationale for change
Based upon analysis at the end of Part 1, selection of 
dose for Part 2 and changes to design of study.
Section to be changed
Section 8.1
Description of change 54
Updated wording for PK sample collection in part 2: 
An optional assessment of pharmacokinetics also is 
planned to be performed in at least 10 patients per 
dose group in Part 1 and in approximately 17 
patients in Part 2
Rationale for change
Updated number of PK patients in Part 2 reflecting 
number of collected PK samples in Part 1
Section to be changed
Section 8.4
Description of change 55
Added: The rules for Adverse Event Reporting 
exemptions still apply, please see Section 5.2.6.2.4.
Rationale for change
Reflecting update on exemptions for Adverse Event 
reporting
Section to be changed
Section 8.7
Description of change 56
Added: The DMC will also review at specified 
intervals the exempted events to SAE reporting as 
specified in Section 5.2.6.2.4.
Rationale for change
Update of DMC responsibilities reflecting update of 
exempted events to SAE reporting 
Section to be changed
Section 9.1
Description of change 57
Added new references
Rationale for change
New references added in the Protocol
Section to be changed
Section 9.2 
Description of change 58
Added unpublished reference


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Rationale for change
Synoptic CTR part 1 completed
Section to be changed
Section 10 Appendices
Description of change 59
Appendix 10.1 Glasgow coma score removed
Rationale for change
Not planned to be used in Part 2
Section to be changed
Appendix 10.4 Monitoring
Description of change 60
Added:
Due to the nature of this trial and the risks 
associated with Covid-19, site monitoring visits as 
physical on-site interaction may not always be 
possible. The focus will be on centralized 
oversight/monitoring. If not restricted by local 
Covid-19 regulations, we recommend combination 
of remote and on-site visits, mainly for initiation and 
close-out visits. Regular interim monitoring visits 
will occur mainly remotely unless the trial team 
requires a visit to be done on-site due to an 
identified risk where the required mitigation may 
need to be performed on-site.
Trial monitoring intervals may be adjusted 
depending on enrolment rates, data quality, 
recruitment and conduct phases, timelines for CRF 
completion and/or site quality. 
Rationale for change
Use of mixture of remote and on-site visits to 
increase quality of data collection.
Section to be changed
Appendix 10.4
Description of change 61
Dosing regimen A (lower dose) dosing table (only 
applicable in Part 1 of the trial)
Dosing regimen B (higher dose) dosing table 
(applicable in both Parts of the trial)
Rationale for change
Reflecting decision on the trial – dosing regimen B
for Part 2
Section to be changed
Appendix 10.5
Description of change 62
A list of guideline-recommended therapies for 
COVID-19 specifically, and for critically ill patients 
in general was added
Rationale for change
Standardization of SOC therapy across participating 
study sizes by providing guideline recommended 
treatment recommendations 


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11.3
GLOBAL AMENDMENT 3
Date of amendment
17 Feb 2022
EudraCT number
EU number
2020-002913-16
BI Trial number
0135-0347
BI Investigational Product(s)
Alteplase (recombinant tissue-type plasminogen 
activator, rt-PA)
Title of protocol
The TRISTARDS trial - ThRombolysIS Therapy 
for ARDS
A Phase IIb/III operationally seamless, open-label, 
randomised, sequential, parallel-group adaptive 
study to evaluate the efficacy and safety of daily 
intravenous alteplase treatment given up to 5 days 
on top of standard of care (SOC) compared with 
SOC alone, in patients with acute respiratory 
distress syndrome (ARDS) triggered by COVID-
19.
Global Amendment due to urgent safety reasons
Global Amendment
X
Section to be changed
Protocol synopsis
Description of change
D-Dimer levels (≥3-fold to <5-fold ULN, versus 
≥5-fold ULN) updated to
Randomisation will be stratified by D-Dimer levels 
(≥ULN to <5-fold ULN, versus ≥5-fold ULN) 
updated from  
Rationale for change
Stratification factor adapted based on the changed 
inclusion criterion of D-Dimer level ≥ ULN.
Section to be changed
Protocol synopsis
Description of change
Inclusion criteria #4 updated: Fibrinogen level  ≥ 
lower limit of normal
Rationale for change
Alteplase results in a modest decrease of the 
circulating plasma fibrinogen levels. Fibrinogen 
levels ≥ULN were previously required as an 
inclusion criterion to ensure that fibrinogen levels
are high enough and are less likely to decreaseto a 
critical level below 150 mg/dL which have been 


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associated with an elevated risk of bleeding events. 
A recommendation for temporary interruption of 
alteplase daily infusions if the fibrinogen level 
decreases to levels <150 mg/dL was implemented. 
The inclusion criterion of fibrinogen levels ≥ 
lower limit of normal is adequate in combination 
with the daily monitoring of fibrinogen levels and 
the implementation of a stopping rule for the 
alteplase administration if fibrinogen level 
decreases to levels <150 mg/dL.
Section to be changed
Protocol synopsis
Description of change
Inclusion criteria#5 update from: D-Dimer ≥ 3-fold 
of upper limit of normal (ULN) according to local 
laboratory to D-Dimer ≥upper limit of normal 
(ULN) according to local laboratory
Rationale for change
Increased plasma D-dimers values are associated 
with an unfavorable prognosis in COVID-19. An 
increase in plasma D-dimers concentration with 
ongoing thromboprophylaxis should give 
consideration of the administration of higher doses 
of anticoagulants. The target patient population are 
patients with microvascular thombi. Hence,
patients with suspected and confirmed pulmonary 
embolism are excluded from the trial. D-Dimer 
values ≥ ULN are more appropriate for a patient 
population with microvascular thombi and fibrin 
deposition within alveoli but without a high risk of
thromboembolic events and with a high need 
anticoagulants in therapeutic dosages (which are
restricted during the treatment period with 
alteplase).
Section to be changed
Protocol synopsis
Description of change
Exclusion criteria#1 updated:  Massive, confirmed 
PE with haemodynamic instability at trial entry, or 
any (suspected or confirmed) PE that is expected to 
require therapeutic dosages of anticoagulants 
during the treatment period
Rationale for change
During the treatment period with Alteplase 
concomitant therapy with anticoagulants is allowed 
only in prophylactic dosages. The diagnosis of 
major thrombosembolic events such as pulmonary 
embolism requires therapeutic levels of 
anticoagulation. Therefore, patients with suspected 
or confirmed pulmonary embolism are excluded.


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Section to be changed
Flowchart
Description of change
Dispense trial drug at V5/EOT removed
Rationale for change
Mistakenly ticked, no IMP dispense at V5
Section to be changed
Flowchart, Footnote 3
Description of change
The time-point of obtaining D-Dimer levels can be 
as long as four calendar days before screening. 2 
days prior to randomization replaced by 4 days
Rationale for change
Increase flexibility for D-dimer level assessment to 
confirm eligibility of patients
Section to be changed
Flowchart, Footnote 7
Description of change
D-dimer levels shall be measured at Day 0 (or no 
more than 4 calendar days before screening),
days 3, 5, 7 and 28
Rationale for change
Increase flexibility for D-dimer level assessment to 
confirm eligibility of patients
Section to be changed
Section 1
Description of change
Phase III corrected at different places to results of 
phase II
Rationale for change
Typos correction
Section to be changed
Section 1.1.
Description of change
Efficacy and safety results summary from part 1, 
phase II of the trial added
Rationale for change
Comprehensive results of safety and efficacy are 
provided based on the final data base lock to 
inform investigators and authorities.
Section to be changed
Section 3.1
Description of change
Randomisation in Part 2 will be 2:1 to dosing 
regimen B of alteplase (initial i.v. infusion of 
alteplase 0.6mg/kg/2h (Day 1)) followed by daily 
i.v. long-term infusion of 0.04 mg/kg/h over 12 
hours or to standard of care, and will be stratified 
by type of ventilation (non-invasive or invasive 
mechanical) and by D-dimer levels (≥ULN to <5-
fold ULN, versus ≥5-fold ULN). D-dimer levels 
updated from (≥3-fold to <5-fold ULN, versus ≥5-
fold ULN). 
Rationale for change
Stratification factor adapted based on the changed 
inclusion criterion of D-Dimer level ≥ ULN.
Section to be changed
Section 3.3


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Description of change
The time-point of obtaining D-Dimer levels can be 
as long as four calendar days before screening.
Rationale for change
To align with update in Flowchart
Section to be changed
Section 3.3.2 Inclusion criteria
Description of change
Inclusion criteria #4 updated to:
Fibrinogen level ≥ lower limit of normal
Inclusion criteria #5 updated to:
D-Dimer ≥upper limit of normal (ULN) according 
to local laboratory
Rationale for change
Please refer to changes to the protocol synopsis
Section to be changed
Section 3.3.3 Exclusion criteria
Description of change
Exclusion criteria #1updated to: Massive, 
confirmed PE with haemodynamic instability at 
trial entry, or any (suspected or confirmed) PE that 
is expected to require therapeutic dosages of 
anticoagulants during the treatment period
Rationale for change
Please refer to changes to the protocol synopsis
Section to be changed
Section 4.1.3 Method of assigning patients to 
treatment arms
Description of change
D-dimers adaptation included: In Part 1 the 
randomisation will be stratified by the type of 
ventilation support (invasive mechanical / non-
invasive) and in Part 2 by both type of ventilation 
support and by baseline D-dimer level (≥ULN to 
<5-fold ULN, versus ≥5-fold ULN)
Rationale for change
Consistency with section 3.1
Section to be changed
Section 4.2.1 Other treatments and emergency 
procedures
Description of change
In Section Alteplase arm Day 1to 5 (treatment 
period) added recommendation for UFH:
UFH dosage up to 5000 IU, SC BID (twice daily) 
can be given alternatively.
Rationale for change
Changes according to guideline on the use of
anticoagulation for thromboprophylaxis in patients 
with COVID-19.
Section to be changed
Section 5.1.3 PaO2/FiO2 ratio
Description of change
Appendix 10.3 added:
Fraction of Inspired Oxygen (FiO2) for common
oxygen delivery systems are provided (Appendix 


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10.3)
Rationale for change
Additional instruction for the estimation of FiO2 
based on oxygen flow applied for common oxygen 
delivery systems provided.
Section to be changed
Section 7 Statistical Methods and Determination of 
Sample Size
Description of change
Updated wording for D-dimers: baseline D-Dimer 
level (≥ULN to <5 vs ≥5-fold ULN).
Rationale for change
Consistency with section 3.1 and 4.1.3
Section to be changed
Section 7.2.2 Primary Endpoint Analyses
Description of change
Added : Following the amendment to the inclusion 
criteria (CTP Version 4.0), whereby the original D-
Dimer level changed from ≥3-ULN to ≥ULN, 
some additional analyses will be performed. 
Firstly, a sensitivity analysis will be added, similar 
to the primary analysis, just replacing the baseline 
D-Dimer status on the two levels with that on the 
three levels (≥ULN to <3, ≥3 to <5, ≥5-ULN). 
Secondly, a supplementary analysis will be added, 
similar to the primary analysis, but removing all 
patients with baseline D-dimer status ≥ULN to <3-
ULN, which is according to the expanded inclusion 
criteria.
Rationale for change
Consistent with section 3.1, 4.1.3 and 7
The sensitivity and supplementary analyses have 
been added, to account for the change to the study 
population, and to adjust for or assess the impact of 
the changes.
Section to be changed
Section 7.4 Randomization
Description of change
Updated wording reflecting D-dimers update:
Randomisation in Part 2 will be to alteplase and to 
standard of care, with an allocation ratio of 2:1, 
with stratification by the following factors: 

Type of ventilation (non-invasive or 
invasive mechanical)

D-Dimer levels (≥ULN to <5-fold 
ULN, versus ≥5-fold ULN


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Rationale for change
Reflecting update for D-dimer level requirements 
Section to be changed
Section 8.3.2
Description of change
Updated: During the conduct of the study, it may 
not be possible for the investigator / institution to 
allow site trial-related monitoring, audits, IRB/EC 
review or regular inspections due to COVID-19
restrictions. 
Rationale for change
Typo for IRB and added restrictions for COVID-19 
for clarity
Section to be changed
Appendix 10.1 
Description of change
Glasgow coma scale added
Rationale for change
Mistakenly removed in previous version
Section to be changed
Appendix 10.3 
Description of change
Added Appendix: FiO2 ranges for common oxygen 
delivery devices
Rationale for change
Additional instructions provided, consistency with 
Section 5.1.3
Section to be changed
Table10.6:1 Treatment Recommendations for 
COVID-19 ARDS (with a focus on NIV Patients)
Description of change
New references added, updated treatment 
recommendations for IL-6 inhibitors, JAK-
inhibitors, oxygenation, ventilation and 
awakeprone positioning updated
Rationale for change
Updated based on up-to-date treatment 
recommendations


 
 
 
 
APPROVAL / SIGNATURE PAGE 
 
Document Number: 
 
Technical Version Number:
 
Document Name: 
 
 
 
                                                                                                                 
 
Title: 
 
 
 
 
                                                                                                                   
 
Signatures (obtained electronically) 
 
Meaning of Signature  
Signed by 
Date Signed 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
c32434345
4.0
clinical-trial-protocol-version-04
The TRISTARDS trial - ThRombolysIS Therapy for ARDS. A Phase IIb/III
operationally seamless, open-label, randomised, sequential, parallel-group adaptive
study to evaluate the efficacy and safety of daily intravenous alteplase treatment
given up to 5 days on top of standard of care (SOC) compared with SOC alone, in
patients with acute respiratory distress syndrome (ARDS) triggered by COVID-19.
Approval-Clinical Trial Leader
18 Feb 2022 09:24 CET
Approval-Team Member Medicine
18 Feb 2022 10:29 CET
Author-Statistician
18 Feb 2022 11:35 CET
Verification-Paper Signature
Completion
18 Feb 2022 11:58 CET


Boehringer Ingelheim 
Page 2 of 2
Document Number: 
 Technical Version Number:
 
 
 
(Continued) Signatures (obtained electronically) 
 
Meaning of Signature  
Signed by 
Date Signed 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
c32434345
4.0

SAP_001.pdf: 
001-MCS-50-415_RD-01 (2.0) / Saved on: 06 Dec 2019
TITLE PAGE
TRIAL STATISTICAL ANALYSIS PLAN
(PART 1 ANALYSIS)
c34525499-01
BI Trial No.:
0135-0347
Title:
A Phase IIb/III operationally seamless, open-label, randomised, 
sequential, parallel-group adaptive study to evaluate the efficacy 
and safety of daily intravenous alteplase treatment given up to 5 
days on top of standard of care (SOC) compared with SOC alone, 
in patients with acute respiratory distress syndrome (ARDS) 
triggered by COVID-19.
Investigational
Product(s):
Alteplase (recombinant tissue-type plasminogen activator, rt-PA)
Responsible trial 
statistician(s):
Tel.:
Fax: 
Date of statistical 
analysis plan:
03 May 2021 SIGNED
Version:
Version 2.0
Page 1 of 37
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2021 Boehringer Ingelheim International GmbH or one or more of its affiliated companies. All rights 
reserved.
This document may not - in full or in part - be passed on, reproduced, published or otherwise used without prior written permission.


