A Phase Ib Trial of Inhaled Carbon Monoxide for the Treatment of Pneumonia and Sepsis-Induced Acute Respiratory Distress Syndrome (ARDS)
Phase: PHASE1
Conditions: Acute Respiratory Distress Syndrome, Sepsis
Sponsor: Brigham and Women's Hospital


January 5, 2026
NCT04870125
A Phase 1b Study of Inhaled CO for the Treatment of Sepsis-Induced ARDS
Submission of Protocol/Statistical Analysis Plan (dated July 21, 2023) to allow us to submit study 
results
We are ready to submit our final results (and we need to do so as soon as possible), and we 
understand that you first need to approve and release our Study Protocol (dated/approved July 21, 
2023) that includes our SAP.  We attach these documents and hope that these can be approved and 
released as soon as possible to enable us to submit our final results by the required deadline.
Sincerely yours,

Rebecca M. Baron, M.D.

A Phase Ib Trial of Inhaled Carbon Monoxide for the 
Treatment of Pneumonia and Sepsis-Induced Acute 
Respiratory Distress Syndrome (ARDS) 
 
National Clinical Trial (NCT) Identified Number: NCT04870125 
Principal Investigator: Rebecca Baron, MD 
IND Sponsor: Mark Perrella, MD 
 
Funded by: National Heart, Lung, and Blood Institute (NHLBI) 
Version Number: v.3.0 
July 21, 2023 
 
Summary of Changes from Previous Version: 
 
 
Affected 
Section(s) 
Summary of Revisions Made 
Rationale 
Cover page 
The protocol title, version, and version date have 
been updated 
Administrative changes 
Multiple 
Sections 
Expanded inclusion criteria to include 3 strata: 1) 
ARDS or 2) pneumonia (unilateral or bilateral 
pulmonary infiltrates) without ARDS or sepsis, or 3)  
pneumonia (unilateral or bilateral pulmonary 
infiltrates) with sepsis, without ARDS. 
Outcomes of patients with lung infections 
related to pneumonia have been reported 
to be similar with unilateral as for bilateral 
infiltrates, and this permits us to expand 
inclusion criteria to improve subject 
enrollment as well as conform to our 
preclinical primate FDA-enabling studies. 
1.1 Synopsis 
Pneumonia was added to the title, study 
description, objectives, and study population. 
Change made to update the study 
population.  
1.4 Definitions 
The following language was added: Pneumonia: 
Unilateral or bilateral pulmonary infiltrates in a 
patient with suspected respiratory infection. 
 
This change was made to clarify the 
definition of pneumonia. 
2.1 Background 
Added: In April 2023, we elected to expand 
inclusion criteria to include intubated patients with 
pneumonia (unilateral or bilateral pulmonary 
infiltrates consistent with lung infection), given 
reported similar outcomes in critically ill patients 
with unilateral and bilateral infiltrates (LUNG SAFE 
study). 
 
 
This change was made to support the 
rationale for adding intubated patients 
with pneumonia to the study population. 
2.2.1 
Introduction 
Added: Pneumonia represents a lung infection with 
unilateral or bilateral infiltrates, and intubated 
patients with pneumonia not formally meeting 
ARDS criteria have been reported to have similar 
This change was made to support the 
rationale for adding intubated patients 
with pneumonia to the study population. 

NIH-FDA Phase 2 and 3 IND/IDE Clinical Trial Protocol Template   
NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
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outcomes with unilateral or bilateral infiltrates.  
Pneumonia in intubated patients represents an 
important spectrum of illness in ARDS, and in fact 
the most recent NIH lung injury consortium to 
improve phenotyping in critical illness will enroll 
subjects with pneumonia, sepsis and ARDS.  
Furthermore, our preliminary data demonstrating 
efficacy and safety of inhaled CO in pneumonia 
baboons is presented below. 
3.0 Objectives, 
4.1 Overall 
Study Design, 
4.2 Scientific 
Rationale 
Objectives were updated to include pneumonia 
and/or sepsis in the study population.  
Change made to update the study 
population. 
5.1 Inclusion 
Criteria 
The following changes were made to this section.  
All patients (age 18 and older) will be eligible for 
inclusion if they meet all of the following consensus 
criteria for sepsis and ARDS3,or if they meet the 
criteria for pneumonia. 
The following inclusion criteria was added: 
• Pneumonia (without ARDS or sepsis) will be 
defined as a unilateral or bilateral lung 
infiltrate on chest X-ray or chest CT (not fully 
explained by effusions, lobar/lung collapse or 
nodules) in the setting of  receiving mechanical 
ventilation, a new suspected respiratory 
infection, an increase in SOFA score less than 2 
at the time of randomization (baseline).  
Pneumonia with bilateral opacities, PaO2/FiO2 
ratio ≤ 300, or an increase in SOFA score 
greater than or equal to 2 over baseline will 
continue to be considered ARDS and sepsis. 
• Pneumonia (with sepsis, without ARDS) 
will be defined as a unilateral or bilateral 
lung infiltrate on chest X-ray or chest CT 
(not fully explained by effusions, 
lobar/lung collapse or nodules) in the 
setting of receiving mechanical ventilation 
and a new suspected respiratory infection 
with an increase in SOFA score of ≥ 2 over 
baseline at the time of randomization. 
Pneumonia with bilateral opacities, 
PaO2/FiO2 ratio ≤ 300, or an increase in 
SOFA score greater than or equal to 2 over 
baseline will continue to be considered 
ARDS and sepsis.   
Inclusion criteria revised to include 
intubated patients with pneumonia.  

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NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
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5.4 Strategies 
for Recruitment 
and Retention 
The following language was added to this section: 
We have expanded inclusion criteria to include 
subjects with unilateral or bilateral infiltrates 
intubated for suspected lung infection 
(pneumonia), given similar outcomes from 
unilateral or bilateral lung infiltrates in critically ill 
patients, consistency with our preclinical baboon 
model of ALI due to pneumococcal pneumonia that 
had improved outcomes with inhaled CO, and the 
need to expand enrollment for this study. 
This phase 1b protocol will also include 
subjects with unilateral or bilateral 
infiltrates intubated for suspected lung 
infection (pneumonia). This was added 
given similar outcomes from unilateral or 
bilateral lung infiltrates in critically ill 
patients and for consistency with our 
preclinical baboon model of ALI due to 
pneumococcal pneumonia that had 
improved outcomes with inhaled CO. This 
may also help with enrollment in this 
study. 
9.1 Statistical 
Hypothesis 
The following changes were made to this section. 
Hypothesis: We hypothesize that low dose iCO will 
be safe and well-tolerated and that the CFK 
equation-based iCO personalized dosing algorithm 
will be accurate in achieving a target COHb level of 
6-8% in patients with pneumonia (without ARDS 
and sepsis criteria, and with sepsis but without 
ARDS criteria) and sepsis-induced ARDS at study 
entry.  
We further hypothesize that precision-based iCO 
therapy will reduce the severity of pneumonia and 
ARDS and organ failure by suppressing 
mitochondrial dysfunction, inhibiting inflammasome 
activation and necroptosis, and accelerating 
resolution of inflammation. 
This change was made to incorporate the 
additional strata into the statistical plan.  
9.4.2 Analysis of 
the Primary 
Efficacy 
Endpoint(s), 
9.4.5 Safety 
Analysis 
The following language was added: ANOVA will be 
repeated to assess the effects of treatment, time 
(over 3 days), entry criteria (ARDS/Sepsis vs. Non-
ARDS/Sepsis Pneumonia vs. Non-ARDS/non-Sepsis 
Pneumonia), and two-way/three-way interactions 
of time, treatment and entry criteria. 
This change was made to incorporate the 
additional strata into the statistical plan. 
9.4.3 Analysis of 
the Secondary 
Endpoint(s) 
This section was updated to add ARDS/Sepsis, Non-
ARDS/Sepsis Pneumonia, and Non-ARDS/non-Sepsis 
Pneumonia to the analysis description for the 
secondary endpoints. 
This change was made to incorporate the 
additional strata into the statistical plan. 

  
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ii 
CONTENTS 
STATEMENT OF COMPLIANCE ....................................................................................................................................... 1 
1 
PROTOCOL SUMMARY ...................................................................................................................................... 2 
1.1 
Synopsis ................................................................................................................................................. 2 
1.2 
Schema ................................................................................................................................................... 3 
1.3 
Schedule of Activities (SoA) ................................................................................................................... 4 
1.4 
Definitions .............................................................................................................................................. 6 
2 
INTRODUCTION ................................................................................................................................................ 7 
2.1 
Study Rationale ...................................................................................................................................... 7 
2.2 
Background ............................................................................................................................................ 7 
2.2.1 
Introduction ....................................................................................................................... 7 
2.2.2 
CO is Endogenously Produced Gas with Pleiotropic Biological Functions .......................... 8 
2.2.3 
Administration of Inhaled CO Protects Against Sepsis and Lung Injury in Animal Models 8 
2.2.4 
Lung Protective Effects of Carbon Monoxide ................................................................... 10 
2.2.5 
CO Delivery via an Inhaled Route is Safe in Human Subjects ........................................... 10 
2.2.6 
Steady State Diffusing Capacity and the Safety of Inhaled Carbon Monoxide ................. 13 
2.2.7 
Delivery of Inhaled CO to Mechanically Ventilated Subjects ........................................... 14 
2.2.8 
Testing the CO Delivery System in Baboon Model of Pneumoccal Pneumonia ............... 14 
2.2.9 
CO Dosing Strategy Using CFK Equation ........................................................................... 16 
2.3 
Risk/Benefit Assessment ...................................................................................................................... 18 
2.3.1 
Known Potential Risks ...................................................................................................... 18 
2.3.2 
Known Potential Benefits ................................................................................................. 19 
2.3.3 
Assessment of Potential Risks and Benefits ..................................................................... 20 
3 
OBJECTIVES AND ENDPOINTS ......................................................................................................................... 21 
4 
STUDY DESIGN ................................................................................................................................................ 22 
4.1 
Overall Design ...................................................................................................................................... 22 
4.2 
Scientific Rationale for Study Design ................................................................................................... 22 
4.3 
Justification for Dose ........................................................................................................................... 23 
4.4 
End of Study Definition ........................................................................................................................ 23 
5 
STUDY POPULATION ....................................................................................................................................... 24 
5.1 
Inclusion Criteria .................................................................................................................................. 24 
5.2 
Exclusion Criteria ................................................................................................................................. 24 
5.3 
Screen Failures ..................................................................................................................................... 26 
5.4 
Strategies for Recruitment and Retention ........................................................................................... 26 
6 
STUDY INTERVENTION .................................................................................................................................... 27 
6.1 
Study Intervention(s) Administration .................................................................................................. 28 
6.1.1 
Study Intervention Description ........................................................................................ 28 
6.1.2 
CO or Placebo Cylinders ................................................................................................... 28 
6.1.3 
Dosing and Administration ............................................................................................... 28 
6.1.4 
CO Monitoring .................................................................................................................. 29 
6.1.5 
CO Dosing Algorithm Using the CFK Equation .................................................................. 30 
6.1.6 
Daily Hold Parameters Prior to Study Drug Administration ............................................. 30 
6.1.7 
Interruption of Dosing During Study Drug Administration ............................................... 32 
6.1.8 
Permanent Discontinuation of Study Drug ...................................................................... 32 
6.1.9 
Completion of the Study Drug Administration ................................................................. 33 
6.1.10 
Treatment in COVID-19 Subjects ...................................................................................... 33 

  
NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
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6.2 
Preparation/Handling/Storage/Accountability .................................................................................... 34 
6.2.1 
Acquisition and Accountability ......................................................................................... 34 
6.2.2 
Formulation, Appearance, Packaging, and Labeling ........................................................ 34 
6.2.3 
Product Storage and Stability ........................................................................................... 35 
6.2.4 
Preparation ....................................................................................................................... 35 
6.3 
Measures to Minimize Bias: Randomization and Blinding ................................................................... 35 
6.4 
Study Intervention Compliance ........................................................................................................... 36 
6.5 
Concomitant Therapy .......................................................................................................................... 36 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT DISCONTINUATION/WITHDRAWAL ............. 37 
7.1 
Discontinuation of Study Intervention ................................................................................................. 37 
7.2 
Participant Discontinuation/Withdrawal From the Study ................................................................... 37 
7.3 
Lost to Follow-Up ................................................................................................................................. 38 
8 
STUDY ASSESSMENTS AND PROCEDURES ...................................................................................................... 38 
8.1 
Description of Study Procedures ......................................................................................................... 38 
8.1.1 
Study Sample Collection ................................................................................................... 40 
8.2 
Screening Procedures .......................................................................................................................... 42 
8.2.1 
Assigning a Screening ID ................................................................................................... 42 
8.2.2 
Screened, Not Enrolled ..................................................................................................... 42 
8.3 
Randomization Procedures (Day 0) ..................................................................................................... 43 
8.3.1 
Randomization ................................................................................................................. 43 
8.3.2 
Assigning a Randomization ............................................................................................... 44 
8.3.3 
Ventilator Procedures ...................................................................................................... 44 
8.4 
Baseline Assessments .......................................................................................................................... 44 
8.5 
Study Drug Administration Days 1 – 3 ................................................................................................. 45 
8.5.1 
Reference Measurements (Days 1 – 3) ............................................................................ 46 
8.5.2 
Pre-Treatment Assessments (Day 1 – 3) .......................................................................... 47 
8.5.3 
Study Drug Administration and Monitoring ..................................................................... 48 
8.6 
Post-Treatment Assessments .............................................................................................................. 49 
8.6.1 
Study Days 4, 5, and 7 ...................................................................................................... 49 
8.6.2 
Study Day 14 ..................................................................................................................... 49 
8.6.3 
Study Day 28 ..................................................................................................................... 50 
8.6.4 
Study Day 60 ..................................................................................................................... 50 
8.6.5 
Month 3 and 6 Assessments ............................................................................................ 50 
8.7 
Adverse Events and Serious Adverse Events ....................................................................................... 50 
8.7.1 
Definition of Adverse Events (AE) .................................................................................... 50 
8.7.2 
Definition of Serious Adverse Events (SAE) ...................................................................... 51 
8.7.3 
Definition of Administration Related Adverse Events ...................................................... 51 
8.7.4 
Classification of an Adverse Event .................................................................................... 52 
8.7.5 
Time Period and Frequency for Event Assessment and Follow-Up .................................. 53 
8.7.6 
Adverse Event Reporting .................................................................................................. 54 
8.7.7 
Serious Adverse Event Reporting ..................................................................................... 54 
8.8 
Unanticipated Problems ...................................................................................................................... 55 
8.8.1 
Definition of Unanticipated Problems (UP) ...................................................................... 55 
8.8.2 
Unanticipated Problem Reporting .................................................................................... 55 
9 
STATISTICAL CONSIDERATIONS ...................................................................................................................... 55 
9.1 
Statistical Hypotheses .......................................................................................................................... 55 

  
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9.2 
Sample Size Determination .................................................................................................................. 56 
9.3 
Populations for Analyses ...................................................................................................................... 57 
9.4 
Statistical Analyses ............................................................................................................................... 57 
9.4.1 
General Approach ............................................................................................................ 57 
9.4.2 
Analysis of the Primary Safety Endpoint(s) ...................................................................... 57 
9.4.3 
Analysis of the Secondary Endpoint(s) ............................................................................. 58 
9.4.4 
Analysis of Exploratory Endpoints (Biomarkers) .............................................................. 59 
9.4.5 
Safety Analyses ................................................................................................................. 60 
9.4.6 
Planned Interim Analyses ................................................................................................. 61 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS ...................................................... 61 
10.1 
Regulatory, Ethical, and Study Oversight Considerations .................................................................... 61 
10.1.1 
Informed Consent Process ............................................................................................... 61 
10.1.2 
Study Discontinuation and Closure .................................................................................. 64 
10.1.3 
Confidentiality and Privacy ............................................................................................... 64 
10.1.4 
Future Use of Stored Specimens and Data ....................................................................... 66 
10.1.5 
Key Roles and Study Governance ..................................................................................... 66 
10.1.6 
Safety Oversight ............................................................................................................... 68 
10.1.7 
Clinical Monitoring ........................................................................................................... 69 
10.1.8 
Quality Assurance and Quality Control ............................................................................ 69 
10.1.9 
Data Handling and Record Keeping .................................................................................. 70 
10.1.10 
Protocol Deviations .......................................................................................................... 70 
10.1.11 
Publication and Data Sharing Policy ................................................................................. 71 
10.1.12 
Conflict of Interest Policy ................................................................................................. 71 
10.2 
Abbreviations ....................................................................................................................................... 71 
10.3 
Protocol Amendment History .............................................................................................................. 74 
11 
REFERENCES .................................................................................................................................................... 75 
 
12 APPENDICES 
APPENDIX A: CO DELIVERY SYSTEM OPERATOR’S MANUAL.................................................................................  
APPENDIX B: SCORING INSTRUCTIONS.................................................................................................................. 
APPENDIX C: LONG TERM OUTCOMES ASSESSMENT............................................................................................   
APPENDIX D: VENTILATOR PROCEDURES..............................................................................................................  
 
