Conditions: COVID
Allocation: NON_RANDOMIZED | Intervention model: SEQUENTIAL
Enrollment: 35
Healthy volunteers: no

Brief summary:
Some patients infected with COVID-19 develop a severe form of a lung disease called acute respiratory distress syndrome (ARDS). In these patients, the lungs become severely inflamed because of the virus. The inflammation causes fluid from nearby blood vessels to leak into the tiny air sacs in the lungs, making breathing increasingly difficult. This fluid forms small clots in the air sacs. In some patients, these clots do not disappear in a timely fashion. Furthermore, the small clots in the air sacs obstruct the air and oxygen getting deep into the lungs, interfering with ventilation. The trial recruited patients with COVID-19 induced ARDS. Eligible patients (or if patients lack capacity, their legal representative) were provided with an information sheet and informed consent was sought. Eligibility was mainly assessed via routine clinical assessments. Patients received a nebulised version of a type of drug called tissue plasminogen activator (rt-PA) that was inhaled using a nebuliser. This is normally a drug used to break down blood clots. In the nebulised form, we hypothesised that it may be useful for stopping clots forming in the lungs. The study ran two cohorts sequentially. In cohort 1, 9 consented patients received nebulised rtPA in addition to SOC. As an observational arm, matched historical controls who received SOC were also recruited at a ratio of 2 controls to every 1 treatment arm patient, resulting in 18 historical controls. After the first wave of COVID-19 cases decreased in August 2020 in the UK, it became difficult to continue recruiting, so recruitment was closed for cohort 1. With a second surge in early 2021, cohort 2 opened with the aim to recruit more patients to provide more data on the safety of rt-PA. In cohort 2, fewer timepoints were collected, which allowed for more rapid recruitment without compromising safety monitoring. A more flexible dosing regimen for rtPA was utilised. 26 patients were recruited in total, 12 in the IMV arm and 14 in the NIV/NIRS arm. To evaluate drug efficacy, the improvement of oxygen levels over time and safety were monitored throughout. Blood samples were taken to measure markers of clotting and inflammation in both cohorts. From the end of the treatment phase, both groups were followed up in accordance with SOC up to a maximum of 28 days, starting from the day of first dose of rt-PA.

Detailed description:
This is a phase II, open label, uncontrolled, repeated dose, pilot trial of nebulised rt-PA in patients with COVID-19 ARDS.

The study recruited patients requiring either invasive mechanical ventilation (IMV) or non-invasive ventilation/non-invasive respiratory support (NIV/NIRS) between April 2020 and February 2021. Eligible patients (or if patients lacked capacity, their legal representative) were provided with an information sheet and informed consent was sought. Eligibility was assessed via routine clinical assessments, which may have been done prior to consent. The only exceptions were pregnancy tests (blood or urine), and possibly any assessments that were not done as per routine care. If required, these would have been done following consent, and all screening assessments must have been done during the 24-hour period before dosing with rt-PA.

In the rt-PA treatment group of cohort 1, 9 consented patients received nebulised rt-PA in addition to standard of care (SOC). Out of the 9 patients, 6 were on the IMV arm and another 3 were on the NIV arm. As an observational arm, matched historical controls who received only SOC were also recruited at a ratio of 2 controls to every 1 treatment arm patient, to ensure that any changes were not entirely due to disease resolution. A total of 18 patients were recruited to the historical matched control arm of cohort 1. Therefore a total of 27 patient constituted cohort 1.

For patients in the treatment group, 10 mg of rt-PA dissolved in 5 ml of diluent was given every 6 hrs for 3 days. This was extended to up to 14 days with a subsequent protocol amendment. Dose modifications were not permitted. Efficacy was described as the change in PaO2/FiO2 ratio from baseline, daily during treatment, end of treatment, 3 days post end of treatment and 5 days post end of treatment.

With a second surge of COVID-19 in early 2021, cohort 2 was opened to recruit more patients to provide additional data on the safety of rt-PA. Fewer timepoints were collected, which allowed for more rapid recruitment while at the same time not compromising safety monitoring. A more flexible dosing regimen for rt-PA was utilised. Patients on IMV received 60mg daily over three doses for up to 14 days. NIV patients received 60mg daily over 3 doses for two days, followed by 40mg daily over two doses for up to 12 days. A total of 26 patients were recruited in cohort 2, 12 on the IMV arm and 14 on the NIV/NIRS arm.

From the end of the treatment phase (after Day 14), patients were followed up in accordance with SOC for 28 days from the day of first dose of rtPA.

Safety monitoring was performed by assessment of the incidence and severity of bleeding events, and by the monitoring of plasma fibrinogen levels and routine coagulation parameters. Additional samples were taken for exploratory assessment of potential biomarkers, including, but not restricted to, PAI- 1, alpha 2 antiplasmin and a range of inflammatory cytokines and coagulation proteins. All other monitoring was done as per SOC.

A Data Monitoring Committee (DMC) was set-up to review safety data within the trial along with the final study results and advise on progressing from a pilot study to a randomised control trial based on the safety and efficacy data that was to be collected. If a patient had major pulmonary bleeding at any time, further dosing of patients was to be stopped and an ad-hoc DMC review to be arranged before resuming dosing (see section 12.2 Data Monitoring Committee).

Although there is extensive experience with the use of nebulised rt-PA in the context of the underlying inflammation, safety measures were included in the study. A gap of 24hrs was maintained between the first and second patient. At 24hrs, if patient 1 had no evidence of major pulmonary bleeding suggesting exaggerated alveolar fibrinolysis and no evidence of fibrinogen reduction of more than 50% (suggestive of systemic absorption), then the second patient was to be dosed. Both patients were evaluated for 72 hrs post dose for any major pulmonary bleeding and if no bleeding was noticed, then the rest of the cohort were to be recruited after the review of the safety data by the trial management group comprised of the investigators. Any concerns with the data were referred to the DMC for review. If the safety profile was acceptable, dosing of the third and subsequent patients in the rt-PA group would resume with no required interval between patients.

A statistically powered randomised controlled trial (RCT) would be ideal to define the magnitude of benefit and impact on overall survival and is the typical design in patients with ARDS. Although there is clinical data on the safety of nebulised rt-PA, there is no data in this clinical condition to facilitate a sample size calculation. There is data from a small RCT that has used another fibrinolytic agent, but the doses were not comparable. When a patient is randomised to receive no treatment, this precludes participation in other interventional studies which might decrease the risk of mortality. The most recent figures suggest a 50% mortality in patients receiving IMV. Currently there is a concerted effort to introduce multiple therapies based on our understanding of the pathophysiologic basis of the disorder. Further, if this pilot study shows significant effect in a subset of patients, there would be justification to progress to a statistically powered RCT. In the event of major adverse drug reactions or minor improvement in the oxygenation as assessed by PaO2/FiO2 , there are minimal gains to be had with a larger randomised control study.

Arms:
arm 1: Patients in the rt-PA group received the first dose as soon as possible after registration.

Initial dosing regime: 10 mg of rt-PA dissolved in 5 ml of diluent given every 6 hrs (resulting in a total daily dose of 40mg) for a maximum of 66 hrs, in addition to standard of care for COVID-19 acute respiratory distress syndrome (ARDS). Following protocol amendment, dosing duration was increased from 3 days to up to 14 days of rt-PA treatment. Six patients were receiving Invasive mechanical ventilation and 3 were receiving non-invasive ventilation. arm 2: Historical matched controls were recruited at a ratio of 2 controls to every 1 rtPA + SOC arm patient, and were matched according to the following characteristics:

1. Ventilation and oxygen type (IMV and non-invasive oxygen support)
2. Severity as determined by PaO2/FiO2 ratio
3. Gender
4. Age (+/- 2 years, up to a maximum of 10 years)
5. Ethnicity arm 3: In cohort 2, fewer timepoints were collected, which allowed for more rapid recruitment while at the same time not compromising safety monitoring. A more flexible dosing regimen for rtPA was utilised.

Patients on IMV received 60mg daily over three doses for up to 14 days arm 4: n cohort 2, fewer timepoints were collected, which allowed for more rapid recruitment while at the same time not compromising safety monitoring. A more flexible dosing regimen for rtPA was utilised.

Patients on NIV received 60mg daily for over 3 doses for two days, followed by 12 days receiving 40mg daily over two doses.

Interventions: intervention 1: Nebulised recombinant tissue-Plasminogen Activator (rt-PA) - Cohort 1 intervention 2: Nebulised recombinant tissue-Plasminogen Activator (rt-PA) - Cohort 2 IMV intervention 3: Nebulised recombinant tissue-Plasminogen Activator (rt-PA) - Cohort 2 NIV
Intervention descriptions: intervention 1: Patients in the rt-PA group received the first dose as soon as possible after registration.

Initial dosing regime: 10 mg of rt-PA dissolved in 5 ml of diluent given every 6 hrs (resulting in a total daily dose of 40mg) for a maximum of 66 hrs, in addition to standard of care for COVID-19 acute respiratory distress syndrome (ARDS). Following protocol amendment, dosing duration was increased from 3 days to up to 14 days of rt-PA treatment. intervention 2: Patients on IMV received 60mg daily over three doses for up to 14 days intervention 3: NIV patients received 60mg daily over 3 doses for two days, followed by 12 days receiving 40mg daily over two doses.

Protocol document:
SAP_002.pdf: 
Page 1 of 13 
 
 
 
        
 
Nebulised rt-PA for ARDS due to COVID-19 – The PACA trial 
 
Statistical Analysis Plan 
 
Version History Log 
Version 
Date 
Changes 
0.1 
28/04/2020 
 
0.2 
29/04/2020 
Minor edits 
0.3 
01/05/2020 
Added details on the nature & analyses of outcomes 
0.4 
26/10/2020 
Modified to account for historical controls 
0.5 
05/11/2020 
Minor edits 
0.6 
16/11/2020 
Added NEWS 2 score  
1.0 
18/11/2020 
Minor edits 
 
 
 
 
 
 


Page 2 of 13 
 
 
Contents 
1. 
Study Summary ............................................................................................................................... 3 
2. 
Introduction .................................................................................................................................... 4 
2.1 
Purpose and scope of the statistical analysis plan ............................................................ 5 
2.2 
Analysis organisation .......................................................................................................... 5 
2.3 
Data checking ....................................................................................................................... 5 
3 
Trial Objectives (from Protocol) ...................................................................................................... 5 
3.1 
Primary objectives ............................................................................................................... 5 
3.2 
Secondary objectives ........................................................................................................... 5 
3.3 
Exploratory objective .......................................................................................................... 6 
4 
Trial Design ...................................................................................................................................... 6 
4.1 
Sample size calculation ........................................................................................................ 6 
4.2 
Randomization ..................................................................................................................... 7 
5 
Data collection ................................................................................................................................ 7 
5.1 
Primary outcome measures ................................................................................................ 7 
5.2 
Secondary outcome measures ............................................................................................ 7 
5.3 
Exploratory endpoints ......................................................................................................... 8 
5.5 
Duration of the intervention period and frequency of follow up ..................................... 8 
6 
Data analysis plan ........................................................................................................................... 8 
6.1 General statistical considerations ................................................................................................... 8 
6.2 
Summary of baseline characteristics .................................................................................. 8 
6.3 
Analysis of primary outcome .............................................................................................. 9 
6.4 
Analysis of secondary outcomes ....................................................................................... 10 
6.5 
Sensitivity analysis................................................................ Error! Bookmark not defined. 
6.6 
Model checking...................................................................... Error! Bookmark not defined. 
7 
Software ........................................................................................................................................ 10 
8 
References .................................................................................................................................... 10 
Appendix – Schedule of assessment ..................................................................................................... 11 
 
 
 


Page 3 of 13 
 
 
1. 
Study Summary 
 
For full details see the protocol (version 7.0, 30/10/2020). 
Title 
A pilot, open label, phase II clinical trial of nebulised recombinant tissue-
Plasminogen Activator (rt-PA) in patients with COVID-19 ARDS: The 
Plasminogen Activator COVID- 19 ARDS (PACA) trial.  
Short title 
Nebulised rt-PA for ARDS due to COVID-19 – The PACA trial 
Chief Investigator Pratima Chowdary 
Statisticians 
Aidan O’Keeffe & Federico Ricciardi  
Health economist … 
Design 
Phase II, open label, multi-centre, pilot interventional study.  
Primary objectives 
1. Efficacy: Investigate the potential for efficacy of nebulised rtPA in 
patients presenting with severe COVID-19 requiring IMV or Non-
invasive support with NIV OR continuous positive airway pressure 
(CPAP) OR high flow oxygen OR standard oxygen therapy. 
2. Safety: Evaluate the safety of nebulised rtPA treatment. 
Primary Outcomes  Efficacy 
1. Change in PaO2/FiO2 ratio from baseline (defined as 3 days prior to 
treatment), daily during treatment, end of treatment and 3 and 5 
days post treatment in the groups receiving rtPA 
Safety 
1. Incidence and severity of major bleeding events directly 
attributable to the study drug 
2. Decrease in fibrinogen levels to < 1.5 gm/L during treatment period 
and 48 hrs after the last dose of treatment 
3. Number and nature of serious adverse events causally related to the 
treatment 
Population 
Inclusion criteria 
1. Patients with COVID-19 confirmed by PCR 
2. ≥16 years 
3. Willing and able to provide written informed consent or where 
patient doesn’t have capacity, consent obtained from a legal 
representative 
4. Patients on IMV must meet both the following criteria: 
a. PaO2/FiO2 of ≤ 300 
b. Intubated > 6 hrs 
5. Patients on NIV must meet all the following criteria: 
a. PaO2/FiO2 ≤ 300 or equivalent imputed by non-linear 
calculation from SpO2/FiO2 
b. In-patient > 6 hours and being actively treated 


Page 4 of 13 
 
 
c. On support with non-invasive ventilation OR continuous 
positive airway pressure (CPAP) OR high flow OR standard 
oxygen therapy. 
Exclusion criteria 
1. Females who are pregnant 
2. Concurrent involvement in another experimental investigational 
a. medicinal product 
3. Known allergies to the IMP or excipients of IMP 
4. A pre-existing bleeding disorder (e.g. severe haemophilia) 
5. Pre-existing severe cardiopulmonary disease (e.g. incurable lung 
cancer, severe chronic obstructive lung disease, cardiomyopathy, heart 
failure or impaired contractility <estimated 40% LVEF or RVEF ) 
6. Fibrinogen < 2.0 g/L at time of screening 
7. Patients considered inappropriate for critical care (prior decision 
re ceiling of care established) 
8. Patients with active bleeding in the preceding 7 days 
9. Patients who in the opinion of the investigator are not suitable. 
 
Sample size  
36: 12 participants receiving rtPA + standard of care (SOC), and 24 matched 
historical controls receiving SOC only. 
Randomisation 
Not applicable – study is open label.  
Sponsor protocol 
number 
132151 
Funder (s): 
Royal Free Charity 
EudraCT No  
2020-001640-26 
ISRCTN / 
Clinicaltrials.gov 
no: 
NCT04356833 
 
List of Abbreviations 
CPAP  
Continuous positive airway pressure 
CRF 
 
Case report form 
IMP 
 
Investigational medicinal product 
IMV 
 
Invasive mechanical ventilation 
L/RVEF 
Left/Right ventricular ejection fraction 
NIV 
 
Non-invasive ventilation 
PCR 
 
Polymerase chain reaction 
rt-PA  
Recombinant tissue plasminogen activator 
SAE  
 
Serious adverse event 
SAP 
 
Statistical analysis plan 
SOC 
 
Standard of care 
SOFA  
Sequential organ failure assessment 
WHO  
World Health Organization 
 
 


Page 5 of 13 
 
 
2. 
Introduction 
2.1 
Purpose and scope of the statistical analysis plan  
This document describes the main statistical analyses to be applied to the data from PACA Trial. 
This Statistical Analysis Plan (SAP) was written by Federico Ricciardi (FR) and Aidan O’Keeffe 
(AOK).  
The SAP does not preclude the undertaking of further analyses nor the amendment of any part of 
the study analysis, should a situation arise in which an amendment or further analysis is deemed 
necessary. Any changes to the SAP shall be documented and justified.   
 
2.2 
Analysis organisation 
Unmasking of the data and analysis will be initiated after the last participant has completed 
follow-up, all relevant data has been entered, checked and locked, the analysis plan has been 
finalised and approved, and the analysis programs prepared as much as possible. The primary 
analysis will be performed independently by two statisticians (FR and AOK) to ensure its 
accuracy. 
 
2.3 
Data checking 
Before analysis, basic checks will be performed to check the quality of the data.  Incomplete or 
inconsistent data include: 
• 
Missing data; 
• 
Data outside expected range; 
• 
Other inconsistencies between variables, e.g., in the dates the questionnaires were 
completed. 
If any inconsistencies are found, the corresponding values will be double checked with the 
researchers and corrected if necessary. All changes will be documented by the trial statistician. 
 
3 
Trial Objectives (from Protocol) 
3.1 
Primary objectives 
1. Efficacy: Investigate the potential for efficacy of nebulised rtPA in patients presenting with 
severe COVID-19 requiring IMV or non-invasive support with NIV OR continuous positive 
airway pressure (CPAP) OR high flow oxygen OR standard oxygen therapy; 
2. Safety: Evaluate the safety of nebulised rtPA treatment. 
 
3.2 
Secondary objectives 
1. Investigate the impact on patient’s clinical status over time using the WHO ordinal scale of 
clinical improvement; 
2. Investigate the effect of nebulised rtPA on other respiratory markers (such as lung 
compliance) and organ dysfunction; 
3. Investigate the impact on in hospital mortality and resource utilisation. 


Page 6 of 13 
 
 
3.3 
Exploratory objective 
Changes to coagulation and inflammatory markers concerning intervention and response to 
treatment. 
 
4 
Trial Design 
This study is a phase II, open label, multicentre, uncontrolled, repeated dose, pilot trial of 
nebulised rt-PA in patients with COVID-19 ARDS.  
The study will recruit patients requiring either IMV or non-invasive oxygen support. Eligible 
patients (or if patients lack capacity, their legal representative) will be provided with an 
information sheet and informed consent will be sought. Eligibility will be assessed via routine 
clinical assessments, which may have been done prior to consent. The only exceptions are a 
pregnancy test (blood or urine), and possibly any assessments that were not done as per routine 
care. These must be done following consent, and all screening assessments must have been done 
during the 24-hour period before dosing with rtPA. Patients will be followed up until discharge, 
death, or day 28 whichever is sooner. 
The study will recruit historical controls at a ratio of 2 controls to every 1 rtPA + SOC arm patient, 
and will be matched according to the following characteristics:  
1. Ventilation and oxygen type (IMV and non-invasive oxygen support) 
2. Severity as determined by PaO2/FiO2 ratio 
3. Gender 
4. Age (+/- 2 years, up to a maximum of +/- 10 years) 
5. Ethnicity 
 
Matching will be done in the order that the characteristics are presented above.  
Deceased patients can be considered for matching as controls. However, as a certain number of 
observations are required for developing a PaO2/FiO2 ratio of the curve, patients who died within 
2 to 3 days of admission are excluded. 
For characteristics 1 and 2, baseline data during admission will be used for matching.  
An age range of +/- 2 years was used in the first instance, and this was widened until two eligible 
patients were identified that matched the treatment patient’s ethnicity, up to a maximum of +/- 
10 years. 
Where more than two controls are identified, two controls will be drawn at random without 
replacement among all considered historical patients that match the age, gender and severity the 
case. 
 
4.1 
Sample size calculation 


Page 7 of 13 
 
 
The sample size for this pilot study is not based on a statistical consideration. The recruitment 
target of 12 patients treated with rtPA and SOC and 24 historical controls is based on feasibility 
and the planned recruitment rate. 
 
4.2 
Randomization  
Participants in this trial will not be randomized, due to practical and ethical considerations. The 
first 12 consented patients will receive nebulised rtPA in addition to SOC.  
 
5 
Data collection 
Data will be collected on study-specific case report forms (CRFs). Source data are contained in 
source documents (medical notes and laboratory reports) and must be accurately transcribed on 
to the CRF. 
Demographic information, medical history, concomitant medication and other baseline 
information will be collected.  
 
5.1 
Primary outcome measures 
Efficacy 
• 
PaO2/FiO2 measured at multiple timepoints over the study period: baseline, during 
treatment, end of treatment, 3 days post end of treatment and 5 days post end of 
treatment. 
Safety 
• 
Incidence and severity of major bleeding events; 
• 
Decrease in fibrinogen levels to < 1.5 gm/L during treatment period and 48 hrs after 
the last dose of treatment; 
• 
Number and nature of serious adverse events causally related to the treatment. 
 