Boehringer Ingelheim
TSAP for BI Trial No: 0135.0347 (Part 1 analysis)
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1.
TABLE OF CONTENTS
TITLE PAGE ...........................................................................................................................1
1.
TABLE OF CONTENTS......................................................................................2
LIST OF TABLES...................................................................................................................4
2.
LIST OF ABBREVIATIONS ..............................................................................5
3.
INTRODUCTION.................................................................................................8
4.
CHANGES IN THE PLANNED ANALYSIS OF THE STUDY......................9
5.
ENDPOINTS(S) ..................................................................................................10
5.1
PRIMARY ENDPOINT(S) ................................................................................10
5.2
SECONDARY ENDPOINT(S) ..........................................................................11
5.2.1
Key secondary endpoint(s).................................................................................11
5.2.2
Secondary endpoint(s) ........................................................................................11
6.
GENERAL ANALYSIS DEFINITIONS ..........................................................17
6.1
TREATMENT(S)................................................................................................17
6.2
IMPORTANT PROTOCOL DEVIATIONS....................................................17
6.3
SUBJECT SETS ANALYSED...........................................................................19
6.5
POOLING OF CENTRES .................................................................................21
6.6
HANDLING OF MISSING DATA AND OUTLIERS....................................21
6.7
BASELINE, TIME WINDOWS AND CALCULATED VISITS ...................22
7.
PLANNED ANALYSIS......................................................................................23
7.1
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS.........23
7.2
CONCOMITANT DISEASES AND MEDICATION .....................................24
7.3
TREATMENT COMPLIANCE........................................................................24
7.4
PRIMARY ENDPOINT(S) ................................................................................24
7.4.1
Primary analysis of the primary endpoint(s) ...................................................24
7.4.2
Sensitivity analysis, subgroup analysis, exploratory analysis of the 
primary endpoint(s)............................................................................................25
7.5
SECONDARY ENDPOINT(S) ..........................................................................27
7.5.1
Key secondary endpoint(s).................................................................................27
7.5.2
Secondary endpoint(s) ........................................................................................27
7.7
EXTENT OF EXPOSURE.................................................................................28
7.8
SAFETY ANALYSIS..........................................................................................28
7.8.1
Adverse Events ....................................................................................................29
7.8.2
Laboratory data ..................................................................................................30
7.8.3
Vital signs.............................................................................................................30


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7.8.4
ECG......................................................................................................................31
7.8.5
Others...................................................................................................................31
8.
REFERENCES....................................................................................................34
10.
HISTORY TABLE..............................................................................................36


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LIST OF TABLES
Table 5.1: 1
Censoring of the primary endpoint for the main estimand...............................10
Table 6.2: 1
Handling of iPDs ..............................................................................................17
Table 6.3: 1
Subject sets analysed ........................................................................................20
Table 7.4.2: 1 Censoring of the primary endpoint for the treatment policy and composite 
estimands ..........................................................................................................26
Table 10: 1
History table .....................................................................................................36


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2.
LIST OF ABBREVIATIONS
Term
Definition / description
ACM
All cause mortality
AE
Adverse events
ALT
alanine transaminase
AME
Average marginal effect
AMI
Acute myocardial infarction
ANCOVA
Analysis of Covariance
APACHE
Acute Physiology And Chronic Health Evaluation
ARDS
Acute respiratory distress syndrome
AST
aspartate transaminase
AUC
Area under the curve
BMI
Body mass index
CI
Confidence interval
CRF
Case report form
CRP
Creatinine reactive protein
CTP
Clinical Trial Protocol
CWRES
Conditional weighted residuals
DVT
Deep Vein Thrombosis
ECMO
Extracorporal membrane oxygenation
eGFR
Estimated glomerular filtration rate
EMA
European medicines agency
EU
European union
EudraCT
European Union Drug Regulating Authorities Clinical Trials Database
FAS
Full Analysis Set
GGT
Gamma Glutamyl Transferase
HR
Hazard ratio
ICU
Intensive care unit
INR
International normalised ratio
iPD
Important protocol deviation
IRT
Interactive recognition technology
ISTH
International Society on Thrombosis and Haemostasis


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Term
Definition / description
LMWH
Low molecular weight heparin
MBE
Major Bleeding Event
MedDRA
Medical Dictionary for Regulatory Activities
MI
Myocardial infarction
MV
Mechanical Ventilation
NIV
Non invasive Ventilation
PAI-1
Plasminogen Activator Inhibitor Type 1
pcVPC
Predicted corrected visual predictive check
PE
Pulmonary Embolism
PK
Pharmacokinetic
PKS
Pharmacokinetic Set
PPS
Per Protocol Set
PRED
Predications
RBC
Red blood cells
RD
Risk difference
REML
Restricted maximum likelihood
REP
Residual effect period
RPM
Report planning meeting
RS
Randomised Set
rt-PA
recombinant tissue-type plasminogen activator
SCR
Screening Set
SD
Standard deviation
SE
Standard error
SOC
Standard of Care
SOFA
Sequential (sepsis-related) Organ Failure Assessment
TEAE
Treatment emergent adverse events
TPA
Tissue Plasminogen activator
TS
Treated Set
TSAP
Trial Statistical Analysis Plan
UFH
Unfractionated heparin
ULN
Upper limit of normal
VFD
Ventilator Free Days


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Term
Definition / description
WBC
White blood cells
WHO
World health organisation


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3.
INTRODUCTION
As per ICH E9 (1), the purpose of this document is to provide a more technical and detailed 
elaboration of the principal features of the analysis, as described in the CTP. It will detail the 
procedures for executing the statistical analysis of the primary and secondary endpoints, and 
other data, and will be limited to the analysis of Part 1 of BI trial 0135-0347 only.
A database snapshot will be made when all patients have completed the Day 28 visit and 
when the data has been cleaned. At this point, all analyses outlined in this Trial Statistical 
Analysis Plan (TSAP) will be performed, except for one, all cause mortality (ACM) at Day 
90. This is a further endpoint that will be available at a later timepoint. When this data is 
available, then another snapshot will be taken and this endpoint analysed.
The results of Part 1 will be used for decision making and for progressing to Part 2, as 
outlined in the CTP.
An updated TSAP will later be produced to include the handling of the additional data of Part 
2.
This TSAP assumes familiarity with the CTP, including Protocol Amendments. In particular,
the TSAP is based on the planned analysis specification as written in CTP Section 7 
“Statistical Methods and Determination of Sample Size”. Therefore, TSAP readers may 
consult the CTP for more background information on the study, e.g., on study objectives, 
study design and population, treatments, definition of measurements and variables, planning 
of sample size and randomization.
The trial data is stored in the BI Rave (BRAVE) database system.
SAS® Version 9.4 or later will be used for all analyses, except for population PK as outlined 
in Section 7.8.5.


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4.
CHANGES IN THE PLANNED ANALYSIS OF THE STUDY
Whilst in the CTP there is a reference to the determination of the Per Protocol Set (PPS), this 
will only be done for Part 2 of the study. This is not considered to be a change from the 
protocol, since it will be done for Part 2. This is considered to be a clarification.
Regarding the protocol definition of the primary endpoint, time to clinical improvement or 
hospital discharge, there has been no detail added regarding patients potentially discontinuing 
the study early due to withdrawal of consent. In the unlikely event that a patient does 
discontinue from the study due to withdrawal of consent, prior to being able to determine if he 
had clinical improvement or hospital discharge, or had intake of bail-out therapy or death, he 
will be censored at the time of withdrawal. This is not considered to change the protocol 
definition, rather a clarification.
The randomisation stratification factor baseline ventilation status (Non Invasive Ventilation 
((NIV) / Mechanical Ventilation (MV)) is correlated with the baseline status on the world 
health organisation (WHO) eleven-point clinical progression scale (5, 6, 7, 8 or 9). That is to 
say, NIV take on values of 5 and 6, whilst MV takes on values of 7, 8 and 9 on the WHO 
clinical progression scale. Only the randomisation stratification factor ventilation status will 
be used in the primary model to avoid over parameterising. This is not considered to be a 
change from the protocol, since this still includes the baseline WHO status.
The secondary endpoint Improvement of Sequential (sepsis-related) Organ Failure 
Assessment (SOFA) score by ≥2 points from baseline to end of Day 6 is to be determined 
based upon assessments at Day 6 / hospital discharge. This is not considered to change the 
protocol definition, rather a clarification.
In Section 6.7 the definition of ‘on treatment’ has been modified, but is not inconsistent with 
the CTP. That is to say, alteplase ‘on treatment’ starts from first administration of alteplase 
and ends 288 h later, or longer for patients with more than 5d of infusion. For the SOC group, 
‘on treatment’ starts from randomization and ends 288 h later.
Unadjusted estimates of the treatment effect for the primary endpoint, main estimand, will be 
produced as outlined in Section 7.4.2. For the secondary efficacy endpoints, which are binary 
in nature, and for the continuous efficacy endpoint number of VFD at Day 28, the unadjusted 
estimates of the treatment effect will be created in addition. For the continuous efficacy 
endpoint PaO2/FiO2 ratio at Day 6, the adjusted treatment effect will be created. Details are 
outlined in Section 7.5.2.
Subgroup analyses not included in the CTP are outlined in Sections 6.7, 7.4.2 and 7.5.2 for 
the primary endpoint and one of the secondary endpoints. 
Any changes to the TSAP from Version 1 to Version 2 are summarized in Section 10.


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5.
ENDPOINTS(S)
5.1
PRIMARY ENDPOINT(S)
The primary endpoint as defined in Section 2.1.2 of the protocol will be used and will be 
analysed on the Full analysis set (FAS). This is based on the WHO Clinical Progression scale
(10), which ranges from 0 to 10. Clearly, a shorter time to clinical improvement or hospital 
discharge reflects a better outcome.
“Time to clinical improvement or hospital discharge up to Day 28, defined as the time 
from randomisation to either an improvement of two points on the 11-point WHO 
Clinical Progression Scale or discharge from the hospital, whichever comes first.”
However, there are several elements which have to be taken into account, when deriving the 
endpoint, and in order to address the hypothetical estimand. These are already specified in the 
protocol and summarized in the table below. 
In addition, in the unlikely event that a patient discontinues from the study due to withdrawal 
of consent, prior to being able to determine if he had clinical improvement or hospital 
discharge, or had intake of bail-out therapy or death, he will be censored at the time of study 
withdrawal. This has not been included in the protocol definition but is considered only as a 
clarification.
Bail-out therapy is as defined in protocol Section 4.2.2.1 Ultima ratio situation - Bail-out, 
which is ‘marketed Actilyse®’. The various scenarios are summarized in the table below:
Table 5.1: 1
Censoring of the primary endpoint for the main estimand
Scenarios
Main Estimand: 
Hypothetical situation
Bail-out then death
Censor at Bail-out
Bail-out then failure to improve#
Censor at Bail-out
Bail-out then clinical improvement / hospital 
discharge
Censor at Bail-out
Bail-out then clinical improvement / hospital 
discharge then death
Censor at Bail-out
Clinical improvement / hospital discharge 
regardless of subsequent events
Positive event at clinical 
improvement / hospital discharge
Death only, prior to D28
Censor at D28
Failure to improve# by D28
Censor at D28
Discontinued from study due to withdrawal of 
consent prior to clinical improvement / hospital 
discharge or Bail-out/Death
Censor at time of withdrawal


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Clinical improvement / hospital discharge refers to the first of clinical improvement or 
hospital discharge, whichever comes first.
#Failure to improve is failure to have clinical improvement or hospital discharge.
The planned analyses of the primary endpoint are outlined in Section 7.4.1, and the 
supplementary and sensitivity analyses of this endpoint in Section 7.4.2.
5.2
SECONDARY ENDPOINT(S)
5.2.1
Key secondary endpoint(s)
Not applicable.
5.2.2
Secondary endpoint(s)
The secondary endpoints as defined in Section 2.1.3 of the protocol will be used. Efficacy 
endpoints will be analysed on the FAS, and safety endpoints on the TS. In the case of the 
secondary endpoints, only Major bleeding event (MBE) up to Day 6 is defined as a safety 
endpoint.
The planned analyses of the secondary endpoint are outlined in Section 7.5.2.
Improvement of Sequential (sepsis-related) Organ Failure Assessment (SOFA) score by ≥2 
points from baseline to end of Day 6, FAS, binary endpoint
The SOFA score is made up of 6 individual components (Pulmonary, Coagulation, Hepatic, 
Circulatory, Neurologic, Renal), each with an integer value in the range of 0 to 4. 
The overall score comprises the sum of the individual components at that visit, the maximum 
value of which can be 24, and the lower the overall score, the better the health status of the 
patient. It is to be measured on days 0, 3, 5 or 6, or at hospital discharge if this is prior to day 
6.
If the visit was conducted but there are missing components, then the last observation will be 
carried forward from the previous assessment, in order to determine the overall score. All 
efforts will be made to have minimal or no missing data.
If the patient does not have a value at Day 6 due to being discharged from hospital prior to 
this, then it will be considered that the patient had an improvement ≥2 points in SOFA score.
If the patient does not have a value at Day 6 due to death, then it will be considered that the 
patient did not have an improvement ≥2 points in SOFA score.
If day 6 total value is missing but day 5 total value is available, then day 5 total value will be 
compared to baseline to check for an improvement of ≥2 points.
Otherwise, SOFA improvement will be left as missing and this patient not included in the 
denominator.
The overall score will be compared to baseline (Day 0) to see if there is an improvement ≥2 
points. Specifically, if value – baseline value ≤-2 then the patient is considered to have had an 
improvement ≥2 points in SOFA score at end of Day 6.


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MBE (according to International Society on Thrombosis and Haemostasis [International 
Society on Thrombosis and Haemostasis (ISTH) definition (11)] until Day 6, TS, binary 
endpoint
If the patient has an MBE, at any timepoint up to end of Day 6 then he has met this 
unfavorable endpoint. If it is unknown whether the patient had an MBE up to end of Day 6, 
regardless of the reason, then it will be assumed that the patient did not have an MBE by the 
end of Day 6. This is a safety defined endpoint and the starting point for consideration should 
be consistent with the ‘on treatment’ definition in Section 6.7, however only including events 
up to midnight of Day 6.
ACM at Day 28, FAS, binary endpoint
If it is unknown whether the patient was dead at end of Day 28, then it will be assumed that 
the patient did not die up to Day 28, regardless of the reason. This unfavorable endpoint is 
met if:

the last known status of the patient is 10 on the WHO clinical progression scale by the end 
of Day 28, or

vital status is dead within 28 days
ACM / MV at Day 28, FAS, binary endpoint
If it is unknown whether the patient was on MV at Day 28, it will be assumed that the patient 
was not on MV at Day 28, regardless of the reason. This unfavorable endpoint is met if:

the last known status of the patient has a value of ≥7 on the WHO clinical progression 
scale, up to end of Day 28, or

vital status is dead within 28 days
Daily average PaO2/FiO2 ratio (or inferred PaO2/FiO2 ratio from SpO2) change from
baseline to Day 6, FAS, continuous endpoint
This assessment is to be measured approximately 3-times daily on each of days 0 to 7, and 
again on day 28, but only whilst the patients is still in hospital. All available values on each of 
these days, regardless of the position of the patient when being measured, will be averaged in 
order to determine the daily average PaO2/FiO2 ratio for that patient. The higher the value the 
better the health status of the patient.