 
 
 
 

A Phase Ib Trial of Inhaled CO for the Treatment of Pneumonia and of Sepsis-Induced ARDS       
Version 3.0 
NCT04870125 
21 July 2023 
NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
 
1 
STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonisation Good Clinical 
Practice (ICH GCP) and the following:  
 
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 46, 21 CFR 
Part 50, 21 CFR Part 56 and 21 CFR Part 312)  
 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are responsible for 
the conduct, management, or oversight of NIH-funded clinical trials have completed Human Subjects 
Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both the protocol 
and the consent form must be obtained before any participant is enrolled.  Any amendment to the 
protocol will require review and approval by the IRB before the changes are implemented to the study.  
In addition, all changes to the consent form will be IRB-approved; a determination will be made regarding 
whether a new consent needs to be obtained from participants who provided consent, using a previously 
approved consent form. 
 
I confirm that I have read and understand the protocol referenced above. I agree to carry out the study 
by the criteria written in the protocol and understand that no changes can be made to this protocol 
without written permission from the Protocol Chairs and the IRB.   
 
Institution Name: _______________________________ 
 
 
Site Principal Investigator Name: ____________________________ 
 
 
Site Principal Investigator Signature: _______________________________            
 
 
Date: ________________ 
 
 
 
 
 
 
 
 
 
 
 

A Phase Ib Trial of Inhaled CO for the Treatment of Pneumonia and of Sepsis-Induced ARDS       
Version 3.0 
NCT04870125 
21 July 2023 
NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
 
2 
1 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS  
Title: 
A Phase Ib Trial of Inhaled Carbon Monoxide for the Treatment of 
Pneumonia and Sepsis-induced Acute Respiratory Distress Syndrome 
(ARDS) 
 
Study Description: 
Multi-center, prospective, randomized, partially double-blind, placebo-
controlled Phase Ib safety clinical trial of inhaled CO (iCO) for the treatment 
of pneumonia and sepsis-induced ARDS. 
 
Hypothesis: We hypothesize that low dose iCO will be safe and well-
tolerated and that the Coburn-Forster-Kane (CFK) equation-based iCO 
personalized dosing algorithm will be accurate in achieving a target 
carboxyhemoglobin (COHb) level of 6-8% in intubated patients with 
pneumonia and sepsis-induced ARDS. 
 
Objectives: 
 
Primary Objective:  To evaluate the safety and accuracy of a CFK equation-
based personalized iCO dosing algorithm in intubated patients with 
pneumonia and sepsis-induced ARDS. 
 
 
Secondary Objectives: To examine the effects of low dose iCO therapy on 
biologic readouts in intubated patients with pneumonia and sepsis-induced 
ARDS. 
 
Endpoints: 
Primary Endpoints: The primary endpoints are: 
1. Safety of iCO, defined by the incidence of pre-specified administration-
related adverse events (AEs) and serious adverse events (SAEs). 
Administration-related SAEs are: 1) myocardial infarction; 2) stroke; 3) new 
onset arrhythmia requiring DC cardioversion; 4) worsening hypoxemia1,2; 5) 
COHb ≥ 10%; and 6) increase in lactate by ≥ 2 mmol/L. 
2. Accuracy of the CFK equation-based personalized iCO dosing algorithm 
to achieve a COHb level of 6-8%. 
 
Secondary Endpoints:  
1. Lung Injury Score (LIS) on days 1-5 and day 7 
2. PaO2/FiO2 on days 1-5 and day 7 
3. Oxygenation index (OI) on days 1-5 and day 7  
4. Dead space fraction (Vd/Vt) on days 1-3 and day 7 
5. Sequential Organ Failure Assessment (SOFA) scores on days 1-5, 7, 14, 
and 28 
6. Ventilator-free days at day 28 
7. ICU-free days at day 28 
8. Hospital-free days to day 60 
9. Hospital mortality to days 28 and 60  
10. Neurocognitive function at 3 and 6 months 
 
Exploratory Endpoints: 
1. Blood and urine biomarkers of mitochondrial dysfunction (mtDNA), 
inflammasome activation (IL-18) and necroptosis (RIPK3) 

A Phase Ib Trial of Inhaled CO for the Treatment of Pneumonia and of Sepsis-Induced ARDS       
Version 3.0 
NCT04870125 
21 July 2023 
NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
 
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2. Lipid mediators (LM) and specialized pro-resolving mediators (SPMs)  
Study Population: 
This study will enroll 36 adult subjects. All intubated patients > 18 years old 
who meet criteria for pneumonia and/or sepsis and ARDS according to 
consensus criteria3,4 will be potentially eligible for inclusion. Subjects will be 
recruited from the medical, surgical, and cardiac intensive care units (ICUs) 
at each center. 
 
Phase: 
Phase Ib 
 
Description of 
Sites/Facilities Enrolling 
Participants: 
The trial will be conducted at 6 tertiary care medical centers including Weill 
Cornell Medicine/New York-Presbyterian Hospital, Brigham and Women’s 
Hospital (BWH), Massachusetts General Hospital (MGH), Duke (Duke 
University Hospital/ Durham Regional Hospital), Washington University, 
and New York-Presbyterian Brooklyn Methodist Hospital. 
 
Description of Study 
Intervention: 
Participants will receive algorithm-specified iCO dose (not to exceed 500 
ppm) or medical grade air, to achieve COHb of 6-8% over 90 minutes for 
three consecutive days. 
 
Study Duration: 
Enrollment will take place over 27 months followed by 9 months for data 
analysis and dissemination of study results.  
 
Participant Duration: 
6 months 
1.2 
SCHEMA 
 
 
 
 

A Phase Ib Trial of Inhaled CO for the Treatment of Pneumonia and of Sepsis-Induced ARDS       
Version 3.0 
NCT04870125 
21 July 2023 
NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
 
4 
1.3 
SCHEDULE OF ACTIVITIES (SOA) 
 
Measurement/Event 
Baseline 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
7 
Day 
14 
Day 
28 
Day 
60 
3M 
6M 
Post- 
discharge 
CallK 
Demographic/Admission Data/Medical Hx  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Etiology of ARDS 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Height/Body weight 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Time on ventilator prior to enrollment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy test  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Vital Signs (HR, SBP, DBP, MAP, T) B 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Central venous pressure B 
A 
A 
A 
A 
A 
A 
 
 
 
 
 
 
 
Electrocardiogram (EKG) 
X 
X 
X 
X 
A 
A 
X 
 
 
 
 
 
 
Ventilator parameters B, C 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
Chest X-ray (CXR) review D  
X 
AD 
AD 
AD 
AD 
AD 
X 
 
 
 
 
 
 
Lung Injury Score (LIS) 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
APACHE II Score 
X 
 
 
 
 
 
 
 
 
 
 
 
 
SOFA Score E 
X 
X 
X 
X 
X 
X 
X 
X 
XF 
 
 
 
 
Glasgow Coma Score (GCS) 
X 
X 
X 
X 
X 
X 
X 
X 
XF 
 
 
 
 
Richmond Agitation Sedation Scale (RASS) 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Arterial Blood Gas (ABG) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
ABG; Venous Blood Gas if ABG not available C 
 
X 
X 
X 
A 
A 
A 
A 
 
 
 
 
 
Sp02 
X 
X 
X 
X 
X 
X 
X 
A 
 
 
 
 
 
Scv02 
A 
A 
A 
A 
A 
A 
 
 
 
 
 
 
 
Complete blood count (CBC) C 
X 
X 
X 
X 
A 
A 
A 
A 
A 
 
 
 
 
Basic metabolic panel C 
X 
A 
A 
A 
A 
A 
A 
A 
A 
 
 
 
 
PT/PTT/INR C 
A 
A 
A 
A 
A 
A 
A 
 
 
 
 
 
 
Serum CK, AST, ALT, Albumin, Total Protein C  
A 
A 
A 
A 
A 
A 
A 
 
 
 
 
 
 
Total Bilirubin C 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Lactate C 
X 
X 
X 
X 
A 
A 
A 
 
 
 
 
 
 
CO Administration Parameters / Treatment 
 
XG 
XG 
XG 
 
 
 
 
 
 
 
 
 
Pulmonary Dead Space (Vd/Vt) Measurement  
 
X 
X 
X 
 
 
X 
 
 
 
 
 
 
COHb and SpCO  
 
XG 
XG 
XG 
 
 
 
 
 
 
 
 
 
Vasopressors and inotropes B  
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Fluid intake and output B 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Renal replacement therapy status 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Concomitant medications  
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
Adverse event monitoring H 
 
X 
X 
X 
X 
X 
X H 
 
 
 
 
 
 

A Phase Ib Trial of Inhaled CO for the Treatment of Pneumonia and of Sepsis-Induced ARDS       
Version 3.0 
NCT04870125 
21 July 2023 
NIH-FDA Clinical Trial Protocol Template – v1.0 7 Apr 2017 
 
5 
Measurement/Event 
Baseline 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
7 
Day 
14 
Day 
28 
Day 
60 
3M 
6M 
Post- 
discharge 
CallK 
Plasma isolation  
 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
RNA banking  
 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
DNA banking – Cell pellet isolation 
 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
Primary cell (PBMC) isolation 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
Urine sample collection 
 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
Discarded BAL and plasma I  
A 
A 
A 
A 
A 
A 
A 
 
 
 
 
 
 
Microbiological results J 
A 
A 
A 
A 
A 
A 
A 
A 
A 
 
 
 
 
Vital Status 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
Discharge Status 
 
 
 
 
 
 
 
 
X 
X 
 
 
X 
Ventilator Status 
 
 
 
 
 
 
 
 
X 
X 
 
 
X 
Vasopressor Status 
 
 
 
 
 
 
 
 
X 
X 
 
 
X 
Neurocognitive Outcomes-MoCA-BLIND, 
Hayling 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
 
X  - Required  
 
A  - When available 
 
 
 
 
 
 
B  - Data gathered at times indicated or until patient achieves 48 hours of unassisted breathing, whichever is 
sooner. Data will be obtained pre and post-treatment on study drug administration days. 
C  - Measure during reference period (03:00-10:00), obtain at time closest to study drug administration if 
applicable 
D  - CXRs done for clinical purposes will be reviewed on days 1-5. If the subject is extubated during days 1-5, a chest 
X-ray will be ordered by the study team the day following extubation, if not ordered by the clinical team. 
E  - Record clinically available creatinine, platelets, bilirubin, SBP and vasopressor use 
F  - Gathered on day 28 or on discharge date 
G  - Daily on days of study drug administration 
H  - AEs will be captured until study day 7 
 
 
I  - Obtained from discarded biobank (site specific) if available 
J  - Microbiological results recorded as they become available 
K– If the subject is discharged home or to a long-term acute care facility before Day 28 or 60, he or she will be 
contacted by phone.  
 
 

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1.4 
DEFINITIONS 
Acute Kidney Injury: Acute kidney injury network Stage 3 disease, defined as a threefold increase in 
creatinine from baseline or the need for dialysis 
Completing 48 hours of UAB (from weaning form): Defined as the date (calendar day) that the subject 
reaches exactly 48 hours of UAB. Example: if subject meets UAB at 1900 on 6/1/06 and does not return 
to AB, then the date of completing 48 hours of UAB would be 6/3/06. 
Date of first UAB (from Study Termination form): Defined as the first day that the subject is on UAB from 
midnight to midnight. Example: if subject meets UAB at 1900 on 6/1/06, then the date of first UAB would 
be 6/2/06, as long as subject does not return to AB on 6/2/06. 
Day zero: Defined as day of randomization. Study days are calendar days.  
Drug held/hold drug: Study medication withheld for 24 hours.  
Drug permanently discontinued: Study medication stopped for remainder of the trial. 
Enrollment: All randomized participants will be included in the intent-to-treat (ITT) population. All 
randomized subjects who receive at least one dose of study product (CO vs. placebo) will be included in 
the safety analyses (ie. modified ITT). For secondary and exploratory endpoints, subjects may also be 
analyzed according to the number of study drug doses completed. Because some patients may withdraw 
or have a change in clinical status precluding dosing of study drug (eg. post- randomization lactic acidosis), 
we may randomize additional patients to achieve the enrollment goal of 36 patients who are randomized 
and treated with at least one dose of the study drug. We expect this to be an uncommon occurrence. 
Patients who are randomized but do not undergo any study procedures or study treatment will not be 
included in the study analysis. 
Extubation: Removal of an orotracheal, nasotracheal tube, or unassisted breathing with a tracheostomy. 
Home: level of residence or health care facility where the patient was residing prior to hospital admission 
Hospital Mortality to Day 60: This primary endpoint includes all deaths following randomization in any 
health care facility prior to discharge “home” until study day 60. Study subjects still in a health care facility 
at study day 61 are considered alive for this endpoint. 
Interruption of Dosing During Drug Administration: Study medication prematurely stopped prior to 90 
minutes. 
NYHA: New York Heart Association Class IV subjects (defined as subjects who have cardiac disease 
resulting in inability to carry out physical activity without discomfort. Symptoms of cardiac insufficiency 
or an anginal syndrome may be present even at rest. If any physical activity is undertaken, discomfort is 
increased). 
Pneumonia: Unilateral or bilateral pulmonary infiltrates in a patient with suspected respiratory infection. 
Sepsis: Life-threatening organ dysfunction caused by a dysregulated host response to infection: 
1. Suspected or proven infection: Sites of infection include thorax, urinary tract, abdomen, skin, sinuses, 
central venous catheters, and central nervous system. 
2. Increase in SOFA score greater than or equal to 2 over baseline. 
Study Drug: Defined as inhaled carbon monoxide or placebo. 
Study hospital: Defined as the hospital where the patient was enrolled. 
Study withdrawal: Defined as permanent withdrawal from study before completion of study activities. 

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This does not include those subjects who have completed the protocol procedures or stopped procedures 
because they have reached unassisted breathing. If a patient or surrogate requests withdrawal from the 
study, the investigators will seek explicit permission to continue data collection. 
UAB (Unassisted Breathing): Spontaneously breathing with face mask, nasal prong oxygen, or room air, 
T-tube breathing, tracheostomy mask breathing, or CPAP ≤ 5 without PS or IMV assistance, or the use of 
noninvasive ventilation solely for sleep-disordered breathing. Assisted breathing is any level of ventilator 
support at pressures higher that the unassisted breathing. 
 
2 
 
INTRODUCTION 
 
2.1 
STUDY RATIONALE  
The purpose of this study is to assess the safety and efficacy of an algorithm-specified dose of inhaled 
carbon monoxide (iCO) to achieve a target COHb level of 6-8% in mechanically ventilated patients with 
sepsis-induced ARDS. By studying subjects with ARDS, we have targeted a disease that has been well 
studied in rodent and non-human primate preclinical models. Furthermore, by focusing on intubated 
subjects with ARDS, we have chosen a group with higher disease burden and thus likely to have both 
increased mortality and an increased opportunity for benefit, including a reduction in the requirement for 
mechanical ventilation. In April 2023, we elected to expand inclusion criteria to include intubated patients 
with pneumonia (unilateral or bilateral pulmonary infiltrates consistent with lung infection), given 
reported similar outcomes in critically ill mechanically ventilated patients with unilateral and bilateral 
infiltrates (LUNG SAFE study). 
 