5.2 
Secondary outcome measures 
• 
Changes in lung compliance (defined as tidal volume / (peak inspiratory pressure – 
PEEP)) from baseline and absolute values at day 5 (96 hrs ± 2 hrs) and day 7 (144 hrs 
± 4hrs); 
• 
Clinical status as assessed by a 7-point WHO ordinal scale at baseline and daily up to 
5 days post end of treatment and at day 28, discharge or death (whichever comes 
first); 
• 
Mean daily Sequential Organ Failure Assessment (SOFA) score at baseline through up 
to 5 days post end of treatment; 
• 
Mean daily NEWS 2 score at baseline through up to 5 days post end of treatment, for 
NIV patients only; 
• 
In follow up period, number of oxygenation free days, ventilator free days, intensive 
care stay, up to 28 days or death or discharge, whichever occurs first; 


Page 8 of 13 
 
 
• 
Incidence and number of days of new oxygen use, non-invasive ventilation or high 
flow oxygen devices in the first 28 days; 
• 
Incidence and duration of new mechanical ventilation use during in the first 28 days; 
• 
In hospital mortality; 
• 
Length of ICU stay post treatment; 
• 
Length of ventilation; 
• 
Use of vasopressors. 
 
Further details on the outcomes, including a list of references, can be seen in the protocol. 
 
5.3 
Exploratory endpoints 
Change in levels of plasma biomarkers of pro- and anti-inflammatory markers (interleukin-6, 
interleukin-8, interleukin-10 and interleukin-1Ra), endothelial injury (plasma von Willebrand 
factor) coagulation (procoagulants, anticoagulants, and fibrinolytic pathway) measure of tissue 
damage. Historical controls will not provide data for exploratory endpoints. 
 
5.5 
Duration of the intervention period and frequency of follow up 
The trial duration per participant is 28 days. Participants will complete assessments at baseline 
and daily after baseline. Respiratory status of patients will be assessed every 4 hours for the first 
week, and then daily. The appendix provides a summary of the times at which data are collected.  
For historical controls, data will be collected where available for 28 days from the date of 
admission to hospital. See section 7.5.1 of the protocol for more details. 
 
6 
Data analysis plan 
6.1 
General statistical considerations 
All analyses will be descriptive in nature and should be considered as hypothesis generating 
rather than providing firm conclusions. All statistical tests and confidence intervals will be 2-
sided.  Significance will be considered at the 5% level and confidence intervals will be at the 95% 
level.  
Analyses for both primary and secondary outcomes will be conducted following an Intention To 
Treat (ITT) approach, and non-compliance will be reported at the individual patient level. No 
adjustments for missing data will be made. 
 
6.2 
Summary of baseline characteristics 
All baseline covariates and baseline valued of the outcomes will be summarised using descriptive 
statistics. For patients treated with rtPA + SOC, the last available value prior to the first 
administration of IMP will be considered the baseline value. For historical controls receiving SOC 
only, the last available value prior to enrolment, as defined in section 8.2, will be considered the 
baseline value. Categorical variables shall be reported as frequencies and percentages. Reports of 
continuous variables shall include mean or median and standard deviation or interquartile range 
(IQR) as appropriate, as well as minimum and maximum values.  


Page 9 of 13 
 
 
Summary measures for the baseline characteristics will be presented both overall and by group. 
We will compare baseline characteristics visually to assess whether balance has been achieved. 
No significance testing will be used. The number of missing observations will be reported.  
A CONSORT flow chart will be provided (Schulz et al., 2010). This will include the number of 
eligible participants, number of participants agreeing to enter the trial, then by intervention arm: 
the number of patients in each group that were enrolled, assessed for eligibility, excluded (with 
reasons listed), assigned to a group, treated with rtPA, followed up, discontinued (with reasons 
such as lost to follow-up, withdrawn, or death), and number of patients analysed in each group, 
and where applicable excluded from analyses (with reasons). 
 
6.3 
Analysis of primary outcome 
The primary outcome for efficacy is oxygenation, measured using the PaO2/FiO2 ratio over time. 
The distributions of the primary outcomes will be summarised, by group, over time. Change in 
PaO2/FiO2 ratio from baseline will be checked visually using means & SE plots. If necessary, 
appropriate transformations of the data will be performed (e.g., using the natural logarithm). 
Subject to the final sample size and data quality, PaO2/FiO2 ratio will also be compared between 
the SOC group (historical controls) and rtPA group using appropriate regression modelling, 
controlling for baseline oxygenation ratio: 
𝑂𝑋𝑌𝑖=  𝛽0 +  𝛽1 ∙𝑇𝑖+  𝛽2 ∙𝑂𝑋𝑌0𝑖+ 𝜀𝑖, 
where the i subscript denotes the ith patient and  
• 
𝑂𝑋𝑌𝑖= PaO2/FiO2 ratio (oxygenation) at the end of treatment;  
• 
𝑇𝑖= group indicator (= 1 if patient is in the rtPA group, = 0 otherwise); 
• 
𝑂𝑋𝑌0𝑖= PaO2/FiO2 ratio (oxygenation) at baseline;  
• 
𝜀𝑖~𝑁(0, 𝜎2) = Normally distributed error term. 
The estimate of the parameter 𝛽1 and associated 95% confidence interval shall be reported, as 
well as a p-value pertaining to the hypothesis test of  
𝐻0: 𝛽1 = 0  versus  𝐻1: 𝛽1  ≠0. 
Similarly, the use of models using oxygenation at other meaningful timepoints as outcome 
measure will be evaluated. Owing to a limited sample size, models shall not adjust for other 
baseline characteristics. Fitted models shall be checked, where appropriate, to ensure that 
modelling assumptions have been satisfied.  Appropriate transformations of outcomes or other 
suitable approaches (e.g., robust or non-parametric methods) shall be explored if model 
assumptions are not satisfied.  
For treatment safety, the incidence and severity (either major or minor) of bleeding events will 
be summarised with frequencies and percentages in each group. Fibrinogen levels will also be 
summarised graphically using plots of the mean, by group, over time. Finally, the number and 
nature of serious adverse events (SAEs) will be summarised. These events are defined in section 
8 of the protocol. 
 


Page 10 of 13 
 
 
6.4 
Analysis of secondary outcomes 
All secondary outcomes will be summarized by group. Categorical variables shall be reported as 
frequencies and percentages. Reports of continuous variables shall include mean or median and 
standard deviation or interquartile range (IQR) as appropriate, as well as minimum and 
maximum values. 
In addition, we will make use of the daily measurements of clinical status, measurements of lung 
compliance, NEWS 2 and SOFA score, summarising them at each post baseline timepoint by 
group. The evolution of these outcomes over time will also be summarised graphically. 
 
7 
Software 
The statisticians will download the data from the trial specific database into a format suitable to 
be read by Stata. All the statistical analysis will be performed using Stata version 15 (or above) 
and/or R version 4.0.2 (or above). 
 
8 
References 
• 
Schulz KF, Altman DG, Moher D. (2010). CONSORT 2010 Statement: updated guidelines for 
reporting parallel group randomised trials. BMC Med, 8(18). doi:10.1186/1741-7015-8-18


Page 11 of 13 
 
 
Appendix – Schedule of assessment (from Protocol) 
 
Screening/
3 day 
baseline 
perioda 
During treatment until 5 days after 
last dose 
From 6 days after 
last dose until day 
28/discharge/death 
Day 
28/discharge/death 
 
 
Every 4 
hours 
Every 6 
hours 
Once 
Daily 
 
 
 
 
24 and 
48 
hours, 
then 
twice 
weekly 
 
 
 
Once daily 
 
Informed consent 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Review of medical history 
(including inpatient history and 
COVID-19 diagnosis) 
X 
 
 
 
 
 
 
Eligibility review 
X 
 
 
 
 
 
 


Page 12 of 13 
 
 
Pregnancy test (blood or urine) 
for female patients of 
childbearing potential 
X 
 
 
 
 
 
 
Review of concomitant 
medication 
Xf 
 
 
X 
 
X 
X 
Review of adverse 
events/adverse reactions 
(including bleeding episodes) 
 
 
 
X 
 
X 
X 
Directed physical examination 
including vital signs (heart rate, 
temperature, blood pressure 
and body weight) 
X 
 
 
X 
 
X 
X 
Documentation of respiratory 
statusa 
X 
X 
 
 
 
X 
X 
Local blood testingb 
X 
 
 
X 
 
as required 
X 
Research blood samplingc 
X 
 
 
 
X 
X – weekly 
 
Administration of rtPAd 
 
 
X 
 
 
 
 
Clinical status as per 7-point 
ordinal scale 
X 
 
 
X 
 
 
X 
Sequential Organ Failure 
Assessment (SOFA) score 
X 
 
 
X 
 
 
 


Page 13 of 13 
 
 
Glasgow Coma scale 
X 
 
 
X 
 
 
 
End of study data collection e 
 
 
 
 
 
 
X 
 
a Respiratory status of patients is monitored routinely hourly, but data points will be extracted for every four hours where available until 5 days after the 
last dose (day 7 for SOC patients), but one data point per day is required as a minimum. After this period, respiratory status is to be documented weekly 
(+/- 1 day) until discharge, death, or 28 days whichever occurs first. See section 7.5 for variables required.  
b Local blood testing as per section 7.6.2 are to be done at a minimum once a day until 5 days after the last dose, and then as required until day 28, 
discharge or death whichever occurs first. 
c Research blood sampling will be done once at baseline at 48 hours (+/- 24 hours), 96 hours (+/- 24 hours), and then twice weekly as per section 7.6.2. 
Twice weekly testing should be evenly spaced across weekdays if possible.  
d Dosing schedule as per section 7.3 
e End of study data will be collected at day 28, discharge or death whichever occurs first as per section 7.5. 
f to be taken where available once daily for 3 days prior to dosing. Screening assessments can be used as part of this baseline period.  
 

Prot_000.pdf: 
Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 1 of 71 
 
 
 
 
 
Full title of trial 
 
A pilot, open label, phase II clinical trial 
of nebulised recombinant tissue-
Plasminogen Activator (rtPA) in 
patients with COVID-19 ARDS: The 
Plasminogen Activator COVID-19 ARDS 
(PACA) trial 
Short title 
 
Nebulised rtPA for ARDS due to COVID-
19 – The PACA trial 
Sponsor 
University College London (UCL) 
Sponsor protocol number 
132151 
EudraCT No  
2020-001640-26 
ISRCTN / Clinicaltrials.gov no: 
NCT04356833 
Document name: 
 
PACA protocol version 10.0 
(2Feb2021) redacted 
Document date:  
02 Feb 2021 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 2 of 71 
 
 
 
A pilot, open label, phase II clinical trial of nebulised recombinant tissue-Plasminogen Activator (rtPA) 
in patients with COVID-19 ARDS: The Plasminogen Activator COVID-19 ARDS (PACA) trial 
 
Full title of trial 
 
A pilot, open label, phase II clinical trial of 
nebulised recombinant tissue-Plasminogen 
Activator (rtPA) in patients with COVID-19 
ARDS: The Plasminogen Activator COVID-
19 ARDS (PACA) trial 
Short title 
 
Nebulised rtPA for ARDS due to COVID-19 
– The PACA trial 
Version and date of protocol 
 
Version 10.0, 2 Feb 2021 
Sponsor: 
University College London (UCL) 
Sponsor protocol number 
132151 
Funder (s): 
Royal Free Charity 
EudraCT No  
2020-001640-26 
ISRCTN / Clinicaltrials.gov no: 
NCT04356833 
ACTIVE IMP(s): 
Nebulised recombinant tissue plasminogen 
activator (rtPA) alteplase  
Phase of trial 
Phase II 
Sites(s) 
Multi Site  
Chief investigator: 
Pratima Chowdary 
The Katharine Dormandy 
Haemophilia and Thrombosis Centre 
Royal Free Hampstead NHS Trust 
Royal Free Hospital, Pond Street 
Hampstead, London NW3 2QG 
Sponsor Representative: 
Joint Research Office, UCL,  
Office address: 
UCLH/UCL Joint Research Office, part of 
the Research Directorate 
4th  Floor,  West 
250 Euston Road 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 3 of 71 
 
 
 
London 
NW1 2PG 
 
 
Protocol Version History 
Version 
Number 
Date 
Protocol Update 
Finalised By 
(insert name of 
person):  
Reasons for Update 
2.0 
14 
April 
2020 
Alison Evans 
Update to inclusion criteria 4b (removal of 7 day timeline for 
intubation in IMV patients)and removal of exclusion criteria 
‘Patients receiving anticoagulation with therapeutic doses’ 
3.0 
17 Apr 
2020 
Alison Evans 
Updates to section 2.5 (assessment and management of 
risk), 7.10 (stopping criteria), 10.5 (Randomisation methods) 
and 12.2 (IDMC) in response to GNA from MHRA and 
comments from MHRA EAG. 
In addition: 
Updated the inclusion criteria to remove the upper age limit 
Updated inclusion criteria no 5 to remove HFO definition 
(and reference to HFO throughout protocol 
Updated the schedule of collection for research blood 
samples to baseline, Day 2, 4, 7 and then every week until 
the end of the trial. 
4.0 
29 Apr 
2020 
 
Amendments made to inclusion/exclusion criteria for 
consistency. 
Updated exclusion criteria no 7 to ‘Patients considered 
inappropriate for critical care (prior decision re ceiling of 
care established e.g. being considered for palliative care). 
Certain sections have been corrected regarding fibrinogen 
monitoring and the action to be taken. In particular, it was 
unclear whether replacement therapy should be given when 
fibrinogen levels are <1.5 g/L or <1.0 g/L. This has been 
clarified. 
A drop in fibrinogen <1.5 g/L is not a stopping criteria for the 
trial and accordingly this has not been listed in the Stopping 
Criteria section (7.10) as the intention has always been that 
this was a reason for treatment discontinuation in an 
individual patient.  The wording in the protocol has been 
clarified around this. 
Updated stopping criteria to remove: 
•     Any major bleeding as reported by ISTH criteria  
and replace with: 
•         Any fatal bleeding  
•         Symptomatic bleeding in a critical area or organ, 
such as intracranial, intraspinal,  pericardial, 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 4 of 71 
 
pulmonary or intramuscular with compartment 
syndrome.  
therefore not including the following as a stopping criteria: 
‘Bleeding causing a fall in haemoglobin level of 20 g/L or 
more, or leading to transfusion of two or more units of 
whole blood or red cells.’ 
We have also added the caveat that the bleeding events 
specified in the stopping criteria should occur ‘within start of 
treatment and 30 h after the last dose’.  
 
Clinicaltrials.gov number added 
5.0 
20 
May 
2020 
 
To update the rtPA dosing schedule and relevant endpoints 
6.0 
18 
Sep 
2020 
 
Use of historical matched controls instead of standard of 
care arm at a ratio of two controls to every one treatment 
arm.  
Allowance for telephone consent for matched historical 
controls. 
Use of deceased patients as historical controls with no 
consent sought. 
Renaming of non-invasive ventilation group to non-invasive 
oxygen support arm (throughout protocol) and amended to 
include standard oxygen therapy.  
Other small changes to the inclusion/exclusion criteria. 
Extension of pre-treatment baseline period to 3 days. 
Minor changes to primary and secondary (objectives and) 
endpoints. 
Addition of University of Surrey laboratory for exploratory 
endpoint analyses. 
7.0 
11 
Nov 
2020 
 
To document that we will not be collecting consent for 
historical controls, following discussion with CAG. 
Updates to data collected for historical control patients to 
allow for a more complete comparison between treatment 
patients and control patients (section 7.5.1). Also updates to 
the criteria to be used for matching of control patients to 
allow for better matching (section 10.4.2). 
To make minor clarifications/corrections. 
8.0 
8 Jan 
2021 
 
To include an additional cohort of up to 30 patients to allow 
further accrual of safety and efficacy data.  
 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 5 of 71 
 
9.0 
14 Jan 
2021 
 
To correct error in the wording in last row of dosing table for 
NIV patients (section 7.3.2) to clarify that dosing will 
continue every 12 h (+/- 2 h) on Days 6-14. 
To make updates to schedule of assessments table in 
Appendix 1 (section 20) to further clarify assessments to be 
done in Cohort 2 (in line with updates made to section 7.5 in 
protocol version 8.0). 
10.0 
2 Feb 
2021 
 
To document that should a patient’s condition change such 
that their ventilation type is changed whilst receiving rtPA 
treatment (that is, from IMV to NIV, or vice versa) they will 
switch to the treatment schedule that is applicable to the 
new ventilation type (section 2.5 and 7.4). 
To increase the window of flexibility in timing of the dose 
from 2 hours to 4 hours when dosing is twice a day (section 
7.3.2). 
Section 5.1 – to add in that the IMP may be supplied as 20 
mg vials, in addition to 10 mg vials. 
Section 10.6 Have updated patient numbering of individual 
groups  in line with the patient registration SOP and clarified 
the definition of the groups.Section 10.6 Clarified that 
additional descriptions of the results may be performed 
taking into account changes in ventilation type.  
 
 
 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 6 of 71 
 
Signatures 
The Chief Investigator and the JRO have discussed this protocol. The investigator agrees to perform the 
investigations and to abide by this protocol 
The investigator agrees to conduct the trial in compliance with the approved protocol, EU GCP and UK 
Regulations for CTIMPs (SI 2004/1031; as amended), the UK Data Protection Act (1998), the Trust 
Information Governance Policy (or other local equivalent), the current UK Policy Framework for Health 
and Social Care Research, the Sponsor’s SOPs, and other regulatory requirements as amended. 
 