If the patient is still in hospital during day 6 then the day 6 daily average value will be 
used, if available. 

If the patient has been discharged from hospital prior to day 6 then the daily average at the 
time of hospital discharge will be used as a surrogate for day 6, if available.

If the patient has died prior to day 6 then there will be no imputation but the death handled 
as failure in the determination of the difference in medians and 95% CI.
Based upon this, the change from baseline for each patient will be calculated and used for the 
analysis.
Number of ventilator-free days (VFD) from start of treatment to Day 28, FAS, continuous 
endpoint
For this endpoint, ‘ventilator’ is defined as ‘assisted breathing’ but it refers to mechanical 
invasive ventilation. The number of VFDs starts from when the patient has a ‘lasting’ value 
on the WHO clinical progression scale of ≤6, and ends on Day 28. A lasting value of ≤6
means that the value cannot exceed 6 at a later timepoint. If the patient is liberated from the 


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ventilator on Day x, then the number of VFDs is 28-x. If a patient has withdrawn consent 
prior to day 28 then he will have a missing value for VFD. In any case, if the status of the 
patient at Day 28 is death, as determined from the vital status page then the VFD=0.


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6.
GENERAL ANALYSIS DEFINITIONS
6.1
TREATMENT(S)
For the purposes of the statistical analyses, treatments will be referred to in the same context 
as in Section 7.4 of the protocol.

“Alteplase low”, which is alteplase 0.3mg/kg over 2 hours followed by 0.02 mg/kg/h over 
12 hours

“Alteplase high”, which is alteplase 0.6 mg/kg over 2 hours followed by 0.04 mg/kg/h 
over12 hours

Standard of Care (SOC)
Alteplase treatment is planned to be up to end of Day 5 but all patients should be taking SOC 
in the background. On day 1 there is an initial 2 h infusion for all, followed up by a longterm 
12 h infusion. On days 2-5 the patients can receive an optional 2h infusion (based on 
investigator decision), but only on one of these days. Each patient should receive a longterm 
12h infusion on these remaining 4 days. 
6.2
IMPORTANT PROTOCOL DEVIATIONS
Whilst is it not planned to conduct a Per Protocol analysis for Part 1 of this study (too few 
patients), patients with any of the following important protocol deviations (iPDs) will be
reviewed on a case by case basis (2) and (3). The review of the iPDs will be used to determine 
criteria for a PPS for Part 2 of the study.
Note that this is a working list and may be updated again prior to the final Report Planning 
Meeting (RPM) prior to the snapshot related to end of Part 1. Inclusion criteria/exclusion 
criteria numbers refer to the definition given in the protocol or CRF.
Table 6.2: 1
Handling of iPDs
iPD code
iPD Category & Brief Description
A
Entrance Criteria Not Met
A1
Inclusion Criteria not met
A1.1
Patient too young (IC1)
A1.2
Diagnosis of ARDS questionable (IC2,4,5)
A1.3
Patient does not test positive for SARS-CoV-2 (IC3)
A2
Exclusion Criteria not met
A2.1
Patient has baseline conditions that are not permitted for safety reasons 
(EC1-31)


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Table 6.2: 1
Handling of iPDs (continued)
A2.2
Patient has baseline conditions that prevent patient from complying with 
study protocol (EC1-31)
A2.3
Patient has had previous conditions or procedures that are not permitted 
(EC1-31)
A2.4
Patient has had forbidden previous therapy (EC1-31)
A2.5
Patient pregnant (EC25)
B
Informed Consent
B1
Informed consent not available (IC6)
B2 
Informed consent not signed at screening visit 1 (IC6)
B3 
Informed consent not available or not available at visit 2a (pre-dose)  (IC6)
C
Trial medication and randomisation
C1
Method of assigning patients to treatment arms
C1.1
Randomisation not followed according to protocol
C1.2
Patients do not receive the initial treatment they were randomised / allocated 
to
C1.3
Patient assignment not followed
C1.4
Timeframe > 1 day between randomisation and first drug intake
C2
Non-compliance
C2.1
Overall Compliance not between 80% and 120% inclusive – see definition in 
Section 5.4.
C2.2
Patient takes the optional 2h infusion on more than one occasion after the 
initial 2 h infusion.
C3
Drug assignment and administration of doses for each patient
C3.1
Dosage and treatment schedule not given according to protocol for initial iv 
infusion
C3.2
Dosage and treatment schedule not given according to protocol for long term 
iv infusion 
C3.3
Drug not permanently discontinued according to Section 3.3.4.1 of CTP
C3.4
Drug not temporarily interrupted according to Section 3.3.4.1 of CTP


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Table 6.2: 1
Handling of iPDs (continued)
D
Concomitant medication
D1
Intake of restricted concomitant medication according to protocol section 
4.2.2.1 and Section 9.2
D2
Intake of marketed Actilyse® (non-study drug) not as ultima-ratio or bail-out
G
Other trial specific important deviations
G1
Other protocol violations affecting patient rights or safety (manual PDs to be 
captured)
6.3
SUBJECT SETS ANALYSED
The subject sets as defined in protocol Section 7 for the Randomised Set (RS), Full Analysis 
Set (FAS) and Treated Set (TS) will be used.
The Screened set (SCR), not outlined in the CTP, will include all patients screened.
The RS will consist of all randomised patients.
The FAS will consist of all randomised patients with at least one baseline and one post 
baseline assessment relating to the primary endpoint of interest. That is to say, they need to 
have at least one assessment post baseline on the 11-point WHO Clinical Progression Scale.
The TS will consist of all patients who were randomised and, for patients in the alteplase 
groups, treated with at least one dose of trial drug.
The Pharmacokinetic set (PKS), not outlined in protocol, will comprise all subjects in the TS 
who provided at least one PK endpoint and had no important protocol violations relevant to 
the evaluation of PK.


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Table 6.3: 1
Subject sets analysed
Subject set
Class of endpoint
SCR / RS
TS
FAS
PKS
Primary endpoint 
primary and 
supplementary 
analyses
Secondary and further EFFICACY 
endpoints 
X
Secondary and further SAFETY 
endpoints
X
Treatment exposure
X
Disposition
X
Demographic/baseline 
X
X
Concomitant medications and 
compliance
X
X
All other safety analyses
X
PK analyses
X


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6.5
POOLING OF CENTRES
Part 1 of this study is planned to be carried out in approximately 35 centres. However, not all 
centres have the same SOC procedures. During the course of the study, and gathering 
information (retrospectively) from each of the centres with regard to their SOC procedures, 
they will be grouped into relatively homogeneous groupings.
Because the groupings will only be made after randomisation of a patient within a centre, this 
will be a retrospective and exploratory analysis, albeit planned. It is planned only to 
investigate the primary endpoint with this centre grouping in a sensitivity analysis – See 
Section 7.4.2.
6.6
HANDLING OF MISSING DATA AND OUTLIERS
All efforts will be made to avoid having missing data in the database, however it is inevitable 
that there will be missing data, by design:

For patients that die, there will be no further data. 

For patients that are discharged from hospital before day 28, the only additional 
efficacy assessment is the vital status at days 28 and 90.
•
For the daily average PaO2/FiO2 ratio at Day 6: if no value available due to hospital 
discharge, then use value at hospital discharge; if no value available due to death then 
do not impute but handle as failure in analysis to determine difference in median with 
95% CI. Otherwise, outcome unknown and not included in denominator.


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•
For SOFA improvement: if no value available due to hospital discharge the 
improvement=yes; if no value available due to death then improvement=No; if 
missing components but assessment conducted, the previous components can be 
carried forward; if day 5 value available then this can be used in place of day 6; then 
improvement from baseline determined. Otherwise, outcome unknown and not 
included in denominator.
•
For Evolution of APACHE II and daily average PaO2/FiO2: carry forward missing 
APACHE II components if possible (APACHE II); otherwise no imputation but 
present the number of cumulative hospital discharges and cumulative deaths in the 
same table.
Missing or incomplete adverse event (AE) dates will be computed using the BI handling 
rules (4).
6.7
BASELINE, TIME WINDOWS AND CALCULATED VISITS
Randomisation is defined as Day 1 and it is planned that the alteplase patients are 
administered their initial 2h infusion of study medication within 6 hours of randomisation, on 
Day 1 (Visit 2a). The last planned administration of study medication for the alteplase patients 
is the long term 12h infusion on Day 5. 
Since all patients are on SOC and there is no clear start or stop of SOC, a fixed treatment 
duration will be used for all patients, for the purposes of the analyses. Therefore, endpoints 
referred to as ‘on treatment’ will include all relevant events from the relevant start point plus 
288 hours (last planned treatment on Day 5 + 7 days of REP = 12 days = 288 hrs). The 
starting point for the alteplase patients is date and time of first administration of alteplase, 
whilst the starting point for the SOC patients is the date and time of randomisation. For 
alteplase patients which have more than 5 days of infusion, the end date of ‘on treatment’ will 
be the date and time of last intake of alteplase plus 168h (=7 days). 
According to CTP Section 1.2, the residual effect period (REP) is 7 days.
According to CTP Section 7.2.1, the term "baseline" refers to the last observed measurement 
prior to randomisation on Day 1, which will be Visit 1, the screening visit. It cannot be 
determined with certainty if a value measured on Day 1 is prior to randomisation, so the 
screening value will be used consistently throughout. Only if otherwise indicated, will the 
assessment on Day 1 be used, where the time of the assessment can be compared to the time 
of the randomisation.


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7.
PLANNED ANALYSIS
The tables that are created from the statistical analysis will follow the format of the standard 
within BI (5).
The overall disposition of the patients in the trial will be presented, together with the number 
of patients discontinuing prior to day 28, and vital status at day 90.
The number of patients participating in the study by country and the number of sites, for each 
of the patient populations will be summarised. 
Demography and baseline characteristics will be presented, as well as the stratification used at 
the time of the patient randomisation. 
A summary of the iPDs will be provided.
Where statistical comparisons are indicated, they will be done by comparing the following 
groups:

alteplase low versus SOC

alteplase high versus SOC
For efficacy analyses, patients will be analysed according to the patient information 
(ventilation status) given in the CRF (NIV / MV), in the case of potentially erroneous data 
entered into the interactive recognition technology (IRT). 
In general, for End-Of-Text tables, the set of summary statistics is: N (number of patients 
with non-missing values) / Mean / SD / standard error (SE) / Min / Q1 (lower quartile)/ 
Median / Q3 (upper quartile)/ Max. 
Statistical parameters will be displayed to a defined number of decimal places as specified in 
the BI guideline "Reporting of Clinical Trials and Project Summaries" (6).
Tabulations of frequencies for categorical data will include all possible categories and will 
display the number of observations in a category as well as the percentage (%) relative to the 
respective treatment group (unless otherwise specified, all patients in the respective patient set 
whether they have non-missing values or not). Percentages will be rounded to one decimal 
place. The category missing will be displayed only if there are actually missing values. 
7.1
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS
Only descriptive statistics are planned for this section of the report. See Section 5.4 for 
specific definitions.


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7.2
CONCOMITANT DISEASES AND MEDICATION
Medical history, COVID-19 signs and symptoms and concomitant diagnoses will be 
presented and coded using MeDRA System Organ Class and Preferred Term where possible.
Regarding concomitant medications, there is a specific question asking if the treatment 
belongs to standard of care, which is relevant for all patients since they all receive SOC in the 
background. This question is different to the randomised treatment assignment. Therefore, all 
concomitant medication analyses or displays will be split by ‘Standard of Care use’ and ‘non-
Standard of Care use’. All concomitant medications will be coded using the WHO Drug 
dictionary. 
Medications of special interest for COVID (as indicated by WHO, see Section 9.1), and 
restricted medications within this study (as outlined in CTP Section 4.2.2.1, see Section 9.2) 
will also be summarized and split by Standard of Care use and non-Standard of Care use. 
Use of heparin will be summarized and presented.
Therefore, only descriptive statistics are planned for this section of the report.
7.3
TREATMENT COMPLIANCE
Only descriptive statistics are planned for this section of the report, and due to the nature of 
the study, compliance will be determined for the alteplase treatment groups only
Compliance to intake of infusion will be assessed in terms of % taken, as measured by 
volume, at each planned infusion. Overall compliance will be as an average measure of the 
%taken. The detailed definitions are provided in Section 5.4.
Compliance with preparation of the infusion in terms of concentration, as indicated in CTP 
Table 10.5 are also assessed, and detailed definitions provided in Section 5.4.
7.4
PRIMARY ENDPOINT(S)
7.4.1
Primary analysis of the primary endpoint(s)
For the primary endpoint, a cox proportional hazards model will be used to estimate the 
hazard ratio (HR) comparing the alteplase groups to SOC. From the model, the 95% 
confidence intervals (CI) and corresponding Wald p-values for the HRs will be produced. The 
model will include fixed effects for treatment (alteplase group of interest, SOC), the 
randomisation stratification factor type of ventilation (NIV, MV) and age (continuous).
Baseline ventilation status (NIV/MV) and baseline status on the WHO eleven-point clinical 
progression scale (5, 6, 7, 8 or 9) are correlated. That is to say, NIV take on values of 5 and 6, 
whilst MV takes on values of 7, 8 and 9 on the WHO clinical progression scale. Of these two, 
only the randomisation stratification factor ventilation status will be used in the primary 
model to avoid over parameterising. This is not considered to be a change from the CTP, 
since this still includes the baseline WHO status.


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Kaplan Meier estimates will be presented by treatment (low alteplase, high alteplase, SOC)
for this endpoint up to Day 28, and additionally by:

treatment and type of ventilation (NIV, MV)

treatment and baseline status on the WHO eleven-point clinical progression scale (5, 6, 7, 
8 or 9)

treatment and age groups (<=median, >median)
From the KM estimates stratified by treatment, and at various timepoints (days 6, 8, 12, 16, 
20, 24 and 28) the risk difference (RD) with 95% CI will be estimated by taking the 
difference of the individual probabilities. Since the observations in the two treatment groups 
are independent, the variance of the Kaplan-Meier probability difference is equal to the sum 
of the Kaplan Meier probabilities’ variances found by Greenwood's method. The 95% CI of 
the RD is therefore calculated accordingly.
7.4.2
Sensitivity analysis, subgroup analysis, exploratory analysis of the primary 
endpoint(s)
Two non-confirmatory supplementary analyses will be conducted, in anticipation of the rare 
situation where bail-out therapy may be used (as defined in CTP Section 4.2.2). They will be 
performed on the FAS.
The first of these supplementary analyses will address the treatment policy estimand, whereby 
there is no consideration for the intake of bail-out therapy.
The second of these supplementary analyses will address a composite estimand, whereby 
patients receiving bail-out therapy will be censored at Day 28, rather than on the day of bail-
out. Here, bail-out represents treatment failure and thereby, by definition, precludes any 
response after bail-out. The various scenarios are summarized in the table below.
In both cases, and in the unlikely event that a patient discontinues from the study, prior to 
being able to determine if he had clinical improvement or hospital discharge, or had intake of 
bail-out therapy or death, he will be censored at the time of withdrawal. 