Mitochondrial dysfunction is associated with increased disease severity and poor outcomes during sepsis 
and may be a key mechanism underlying ARDS and multiple organ dysfunction syndrome in critically ill 
patients5. Furthermore, early activation of mitochondrial biogenesis has been associated with improved 
survival in critical illness6. We and others have demonstrated that circulating mitochondrial DNA (mtDNA) 
levels are significantly increased in patients with sepsis and ARDS and correlate with mortality in critically 
ill patients5,7,8. We have also shown that CO can inhibit mitochondrial reactive oxygen species (ROS) 
generation, inhibit translocation of mtDNA into the cytosol, and preserve mitochondrial function in 
macrophages9. CO also has been shown to activate mitochondrial biogenesis in skeletal muscle in 
humans10. Taken together, these findings suggest that preservation of mitochondrial function may be a 
key mechanism by which CO protects against ARDS and multiple organ failure in critical illness.  
 
2.2 
BACKGROUND  
2.2.1 INTRODUCTION 
Acute respiratory distress syndrome (ARDS) is a syndrome of severe acute lung inflammation and 
hypoxemic respiratory failure with an incidence of 180,000 cases annually in the United States11,12. Despite 
recent advances in critical care management and lung protective ventilation strategies13-16, ARDS 
morbidity and mortality remain unacceptably high3,17,18. Furthermore, no specific effective pharmacologic 
therapies currently exist. Sepsis, life-threatening organ dysfunction caused by a dysregulated host 
response to infection, represents a major risk for the development of ARDS and multi-organ dysfunction 
syndrome (MODS). In recent years, the number of patients with severe sepsis has risen to 750,000 per 
year in the U.S.19-21, which bears an alarming forecast for critically ill patients in the intensive care unit 
with significant risk for the development of ARDS. The lack of specific effective therapies for pneumonia 

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and ARDS indicates a need for new treatments that target novel pathways.  Pneumonia represents a lung 
infection with unilateral or bilateral infiltrates, and intubated patients with pneumonia not formally 
meeting ARDS criteria have been reported to have similar outcomes with unilateral or bilateral infiltrates.  
Pneumonia in intubated patients represents an important spectrum of illness in ARDS, and in fact the 
most recent NIH lung injury consortium to improve phenotyping in critical illness will enroll subjects with 
pneumonia, sepsis, and ARDS.  Furthermore, our preliminary data demonstrating efficacy and safety of 
inhaled CO in unilateral pneumonia in baboons is presented below.  Carbon monoxide (CO) represents a 
novel therapeutic modality in pneumonia and sepsis-induced ARDS based on data obtained in 
experimental models of pneumonia, sepsis, and ARDS over the past decade.  
2.2.2 CO IS ENDOGENOUSLY PRODUCED GAS WITH PLEIOTROPIC BIOLOGICAL FUNCTIONS 
CO is endogenously produced in the body by heme oxygenase-1 (HO-1), one of the few inducible 
molecules that can protect the lung from an increased oxidant burden under circumstances of stress22. 
HO-1 is ubiquitously expressed, and is responsible for degradation of heme to biliverdin, free iron, and 
CO. While all three products of its activity have been shown to possess cytoprotective properties, CO is 
the product that has been most extensively studied with respect to lung disease. This colorless, odorless 
diatomic gas has been shown to exert biological functions as diverse as protection against oxidative 
injury23-25, inflammation26, and cell death27,28, inhibition of cell proliferation29, suppression of matrix 
production30, increased fibrinolysis31, as well as enhanced phagocytosis32,33 and macrophage 
efferocytosis34, all of which may be important in the pathogenesis of sepsis and ARDS. Recently, we have 
demonstrated several mechanisms by which CO exerts these beneficial effects including activation of 
mitochondrial biogenesis35-38, enhancement of autophagy33,39,40, suppression of mitochondrial dysfunction 
and inflammasome activation9, as well as acceleration of resolution of inflammation via production of 
specialized pro-resolving lipid mediators (SPMs)34,38,41-43. 
2.2.3 ADMINISTRATION OF INHALED CO PROTECTS AGAINST SEPSIS AND LUNG INJURY IN 
ANIMAL MODELS 
Our laboratory and others have demonstrated that CO at a low physiological dose confers cytoprotection 
via potent anti-inflammatory, anti-apoptotic, and anti-proliferative effects23-29. These responses are 
regulated by cell- or tissue-specific, and stimuli- or stress-specific signaling pathways44. We and others 
worldwide have demonstrated that CO mediates cytoprotection in a variety of tissue injury models 
including experimental models of sepsis and acute lung injury (ALI) (Table 1)24-26,28,30,32,33,38,45-55. These 
studies have shown that low dose CO confers tissue protective effects in experimental ALI models 
including hyperoxia and endotoxin exposure, bleomycin, ischemia/reperfusion, and ventilator-induced 
lung injury (VILI)24,25,30,45-50.  
 
We have also recently shown that low dose iCO accelerates resolution of ALI in a clinically-relevant baboon 
model of pneumococcal pneumonia38,41,42,56. Administration of iCO at 200 ppm for 60 minutes at 48 hours 
following S. pneumoniae inoculation significantly attenuated histologic lung injury scores and reduced 
lung wet-to-dry ratios at 8 days38. CO-treated animals had significantly increased expression of citrate 
synthase and increased ATP synthase staining in alveolar type 2 cells and macrophages suggesting that 
CO induces mitochondrial biogenesis38. We also found that CO had systemic anti-oxidant effects with 
augmentation of SOD2 expression in the kidney, which co-localized with cytochrome c consistent with 
attenuated mitochondrial injury and cytochrome c retention in intact mitochondria38. CO also reduced 
pro-inflammatory urinary cysteinyl leukotrienes42 and partially restored levels of circulating SPMs41. 
 

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In addition, we and others have demonstrated that CO decreases inflammation, enhances phagocytosis, 
and improves mortality in models of sepsis including endotoxemia26,51,52, hemorrhagic shock53 and cecal 
ligation and puncture (CLP)32,33. Furthermore, CO has been shown to have beneficial therapeutic effects 
in pre-clinical models of other diseases including pulmonary hypertension57-60, vascular injury61-65, and 
transplantation48,49,66-74 (Table 1). 
 
 
 
 
Table 1: Pre-clinical studies of inhaled CO 
Model 
Year 
Species 
CO (ppm) 
Outcome 
Reference 
Lung injury (VILI, 
acid, hyperoxia, 
LPS, pneumonia) 
1999, 2003, 
2004, 2008, 
2009, 2010, 2015 
mouse, rat, 
macaques, 
baboon 
10-250, 500 
Less inflammation & 
lung injury 
24,25,38,45-
47,54 
Bleomycin lung 
fibrosis 
2005 
Mouse 
250 
Decreased lung 
hydroxyproline, 
fibronectin, collagen 
30 
Ischemia-
reperfusion (hind 
leg, lung) 
2001, 2003, 
2006, 2007, 2009 
mouse, rat 
250, 500, 
1000 
Less remote organ 
inflammation, less 
apoptosis, improved 
survival 
28,31,50,75-77 
Transplantation 
(liver, lung, 
kidney, heart, 
intestine) 
2001, 2003, 
2004, 2006, 
2007, 2008, 2009 
mouse, rat 
20, 250, 400, 
500 
Improved survival & 
graft function, less 
inflammation & 
apoptosis 
48,49,66-69,71-
74,78 
Endotoxemia 
2000, 2003, 2004 
rat, mouse 
10-250 
Improved survival, 
decreased 
inflammation 
26,51,52 
Hemorrhagic 
shock 
2005 
Mouse 
250 
Decreased end 
organ 
injury/ischemia 
53 
Cecal ligation and 
puncture 
2008, 2014 
Mouse 
250 
Improved survival, 
decreased 
inflammation, 
enhanced 
phagocytosis 
32,33 
Pulmonary arterial 
hypertension 
2006 
rat, mouse 
50, 250 
Reversal of 
established PAH & 
reversal of 
remodeling 
57 
Vascular injury 
2003, 2005, 
2006, 2007 
mouse, pig 
100, 250-500 
less intimal 
hyperplasia, 
reduced thrombosis 
61-65 
Cardiopulmonary 
bypass 
2004, 2008, 2009 
Pig 
250 
Less lung injury, 
decreased cardiac 
edema, apoptosis 
79-82 

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Model 
Year 
Species 
CO (ppm) 
Outcome 
Reference 
Doxorubicin 
cardiomyopathy 
2007 
Mouse 
500 
Improved cardiac 
function 
83,84 
Asthma 
2003 
Mouse 
250-1000 
Reduced 
inflammation & 
bronchoconstriction 
30,85,86 
Cerebral malaria 
2007 
Mouse 
250 
Reduced incidence 
of cerebral malaria 
87 
Hepatitis 
2003, 2007 
Mouse 
100, 250, 500 
Improved survival, 
decreased apoptosis 
88,89 
Colitis, ileus 
2005 
mouse, rat, 
pig 
250 
Reduced injury & 
inflammation, 
improved motility 
57,90-92 
Sickle cell disease 
2009 
Mouse 
250 
Reduced 
leukocytosis 
93 
Collagen-induced 
arthritis 
2009 
Mouse 
200 
Improved arthritis 
score, less 
inflammation 
94 
Ureteral 
obstruction 
2008 
Mouse 
250 
Reduced renal 
fibrosis 
95 
 
2.2.4 LUNG PROTECTIVE EFFECTS OF CARBON MONOXIDE 
Numerous studies have examined the protective effects of low concentrations of CO on the pulmonary 
parenchyma and vasculature. Inhaled CO prolongs survival and prevents tissue injury and epithelial cell 
death in rodents subjected to high oxygen stress24. CO also reduces lung cell apoptosis during lung 
ischemia-reperfusion injury in mice50 and prevents tissue injury during mechanical ventilation in mice by 
preventing alveolar-capillary barrier dysfunction and reducing inflammation45-47. Low concentration iCO 
can also reverse established pulmonary hypertension in rats57 and has been shown to protect against 
endothelial apoptosis27. In addition, CO has been shown to de-repress fibrinolysis and to inhibit expression 
of plasminogen activator inhibitor-1, which could alter the progression of fibrosis31. CO has also been 
shown to confer protection in a number of additional disease models, including asthma, vascular injury, 
transplantation and fibrosis (Table 1). These studies collectively have provided a rationale for pursuing 
the clinical applications of CO, including the trials outlined below.  
2.2.5 CO DELIVERY VIA AN INHALED ROUTE IS SAFE IN HUMAN SUBJECTS 

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CO has proven to be an ideal gas for developing 
theoretical uptake equations. The formation of 
carboxyhemoglobin (COHb) on the basis of CO 
exposure is well described by a physiologically 
based pharmacokinetics model developed by 
Coburn in 1965 and is referred to as the Coburn-
Foster-Kane equation (typically identified as the 
CFK or CFKE) in the literature96. This model has 
been tested and confirmed in humans for varying 
inspired CO concentrations and durations of 
exposure97-103 (Figure 1).  
 
Extensive data is available regarding the safety 
and tolerability of low dose iCO in healthy 
volunteers10,101,104-108 and more recently, in 
subjects with chronic obstructive pulmonary 
disease (COPD)109 and idiopathic pulmonary 
fibrosis (IPF) (Table 2). Previous studies have 
carefully measured COHb levels in response to iCO 
and demonstrated that low dose iCO is safe in 
healthy normal volunteers10,35,101,104-108. Stewart et al. performed 25 exposures to known CO 
concentrations in healthy volunteers; in this study, exposure to 100 ppm CO for 8 hours resulted in COHb 
levels of 11-13% with no adverse effects in time estimation, steadiness, manual dexterity, EEG, and evoked 
potentials106. In a study aiming to simulate cigarette smoke inhalation, Zevin et al. exposed healthy 
volunteers to iCO (1200-1500 ppm) for 10 minutes, repeating every 45 minutes for 16 hours per day for 7 
days107. In this study, mean COHb levels were 5 ± 1% and no adverse events (AEs) were reported107. 
Pecorella et al. has also demonstrated safety (COHb 5.4 ± 0.79%) and activation of mitochondrial 
biogenesis in healthy individuals after CO inhalation at 200 ppm for 1 hour per day for 5 consecutive 
days35. 
      Table 2: Clinical Trials of CO in Human Subjects 
 
Study 
CO Exposure 
COHb 
Adverse Effects 
Stewart (1970)106 
100 ppm for 8 h 
11-13% 
No adverse effects 
Peterson (1975)101 
50-200 ppm, up to 5.25 h  
1-20% 
None noted 
Hausberg (1997)104 
1000 ppm x 30 min, then 
100 ppm x 30 min  
8.3 ± 0.5% 
None reported 
Zevin (2001)107 
1500 ppm x 10 min, then 
every 45 min x 16 h for 7 
days 
5 ± 1% 
None reported 
Ren (2001)108 
4000 ppm until COHb ~ 
10%, then repeated to 
keep COHb ~ 10% for 8 h 
9.7 ± 0.1% 
None reported 
Mayr (2005)105 
500 ppm for 1 h  
6.5-7.7% 
Mild headache in 1 subject 
Figure 1. COHb levels as a function of exposure 
duration and CO concentration as determined by 
the CFK equation. (Peterson et al., 1975) 

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Rhodes (2009)10 
100 ppm for 1 h for 5 days 
3.3 ± 0.6% 
No adverse effects 
Pecorella (2015)35 
200 ppm for 1 h for 5 days 
5.4 ± 0.79% 
No adverse effects 
Bathorn (2007)109 
125 ppm for 2 h, 4 
consecutive days (COPD) 
2.1-3.4% 
2 COPD exacerbations, judged 
unrelated 
NCT00094406 (IND# 
70,694) 
100 ppm for 6 h 
6.5 ± 1.7% 
No adverse effects 
Rosas (2017)110 
100-200 ppm x 2 h, 2 times 
weekly x 12 wks (IPF) 
3-4% 
Well tolerated, no SAEs related 
to CO  
Fredenburgh 
(2018)111,112 
100 or 200 ppm for 90 min 
for up to 5 days 
3.48 ± 0.7% (100 ppm) 
4.9 ± 0.28% (200 ppm) 
Well tolerated, no SAEs related 
to CO 
 
In a study evaluating the effects of hypoxemia, hemodilution, and carboxyhemoglobinemia on respiratory 
control, Ren et al. exposed 11 normal volunteers to an iCO treatment regimen aiming to maintain a COHb 
level of 10% for 8 hours108. COHb levels ranged from 9.1 to 10.5% (mean 9.7%) and no AEs were 
reported108. Similar results have been published in a number of other studies, and none have reported 
AEs10,35,101,104,105. In fact, baseline COHb levels of 3% have been reported in some urban areas106 and levels 
as high as 10-15% may be observed in asymptomatic chronic smokers113-115. 
 
We have conducted three studies, one in normal human subjects (NCT00094406), one in subjects with IPF 
(NCT01214187)110, and one recently completed Phase I trial in sepsis-induced ARDS (NCT02425579)112. In 
our placebo-controlled study of 24 healthy volunteers (11 females; mean age 26.2 ± 5.2 yrs), individuals 
were randomized to treatment with room air or iCO (100 ppm) for 6 hours following endotoxin instillation. 
CO treatment was well tolerated and mean COHb levels were 6.5% ± 1.7% in CO-treated subjects. There 
were no significant differences in vital signs, neurocognitive studies (including immediate and delayed 
memory, attention, language and visuospatial/constructional function), or AEs (Table 3).  
 
                   Table 3: Vital Signs and Laboratory Parameters in CO-Treated Healthy Volunteers. 
Parameter 
Room Air 
CO (100 ppm) 
p value 
Respiratory rate 
17.7 ± 3.6 
16.7 ± 4.0 
0.5 
Heart rate 
84.5 ± 14 
83.8 ± 15 
0.9 
Temperature °C 
36.9 ± 0.4 
37.2 ± 0.4 
0.15 
Mean arterial pressure (mm Hg) 
92.8 ± 11 
98.7 ± 11 
0.21 
Carboxyhemoglobin (%) 
1.1 ± 0.7 
6.5 ± 1.7 
0.001 
Oxyhemoglobin (%) 
96.3 ± 2.1 
92.3 ± 1.9 
0.002 
PaO2 
96.5 ± 11 
94 ± 7.3 
0.54 
Arterial pH 
7.4 ± 0.02 
7.41 ± 0.02 
0.29 
Lactate (mmol/L) 
0.7 ± 0.26 
0.62 ± 0.19 
0.41 
 
In addition to studies in healthy volunteers, prior work has demonstrated the feasibility of administering 
low dose iCO to subjects with COPD109. In this study, ex-smoking subjects with stable COPD were treated 
with iCO at 100-125 ppm for 2 hours per day on 4 consecutive days. This led to COHb levels of 2.1-3.4% 
with a maximal individual COHb level of 4.5%. Inhalation of CO by subjects with stable COPD led to trends 
in reduction of sputum eosinophils and improvement in methacholine responsiveness109. In our recently 
completed 8-center Phase I/II trial of low dose iCO in IPF110,116, 58 subjects were randomized to treatment 
with iCO (100-200 ppm) or 21% O2 for 2 hours, twice weekly for 12 weeks. Inhaled CO was well-tolerated 

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with no significant difference in AEs or neurocognitive function by the Montreal Cognitive Assessment 
(MoCA) between iCO-treated and control subjects110. 
 