Chief investigator 
Dr Pratima Chowdary 
 
 
UCL 
Signature 
Date 
Sponsor 
Dr Rajinder Sidhu  
 
 
UCL 
Signature 
Date 
 
 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 7 of 71 
 
Table of contents 
1 
Summary ................................................................................................................................ 15 
2 
Background and Rationale ....................................................................................................... 19 
2.1 
Background ................................................................................................................................................... 19 
2.2 
Pathophysiological basis for the use of  inhaled rtPA by nebulisation ......................................................... 20 
2.3 
Rationale for fibrinolytic therapy delivered by inhalation ............................................................................ 21 
2.4 
Rationale for rtPA dose – Cohort 1 ............................................................................................................... 21 
2.5 
Dosing schedule of rtPA for cohort 2 – rationale .......................................................................................... 24 
2.6 
Assessment and management of risk ........................................................................................................... 25 
2.7 
Safety data from cohort 1 ............................................................................................................................ 28 
3 
Objectives ............................................................................................................................... 29 
3.1 
Primary Objectives ........................................................................................................................................ 29 
3.2 
Secondary Objectives .................................................................................................................................... 29 
3.3 
Exploratory Objectives .................................................................................................................................. 30 
4 
Trial design ............................................................................................................................. 30 
4.1 
Overall design ............................................................................................................................................... 30 
4.2 
Schematic diagram(s) of overall trial design ................................................................................................ 32 
5 
Investigational Medicinal Products and Non-Investigational Medicinal Products ...................... 32 
5.1 
Name and description of IMP(s) - rtPA, alteplase (Actilyse®) ....................................................................... 32 
5.2 
Source of IMP, Manufacture, Distribution and Storage ............................................................................... 32 
5.3 
Storage and handling of IMP(s) at site ......................................................................................................... 33 
5.4 
Accountability of IMP(s) ............................................................................................................................... 33 
5.5 
Concomitant medication .............................................................................................................................. 33 
5.6 
Post-trial IMP arrangements ........................................................................................................................ 34 
6 
Selection of Participants .......................................................................................................... 34 
6.1 
Eligibility of trial participants ....................................................................................................................... 34 
6.2 
Recruitment .................................................................................................................................................. 35 
6.3 
Informed consent procedure ........................................................................................................................ 36 
6.4 
Consent for historical standard of care controls (cohort 1 only) .................................................................. 36 
7 
Trial procedures ...................................................................................................................... 37 
7.1 
Pre-treatment Assessments .......................................................................................................................... 37 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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7.2 
Registration Procedures ............................................................................................................................... 38 
7.3 
Treatment Schedule for nebulisation ........................................................................................................... 39 
7.4 
Dose Modifications ....................................................................................................................................... 43 
7.5 
Visit schedule and assessments post initiation of treatment ....................................................................... 43 
7.6 
This data will be collected where available for 28 days from the date of admission to hospital. Laboratory 
Assessments and Procedures ................................................................................................................................. 45 
7.7 
Clinical Procedures and Data Collection ....................................................................................................... 46 
7.8 
Discontinuation/withdrawal of participants ................................................................................................ 49 
7.9 
Replacements ............................................................................................................................................... 50 
7.10 
Stopping rules .......................................................................................................................................... 50 
7.11 
Definition of End of Trial .......................................................................................................................... 50 
8 
Recording and reporting of adverse events and reactions ........................................................ 50 
8.1 
Definitions .................................................................................................................................................... 51 
8.2 
Recording adverse events ............................................................................................................................. 52 
8.3 
Assessments of Adverse Events .................................................................................................................... 52 
8.4 
Procedures for recording and reporting Serious Adverse Reactions............................................................. 53 
8.5 
Notification of deaths ................................................................................................................................... 53 
8.6 
Reporting of SUSARS..................................................................................................................................... 54 
8.7 
Development Safety Update Reports ........................................................................................................... 54 
8.8 
Pregnancy ..................................................................................................................................................... 54 
8.9 
Reporting Urgent Safety Measures and other safety events ........................................................................ 54 
8.10 
Notification of Serious Breaches to GCP and/or the protocol (SPON/S15) .............................................. 54 
9 
Data management and quality assurance ................................................................................ 54 
9.1 
Confidentiality .............................................................................................................................................. 54 
9.2 
Data collection tools and source document identification ........................................................................... 55 
9.3 
Completing Case Report Forms .................................................................................................................... 55 
9.4 
Data handling and analysis .......................................................................................................................... 55 
10 
Statistical Considerations ........................................................................................................ 55 
10.1 
Primary endpoints .................................................................................................................................... 55 
10.2 
Secondary endpoints ................................................................................................................................ 55 
10.3 
Exploratory endpoints .............................................................................................................................. 56 
10.4 
Sample size and recruitment ................................................................................................................... 56 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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10.5 
Randomisation methods .......................................................................................................................... 57 
10.6 
Statistical analysis plan ............................................................................................................................ 57 
11 
Record keeping and archiving .................................................................................................. 60 
12 
Oversight Committees ............................................................................................................. 60 
12.1 
Trial Management Group (TMG) ............................................................................................................. 60 
12.2 
Data Monitoring Committee (DMC) ........................................................................................................ 60 
13 
Direct Access to Source Data/Documents ................................................................................ 61 
14 
Ethics and regulatory requirements ......................................................................................... 61 
15 
Monitoring requirement for the trial ....................................................................................... 61 
16 
Finance ................................................................................................................................... 61 
17 
Insurance ................................................................................................................................ 62 
18 
Publication policy .................................................................................................................... 62 
19 
References .............................................................................................................................. 62 
20 
Appendix 1 - Schedule of assessments ..................................................................................... 66 
21 
Appendix 2 - Lookup table for imputed PaO2 for a given SpO2 based on non-linear equation 
(from Brown SM et al. Nonlinear Imputation of PaO2/FIO2 From SpO2/FIO2 Among Mechanically 
Ventilated Patients in the ICU: A Prospective, Observational Study. Crit Care Med. 2017; 
Aug;45(8):1317-1324) ...................................................................................................................... 69 
22 
Appendix 3 – Oxygen flow rate calculation (used for calculating FiO2 in patients receive non-
invasive oxygen support .................................................................................................................. 71 
 
 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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List of abbreviations 
ACDP  
Advisory Committee of Dangerous Pathogens  
ACE 
Angiotensin-converting-enzyme 
AE 
Adverse Event 
AESI 
Adverse Event of Special Interest  
ALI 
Acute Lung Injury  
APR 
Annual Progress Report  
APTT  
Activated Partial Thromboplastin Time 
AR 
Adverse Reaction 
ARDS 
Acute Respiratory Distress Syndrome 
AT 
Antithrombin  
BNP  
Brain Natriuretic Peptide  
CA 
Competent Authority 
CI 
Chief Investigator 
COVID-19 
Coronavirus Disease 2019 
CPAP 
Positive Airway Pressure  
CRF 
Case Report Form 
CRO 
Contract Research Organisation 
CRP 
C-Reactive Protein  
CTA 
Clinical Trial Authorisation 
CTIMP 
Clinical Trial of Investigational Medicinal Product 
DH  
Department of Health  
DI 
Designated Individual 
DIBD 
Developmental International Birth Date  
DMC 
Data Monitoring Committee 
DNA  
DeoxyriboNucleic Acid  
DP 
Driving Pressure  
DSUR 
Development Safety Update Report 
EC 
European Commission 
ECMO 
Extracorporeal Membrane Oxygenation  
ED 
Emergency Department  
EMA 
European Medicines Agency 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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EU 
European Union 
EUCTD 
European Clinical Trials Directive 
EudraCT 
European Clinical Trials Database 
EudraVigilance 
European database for Pharmacovigilance 
FDF 
Fibrin Degradation Product  
FiO2 
Fraction of inspired Oxygen 
GAfREC 
 
Governance Arrangements for NHS Research Ethics 
GCP 
Good Clinical Practice 
GMO 
Genetically Modified Organisms 
GMP 
Good Manufacturing Practice 
GP 
General Practitioner  
HFO 
High Flow Oxygen  
HSL  
Health Services Laboratories  
HTA 
Human Tissue Authority 
IB 
Investigator Brochure 
ICF 
Informed Consent Form 
ICU 
Intensive Care Unit  
IDMC 
Independent Data Monitoring Committee 
IDW 
Ideal Body Weight 
IMP 
Investigational Medicinal Product 
IMPD 
Investigational Medicinal Product Dossier 
IMV 
Invasive mechanical ventilation  
ISF 
Investigator Site File 
ISRCTN 
International Standard Randomised Controlled Trial Number  
ISTH 
International Society on Thrombosis and Haemostasis  
ITT 
Intention to treat  
IU 
International Units  
IV 
Intravenous  
JRO 
Joint Research Office  
KD 
Katharine Dormandy 
LHD 
Lactate Dehydrogenase  
LMWH 
Low Molecular Weight Heparin  


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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LVEF 
Left Ventricular Ejection Fraction  
MA 
Marketing Authorisation 
MHRA 
Medicines and Healthcare products Regulatory Agency 
MRC 
Medical Research Council  
MS 
Member State 
NEWS 
National Early Warning Score  
NHS 
National Health Service  
NHS R&D 
National Health Service Research & Development 
NIMP  
Non Investigational Medicinal Product  
NIV 
Non-invasive ventilation  
PAA 
Plasminogen Activator Activity  
PACA 
Plasminogen Activator COVID-19 ARDS Trial  
PAI-1 
Plasminogen Activator Inhibitor-1 
PaO2 
Partial Pressure of Oxygen  
PEEP 
Positive and Expiry Pressure  
PI 
Principal Investigator 
PIS 
Participant Information Sheet 
PL 
Product License 
PT 
Prothrombin Time  
QA 
Quality Assurance 
QC 
Quality Control 
QP 
Qualified Person (for release of trial drug) 
RCT 
Randomised Controlled Trial 
REC 
Research Ethics Committee 
RSI 
Reference Safety Information  
rtPA 
recombinant tissue plasminogen activator, alteplase  
RVEF 
Right Ventricular Ejection Fraction  
SAE 
Serious Adverse Event 
SaO2 
Oxygen Saturation measured by arterial blood gas  
SAR 
Serious Adverse Reaction 
SARS  
Severe Acute Respiratory Syndrome  
SDV 
Source Document Verification 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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SI 
Statutory Instrument  
SOC 
Standard of care  
SOFA 
Sequential Organ Failure Assessment  
SOP 
Standard Operating Procedure 
SmPC 
Summary of Product Characteristics 
SpO2 
Oxygen saturation measured by pulse oximeter  
SPON 
Sponsor  
SSA 
Site Specific Assessment 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TMG 
Trial Management Group 
tPA 
tissue Plasminogen Activator 
TSC 
Trial Steering Committee 
TV 
Tidal Volume  
UCL 
University College of London 
UK 
United Kingdom  
uPA 
Urokinase Plasminogen Activator  
USA 
United States of America  
WBC 
White Blood Cells  
WHO  
World Health Organization  
 
 
 
 
 
 
 
 
 
 
 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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Trial personnel 
See protocol cover page for Chief Investigator and Sponsor contact details. 
Statistician 
Cohort 1: To be supported by the UCL/UCLH Biostatistics 
Group (Federico Ricciardi and Aidan O’Keeffe) 
Cohort 2: To be supported by WStats Limited (Marie 
Watissée) 
 
Central 
laboratories 
Ms Anne Riddell  
Head of specialist coagulation lab 
KD Haemophilia and Thrombosis Centre 
Royal Free Hospital, Pond St 
London NW3 2QG 
Email: anne.riddell@nhs.net 
tel:  0207 472 6835  
 
fax: 0207 472 6759 
 
 
 
 
 
 
 
 
 
 
 
 
   
 
 
 
 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 15 of 71 
 
1 
Summary 
 
Objectives: 
The overall objective of the pilot study is to investigate the potential 
for clinical efficacy and safety of nebulised recombinant tissue 
plasminogen activator (rtPA) in patients hospitalised with Severe 
COVID-19 complicated by mild to severe ARDS as assessed by an 
improvement in oxygen saturation and incidence of major bleeding 
events.  
Type of trial: 
Phase II, open label, single centre, pilot interventional study of 
repeated doses of nebulised rtPA in patients with COVID-19 ARDS. 
Trial design and methods: 
Investigation of safety and efficacy includes dose finding and safety of 
the final selected dose. The initial first cohort will enable identification 
of a dose to be further studied in a second cohort. This will allow 
further accrual of safety and efficacy data at the final selected dose.  
The first cohort will include a treatment group (rtPA + standard of care 
(SOC)) and a group of matched historical controls that received 
standard of care alone.   
The first group of 12 eligible patients will receive nebulised rtPA for 14 
days in addition to SOC.  Each group will comprise six patients 
receiving invasive mechanical ventilation (IMV) for moderate to severe 
ARDS, and six patients with mild ARDS requiring non-invasive 
ventilation (NIV) or standard oxygen therapy. NIV includes continuous 
positive airway pressure (CPAP) OR high flow oxygen  
The primary efficacy assessment will be the change in arterial oxygen 
saturation levels, specifically arterial hypoxemia using the arterial 
oxygen to inhaled oxygen ratio (PaO2/FiO2 or SaO2/FiO2)  daily during 
treatment,  at the end of treatment and at 5 days post end of 
treatment. Primary safety assessments will include monitoring for 
bleeding events and plasma fibrinogen levels. Patients will be followed 
up until day 28, discharge from hospital or death - whichever occurs 
first. 
These 12 patients will be compared against 24 matched historical 
controls who received SOC alone. A DMC will be set-up to review 
emergent safety data during the trial and review and advise on the 
final study results progression from a pilot study to a statistically 
powered randomised control trial (RCT) based on the safety and 
efficacy data that is collected. 
The second cohort will include a treatment group only. 30 patients will 
be recruited, which will include a minimum of 10 patients receiving 
IMV and 10 patients receiving NIV or standard oxygen therapy.  


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 16 of 71 
 
Trial duration per 
participant: 
Informed consent to day 28, discharge or death whichever occurs first. 
Estimated total trial 
duration: 
 12 months 
Planned trial sites: 
Multi site: 
Royal Free Hospital (Royal Free London NHS Foundation Trust) 
Barnet Hospital (Royal Free London NHS Foundation Trust) 
Total number of participants 
planned: 
Cohort 1 - 12 participants receiving rtPA + standard of care (SOC), and 
24 matched historical controls receiving SOC only 
Cohort 2 – 30 participants receiving rtPA + SOC or participating in 
other trials of investigational medicinal products  
Main inclusion/exclusion 
criteria: 
Cohort 1  
Inclusion Criteria: 
1. Patients with COVID-19 (confirmed by PCR or radiologically) 
2. ≥16 years  
3. Willing and able to provide written informed consent or where 
patient doesn’t have capacity, consent obtained from a legal 
representative  
4. Patients on IMV must meet both the following criteria: 
1. PaO2/FiO2 of ≤ 300 
2. Intubated >   6  hrs  
5. Patients not intubated must meet all the following criteria: 
1. PaO2/FiO2 ≤ 300 or equivalent imputed by non-linear 
calculation from SpO2/FiO2  (see look-up table in 
appendices) 
2. In-patient >6hours and being actively treated 
3. On support with non-invasive ventilation OR continuous 
positive airway pressure (CPAP) OR high flow OR 
standard oxygen therapy  
Exclusion criteria 
6. Females who are pregnant 
7. Concurrent involvement in another experimental investigational 
medicinal product 
8. Known allergies to the IMP or excipients of IMP 
9. A pre-existing bleeding disorder with no definitive treatment 
(e.g. severe haemophilia) 
10. Pre-existing severe cardiopulmonary disease (e.g. incurable lung 
cancer, severe chronic obstructive lung disease, cardiomyopathy, 
heart failure or impaired contractility <estimated 40% LVEF or 
RVEF) 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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11. Fibrinogen < 2.0 g/L at time of screening 
12. Patients considered inappropriate for active treatment (e.g. 
being considered for palliative care) 
13. Patients with active bleeding in the preceding 7 days 
14. Patients who in the opinion of the investigator are not suitable 
Cohort 2  
Inclusion criteria are the same as for cohort 1  
Exclusion criteria  
1. Known allergies to the IMP or excipients of IMP 
2. Fibrinogen < 1.5 g/L at time of screening 
3. Patients considered inappropriate for active treatment (e.g. 
being considered for palliative care) 
4. Patients who in the opinion of the investigator are not suitable 
5. Females who are pregnant 
Statistical methodology and 
analysis: 
Cohort 1  
Data will be presented primarily using standard descriptive statistics. 
In the treatment group (rtPA + SOC) outcomes will be summarised  
separately for the six treated patients receiving invasive mechanical 
ventilation (IMV), and for the other six treated patients requiring 
support with  non-invasive ventilation (NIV) Or  continuous positive 
airway pressure (CPAP) OR high flow oxygen or standard oxygen 
therapy. Similar descriptive statistics will also be presented for the 24 
historical controls. If appropriate, outcomes will be compared 
between the historical SOC group and intervention group using 
appropriate regression methods, making an adjustment for baseline 
measurements.  
Cohort 2 
Data will be presented primarily using standard descriptive statistics. 
Outcomes will be summarised  separately for the treated patients 
receiving invasive mechanical ventilation (IMV), and for the treated 
patients requiring support with  non-invasive ventilation (NIV) Or  
continuous positive airway pressure (CPAP) OR high flow oxygen or 
standard oxygen therapy. 
 
Given the study is not designed or powered to definitively show 
efficacy, all analyses should be considered as hypothesis generating 
rather than providing firm conclusions. 
Primary objectives  
1. Efficacy: Investigate the potential for efficacy of nebulised rtPA in 
patients presenting with severe COVID-19 requiring IMV or Non-
invasive support with NIV OR continuous positive airway pressure 
(CPAP) OR high flow oxygen Or standard oxygen therapy   
2. Safety: Evaluate the safety of nebulised rtPA treatment. 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 18 of 71 
 
Primary endpoints  
Efficacy 
1. Change in PaO2/FiO2 ratio from baseline(as defined in sections 
7.1.2 and 7.1.3, daily during treatment, end of treatment and 3 
and 5 days post treatment in the groups receiving rtPA 
Safety 
1. Incidence and severity of major bleeding events directly 
attributable to the study drug 
2. Decrease in fibrinogen levels to < 1.0 gm/L during treatment 
period and 48 hrs after the last dose of treatment 
3. Number and nature of serious adverse events causally related to 
the treatment 
 
Secondary objectives 
1. Investigate the impact on patient’s clinical status over time using 
the WHO ordinal scale of clinical improvement. 
2. Investigate the effect of nebulised rtPA on other respiratory 
markers (such as lung compliance) and organ dysfunction  
3. Investigate the impact on in hospital mortality and resource 
utilisation  
4.  
Secondary endpoints  
 
1. Changes in lung compliance  (defined as tidal volume / (peak 
inspiratory pressure – PEEP) ) from baseline (as defined in sections 
7.1.2 and 7.1.3) and absolute values at day 5, day 7, end of 
treatment, 3 days post end of treatment and 5 days post end of 
treatment 
2. Clinical status as assessed by a 7-point WHO ordinal scale at 
baseline (as defined in sections 7.1.2 and 7.1.3), daily up to 5 days 
post end of treatment and at day 28, discharge or death 
(whichever comes first) 
3. Mean daily Sequential Organ Failure Assessment (SOFA) score at 
baseline (as defined in sections 7.1.2 and 7.1.3) and daily up to 5 
days post end of treatment. 
4. In follow up period, number of oxygenation free days, ventilator 
free days, intensive care stay, up to 28 days or death or discharge, 
whichever occurs first.  
5. Incidence and number of days of new oxygen use, non-invasive 
ventilation or high flow oxygen devices in the first 28 days. 
6. Incidence and number of days of new mechanical ventilation use 
during in the first 28 days 
7. In hospital mortality 
Exploratory objectives  
Changes to coagulation and inflammatory markers concerning 
intervention and response to treatment. 


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Exploratory endpoints  
Change in levels of plasma biomarkers of pro- and anti-inflammatory 
markers (e.g. interleukin-6, interleukin-8, interleukin-10 and 
interleukin-1Ra), endothelial injury (plasma von Willebrand factor) 
coagulation (procoagulants, anticoagulants, and fibrinolytic pathway) 
measure of tissue damage 
2 
Background and Rationale 
2.1 
Background 
A novel coronavirus, designated SARS-CoV-2 results in a respiratory illness termed COVID-19 pneumonia 
and is responsible for the current pandemic. The infection is in the majority of people a self-limiting 
illness, but in a significant proportion of the population, particularly the elderly, results in acute 
respiratory distress syndrome (ARDS), with severe progressive pneumonia, multiorgan failure, and death 
(Yang et al. 2020; Guan et al. 2020). In one study from Wuhan, China 31% of adult hospitalized patients 
with COVID-19 pneumonia developed ARDS. 
Several therapeutic interventions for ARDS caused by SARS-CoV-2 are in clinical trials, including anti-viral 
(e.g. Remesdivir, EudraCTs: 2020-000936-23, 2020-000842-32 and 2020-000841-15), and anti-
inflammatory agents (e.g.Tocilizumab, clinicaltrials.gov: NCT04317092) which have completed 
recruitment  and licensed for use. 
The current standard of care for ARDS includes conservative fluid strategies for patients, early empirical 
antibiotics for suspected bacterial co-infection, lung-protective ventilation and prone positioning. The 
mortality rate is 25 - 60% with the current standard of care (Aranda-Valderrama and Kaynar 2018).  
 
Currently, the only intervention that has a positive recommendation for routine use is corticosteroids 
(Siemieniuk et al. 2020) with remdesvir and tocilizumab being used in a selected patient population. We 
propose that therapeutic interventions targeting different components of the pathophysiological 
process in patients hospitalised for severe COVID-19 is essential for rapid control of the disease. In 
addition to antivirals and anti-inflammatory strategies, interventions aimed at salvaging patients with 
severe disease or with decreased reserved is essential if the overall mortality and morbidity are to be 
reduced.   


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2.2 
Pathophysiological basis for the use of  inhaled rtPA by nebulisation 
The exact mechanism for this rapid acute lung injury in these patients is not fully understood, but diffuse 
alveolar damage typically marks the onset of ARDS. Viral replication in alveolar epithelium associated 
with an intense dysregulated local inflammatory response at the alveolar-capillary level, mediated by 
various cytokines, chemokines and other inflammatory mediators appears to play a crucial role. This 
early phase of epithelial and endothelial injury is characterised 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 (Bhargava and Wendt 2012). 
This procoagulant state stems from increased tissue factor expression and suppression of fibrinolytic 
activity due to a rise in plasminogen activator inhibitor -1 (PAI-1)(Idell, James, and Coalson 1992). This 
leads to the formation of microthrombi in the lungs, as demonstrated at autopsy (Xu et al. 2020), 
further compromising gas exchange. Emerging evidence indicates that activation of coagulation as 
demonstrated by elevated D-dimer and fibrin degradation product (FDP) levels are associated with 
poorer outcomes in novel coronavirus (Tang et al. 2020). Furthermore, patients treated with heparin 
show improved outcomes (Tang et al.). The use of concurrent anticoagulation or antiplatelet agents is 
not forbidden in the trial.  
Additionally, fibrin formation within the lungs starts a vicious cycle in which leukocytes localised to the 
clot through integrin binding sites within the fibrinogen molecule (Cooper, Lo, and Malik 1988) 
exacerbate inflammation and fibrinolysis (Barrett et al. 2017).  The sequestration of neutrophils to fibrin 
in the lung has been observed in both pre-clinical (Cooper, Lo, and Malik 1988)  and clinical contexts and 
have been shown to induce inflammation and tissue injury in the context of both viral (Ichikawa et al. 
2013; Narasaraju and Harshini 2016), and non-viral (Ichikawa et al. 2013; Conhaim et al. 2014) lung 
injury. The interplay of neutrophils with fibrin in the clot may further exacerbate clotting through the 
generation of neutrophil extracellular traps (NETs) which slow down fibrinolysis (Varjú et al. 2015) and 
further increases the risk of thrombosis. 