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Table 7.4.2: 1 Censoring of the primary endpoint for the treatment policy and composite 
estimands
Scenarios
Treatment policy
estimand
Composite estimand
Bail-out then death
Censor at D28 
Censor at D28 
Bail-out then failure to improve#
Censor at D28 
Censor at D28 
Bail-out then clinical improvement 
/ hospital discharge
Positive event at clinical 
improvement / hospital 
discharge
Censor at D28 
Bail-out then clinical improvement 
/ hospital discharge then death
Positive event at clinical 
improvement / hospital 
discharge
Censor at D28 
Clinical improvement / hospital 
discharge regardless of subsequent 
events
Positive event at clinical 
improvement / hospital 
discharge
Positive event at clinical 
improvement / hospital 
discharge
Death only, prior to D28
Censor at D28 
Censor at D28 
Failure to improve# by D28
Censor at D28 
Censor at D28 
Discontinued from study due to 
withdrawal of consent prior to 
clinical improvement / hospital 
discharge or Bail-out/Death
Censor at time of study 
discontinuation
Censor at time of 
withdrawal
Clinical improvement / hospital discharge refers to the first of clinical improvement or 
hospital discharge, whichever comes first.
#Failure to improve is failure to have clinical improvement or hospital discharge.
The analyses performed will be identical to that outlined for the primary endpoint in Section 
7.4.1.
Based on the main estimand, the treatment effect will be estimated using the cox proportional 
hazard model, containing only treatment in the model. The HR (95%CI) and p-value 
estimating the unadjusted treatment effect will be produced.
The effect of the subgroups outlined in Section 6.4, related to the primary endpoint, main 
estimand, will be assessed descriptively and using Kaplan Meier estimates. 
A covariate adjusted sensitivity analysis of the primary endpoint, main estimand, will be 
conducted, to determine the effect of the grouping of centres according to similar types of 
Standard of care. This will be the same analysis as outlined in Section 7.4.1 with this 
additional covariate.


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7.5
SECONDARY ENDPOINT(S)
7.5.1
Key secondary endpoint(s)
Not applicable.
7.5.2
Secondary endpoint(s)
Secondary endpoints are defined in Section 5.2.2. In all cases, standard descriptive statistics 
will be presented, whether the endpoint is binary or continuous.
Binary efficacy endpoints
For the binary efficacy endpoints (ACM, ACM/MV, SOFA), analysed on the FAS, the RD
will be estimated between the two treatment arms using logistic regression. In addition to 
treatment, adjustment will be for the randomization stratification factor (NIV/MV) and age. 
RD and CIs (95%) for the RD will be obtained using the Delta method and the average 
marginal effect (AME) method.
As sensitivity analysis for each of these binary efficacy endpoints, the unadjusted treatment 
estimates will be produced, using the same logistic regression model containing only 
treatment.
Binary safety endpoint
For the binary safety endpoint MBE, analysed on the TS, there will be no adjustment for 
covariates. Therefore, the RD will be the difference between the two incidences and the 95% 
CI will be determined using the Chan and Zhang exact method.
Continuous efficacy endpoint
The change from baseline in the continuous efficacy endpoint, daily average PaO2/FiO2 ratio 
at Day 6 is considered non-parametric in nature. Missing values for missing visits will not be 
imputed, and left as missing. Missing values due to death or post hospital discharge will be 
carried out as described in Section 5.2.2. The difference in medians will be presented, 
together with 95% CIs, based upon the methodology of Hodges-Lehmann and the Wilcoxon 
rank sum test. There will be no adjustment for covariates in this non-parametric analysis.
As a sensitivity analysis for this PaO2/FiO2 endpoint, an ANCOVA analysis adjusting for 
baseline ventilation status and age will be performed. For patients that have died, the last 
value prior to death will be used to determine the change from baseline. Note, there are 
expected to be very few deaths prior to Day 6. Missing values due to hospital discharge will 
be handled as in the main analysis. 
For the continuous efficacy endpoint, number of VFDs at Day 28, there are not expected to be 
any missing values by definition. This endpoint will be analysed using a restricted maximum 
likelihood (REML) based ANCOVA comparing the number of VFDs after 28 days, by 
treatment. The ANCOVA model will be adjusted for the randomization stratification factor 
(NIV/MV) and age. The treatment difference with 95% CI will be presented. 


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As a sensitivity analysis for VFD at Day 28, the unadjusted treatment effect will be estimated, 
using the same approach as the main analysis, but with only treatment in the model. In 
addition, subgroup analyses of VFD at Day 28 will be conducted using the subgroups as 
outlined in Section 6.4. An ANCOVA model will contain the subgroup and the interaction 
between subgroup and treatment.
7.7
EXTENT OF EXPOSURE
Exposure will be handled descriptively. 
The number of patients with initial 2h infusion on day 1, optional 2h between days 2 and 5, 
and longterm 12h infusion over the five days of treatment will be summarized, as well as the 
rate of infusion (mL/kg/h) and duration of infusion (h).
The cumulative exposure will be presented, in terms of Total number of days of infusion.
Exposure is relevant for the alteplase treatment groups only.
7.8
SAFETY ANALYSIS
The safety analysis will be performed as outlined in the CTP, according to BI standards and 
on the TS. For further details please see guideline 'Handling and summarization of AE data 
for clinical trial reports and integrated summaries' (7).
The definition of ‘on treatment’ is outlined in Section 6.7. Any potential events starting 
between the time of randomization and the start of alteplase treatment, will be indicated as 
such in the listings.
In addition, to fulfil the requirements of the European Union (EU) Regulation 536/2014 
(Annex V, Point 6) (8), the following information should be made available, for the lay 
summary or CTR:


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
frequency of serious drug-related AEs by treatment, primary system organ class and 
preferred term.
7.8.1
Adverse Events
Unless otherwise specified, the analyses of AE will be descriptive in nature. All analyses of 
AEs will be based on the number of subjects with AEs and NOT on the number of AEs.
AEs will be coded using the most recent version of the Medical Dictionary for Regulatory
Activities (MedDRA) coding dictionary
The analysis of AEs will be based on the concept of treatment emergent adverse events
(TEAE). That means that all adverse events occurring ‘on treatment’ as outlined in Section 
6.7 will be considered as TEAE. 
All AEs occurring before ‘on treatment’ will be assigned to ‘screening’ and all AEs
occurring after ‘on treatment’ will be assigned to ‘post-treatment’. In general, AEs attributed 
to ‘Screening’ or ‘Post-treatment’ will be listed only.
An overall summary of AEs will be presented.
Frequencies [N (%)] of patients with AEs will be summarised by treatment, primary system 
organ class and preferred term (using MedDRA). Separate tables will be provided for 
patients with serious adverse events.
The system organ classes will be sorted according to the standard sort order specified by 
European medicines agency (EMA), preferred terms will be sorted by frequency (within 
system organ class). The severity of AEs will be summarised by the maximum intensity of 
the events each patient had. This will show the number and percent of patients who had at 
most mild, moderate, severe, and life threatening events. Severe AEs is defined as an AE 
with mild, moderate, severe grade.
Additionally, the following analyses will be reported for disclosure on European Union Drug 
Regulating Authorities Clinical Trials Database (EudraCT) and clinicaltrials.gov:

Frequency [N(%)] of patients with non-serious AEs occurring with
incidence in preferred term greater than 5% by treatment,

AEs per arm for disclosure on EudraCT by treatment

Non-serious AEs for disclosure on EudraCT by treatment

Serious AEs for disclosure on EudraCT by treatment
Adverse events of interest
All endpoints related to safety, outlined in Section 5.2.2 and Section 5.3, are clearly marked 
as such. In particular, endpoints related to MBE, Stroke, MI, PE and DVT. There is no further 
description below on how they should be analysed, however, they will be summarized with 
the safety data under the topic of ‘AEs of special interest’.


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There are two other AEs of special interest, which are not mentioned in Sections 5.2.2 and 
5.3:

Transient ischemic attack

Systemic embolism
AEs recorded on the AE page of the CRF, indicated as such via the relevant tick box, will be 
grouped and summarized using frequency tables.
7.8.2
Laboratory data
Changes in coagulation and inflammatory markers on treatment, as outlined in Section 5.3, 
are the laboratory parameters of special interest in this trial. They will be analysed in the 
efficacy section.
The analyses of laboratory data will be descriptive in nature and will be based on BI standards
“Handling, Display and Analysis of Laboratory Data” (9).
The following laboratory parameters are considered safety and hence processed according to 
standard safety processing rules:

Haematology (Haematocrit, Haemoglobin,  Red blood cells (RBC) / Erythrocytes, 
White Blood Cells (WBC) / Leukocytes, Platelet Count / Thrombocytes)

Clinical Chemistry (Albumin,  Alkaline phosphatase,  ALT (alanine transaminase), 
AST (aspartate transaminase), GGT ( Gamma Glutamyl Transferase), Bilirubin total,  
Bilirubin direct, Creatinine, eGFR, Creatine kinase,  Troponin, Lactate dehydrogenase, 
Glucose, Potassium, Sodium, Calciom, Magnesium,  Urea (BUN),  NT-proBNP, 
Cystatin C, Total Cholesterol, LDL- cholesterol, HDL- cholesterol, Triglycerides, 
Protein total, Glucose, Hba1c, C-peptide.
For continuous safety laboratory parameters standardized values will be derived as well as the 
differences to baseline. Laboratory parameters will be shown in SI units.  
Laboratory values will be compared to their reference ranges and frequency tables will be 
provided for the number of patients within and outside the reference range at baseline, each 
visit and the last measurement on treatment. Descriptive statistics will be provided by 
treatment group for baseline, each visit and last value on-treatment and for changes from 
baseline to each respective visit and last value on treatment. The laboratory parameters will be 
summarized by day of assessment.
7.8.3
Vital signs
The following vital signs will be descriptively analysed: systolic and diastolic blood pressure, 
body temperature, respiratory rate and pulse rate. Both absolute values and change from 
baseline are planned for this section of the report. 


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7.8.4
ECG
Not applicable.
7.8.5
Others
Pharmacokinetic analysis
Descriptive statistics of all PK parameters will be presented and will be performed on the 
PKS as outlined in Section 6.3. In particular, the parameters / endpoints are:

Css,1 (plasma concentration of alteplase at steady state of 2 hour infusion)

Css,2 (plasma concentration of alteplase at steady state of long-term infusion)
Individual endogenous Tissue Plasminogen activator (TPA) will be determined for each 
patient at baseline using the first PK sample. Baseline levels will then be subtracted from all 
further concentration measurements (second and third samples), for each patient, in order to 
determine the Css,1 and Css,2 endpoints. In case the resulting concentration value is negative, 
it will then be set to “BLQ”.
As outlined in CTP Section 7.2.4, a population PK analysis is planned to be conducted if 
sufficient data are available. The objectives of the population PK analysis are:-
1) to compare the alteplase concentrations in study 0135-0347 to PK simulations based on a 
previously developed population PK model for patients with acute myocardial infarction 
(AMI)
2) to derive model-based area under the curve (AUC) estimates for patients with ARDS 
based on the sparse PK measurements of study 0135-0347
Dataset preparation
Alteplase plasma concentrations, sampling times, infusion times, and covariates (e.g., age, 
weight, sex, and race) will be assembled and formatted for analysis. Detailed dataset
specifications will be provided in a separate document.
Handling of missing data
Handling of missing data will be described in the dataset specification document.
Handling of outliers
Alteplase concentrations and covariate values will be included in the analysis whenever 
possible. However, for methods based on least-squares estimation and normal theory it is not 
good practice to include extreme values. Thus, outlying data points in the dependent variable 
may be excluded if they are infrequent, occur randomly and are spurious. Furthermore, it may 
be justified to exclude outlying individuals from model development. Also, extreme covariate 
values might be excluded.  If more than 5% of alteplase plasma concentrations are excluded, a 
sensitivity analysis will be performed to evaluate the impact of the excluded data points on the 
final model. All outliers which are excluded from the analysis will be documented along with 
reasons for their exclusion.
Pharmacokinetic modeling strategy


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In the following the framework of the PK modelling strategy is outlined, this may be adjusted 
as deemed necessary.
The pharmacokinetics of alteplase follows a 3-compartment distribution. A 3-compartment 
model cannot be developed using the information from study 0135-0347 alone as only sparse 
PK data is collected in a subset of the study population. Therefore, a previously developed 
population PK model for alteplase will be used to inform the current analysis.  The population 
PK model for patients with AMI which is based on studies 0135.30 and 0135.53 is the 
preferred starting model. The pharmacokinetics of alteplase in these AMI studies were best 
described by a linear 3 compartment model. A baseline t-PA concentration was estimated to 
account for endogenous t-PA in plasma. Weight was included as covariate affecting baseline 
t-PA concentration and clearance of alteplase. Age was included as covariate affecting 
clearance of alteplase.
First, the alteplase plasma concentrations in study 0135-0347 will be compared to PK model 
simulations. Simulations will be performed using the previously developed population PK 
model for alteplase in patients with AMI. In the simulations, inter-individual and residual 
variability will be considered. The alteplase plasma concentration-time profiles will be 
simulated 1,000 times for each subject in study 0135-0347 assuming its actual infusion 
regimen, sampling scheme and weight. The simulated alteplase plasma concentrations at the 
different time points will be used to calculate the median concentration-time profile as well as 
it’s 90% prediction interval. This simulation results will be graphically compared to the 
observed data in study 0135-0347.
Given that the model-simulated plasma PK is in the same range as the observations, it will be 
further investigated whether the population PK model for patients with AMI can describe the 
individual alteplase concentrations in ARDS patients. Individual post-hoc estimates will be 
derived for study 0135-0347 using the parameters of the population PK model for patients 
with AMI. The distribution of the post-hoc estimates around the typical parameters and the 
description of the individual plasma concentrations will be investigated. 
Provided that the population PK model for patients with AMI successfully describes the 
individual profiles of patients with ARDS, the individual PK parameters will be determined. 
In addition, AUC will be derived within NONMEM® from the individual PK parameters 
considering the patients’ actual alteplase doses, infusion regimen, and covariates. In case, the 
structural PK model of patients with AMI cannot describe the PK of patients with ARDS, 
necessary adjustments (e.g. re-estimation of clearance) might be implemented into the model. 
The population PK model for patients with AMI comprises the impact of weight and age on 
the PK of alteplase. Assessment of any remaining trends for weight, age, sex, and race will be 
conducted e.g. by graphical inspection plotting empirical Bayes estimate versus covariate 
values and conditional weighted residuals (CWRES) versus covariate values. 
Inter-subject variability will be modeled using exponential random effect models. Residual 
variability will be modeled starting with the same error model structure as in patients with 


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AMI; adjustments will be made if necessary. The set of inter individual (η) and residual (ε) 
variability values are assumed to be symmetrically distributed around 0 with variances ω2 and 
σ2, respectively. 
Evaluation 
If model development is necessary, objective function values (for nested models) as well as 
the criteria listed below will be considered:

A “successful minimization” statement by the NONMEM® program at the end of the
estimation step.