More recently, we completed a Phase I trial of low 
fixed-dose iCO in patients with sepsis-induced 
ARDS111,112. Twelve participants were randomized 
to iCO or placebo air 2:1 in two cohorts. Four 
subjects each were administered iCO (100 ppm in 
Cohort 1 or 200 ppm in Cohort 2) or placebo for 90 
minutes for up to 5 consecutive days. CO treatment 
was well tolerated and no participants exceeded a 
COHb level of 10%. There were no administration-
associated AEs or study-related serious AEs (SAEs). 
Baseline COHb levels were not different across 
treatment groups. CO-treated participants had a 
significant increase in COHb (3.48 ± 0.7% [Cohort 
1]; 4.9 ± 0.28% [Cohort 2]) compared with placebo-
treated subjects (1.97 ± 0.39%), which did not differ 
from baseline levels (Figure 2). The maximum COHb 
level in Cohort 1 was 4.4% and 6.87% in Cohort 2. The maximum change in COHb from baseline to 90 
minutes (Δ 90 min COHb) was 2.5% in Cohort 1 and 4% in Cohort 2. There was no significant increase in 
COHb in placebo subjects.  
 
Taken together, these findings demonstrate that experimental administration of several different 
concentrations of CO is well tolerated and that low dose iCO can be safely administered to subjects in a 
controlled research environment.  
2.2.6 STEADY STATE DIFFUSING CAPACITY AND THE SAFETY OF INHALED CARBON MONOXIDE 
CO is also a diagnostic gas that has been used for more than a century to evaluate lung function, and in 
particular, in the steady state diffusing capacity test to determine the function of the alveolar-capillary 
membrane. The steady state method of measuring the diffusing capacity of the lung dates back more than 
100 years, based on the research of Haldane and Smith. In the early 1900s, Krogh and Barcroft developed 
it into a standard test procedure for both understanding alveolar membrane function, as well as for 
diagnosing diseases of the alveolar-capillary membrane. Up until the 1970s, steady state diffusing capacity 
was the standard diagnostic test for pulmonary laboratories worldwide, at which point it was replaced by 
the single breath diffusing capacity test due to its better accuracy and being less time consuming.  
 
The steady state diffusing capacity test is still used today in some instances where the single breath 
procedure is not as practical, such as measurements during exercise. The procedure for steady state 
diffusing capacity testing entails patients inhaling 0.1% CO (1000 ppm) for seven minutes117. As duplicate 
or triplicate measurements are required for most lung function tests, this suggests that hundreds of 
thousands of people have inhaled 1000 ppm for 14 to 21 minutes with no known reports of adverse events 
associated with the test. Based on the curves from the CFK equation (Figure 1), it is likely that this same 
number of people had their COHb levels raised above 3-6% following the diagnostic procedure. This 
longstanding diagnostic procedure reinforces that inhaling a constant concentration of low dose CO can 
be safely done without significant adverse events. 

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2.2.7 DELIVERY OF INHALED CO TO MECHANICALLY VENTILATED SUBJECTS 
In order to study inhaled CO in mechanically ventilated 
subjects with pneumonia and sepsis-induced ARDS, a 
Carbon Monoxide Delivery System (COventDS) (Figure 3) 
developed by 12th Man Technologies will be used 
(Appendix A- CO Delivery System). The CO Delivery 
System 
is 
a 
microprocessor-based 
constant 
gas 
concentration delivery system that can be used to deliver 
operator-specified concentrations of CO to mechanically 
ventilated patients. The CO Delivery System delivers an 
operator set, constant concentration of CO gas into the 
inspiratory path of the patient breathing circuit 
independent of the patient’s inspiratory flow, while the 
patient’s respiration is supported by a ventilator. For safety 
reasons, the CO Delivery System has twin microprocessors 
such that the division of control is split between a closed loop proportional-integral-derivative (PID) 
controlled mixing module that is only involved with monitoring the patient flow and mixing of the gases, 
and an interface module that is the working face to the user for control and monitoring functions. This 
second microprocessor watches the delivery subsystem, monitors the inspired gas for deviations from the 
set concentration, and monitors inspired oxygen.  
 
The heart of the system is comprised of three components including an inspiratory flow monitor with a 
ratio-metric matching CO injector module, an inhaled gas monitoring module, and a gas mixing subject 
interface/breathing valve for spontaneously breathing subjects. It is a variable inspiratory flow delivery 
system that matches the patient’s inspiratory flow with the injected 5000 ppm CO to deliver the operator 
set CO concentration on the LCD user interface. It is a breathing-initiated delivery system and the CO is 
blended into the inspiratory gas stream only as long as flow is being delivered to the patient and at the 
exact proportions to maintain the desired concentration, independent of any change in breathing pattern, 
flow rate, respiratory rate, or tidal volume. The analyzer, with alarm functions, monitors the inhaled CO 
and O2 concentrations. 
 
The CO Injector is a core component for the delivery of CO. The CO Injector is constructed of CO-
compatible materials and consists of a pressure regulation circuit that reduces the 40-60 psig inlet CO gas 
source to the optimal pressure for its proportional flow control valve. Upon sensing inspiratory flow by 
the patient with the flow monitoring interface, the injector module will track the flow and match the 
volume of CO injected to the volume of inspired gas to keep the concentration of CO constant 
independent of the patient’s pattern of inspiratory flow. The PID controlled mixing module’s sole function 
is to read the patient’s inspiratory flow and inject CO proportional to that flow in 1 millisecond intervals. 
Alarms will sound on high or low CO or O2 concentrations. Should the inspired CO concentration rise above 
660 ppm, in addition to the alarms, the system will stop injecting CO into the circuit. 
2.2.8 TESTING THE CO DELIVERY SYSTEM IN BABOON MODEL OF PNEUMOCCAL PNEUMONIA 
Figure 3. CO Delivery System (12th Man 

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We evaluated the safety and efficacy of the CO Delivery System developed by 12th Man Technologies in a 
baboon model of S. pneumoniae pneumonia (Figure 4). Five juvenile, male colony-bred baboons (Papio 
cynocephalus) were intubated, sedated, and mechanically ventilated38. The animals underwent 
bronchoscopy and a baseline bronchoalveolar lavage (BAL) was performed followed by instillation of 
Streptococcus pneumoniae (108 - 109 CFU) in the right and left lower lung zones. At 24 or 48 hours post-
inoculation, animals were sedated, intubated, ventilated, and underwent a repeat bronchoscopy and BAL. 
The CO Delivery System was calibrated using 100 ppm and 400 ppm CO tanks and readied for use with a 
5000 ppm CO source cylinder. All CO tanks contained CO gas at the specified concentration in air. Ambient 
CO levels were monitored during the CO delivery device assembly, calibration, and continuously 
throughout the experiment using a CO detector.  
 
Following bronchoscopy, animals were administered iCO at 200 ppm through the ventilator via the CO 
Delivery System for 60-90 minutes. After iCO treatment was completed, animals were administered 
supplemental FiO2 for 60-90 minutes until COHb levels returned to near baseline levels. Arterial blood was 
drawn before, during, and after CO delivery at 10-15 minute intervals and arterial blood gas (ABG) and 
COHb measurements were performed. In certain experiments, both venous and arterial blood samples 
were drawn simultaneously for measurement of venous and arterial COHb levels respectively (Figure 5). 
COHb levels were measured using the IL 682 Co-oximeter and, in certain experiments, using the 
AVOXimeter 4000 Co-oximeter. After CO exposure, animals were administered ceftriaxone daily for a total 
of 3 days.  
 

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After one hour of iCO administration at 200 ppm, animals achieved the pre-specified goal COHb level of 
6-8%. At baseline, arterial COHb levels were 1.1 ± 0.2% 
and increased linearly to 2.4 ± 0.6%, 3.7 ± 0.5%, 4.5 ± 
0.7%, 5.5 ± 0.8%, 6.6 ± 0.9%, and 7.6 ± 1% at 10, 20, 
30, 40, 50 and 60 minutes of iCO administration38, 
respectively (p<0.0001) (Figure 4A). One animal was 
intentionally given a prolonged (90 minute) exposure 
which similarly demonstrated a linear rise in COHb to 
7.8%, 8.8%, 9.7%, and 10.3% at 60, 70, 80, and 90 
minutes, respectively. Peak COHb levels decreased 
following administration of FiO2 1.0, returning to 
near baseline levels after 82 ± 9.5 minutes38 (Figure 
4B). Throughout the exposure, ambient CO levels 
remained ≤1 ppm. Low dose iCO treatment was 
well-tolerated with no significant differences in pre- 
and 
post-CO 
heart 
rate, 
blood 
pressure, 
temperature, PaO2, or minute ventilation.  
 
Using the IL 682 Co-oximeter, we found that venous and arterial COHb levels were highly correlated 
(r=0.9904, p<0.0001)38. Modeling these data with type II linear regression, we found that the regression 
line was a near-perfect diagonal with a slope of 1.003 (95% CI [0.952 – 1.05]) and a y-intercept of -0.064 
(95% CI [-0.2941 – 0.1661])38 (Figure 5). This tight correlation argues that venous measurements are as 
accurate and reliable as arterial measurements. To determine the accuracy and precision of the point-of-
care AVOXimeter 4000 Co-oximeter relative to the gold-standard IL 682 Co-oximeter, we measured COHb 
levels using both devices on synchronously drawn arterial blood. Compared with the IL 682 Co-Oximeter, 
the AVOXimeter 4000 Co-oximeter was substantially less sensitive as COHb was not detected until levels 
had reached 4-5%. Furthermore, the AVOXimeter consistently reported COHb levels that were four 
percentage points lower than the gold standard (Figure 6).  
Ambient CO levels in the experiment room remained at 0-1 ppm during assembly, calibration, and use of 
the CO delivery device. These levels are well below the OSHA permissible exposure limit of 50 ppm as an 
8 hour time-weighted average (U.S. Department of Labor, Occupational Safety & Health Administration, 
https://www.osha.gov/chemicaldata/chemResult.html?RecNo=462).  
2.2.9 CO DOSING STRATEGY USING CFK EQUATION 
In order to develop a safe and effective dosing strategy based on an initial short exposure to inhaled CO, 
we also evaluated the accuracy of the Coburn-Forster-Kane (CFK) equation (below)96,101 to predict COHb 
levels in baboons using measured COHb levels following a 10 and 20 minute CO exposure. Using the 10 
minute COHb, we found that 
there was good correlation 
between measured COHb levels 
and the COHb levels predicted 

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by the CFK equation (r=0.9038, p<0.0001)38. However, there was superior correlation between measured 
COHb levels and COHb levels predicted by the CFK equation using the 20 minute COHb (r=0.9828, 
p<0.0001)38 (Figure 7A).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Modeling these data with linear regression, we found that the regression line was a near-perfect diagonal 
with a slope of 0.9373 (95% CI [0.8885 – 0.9860]), a y-intercept of 0.7291 (95% CI [0.3976 – 1.061]), and 
goodness-of-fit R2=0.9864 (p<0.0001)38. Furthermore, the 20-minute measured COHb was highly accurate 
in predicting the 60 minute COHb with a difference between predicted and measured COHb of 0.24 ± 
0.33% (95% CI [-0.17 – 0.66])38 (Figure 7B). Taken together, by inputting the 20-minute measured COHb 
level into the CFK equation, we can predict the COHb level after 60 minutes of CO exposure with high 
accuracy.  
 
In our recently completed Phase I fixed dose iCO trial, we also found an excellent correlation between 
measured and predicted COHb levels using the CFK equation112. While the CFK equation is a well-validated 
model of CO uptake that accurately predicts the rise in COHb following CO exposure in humans with 
normal lung function97-103, it had not been previously used in ARDS patients with impaired gas exchange 
before. We found an excellent correlation in participants treated with 100 ppm iCO (Spearman r= 0.8614; 
p<0.0001), but an even stronger correlation in subjects treated with 200 ppm iCO (Spearman r=0.916; 
p<0.0001) (Figure 8A). Modeling the 200 ppm Cohort 2 data with linear regression revealed a slope of 
1.112 (95% confidence interval [CI] [1.003, 1.22]), a y-intercept of –0.6632 (95% CI [–1.174, –0.1522]), and 
goodness-of-fit R2=0.9204 (p<0.0001). Bland-Altman plots also demonstrated excellent agreement 
between measured and CFK equation-predicted COHb levels (Figure 8B) with a mean difference between 
measured and predicted COHb levels of 0.1467 ± 0.2738 in Cohort 2 (200 ppm).  
 
The above predictions using the CFK equation were made using a MATLAB-generated computer program 
to estimate DLCO using the baseline and 20 minute COHb. The estimated DLCO was then input into the 
programmed CFK equation and used to predict the 60, 75, and 90 minute COHb levels. This program was 
validated by generating the previous published curves (Figure 1) that were derived from predicted values 
using the CFK equation101.  

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CFK Equation For Precision-Based iCO Dosing  
Our Phase I fixed-dose iCO study showed that 
precise delivery of low dose iCO is feasible and safe 
in mechanically ventilated ARDS patients. However, 
as anticipated, patients with ARDS of varying 
severity had different degrees of CO uptake and did 
not achieve the target COHb level of 6-8% with fixed 
iCO dosing, as was observed in our baboon ALI 
model. These results were predictable, based on key 
variables in the CFK equation that influence CO 
uptake. These data suggest that patients with ARDS 
require a personalized approach to iCO dosing to ensure optimal, consistent, and safe CO uptake in the 
setting of variable gas exchange. To estimate the range of iCO doses that may be required in ARDS 
patients, we used the CFK equation to predict COHb levels for a range of CO concentrations and exposure 
times using mean values for DLCO, VA, weight118-123, Hgb, and FiO2 in patients with ARDS (Table 4). This 
modeling demonstrated that the average ARDS subject will likely require doses in the range of 200-500 
ppm to achieve a target COHb level of 6-8%. 
 
Given the severe diffusion impairment and variable degree of CO uptake in patients with ARDS, we have 
developed an iCO dosing strategy using the CFK equation to personalize iCO dosing based on each 
patient’s unique lung physiology. Our prior Phase I fixed dose iCO trial results show that the CFK equation 
is highly accurate at predicting COHb levels, suggesting that the CFK equation can be used in a novel 
personalized medicine approach to determine in real-time an optimal and safe iCO dose to achieve a 
target COHb level of 6-8% in ARDS patients with varying degrees of impaired gas exchange. This 
randomized, partially double-blind, placebo-controlled Phase Ib trial will evaluate the safety and accuracy 
of a novel CFK equation-based personalized iCO dosing approach in patients with sepsis-induced ARDS. 
 
2.3 
RISK/BENEFIT ASSESSMENT   
 
2.3.1 KNOWN POTENTIAL RISKS  
 

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Potential risks of active study drug include headache and tachycardia. In cases of overdose, patients can 
have nausea, vomiting, seizures, problems thinking, coma, cardiopulmonary arrest, and death. These 
adverse effects are seen at doses much higher than those proposed in this study. Subjects will be vigilantly 
monitored for side effects during study drug administration and COHb and lactate levels will be carefully 
monitored throughout the study. There may be other risks of iCO in patients with ARDS that are currently 
unknown. Subjects will be monitored closely throughout their participation in the trial. In our recently 
completed Phase I trial, no protocol-specified iCO administration-related AEs occurred in 12 subjects, and 
the Data and Safety Monitoring Board (DSMB) has recommended moving forward with Phase Ib and Phase 
II studies after three interim analyses. 
 
Although there is a risk of toxicity associated with inhalation of high concentrations of CO, low dose iCO 
has been shown to be protective in animal models and safe in human subjects. Data from animal studies 
demonstrate that iCO has beneficial effects on outcomes in sepsis and ALI. Furthermore, extensive data 
is available regarding the safety and tolerability of low dose iCO in healthy volunteers and more recently, 
in subjects with COPD and IPF. Subjects enrolled in the study will receive the same standard of care that 
is otherwise available to non-study patients in an ICU setting.  
 