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2.3 
 Rationale for fibrinolytic therapy delivered by inhalation 
There is accumulating data in animal, and clinical models that both intravenous and inhalation 
thrombolytic drugs are associated with increased oxygenation and improved rates of mortality, as a 
result of clearance of fibrin from the alveolar space (Hofstra et al. 2013). Following an initial case report 
demonstrating the success of nebulised tissue plasminogen activator (rtPA) (Gram et al. 1999), a recent 
randomised controlled trial demonstrated that streptokinase improved survival compared to standard 
of care with the restoration of oxygenation to normal levels within three days (Abdelaal Ahmed 
Mahmoud et al. 2020). 
A recent meta-analysis of pre-clinical data demonstrated an apparent reduction in inflammation 
(alveolar neutrophils) when rtPA was directly administered to the lung as a nebulised therapy (Liu et al. 
2018; Foley 2017). Although rtPA is widely used in clinical practice as a therapy for clot dissolution in 
thrombotic cardiovascular and cerebrovascular events but hasn’t been widely investigated for this 
indication. 
We propose to undertake an interventional study using nebulised rtPA, which has a proven favourable 
safety profile. Importantly, this molecule targets the pathway at a point with no proposed interventional 
studies. The molecule can be administered either intravenously or nebulised. 
Intravenous administration of rtPA can result in diffusion across the disrupted barrier to facilitate the 
resolution of the accumulated debris. This diffusion is not predictable, and importantly the intravenous 
route has an increased risk of bleeding and requires intensive monitoring. In contrast, nebulised rtPA 
targets the area where it can overcome the increased local PAI-1 activity. Besides, diffusion into the 
vasculature results in improvement of the pulmonary vascular thrombosis. Anticoagulation can address 
the issue of pulmonary vessel thrombosis but does not address the primary issue of the fibrin 
membranes on the alveoli. 
We propose to undertake an interventional pilot study using inhaled rtPA by nebulisation. We 
hypothesise that this route of administration will result in more rapid resolution with the potential to 
avoid secondary inflammation and cytokine storm triggered by the non-resolving fibrin membranes. 
This intervention study will examine whether there is an indication that targeted clearing of fibrin and 
the resulting leukocytes from the alveolar space early in the progression of COVID-19, would have a 
protective effect on inflammation, facilitating the restoration of alveolar-capillary barrier and survival. 
The trial, through accelerated clearance, has the potential to decrease the need for ventilatory support, 
decreased length of ICU stay and decreased mortality. 
 
2.4 
Rationale for rtPA dose – Cohort 1  
Rehberg et al. USA (Rehberg et al. 2014) in a sheep model of lung injury  post burn and smoke inhalation 
injury randomly assigned sheep to intravenous saline plus saline nebulization (control), intravenous 
recombinant human antithrombin (AT, 6 IU/kg/h) started 1 hour after injury plus saline nebulization or 
intravenous AT combined with nebulized heparin (10,000 IU every 4 hours, started 2 hours after injury), 
and nebulized tissue plasminogen activator (2 mg every 4 hours, started 4 hours after injury, a total of 
22mg) (triple therapy, n = 6 each) with animals mechanically ventilated and fluid resuscitated according 
to standard protocols during the 48-hour study period. Triple therapy resulted in improved PaO2/FiO2 


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ratio (p = 0.007), attenuated pulmonary obstruction (p = 0.02) and shunting ( p = 0.025), as well as 
reduced ventilatory pressures versus AT i.v. at 48 hours. 
Hofstra et al, Netherlands (Hofstra et al. 2013) in a rat model of bacterial infection administered 
nebulised rtPA (1.25 mg/kg, prophylactically and at 6 and 12 h). Nebulized rtPA  enhanced the 
bronchoalveolar fibrinolytic system, as reflected by a significant reduction of PAI–1 activity levels in 
bronchoalveolar lavage fluid, and a consequent increase in plasminogen activator activity (PAA) levels to 
supranormal levels and both treatments also significantly affected systemic fibrinolysis as reflected by a 
significant increase in PAA levels in plasma to supranormal levels. Neither nebulized rtPA nor anti–PA1–
1 affected pulmonary inflammation as evaluated by the neutrophil and mononuclear cell count. 
In a metanalysis of 22 studies (Liu et al. 2018) evaluating the potential effects of fibrinolytics on animal 
models of ALI, both large and small animals who received rtPA, uPA, and plasmin by various routes 
including inhalational and intravenous were included.  Fibrinolytics significantly increased the fibrinolytic 
activity both in the plasma and bronchoalveolar lavage fluid. 
Stringers group  (Lackowski et al. 2010)  have demonstrated the feasibility of inhaled tPA as evidenced 
by the retention of protein stability and activity following nebulization.  of pulmonary formulation of 
mouse tPA. In these studies mice received either a low dose (0.30 mg/kg/d) or a high dose (0.60 
mg/kg/d) or saline every 12 h for 28 days. Although male mice had a lower incidence of bleeding at 8%, 
these events occurred at lower mean doses (± SE) of 1.6 (± 0.02) mg/kg/d compared to females who 
received doses of 1.48 (± 0.03) mg/kg/d and a bleeding incidence of 16%.  Further bleeding was seen 
about six and 12 days in male and female mice, respectively, after the initiation of dosing suggesting 
potential accumulation of tPA.  
Sulfur mustard (SM) inhalation causes life-threatening plastic bronchitis, characterized by bronchial cast 
formation, resulting in severe airway obstruction that can lead to respiratory failure and death. 
Mortality in those requiring intubation is greater than 80%. In a rat model exposed to inhalation of the 
toxic SM analog, 2-chloroethyl ethyl sulfide (CEES) were rescued by intratracheal tPA (0.15 to 0.7 
mg/kg)(Veress et al. 2013). Dosing was delayed until 5.5 hrs as case formation starts around 4 hrs with a 
reduction in saturation to below 90% at 5 hrs post exposure.  A second dose was administered an hour 
later. A dose-dependent improvement in SpO2 was noted with tPA relative to controls with only the 
highest tPA (0.7 mg/kg) used resulting in near-baseline oxygen saturations (>90%) at 12 hours. 
Remarkably, tPA completely eliminated mortality in this model. Deaths in control groups occurred 
mainly between 8 and 28 hours after CEES exposure, and mortality was about 90%. The same group in 
another rat model exposed to SM, administered intratracheal t-PA (0.7 mg/kg ) or placebo every 4 hrs 
for 48 hrs when oxygen saturation reached less than 85% (median: 6.5 h).  An improvement was seen 
after the first dose with normalisation at the end of 48 hrs (Veress et al. 2015). Further, at the time of  
euthanasia, well in excess of 50%–65% of the total luminal areas of central conducting airways were 
obstructed with fibrin casts in both the nontreated and placebo-treated rats, whereas the degree of 
airway obstruction was nonsignificant in tPA-treated animals. Further no bleeding was observed unlike 
that seen with nebulised heparin. 
Normal rat airways possess endogenous fibrinolytic activity, and none of three native inhibitors of 
fibrinolysis, namely PAI-1, apha 2 antiplasmin (α2AP), or thrombin activable fibrinolysis inhibitor (TAFI), 
was detectable. Following exposure to CEES, the fibrin-degrading and plasminogen-activating 
capabilities of the airways become impaired and analysis demonstrates increased levels of PAI-1, TAFI, 
and α2AP. Further, RT-PCR analysis indicates a minimal production of TAFI or α2AP mRNA, an indication 


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that their presence in airways is due to vascular permeability and leak. PAI-1 mRNA expression was 
increased suggesting an upregulation (Rancourt et al. 2014). 
In fact, in the absence of fibrin, human tPA is generally considered a weak protease(Longstaff et al. 
2011) and it is reasonable to expect that the dose of rtPA in the clinical situation of ALI/ARDS or plastic 
bronchitis may be lower. Of note is the limited amount of plasminogen in the exudate and high levels of 
PAI- 1 all which can all limit the activity. 
In patients presenting with plastic bronchitis, nebulised rtPA has been used extensively. A range of 
doses have been reported in children, ranging from 5mg,  four times a day for seven days (Colaneri et al. 
2014) to 2 mg/kg in divided doses (Grutter et al. 2012) without any bleeding complications.  In single 
case report of lupus induced ARDS prompt resolution of ARDS was seen with ultrasonic nebulized rtPA 
(30 mg) and intravenous rtPA (20 mg) over 2 h followed by continuous treatment with ultrasonic 
nebulized unfractionated heparin (15,000 IU/day) (Gram et al. 1999). A more recent small randomised 
controlled study of saline, nebulised heparin and nebulised streptokinase showed that patients receiving 
streptokinase achieved a PaO2/FIO2 ratio >100 at day 1, PaO2/FiO2 >200 at day 5, and PaO2/FiO2 >300 at 
day 7 when compared to standard of care group who did not achieve a PaO2/FiO2 ratio >100 after eight 
days of conservative management (Abdelaal Ahmed Mahmoud et al. 2020). In this study patients 
received 250,000 IU/4 h by a nebulizer, with a total daily dose of nebulized streptokinase of 1,500,000 
IU. A personal communication from the authors reveals that most patients did not require treatment 
beyond 3 to 4 days. Currently there is clinical trial of Inhaled Tissue Plasminogen Activator for Acute 
Plastic Bronchitis in the USA. (https://clinicaltrials.gov/ct2/show/NCT02315898). 
We communicated with Kathleen A. Stringer, University of Michigan who has undertaken a considerable 
amount of animal work and she confirmed the safety and efficacy of 5 mg of tPA reconstituted per 
instructions, nebulised every 6 hrs for 3 days was adequate in their paediatric patients.  
In the context of ARDS higher PAI-I activity associated with ARDS may require a higher dose, further 
compared to children adults also require higher doses. Therefore, a dose of 10 mg every 6 hrs for 72 hrs 
was initially chosen (now updated – see below*), although this dose is not equivalent to the dose used 
in the study by Ahmed Abdelaal Ahmed Mahmoud M. Alkhatip et al. This dose was finalised in 
consultation with Professor Kathleen A. Stringer, PharmD, University of Michigan. 
*Please note protocol version 5.0 has updated this dosing schedule – please see rationale below. 
Further, dosing for the first two patients will be staggered to allow adequate safety monitoring (see 
section 4.1), before enrolling the rest of the cohort. 
An autopsy study on COVID-19 patients has shown that COVID-19 ARDS is characterised in the early 
stages by a lymphocytic infiltrate, but subsequently an increasingly extensive  intra-alveolar  fibrin 
deposition accumulation (or fibrin “balls”)(Copin et al. 2020) ). However, it is not possible to evaluate 
the fibrin burden in an individual patient, so we believe that treatment should be extended to a total of 
14 days unless they meet a stopping criterion.  The treatment duration is based on another trial of 
nebulised heparin which was conducted for 14 days with no evidence of excessive bleeding (Dixon et al. 
2010). Similarly, pre-clinical data in rats show intratracheal rtPA administered over 28 days was 
associated with bleeding if the dose exceeded 1mg/kg/day(Lackowski et al. 2010).  
 
In this trial, one patient on non-invasive ventilation responded to the treatment given for 72 hrs and was 
discharged from ICU to ward on nasal oxygen (2 litres per minute) at 96hrs. 36 hrs after the last dose his 


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oxygenation has decreased again and the trial management group was of the opinion that the patient 
required additional treatment in view of the previous response.  
 
This led to the implementation of an urgent safety measure in this one patient (patient PACA-201) to 
allow re-treatment. Retreatment consisted of a second 3 day block of treatment with rtPA, in line with 
the treatment schedule as already described in the protocol. During retreatment, the patient’s 
PaO2/FiO2 ratio increased and showed periods of normalisation. After treatment was discontinued for a 
second time, the patient’s oxygenation has decreased but not as dramatically as it had when treatment 
was discontinued for the first time.  
 
The protocol (version 5.0) has now been updated with a new dosing schedule to allow treatment for 
maximum of 14 days treatment (see section 7.3 for details of schedule). This updated dosing schedule 
has been discussed and agreed with the TMG and IDMC.  
 
2.5 
Dosing schedule of rtPA for cohort 2 – rationale 
During the first surge 9 patients were recruited to the pilot study, with 6 patients receiving IMV and 3 
patients receiving non-invasive oxygen support. When the first surge died out, recruitment was not 
feasible and following discussion with DMC and TMG cohort 1 was closed for further recruitment and 
historical controls were recruited for evaluation of efficacy to enable a sample size calculation for a 
larger phase 3 study.  
 
None of the bleeding events were considered related to the tPA. See preliminary safety data for cohort 
1 in section 2.7.   Final analysis of data is awaited; preliminary review of data is described here. 
Preliminary data from the 9 patients from cohort 1 indicated a potential increase in arterial oxygen 
saturation levels, and importantly no evidence of consumption of fibrinogen nor a decrease in 
plasminogen levels was observed unlike the changes associated with intravenous administration.  
Protocol v8.0 has been prepared at the start of the second UK surge. 30 patients will be recruited to this 
second cohort, with a minimum recruitment of 10 IMV patients and minimum of 10 patients receiving 
non-invasive ventilation. Early initiation of treatment appears to more effective, and this may be related 
either to lower fibrin burden or higher amount of plasminogen from the plasma exudate the substrate 
for rtPA. In the NIV and standard oxygen therapy group, a loading dose of 20 mg three times a day for 2 
days followed by 20 mg twice a day is reasonable as intermittent fibrinolysis is effective. The loading 
dose potentially can help overcome the accumulation of PAI-1 early in the disease process, and is strong 
inhibitor of fibrinolysis.  
The effect on arterial oxygen saturation levels appears to be less in IMV patients and drug losses can be 
expected because of the dead space created by the ventilator circuit. To account for this, we propose 20 
mg every 8 hrs in patients receiving IMV.  
Although the pilot study has used nebulisation every 6hrs, it is very reasonable to decrease the 
frequency to either twice a day for the non-invasive oxygen support group, or three times a day for the 
IMV group. The less frequent administration should make it easier for delivery teams.   
The maximum duration of treatment will continue to be 14 days with a provision for early termination 
as described in section 7.3.3.   


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Should a patient’s condition change such that their ventilation type is changed whilst receiving rtPA 
treatment (that is, from IMV to NIV, or vice versa) they will switch to the treatment schedule that is 
applicable to the new ventilation type. 
 
2.6 
Assessment and management of risk 
Deposition and removal of fibrin are critical processes in reducing blood loss, wound healing and repair. 
Thrombin is the final enzyme of the coagulation cascade and converts soluble fibrinogen into a fibrin 
meshwork that traps red cells and other cellular debris. The removal of the fibrin meshwork occurs via 
fibrinolysis that requires plasmin generated from zymogen plasminogen that normally circulates in 
plasma and is incorporated into the clot at the time of formation. Plasminogen is activated by two 
proteases: urokinase‐type plasminogen activator (uPA) or tissue plasminogen activator (tPA), both of 
which are primarily synthesized by the endothelium (Weisel and Litvinov 2014). Fibrinolysis is tightly 
regulated under normal physiological conditions. The catalytic activities of tPA and uPA are curtailed 
through the inhibitory proteins plasminogen activator inhibitor‐1 (PAI‐1) and α2-antiplasmin which 
inhibits free tPA but not that bound to a fibrin clot. Finally, circulating tPA has a very short half‐life of 5–
8 min (Sheehan and Tsirka 2005). 
Recombinant tPA (rtPA, alteplase) is licensed for the thrombolytic treatment of acute myocardial 
infarction, massive pulmonary embolism with haemodynamic instability and acute ischaemic stroke. The 
dosages used in these conditions are typically around 0.9 to 1.2 mg/Kg administered over 1 to 2 hrs 
(Aleplase June 2019). rtPA administered by the normal intravenous route carries a risk of bleeding and 
the extent of this risk is related to the drug dose (Lee et al. 2015). The highest number of bleeding 
events were in seen in trials involving acute ischemic stroke where the reported complications were 
symptomatic intracranial haemorrhage (6%), other major haemorrhage (2%), and angioedema (5%) 
(Miller, Simpson, and Silver 2011). Angioedema is related to the rapid generation of bradykinin from 
high-molecular-weight kininogen by plasmin (Fugate, Kalimullah, and Wijdicks 2012). The risk factors for 
symptomatic intracranial haemorrhage include age, male gender, obesity, increased stroke severity, 
diabetes, hyperglycemia, uncontrolled hypertension, combination antiplatelet use, large areas of early 
ischemic change, atrial fibrillation, congestive heart failure, and leukoaraiosis. A risk factor for 
angioedema is the use of angiotensin-converting enzyme inhibitor. 
Similarly in patients with acute myocardial infarction, the risk of intracranial haemorrhage is <1%, and in 
multivariable models the main risk factors were older age, female sex, black ethnicity, systolic blood 
pressure of 140 mm Hg or more, diastolic blood pressure of 100 mm Hg or more, history of stroke, tPA 
dose more than 1.5 mg/kg, and lower body weight(Gurwitz et al. 1998). In another study where the 
dose of r-tPA was investigated major or minor haemorrhagic events were associated with the extent of 
fibrinogen breakdown, peak rtPA levels, thrombocytopenia, prolongation of the activated partial 
thromboplastin time (APTT) to more than 90 seconds, weight of 70 kg or less, female gender, and 
physical signs of cardiac decompensation (Bovill et al. 1991). The outcomes in patients with stroke and 
acute myocardial infarction, is related to both the indication for thrombolysis and potentially fatal brain 
haemorrhages. 
The other common site of bleeding is gastrointestinal bleeding, which is often not fatal and can be 
managed as per standard protocols. There has been an increasing interest in catheter directed 
thrombolysis over extended duration in venous thrombosis to decrease the risk of bleeding with 


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potential similar efficacy. In a randomised trial of pharmacomechanical-thrombolysis, in the treatment 
group major bleeding was seen in 1.7% of patients (6 of 336 treated patients) which consisted of two 
patients with gastrointestinal bleeding, two patients with retroperitoneal bleeding and two patients 
with bleeding at the site of the procedure. All patients were receiving concomitant anticoagulation 
(Vedantham et al. 2017). In another retrospective registry of around 500 patients, one intracranial 
haemorrhage was seen with the majority of major bleeding limited to the site of procedure. 
The potential for pulmonary haemorrhage has been carefully considered during the study design. This 
risk is probably related to the generation of plasmin in the alveoli, responsible for the fibrinolysis. To 
date no bleeding has been seen in anecdotal reports using 1 to 2mg/Kg of nebulised rtPA per day, over 3 
- 14 days in patients with plastic bronchitis. Plasmin generation, in addition to the amount of rtPA 
inhaled, is also related to the availability of plasminogen in the alveoli, which is limited by the amount 
that can cross the alveolar capillary barrier. Indeed this limited availability has the potential to result in 
therapeutic failure. The other factor that  may contribute to the bleeding is the use of therapeutic 
anticoagulation.  Concurrent therapeutic anticoagulation is common with the use of rtPA as it prevents  
recurrence of thrombosis. There is emerging evidence that these patients have pulmonary thrombin at 
the segmental and sub segmental , and importantly they appear to a very strong prothrombotic 
tendency. It is important to note that the bleeding risk in the literature typically includes the concurrent 
use of anticoagulants. A strong prothrombotic tendency can result in relative resistance to the action of 
anticoagulants. Indeed patients presenting with elevated troponin in our organisation have been noted 
to have atypical coronary thrombosis with regards extent and clot burden .  
We hypothesise that this risk will be much reduced if administered by inhalation. This is because 
relatively little of the drug is expected to cross the alveolar-capillary interface and enter the 
bloodstream. The doses being used in this study are lower than that used in studies for myocardial 
infarction and stroke and similar to that used in venous thrombosis. Additionally, as the absorption rate 
is slow, the rapid inactivation of rtPA in the circulation will prevent accumulation to the level normally 
associated with bleeding. The background risk of pulmonary bleeding in COVID-19 pneumonia is low, 
with this symptom reported in only 5% of cases (Huang et al. 2020). The bleeding risk is accurately 
predicted from the plasma fibrinogen level which provides a measure of the rtPA absorbed. This will be 
monitored and if plasma fibrinogen levels fall to <1.0 g/L, the further dosing of rtPA in that patient will 
be stopped. Replacement therapy, either in the form of fibrinogen concentrate or cryoprecipitate will be 
administered to return fibrinogen to >1.0 g/L. 
The table below summarises the risks, frequencies and mitigations of the IMP(s) and NIMP(s):  
Name of IMP(s)  
Potential risk 
Risk Frequency 
Risk Management 
rtPA (alteplase) 
Pulmonary haemorrhage  
Uncommon when 
administered 
intravenously, 
unknown when 
administered by 
ventilation 
Stop rtPA nebulisation for 
this patient, and administer 
fibrinogen concentrate or 
cryoprecipitate if fibrinogen 
is < 1.0 g/L, and other 
supportive treatment 
including IV tranexamic 
acid.  May trigger stopping 
rule for trial if considered a 