The final parameter estimates have a number of significant digits of 3 for all θ’s.

Estimates of θ’s not close to a boundary.

Correlation of the uncertainty of the estimates of fixed effect parameters < 0.95 or >-0.95.

No significant trends in the basic goodness-of-fit plots (predictions (PRED), individual 
predictions (IPRED) vs. DV)

Residuals (CWRES) scatter randomly and uniformly around zero when plotted against 
population prediction and time.

ETAs are normally distributed around 0.

No significant model misspecification in the individual predicted concentration-time 
profiles when compared to the observed profiles.
If a model is accepted as final model, although one or more of the criteria above are not 
fulfilled, the reasons for its acceptance need to be given in the report.
If model development is necessary, the final model will be evaluated using a prediction 
corrected visual predictive check (pcVPC, 12). A visual predictive check shows the ability of 
the model to simulate data that are similar to the observed data that were used for model 
development. The concentration-time profiles will be simulated 1,000 times using the same 
actual doses, infusion regimen, subjects’ covariates, and study design as in the analysis data 
set. In order to adjust for the differences in e.g. actual doses, a pcVPC will be performed in 
which the observed and simulated DV will be normalized based on the typical population 
prediction. 


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8.
REFERENCES
1.
CPMP/ICH/363/96: "Statistical Principles for Clinical Trials", ICH Guideline Topic 
E9, Note For Guidance on Statistical Principles for Clinical Trials, current version.
2.
001-MCS-40-413: Identify and Manage Important Protocol Deviations (iPD)”, current 
version, Group “Clinical Operations”, IDEA for CON.
3.
001-MCS-40-135_RD-01: “Integrated Quality and Risk Management Plan”, current 
version, Group “Clinical Operations”, IDEA for CON.
4.
001-MCG-156_RD-01: "Handling of missing and incomplete AE dates", current 
version; IDEA for CON.
5.
001-MCG-159_RD-06: “Standard table shells for inferential and descriptive Company 
Standard Displays (CSD-Catalogue)”, current version; IDEA for CON.
6.
001-MCG-159: "Reporting of Clinical Trials and Project Summaries", current version; 
IDEA for CON. 
7.
001-MCG-156: "Handling and summarization of adverse event data for clinical trial 
reports and integrated summaries", current version; IDEA for CON.
8.
REGULATION (EU) No 536/2014 OF THE EUROPEAN PARLIAMENT AND OF 
THE COUNCIL of 16 April 2014 on clinical trials on medicinal products for human 
use, and repealing Directive 2001/20/EC, European Commission webpage.
9.
001-MCG-157: "Handling, Display and Analysis of Laboratory Data", current version; 
IDEA for CON.
10.
WHO Working Group on the Clinical Characterisation and Management of
COVID-19 infection. A minimal common outcome measure set for
COVID-19 clinical research. Lancet Infectious Diseases, Published Online
June 12, 2020, doi: 10.1016/S1473-3099(20)30483-7; 2020. [R20-2002]
11.
Schulman S, Kearon C. Subcommittee on Control of Anticoagulation of the Scientific 
and Standardization Committee of the International Society on Thrombosis and
Haemostasis. Definition of major bleeding in clinical investigations of antihemostatic
medicinal products in non-surgical patients. J Thromb Haemost. 2005. 3: 692-694.
[R05-0344]
12.
Bergstrand M, Hooker AC, Wallin JE, Karlsson MO Prediction-corrected visual 
predictive checks for diagnosing nonlinear mixed-effects models. AAPS J 2011 ; 13(2) ; 
143-151. [R13-0300]


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10.
HISTORY TABLE
Table 10: 1
History table
Version
Date
(DD-MMM-YY)
Author
Sections 
changed
Brief description of change
1.0
17-DEC-2020
None
This is the final TSAP.
2.0
03-May-2020
2
Administrative: Added 
abbreviation for APACHE.
4
Administrative: Included all 
changes, including also for 
TSAP V1.
6.6
Administrative: Addition of 
clarification sentence for 
SOFA.
7.8.1
Administrative: Remove text 
on collapsing of AEs to be in 
line with new template.
8
Administrative: Reference 
format same for all.
7.4.2
Addition of sensitivity 
analysis: Estimate of 
unadjusted treatment effect 
for primary endpoint, main 
estimand.
7.5.2
Addition of sensitivity 
analyses: Estimates of 
unadjusted treatment effects 
for MBE, ACM, ACM/MV, 
VFD at Day 28.
Estimate of adjusted 
treatment effect for 
PaO2/FiO2 ratio at Day 6. 
4,
5.2.2
Clarification: TEAE text 
added to the secondary 
endpoint ‘MBE until Day 6’ 
4,
5.3,
7.4.1
Clarification: Patients with 
WHO=9 at baseline are 


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accepted and text updated 
accordingly.
4,
6.7
Clarification: Definition of 
‘on treatment’ to start from 
first administration of 
alteplase / randomisation for 
active / SOC groups, and to 
last for 288h (=12days). For 
alteplase with >5d then end 
of ‘on treatment’ is last 
infusion + 168h (7d).
4,
6.4,
7.4.2
Addition of sensitivity 
analyses, requested by 
Brazilian authorities: New 
subgroups for investigation 
of primary endpoint:
1) Participation in more than 
1 stud for COVID, started 
either prior to or after 
randomisation
2) Intake of conmeds for 
COVID-19 (yes/no)
3) Type of heparin intake, 
either UFH, LMWH, then 
low /  mod / high dose 
intensity.
Patients with comorbidities, 
e.g. diabetes, overweight, 
age, cardiac comorbidities.


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TITLE PAGE
TRIAL STATISTICAL ANALYSIS PLAN
(PART 2 ANALYSIS)
c37258408-01
BI Trial No.:
0135-0347
Title:
The TRISTARDS trial - ThRombolysIS Therapy for ARDS
A Phase IIb/III operationally seamless, open-label, randomised, 
sequential, parallel-group adaptive study to evaluate the efficacy 
and safety of daily intravenous alteplase treatment given up to 5 
days on top of standard of care (SOC) compared with SOC alone, 
in patients with acute respiratory distress syndrome (ARDS) 
triggered by COVID-19.
Protocol Version:
03 (05Oct2021)
Investigational
Product(s):
Alteplase (recombinant tissue-type plasminogen activator, rt-PA)
Responsible trial 
statistician(s):
Tel.:
Fax: 
Date of statistical 
analysis plan:
26 OCT 2021 SIGNED
Version:
1
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1.
TABLE OF CONTENTS
TITLE PAGE ...........................................................................................................................1
1.
TABLE OF CONTENTS......................................................................................2
LIST OF TABLES...................................................................................................................4
2.
LIST OF ABBREVIATIONS ..............................................................................5
3.
INTRODUCTION.................................................................................................8
4.
CHANGES IN THE PLANNED ANALYSIS OF THE STUDY......................9
5.
ENDPOINTS .........................................................................................................9
5.1
PRIMARY ENDPOINT .......................................................................................9
5.2
SECONDARY ENDPOINTS.............................................................................10
5.2.1
Key secondary endpoints....................................................................................10
5.2.2
Other secondary endpoints ................................................................................11
6.
GENERAL ANALYSIS DEFINITIONS ..........................................................16
6.1
TREATMENTS...................................................................................................16
6.2
IMPORTANT PROTOCOL DEVIATIONS....................................................16
6.3
SUBJECT SETS ANALYSED...........................................................................18
6.5
POOLING OF CENTRES .................................................................................21
6.6
HANDLING OF MISSING DATA AND OUTLIERS....................................22
6.7
BASELINE, TIME WINDOWS AND CALCULATED VISITS ...................22
7.
PLANNED ANALYSIS......................................................................................24
7.1
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS.........25
7.2
CONCOMITANT DISEASES AND MEDICATION .....................................25
7.3
TREATMENT COMPLIANCE........................................................................26
7.4
PRIMARY ENDPOINT .....................................................................................26
7.4.1
Primary analysis of the primary endpoint........................................................26
7.4.2
Sensitivity analysis, subgroup analysis, exploratory analysis of the 
primary endpoint ................................................................................................27
7.5
SECONDARY ENDPOINTS.............................................................................29
7.5.1
Key secondary endpoints....................................................................................29
7.5.1.1
Primary analysis of the key secondary endpoints .................................................29
7.5.1.2
Sensitivity analysis, subgroup analysis, exploratory analysis of the key 
secondary endpoints..............................................................................................29
7.5.2
Other Secondary endpoints................................................................................30
7.7
EXTENT OF EXPOSURE.................................................................................31
7.8
SAFETY ANALYSIS..........................................................................................31
7.8.1
Adverse Events ....................................................................................................32
7.8.2
Laboratory data ..................................................................................................33
7.8.3
Vital signs.............................................................................................................33


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7.8.4
ECG......................................................................................................................34
7.8.5
Others...................................................................................................................34
7.8.5.1 Pharmacokinetic analysis.............................................................................................34
8.
TIMEPOINT OF RELEASE OF TREATMENT INFORMATION.............37
8.1
HANDLING OF INDIVIDUAL PATIENT TREATMENT
INFORMATION.................................................................................................37
8.2
HANDLING OF AGGREGATED TREATMENT INFORMATION...........37
9. 
REFERENCES....................................................................................................38
11.
HISTORY TABLE..............................................................................................41


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LIST OF TABLES
Table 5.1: 1
Censoring of the primary endpoint for the main estimand...............................10
Table 5.2.2: 1 Time period for counting MBE up to day 6 .....................................................11
Table 6.2: 1
Handling of iPDs ..............................................................................................16
Table 6.3: 1
Subject sets analysed ........................................................................................19
Table 6.7: 1
Time period for counting on treatment safety events.......................................23
Table 7.4.2: 1 Censoring of the primary endpoint for the treatment policy and composite 
estimands ..........................................................................................................28
Table 11: 1
History table .....................................................................................................41


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2.
LIST OF ABBREVIATIONS
Term
Definition / description
ACM
All cause mortality
AE
Adverse events
ALT
alanine transaminase
AME
Average marginal effect
AMI
Acute myocardial infarction
ANCOVA
Analysis of Covariance
aPTT
activated partial thromboplastin time
ARDS
Acute respiratory distress syndrome
AST
aspartate transaminase
AUC
Area under the curve
BRAVE
BI Rave
BMI
Body mass index
CI
Confidence interval
COVID-19
coronavirus disease 2019
CRF
Case report form
CRP
C-reactive protein
CTP
Clinical Trial Protocol
CWRES
Conditional weighted residuals
DVT
Deep Vein Thrombosis
ECMO
Extracorporal membrane oxygenation
eGFR
Estimated glomerular filtration rate
EMA
European Medicines Agency
EU
European Union
EudraCT
European Union Drug Regulating Authorities Clinical Trials Database
FAS
Full Analysis Set
GGT
Gamma-Glutamyl Transferase
HR
Hazard ratio
ICU
Intensive care unit
IMV
Invasive mechanical ventilation
INR
International normalised ratio


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Term
Definition / description
iPD
Important protocol deviation
IRT
Interactive response technology
ISTH
International Society on Thrombosis and Haemostasis
LMWH
Low molecular weight heparin
MBE
Major Bleeding Event
MedDRA
Medical Dictionary for Drug Regulatory Activities
MI
Myocardial infarction
MV
Mechanical Ventilation
NIV
Non invasive Ventilation
OFD
Oxygen-free days
PaO2/FiO2
Oxidation Index (Horowitz Index)
pcVPC
Predicted corrected visual predictive check
PE
Pulmonary Embolism
PK
Pharmacokinetic
PKS
Pharmacokinetic Set
PPS
Per Protocol Set
PRED
Predictions
RBC
Red blood cells
RD
Risk difference
REML
Restricted maximum likelihood
REP
Residual effect period
RPM
Report planning meeting
RS
Randomised Set
rt-PA
recombinant tissue-type plasminogen activator
SARS-CoV-2
severe acute respiratory syndrome coronavirus 2
SCR
Screening Set
SD
Standard deviation
SE
Standard error
SOC
Standard of Care
SOFA
Sequential (sepsis-related) Organ Failure Assessment
SpO2
Oxygen saturation
TEAE
Treatment emergent adverse events


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Term
Definition / description
TPA
Tissue Plasminogen activator
TS
Treated Set
TSAP
Trial Statistical Analysis Plan
UFH
Unfractionated heparin
ULN
Upper limit of normal
VFD
Ventilator Free Days
WBC
White blood cells
WHO
World Health Organisation


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3.
INTRODUCTION
As per ICH E9 (1), the purpose of this document is to provide a more technical and detailed 
elaboration of the principal features of the analysis, as described in the clinical trial protocol 
(CTP) Version 3 (c32434345-03, 2). It will detail the procedures for executing the statistical 
analysis of the primary, all secondary endpoints, and other data, and will be limited to the 
analysis of Part 2 of BI trial 0135-0347.
CTP Version 3 is the updated CTP, following the interim results of Part 1. This TSAP 
assumes familiarity with CTP Version 3. In particular, the TSAP is based on the planned 
analysis specifications as written in CTP Version 3 Section 7 “Statistical Methods and 
Determination of Sample Size”. Therefore, TSAP readers may consult the CTP Version 3 for 
more background information on the study, e.g., on study objectives, study design and 
population, treatments, definition of measurements and variables, and planning of sample size 
and randomisation.
The NIV (non-invasive ventilation) patient cohort are those with a baseline WHO value of 6, 
and the IMV (invasive mechanical ventilation) patient cohort are those patients with a value 
of 7, 8 or 9 at baseline. All analyses will be performed within the NIV patient cohort as the 
main cohort for confirmatory purposes, and separately within the IMV patient cohort for 
exploratory purposes. This is consistent with the hierarchy testing approach following the 
primary and key secondary objectives of the statistical analysis, as outlined in CTP version 3
Section 7. Select analyses will be performed on the NIV and IMV cohorts pooled and the 
extent of the analyses will depend on the homogeneity observed of the two groups.
A database snapshot will be made when all patients have completed the day 28 visit and when 
the data has been cleaned. At this point, all analyses outlined in this Trial Statistical Analysis 
Plan (TSAP) will be performed, except for all cause mortality (ACM) at day 90. This further 
endpoint will be available at a later timepoint. When this data is available, then another 
snapshot will be taken, the database will be locked, and this endpoint will be analysed.
This is an open-label study, therefore no blinding will apply for patients, investigators and 
personnel at the site involved in trial conduct. The access to the randomisation code list will 
be kept restricted until it is released for analysis. While the study is in progress, access to 
tabular results of study outcomes by treatment allocation will not be made available to the 
patients, investigators and personnel at the site, to the trial statistician, clinical team, or 
members of the steering committee (unless the DMC advises otherwise). The DMC and DMC
statisticians will be unblinded.
A separate abbreviated TSAP will be written to cover pooled analyses of Part 1 and Part 2 for 
SOC alone and the common alteplase group. Within that abbreviated TSAP, efficacy analyses 
will be limited to primary and secondary efficacy endpoints (as defined in Part 2) and to the 
NIV patient cohort.
The trial data is stored in the BI Rave (BRAVE) database system. SAS® Version 9.4 or later 
will be used for all analyses, except for population PK as outlined in Section 7.8.5.1.