Risks of blood draws 
All subjects will have blood drawn for research purposes. Most blood will be drawn through existing 
indwelling catheters. Risks of drawing blood percutaneously are uncommon and include bleeding and 
bruising.  
 
Risks of radiation exposure 
Most chest X-rays will be performed daily as part of usual ICU care in intubated patients. However, if not 
performed, we will obtain chest X-rays for endpoint assessment on study days 1-5 and study day 7. The 
risk of ionization radiation from routine chest X-rays is generally considered minimal due to the low dose 
(~0.1 milliSievert [mSv]).  
 
Risks to privacy and/or confidentiality 
There is a very low risk of loss of confidentiality but this should be mitigated by the exclusive use of coded 
samples. All study personnel with access to samples or data will be required to undergo HIPAA and CITI 
training as per institutional policies. Subject confidentiality will be protected throughout the study and no 
subject-identifying information will be released to anyone outside the study. Confidentiality will be 
maintained as detailed above. 
 
Subjects will not be identified by name in any reports or publications, nor will the data be presented in 
such a way that the identity of individual subjects can be inferred. Analysis files created for further study 
by the scientific community will have no subject identifiers. Clinical information will not be released 
without the written permission of the subject, except as necessary for monitoring by the Clinical 
Coordinating Center (CCC), Data Coordinating Center (DCC), single IRB (sIRB), NHLBI, FDA or other 
authorized federal agencies. We will use methods that are scientifically rigorous and valid and in 
accordance with GCP guidelines. All research personnel have completed training in the Responsible 
Conduct of Research and the Protection of Human Research Subjects including GCP training.  
 
2.3.2 KNOWN POTENTIAL BENEFITS  
 
Study subjects may or may not receive any direct benefits from their participation in this study. Our 
preclinical data demonstrate that low dose iCO is protective in experimental models of sepsis and ALI. 

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Numerous studies have shown CO to be cytoprotective in animal models of lung injury including hyperoxia 
and endotoxin exposure, bleomycin, ischemia/reperfusion, pneumococcal pneumonia, and VILI24,25,30,45-
50,54. Furthermore, CO has been shown to decrease inflammation, enhance phagocytosis, and improve 
mortality in models of sepsis including endotoxemia26,51,124, hemorrhagic shock53,125-127, and CLP32,33,43. In 
addition, CO has been shown to have beneficial therapeutic effects in pre-clinical models of other diseases 
including 
traumatic 
brain 
injury 
(TBI)128, 
kidney 
fibrosis95, 
colitis90,92, 
arthritis94, 
organ 
transplantation48,49,66-74, hepatitis88,89, and vasculopathies of the heart and lung57,62-64,84. 
 
Potential benefits from the administration of iCO include decreased requirement for ventilatory support, 
decreased days spent in the ICU, increased organ failure free days, and enhanced survival.  
 
In addition to the potential benefit of iCO administration, all subjects, including those randomized to 
placebo, will benefit from protocolized low tidal volume ventilation and weaning protocols, as these have 
been shown to be beneficial in prior studies and are recommended in evidence-based consensus 
guidelines as best practice. As this protocol incorporates best practice guidelines, participation in the 
study will reduce the unexplained or potentially harmful variability in the application of such guidelines, 
and thus likely improve care, for all participants in our trial, including those participants randomized to 
placebo. Furthermore, there may be salutary effects on all participants given the additional clinical 
personnel and monitoring that will occur during administration of the study products. 
 
Finally, there are potential benefits to society as the discovery of effective therapies that can reduce the 
substantial morbidity and mortality of ARDS or the identification of biomarkers that might improve 
prediction and monitoring of ARDS disease severity would enhance the health of society. 
 
2.3.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
 
Federal regulations at 45 CFR 46. 111 (a)(2) require that “the risks to subjects are reasonable in relation 
to anticipated benefits, if any, to subjects, and the importance of the knowledge that may reasonably be 
expected to result.” There are several elements of study design in the present protocol that meets this 
human subject protection requirement. Several of the exclusion criteria prohibit participation of patients 
who might be at increased risk from the effects of iCO. These include individuals with severe hypoxemia, 
acute myocardial infarction (MI), stroke within 1 month, cardiac arrest requiring CPR within 72 hours, or 
inability to assess mental status following cardiac arrest, angina pectoris with activities of daily living, 
cardiopulmonary disease (NYHA class IV), as well as women who are pregnant or breastfeeding. In 
addition, to ensure the safety of subjects and minimize potential risks related to the study drug 
administration, our study protocol includes stringent monitoring of subjects, provisions for daily hold 
parameters, criteria for interruption of dosing, and permanent discontinuation of the study drug. 
 
Based on the preceding assessment of risks and potential benefits, the risks to subjects are reasonable in 
relation to anticipated benefits, and commensurate with those inherent in the subjects' expected critical 
illness requiring ICU care. There are currently no alternative available approaches to treat intubated 
patients with pneumonia or ARDS itself, besides supportive care, treatment of the underlying disease (eg. 
infection), and lung protective ventilator strategies. In addition, there are currently no biomarkers to 
assess disease severity and outcomes in pneumonia or ARDS. The potential risks of participation include 
those related to blood draws, chest X-rays, and those related to iCO treatment.  
 
CO Treatment: Although there is a risk of toxicity associated with inhalation of high concentrations of CO, 
low dose iCO has been shown to be protective in animal models and safe in humans. No protocol-specified 

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iCO administration-related adverse events occurred in 12 subjects in our prior Phase I fixed-dose iCO trial, 
and the DSMB recommended advancing iCO to Phase Ib and Phase II sepsis and ARDS studies after three 
interim analyses. Data from animal studies demonstrate that iCO has beneficial effects on outcomes in 
sepsis and ALI. There is potential benefit to society and individual patients should iCO treatment prove to 
be beneficial for future patients with pneumonia,  sepsis, and ARDS. 
 
Subjects will be vigilantly monitored for side effects during drug administration and COHb and lactate 
levels will be carefully monitored as outlined. There may be other risks of iCO in patients with ARDS that 
are currently unknown. Subjects will be monitored closely throughout their participation in the trial. 
 
Blood draws: All enrolled subjects will have blood drawn for both safety monitoring and research 
purposes. Most blood will be drawn through indwelling catheters. Risks of drawing blood percutaneously 
are uncommon and include bleeding and bruising. The risks associated with this common clinical practice 
are small, whereas the knowledge gained in furthering our understanding of ARDS and development of a 
potential new therapy for patients with ARDS may be substantial. 
 
Radiation Exposure: Most chest X-rays will be performed as part of usual ICU care. The risk of ionization 
radiation from routine chest X-rays is generally considered minimal due to the low dose (~0.1 milliSievert 
[mSv]).  
 
3 
OBJECTIVES AND ENDPOINTS 
 
OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
To evaluate the safety and 
accuracy of a CFK equation-
based personalized iCO dosing 
algorithm in patients with 
pneumonia and/or sepsis-
induced ARDS. 
 
 
1. Safety of iCO, defined by the incidence 
of pre-specified administration-related 
AEs and SAEs. 
 
Administration-related SAEs are: 1) 
myocardial infarction; 2) stroke; 3) new 
onset arrhythmia requiring DC 
cardioversion; 4) worsening 
hypoxemia1,2; 5) COHb ≥ 10%; and 6) 
increase in lactate by ≥ 2 mmol/L. 
 
2. Accuracy of the CFK equation-based 
personalized iCO dosing algorithm. This 
will be assessed by comparing the 
measured COHb level at 90 minutes and 
the target COHb level of 6-8%. 
1. Safety of iCO: These 
administration-related SAEs 
were used in our prior Phase 
I fixed-dose iCO trial and are 
SAEs that could potentially 
occur if there were 
decreased delivery of 
oxygen. A COHb level ≥ 10% 
was chosen as an 
administration-related SAE as 
the goal COHb is 6-8%. 
 
2. Accuracy of personalized 
iCO dosing approach: The 
accuracy of the iCO dosing 
algorithm to achieve the 
target COHb level (6-8%) will 
be evaluated. 
Secondary 
 
 
To determine the effect of low 
dose iCO therapy on clinical 
outcomes in patients with 
1. Lung Injury Score (LIS) on days 1-5 and 
day 7 
2. PaO2/FiO2 on days 1-5 and day 7 
LIS, PaO2/FiO2, OI, and Vd/Vt 
have been shown to 
correlate with VFDs and 

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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
pneumonia and/or sepsis-
induced ARDS. 
  
3. Oxygenation index (OI) on days 1-5 
and day 7 
4. Dead space fraction (Vd/Vt) on days 1-
3 and day 7 
5. Sequential Organ Failure Assessment 
(SOFA) scores on days 1-5, 7, 14, and 28 
6. Ventilator-free days (VFDs) to day 28 
7. ICU-free days to day 28 
8. Hospital-free days to day 60 
9. Hospital mortality to days 28 and 60  
10. Neurocognitive function at 3 and 6 
months 
mortality in prior ARDS 
studies. 
 
Exploratory  
 
 
To examine the effects of low 
dose iCO therapy on biologic 
readouts in patients with 
pneumonia and/or sepsis-
induced ARDS. 
Blood and urine biomarkers of 
mitochondrial dysfunction (mtDNA), 
inflammasome activation (IL-18) and 
necroptosis (RIPK3). 
Lipid mediators (LM) and specialized pro-
resolving mediators (SPMs). 
Our preliminary data suggest 
that CO may modulate these 
biomarkers. 
 
4 
STUDY DESIGN  
 
4.1 
OVERALL DESIGN 
This is a multi-center, randomized, partially double-blind, and placebo-controlled Phase Ib safety clinical 
trial of inhaled CO (iCO) for the treatment of pneumonia and/or sepsis-induced ARDS. We hypothesize 
that low dose iCO will be safe and well-tolerated and that the CFK equation-based iCO personalized dosing 
algorithm will be accurate in achieving a target COHb level of 6-8% in patients with pneumonia and/or 
sepsis-induced ARDS. 
 
Intubated subjects (n=36) with pneumonia and/or sepsis-induced ARDS who have consented to 
participate will be randomized in a 2:1 ratio to treatment with iCO (n=24) or medical grade air (n=12) for 
90 minutes daily for 3 consecutive days. Subjects will be administered iCO or placebo air via inhalation 
through the mechanical ventilator using the CO Delivery System (COventDS, 12th Man Technologies). 
Subjects randomized to CO will be administered iCO at an individualized dose between 200-500 ppm as 
determined by a personalized iCO dosing algorithm to achieve a COHb level of 6-8%. Subjects randomized 
to placebo will receive medical grade air through the COventDS from identical appearing gas cylinders. 
The study drug will be administered for up to 3 days following randomization or until discontinuation of 
mechanical ventilation, whichever occurs first. For patients who have a tracheostomy, the equivalent of 
extubation for the purposes of this protocol will be breathing via tracheostomy with unassisted breathing.  
 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 
 
Low dose iCO has been shown to be safe and well-tolerated in Phase I/II trials10,35,101,104-110. Our group has 
developed and tested a ventilator-compatible CO Delivery System and a CO dosing strategy in a non-

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human primate model of pneumonia-induced ALI38. We demonstrated that low dose iCO can be safely 
delivered to mechanically ventilated baboons with ALI, and that COHb levels can be accurately predicted 
using the CFK equation38. We recently completed a fixed dose Phase I trial of iCO in sepsis-induced ARDS 
(NCT02425579), which showed that precise delivery of low dose iCO is feasible and safe in mechanically 
ventilated ARDS patients112. As anticipated, we found that patients with ARDS of varying severity had 
different degrees of CO uptake with fixed iCO dosing. We demonstrated that the CFK equation is highly 
accurate at predicting COHb levels, suggesting that the CFK equation can be used to individually titrate 
iCO dosing to ensure consistent and safe systemic uptake in ARDS patients with varying degrees of 
impaired gas exchange.  
 
This Phase Ib safety trial is to evaluate a novel CFK equation-based iCO dosing algorithm to safely and 
accurately administer low dose iCO in a personalized medicine approach in patients with pneumonia 
and/or sepsis-induced ARDS that follows our pre-clinical animal studies submitted to the FDA, as well as 
our published phase 1 safety study. This innovative approach will allow us to determine an optimal and 
safe dose in real-time for patients with pneumonia, sepsis, and ARDS. Importantly, this study affords a 
unique opportunity to investigate functional biological signatures underlying the beneficial effects of iCO 
and their correlation with clinical outcomes in pneumonia, sepsis, and ARDS. We hypothesize that 
precision-based iCO therapy will be safe, well-tolerated, and reduce the severity of pneumonia and ARDS 
and organ failure by suppressing mitochondrial dysfunction, inhibiting inflammasome activation and 
necroptosis, and accelerating resolution of inflammation.  
 
4.3 
JUSTIFICATION FOR DOSE 
 
Subjects randomized to CO will be administered algorithm-specified iCO dose (not to exceed 500 ppm) to 
achieve a COHb of 6-8% for up to 90 minutes for three consecutive days. The decision to target a COHb 
level of 6-8% is based on animal studies demonstrating protection in sepsis and ALI models (Table 1)24-
26,28,30,32,33,38,45-55 and human studies demonstrating safety (Table 2)35,101,104,105,107,108,129 at these COHb 
levels. 
 
Given diffusion impairment in patients with ARDS, it is anticipated, based on CFK equation predictions 
(Table 4), that subjects will require doses in the range of 200-500 ppm to achieve COHb levels of 6-8%. 
The decision to limit the maximal dose to 500 ppm is based on concern for potential epithelial toxicity at 
inhaled concentrations above 500 ppm. Previous studies have demonstrated safety of CO inhalation at 
500 ppm in humans105 and reduction of pulmonary neutrophilia in non-human primates54, therefore we 
do not anticipate epithelial toxicity at our maximal dose. 
 
The rationale for once-daily dosing as opposed to continuous inhalation, as with inhaled NO or other 
pulmonary vasodilators, is based on pre-clinical studies showing that CO treatment for short periods of 
time 
induces 
transcriptional 
programs 
of 
mitochondrial 
biogenesis36,40,41,130 
and 
induces 
autophagy33,39,40,131-133 leading to downstream protection without the need for continuous inhalation. In 
addition, prior work from our group demonstrated safety and activation of mitochondrial biogenesis in 
healthy individuals after three consecutive days of treatment10,35. Once-daily iCO dosing will also optimize 
subject safety by ensuring that COHb levels do not exceed our target range of 6-8%, and that CO 
accumulation will not occur as treatments are separated by 4-6 half-lives for CO elimination134-136.  
 
4.4 
END OF STUDY DEFINITION 

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A participant is considered to have completed the study if he or she has completed all phases of the study 
including the last visit or the last scheduled procedure shown in the Schedule of Activities (SoA), Section 
1.3. The end of the study is defined as completion of the last visit or procedure shown in the SoA in the 
trial globally. 
 
5 
STUDY POPULATION 
 
5.1 
INCLUSION CRITERIA 
All patients (age 18 and older) will be eligible for inclusion if they meet all of the following consensus 
criteria for sepsis and ARDS3,4 or if they meet the criteria for pneumonia as described below.  
• 
Patients with sepsis are defined as those with life-threatening organ dysfunction caused by a 
dysregulated host response to infection: 
1. Suspected or proven infection: Sites of infection include thorax, urinary tract, abdomen, 
skin, sinuses, central venous catheters, and central nervous system 
2. Increase in Sequential Organ Failure Assessment (SOFA) Score ≥ 2 over baseline 
 
• 
ARDS is defined when all four of the following criteria are met:  
1. A PaO2/FiO2 ratio ≤ 300 with at least 5 cm H2O positive end-expiratory airway pressure 
(PEEP) 
2. Bilateral opacities on frontal chest radiograph (not fully explained by effusions, lobar/lung 
collapse, or nodules) within 1 week of a known clinical insult or new or worsening 
respiratory symptoms  
3. A need for positive pressure ventilation by an endotracheal or tracheal tube  
4. Respiratory failure not fully explained by cardiac failure or fluid overload; need objective 
assessment (e.g., echocardiography) to exclude hydrostatic edema if no risk factor is 
present  
 
• 
Pneumonia (without ARDS or sepsis) will be defined as a unilateral or bilateral lung infiltrate on 
chest X-ray or chest CT (not fully explained by effusions, lobar/lung collapse or nodules) in the 
setting of receiving mechanical ventilation, a new suspected respiratory infection, an increase in 
SOFA score less than 2 at the time of randomization (baseline).   
 