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major bleed by ISTH criteria 
(see section 7.10) 
rtPA (alteplase) 
Minor bleeding 
particularly traumatic – 
suction through 
endotracheal tube or at 
site of venepuncture  
Common when 
administered 
intravenously, 
unknown when 
administered by 
ventilation 
Supportive treatment, and 
use caution during suction  
rtPA (alteplase) 
hypersensitivity reactions 
(e.g. rash, urticaria, 
bronchospasm, angio-
oedema, hypotension, 
shock)* 
Rare when 
administered 
intravenously, 
unknown when 
administered by 
ventilation 
Anti- allergic medications  
rtPA (alteplase) 
serious anaphylaxis 
very rare when 
administered 
intravenously, 
unknown when 
administered by 
ventilation 
Treat per local protocols 
and stop further 
administration of rtPA for 
this patient.   
rtPA (alteplase) 
Major Bleeding including 
intracerebral 
haemorrhage  
Common when 
administered 
intravenously, 
unknown when 
administered by 
ventilation.  
Rapid absorption is 
required to achieve high 
enough levels to result in 
bleeding. the plasma half-
life is 5 minutes. 
Stop rtPA nebulisation for 
that patient, and administer 
fibrinogen concentrate or 
cryoprecipitate if fibrinogen 
is < 1.0 g/L. May trigger 
stopping rule for trial (see 
section 7.10) 
 
The table below summarise the risks and mitigations of all test above standard care that are being 
performed in a table: 
Intervention  
Potential risk 
Risk Management 
Additional blood 
samples  
Related to venepuncture, which is 
done as standard of care.  
Press on the site of blood tests 
 


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2.7 
Safety data from cohort 1  
Preliminary data from the 9 treatment arm patients is summarised below (this data is preliminary data 
and is not the final analysis): 
All bleed events were identified as adverse events of special interest and evaluated for severity (mild, 
moderate and severe as described in section 8.3.1)  and causality. In addition, the ISTH (International 
Society of Haemostasis and Thrombosis) classification for bleed severity as used for anticoagulation 
studies was implemented.  
In total, 7 bleeding events were identified. A bleed was considered not related to alteplase if the event 
happened 30 hrs after the last dose of treatment, alteplase exposure time period. Six of the seven 
bleeds were beyond alteplase exposure time period, and one was within the time period.   
One bleed each met the criteria for ISTH major and minor bleed. Five bleeds did not meet the ISTH 
criteria for minor or major bleeds.  The severity of bleeds as assessed for adverse event reporting 
resulted in five bleeds being categorised as mild and two events as moderate.  
The severity assessment as used for AE reporting was used to further describe the bleeds.   
2.7.1 
Mild bleeds  
These bleeds did not require any intervention, and neither did they meet the criteria for minor or major 
ISTH bleeding events. All patients were on therapeutic anticoagulation, and no intervention was 
required.  
• 
Two mild bleeds were due to collection of blood around the site of insertion of the jugular line 
under mepore transparent dressing. Both were precipitated by proning of patients. One of the 
bleed around the insertion site was within alteplase exposure time window and was considered 
unrelated to use of alteplase.    
• 
One mild bleed in from the oral cavity followed an injury from a tracheal tube bitten by the 
patient and presented as blood-streaked saliva.   
• 
One mild bleed presented as blood-streaked thick secretions on one occasion.  
• 
One mild event was due to bleeding from the tracheostomy site along with a small clot in the 
tube.  
2.7.2 
Moderate bleeds 
• 
Two moderate bleeds were noted during the trial, both of which required interruption of 
anticoagulation and additional interventions. Both were outside the alteplase exposure time 
period.   
• 
One moderate bleed was gastrointestinal bleed secondary to ulceration and met the criteria for 
major bleed as per ISTH classification. In addition to the interruption of anticoagulation, the 
patient required red cell transfusion and endoscopy.   
• 
One moderate bleed for two days was intermittent bleeding around the tracheostomy site, that 
increased over time. Interruption of anticoagulation was initiated on the second day of the 
bleed, and the patient also required intravenous tranexamic acid and local measures.  


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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2.7.3 
Lab safety data  
Trial stopping criteria included a decrease in fibrinogen to < 1.5gm/L at any time, and more than 50% 
decrease over 24hrs in the first couple of patients. No patient had <1.5gm/L during cohort 1, and 
notably, no reductions of fibrinogen as described above were seen during the treatment period and 
48hrs after the last dose.  
2.7.4 
Exploratory biomarkers for fibrinolytic pathway  
The plasma fibrinolytic pathway was evaluated through measurement of plasminogen, the precursor of 
plasmin and the alpha-2 antiplasmin its major inhibitor, and these were within normal limits. 
Preliminary analysis of the tPA data show no changes were seen during tPA administration. Similarly, no 
changes in concentration of tPA antigen, PAI-1 antigen and tPA-PAI-1 complexes were seen. The 
abnormalities were consistent with COVID -19 with no evidence of any significant absorption of tPA or 
activation of the fibrinolytic pathway.  
 
baseline  
day 3  
day 5  
day 
13 
Median  
10% - 
90%  
Median  
10% - 
90%  
Median  
10% - 90%  
No. of patients  
9 
9 
8 
2 
Plasminogen 
activity IU/dL 
108 63.5 - 
124.7 
111 36.2 - 
127.8 
97.5 76.5 - 
130 
70 -
112 
Alpha 2 
antiplasmin IU/dL 
112.5 103.6 - 
129 .4 
116 104.6 - 
128 
119 106.1 - 
139 
112 - 
116 
PAI -1 antigen 
ng/mL 
41 31.6 - 
58.4 
39 28 - 
54.2 
38 26.8 - 
92.2 
25 -
35 
tPA antigen 
ng/mL 
16.5 7.8 - 
37.2 
14 9 - 23 
14 11.1 - 
40 
26 - 
10 
 
In accordance with the MRC/DH/MHRA Joint Project Risk-adapted Approaches to the Management of 
Clinical Trials of Investigational Medicinal Products, this trial is categorised as: 
• 
Type B = Somewhat higher than the risk of standard medical care 
3 
Objectives 
3.1 
Primary Objectives 
1. Efficacy: Investigate the potential for efficacy of nebulised rtPA in patients presenting with 
severe COVID-19 requiring IMV or non invasive support with NIV OR continuous positive airway 
pressure (CPAP) OR high flow oxygen OR standard oxygen therapy  
2. Safety: Evaluate the safety of nebulised rtPA treatment. 
3.2 
Secondary Objectives 
1. Investigate the impact on patient’s clinical status over time using the WHO ordinal scale of clinical 
improvement. 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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2. Investigate the effect of nebulised rtPA on other respiratory markers (such as lung compliance) and 
organ dysfunction   
3. Investigate the impact on in hospital mortality and resource utilisation  
3.3 
Exploratory Objectives 
1. Changes to coagulation and inflammatory markers concerning intervention and response to 
treatment. 
4 
Trial design 
4.1 
Overall design 
This is a phase II, open label, single centre, uncontrolled, repeated dose, pilot trial of nebulised rtPA in 
patients with COVID-19 ARDS. 
The study will recruit patients requiring either IMV or non-invasive oxygen support. Eligible patients (or 
if patients lack capacity, their legal representative) will be provided with an information sheet and 
informed consent will be sought (see section 6.3). Eligibility will be assessed via routine clinical 
assessments, which may have been done prior to consent. The only exceptions are a pregnancy test 
(blood or urine), and possibly any assessments listed in section 7.1 that were not done as per routine 
care. These must be done following consent, and all screening assessments must have been done during 
the 24-hour period before dosing with rtPA. 
The study will run two cohorts sequentially. In cohort 1, 9 consented patients received nebulised rtPA in 
addition to SOC. 6 patients were receiving IMV and 3 were receiving non invasive support with NIV or 
CPAP or high flow oxygen or standard oxygen therapy. As an observational arm, matched historical 
controls who received standard of care were also recruited at a ratio of 2 controls to every 1 treatment 
arm patient, resulting in 18 historical controls. Originally, the study aimed to recruit 12 patients with 6 
on each ventilation type (IMV and non-invasive oxygen support). This would have resulted in 24 
historical controls. After the first wave of COVID-19 cases decreased in August 2020 in the UK it became 
difficult to continue recruitment, so recruitment closed for cohort 1.  
With a second surge underway in early 2021, cohort 2 will aim to recruit more patients during this 
period to provide more data on the safety of rtPA. Based on the analysis of cohort 1, fewer timepoints 
will be collected (see sections 7.1.2 and 7.5.2), which will allow for more rapid recruitment while at the 
same time not compromising safety monitoring. A more flexible dosing regimen for rtPA will be utilised 
which is described in section 7.3.2. 30 patients will be recruited in total, with an aim to recruit a 
minimum of 10 IMV patients and 10 patients on non-invasive oxygen support. 
A statistically powered randomised controlled trial (RCT) would be ideal to define the magnitude of 
benefit and impact on overall survival and is the typical design in patients with ARDS. Although there is 
clinical data on the safety of nebulised rtPA, but there is no clinical data in this clinical condition to 
facilitate a sample size calculation. There is data from a small RCT that has used another fibrinolytic 
agent, but the doses are not comparable(Abdelaal Ahmed Mahmoud et al. 2020). When a patient is 
randomised to receive no treatment, this precludes participation in other interventional studies which 
might decrease the risk of mortality. The most recent figures suggest a 50% mortality in patients 
receiving IMV there is 50% mortality. (https://www.icnarc.org/About/Latest-News/2020/04/04/Report-


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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On-2249-Patients-Critically-Ill-With-Covid-19) Currently there is a concerted effort to introduce multiple 
therapies based on our understanding of the pathophysiologic basis of disorder. Further, if this pilot 
study shows significant effect in a subset of patients, there would be justification to progress to a 
statistically powered RCT. We would progress to a RCT only after submission of a substantial 
amendment to this trial protocol or submission of a new clinical trial application. In the event of major 
adverse drug reactions or minor improvement in the oxygenation as assessed by PaO2/FiO2, there are 
minimal gains to be had with a larger randomised control study. 
For patients in the rtPA group, 10 mg of rtPA dissolved in 5 ml of diluent will be given every 6 hrs for a 
maximum of 14 treatment days.  Efficacy will be assessed by the monitoring of arterial oxygen 
saturation. 
Safety monitoring will be performed by assessment of the incidence and severity of bleeding events, 
and by the monitoring of plasma fibrinogen levels and routine coagulation parameters. Additional 
samples will be taken for exploratory assessment of potential biomarkers, including (but not restricted 
to) PAI- 1, alpha 2 antiplasmin and a range of inflammatory cytokines and coagulation proteins. All other 
monitoring will be done as per SOC. From the end of the treatment phase  both groups will be followed 
up in accordance with SOC. 
Although there is extensive experience with the use of nebulised rtPA in the context of the underlying 
inflammation, safety measures been included. A gap of 24hrs will be maintained between first and 
second patient. At 24hrs if patient 1 has no evidence of major pulmonary bleeding suggesting 
exaggerated alveolar fibrinolysis and no evidence of fibrinogen reduction of more than 50%, suggestive 
of systemic absorption a second patient will be dosed. Both patients will be evaluated for 72 hrs. If no 
major bleeding is noticed and fibrinogen levels have not decreased more than 50% in either patient (in 
line with stopping criteria – section 7.10), and no other stopping criteria have been met, then the rest of 
the cohort can be recruited after the review of the safety data by the trial management group 
comprised of the investigators. If there are any concerns with the data this will be referred to the DMC 
for review. If the safety profile is acceptable, dosing of the third and subsequent patients in the rtPA 
group will resume with no required interval between patients. 
Efficacy will be described as a change in PaO2/FiO2 (or SaO2/FiO2) ratio assessed daily, end of treatment 
and 5 days post treatment in relation to baseline.  A Data Monitoring Committee (DMC) will be set-up to 
review safety data within the trial along with the final study results and advise on progressing from a 
pilot study to a randomised control trial based on the safety and efficacy data that is collected. The DMC 
will receive weekly reports on bleeding complications, both major and minor along with fibrinogen 
levels. If at any time a patient has major pulmonary bleeding, further dosing of patients will be stopped 
and an adhoc DMC review will be arranged before resuming dosing (see section 12.2 Data Monitoring 
Committee). 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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4.2 
Schematic diagram(s) of overall trial design 
 
5 
Investigational Medicinal Products and Non-Investigational 
Medicinal Products 
5.1 
Name and description of IMP(s) - rtPA, alteplase (Actilyse®) 
Actilyse 10 mg powder and solvent for solution for injection and infusion or Actilyse 20 mg powder and 
solvent for solution for injection and infusion. 
1 vial with powder contains: 
• 
10 mg rtPA (alteplase) (corresponding to 5,800,000 IU) or 
• 
20 mg rtPA (alteplase) (corresponding to 11,600,000 IU) 
Alteplase is produced by recombinant DNA technique using a Chinese hamster ovary cell-line. 
The powder is presented as a colourless to pale yellow lyophilizate cake. The reconstituted preparation 
is a clear and colourless to pale yellow solution. 
The reconstitution solvent is water for injections. 
Actilyse is authorised as an intravenous formulation: 
• 
Thrombolytic treatment in acute myocardial infarction 
• 
Thrombolytic treatment in acute massive pulmonary embolism with haemodynamic 
instability 
• 
Fibrinolytic treatment of acute ischaemic stroke 
Actilyse is not authorised for administration via a nebulised route. 
5.2 
Source of IMP, Manufacture, Distribution and Storage 
Actilyse will be supplied from existing hospital stock and replenished as required free-of-charge directly 
to the pharmacy by the manufacturer (Boehringer Ingelheim Ltd). 
In this study Actilyse will be administered by nebulisation rather than intravenous injection. The 
rationale for selecting this route of administration is described in section 3.3. 
Identify matched 
historical controls 
(2:1 ratio) 
Patient 
admitted for 
COVID-19 
ARDS 
Begin 
cohort 1: 
ICU 1
st 
Patient 
ICU 2
nd 
Patient 
TMG 
Review 
No safety 
concerns 
Obtain data for 
historical controls 
Wait 24 hours after 
1
st dose 
No major bleeding 
events or safety 
concerns 
Wait 72 hours after 1
st 
dose of 2
nd patient 
No major bleeding 
events or safety 
concerns 
Begin recruitment 
into cohort 2 (n=30) 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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A proof-of-concept nebulisation study (personal communication from H. Wachtel, Boehringer 
Ingelheim) indicated that at the planned concentration of 2 mg/mL nebulisation produces a fine particle 
fraction of approximately 60%. This means that, in principle, 60% of the dose is able to reach the deep 
lungs. 
The potential irritant effect of nebulised rtPA (with excipients: arginine, phosphoric acid for pH-
adjustment, polysorbate 80) on the lungs is not known, but experience within the critical care setting 
indicates that risk will be low and manageable. 
5.3 
Storage and handling of IMP(s) at site 
All IMP aspects of the trial at participating sites are the responsibility of the PI, who may delegate this 
duty to the local pharmacist or other appropriately trained personnel. The delegation of duties must be 
recorded on the Staff Signature and Delegation of Tasks. 
Storage must be in the original package in order to protect from light. Do not store above 25 °C. 
Actilyse will be reconstituted with water for injection in accordance with the instructions in the 
Summary of Drug Arrangements (SoDa) for the trial.  
The reconstituted solution has been demonstrated to be stable for 24 hours at 2 °C – 8 °C and for 8 
hours at 25 °C. From a microbiological perspective, the product should be used immediately after 
reconstitution. If not used immediately, in-use storage times and conditions prior to use are the 
responsibility of the user and would normally not be longer than 24 hours at 2 to 8°C. 
For cohort 1, 10 mg of Actilyse reconstituted in 5ml of diluent provided will be used in an ultrasonic 
nebuliser available in the hospital. For cohort 2, 20mg will be reconstituted as directed in the SoDa, and 
will be used in an ultrasonic nebuliser available in the hospital.  
Detailed instructions are contained in the summary of drug arrangements. 
5.4 
Accountability of IMP(s) 
IMP will be supplied by Boehringer Ingelheim Ltd to the hospital site. Details of initial supply and 
resupply will be detailed in the summary of drug arrangements. 
The IMP Drug Accountability Log must be completed to record each dose of IMP dispensed for each trial 
participant. This log must be retained in the relevant section of the Pharmacy Site File, and a copy must 
be submitted to the sponsor upon request. It is the responsibility of the Pharmacy Lead to maintain drug 
accountability records. 
All used/unused IMPs dispensed will be destroyed and not returned to pharmacy. 
Detailed instructions are contained in the summary of drug arrangements. 
5.5 
Concomitant medication 
Concomitant medications will be recorded in the Participant’s medical records/CRF. 
No formal interaction studies with Actilyse and medicinal products commonly administered in patients 
with acute myocardial infarction have been performed. 
Drugs affecting coagulation/platelet function 
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 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
Sponsor code: 132151 
Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
Page 34 of 71 
 
administered (before, during or within the first 24 hours after treatment with Actilyse). This is 
particularly the case with intravenous administration; however, bleeding risk associated with nebulised 
administration, where absorption and systemic exposure is expected to be low, is not known. 
There are now reports that standard prophylactic doses of anticoagulation have a high failure rate in 
COVID-19 pneumonia. As a result higher than standard doses are being used (including therapeutic 
anticoagulation) in these patients(Klok et al. 2020; Bikdeli et al. 2020) As the plasma concentration of 
rtPA is expected to be relatively low after nebulised administration, we do not expect concomitant 
administration of higher than standard doses of thromboprophylaxis to result in higher bleeding rates 
during this study. Use of anticoagulation at higher than standard doses is therefore not an exclusion 
criteria. 
ACE inhibitors 
Concomitant treatment with ACE inhibitors may enhance the risk of suffering a hypersensitivity reaction 
(see section 4.4). 
Concomitant use of GPIIb/IIIa antagonists increases the risk of bleeding. 
Where bronchoscopy is undertaken, diagnostic or therapeutic, it is reasonable to use topical tPA at a 
dose not exceeding 10 mg for the session.  
5.6 
Post-trial IMP arrangements 
There will be no arrangements to provide IMP to patients after trial participation as patients would have 
either recovered or succumbed to their disease. 
6 
Selection of Participants 
6.1 
Eligibility of trial participants  
6.1.1 
Trial participant inclusion criteria – Cohort 1 and Cohort 2  
All patients must meet the following criteria (ward or ICU based): 
1. Patients with COVID-19 (confirmed by PCR  or radiologically) 
2. ≥16 years  
3. Willing and able to provide written informed consent or where patient doesn’t have capacity, 
consent obtained from a legal representative if available 
4. Patients on mechanical ventilation must meet the following criteria: 
a. PaO2/FiO2 of ≤ 300 
b. Intubated > 6 hrs  
5. Patients not intubated must meet the following criteria: 
a. PaO2/FiO2 ≤ 300 or equivalent imputed by non-linear calculation from SpO2/FiO2 (see 
look-up table in appendices) 
b. In-patient >6 hours and being actively treated 
c. On  support with non-invasive ventilation OR  continuous positive airway pressure (CPAP) 
OR high flow OR standard oxygen therapy 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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6.1.2 
Trial participant exclusion criteria – Cohort 1  
None of the following criteria must apply: 
1. Females who are pregnant 
2. Concurrent involvement in another experimental investigational medicinal product 
3. Known allergies to the IMP or excipients of IMP 
4. A pre-existing bleeding disorder (e.g. severe haemophilia) with no definitive treatment 
5. Pre-existing severe cardiopulmonary disease (e.g. incurable lung cancer, severe chronic 
obstructive lung disease, cardiomyopathy, heart failure or impaired contractility <estimated 
40% LVEF or RVEF) 
6. Fibrinogen < 2.0 g/L at time of screening 
7. Patients considered inappropriate for active treatment ( e.g. being considered for palliative 
care) 
8. Patients with active bleeding in the preceding 7 days 
9. Patients who in the opinion of the investigator are not suitable 
6.1.3 
Eligibility of matched historical controls 
Historical controls with COVID-19 (confirmed either via PCR or radiologically) will be selected on the 
basis of the matching criteria stated in section 10.4.2 only.  Historical controls must be aged 16 years or 
over. 
6.1.4 
Trial participant exclusion criteria – Cohort 2  
1. Females who are pregnant 
2. Known allergies to the IMP or excipients of IMP 
3. Fibrinogen < 1.5 g/L at time of screening 
4. Patients considered inappropriate for active treatment (e.g. being considered for palliative care) 
5. Patients who in the opinion of the investigator are not suitable 
 