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4.
CHANGES IN THE PLANNED ANALYSIS OF THE STUDY
The screened set (SCR) and pharmacokinetic set (PKS), not outlined in CTP Version 3, are 
now defined in Section 6.3. They are needed to summarise those patients to be included in the 
disposition flow and the PK analysis.
Subgroup analyses not included in CTP Version 3 are now outlined in Sections 6.4,  7.4.2 and 
7.5.2 for the primary endpoint and one of the secondary endpoints. These analyses are to be 
used to test the robustness of the main analyses for that particular endpoint and to investigate 
if any of the subgroups are having an overwhelming influence on the study results.
5.
ENDPOINTS
5.1
PRIMARY ENDPOINT
The primary endpoint as defined in CTP Version 3 Section 2.1.2 will be used and will be 
analysed on the Full analysis set (FAS). This is based on the daily assessment of the WHO 
Clinical Progression scale (3), which ranges from 0 to 10. Clearly, a shorter time to clinical 
improvement or hospital discharge reflects a better outcome.
“Time to clinical improvement or hospital discharge up to day 28, defined as the time 
from randomisation to either an improvement of two points on the 11-point WHO 
Clinical Progression Scale or discharge from the hospital, whichever comes first.”
However, there are several elements which have to be taken into account, when deriving the 
endpoint, and in order to address the hypothetical estimand. These are already specified in the 
CTP Version 3 and summarized in Table 5.1: 1 below. 
In addition, in the unlikely event that a patient discontinues from the study due to withdrawal 
of consent, prior to being able to determine if he had clinical improvement or hospital 
discharge, or had intake of bail-out therapy or death, he will be censored at the time of study 
withdrawal. This has not been included in the protocol definition but is considered only as a 
clarification.
Bail-out therapy is as defined in CTP Version 3 Section 4.2.2.1 Ultima ratio situation - Bail-
out, which is ‘marketed Actilyse®’. The various scenarios for handling of intake of bail-out 
therapy are summarized in Table 5.1: 1 below:


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Table 5.1: 1
Censoring of the primary endpoint for the main estimand
Scenarios
Main Estimand: 
Hypothetical situation
Bail-out then death
Censor at Bail-out
Bail-out then failure to improve#
Censor at Bail-out
Bail-out then clinical improvement / hospital 
discharge
Censor at Bail-out
Bail-out then clinical improvement / hospital 
discharge then death
Censor at Bail-out
Clinical improvement / hospital discharge 
regardless of subsequent events
Positive event at clinical 
improvement / hospital discharge
Death only, prior to day 28
Censor at day 28
Failure to improve# by day 28
Censor at day 28
Discontinued from study due to withdrawal of 
consent prior to clinical improvement / hospital 
discharge or Bail-out/Death
Censor at time of withdrawal
Clinical improvement / hospital discharge refers to the first of clinical improvement or 
hospital discharge, whichever comes first.
#Failure to improve is failure to have clinical improvement or hospital discharge.
The planned analyses of the primary endpoint are outlined in Section 7.4.1, and the 
supplementary and sensitivity analyses of this endpoint in Section 7.4.2.
5.2
SECONDARY ENDPOINTS
5.2.1
Key secondary endpoints
The two key secondary endpoints as defined in CTP Version 3 Section 2.1.3 will be analysed 
on the FAS, and these are both based on the daily assessment of the WHO Clinical 
Progression scale.
The planned analyses of the key secondary endpoints are outlined in Section 7.5.1.
Treatment failure (ACM / MV) at day 28, FAS, binary endpoint
This unfavorable endpoint is met if:

the last known status of the patient has a value of ≥7 on the WHO clinical progression 
scale, up to end of day 28, or

vital status is dead within 28 days


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ACM at day 28, FAS, binary endpoint
This unfavorable endpoint is met if:

the last known status of the patient is 10 on the WHO clinical progression scale by the end 
of day 28, or

vital status is dead within 28 days
5.2.2
Other secondary endpoints
The secondary endpoints as defined in CTP Version 3 Section 2.1.3 which are not listed under 
“Key secondary endpoints” will be referred to as “Other secondary endpoints”. Efficacy 
endpoints will be analysed on the FAS, and the one safety endpoint “Major bleeding event 
(MBE) until day 6” will be analysed on the Treated Set (TS). 
The planned analyses of the secondary endpoints are outlined in Section 7.5.2.
MBE (according to International Society on Thrombosis and Haemostasis [International 
Society on Thrombosis and Haemostasis (ISTH) definition (4)])  until day 6, TS, binary 
endpoint
If the patient has an MBE, at any timepoint up to end of day 6 then he has met this 
unfavorable endpoint. If it is unknown whether the patient had an MBE up to end of day 6, 
regardless of the reason, then it will be assumed that the patient did not have an MBE by the 
end of day 6. This is a safety defined endpoint and the starting point for consideration should 
be consistent with the ‘on treatment’ definition in Section 6.7. The time period for counting 
events is defined in Table 5.2.2: 1 below:
Table 5.2.2: 1 Time period for counting MBE up to day 6
MBE until day 6
Start point
End date
SOC patients
Randomisation date/time
Start point + 144 hrs
Alteplase patients
First administration of 
alteplase date/time
Latest time of:
o
Start point + 144 hrs
o
Last administration of alteplase date/time + 24 hrs
[Note: 144 hrs represents 6 days]
PaO2/FiO2 ratio (or inferred PaO2/FiO2 ratio from SpO2) change from baseline to day 6, FAS, 
continuous endpoint
This assessment is planned to be measured on each of days 0 to 7, and again on day 28, but 
only whilst the patients is still in hospital. For this endpoint, the worst (lowest) daily 
measurement will be used and the higher the value the better the health status of the patient.

If the patient is still in hospital during day 6 then the day 6 value will be used 

If the patient has been discharged from hospital prior to day 6 then the value at the time of 
hospital discharge will be used 

If the patient has died prior to day 6 then the last value prior to death will be used

If day 6 value is missing but Day 5 value available, the day 5 value will be used


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
If day 6 value is missing, no Day 5 value available, but day 7 available, then  day 7 value 
will be used

Otherwise value set to missing for that patient.
Based upon this, the change from baseline for each patient will be calculated and used for the 
analysis.
Number of oxygen-free days (OFD) up to day 28, FAS, continuous endpoint
For this endpoint, ‘oxygen-free‘ is defined as free from assistance from oxygen support. The 
number of oxygen-free days starts from when the patient has a ‘lasting’ value on the WHO 
clinical progression scale of ≤4 and ends on Day 28. A lasting value of ≤4 means that the 
value cannot exceed 4 at a later timepoint. If the patient is liberated from oxygen on Day x, 
then the number of OFDs is 28-x. If a patient has withdrawn consent prior to day 28 then he 
will have a missing value for OFD. In any case, if the status of the patient at Day 28 is death, 
as determined from the vital status page then the OFD=0. 
Length of hospital stay up to day 28, FAS, continuous endpoint
This will be determined based upon the first hospital discharge date, or discharge to another 
care facility. If the patient dies within the first 28 day period, then length of hospital stay is 
28.


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6.
GENERAL ANALYSIS DEFINITIONS
6.1
TREATMENTS
For the purposes of the statistical analyses, treatments will be referred to in the same context 
as in CTP Version 3 Section 7.4.

“Alteplase”, which is alteplase 0.6 mg/kg over 2 hours followed by 0.04 mg/kg/h over 12 
hours

Standard of Care (SOC)
Alteplase treatment is planned to be up to end of Day 5 but all patients should be taking SOC 
in the background. On day 1 there is an initial 2 h infusion for all, followed up by a longterm 
12 h infusion. On days 2-5 the patients can receive an optional 2h infusion (based on 
investigator decision), but only on one of these days. Each patient should receive a longterm 
12h infusion on these remaining 4 days. Some patients may have a temporary interruption, as 
advised by CTP Version 3 Section 3.3.4.1.
6.2
IMPORTANT PROTOCOL DEVIATIONS
Patients with any of the following important protocol deviations (iPDs) will be reviewed on a 
case by case basis (5) and (6). The review of the iPDs will be used to determine criteria for a 
PPS.
Note that this is a working list and may be updated again prior to the final Report Planning 
Meeting (RPM) prior to the snapshot related to end of Part 2. Inclusion criteria/exclusion 
criteria numbers refer to the definition given in the CTP Version 3 or CRF.
Table 6.2: 1
Handling of iPDs 
iPD 
code
iPD Category & Brief Description
A
Entrance Criteria Not Met
A1
Inclusion Criteria not met
A1.1
Patient too young (IC1)
A1.2
Diagnosis of ARDS questionable (IC2,4,5)
A1.3
Patient does not test positive for SARS-CoV-2 (IC3)
A2
Exclusion Criteria not met
A2.1
Patient has baseline conditions that are not permitted for safety reasons (EC1-33)


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Table 6.2: 1
Handling of iPDs (continued)
iPD 
code
iPD Category & Brief Description
A2.2
Patient has baseline conditions that prevent patient from complying with study 
protocol (EC1-33)
A2.3
Patient has had previous conditions or procedures that are not permitted (EC1-33)
A2.4
Patient has had forbidden previous therapy (EC1-33)
A2.5
Patient pregnant (EC27)
B
Informed Consent
B1
Informed consent not available (IC6)
B2 
Informed consent not signed at screening visit 1 (IC6)
B3 
Informed consent not available or not available at visit 2a (pre-dose)  (IC6)
C
Trial medication and randomisation
C1
Method of assigning patients to treatment arms
C1.1
Randomisation not followed according to protocol
C1.2
Patients do not receive the initial treatment they were randomised / allocated to
C1.3
Patient assignment not followed
C1.4
Timeframe > 1 day between randomisation and first drug intake
C2
Non-compliance
C2.1
Overall Compliance not between 80% and 120% inclusive – see definition in 
Section 5.4.
C2.2
Patient takes the optional 2h infusion on more than one occasion after the initial 2h 
infusion.
C3
Drug assignment and administration of doses for each patient
C3.1
Dosage and treatment schedule not given according to protocol for initial iv 
infusion
C3.2
Dosage and treatment schedule not given according to protocol for long term iv 
infusion 
C3.3
Drug not permanently discontinued according to CTP Version 3 Section 3.3.4.1 
C3.4
Drug not temporarily interrupted according to CTP Version 3 Section 3.3.4.1 


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Table 6.2: 1
Handling of iPDs (continued)
iPD code
iPD Category & Brief Description
D
Concomitant medication
D1
Intake of restricted concomitant medication according to CTP Version 3 
Section 4.2.2.1 and Section 10.2
D2
Intake of marketed Actilyse® (non-study drug) not as ultima-ratio or bail-out
G
Other trial specific important deviations
G1
Other protocol violations affecting patient rights or safety (manual PDs to be 
captured)
6.3
SUBJECT SETS ANALYSED
The subject sets as defined in CTP Version 3 Section 7 for the Randomised Set (RS), Full 
Analysis Set (FAS) and Treated Set (TS) will be used.
The Screened set (SCR), not outlined in the CTP Version 3, will include all patients 
screened.
The RS will consist of all randomised patients.
The FAS will consist of all randomised patients with at least one baseline and one post 
baseline assessment relating to the primary endpoint of interest. That is to say, they need to 
have at least one assessment post baseline on the 11-point WHO Clinical Progression Scale.
The TS will consist of all patients who were randomised and, for patients in the alteplase 
group, treated with at least one dose of trial drug.
Per Protocol Set (PPS) will consist of all patients in FAS who were without important
protocol deviations (IPDs) for efficacy, as outlined in Table 6.2: 1.
The Pharmacokinetic set (PKS), not outlined in CTP Version 3, will comprise all subjects in 
the TS who provided at least one PK endpoint and had no important protocol violations 
relevant to the evaluation of PK.
Table 6.3: 1 below outlines which analysis set is to be used for the various endpoints or 
summaries.


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Table 6.3: 1
Subject sets analysed
Subject set
Class of endpoint
SCR / RS
TS
FAS
PPS
PKS
Primary endpoint 
primary and 
supplementary 
analyses
Primary 
analyses 
Key secondary endpoints
x
Primary 
analyses
Other secondary EFFICACY and 
further EFFICACY endpoints 
X
Secondary SAFETY and further 
SAFETY endpoints
X
Treatment exposure
X
Disposition
X
Demographic/baseline 
X
X
x
Concomitant medications and 
compliance
X
X
All other safety analyses
X
PK analyses
X


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6.5
POOLING OF CENTRES
Part 2 of this study is planned to be carried out in approximately 110-130 centres in 
approximately 22 countries. However, not all centres have the same SOC procedures. During 
the course of the study, and gathering information (retrospectively) from each of the centres 


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with regard to their SOC procedures, they will be grouped into relatively homogeneous 
groupings. The same process as done for Part 1 will be carried out again for Part 2.
Because the groupings will only be made after randomisation of a patient within a centre, this 
will be a retrospective and exploratory analysis, albeit planned. It is planned only to 
investigate the primary endpoint with this centre grouping in a sensitivity analysis – see 
Section 7.4.2.
6.6
HANDLING OF MISSING DATA AND OUTLIERS
All efforts will be made to avoid having missing data in the database, however it is inevitable 
that there will be missing data, by design:

For patients that die, there will be no further data, however this irreversible 
unfavourable state will be taken into consideration in the determination of each of the
endpoints.

For patients that are discharged from hospital before day 28, the only additional 
efficacy assessment is the vital status at days 28 and 90. The fact that the patient has 
improved in order to be discharged from hospital prior to day 28 will be taken into 
account in the determination of each of the endpoints.
Handling of missing data for the endpoints are outlined in Section 5, in line with the 
definitions of the endpoints.
Missing or incomplete adverse event (AE) dates will be computed using the BI handling 
rules (7).
6.7
BASELINE, TIME WINDOWS AND CALCULATED VISITS
Randomisation is defined as Day 1 and it is planned that the alteplase patients are 
administered their initial 2h infusion of study medication within 6 hours of randomisation, on 
Day 1 (Visit 2a). The last planned administration of study medication for the alteplase patients 
is the long term 12h infusion on Day 5. 
Since all patients are on SOC and there is no clear start or stop of SOC, a fixed treatment 
duration will be used for all patients, for the purposes of the analyses. Therefore, endpoints 
referred to as ‘on treatment’ will include all relevant events from the relevant start point plus 
288 hours (last planned treatment on Day 5 + 7 days of REP = 12 days = 288 hrs).
However, to take account of the fact that some patients may have had treatment interruptions, 
the residual effect period should be in relation to the last intake of alteplase infusion. Table 
6.7: 1 below outlines the time period definitions for counting on treatment safety event.


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Table 6.7: 1
Time period for counting on treatment safety events
On treatment
Start point
End date
SOC patients
Randomisation date/time
Start point + 288 hrs
Alteplase patients
First administration of 
alteplase date/time
Latest of:
o
Start point + 288 hrs 
o
Last administration of alteplase date/time + 168 hrs
[Note: 288 hrs represents 12 days, 168 hrs represents 7 days]
According to CTP Version 3 Section 1.2, the residual effect period (REP) is 7 days.
As outlined in CTP Version 3 Section 7.2.1, the term "baseline" refers to the last observed 
measurement prior to randomisation on Day 1.