• 
Pneumonia (with sepsis, without ARDS) will be defined as a unilateral or bilateral lung infiltrate 
on chest X-ray or chest CT (not fully explained by effusions, lobar/lung collapse or nodules) in 
the setting of receiving mechanical ventilation and a new suspected respiratory infection with 
an increase in SOFA score of ≥ 2 over baseline at the time of randomization. Pneumonia with 
bilateral opacities, PaO2/FiO2 ratio ≤ 300, or an increase in SOFA score greater than or equal to 2 
over baseline will continue to be considered ARDS and sepsis.   
 
5.2 
EXCLUSION CRITERIA 
An individual who meets any of the following criteria will be excluded from participation in this study: 
1. Age less than 18 years 
2. Greater than 168 hours since ARDS or pneumonia with mechanical ventilation onset  
3. Pregnant or breastfeeding  
4. Prisoner 

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5. Patient, surrogate, or physician not committed to full support (exception: a patient will not be 
excluded if he/she would receive all supportive care except for attempts at resuscitation from 
cardiac arrest) 
6. No consent/inability to obtain consent or appropriate legal representative not available  
7. Physician refusal to allow enrollment in the trial 
8. Moribund patient not expected to survive 24 hours 
9. No arterial line or central line/no intent to place an arterial or central line 
10. No intent/unwillingness to follow lung protective ventilation strategy for subjects with ARDS 
11. Severe hypoxemia defined as SpO2 < 95 or PaO2 < 90 on FiO2 ≥ 0.9 
12. Hemoglobin < 7.0 g/dL  
13. Subjects who are Jehovah’s Witnesses or are otherwise unable or unwilling to receive blood 
transfusions during hospitalization 
14. Acute myocardial infarction (MI) or acute coronary syndrome (ACS) within the last 90 days 
15. Coronary artery bypass graft (CABG) surgery within 30 days 
16. Angina pectoris or use of nitrates with activities of daily living 
17. Severe cardiopulmonary disease classified as New York Heart Association (NYHA) class IV 
18. Stroke (ischemic or hemorrhagic) within the prior 1 month, cardiac arrest requiring CPR within 
the prior 72 hours, or inability to assess mental status following cardiac arrest  
19. Burns > 40% total body surface area 
20. Severe airway inhalational injury 
21. Use of high frequency oscillatory ventilation 
22. Use of extracorporeal membrane oxygenation (ECMO) 
23. Use of inhaled pulmonary vasodilator therapy (eg. nitric oxide [NO] or prostaglandins) 
24. Diffuse alveolar hemorrhage from vasculitis 
25. Concurrent participation in other investigational drug study  
 
Reasons for Exclusions: 
Patients less than 18 years of age are excluded because the participating ICUs do not typically admit 
pediatric patients and we believe the benefit/risk ratio for children to receive iCO is not appropriate at 
this early stage. Patients with ARDS or pneumonia with mechanical ventilation for more than 168 hours 
are excluded in order to evaluate more clearly the effects of iCO early in the course of lung injury. Patients 
with severe hypoxemia or need for ECMO are excluded because they may not have adequate reserve to 
tolerate the reduction in oxygen carrying capacity. The parameters of SpO2 < 95 or PaO2 < 90 on FiO2  ≥ 0.9 
are used rather than a simpler PaO2/FiO2 ratio of 100 to define severe hypoxemia for both enrollment and 
daily treatment to better account for factors affecting arterial oxygen content and to ensure an additional 
safety margin during treatment of enrolled subjects. These criteria exclude patients with PaO2/FiO2 ≤ 100 
(PaO2 90/FiO2 0.9), but also explicitly specify a safety margin of 0.1 for FiO2. These criteria are currently 
approved by the Institutional Review Boards (IRBs) at our participating clinical centers, our DSMB, and the 
FDA for our Phase II CO ARDS trial (NCT03799874).  
 
Patients with hemoglobin < 7.0 g/dL, Jehovah’s witnesses, or patients otherwise unable or unwilling to 
receive blood transfusions during hospitalization are excluded because of the volume of blood drawn for 
monitoring during iCO therapy may place these patients at greater risks from complications of anemia. 
Moribund patients and patients with extensive body surface area burns have a high incidence of AEs and 
lactic acidosis that will confound the safety assessment. Pregnancy, recent stroke, or cardiac arrest are 
exclusions because iCO may reduce oxygen delivery to the fetus and recently injured brain respectively. 
Patients with alveolar hemorrhage from vasculitis are excluded because the mechanism of lung injury is 

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different from ARDS and diffuse alveolar damage. Patients with acute MI within 90 days, recent CABG, 
and angina pectoris are excluded because of a potential excess risk of reducing oxygen delivery to the 
myocardium. Patients with NYHA class IV cardiopulmonary disease are excluded because of concerns 
about ventricular arrhythmias and high mortality. Patients ventilated with high frequency oscillatory 
ventilation are excluded because administration of iCO with this mode of ventilation may be potentially 
unreliable. Patients on ECMO are also excluded because the effects of CO uptake and elimination from 
the blood during ECMO are unknown. Patients on concurrent inhaled pulmonary vasodilator therapy are 
excluded as these inhaled medications may interfere with the dosing of iCO. 
Patients who are cigarette smokers, but otherwise eligible for inclusion, will not be excluded from 
potential enrollment in the study. Rather, enrolled subjects will be screened with daily COHb levels and 
study drug treatment held if COHb ≥ 3%. This criterion was implemented for our Phase Ia study and will 
be used in this trial as smoking history obtained from surrogate decision makers may be unreliable and 
the average COHb half life is 240 minutes when breathing room air and less on supplemental oxygen134-
136.  
 
Exclusion on the basis of smoking history would preclude many patients from receiving this potentially 
beneficial treatment. Instead, this protocol incorporates treatment holds on the basis of daily COHb levels, 
which will allow otherwise eligible patients to be treated safely and have the opportunity to benefit from 
this potentially new promising therapy. We anticipate enrolling smokers at a frequency similar to their 
proportion in our target populations. 
 
5.3 
SCREEN FAILURES 
 
Screen failures are defined as participants who consent to participate in the clinical trial but are not 
subsequently randomly assigned to the study intervention or entered in the study. A minimal set of screen 
failure information is required to ensure transparent reporting of screen failure participants, to meet the 
Consolidated Standards of Reporting Trials (CONSORT) publishing requirements and to respond to queries 
from regulatory authorities. Minimal information includes demography, screen failure details, eligibility 
criteria, and any serious adverse event (SAE). Subjects who are screened but fail to be enrolled in the 
study for any reason will be documented on the screening log along with the reason for screen failure. 
 
Individuals who do not meet the criteria for participation in this trial (screen failure) because of because 
of severe hypoxemia (Exclusion Criterion 11), low hemoglobin (Exclusion Criterion 12), use of high 
frequency oscillatory ventilation (Exclusion Criterion 21), or use of inhaled pulmonary vasodilators 
(Exclusion Criterion 23) may be rescreened. Rescreened participants should be assigned the same 
participant number as for the initial screening. 
 
5.4 
STRATEGIES FOR RECRUITMENT AND RETENTION 
 
Thirty-six inpatient subjects will be recruited for this study at four enrolling medical centers. Subjects will 
be identified by prospective medical record review. We anticipate that the distribution of gender, race, 
and ethnicity of the subjects enrolled will be similar to the demographic characteristics of ICU patients 
admitted to each of our participating centers and be proportional to the enrollment distribution. No 
gender, race, and/or ethnicity group will be excluded from participation in our proposed study. 
 
Study coordinators at each site will screen inpatient ICUs daily to identify all potential subjects based on 
the pre-defined inclusion and exclusion criteria. The recruitment process will consist of electronic medical 
record (EMR) review of all intubated patients in the ICUs at each site to determine if a potential subject 

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meets study eligibility criteria. EMR alerts may also be used to assist research coordinators in the early 
identification of potential participants that meet study eligibility criteria. The research coordinators at 
each site will identify potential participants and review the study eligibility criteria in detail with the site 
PI or designee. No recruitment or advertisement materials will be utilized to recruit patients, however 
flyers may be posted in ICU staff areas to educate colleagues and other health care providers about study 
eligibility criteria and the importance of participation in clinical research. We will also engage the ICU 
clinical communities through conferences and other educational resources at each site as our ICU 
colleagues will play a critical role in the recruitment, retention, and overall conduct of the clinical trial. 
Participants will be hospitalized in the ICU at the time of informed consent and throughout the treatment 
period of the study protocol, thus we do not foresee difficulties in adherence with the study drug 
treatment. For participants who are extubated prior to completion of the 3-day treatment portion of the 
protocol, all data will be analyzed on a modified intention-to-treat (MITT) basis. For long-term follow-up 
outcomes, we will use web-based investigator tools for messaging and scheduling reminders to contact 
participants.   
 
Several engagement strategies will be adopted to ensure retention of study participants for assessment 
of long-term outcomes following hospitalization. First, we have designed the study protocol to minimize 
participant burden in order to optimize subject retention in the trial. We have designed the protocol such 
that the long-term follow up requires no travel or costs and entails four brief (2-10 minutes) telephone 
calls at day 28, day 60, 3 months, and 6 months. Second, we have established an efficient tracking system 
for long-term follow-up. At the time of consent, participants and/or their legally authorized representative 
(LAR) will be asked to provide email addresses and several phone numbers (home, work, cell) and to 
indicate the best times to contact them and whether voice and/or text messages can be left.  
 
As many ICU patients are discharged to long-term care facilities following hospitalization, we will consent 
study participants for permission to contact them at a long-term care facility if applicable. The names and 
contact information of up to three people who can be contacted for assistance with locating participants 
will also be requested. Written informed consent to contact these individuals will be obtained, including 
a description of the scope of the information to be obtained (eg. forwarding addresses versus questions 
about the clinical status of the participant). In the event of being unable to contact study subjects, we will 
send letters and/or emails to participants who are unresponsive to personal contact. We will also obtain 
consent to use social security numbers to determine vital status of participants lost to follow-up. 
 
To mitigate retention risks at the time of consent, we will educate subjects (and their LARs) about their 
important role as research participants, the requirements of the study, and clearly orient participants to 
the study demands, tasks, and responsibilities. Most of the patients approached for participation in this 
research protocol will have limitations of decision-making abilities due to their critical illness or need for 
sedating medications. Hence, most patients will not be able to provide informed consent and accordingly, 
informed consent will be sought from the potential subject’s LAR. See Section 10.1.1 for additional 
information.  
 
We have expanded inclusion criteria to include subjects with unilateral or bilateral infiltrates intubated 
for suspected lung infection (pneumonia), given similar outcomes from unilateral or bilateral lung 
infiltrates in critically ill patients, consistency with our preclinical baboon model of ALI due to 
pneumococcal pneumonia that had improved outcomes with inhaled CO, and the need to expand 
enrollment for this study. 
6 
STUDY INTERVENTION 

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6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
Inhaled CO or placebo air will be administered to mechanically ventilated subjects using a mechanical 
ventilator approved for inhaled NO delivery and the CO Delivery System developed by 12th Man 
Technologies (Figure 3, Appendix A). See Appendix A for details of the CO Delivery Device testing, 
assembly, calibration, and standard operating procedures (SOP). The CO Delivery System will be calibrated 
and connected to the ventilator (Figure 9) as described in Appendix A. As per the SOP and illustrated in 
the schema in Figure 9, the injector module will be connected between the inspiration port of the 
ventilator and inlet port of the humidifier. The gas sampling line will be placed between the outlet port of 
the humidifier and the patient wye as shown. 
The study drug will be administered by an RT and a 
physician study staff member OR a physician study 
staff member alone. A physician study staff 
member will be present at the bedside during and 
immediately available for an additional 90 minutes 
after administration of the study drug to address 
any clinical concerns that arise, or provide any 
necessary emergency care in conjunction with the 
subject’s clinical ICU team. The administering RT 
and physician study staff member OR the physician 
study staff member alone will be unblinded to the 
treatment assignments and will conceal the gas 
cylinders and COventDS to assure that the study 
coordinator and treating ICU team remain blinded 
to the study drug assignment.  
 
6.1.2 CO OR PLACEBO CYLINDERS 
The gas cylinders for this clinical trial are AG or A3 aluminum cylinders with a CGA 500 valve and will be 
supplied by Praxair Healthcare Services (Morrisville, PA). The cylinder contains approximately 360 liters of 
5000 ppm (0.5%) CO in room air (21% oxygen) and poses no increased flammability risk. Placebo tanks 
will contain medical grade air in identical appearing cylinders. The gas cylinder’s nominal size is 5 inches 
in diameter by 17 inches tall. Given the flow limitation from the regulator of ~7 liters per minute and a 
minimum of 6 air exchanges per hour in an average 15x15x10 ICU room, we do not expect ambient CO 
levels to exceed the OSHA permissible exposure limit (PEL) of 50 ppm as an 8 hour time-weighted average. 
We measured ambient CO levels during our animal studies using the COventDS and were unable to detect 
increases in ambient CO levels throughout assembly, calibration, and delivery of CO to the animals. In 
addition, we simulated CO administration with an ICU ventilator (Puritan-Bennett 840), CO Delivery 
System, and lung model in an ICU room at Massachusetts General Hospital at 500 ppm for over 2 hours 
and ambient CO levels were near zero and well below the OSHA PEL. 
6.1.3 DOSING AND ADMINISTRATION 
 
Figure 9. Schema of CO Delivery System 
and ventilator. 

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Inhaled CO or placebo air will be administered to participants via the COventDS, which will be calibrated 
and connected to the ventilator (Figure 9). Ventilator management will follow the ARDS Network lower 
tidal volume (6 mL/kg PBW) protocol13. Subjects randomized to CO will be administered iCO at 200 ppm 
for 20 min as a “test dose” on each treatment day to determine in real-time each subject’s CFK equation-
determined personalized dose.  
 
COHb levels will be measured before and after the 20 min test dose, and these two levels entered into 
the CFK equation calculator program to estimate DLCO and compute the iCO dose to achieve a COHb level 
of 7% (target 6-8%) at 90 min. After the iCO dose is determined, subjects will be administered iCO at the 
CFK equation-determined personalized dose (200-500 ppm) but not to exceed 500 ppm or 90 min total 
(including test dose). Subjects randomized to placebo will be administered medical grade air.  
 
The study drug will be administered for up to 90 minutes daily for three days following randomization or 
until discontinuation of mechanical ventilation, whichever occurs first. For patients who have a 
tracheostomy, the equivalent of extubation for the purposes of this protocol will be breathing via 
tracheostomy with unassisted breathing. 
 
The study drug concentration will be monitored continuously with the COventDS built-in analyzer and 
blood will be drawn at serial time points for arterial blood gas (ABG) analysis and safety monitoring of 
COHb levels as described in Section 6.1.4.  
6.1.4 CO MONITORING 
 
The study drug concentration will be monitored continuously with the COventDS built-in analyzer to 
assure that the accurate dose is delivered. A physician investigator will assess subjects prior to daily iCO 
treatment. Subjects will have blood drawn daily for measurement of COHb and lactate prior to study drug 
administration. If COHb ≥ 3% (assessed by unblinded physician) or lactate ≥ 4 mmol/L, the study drug will 
be held and the subject will be reassessed for treatment the following day. An electrocardiogram (EKG) 
will also be performed daily prior to study drug administration to evaluate for cardiac exclusion criteria. 
Blood COHb levels will be measured with an IL GEM Premier Co-oximeter and SpCO measured with a 
noninvasive pulse oximeter (Masimo Radical-7).  
 
All CO monitoring will be carried out by the administering RT and physician study staff member OR 
physician study staff member alone and concealed from the study coordinator and clinical team. The 
physician study staff member will be responsible for maintaining a separate password-protected limited-
access project within the database with the subject identification number and CO-related measurements 
(SpCO, COHb, ambient CO) which the study coordinator will not have access to. 
 