6.2 
Recruitment 
Patients will be identified by the clinical team involved in the patient’s routine care in either wards or 
ICU. As described in section 6.3 patients will either go through the consenting process themselves or, if 
they do not have the capacity, consent will be obtained from their legal representative. 
All trial related activities will ideally be carried out by site staff delegated to the trial. As nebulisation will 
need to be regularly undertaken over 14 days, trained nurses who are used to nebulisation will be 
permitted to undertake this procedure, with two nurses double checking the dilution and amount of 
rtPA being reconstituted. 
 The only procedures that may be performed in advance of written informed consent being taken are 
those that would have been performed on all participants in the same situation as routine clinical 
practice. 
Participant recruitment at a site will only commence when the trial has been issued with the ‘Open to 
Recruitment’ letter by the sponsor. 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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Protocol Version 10.0 dated 2 Feb 2021 
 
 
 
 
 
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6.3 
Informed consent procedure 
It is the responsibility of the Investigator, or a person delegated by the Investigator to obtain written 
informed consent from each participant prior to participation in the trial, following adequate 
explanation of the aims, methods, anticipated benefits and potential hazards of the trial. 
The person taking consent will be GCP-trained, suitably qualified and experienced, and will have been 
delegated this duty by the CI/ PI on the Staff Signature and Delegation of Tasks. 
“Adequate time” must be given for consideration by the participant/legal representative before taking 
part. Consent will ideally be sought at least 24 hours after being given the study documentation. 
However, given the need to urgently begin treatment in many of these patients, it may not be justifiable 
to seek consent at least 24 hours after providing the study documentation, as the patient’s condition 
may deteriorate over this time. In these instances, consent may be sought less than 24 hours after 
providing the study documentation. It must be recorded in the medical notes when the participant 
information sheet (PIS) has been given to the participant/legal representative. 
The Investigator or designee will explain that participants are under no obligation to enter the trial and 
that they can withdraw at any time during the trial, without having to give a reason. 
However, if the patient lacks capacity to give consent due to the severity of their medical condition (e.g. 
acute respiratory failure or need for immediate ventilation), then consent may be obtained from a 
relative acting as the patient’s personal legal representative. If there is no personal legal representative 
available (which due to restrictions of visitors to the hospital is likely) a professional legal representative 
(independent clinical responsible for the patients’ medical care or other person appointed by the 
hospital) will be asked to provide consent on behalf of the patient. Further consent will then be sought 
from a personal legal representative if one becomes available or from the patient if they recover 
sufficiently. If the patient refuses consent after recovery further assessments will be stopped.  
In the event that a patient loses capacity as a result of treatment for their primary medical condition 
while on the study, an appropriate consultee will be sought as described above. No clinical trial 
procedures will be conducted prior to the participant/legal representative giving consent by signing the 
Consent form. Consent will not denote enrolment into trial. 
A copy of the signed informed consent form will be given to the participant. The original signed form will 
be retained in the trial file at site and a copy placed in the medical notes. 
The PIS and consent form will be reviewed and updated if necessary throughout the trial (e.g. where 
new safety information becomes available) and participants will be re-consented as appropriate. 
6.4 
Consent for historical standard of care controls (cohort 1 only) 
 Patients will be identified as historical controls by reviewing site admissions for cases that match 
treatment arm patients according to the characteristics listed in section 10.4.2. Historical controls can 
be identified from either site (i.e. Royal Free Hospital treatment arm patients can be matched with 
historical controls from Barnet Hospital). There is no limit to how far back in time the search for 
historical controls will go.  
Initial collection of data will be done without patient consent under The Health Service (Control of 
Patient Information) Regulations 2002 and the notices issued by the secretary of state to healthcare 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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organisations, GP’s, local authorities and arm’s length bodies to allow sharing of  information to support 
efforts against coronavirus (COVID-19). 
These notices are currently due to end on 31 March 2021. Consent will not be sought from historical 
controls because the use of their personal identifiable information is covered by this notice.     
The Royal Free trial team will retain a subject ID log that links the patient’s trial identification number 
with their full name and hospital number. The log will indicate whether a patient is alive or dead at the 
time of enrolment.  Because the both hospitals are part of the same Trust (Royal Free London NHS 
Trust), as per routine practice, the study team at the Royal Free Hospital  also have access to the medical 
records of Barnet Hospital patients. The patient’s identifiable information will not be recorded on the 
study database or case report forms, they will only be referred to by their unique trial ID number.  
7 
Trial procedures 
7.1 
Pre-treatment Assessments  
7.1.1 
Screening – Cohorts 1 and 2 
The results from the following routine procedures may be used to assess the participant’s eligibility. 
Where routine results are not available at the time of consent, the procedure(s) will be carried out after 
consent. All must have been done within 24 hours prior undertaken as part of their standard care before 
their first dose of rtPA. These will also constitute the baseline assessments for cohort 2.  
Patients receiving rtPA and SOC will undertake all assessments: 
• 
Informed consent 
• 
Demographics 
• 
Review of medical history (including inpatient history and COVID-19 diagnosis – see section 7.7).  
• 
Eligibility review 
• 
Pregnancy test (blood or urine) for female participants of childbearing potential  
• 
Concomitant medications 
• 
Directed physical examination including vital signs (heart rate, temperature, blood pressure, and 
body weight) 
• 
Documentation of respiratory status: Respiratory status of patients is monitored hourly and data 
points will be extracted for every four hour where available, but one data point is required as a 
minimum. The time of measurement should be documented. The variables include: 
o Details of oxygen and respiratory support including the type of device and Fi02 
Respiratory rate 
o PaO2 
o For IMV patients, details of ventilation including but not exclusively the below 
▪ 
Tidal volume (in mL) 
▪ 
Full sedation or not – not for cohort 2  
▪ 
Paralysis or not – not for cohort 2 
▪ 
Patient position – not for cohort 2 
▪ 
Peak inspiratory pressure (for computing compliance)  
▪ 
PEEP (for computing compliance ) 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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o For non-invasive support patients: 
▪ 
SpO2 
▪ 
Oxygen flow rate 
▪ 
National Early Warning Score (NEWS 2) – not for cohort 2 
o For CPAP patients 
▪ 
Positive end expiry pressure (PEEP) 
• 
Local blood testing as per section 7.6.2 
• 
Research blood sampling as per section 7.6.2 
• 
Clinical status as assessed by 7-point ordinal scale (see section 7.7) 
• 
Sequential Organ Failure Assessment (SOFA) score (see section 7.7) 
• 
Glasgow coma scale (see section 7.7) 
7.1.2 
Baseline assessments (for duration of 3 days prior to treatment) – Cohort 1 
only  
Screening assessments will be combined with daily routine assessments taken from the 3 days prior to 
dosing to establish a baseline: 
• 
Documentation of respiratory status: Respiratory status of patients is monitored hourly and data 
points will be extracted for every four hours where available, but one data point is required as a 
minimum. The time of measurement should be documented. The variables include: 
o Mode of Ventilation 
o Oxygenation index 
o Respiratory rate 
o PaO2 
o 
SpO2 
o 
FiO2 
o For IMV patients, details of ventilation including but not exclusively the below 
▪ 
Tidal volume (in mL) 
▪ 
Full sedation or not 
▪ 
Paralysis or not. 
▪ 
Patient position  
▪ 
Peak inspiratory pressure (collected for computing compliance) 
▪ 
PEEP (collected for computing compliance)  
o For non-invasive oxygen support patients: 
▪ 
National Early Warning Score (NEWS 2) 
o For CPAP patients 
▪ 
Positive end expiry pressure (PEEP) 
• 
Clinical status as assessed by 7-point clinical ordinal scale (see section 7.7) 
• 
Sequential Organ Failure Assessment (SOFA) score (see section 7.7) 
• 
Glasgow coma scale (see section 7.7) 
7.2 
Registration Procedures 
Participant registration will be undertaken by the coordinating trial team at site on a trial specific 
registration form. 


Short title: Nebulised rtPA for ARDS due to COVID-19 – The PACA trial 
 
 
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Participants are considered to be enrolled into the trial following: consent, pre-treatment assessments 
(see section 7.1), confirmation of eligibility, completion of the registration process, allocation of the 
participant trial number. 
7.3 
Treatment Schedule for nebulisation 
Patients in the rtPA and SOC group will receive the first dose as soon as possible after enrolment, and in 
accordance with the dosing schedule below. The time of initiation of the first dose will be recorded and 
the timing of the initiation of subsequent doses will be relative to this time. 10mg rtPA will be 
administered by nebulisation every 6 hours. Treatment with rtPA will be administered at a dose of 10 
mg every 6hrs for a minimum of 5 days initially and a maximum of 14 treatment days overall. 
7.3.1 
Cohort 1 
rtPA treatment schedule 
Study 
Day 
Time 
(hours) 
Dose 
No. 
Dose 
(mg) 
Daily 
Dose 
(mg) 
1 
0 (± 1hr)  
1 
10 
40 
6 (± 1hr) 
2 
10 
12 (± 
1hr) 
3 
10 
18 (± 
1hr) 
4 
10 
2 
24 (± 
1hr) 
5 
10 
40 
30 (± 
1hr) 
6 
10 
36 (± 
1hr) 
7 
10 
42 (± 
1hr) 
8 
10 
3 
48 (± 
1hr) 
9 
10 
40 
54 (± 
1hr) 
10 
10 
60 (± 
1hr) 
11 
10 
66 (± 
1hr) 
12 
10 


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4 
72 (± 
1hr) 
13 
10 
40 
78 (± 
1hr) 
14 
10 
84 (± 
1hr) 
15 
10 
90 (± 
1hr) 
16 
10 
5 
96 (± 
1hr) 
17 
10 
40 
102 (± 
1hr) 
18 
10 
108 (± 
1hr) 
19 
10 
114 (± 
1hr) 
20 
10 
6 to 14 
Patients will continue to be dosed every 6 hours (± 1hr) from the time 
of the first dose, resulting in 56 doses in total being administered.  
 
The duration of therapy can be extended from five days to a maximum of 14 treatment days in total. 
This decision can only be made in the absence of any dose-limiting toxicity. Treatment cannot be given 
for more than 56 doses. 
7.3.2 
Cohort 2  
The treatment schedule will differ depending on the ventilation type 
rtPA treatment schedule for IMV patients 
Study 
Day 
Time 
(hours) 
Dose 
No. 
Dose 
(mg) 
Daily 
Dose 
(mg) 
1 
0   
1 
20 
60 
8 (± 2hrs) 
2 
20 
16 (± 
2hrs) 
3 
20 
2 
24 (± 
2hrs) 
4 
20 
60 
32 (± 
2hrs) 
5 
20 


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40 (± 
2hrs) 
6 
20 
3 
48 (± 
2hrs) 
7 
20 
60 
56 (± 
2hrs) 
8 
20 
64 (± 
2hrs) 
9 
20 
4 
72 (± 
2hrs) 
10 
20 
60 
80 (± 
2hrs) 
11 
20 
88 (± 
2hrs) 
12 
20 
5 to 14 
Patients will continue to be dosed every 8 hours (± 2hrs) from the time 
of the first dose, resulting in 42 doses in total being administered.  
 
rtPA treatment schedule for patients on non-invasive oxygen support 
This dosing schedule will consist of a higher loading dose for the first 2 days.  
Study 
Day 
Time 
(hours) 
Dose 
No. 
Dose 
(mg) 
Daily 
Dose 
(mg) 
1 
0   
1 
20 
60 
8 (± 2hrs) 
2 
20 
16 (± 
2hrs) 
3 
20 
2 
24 (± 
2hrs) 
4 
20 
60 
32 (± 
2hrs) 
5 
20 
40 (± 
2hrs) 
6 
20 
3 
48 (± 
4hrs) 
7 
20 
40 
60 (± 
4hrs) 
8 
20 


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4 
72 (± 
4hrs) 
9 
20 
40 
 
84 (± 4hr) 
10 
20 
5 
96 (± 4hr) 
11 
20 
40 
108 (± 
4hr) 
12 
20 
6 to 14 
Patients will continue to be dosed every 12 hours (± 4hrs) from the time 
of the first dose, resulting in 30 doses in total being administered.  
 
7.3.3 
Treatment discontinuation 
Treatment may be stopped at any point in a patient if any of the following rules are met: 
• 
If plasma fibrinogen levels fall to <1.0 g/L, the further dosing of rtPA in the patient will be 
stopped (see section 7.4).  
• 
Patient maintains saturation on room air that is either normal or normal for them for 48 hours 
as assessed by the investigator 
• 
A trial stopping rule has been met (see section 7.10)    
 
7.3.4 
Re-treatment with rtPA after treatment discontinuation due to improvement 
in patient saturation (as described above) 
If there is a recurrence of COVID-19 ARDS related symptoms or a worsening of PaO2/FiO2 ratio, which 
the investigator considers could be related to treatment discontinuation, treatment can be restarted. 
The decision to re-treat will be made within 5 days (120 hrs) from the last dose of treatment. Treatment 
may not be restarted more than once.  
The rationale for restarting of treatment must be documented in the notes, along with the absence of 
toxicity. The patients monitoring will done according to main treatment phase.  
 
7.3.5 
Re-treatment of patients previously consented and treated on the trial – 
Cohort 1  
For patients that have already been treated under the previous 3 day dosing schedule, if they remain 
eligible for the trial, they will be offered the option to continue treatment as above – the maximum 
number of days of extra treatment for these patients will be 11 days. Patients (or their legal 
representatives) must give consent to this re-treatment. 
The total duration of trial participation for these patients may exceed 28 days (to approximately 5-6 
weeks) to allow us to extend treatment to the maximum 14 days and to allow us to collect 5 days of post 
treatment data. We will inform the patient or their legal representative of this possibility and this will be 
documented in the patient information sheet. 
 


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7.4 
Dose Modifications 
Modifying the dose of rtPA will not be permitted. However, should a patient’s condition change such 
that their ventilation type is changed whilst receiving rtPA treatment (that is, from IMV to NIV, or vice 
versa) they will switch to the treatment schedule that is applicable to the new ventilation type. 
If plasma fibrinogen levels fall to <1.0 g/L, the further dosing of rtPA in the patient will be stopped.  
Replacement therapy, either in the form of fibrinogen concentrate or cryoprecipitate will be 
administered to return fibrinogen to >1.0 g/L. 
7.5 
Visit schedule and assessments post initiation of treatment 
7.5.1 
Cohort 1 and 2  
Any assessments that are done as per routine care do not need to be repeated.  
• 
Documentation of respiratory status: Respiratory status of patients is monitored hourly and data 
points will be extracted for every four hours where available until 5 days after the last dose for 
cohort 1, but one data point per day is required as a minimum. For cohort 2 data will be extracted 
once a day coinciding with the worst PF ratio. As PF ratio is the primary endpoint, in addition to the 
worst PF ratio over the preceding 24 hrs up to a maximum of 6 data points spread across 24 hrs that 
represents patients clinical status will be extracted. Similar to cohort 1 respiratory data will be 
collected until 5 days after the last dose.  After this period, respiratory status is to be documented 
weekly (+/- 1 day) until discharge, death, or 28 days whichever occurs first. The time of 
measurement should be documented. 
• 
The variables include: 
o Details of oxygen and respiratory support including the type of device, FiO2 Respiratory 
rate 
o PaO2 
o For IMV patients, details of ventilation including but not exclusively the below 
▪ 
Tidal volume (in mL) 
▪ 
Full sedation or not – not for cohort 2 
▪ 
Paralysis or not - – not for cohort 2. 
▪ 
Patient position - – not for cohort 2 
▪ 
Peak inspiratory pressure (collected for computing compliance) 
▪ 
PEEP (collected for computing compliance)  
o For non-invasive oxygen support patients: 
▪ 
National Early Warning Score (NEWS 2) – not for cohort 2 
▪ 
Oxygen flow rate 
▪ 
SpO2 
o For CPAP patients 
▪ 
Positive end expiry pressure (PEEP) 
The following tests are to be done at a minimum once a day until 5 days after the last dose (day 7 for 
SOC patients) in cohort 1, a minimum every other day in cohort 2, and then as required until day 28, 
discharge or death whichever occurs first. 
• 
Local blood testing as per section 7.6.2 


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The following tests will be done once at 48 h (+/-24 h) and 96 h (+/-24h) after start of treatment  and 
then twice weekly while on treatment until 5 days after stopping treatment. After this period, testing 
should be weekly (+/- 1 day) until day 28, discharge or death whichever occurs first. Twice weekly 
testing should be evenly spaced across weekdays if possible.  
• 
Research blood sampling as per section 7.6.2 
The following tests are to be done daily until 5 days after the last dose (day 7 for SOC patients), and then 
weekly (+/- 1 day) until day 28, discharge or death whichever occurs first: 
• 
Directed physical examination at baseline, and thereafter weekly only in cohort 2  
• 
 vital signs (heart rate, temperature, blood pressure) 
• 
Concomitant medications 
• 
Review of Adverse events and Adverse drug reactions including bleeding episodes 
The following assessments are to be done daily until 5 days after the last dose (day 7 for SOC patients) 
after the baseline assessments were done for these tests: 
• 
Clinical status as assessed by a 7-point ordinal scale 
• 
SOFA score 
• 
Glasgow coma scale 
The following end of study data will also be collected at day 28, discharge or death whichever occurs 
first: 
• 
Documentation of any Oxygenation free days in the first 28 days 
• 
Documentation of any Ventilator-free days in the first 28 days 
• 
Incidence and duration of new oxygen use, non-invasive ventilation or high flow oxygen devices 
in the first 28 days. 
• 
Documentation of incidence and duration of new mechanical ventilation use during the trial 
• 
Documentation of intensive care unit (ICU) free days in the first 28 days Death and cause of 
death (if applicable) 
• 
Documentation of any Oxygenation free days 
• 
Documentation of any Ventilator free days 
• 
At 28 days on trial (or death or discharge, whichever comes first), a single measurement will be 
taken on the 7-point ordinal scale (see section 7.74) 
7.5.2 
Cohort 1 -Data collection for matched historical controls 
The following data will be collected for matched historical controls: 
• 
Method of COVID-19 confirmation (PCR or radiological) 
• 
Matching criteria (type of oxygen support, age, severity of disease according to PaO2/FiO2 ratio, 
ethnicity, gender 
• 
COVID-19 admission history – to include PCR and CXR findings, date of admission to hospital, 
date of ICU admission, date of oxygen support and the progression of oxygen support to death or 
discharge from the study, presence or absence of pulmonary embolism and deep vein 
thrombosis. Presence of haemophagocytic syndrome will also be recorded, as this impacts on 
fibrinogen levels. 
• 
Date of admission to hospital and duration of stay 
• 
PaO2 


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• 
FiO2 
• 
Patient position  
• 
Ongoing 7-point ordinal scale 
• 
Medical history – medical history to be reviewed and relevant history as assessed by the 
clinician to be recorded in the CRF, including an ISARIC style categorisation for all the major 
organ systems  
• 
Concomitant medications 
• 
SOFA score 
• 
Glasgow coma scale 
• 
Local blood results as per section 7.6.2 
• 
Results from other standard of care assessments may also be collected if they explain the rate of 
change for the PaO2/FiO2 ratio, such as secondary infections, medication changes.  
This data will be collected where available for 28 days from the date of admission to hospital. 
7.6 
Laboratory Assessments and Procedures 
7.6.1 
Sample Collection 
The Advisory Committee of Dangerous pathogens (ACDP) in conjunction with the Health and Safety 
Executive and Public Health England have classified COVID-19 as Hazard Group 3. Work with specimens 
that may contain the virus has been given dispensation to have diagnostic work carried out at less than 
full containment level 3 conditions subject to local risk assessment. The view of ACDP is that blood 
samples from confirmed COVID-19 patients can be handled in CL2 after local risk assessment of the 
possibility of aerosol generation for each kind of test/analyser. 
7.6.2 
Summary of laboratory tests 
The following tests will be carried out at Local Laboratories as part of clinical care. 
1. Confirmatory tests for coronavirus 
2. Blood  
Haematology 
Full blood count including: haemoglobin, WBC, platelet count,  
Serum chemistry 
 
Routine clinical biochemistry including: renal, liver, bone, CRP, 
ferritin, LDH, BNP, troponin T (the last two baseline only)  
Conventional 
coagulation tests 
PT, APTT, Fibrinogen and D-dimers 
3. Research blood tests 
Procoagulants, anticoagulants, fibrinolytic proteins, fibrinolytic inhibitors, chemokines and cytokines 
Research blood samples will be analysed in one of two laboratories:  
1. Haemophilia research lab, Royal Free Hospital. 
2. Department of Biochemical Sciences, University of Surrey, Guildford 
 
Testing frequency will be presented in the schedule of assessments. 