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7.
PLANNED ANALYSIS
The tables that are created from the statistical analysis will follow the format of the standard 
within BI (8).
The overall disposition of the patients in the trial will be presented, together with the number 
of patients discontinuing prior to day 28, and vital status at day 90.
The number of patients participating in the study by country and the number of sites, for each 
of the patient populations will be summarised. 
Demography and baseline characteristics will be presented, as well as the stratification used at 
the time of the patient randomisation. A summary of the iPDs will be provided.
Where statistical comparisons are indicated, they will be done by comparing alteplase versus 
SOC.
All descriptive analyses will be performed:
o
within the NIV patient cohort as the main cohort
o
within the IMV patient cohort as the exploratory cohort
o
on the NIV and IMV cohorts pooled – additional select efficacy analyses for primary 
and secondary efficacy endpoints
For the NIV patient cohort, the default set of covariates for adjustment in analyses are:
o
Baseline D-Dimer status (≥3 to <5,  ≥5-fold ULN)
o
Age
o
Number of days of NIV support
o
Baseline value of the PaO2/FiO2 ratio – only for the endpoint PaO2/FiO2 ratio change 
from baseline to Day 6
For the IMV patient cohort, the default set of covariates for adjustment in analyses are:
o
Baseline D-Dimer status (≥3 to <5,  ≥5-fold ULN)
o
Age
o
Baseline WHO value (7, 8, 9)
o
Baseline value of the PaO2/FiO2 ratio – only for the endpoint PaO2/FiO2 ratio change 
from baseline to Day 6
For efficacy analyses, patients will be analysed according to the patient information given in 
the CRF, in the case of potentially erroneous data entered into the interactive response 
technology (IRT). This is related to the baseline information, ventilaton support (NIV, IMV) 
and D-Dimer level (≥3 to <5, ≥5-fold ULN).
In general, for End-Of-Text tables, the set of summary statistics is: N (number of patients 
with non-missing values) / Mean / SD / standard error (SE) / Min / Q1 (lower quartile)/ 
Median / Q3 (upper quartile)/ Max. 
Statistical parameters will be displayed to a defined number of decimal places as specified in 
the BI guideline "Reporting of Clinical Trials and Project Summaries" (9).


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Tabulations of frequencies for categorical data will include all possible categories and will 
display the number of observations in a category as well as the percentage (%) relative to the 
respective treatment group (unless otherwise specified, all patients in the respective patient set 
whether they have non-missing values or not). Percentages will be rounded to one decimal 
place. The category missing will be displayed only if there are actually missing values. 
7.1
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS
Only descriptive statistics are planned for this section of the report. See Section 5.4 for 
specific definitions.
For the two randomisation stratification factors at baseline, baseline ventilation support (NIV, 
IMV) and baseline D-Dimer level (≥3 to <5,  ≥5-fold ULN), any discrepancies between the 
IRT and the CRF will be documented and summarized. Should there be a discrepancy 
between the value used for the randomisation stratification and the actual value as determined 
on the CRF, the actual value as determined on the CRF will be used in the analyses.
Within this section, all analyses will be performed on the NIV patient cohort, the IMV patient 
cohort and on the NIV and IMV cohorts pooled.
7.2
CONCOMITANT DISEASES AND MEDICATION
Medical history, COVID-19 signs and symptoms and concomitant diagnoses will be 
presented and coded using MeDRA System Organ Class and Preferred Term where possible.
Regarding concomitant medications, there is a specific question asking if the treatment was 
given as part of standard of care for the treatment of COVID-19, which is relevant for all 
patients since they all receive SOC in the background. This question is different to the 
randomised treatment assignment. Therefore, all concomitant medication analyses or displays 
will be split by ‘Standard of Care for the treatment of COVID-19’, yes or no. All concomitant 
medications will be coded using the WHO Drug dictionary. 
Medications of special interest for COVID (as indicated by WHO, see Section 10.1), and 
restricted medications within this study (as outlined in CTP Version 3 Section 4.2.2.1, see 
Section 10.2) will also be summarized and split by whether or not the medication was given 
as part of the Standard of Care for the treatment of COVID-19.
Use of heparin will be summarized and presented.
Therefore, only descriptive statistics are planned for this section of the report. 
Within this section, all analyses will be performed on the NIV patient cohort, the IMV patient 
cohort and on the NIV and IMV cohorts pooled.


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7.3
TREATMENT COMPLIANCE
Only descriptive statistics are planned for this section of the report, and due to the nature of 
the study, compliance will be determined for the alteplase treatment group only.
Compliance to intake of infusion will be assessed in terms of % taken, as measured by 
volume, at each planned infusion. Overall compliance will be as an average measure of the 
%taken. The detailed definitions are provided in Section 5.4.
Compliance with preparation of the infusion in terms of concentration, as indicated in CTP 
Version 3 Table 10.4 are also assessed, and detailed definitions provided in Section 5.4.
Within this section, all analyses will be performed on the NIV patient cohort, the IMV patient 
cohort and on the NIV and IMV cohorts pooled.
7.4
PRIMARY ENDPOINT
Within this section, all descriptive analyses will be performed on the NIV patient cohort, the 
IMV patient cohort and on the NIV and IMV cohorts pooled. 
7.4.1
Primary analysis of the primary endpoint
For the primary endpoint, a Cox proportional hazards model will be used to estimate the 
hazard ratio (HR) comparing alteplase to SOC. From the model, the 95% confidence intervals 
(CI) and corresponding Wald p-values for the HRs will be produced. The model will include 
fixed effects for treatment (alteplase, SOC), and the default set of covariates for the respective 
NIV or IMV patient cohorts, as outlined in Section 7.
Kaplan Meier estimates will be presented by treatment (alteplase, SOC) for this endpoint up 
to day 28, and additionally, for the NIV patient cohort:

treatment and baseline D-Dimer status (≥3 to <5,  ≥5-fold ULN)

treatment and age  (≤median, >median)

treatment and number of days of NIV support (≤median, >median)
and for the IMV patient cohort:

treatment and baseline D-Dimer status (≥3 to <5,  ≥5-fold ULN)

treatment and age  (≤median, >median)

treatment and baseline WHO status (7, 8, 9)
From the KM estimates at various timepoints (days 6, 8, 12, 16, 20, 24 and 28) the risk 
difference (RD) for the treatments with 95% CI will be estimated by taking the difference of 
the individual probabilities. Since the observations in the two treatment groups are 


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independent, the variance of the Kaplan-Meier probability difference is equal to the sum of 
the Kaplan Meier probabilities’ variances found by Greenwood's method. The 95% CI of the 
RD is therefore calculated accordingly.
7.4.2
Sensitivity analysis, subgroup analysis, exploratory analysis of the primary 
endpoint
Two non-confirmatory supplementary analyses will be conducted, in anticipation of the rare 
situation where bail-out therapy may be used (as defined in CTP Version 3 Section 4.2.2). 
They will be performed on the FAS.
The first of these supplementary analyses will address the treatment policy estimand, whereby 
there is no consideration for the intake of bail-out therapy.
The second of these supplementary analyses will address a composite estimand, whereby
patients receiving bail-out therapy will be censored at day 28, rather than on the day of bail-
out. Here, bail-out represents treatment failure and thereby, by definition, precludes any 
response after bail-out. The various scenarios are summarized in Table 7.4.2: 1 below.
In both cases, and in the unlikely event that a patient discontinues from the study, prior to 
being able to determine if he had clinical improvement or hospital discharge, or had intake of 
bail-out therapy or death, he will be censored at the time of withdrawal. 


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Table 7.4.2: 1 Censoring of the primary endpoint for the treatment policy and composite 
estimands
Scenarios
Treatment policy 
estimand
Composite estimand
Bail-out then death
Censor at day 28 
Censor at day 28
Bail-out then failure to improve#
Censor at day 28
Censor at day 28
Bail-out then clinical improvement 
/ hospital discharge
Positive event at clinical 
improvement / hospital 
discharge
Censor at day 28
Bail-out then clinical improvement 
/ hospital discharge then death
Positive event at clinical 
improvement / hospital 
discharge
Censor at day 28
Clinical improvement / hospital 
discharge regardless of subsequent 
events
Positive event at clinical 
improvement / hospital 
discharge
Positive event at clinical 
improvement / hospital 
discharge
Death only, prior to day 28
Censor at day 28
Censor at day 28
Failure to improve# by day 28
Censor at day 28
Censor at day 28
Discontinued from study due to 
withdrawal of consent prior to 
clinical improvement / hospital 
discharge or Bail-out/Death
Censor at time of study 
discontinuation
Censor at time of 
withdrawal
Clinical improvement / hospital discharge refers to the first of clinical improvement or 
hospital discharge, whichever comes first.
#Failure to improve is failure to have clinical improvement or hospital discharge.
The analyses performed will be identical to that outlined for the primary endpoint in Section 
7.4.1.
Based on the main estimand, the treatment effect will be estimated using the Cox proportional 
hazard model, containing only treatment in the model. The HR (95%CI) and p-value 
estimating the unadjusted treatment effect will be produced.
The effect of the subgroups in the NIV patient cohort, as outlined in Section 6.4, related to the 
primary endpoint, main estimand, will be assessed descriptively and using Kaplan Meier 
estimates. Using the Cox proportional hazard model, hazard ratio estimates with 95% CI will 
be estimated from the model in two different methods:
o
Firstly, in a model containing treatment, subgroup and the treatment * subgroup 
interaction. 
o
Secondly, in the model containing the default set of covariates as outlined in Section 
7.4.1 with this additional covariate added. 


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A covariate adjusted sensitivity analysis of the primary endpoint, main estimand, will be 
conducted, to determine the effect of the grouping of centres according to similar types of 
Standard of care. This will be the same analysis.
For the NIV and IMV cohorts pooled, the Cox proportional hazard model will be used, 
including the fixed effects for treatment (alteplase, SOC), baseline D-Dimer status (≥3 to <5,  
≥5-fold ULN), age and baseline ventilation status (NIV, IMV). From this model, the HR and 
95% CI will be produced. In addition to this model, the interaction term for the baseline 
ventilation status and treatment will be included, and the p-value for this additional term will 
be used as a test of homogeneity. If the p-value is >0.05 the it can be considered that the two 
groups are homogeneous, with respect to this primary endpoint.
7.5
SECONDARY ENDPOINTS
Within this section, all descriptive analyses will be performed on the NIV patient cohort, the 
IMV patient cohort and on the NIV and IMV cohorts pooled. 
7.5.1
Key secondary endpoints
The two key secondary efficacy endpoints, outlined in Section 5.2.1, are binary in nature and 
will be analysed on the FAS for both the NIV patient cohort and the IMV patient cohort.
7.5.1.1
Primary analysis of the key secondary endpoints
The risk difference (RD) will be estimated between the two treatment arms using logistic 
regression. In addition to treatment, adjustment will be made for the default set of covariates, 
according to the NIV or IMV patient cohort as outlined in Section 7. RD and CIs (95%) for 
the RD will be obtained using the Delta method and the average marginal effect (AME) 
method.
7.5.1.2
Sensitivity analysis, subgroup analysis, exploratory analysis of the key secondary 
endpoints
As sensitivity analysis for each of these binary efficacy endpoints, the unadjusted treatment 
estimates will be produced, using the same logistic regression model containing only 
treatment.
For the NIV and IMV cohorts pooled, the RD and 95% CI will be estimated between the two 
treatment arms using logistic regression, the Delta and the average marginal effect (AME) 
method. Adjustment will be made for treatment (alteplase, SOC), baseline D-Dimer status (≥3 
to <5,  ≥5-fold ULN), age and baseline ventilation status (NIV, IMV). In addition to this 
model, the interaction term for the baseline ventilation status and treatment will be included, 
and the p-value for this additional term will be used as a test of homogeneity. If the p-value is 
>0.05 the it can be considered that the two groups are homogeneous, with respect to these two 
key secondary efficacy endpoints.


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7.5.2
Other Secondary endpoints
Other secondary endpoints are defined in Section 5.2.2. In all cases, standard descriptive 
statistics will be presented, whether the endpoint is binary or continuous. 
Binary safety endpoint
For the binary safety endpoint MBE up to day 6, analysed on the TS, there will be no 
adjustment for covariates. Therefore, the RD will be the difference between the two 
incidences and the p-value and 95% CI will be determined using the Chan and Zhang exact 
method.
For the NIV and IMV cohorts pooled, the RD 95% CI will be estimated between the two 
treatment arms using the same approach as for the key secondary efficacy endpoints. 
However, for this pooled investigation of MBE up to day 6, the baseline D-Dimer status (≥3 
to <5,  ≥5-fold ULN) and age covariates will not be included in the model. The p-value for 
homogeneity will determined similarly.
Continuous efficacy endpoints
The three other secondary endpoints which are continuous in nature will be analysed using a 
restricted maximum likelihood (REML) based ANCOVA comparing the outcome, by 
treatment. The ANCOVA model will be adjusted for the default set of covariates as outlined 
in Section 7 for the respective NIV and IMV patient cohorts. The treatment difference with 
95% CI and p-values will be presented.
For the continuous efficacy endpoints, number of OFDs at day 28, and number of days in 
hospital, there are not expected to be any missing values by definition and as outlined in 
Section 5.2.2. 
For the continuous efficacy endpoint change from baseline in the PaO2/FiO2 at day 6, the 
baseline PaO2/FiO2 will also be included in the model as covariate for adjustment. For 
patients that have died, or who have been discharge from hospital, the handling rules are as 
outlined in Section 5.2.2. Note, there are expected to be very few deaths or hospital discharges 
prior to day 6. 
In addition, subgroup analyses of this PaO2/FiO2 endpoint will be conducted in the NIV 
patient cohort only and using the subgroups as outlined in Section 6.4. An ANCOVA model 
will contain the baseline PaO2/FiO2, the subgroup of interest and the interaction between 
subgroup and treatment. 
As sensitivity analyses, the unadjusted treatment effects will be estimated, using the same 
approach as the main analysis, but with only treatment in the model. 
Within this section, all descriptive analyses will be performed on the NIV patient cohort, the 
IMV patient cohort and on the NIV and IMV cohorts pooled. 


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For the NIV and IMV cohorts pooled, the treatment difference and 95% CI will be estimated 
between the two treatment arms using a restricted maximum likelihood (REML) based 
ANCOVA comparing the outcome, by treatment. The ANCOVA model will be adjusted for 
treatment (alteplase, SOC), baseline D-Dimer status (≥3 to <5,  ≥5-fold ULN), age and 
baseline ventilation status (NIV, IMV). In addition to this model, the interaction term for the 
baseline ventilation status and treatment will be included, and the p-value for this additional 
term will be used as a test of homogeneity. If the p-value is >0.05 the it can be considered that 
the two groups are homogeneous, with respect to these three other secondary efficacy 
endpoints.
7.7
EXTENT OF EXPOSURE
Exposure will be handled descriptively. 
The number of patients with initial 2h infusion on day 1, optional 2h between days 2 and 5, 
and longterm 12h infusion over the five days of treatment will be summarized, as well as the 
rate of infusion (mL/kg/h) and duration of infusion (h).
The cumulative exposure will be presented, in terms of Total number of days of infusion. 
Exposure is relevant for the alteplase treatment groups only.
Within this section, all analyses will be performed on the NIV patient cohort, the IMV patient 
cohort and on the NIV and IMV cohorts pooled.
7.8
SAFETY ANALYSIS
The safety analysis will be performed as outlined in the CTP Version 3, according to BI 
standards and on the TS. For further details please see guideline 'Handling and summarization 
of AE data for clinical trial reports and integrated summaries' (10).