Blood for COHb Monitoring 
Blood (arterial or venous) will also be drawn at 20, 40, 60, 75, 90, and 180 minutes for safety monitoring 
of COHb levels. If an arterial line is not available, venous blood will be used for COHb monitoring as we38 
and others137-139 have shown that venous levels are as accurate and reliable as arterial measurements. 
Blood (arterial or venous) will also be drawn for blood gas analysis (using the IL GEM Premier Co-oximeter 
or local clinical lab) at 90 minutes or at the completion of treatment, if discontinued sooner than 90 
minutes. If arterial blood is not available and venous blood gas is used, PaO2/FiO2 will be estimated using 
the formula SpO2/FiO2 = 64 + 0.84 X PaO2/FiO2 , as long as SpO2 ≤ 97% and subject remains intubated as 
described in the Statistical Analysis Plan. 
 

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The unblinded physician will review and adjudicate COHb measurements and communicate any study 
drug treatment holds to the administering study staff. COHb levels will be entered into the electronic case 
report forms (eCRFs) in a separate limited-access project in StudyTRAX by the unblinded RT or physician. 
The study drug will be administered for up to 90 minutes daily for up to 3 days following randomization 
or until discontinuation of mechanical ventilation, whichever occurs first.  
 
COVentDS CO Monitoring 
The concentration of the study drug will be measured by the built-in gas monitor in the COventDS. The 
CO Delivery System contains an inhaled gas monitor, which is an electrochemical device that monitors the 
inhaled gas for concentrations of CO (0-800 ppm) and O2 (15-100%) to assure that safe levels are inhaled. 
The sample pump maintains a constant flow of gas to the sensors. Samples of inspired gases are taken 
with a continuous ~400 mL/min sample pump just proximal to the patient’s airway to reflect actual 
inspired gases. Alarms will sound on high or low CO or O2 concentrations.  
 
Ambient Air Monitoring 
Ambient air CO concentrations will be measured in real time with a Dräger Pac 7000 CO detector to assure 
that ambient levels are maintained within the recommended limits for occupational exposure of a 
maximum of 50 ppm. Ambient air CO detectors will be calibrated every 6 months per the manufacturer’s 
instructions to ensure proper functioning. 
6.1.5 CO DOSING ALGORITHM USING THE CFK EQUATION 
 
For subjects randomized to iCO, iCO (200 ppm) will be administered for up to 20 minutes as a “test dose” 
on each day to determine the subject’s CFK equation-determined dose. The COHb level will be measured 
prior to the test dose and 20 minutes after the test dose. These two COHb levels will be used in the CFK 
equation to determine each subject’s specified dose in order to achieve a COHb level of 6-8%. After the 
CO dose is determined, subjects will be administered inhaled CO at the algorithm-determined dose- not 
to exceed 500 ppm or 90 minutes total (including test dose). Subjects randomized to placebo will be 
administered medical grade air. 
 
1. Arterial or venous blood will be drawn for baseline blood gas analysis and COHb measurements 
prior to iCO or placebo administration. 
2. Subjects randomized to CO will be treated with inhaled CO at 200 ppm for 20 minutes (test dose). 
3. After 20 minutes, blood will be drawn for COHb measurement. 
4. DLCO will be estimated using the baseline and 20 min COHb in the CFK equation calculator 
program, and used to compute the iCO dose to achieve a COHb level of 7% (target 6-8%) at 90 
minutes.  
5. Dose will be adjusted (200-500 ppm) in subjects randomized to CO based on CFK equation-
determined dose. 
6. Blood will be drawn for COHb measurements at 20 min, 40 min, 60 min, 75 min, 90 min, and 3 
hours. Blood will also be drawn for blood gas analysis at 90 min. 
 
On days 1-3, a total of approximately 3 mL of blood will be drawn during the study drug administration 
for safety monitoring of COHb levels and blood gas analysis. 
6.1.6 DAILY HOLD PARAMETERS PRIOR TO STUDY DRUG ADMINISTRATION 
 

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Subjects will be assessed daily prior to study drug administration as described in the Schedule of Activities 
(Section 1.3). 
 
Administration of the study drug will be held if the following criteria are met.  
 
• 
COHb level ≥ 3% 
• 
Lactate ≥ 4 mmol/L 
• 
ST elevation MI or unstable angina/non ST elevation MI concerning for ACS 
• 
Unstable atrial or ventricular arrhythmia 
 
Subjects will have blood drawn daily for measurement of COHb and lactate prior to administration of 
study drug. If COHb ≥ 3% or lactate ≥ 4 mmol/L, the study drug will be held until the next scheduled dose 
the following day. Lactate and COHb will be measured the following day to determine whether the study 
drug will be administered. If the study drug is being held for another reason, COHb levels will not be 
measured on days the study drug is being held. 
A 12 lead EKG will also be performed and reviewed daily by the study staff physician prior to study drug 
administration to evaluate for cardiac exclusion criteria (eg. acute MI, acute coronary syndrome (ACS), or 
unstable atrial or ventricular arrhythmia). If a subject meets the following criteria for ST elevation MI or 
unstable angina/non ST elevation MI concerning for ACS according to the American College of Cardiology 
Foundation/American Heart Association guidelines140-142, they will be excluded from enrollment or further 
study drug administration according to exclusion criteria.  
 
ST elevation MI (STEMI) Criteria: New ST elevation at the J point in at least 2 contiguous leads of ≥ 2 mm 
(0.2 mV) in men or ≥ 1.5 mm (0.15 mV) in women in leads V2–V3 and/or of ≥ 1 mm (0.1 mV) in other 
contiguous chest leads or the limb leads; New or presumably new left bundle branch block (LBBB)140-142. 
 
Unstable angina/non ST elevation MI (NSTEMI): Ischemic ST-segment depression ≥ 0.5 mm (0.05 mV) or 
dynamic T-wave inversion with pain or discomfort; Nonpersistent or transient ST-segment elevation ≥ 0.5 
mm for < 20 minutes. Threshold values for ST-segment depression consistent with ischemia are J-point 
depression 0.05 mV (-0.5 mm) in leads V2 and V3 and -0.1 mV (-1 mm) in all other leads (men and 
women)140-142. 
 
Note, troponin (I or T) may be increased in patients with sepsis and ARDS143 in the absence of an acute MI 
or ACS from coronary artery disease. If, in the judgment of the clinical team, an ARDS patient with elevated 
troponin levels has no other indication of an MI or ACS, the patient may still be eligible for enrollment. 
 
In addition, if a subject develops the following criteria during the study, the study drug will be held until 
resolved: 
 
• 
Severe hypoxemia defined as SpO2 < 95 or PaO2 < 90 on FiO2 ≥ 0.9 
• 
Hemoglobin < 7.0 g/dL  
• 
Diffuse alveolar hemorrhage from vasculitis 
• 
Use of high frequency ventilation 
• 
Use of inhaled pulmonary vasodilator therapy (eg. NO or prostaglandins) 
• 
Use of ECMO 
 

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If study drug hold criteria are met, the study drug will be held and subjects will be evaluated on a daily 
basis and assessed for whether a given criteria has resolved and whether study drug treatment may be 
resumed. 
6.1.7 INTERRUPTION OF DOSING DURING STUDY DRUG ADMINISTRATION 
 
Subjects will have blood drawn for measurement of COHb levels prior to study drug treatment and 20 
min, 40 min, 60 min, 75 min, 90 min, and 180 min after the start of study drug treatment. It is anticipated 
that COHb levels will not exceed the target range of 6-8% given the impaired diffusion capacity in ARDS 
patients and CFK equation-determined personalized iCO dosing algorithm. However, the following 
parameters will be used to shorten the 90 minute treatment duration should CO uptake be higher than 
anticipated: 
 
The study drug will be stopped prior to 90 minutes: 
 
1. If the measured COHb level is > 8% at any time during study drug treatment. 
2. If the investigator, attending physician, the patient, or their surrogate decides that the study drug 
should be discontinued. 
If COHb levels unexpectedly exceed our target level, in addition to discontinuation of the study drug, 
subjects will be placed on 100% O2 to accelerate CO elimination until COHb levels normalize. 
6.1.8 PERMANENT DISCONTINUATION OF STUDY DRUG 
 
Permanent discontinuation of the study drug is defined as cessation of the study drug without the intent 
of restarting the study drug during the three-day treatment period.  
 
Permanent discontinuation of the study drug inhalation will occur in the following situations: 
 
• 
Occurrence of pre-specified administration-related AEs: 
o Acute MI* within 48 hours of study drug administration 
o Acute stroke** within 48 hours of study drug administration 
o New onset atrial or ventricular arrhythmia requiring DC cardioversion within 48 hours of study 
drug administration 
o Increased oxygenation requirements defined as: an increase in FiO2 of ≥ 0.2 AND increase in 
PEEP ≥ 5 cm H2O within 6 hours of study drug administration.  
o Increase in any protocol-specified measurement of COHb ≥ 10%  
o Increase in lactate by ≥ 2 mmol/L within 6 hours of study drug administration  
• 
If the subject experiences serious adverse events (SAEs) related to the study drug   
• 
If the investigator, attending physician, the patient or their surrogate decides that the study drug 
should be discontinued. If this decision is made because of an AE, then appropriate AE reporting 
procedures will be followed (Section 8.7). 
• 
Daily baseline COHb levels ≥ 3% leading to three missed drug doses 
• 
Three or more missed drug doses due to AEs 
 
* Acute MI is defined below according to the Executive Group on behalf of the Joint European Society of 
Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart 
Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction144. We will also rely 
upon subspecialty consultation to confirm the presence or absence of an acute MI. 

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Acute myocardial infarction is defined as acute myocardial injury with clinical evidence of acute 
myocardial ischemia and with detection of a rise and/or fall of cardiac troponin values with at least 1 value 
above the 99th percentile upper reference limit and at least 1 of the following: 
• Symptoms of myocardial ischemia; 
• New ischemic ECG changes; 
• Development of pathological Q waves; 
• Imaging evidence of new loss of viable myocardium or new regional wall motion abnormality 
in a pattern consistent with an ischemic etiology; 
• Identification of a coronary thrombus by angiography or autopsy (not for types 2 or 3 MIs). 
 
** Acute stroke is defined as an acute neurological deficit attributed to an acute focal injury of the central 
nervous system by a vascular cause, including cerebral infarction, intracerebral hemorrhage, and 
subarachnoid hemorrhage based on neuropathological, neuroimaging, and/or other clinical evidence of 
permanent injury145. We will also rely upon subspecialty consultation to confirm the presence or absence 
of an acute stroke. 
 
Subjects who have their study drug permanently discontinued will continue their participation in the 
study, and will be followed to determine their vital status and perform neurocognitive testing at 3 and 6 
months, as outlined in the Schedule of Activities (Section 1.3). 
6.1.9 COMPLETION OF THE STUDY DRUG ADMINISTRATION 
Subjects will be considered to have completed the study drug administration portion of the study when 
one of the following conditions is met, whichever comes first: 
 
1. Three days after study drug administration   
2. Discontinuation of mechanical ventilation 
3. Death 
4. Pre-specified criteria met for permanent discontinuation of study drug (Section 6.1.8 in the 
Study Protocol) 
6.1.10 TREATMENT IN COVID-19 SUBJECTS 
For enrolled pneumonia or ARDS patients with COVID-19 infection, all research and clinical staff will 
conform to each site’s hospital infection control guidelines for COVID-19 positive patients with 
appropriate personal protective equipment (PPE) for enhanced respiratory isolation precautions. All 
research and clinical staff will have appropriate training on PPE, be fit-tested, and wear an N95 respirator 
or powered air-purifying respirator (PAPR) if staff cannot be fit-tested, face shield or other eye protection, 
gloves, and gown when entering the subject’s room for treatment or sample collection. Gown, gloves, and 
eye protection will be doffed upon exiting the subject’s room either just inside or just outside the door, 
and hand hygiene will be performed. Respiratory protection should be removed after exiting the subject’s 
room unless extended use is being practiced. 
 
During study drug treatment, all staff will conform to each site’s hospital infection control guidelines 
regarding treatment of COVID-19 positive mechanically ventilated patients with inhaled therapies. Staff 
will be limited to essential personnel in the room. All equipment will be single-use or dedicated to use of 

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the subject to avoid sharing with other subjects. Reusable equipment will be disinfected with a hospital-
approved disinfectant before use in another subject.  
 
Blood sample collection and handling will be performed according to each site’s hospital infection control 
guidelines for COVID-19. Samples obtained from a subject will be put in a tube by research or clinical staff, 
and the tube will be wiped down with a hospital-approved disinfectant. While holding the tube with the 
wipe, the specimen tube will be carefully placed into a clean specimen bag held by a monitor or nurse 
outside the room. The specimen bag will in turn be wiped down with a hospital-approved disinfectant. A 
member of the research team will pick up the research specimens and transport them to a BSL2+ 
laboratory in a secondary container. Research samples will only be processed in hospital biosafety 
committee approved BSL2+ laboratory facilities. 
 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
Each participating site is responsible for the ordering, storing and dispensing of the investigational 
agent(s). Each study team will be responsible for keeping a Site Drug Accountability Log at the site which 
will include a log of CO/placebo air tanks with lot number/cylinder number, concentration, expiration 
date, and dates received from/returned to Praxair. A subject study drug accountability log will record the 
subject ID, cylinder number, content of tank, dose (ppm), date dispensed, and tank pressure at start/end 
of treatment. 
6.2.2 FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
 
The study drug will be provided for this study by Praxair Healthcare Services. Gas mixtures will be 
manufactured with United States Pharmacopeia (USP)/Ultra High Purity (UHP)/National Formulary (NF) 
components. The mixtures will be prepared as a Certified Standard with a blend tolerance of ± 10% and 
analytical uncertainty of 2%.  
 
The gas mixture provided to subjects randomized to CO will be 5000 ppm CO (balance air) and will be 
provided to study sites by Praxair Healthcare Services in AG or A3 aluminum cylinders with a CGA 500 
valve. The gas cylinder’s nominal size is 5 inches in diameter by 17 inches tall, contains 360 liters of 5000 
ppm (0.5%) CO in room air (21% oxygen), and poses no increased flammability risk. The 5000 ppm CO 
source gas will be administered via the CO Delivery System (12th Man Technologies, Garden Grove, CA) 
and delivered to subjects at a CFK equation-determined individualized dose (200-500 ppm) to achieve a 
target COHb level of 6-8%. 
 
The mixture components include: Carbon Monoxide UHP Grade 3.0 (Assay 99.9%; O2 <10 ppm, H20 <5 
ppm, N2 <900 ppm and CO2 <20 ppm); and Medical Air USP. The source materials for Medical Air USP 
include: Oxygen USP (Assay ≥ 99.2%; Odor- none; Oxygen produced by air liquefaction process, does not 
require CO and CO2 analysis); and Nitrogen NF USP (Assay ≥ 99.2%; Identification- OK, Paramagnetic Assay 
Method; CO ≤ 10 ppm; Odor- none). 
 
The gas mixture provided to subjects randomized to placebo will be medical grade air and will be provided 
to study sites by Praxair Healthcare Services in AG or A3 aluminum cylinders with a CGA 346 valve. With 
the exception of the 346 valve, the placebo gas cylinders will be identical in size and color as the CO 
cylinders. The placebo gas will be administered via the CO Delivery System (12th Man Technologies, 

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Garden Grove, CA) and delivered to subjects in an identical fashion as iCO delivery.  
 
6.2.3 PRODUCT STORAGE AND STABILITY 
The CO and placebo air cylinders will be stored at room temperature in a secure area at each center. The 
cylinders will be stored and handled according to each site’s Compressed Gas Cylinder Storage Guidelines. 
The CO and placebo air cylinders will be returned to Praxair upon expiration. The expiration date is 
generally three years from the date of fill and is clearly indicated on the certificate of analysis for each 
cylinder. 
 
6.2.4 PREPARATION 
As described in the SOP, the CO Delivery System will be calibrated and performance testing conducted 
daily prior to use. The calibration will be performed as described in the Operator’s Manual in Appendix A. 
The calibration consists of a low (21%) and high (100%) range O2 calibration, followed by a low (100 ppm) 
and high (400 ppm) range CO calibration. After completion of the calibration, performance testing will be 
performed as described in the SOP. The CO Delivery System will be assembled, the appropriate gas 
cylinder (CO or air) connected, and the delivery system will be connected to the ventilator as described in 
Appendix A. As shown in Figure 9, the injector module will be connected between the inspiration port of 
the ventilator and inlet port of the humidifier. The gas sampling line will be placed between the outlet 
port of the humidifier and the patient wye as shown.  
 