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7.6.3 
Storage of Sample Aliquots 
Serum and plasma samples will be stored at in the Haemophilia and Thrombosis Laboratory 
Containment level 2 -80°C freezer prior to analysis and any additional analysis, the left-over aliquots will 
be stored in the Haemophilia and Thrombosis Centre Plasma Bank.  
Samples for testing by University of Surrey will be shipped in batches. At the end of the trial, and on 
completion of all analyses, any remaining blood samples and the derivatives at University of Surrey 
thereof will be returned to CI for storage in Biobank/further analysis in ethics approved study. 
Samples will be stored up to 5 years after the end of study for use in other ethically approved research 
projects. 
7.7 
Clinical Procedures and Data Collection 
7.7.1 
Directed physical examination: 
A review of relevant body systems will be conducted by a study clinician. Vital signs will also be 
undertaken; these can be done by a clinician or study nurse. Vital signs include heart rate, temperature, 
blood pressure, and (at baseline only) body weight. 
7.7.2 
Review of medical history (including inpatient history and COVID-19 
diagnosis) and document concomitant medications 
A review of the patient’s relevant medical history must be undertaken and documented in the medical 
notes and CRF. The patient’s COVID-19 diagnosis (clinical or laboratory - confirmed by PCR or 
radiologically) must also be documented. 
Inpatient history must include: 
• 
Hospital admission time 
• 
Hospital admission source (Community vs transfer from other institution) 
• 
ICU admission time 
• 
ICU admission source (ward/ED/OR/Transfer from other institution) 
• 
Planned ICU admission (yes/No) 
All over-the-counter or prescription medication, vitamins, and/or herbal supplements will be recorded. 
7.7.3 
Bleeding Episodes: 
Bleeding episodes will be documented in the medical notes and CRF, and will include: 
• 
Number 
• 
Site, date and time of onset, Aetiology (spontaneous, traumatic, etc) 
• 
Control of Anticoagulation Subcommittee of the International Society on Thrombosis and 
Haemostasis (Schulman and Kearon 2005) recommendation for major and minor bleeding 
will be implemented. 
• 
Major bleeding in non‐surgical patients is said to be present if the bleeding is 
o Fatal bleeding, and/or 
o Symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, 
intraocular, retroperitoneal, intra‐articular or pericardial, pulmonary or 
intramuscular with compartment syndrome, and/or 


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• 
Minor bleed, if any sign or symptom of haemorrhage that does not fit ISTH definition of 
major bleed and one of (requiring medical intervention by a healthcare professional) leading 
to hospitalization or increased level of care prompting a face to face (i.e., not just a 
telephone or electronic communication) evaluation 
• 
Any coagulation treatment required 
7.7.4 
7-point clinical status ordinal scale: 
The ordinal scale is an assessment of the clinical status at a given study day: 
1. Limitation of activities 
2. Hospitalized, no oxygen therapy 
3. Oxygen by mask or nasal prongs 
4. Non-invasive ventilation or high-flow oxygen 
5. Intubation and mechanical ventilation 
6. Ventilation+ additional organ support (vasopressor, RRT, ECMO) 
7. Death 
7.7.5 
Sequential Organ Failure Assessment (SOFA) score: 
The Sequential Organ Failure Assessment (SOFA) is a morbidity severity score and mortality estimation 
tool developed from a large sample of ICU patients throughout the world. The SOFA was designed to 
focus on organ dysfunction and morbidity, with less of an emphasis on mortality prediction. It requires 
the following (worst value within past 24 hours): 
• 
FiO2 
• 
PaO2 
• 
Mechanical ventilation y/n 
• 
Platelets 
• 
Bilirubin 
• 
Glasgow coma scale 
• 
Mean arterial pressure 
• 
Vasopressors y/n 
• 
Creatinine 
• 
Urine output 
This can be calculated by entering the results above into an online tool: 
https://clincalc.com/IcuMortality/SOFA.aspx 
7.7.6 
Glasgow Coma Scale: 
The Glasgow coma scale is used to assess patients in a coma. The initial score correlates with the 
severity of brain injury and prognosis. The Glasgow Coma Scale provides a score in the range 3-15; 
patients with scores of 3-8 are usually said to be in a coma. 


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Scoring is done according to the table below: 
 
1 
2 
3 
4 
5 
6 
Eye 
Does not 
open eyes 
Opens eyes in 
response to 
pain 
Opens eyes in 
response to 
voice 
Opens eyes 
spontaneously 
N/A 
N/A 
Verbal Makes no 
sounds 
Makes sounds 
Words 
Confused, 
disoriented 
Oriented, 
converses 
normally 
N/A 
Motor Makes no 
movements 
Extension to 
painful stimuli 
(decerebrate 
response) 
Abnormal 
flexion to 
painful stimuli 
(decorticate 
response) 
Flexion / 
Withdrawal to 
painful stimuli 
Localizes 
to painful 
stimuli 
Obeys 
commands 
 
Maximum score is 15 which has the best prognosis 
Minimum score is 3 which has the worst prognosis 
Scores of 8 or above have a good chance for recovery 
Scores of 3-5 are potentially fatal, especially if accompanied by fixed pupils or absent oculovestibular 
responses. 
7.7.7 
National Early Warning Score (NEWS - 2) 
The NEWS, like many existing Early Warning Score systems, is based on a simple scoring system in which 
a number is allocated to physiological measurements (Vital Signs) already routinely measured in hospital 
and recorded on the patient clinical chart. The six simple physiological parameters form the basis of the 
scoring system: 
1. Respiratory rate 
2. Oxygen saturations 
3. Temperature 
4. Systolic blood pressure 
5. Heart rate 
6. Level of consciousness. 


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Values are added to each variable using the table below: 
Clinical risk is assigned based on the total score: 
• 
Aggregate score 1-4: Low 
• 
Aggregate score 5-6: Medium 
• 
Aggregate score 7 or more: High 
Refer to https://www.activ8rlives.com/support/data-collected/cardiovascular-and-respiratory/national-
early-warning-score-news/ 
7.8 
Discontinuation/withdrawal of participants 
In consenting to participate in the trial, participants or legal representatives on their behalf are 
consenting to trial treatment, assessments, follow-up and data collection. 
7.8.1 
Discontinuation of Trial Treatment for clinical reasons 
A participant may be withdrawn from trial treatment whenever continued participation is no longer in 
the participant’s best interests, but the reasons for doing so must be recorded. 
The decision to withdraw a participant from treatment must be recorded in the CRF and medical notes, 
and the sponsor when required should be notified in writing. 
7.8.2 
Participant withdrawal from trial treatment and/or follow-up 
If a participant expresses their wish to withdraw from trial treatment or trial follow-up, sites should 
explain the importance of remaining on trial follow-up and seek permission to allow use of routine follow-
up data to be used for trial purposes.  The importance of safety follow-up should be emphasised to the 
participant in the Participant Information Sheet. 
The decision of the participant to withdraw from treatment or follow-up must be recorded in the CRF and 
medical notes. 


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The participant may withhold their reason for withdrawal however, if the participant gives a reason for 
their withdrawal, this should be recorded. 
7.8.3 
Withdrawal of Consent to Data Collection 
If a participant explicitly states, they do not wish to contribute further data to the trial their decision must 
be respected and recorded in the CRF and medical notes. 
7.9 
Replacements 
Replacement of withdrawn patients may occur at the discretion of the Investigator. 
7.10 Stopping rules 
The trial may be stopped before completion for the following reasons: 
• 
On the recommendation of the sponsor and CI 
• 
A major pulmonary bleeding that is symptomatic or fatal that occurs between start of treatment 
and 30 hours after the last dose.  
• 
Cohort 1 - A 50% or greater reduction of fibrinogen at 72 hrs (from baseline value at screening) in 
either of the first 2 patients after dosing. If we see a 50% or greater drop in fibrinogen in a patient, 
it would only be a particular safety concern in that patient if the fibrinogen were to go below 1.5 
g/L (hence why this level is stated as a reason to stop dosing in an individual patient). However, if 
we do see a 50% drop in fibrinogen from baseline values in either of the first 2 patients it might 
suggest that the dose is too high overall with secondary systemic absorption.  As such stopping 
the trial will provide us the opportunity to discuss within the TMG and IDMC to make a decision 
on the dose going forward.  A substantial amendment would be made to the MHRA and REC. 
• 
Any fatal bleeding that occurs between start of treatment and 30 hours after the last dose. 
• 
Symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, pericardial, 
pulmonary or intramuscular with compartment syndrome, that occurs between start of treatment 
and 30 hours after the last dose.  
Further enrolment into the study will be put on hold in the event of any of the stopping rules and a 
substantial amendment will be submitted to temporarily halt the trial. Whenever the study is stopped in 
addition to a review of the data by the trial management group consisting of the investigators, the  DMC 
will also be convened for review of the data to assess the risk benefit of progressing the pilot study. If, 
following an internal safety review by the DMC, the Sponsor deems it appropriate to restart the trial; 
this can only be done following approval by the MHRA and REC and along with any local approvals 
required. 
7.11 Definition of End of Trial 
The end of trial is the date of discharge, death, or day 28 of the last patient whichever is sooner. 
 
8 
Recording and reporting of adverse events and reactions 
Collection, recording and reporting of adverse events (including serious and non-serious events and 
reactions) to the sponsor will be completed according to the sponsor’s SOP (INV/S05). 


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8.1 
Definitions 
Term 
Definition 
Adverse Event (AE) 
Any untoward medical occurrence in a patient or clinical trial 
participant administered a medicinal product and which does not 
necessarily have a causal relationship with this treatment. Therefore 
an AE can be any unfavourable or unintended change in the structure 
(signs), function (symptoms) or chemistry (laboratory data) in a 
participant to whom an IMP or procedural intervention has been 
administered, including occurrences which are not necessarily caused 
by or related to that product. 
Adverse Reaction (AR) 
Any untoward and unintended response in a participant to an 
investigational medicinal product which is related to any dose 
administered to that participant. 
This includes medication errors, uses outside of protocol (including 
misuse and abuse of product) 
Serious Adverse Event 
(SAE), Serious Adverse 
Reaction (SAR) or 
Unexpected Serious 
Adverse Reaction  
Any adverse event, adverse reaction or unexpected adverse reaction, 
respectively, that: 
• 
results in death, 
• 
is life-threatening*, 
• 
requires hospitalisation or prolongation of existing 
hospitalisation**, 
• 
results in persistent or significant disability or 
incapacity, or 
• 
consists of a congenital anomaly or birth defect 
*A life- threatening event, this refers to an event in which the 
participant was at risk of death at the time of the event; it does not 
refer to an event which hypothetically might have caused death if it 
were more severe. 
** Hospitalisation is defined as an in-patient admission, regardless of 
length of stay. Hospitalisation for pre-existing conditions, including 
elective procedures do not constitute an SAE. 
Some medical events may jeopardise the subject or may require an 
intervention to prevent one of the above 
characteristics/consequences. Such important medical events should 
also be considered as serious. 
The term “severe” is often used to describe the intensity of an event 
or reaction (e.g. mild, moderate or severe) and should not be 
confused or interchanged with the term “serious”. 


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Suspected Unexpected 
Serious Adverse 
Reaction (SUSAR) 
 A serious adverse reaction, the nature, severity or outcome of which 
is not consistent with the Reference Safety Information. 
Reference Safety 
Information (RSI) 
A list of medical events that defines which reactions are expected for 
the IMP being administered to clinical trial subjects, and so do not 
require expedited reporting to the Competent Authority. It is 
contained in a specific section in the Summary of product 
characteristics (SmPC) or the Investigator Brochure (IB). 
 
8.2 
Recording adverse events 
All adverse events will be recorded in the medical records in the first instance. Non-serious AEs will not 
be collected in the CRFs for this trial, apart from the adverse events of special interest (AESI) listed 
below: 
• 
Bleeding events 
In addition, any Serious Adverse Events (SAEs) assessed as likely to be related to IMP administration will 
be collected. See section 8.4 for further details on recording and reporting of Serious Adverse Reactions 
(SARs).These AESIs and Serious Adverse Reactions (SARs) will be recorded with clinical symptoms and 
accompanied with a simple, brief description of the event, including dates as appropriate, until the 
participant completes the trial. 
8.3 
Assessments of Adverse Events 
Each adverse event will be assessed for severity, causality, seriousness and expectedness as described 
below. 
8.3.1 
Severity  
Category 
Definition 
Mild 
The adverse event does not interfere with the participant’s daily routine, and 
does not require intervention; it causes slight discomfort 
Moderate 
The adverse event interferes with some aspects of the participant’s routine, or 
requires intervention, but is not damaging to health; it causes moderate 
discomfort 
Severe 
The adverse event results in alteration, discomfort or disability which is clearly 
damaging to health 
8.3.2 
Causality 
The assessment of relationship of adverse events to the administration of IMP is a clinical decision based 
on all available information at the time of the completion of the case report form. 
The following categories will be used to define the causality of the adverse event: 
Category 
Definition 


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Related 
A causal relationship between an IMP/investigational treatment and an 
adverse event is at least a reasonable possibility, i.e., the relationship cannot 
be ruled out. 
Not related 
There is no reasonable possibility of a causal relationship between an 
IMP/investigational treatment and an adverse event. 
8.3.3 
Expectedness 
The RSI for this trial is the Investigator Brochure for Recombinant tissue plasminogen activator (rtPA) – 
alteplase. As the IMP rtPA (Actilyse) is being used outside its authorised indication and route of 
administration all SARs will be assessed as unexpected and classified as SUSARs, and will be subject to 
expedited reporting to the MHRA and REC. 
8.3.4 
Seriousness 
All events are assessed for seriousness as defined for an SAE in section 8.1. 
All SAEs and reported in line with the procedures set-out in section 8.4 apart from regulatory reporting. 
8.4 
Procedures for recording and reporting Serious Adverse Reactions 
All SAEs will be recorded in the medical records in the first instance. Only serious adverse events 
assessed as likely to be related to IMP administration, Serious Adverse Reactions (SARs), will be 
recorded in the CRF, and the sponsor’s SAE log. The SAE log will be reported to the sponsor at the end of 
the study. 
SARs will be recorded from the start of treatment until the end of the study. 
All SARs must be recorded on a SAE Reporting Form. The CI/PI or designated individual will complete the 
sponsor’s SAE form and email to the Sponsor at SAE@ucl.ac.uk immediately and no later than 24 hours 
of his / her becoming aware of the event. The Chief or Principal Investigator will respond to any SAE 
queries raised by the sponsor as soon as possible. 
 
Completed SAE forms must be sent within 24 hours of becoming 
aware of the event to the Sponsor 
Email forms to SAE@ucl.ac.uk 
8.5 
Notification of deaths 
Only deaths assessed as likely related to IMP administration will be reported to Sponsor, see section 9.4 
above. 
Category 
Definition 
Expected 
An adverse event which is consistent with the information about the IMP listed 
in the current approved Reference Safety Information (RSI) for the trial. 
Unexpected 
An adverse event which is not consistent with the information about the IMP 
listed in the current approved Reference Safety Information (RSI) for the trial. 


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8.6 
Reporting of SUSARS 
The sponsor will notify the main REC and MHRA of all SUSARs. SUSARs that are fatal or life-threatening 
must be notified to the MHRA and REC within 7 days after the sponsor has learned of them. Other 
SUSARs must be reported to the REC and MHRA within 15 days after the sponsor has learned of them. 
8.7 
Development Safety Update Reports 
The sponsor will provide the main REC and the MHRA with Development Safety Update Reports (DSUR) 
which will be written in conjunction with the trial team and the Sponsor’s office. The report will be 
submitted within 60 days of the Developmental International Birth Date (DIBD) of the trial each year 
until the trial is declared ended. 
8.8 
 Pregnancy 
Not applicable. All female patients of child bearing potential must have a confirmed negative pregnancy 
test prior to enrolment (within 24hrs) and will remain in-patients in hospital during the course of the 
study.  
8.9 
Reporting Urgent Safety Measures and other safety events 
If any urgent safety measures are taken the CI/ PI shall immediately and in any event no later than 3 
days from the date the measures are taken, give written notice to the MHRA, the relevant REC and 
Sponsor of the measures taken and the circumstances giving rise to those measures. 
8.10 Notification of Serious Breaches to GCP and/or the protocol (SPON/S15) 
A “serious breach” is a breach which is likely to effect to a significant degree – 
(a) the safety or physical or mental integrity of the participants of the trial; or 
(b) the scientific value of the trial. 
The sponsor of a clinical trial shall notify the licensing authority in writing of any serious breach of – 
(a) the conditions and principles of GCP in connection with that trial; or (b) the protocol relating to that 
trial, as amended from time to time, within 7 days of becoming aware of that breach. 
The sponsor will be notified immediately of any case where the above definition applies during the trial 
conduct phase. The sponsor’s SOP on the ‘Notification of violations, urgent safety measures and serious 
breaches’ will be followed. 
9 
Data management and quality assurance 
9.1 
Confidentiality 
All data will be handled in accordance with the UK Data Protection Act 2018. 
The Case Report Forms (CRFs) will not bear the participant’s name or other personal identifiable data. 
The participant’s initials, date of birth and trial identification number, will be used for identification and 
this will be clearly explained to the patient in the Patient information sheet. Patient consent for this will 
be sought. 


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9.2 
Data collection tools and source document identification 
Data will be collected on Trial specific case report forms (CRFs). 
Source data are contained in source documents (medical notes and laboratory reports) and must be 
accurately transcribed on to the CRF. 
It is the responsibility of the investigator to ensure the accuracy of all data entered in the CRFs. The 
delegation log will identify all those personnel with responsibilities for data collection and handling, 
including those who have access to the trial database. 
9.3 
Completing Case Report Forms 
All CRFs must be completed and signed by staff that are listed on the site staff delegation log and 
authorised by the CI/ PI to perform this duty. The CI/ PI is responsible for the accuracy of all data 
reported in the CRF. Once completed the original will be kept with the study team at site. 
9.4 
Data handling and analysis 
A trial specific data management SOP will be in place for the trial. This will contain details of the 
software to be used for the database, the process of database design, data entry, data quality checks, 
data queries, data security, database lock. 
Where data are transferred electronically this will be in accordance with the UK Data Protection Act 
2018 as well as UCL Information Security Policy and Trust Information Governance Policy. There will be a 
documented record of data transfer and measures in place for the recovery of original information after 
transfer. 
10 
Statistical Considerations 
10.1 Primary endpoints 
10.1.1 Efficacy 
1. Change in PaO2/FiO2 ratio from baseline (as defined in sections 7.1.2 and 7.1.3), daily during 
treatment, end of treatment, 3 days post end of treatment and 5 days post end of treatment.  
10.1.2  Safety 
1. Incidence and severity of major bleeding events directly attributable to the study drug 
2. Decrease in fibrinogen levels to < 1.0 gm/L during treatment period and 48 hrs after the last dose of 
treatment 
3. Number and nature of serious adverse events causally related to the treatment 
 
10.2 Secondary endpoints 
1. Changes in lung compliance  (defined as tidal volume / (peak inspiratory pressure – PEEP)) from 
baseline and absolute values at day 5 (96 hrs ± 2 hrs), day 7 (144 hrs ± 4hrs), end of treatment, 3 and 
5 days post end of treatment 


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2. Clinical status as assessed by a 7-point WHO ordinal scale at baseline and daily up to 5 days post end 
of treatment and at day 28, discharge or death (whichever comes first). 
3. Mean daily Sequential Organ Failure Assessment (SOFA) score at baseline through to 5 days post 
end of treatment 
4. In follow up period, number of oxygenation free days, ventilator free days, intensive care stay, up to 
28 days or death or discharge, whichever occurs first.  
5. Incidence and number of days of new oxygen use, non-invasive ventilation or high flow oxygen 
devices in the first 28 days. 
6. Incidence and number of days of new mechanical ventilation use during in the first 28 days 
7. In hospital mortality 
10.3 Exploratory endpoints 
Change in levels of plasma biomarkers of pro- and anti-inflammatory markers (e.g. interleukin-6, 
interleukin-8, interleukin-10 and interleukin-1Ra), endothelial injury (plasma von Willebrand factor) 
coagulation (procogulants, anticoagulants, and fibrinolytic pathway) measure of tissue damage. 
Historical controls will not provide data for exploratory endpoints.  
10.4 Sample size and recruitment 
10.4.1 Sample size calculation 
The sample size for this pilot study is not based on a statistical consideration. The recruitment target of 
12 patients treated with rtPA and SOC is based on feasibility and the planned recruitment rate.  
Similarly, for cohort 2 the sample size of 30 patients is not based on statistical consideration, but has 
been selected on the grounds of what is reasonable amid a second surge of COVID-19 admissions in 
early 2021 and the number of patients we might reasonably accept to recruit across the 2 sites over the 
next 2 to 6 weeks.  
10.4.2 Historical control recruitment – Cohort 1 only 
Historical controls will be recruited at a ratio of 2 controls to every 1 rtPA + SOC arm patient, and will be 
matched according to the following characteristics:  
1. Ventilation and oxygen type (IMV and non-invasive oxygen support) 
2. Severity as determined by PaO2/FiO2 ratio 
3. Gender 
4. Age (+/- 2 years, up to a maximum of 10 years) 
5. Ethnicity 
Matching will be done in the order that the characteristics are presented above.  
Deceased patients can be considered for matching as controls. However, as a certain number of 
observations are required for developing a P/F ratio of the curve, patients who died within 48  hrs of 
admission are excluded.  
 