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The definition of ‘on treatment’ is outlined in Section 6.7. Any potential events starting 
between the time of randomisation and the start of alteplase treatment, will be indicated as 
such in the listings.
In addition, to fulfil the requirements of the European Union (EU) Regulation 536/2014 
(Annex V, Point 6) (11), the following information should be made available, for the lay 
summary or CTR:

frequency of serious drug-related AEs by treatment, primary system organ class and 
preferred term.
Within this section, all analyses will be performed on the NIV patient cohort, the IMV patient 
cohort and on the NIV and IMV cohorts pooled.
7.8.1
Adverse Events
Unless otherwise specified, the analyses of AE will be descriptive in nature. All analyses of 
AEs will be based on the number of subjects with AEs and NOT on the number of AEs.
AEs will be coded using the most recent version of the Medical Dictionary for Regulatory
Activities (MedDRA) coding dictionary
The analysis of AEs will be based on the concept of treatment emergent adverse events 
(TEAE). That means that all adverse events occurring ‘on treatment’ as outlined in Section 
6.7 will be considered as TEAE.
All AEs occurring before ‘on treatment’ will be assigned to ‘screening’ and all AEs 
occurring after ‘on treatment’ will be assigned to ‘post-treatment’. In general, AEs attributed 
to ‘Screening’ or ‘Post-treatment’ will be listed only.
An overall summary of AEs will be presented.
Frequencies [N (%)] of patients with AEs will be summarised by treatment, primary system 
organ class and preferred term (using MedDRA). Separate tables will be provided for 
patients with serious adverse events.
The system organ classes will be sorted according to the standard sort order specified by 
European medicines agency (EMA), preferred terms will be sorted by frequency (within 
system organ class). 
Additionally, the following analyses will be reported for disclosure on European Union Drug 
Regulating Authorities Clinical Trials Database (EudraCT) and clinicaltrials.gov:

Frequency [N(%)] of patients with non-serious AEs occurring with
incidence in preferred term greater than 5% by treatment,

AEs per arm for disclosure on EudraCT by treatment

Non-serious AEs for disclosure on EudraCT by treatment

Serious AEs for disclosure on EudraCT by treatment


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Adverse events of interest
All endpoints related to safety, outlined in Section 5.2.2 and Section 5.3, are clearly marked 
as such, in particular, endpoints related to MBE, Stroke, MI, PE and DVT. There is no further 
description below on how they should be analysed, however, they will be summarized with 
the safety data under the topic of ‘AEs of special interest’.
There are two other AEs of special interest, which are not mentioned in Sections 5.2.2 and 
5.3:

Transient ischemic attack

Systemic embolism
AEs recorded on the AE page of the CRF, indicated as such via the relevant tick box, will be 
grouped and summarized using frequency tables.
7.8.2
Laboratory data
Changes in coagulation and inflammatory markers on treatment, as outlined in Section 5.3, 
are the laboratory parameters of special interest in this trial. They will be analysed in the
efficacy section.
The analyses of laboratory data will be descriptive in nature and will be based on BI standards 
“Handling, Display and Analysis of Laboratory Data” (12).
The following laboratory parameters are considered safety and hence processed according to 
standard safety processing rules:

Haematology (Haematocrit, Haemoglobin,  Red blood cells (RBC) / Erythrocytes, 
White Blood Cells (WBC) / Leukocytes, Platelet Count / Thrombocytes)

Clinical Chemistry (Albumin,  Alkaline phosphatase,  ALT (alanine transaminase), 
AST (aspartate transaminase), Bilirubin total,  Creatinine, eGFR, Creatine kinase,  
Troponin, Lactate dehydrogenase, Glucose, Potassium, Sodium, Calcium, Magnesium,  
Urea (BUN),  NT-proBNP
For continuous safety laboratory parameters standardized values will be derived as well as the 
differences to baseline. Laboratory parameters will be shown in SI units.  
Laboratory values will be compared to their reference ranges and frequency tables will be 
provided for the number of patients within and outside the reference range at baseline, each 
visit and the last measurement on treatment. Descriptive statistics will be provided by 
treatment group for baseline, each visit and last value on-treatment and for changes from 
baseline to each respective visit and last value on treatment. The laboratory parameters will be 
summarized by day of assessment.
7.8.3
Vital signs
The following vital signs will be descriptively analysed: systolic and diastolic blood pressure, 
body temperature, respiratory rate and pulse rate. Both absolute values and change from 
baseline are planned for this section of the report. 


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7.8.4
ECG
Not applicable.
7.8.5
Others
7.8.5.1 Pharmacokinetic analysis
Descriptive statistics of all PK parameters will be presented and will be performed on the 
PKS as outlined in Section 6.3. In particular, the parameters / endpoints are:

Css,1 (plasma concentration of alteplase at steady state of 2 hour infusion)

Css,2 (plasma concentration of alteplase at steady state of long-term infusion)
Individual endogenous Tissue Plasminogen activator (TPA) will be determined for each 
patient at baseline using the first PK sample. Baseline levels will then be subtracted from all 
further concentration measurements (second and third samples), for each patient, in order to 
determine the Css,1 and Css,2 endpoints. In case the resulting concentration value is negative, 
it will then be set to “BLQ”.
As outlined in CTP Version 3 Section 7.2.4, a population PK analysis is planned to be 
conducted if sufficient data are available. The objectives of the population PK analysis are:-
1) to compare the alteplase concentrations in study 0135-0347 to PK simulations based on a 
previously developed population PK model for patients with acute myocardial infarction 
(AMI)
2) to derive model-based area under the curve (AUC) estimates for patients with ARDS 
based on the sparse PK measurements of study 0135-0347
Dataset preparation
Alteplase plasma concentrations, sampling times, infusion times, and covariates (e.g., age, 
weight, sex, and race) will be assembled and formatted for analysis. Detailed dataset 
specifications will be provided in a separate document.
Handling of missing data
Handling of missing data will be described in the dataset specification document.
Handling of outliers
Alteplase concentrations and covariate values will be included in the analysis whenever 
possible. However, for methods based on least-squares estimation and normal theory it is not 
good practice to include extreme values. Thus, outlying data points in the dependent variable 
may be excluded if they are infrequent, occur randomly and are spurious. Furthermore, it may 
be justified to exclude outlying individuals from model development. Also, extreme covariate 
values might be excluded.  If more than 5% of alteplase plasma concentrations are excluded, a 
sensitivity analysis will be performed to evaluate the impact of the excluded data points on the 
final model. All outliers which are excluded from the analysis will be documented along with 
reasons for their exclusion.


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Pharmacokinetic modeling strategy
In the following the framework of the PK modelling strategy is outlined, this may be adjusted 
as deemed necessary.
The pharmacokinetics of alteplase follows a 3-compartment distribution. A 3-compartment 
model cannot be developed using the information from study 0135-0347 alone as only sparse 
PK data is collected in a subset of the study population. Therefore, a previously developed 
population PK model for alteplase will be used to inform the current analysis.  The population 
PK model for patients with AMI which is based on studies 0135.30 and 0135.53 is the 
preferred starting model. The pharmacokinetics of alteplase in these AMI studies were best 
described by a linear 3 compartment model. A baseline t-PA concentration was estimated to 
account for endogenous t-PA in plasma. Weight was included as covariate affecting baseline 
t-PA concentration and clearance of alteplase. Age was included as covariate affecting 
clearance of alteplase.
First, the alteplase plasma concentrations in study 0135-0347 will be compared to PK model 
simulations. Simulations will be performed using the previously developed population PK 
model for alteplase in patients with AMI. In the simulations, inter-individual and residual 
variability will be considered. The alteplase plasma concentration-time profiles will be 
simulated 1,000 times for each subject in study 0135-0347 assuming its actual infusion 
regimen, sampling scheme and weight. The simulated alteplase plasma concentrations at the 
different time points will be used to calculate the median concentration-time profile as well as 
it’s 90% prediction interval. This simulation results will be graphically compared to the 
observed data in study 0135-0347.
Given that the model-simulated plasma PK is in the same range as the observations, it will be 
further investigated whether the population PK model for patients with AMI can describe the 
individual alteplase concentrations in ARDS patients. Individual post-hoc estimates will be 
derived for study 0135-0347 using the parameters of the population PK model for patients 
with AMI. The distribution of the post-hoc estimates around the typical parameters and the 
description of the individual plasma concentrations will be investigated. 
Provided that the population PK model for patients with AMI successfully describes the 
individual profiles of patients with ARDS, the individual PK parameters will be determined. 
In addition, AUC will be derived within NONMEM® from the individual PK parameters 
considering the patients’ actual alteplase doses, infusion regimen, and covariates. In case, the 
structural PK model of patients with AMI cannot describe the PK of patients with ARDS, 
necessary adjustments (e.g. re-estimation of clearance) might be implemented into the model. 
The population PK model for patients with AMI comprises the impact of weight and age on 
the PK of alteplase. Assessment of any remaining trends for weight, age, sex, and race will be 
conducted e.g. by graphical inspection plotting empirical Bayes estimate versus covariate 
values and conditional weighted residuals (CWRES) versus covariate values. 
Inter-subject variability will be modeled using exponential random effect models. Residual 
variability will be modeled starting with the same error model structure as in patients with 


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AMI; adjustments will be made if necessary. The set of inter individual (η) and residual (ε) 
variability values are assumed to be symmetrically distributed around 0 with variances ω2 and 
σ2, respectively. 
Evaluation 
If model development is necessary, objective function values (for nested models) as well as 
the criteria listed below will be considered:

A “successful minimization” statement by the NONMEM® program at the end of the 
estimation step.

The final parameter estimates have a number of significant digits of 3 for all θ’s.

Estimates of θ’s not close to a boundary.

Correlation of the uncertainty of the estimates of fixed effect parameters < 0.95 or >-0.95.

No significant trends in the basic goodness-of-fit plots (predictions (PRED), individual 
predictions (IPRED) vs. DV)

Residuals (CWRES) scatter randomly and uniformly around zero when plotted against 
population prediction and time.

ETAs are normally distributed around 0.

No significant model misspecification in the individual predicted concentration-time 
profiles when compared to the observed profiles.
If a model is accepted as final model, although one or more of the criteria above are not 
fulfilled, the reasons for its acceptance need to be given in the report.
If model development is necessary, the final model will be evaluated using a prediction 
corrected visual predictive check (pcVPC, 13). A visual predictive check shows the ability of 
the model to simulate data that are similar to the observed data that were used for model 
development. The concentration-time profiles will be simulated 1,000 times using the same 
actual doses, infusion regimen, subjects’ covariates, and study design as in the analysis data 
set. In order to adjust for the differences in e.g. actual doses, a pcVPC will be performed in 
which the observed and simulated DV will be normalized based on the typical population 
prediction. 


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8.
TIMEPOINT OF RELEASE OF TREATMENT 
INFORMATION
8.1
HANDLING OF INDIVIDUAL PATIENT TREATMENT INFORMATION
This is an open-label study, therefore no blinding will apply for patients, investigators and
personnel at the site involved in trial conduct. The access to the (planned) randomisation code 
list will be kept restricted until it is released for analysis.
The treatment information for an individual patient will be available in the trial database after 
randomisation.
Select data derivations, mostly related to safety endpoints, are treatment dependent. 
Therefore, the actual treatment codes available on the database will be used to create these 
treatment dependent data derivations, ongoing throughout the duration of the trial.
8.2
HANDLING OF AGGREGATED TREATMENT INFORMATION
While the study is in progress, access to tabular results of study outcomes by treatment 
allocation will not be made available to the patients, investigators and personnel at the site, to 
the trial statistician, clinical team, or members of the steering committee (unless the DMC 
advises otherwise). The DMC and DMC statisticians will be unblinded.
Ongoing tabular results of study outcomes will be produced using mock (dummy) treatment 
codes, throughout the duration of the trial, and until database snapshot.
At the timepoint of the database snapshot, following completion of day 28 visit for all 
patients, the actual treatment codes will be used to unblind the data for analysis.


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9.
REFERENCES
1.
CPMP/ICH/363/96: "Statistical Principles for Clinical Trials", ICH Guideline Topic E9, 
Note For Guidance on Statistical Principles for Clinical Trials, current version.
2.
0135-0347 Clinical Trial Protocol Version 3 (c32434345-03)
3.
WHO Working Group on the Clinical Characterisation and Management of
COVID-19 infection. A minimal common outcome measure set for
COVID-19 clinical research. Lancet Infectious Diseases, Published Online
June 12, 2020, doi: 10.1016/S1473-3099(20)30483-7; 2020. [R20-2002]
4.
Schulman S, Kearon C. Subcommittee on Control of Anticoagulation of the Scientific 
and Standardization Committee of the International Society on Thrombosis and
Haemostasis. Definition of major bleeding in clinical investigations of antihemostatic
medicinal products in non-surgical patients. J Thromb Haemost. 2005. 3: 692-694.
[R05-0344]
5.
001-MCS-40-413: Identify and Manage Important Protocol Deviations (iPD)”, current 
version, Group “Clinical Operations”, IDEA for CON.
6.
001-MCS-40-135_RD-01: “Integrated Quality and Risk Management Plan”, current 
version, Group “Clinical Operations”, IDEA for CON.
7.
001-MCG-156: "Handling and summarization of adverse event data for clinical trial 
reports and integrated summaries", current version; IDEA for CON.
8.
001-MCG-159_RD-06: “Standard table shells for inferential and descriptive Company 
Standard Displays (CSD-Catalogue)”, current version; IDEA for CON.
9.
001-MCG-159: "Reporting of Clinical Trials and Project Summaries", current version; 
IDEA for CON.
10. 001-MCG-156: "Handling and summarization of adverse event data for clinical trial 
reports and integrated summaries", current version; IDEA for CON.
11. REGULATION (EU) No 536/2014 OF THE EUROPEAN PARLIAMENT AND OF THE 
COUNCIL of 16 April 2014 on clinical trials on medicinal products for human use, and 
repealing Directive 2001/20/EC, European Commission webpage.
12. 001-MCG-157: "Handling, Display and Analysis of Laboratory Data", current version; 
IDEA for CON.
13. Bergstrand M, Hooker AC, Wallin JE, Karlsson MO Prediction-corrected visual 
predictive checks for diagnosing nonlinear mixed-effects models. AAPS J 2011 ; 13(2) ; 
143-151. [R13-0300]


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11.
HISTORY TABLE
Table 11: 1
History table
Version
Date
Author
Sections changed
Brief description of change
1 
26-OCT-2021
None
This is the final TSAP