Baseline data during admission will be used for matching.  
 


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An age range of +/- two years was used in the first instance, and this was widened until two eligible 
patients were identified that matched the treatment patient’s ethnicity, up to a maximum of +/- 10 
years. 
Where more than two controls are identified, two controls will be drawn at random without 
replacement among all considered historical patients that match according to the characteristics above.  
10.5 Randomisation methods 
No randomisation will be employed in this pilot study due to practical and ethical  considerations. 
 A statistically powered RCT would be ideal to define the magnitude of benefit and impact on overall 
survival and is the typical design in patients with ARDS Although there is clinical data on the safety of 
nebulised rtPA, but there are no clinical data on this  condition to facilitate a sample size calculation. 
There are data from a small RCT that has used another fibrinolytic agent, but the doses are not 
comparable (Abdelaal Ahmed Mahmoud et al. 2020). If a patient is randomised to receive no treatment, 
this precludes participation in other interventional studies which might increase the risk of mortality. 
The most recent figures suggest a 50% mortality in patients receiving 
IMV.(https://www.icnarc.org/About/Latest-News/2020/04/04/Report-On-2249-Patients-Critically-Ill-
With-Covid-19) Currently there is a concerted effort to introduce multiple therapies based on our 
understanding of the pathophysiologic basis of disorder. Ethical concerns were raised about the 
potential for denying patients new treatment strategies when randomised to standard of care arm. The 
management strategies are evolving on an unprecedented scale with repurposing of existing 
medications, many of which are proof of concept of studies.  Further, if this pilot study shows significant 
effect in a subset of patients, there would be justification to progress to a statistically powered RCT. We 
would progress to a RCT only after submission of a substantial amendment to this trial protocol or 
submission of a new clinical trial application In the event of major adverse drug reactions or minor 
improvement in the oxygenation as assessed by PaO2/FiO2 , there are minimal gains to be had with a 
larger randomised control study. 
A Data Monitoring Committee (DMC) will be set-up to review safety data within the trial along with the 
final study results and advise on progressing from a pilot study to a statistically powered RCT based on 
the safety and efficacy data that is collected.  
10.6 Statistical analysis plan 
All analyses will be descriptive in nature and should be considered as hypothesis generating rather than 
providing firm conclusions. 
Analysis of cohorts 1 and 2 will be undertaken separately.  
 All study data will be listed and all relevant data will be tabulated and summarised separately by cohort 
and by group (rtPA treated, historical controls), separately for the patients receiving IMV, and for other 
patients with mild ARDS requiring non-invasive oxygen support at enrolment, and overall only where 
appropriate, as follows: 
Planned recruitment for Cohort 1: 
Group 1: Patients with ARDS receiving IMV at enrolment treated with rtPA (n=6) 
Group 2: Patients with ARDS requiring non-invasive support at enrolment treated with rtPA (n=6) 


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Group 3: Patients with ARDS receiving IMV - historical controls (n=12) 
Group 4: Patients with ARDS requiring non-invasive support  - historical controls (n=12) 
Planned recruitment for Cohort 2: 
A total of 30 patients will be recruited, with a minimum of 10 from each ventilation type.  
Group 5: Patients with ARDS receiving IMV at enrolment treated with rtPA 
Group 6: Patients with ARDS requiring non-invasive support at enrolment treated with rtPA 
Data will be presented using standard descriptive statistics. Continuous variables will be summarised 
using number of observations, mean, and standard deviation, median, inter-quartile range, minimum, 
and maximum values. Categorical values will be summarised using number of observations and 
percentages. 
A statistical analysis plan will be written and finalised prior to database lock. This plan will give a detailed 
description of all summaries and results that will be presented. Cohort 1 data will be locked separately 
from the cohort 2 data and a separate SAP will be produced for the analysis of the cohort 2 data. 
 
10.6.1 Summary of baseline data and flow of participants 
All pre-treatment assessments data in the protocol will be listed and all relevant data will be tabulated 
for each group separately. 
For patients treated with rtPA + SOC, the last available value prior to the first administration of IMP will 
be considered the baseline value. 
For patients receiving SOC only (historical controls), the last available value prior to enrolment, as 
defined in section 8.2, will be considered the baseline value. 
All data necessary to produce a consort flow diagram (http://www.consort-statement.org/) will be 
summarised: the number of patients in each group that were enrolled, assessed for eligibility, excluded 
(with reasons listed), assigned to a group, treated with rtPA + SOC, followed up, discontinued (with 
reasons such as lost to follow-up, withdrawn, or death), and number of patients analysed in each group, 
and where applicable excluded from analyses (with reasons). 
10.6.2 Primary outcome analysis 
The PaO2/FiO2 ratio measured over time will be described for each group. The change from baseline in 
the PaO2/FiO2 ratio will be summarised at each post baseline timepoint. Plots will be used, where 
appropriate, to show the PaO2/FiO2 summarised for patients over time. 
PaO2/FiO2 will be a calculated field on the trial database derived from the fields for PaO2 and FiO2. For 
cohort 2 data will be extracted once a day coinciding with the worst PF ratio. As PF ratio is the primary 
endpoint, in addition to the worst PF ratio, up to a maximum of 6 data points spread across 24 hrs that 
represents patients clinical status will be extracted. Details of further data derivation and analyses will 
be described in the SAP. 
All patient data will be used, regardless of any intercurrent events that may have occurred after 
enrolment: 


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- 
Data from patients recruited into the rtPA + SOC group will be summarised whether or not 
patients have received all planned doses of the IMP. 
- 
Any change in requirement for oxygenation and ventilation support will be summarised and 
described 
- 
Changes in PaO2/FiO2 ratio over time will be described and interpreted taking into 
consideration that a patient’s condition may have changed and led to their ventilation type 
changed post enrolment. If data allows, additional descriptions of the PaO2/FiO2 ratio over 
time may be performed in each subgroup of patients experiencing such changes (e.g. IMV to 
NIV, or vice versa). 
The incidence and severity of bleeding events will be summarised in each group, and the change in 
fibrinogen levels will also be summarised at each post-baseline timepoint. 
For cohort 1, subject to the final sample size and data quality, outcomes will also be compared between 
the SOC only group and intervention (rtPA + SOC) group using appropriate regression methods to make 
adjustment for baseline characteristics. Appropriate data transformations required for model 
assumptions will be considered if necessary.   
10.6.3 Secondary outcome analysis 
All secondary outcomes measured overtime will be summarised at each post-baseline timepoint, for  
each group, and overall where appropriate: 
• 
Clinical status as assessed by a 7-point ordinal scale and change in clinical status 
• 
Changes in lung compliance  (computed in the database using tidal volume / (peak inspiratory 
pressure – PEEP)) from baseline and absolute values at day 5 (96 hrs ± 2 hrs), day 7 (144 hrs ± 
4hrs), end of treatment, 3 and 5 days post end of treatment. 
• 
The Mean daily Sequential Organ Failure Assessment (SOFA) score and change from baseline 
The following secondary outcomes will also be summarised for each group, and overall, where 
appropriate: 
• 
The number of oxygenation free days in the first 28 days 
• 
Number of ventilator-free days in the first 28 days 
• 
The incidence and number of days of new oxygen use, non-invasive ventilation or high flow 
oxygen devices in the first 28 days. 
• 
Incidence and number of days of new mechanical ventilation use during the trial 
• 
Intensive care unit (ICU) free days in the first 28 days 
• 
In hospital mortality 
• 
Length of ICU stay post treatment 
• 
Length of ventilation 
• 
Use of vasopressors 
10.6.4 Sensitivity and other planned analyses 
A description of plans for sensitivity and other analyses will be provided in the SAPs.  


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11 
Record keeping and archiving 
At the end of the trial, all essential documentation will be archived securely by the CI and trial sites for a 
minimum of 25 years from the declaration of end of trial. 
Essential documents are those which enable both the conduct of the trial and the quality of the data 
produced to be evaluated and show whether the site complied with the principles of Good Clinical 
Practice and all applicable regulatory requirements. 
The sponsor will notify sites when trial documentation can be archived. All archived documents must 
continue to be available for inspection by appropriate authorities upon request. 
12 
Oversight Committees 
12.1 Trial Management Group (TMG) 
The TMG will include the Chief Investigator, co-investigators and trial staff. The TMG will assess the safety 
of the trial specifically at 24 hrs after the first dose (to confirm progression to treatment of the second 
patient) and 72 hrs after the second patient’s first dose (to confirm dosing can continue). If there are any 
safety issues identified the TMG will refer to the DMC for their input. The TMG will be responsible for 
overseeing the trial. The group will meet regularly during the estimated 3-week recruitment / treatment 
period to review efficacy and safety. 
The TMG will review recruitment figures, SAEs and substantial amendments to the protocol prior to 
submission to the REC and/or MHRA. 
During cohort 2, the TMG will convene after every 10 patients have been recruited.  
12.2 Data Monitoring Committee (DMC) 
The role of the DMC is to provide advice on data and safety aspects of the trial and recommend if the trial 
should progress to a statistically powered RCT. We would progress to a RCT only after submission of a 
substantial amendment to this trial protocol or submission of a new clinical trial application. 
Cohort 1: 
If at the 24 hr post dose review of patient one and 72 hr post first dose review of patient two there are 
any safety issues noted, the TMG will refer to the DMC for their input.  
The DMC will receive weekly reports on bleeding complications, both major and minor along with 
fibrinogen levels. If at any time a patient meets one of the stopping criteria, further dosing of patients will 
be stopped and an adhoc DMC review will be arranged before resuming dosing.  
Cohort 2: 
During cohort 2, the DMC will convene after every 10 patients have been recruited. If at any time a patient 
meets one of the stopping criteria (as per section 7.10), further dosing of patients will be stopped and an 
adhoc DMC review will be arranged before resuming dosing.  
 
The DMC will review the final study results and advise on progressing from a pilot study to a statistically 
powered randomised control trial based on the safety and efficacy data that is collected.  
 


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13 
Direct Access to Source Data/Documents 
The investigator/ institution will permit trial-related monitoring, audits, REC review, and regulatory 
inspection(s), providing direct access to source data/documents. Trial participants are informed of this 
during the informed consent discussion. Participants will consent to provide access to their medical 
notes. 
14 
Ethics and regulatory requirements 
The sponsor will ensure that the trial protocol, participant information sheet, consent form, GP letter 
and submitted supporting documents have been approved by the appropriate regulatory body (MHRA 
in UK) and an appropriate research ethics committee and the HRA, prior to any participant recruitment. 
The protocol, all other supporting documents including and agreed amendments, will be documented 
and submitted for ethical and regulatory approval as required. Amendments will not be implemented 
prior to receipt of the required approval(s). 
Before the site may be opened to recruit participants, the Chief Investigator or designee must receive 
confirmation of capability and capacity in writing from the Trust Research & Development (R&D). It is 
the responsibility of the CI to ensure that all subsequent amendments gain the necessary approvals, 
including NHS Permission (where required) at the site. This does not affect the individual clinician’s 
responsibility to take immediate action if thought necessary to protect the health and interest of 
individual participants (see section 8.9 for reporting urgent safety measures). 
An annual progress report (APR) will be submitted to the REC within 30 days of the anniversary date on 
which the favourable opinion was given, and annually until the trial is declared ended. The chief 
investigator will prepare the APR. 
Within 90 days after the end of the trial, the CI/Sponsor will ensure that the main REC and the MHRA are 
notified that the trial has finished. If the trial is terminated prematurely, those reports will be made 
within 15 days after the end of the trial. 
The CI will supply the Sponsor with a report of the clinical trial which complies with the format as 
defined by the EMA. This will then be uploaded to EudraCT for availability to the MHRA and a copy of 
the report will be submitted to the main REC, within 1 year after the end of the trial. 
15 
Monitoring requirement for the trial 
In the context of this pandemic, visits to hospital sites are generally not appropriate as they could 
increase the risks of spreading infection. Therefore, no prospective monitoring will be planned for this 
trial. If necessary, for cause telephone monitoring calls will be undertaken with site and documented. 
16 
Finance 
This trial will be funded by the Royal Free Hospital Charity. 


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17 
Insurance 
University College London holds insurance against claims from participants for injury caused by their 
participation in the clinical trial. Participants may be able to claim compensation if they can prove that 
UCL has been negligent. However, as this clinical trial is being carried out in a hospital, the hospital 
continues to have a duty of care to the participant of the clinical trial. University College London does 
not accept liability for any breach in the hospital’s duty of care, or any negligence on the part of hospital 
employees. This applies whether the hospital is an NHS Trust or otherwise. 
Participants may also be able to claim compensation for injury caused by participation in this clinical trial 
without the need to prove negligence on the part of University College London or another party. 
Participants who sustain injury and wish to make a claim for compensation should do so in writing in the 
first instance to the Chief Investigator, who will pass the claim to the Sponsor’s Insurers, via the 
Sponsor’s office. 
Hospitals selected to participate in this clinical trial shall provide clinical negligence insurance cover for 
harm caused by their employees and a copy of the relevant insurance policy or summary shall be 
provided to University College London, upon request. 
18 
Publication policy 
The sponsor intends to publish the results of this study in accordance with external guidelines regardless 
of whether the outcomes are perceived as positive, neutral, or negative. 
All publications relating to the sponsor’s projects must undergo appropriate review, and require sponsor 
agreement to publish prior to release of information. 
No publication that incorporates the sponsor’s confidential information shall be submitted for 
publication without the sponsor’s prior written agreement to publish, and shall be given to the sponsor 
for review at least 30 days prior to submission for publication. 
19 
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20 
Appendix 1 - Schedule of assessments   
 
Screening/
3 day 
baseline 
perioda 
During treatment until 5 days after 
last dose 
From 6 days after 
last dose until day 
28/discharge/death 
Day 
28/discharge/death 
 
 
Every 4 
hours 
Every 6 
hours 
Once 
Daily 
 
 
 
 
24 and 
48 
hours, 
then 
twice 
weekly 
 
 
 
Once daily 
 
Informed consent 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Review of medical history 
(including inpatient history and 
COVID-19 diagnosis) 
X 
 
 
 
 
 
 
Eligibility review 
X 
 
 
 
 
 
 


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Pregnancy test (blood or urine) 
for female patients of 
childbearing potential 
X 
 
 
 
 
 
 
Review of concomitant 
medication 
Xf 
 
 
X 
 
X - Weekly 
X 
Review of adverse 
events/adverse reactions 
(including bleeding episodes) 
 
 
 
X 
 
X - Weekly 
X 
Directed physical examination 
including vital signs (heart rate, 
temperature, blood pressure 
and body weight) 
X 
 
 
X 
(cohort 
1) 
X – 
weekly 
only 
(cohort 
2) 
X (cohort 1 daily) 
X (cohort 2 weekly) 
X 
Documentation of respiratory 
statusa 
X 
X 
 
 
 
X -  weekly) 
X  
Local blood testingb 
X 
 
 
X 
(cohort 
1 daily) 
X 
(cohort 
2) 
every 
other 
day 
as required 
X 
Research blood samplingc 
X 
 
 
 
X 
X – weekly 
 
Administration of rtPAd 
 
 
X 
 
 
 
 


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Clinical status as per 7-point 
ordinal scale 
X 
 
 
X 
 
 
X 
Sequential Organ Failure 
Assessment (SOFA) score 
X 
 
 
X 
 
 
 
Glasgow Coma scale 
X 
 
 
X 
 
 
 
End of study data collection e 
 
 
 
 
 
 
X 
 
a Cohort 1 baseline period is 3 days, Cohort 2 baseline is screening assessments only. Respiratory status of patients is monitored routinely hourly, but data points 
will be extracted for every four hours where available until 5 days after the last dose (day 7 for SOC patients) for cohort 1, but one data point per day is required 
as a minimum. For cohort 2 data will be extracted once a day coinciding with the worst PF ratio. As PF ratio is the primary endpoint, in addition to the worst PF 
ratio over the preceding 24 hrs up to a maximum of 6 data points spread across 24 hrs that represents patients clinical status will be extracted. Similar to cohort 
1 respiratory data will be collected until 5 days after the last dose.  After this period, respiratory status is to be documented weekly (+/- 1 day) until discharge, 
death, or 28 days whichever occurs first. See section 7.5 for variables required.  
b Local blood testing as per section 7.6.2 are to be done at a minimum once a day for cohort 1, or every other day for cohort 2, until 5 days after the last dose, 
and then as required until day 28, discharge or death whichever occurs first. 
c Research blood sampling will be done once at baseline at 48 hours (+/- 24 hours), 96 hours  (+/- 24 hours), and then twice weekly as per section 7.6.2. Twice 
weekly testing should be evenly spaced across weekdays if possible.  
d Dosing schedule as per section 7.3 
e End of study data will be collected at day 28, discharge or death whichever occurs first as per section 7.5. 
f to be taken where available once daily for 3 days prior to dosing (cohort 1 only). Screening assessments can be used as part of this baseline period.  
 


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21 
Appendix 2 - Lookup table for imputed PaO2 for a given SpO2 
based on non-linear equation (from Brown SM et al. Nonlinear 
Imputation of PaO2/FIO2 From SpO2/FIO2 Among 
Mechanically Ventilated Patients in the ICU: A Prospective, 
Observational Study. Crit Care Med. 2017; Aug;45(8):1317-
1324) 
 
Measured 
SpO2 (%) 
Imputed 
PaO2 
(mmHg) 
Measured 
SpO2 (%) 
Imputed 
PaO2 
(mmHg) 
100* 
167*§ 
84 
49 
99* 
132* 
83 
47 
98* 
104* 
82 
46 
97* 
91* 
81 
45 
96 
82 
80 
44 
95 
76 
79 
43 
94 
71 
78 
42 
93 
67 
77 
42 
92 
64 
76 
41 
91 
61 
75 
40 
90 
59 
74 
39 
89 
57 
73 
39 
88 
55 
72 
38 
87 
53 
71 
37 
86 
51 
70 
37 
85 
50 
 
 
*Generally considered unreliable on the basis of the sigmoidal shape of the haemoglobin-oxygen 
dissociation curve 


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§Based on SpO2 99.5%. 
Imputed values calculated using the following equation:  
 
𝑃𝑂2 = { 11,700
(1 𝑆
⁄ −1)
+ [503 + (11,700
1 𝑆
⁄ −1
) ]
}
+ { 11,700
(1 𝑆
⁄ −1)
 −[503 + (11,700
1 𝑆
⁄ −1
)
2
]
1/2
}
1/3
 
 
 
 
 


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22 
Appendix 3 – Oxygen flow rate calculation (used for calculating 
FiO2 in patients receive non-invasive oxygen support 
 
Oxygen delivery devices 
Device 
Flow rate 
% Oxygen 
delivered 
(approx..) 
Nasal Cannula 
1 L/min 
24% 
2 L/min 
28% 
4 L/min 
36% 
Venturi 
Valve & 
Mask 
Blue 
2-4 L/min 
24% 
White 
4-6 L/min 
28% 
Yellow 
8-10 L/min 
35% 
Red 
10-12 L/min 
40% 
Green 
12-15 L/min 
60% 
Non-rebreather Mask 
15 L/min 
85% 
 

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