6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
 
Eligible participants will be randomized to one of the two treatment arms using a permuted block 
method146 with random block sizes. The randomization ratio will be 2:1 iCO:placebo. The randomization 
will not be stratified by any factors. The study biostatistician will verify all the necessary randomization 
information (blinding method, randomization method, block size, and number of treatment groups) in the 
StudyTRAX randomization configuration module.  
 
Site investigators will review all potential study participants with one of the CCC physician members. 
Following informed consent, the unblinded administering RT or physician or blinded study coordinator 
will randomize subjects in the database using the StudyTRAX randomization tool. The unblinded 
administering RT or physician will obtain the subject’s treatment assignment through the “View 
Treatment” button in the blinded database, a privilege granted to unblinded RTs and physicians only. CO-
related measurements (SpCO, COHb, ambient CO) and drug-related information will be in a separate 
project within the database, which the blinded study coordinators will not have access to. Permission to 
this limited-access project will only be granted to unblinded study personnel by the PI, as it contains 
treatment-specific information such as CO monitoring and study drug administration data.  
 
The study drug assignment will be blinded to the subject, clinical team, study coordinators, and other 
study staff with the exception of the administering study staff (RT and physician OR physician alone), who 
will be unblinded to the treatment assignment to ensure subject safety. We chose this partially double-
blind design to ensure safe iCO administration and an unblinded assessment of COHb levels in order not 
to exceed potentially toxic thresholds. 
 
To maintain blinding, the placebo air cylinders are identical in appearance as the CO cylinders (with the 
exception of different CGA valves required by the manufacturer Praxair). Cylinders will also be shrouded 
in dark sleeves to ensure blinding. The placebo air will be administered via inhalation via the COventDS 

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and delivered to subjects in an identical fashion as those randomized to CO. To ensure that the subject, 
study coordinator, and clinical staff remain blinded to the study drug assignment, the administering RT 
and physician study staff member OR physician study staff member alone will conceal the gas cylinders, 
CO delivery device, and measurements of COHb, SpCO, and ambient CO levels. Unblinded test results will 
be kept in a separate location from the blinded test results and concealed from the study coordinators.  
 
The unblinded administering physician will be responsible for maintaining a separate password-protected 
limited-access project within the database with the subject identification number and CO-related 
measurements (SpCO, COHb, ambient CO) which the study coordinator will not have access to. While the 
administering investigator will be unblinded throughout the study due to safety monitoring, they will only 
be unblinded to the treatment assignment for subjects enrolled at their own site. Investigators will 
otherwise be blinded to the study treatment assignment for subjects enrolled at the other sites. The lead 
RT (Mr. Davies) may become unblinded to the treatment assignments at other sites to assist with study 
drug administration procedures if necessary. 
 
If the clinical team believes that unblinding is medically necessary in order to properly treat the patient, 
the unblinded administering physician can disclose study product assignment. If there is time to do so, 
the unblinded administering physician should discuss this unblinding with the unblinded CCC physician 
prior to the disclosure. All episodes of unblinding, whether deliberate or inadvertent, will be reported to 
the DSMB at the next routine report. 
 
A blinded study physician at each site will review all AEs and will assess their relationship to the study 
intervention. Additional AE adjudication will be provided by one of the Medical Monitors (Dr. North or Dr. 
Thompson). In addition to blinded adjudication of AEs, investigators blinded to treatment allocation will 
assess chest X-rays required for the calculation of the LIS score. De-identified chest X rays at each site will 
be reviewed centrally by blinded investigators at BWH. 
 
6.4 
STUDY INTERVENTION COMPLIANCE 
 
To assess protocol adherence during monitoring visits, the CCC will review the Study Drug Administration 
and CO Administration Monitoring forms, in addition to the Study Drug Accountability Logs. The 
administering RT and physician study staff member OR physician study staff member alone will keep track 
of study drug assignment and dosages, time and duration of treatment, tank lot number, and COHb results 
on Subject Drug Administration and CO Administration Monitoring forms. These forms should be kept in 
a secure place and confidential from blinded study staff. 
 
Each participating site is responsible for the ordering, storing and dispensing of the investigational 
agent(s). Each study team will be responsible for keeping a Site Drug Accountability Log at the site which 
will include a log of CO/placebo air tanks with lot number/cylinder number, concentration, expiration 
date, and dates received from/returned to Praxair. A subject study drug accountability log will record the 
subject ID, cylinder number, content of tank, dose (ppm), date dispensed, and tank pressure at start/end 
of treatment. 
6.5 
CONCOMITANT THERAPY 
Subjects will be excluded from participation in the study if they are receiving concomitant inhaled 
pulmonary vasodilators continuously (such as inhaled nitric oxide [NO] or prostacyclin), as these inhaled 
medications may interfere with the dosing of inhaled CO. If a decision is made by the clinical team to 

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initiate these medications in enrolled subjects following randomization and within the 3-day study 
treatment period, the study drug will be held until subjects are no longer receiving these medications 
concomitantly. The study drug may be reinitiated following cessation of these inhaled medications if there 
are no other protocol-specified reasons that preclude ongoing treatment in the study. Potential subjects 
will also be excluded if they are participating in another investigational drug study. There are otherwise 
no contraindications to concomitant medications in the study. 
 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
 
Discontinuation from study drug does not mean discontinuation from the study, and remaining study 
procedures should be completed as indicated in the study protocol. If a clinically significant finding is 
identified (including, but not limited to changes from baseline) after enrollment, the site investigator or 
designee will determine if any change in participant management is needed. Any new clinically relevant 
finding will be reported as an AE. 
 
Permanent discontinuation of the study drug is defined as cessation of the study drug without the intent 
of restarting the study drug during the three-day treatment period. Subjects who have their study drug 
permanently discontinued will continue their participation in the study, and will be followed to determine 
their vital status and perform neurocognitive testing at 3 and 6 months as outlined in the Schedule of 
Activities (Section 1.3). 
 
Criteria for permanent discontinuation of the study drug are described in Section 6.1.8. In some instances, 
the study drug may be held and the subject may be re-evaluated for treatment on subsequent days. The 
criteria for a study drug hold are described in Section 6.1.6. 
 
If study drug hold criteria are met, the study drug will be held and subjects will be evaluated on a daily 
basis and assessed for whether a given criteria has resolved and whether study drug treatment may be 
resumed. 
 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
 
Participants are free to withdraw from participation in the study at any time upon request. An investigator 
may discontinue or withdraw a participant from the study for the following reasons: 
 
• 
If any clinical AE, laboratory abnormality, or other medical condition or situation occurs such that 
continued participation in the study would not be in the best interest of the participant 
• 
Disease progression which requires discontinuation of the study intervention 
• 
If the participant meets an exclusion criterion (either newly developed or not previously 
recognized) that precludes further study participation 
• 
Participant unable to receive at least one dose of the study drug 
 
If a patient or surrogate requests withdrawal from the study, the investigators will seek explicit permission 
to continue data collection. 
 

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The reason for participant discontinuation or withdrawal from the study will be recorded on the Case 
Report Form (CRF). Subjects who sign the informed consent form and are randomized but do not receive 
the study intervention may be replaced. Subjects who sign the informed consent form, and are 
randomized and receive at least one dose of the study product, and subsequently withdraw, or are 
withdrawn or discontinued from the study will not be replaced. 
 
7.3 
LOST TO FOLLOW-UP 
 
A participant will be considered lost to follow-up if the study staff if unable to verify vital status after the 
subject has been discharged from the hospital. Before a participant is deemed lost to follow-up, the site 
investigator or designee will make every effort to regain contact with the participant or surrogate (where 
possible, 3 telephone calls and, if necessary, a certified letter to the participant’s last known mailing 
address or local equivalent methods). These contact attempts should be documented in the participant’s 
medical record or study file.  
 
We will verify duration of survival for participants lost to follow-up or noted to have died using the Centers 
for Disease Control and Prevention’s National Death Index (National Death Index, 2000). The subject’s 
social security number (SSN) will be used for an exact NDI match. Contact information will be collected 
for the subject and alternative contact information will be collected on up to 3 individuals. Contact 
information and the subject’s SSN will be collected on paper at the time of consent. Contact information 
and SSN will be maintained on paper locally and will not appear in the study database. 
 
8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 
DESCRIPTION OF STUDY PROCEDURES 
All clinical study procedures will be performed in accordance with Good Clinical Practice (GCP) standards.  
Study assessments described below will be done according to the Schedule of Activities in Section 1.3. 
 
APACHE II Score: The APACHE II consists of 12 physiological variables, age, and underlying health. The 
score will be calculated based on the worst values in the 24 hours prior to randomization. Calculation 
variables are described in Appendix B1. 
Blood gas analysis: The following values will be recorded from the arterial blood gas (ABG) results: pH, 
PCO2, PO2, and base excess or deficit. ABG is required for baseline assessment. If no ABG is available on 
subsequent days, record blood gas analysis results from the venous blood gas (VBG). 
Calibration of IL GEM Premier Co-oximeter and COventDS: Calibration will be performed according to 
device manuals and SOP. Documentation of calibration will be kept in study files.  
Central Venous Pressure (CVP): CVP will be recorded if available. 
Calculate Deadspace (Vd/Vt): Dead space fraction will be measured daily by the administering RT or 
physician prior to study drug administration using a NICO or NM3 monitor (Respironics) and the PCO2 
obtained from an arterial or venous blood gas. 
Chest X-ray (CXR): The baseline frontal CXR will be reviewed by two study physician investigators to 
determine study eligibility. Study physician investigators blinded to treatment allocation will review and 
perform the assessment for the Lung Injury Score (LIS). All 4 lung quadrants will be assessed for infiltrates 
and pneumothoraces. If the subject is extubated on days 1-5, the study team will order a chest X-ray the 

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day following extubation, if not ordered by the clinical team. Chest X-rays will also be ordered by the study 
team for endpoint analysis on Day 7, if not ordered by the clinical team.  
Clinical Laboratory Assessments: Clinical labs will be processed at each site’s local laboratory and 
reviewed by a study investigator. If labs are required according to the Schedule of Activities (Section 1.3) 
and are not available as part of standard of care testing, they will be drawn for research. A urine or serum 
pregnancy test will be done for women of childbearing potential. A subject with a positive pregnancy 
result will not be eligible for randomization. 
Concomitant Medications Review: The following medications will be recorded in the study eCRFs: aspirin, 
COX-2 inhibitors, NSAIDs, ACE inhibitors, steroids, phosphodiesterase inhibitors, neuromuscular blockade, 
inhaled NO or prostaglandins, antibiotics, diuretics. TPN use will also be recorded. 
EKG: A 12 lead EKG will be obtained and reviewed daily by the study staff physician prior to study drug 
administration to evaluate for cardiac exclusion criteria (acute MI, ACS, or unstable atrial or ventricular 
arrhythmia). The study drug will not be started until the EKG is obtained and reviewed by the study 
physician investigator for a potential study hold. The EKG will also be officially read locally at each site.  
Fluid Intake/Output: Fluid intake and output (most recent 24 hour value) or mean hourly value for most 
recently available period. Diuretic administration will also be recorded if applicable.  
Glasgow Coma Scale (GCS): The GCS is a determination of the best eye opening, motor, and verbal 
responses at any given time. The GCS is documented by the clinical team as part of routine ICU nursing 
assessment. If not available, the GCS will be assessed by the study staff as described in Appendix B2. 
Lung Injury Score (LIS): The lung injury score will be calculated based on the chest x-ray assessment, PaO2, 
FiO2, PEEP, and ventilator compliance measurement as described in Appendix B3.  
Microbiological Results: Microbiological results will be recorded in the eCRF when ordered for clinical 
purposes.  
a) Blood cultures 
b) Urine cultures 
c) Sputum cultures 
d) BAL cultures 
e) CSF cultures if available 
f) Stool  
g) Other 
Neurocognitive Testing: Neurocognitive testing will be performed via telephone interviews using the 
Montreal Cognitive Assessment (MoCA)-BLIND and Hayling sentence completion test as (Appendix C). 
Oxygenation Index (OI): The OI will be calculated based on the PaO2, FiO2, and mean airway pressure 
obtained from the ventilator parameters. 
Renal replacement therapy status: Renal replacement therapy status within the last 24 hours will be 
recorded.  
Richmond Agitation Scale (RASS): RASS is a scale used to measure the agitation or sedation level of 
a patient. RASS scoring is documented by the clinical team as part of routine ICU nursing assessment. If 
not available, the RASS will be assessed by the study staff as described in Appendix B4. 
ScvO2: ScvO2 will be recorded if available. 
Sequential Organ Failure Assessment (SOFA): The SOFA score will be calculated based on the worst values 
recorded in the past 24 hours. Calculation variables are described in Appendix B5. 

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a) Worst PaO2/FiO2 for that date 
b) Worst creatinine (or urine output), bilirubin, and platelet count for that date 
c) Worst Glasgow Coma Scale for that date  
d) Vasopressor use and maximal dose for that date  
Vasopressors and Inotropes Review: The following vasopressors/inotropes will be recorded in the study 
eCRFs 
(epinephrine, 
norepinephrine, 
phenylephrine, 
vasopressin, 
dopamine, 
dobutamine, 
phosphodiesterase inhibitors, including dose).  
Ventilator Parameters: The study staff will record ventilator parameters prior to starting study drug 
administration. The following settings will be recorded:  
a) Mode of ventilation 
b) FiO2 and PEEP 
c) Tidal volume, Respiratory rate (set rate and total rate), Minute ventilation, Inspiratory flow rate  
d) Peak pressure, Plateau pressure, Compliance, and Mean airway pressure. If on a pressure-cycling 
mode, peak pressure during inspiration will be assumed to be the plateau pressure. 
e) Pressure during inspiration if on a pressure targeted mode (PSV, PCV, etc) 
Vital Signs: Heart rate (beats/min), systemic systolic, diastolic, mean arterial blood pressure (mm Hg), and 
body temperature (°C), will be recorded in the eCRF. 
 
Vital Status: Vital status will be assessed at Days 28, 60, 3 months, and 6 months. Subjects who have been 
discharged from the hospital will be contacted by phone. Additional information will be collected on 
discharge status, ventilator status, and vasopressor status at Days 28 and 60. 
8.1.1 STUDY SAMPLE COLLECTION 
Sample collection will be done according to the Schedule of Activities (SoA) in Section 1.3. 
 
COHb Levels: COHb levels will be measured using the IL GEM Premier Co-oximeter at specified time points 
on Days 1 through 3. See Section 8.5.3. 
 
Plasma Samples: Blood (4 mL) will be collected pre-treatment and post-treatment (90 mins and 3 hrs) on 
days 1-3 in EDTA anti-coagulated tubes for plasma isolation and measurement of biomarkers of 
mitochondrial dysfunction, inflammasome activation, lipid mediators, and cytokines. Blood (4 mL) will 
also be collected for plasma isolation on Day 5. Plasma will be obtained and divided immediately after 
centrifugation into equal aliquots in specified tubes and frozen at –80°C.  
 
RNA Samples: Blood (2.5 mL) will be collected pre-treatment and post-treatment (90 min and 3 hrs) on 
days 1-3 in Paxgene tubes for RNA isolation, and frozen at –80°C. Blood (2.5 mL) will also be collected in 
Paxgene tubes for RNA isolation on Day 5 and frozen at –80°C. 
 
DNA Samples: Cell pellets will be obtained for future DNA isolation from the blood collected in EDTA tubes 
on days 1-3 and on day 5. Cell pellets will be isolated pre-treatment and post-treatment (at 90 min and 3 
hrs) on days 1-3.  
 
Primary Cell Isolation: An additional 8 mL of blood will be drawn for isolation of peripheral blood 
mononuclear cells (PBMCs) pre-treatment on day 1 and post-treatment on day 3. This tube may be used 
to isolate cell pellets (for future DNA isolation) if unable to obtain an adequate pellet from the 4 mL tube 
on days 1 and 5. 
 

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Urine Sample Collection: Between 50-100 mL of urine will be collected in a sterile specimen container on 
days 1-3 and on Day 5. Cell pellets will be isolated pre-treatment and post-treatment (at 90 min and 3 
hours) to obtain RNA and protein, as well as mtDNA. 
 
Discarded Specimens: Discarded bronchoalveolar lavage (BAL) fluid and plasma will be obtained (when 
available) for levels of cytokines, mediators/biomarkers, and protein. Plasma and BAL fluid will be divided 
into equal aliquots in specified tubes and frozen at –80°C.  
 
A total of approximately 90 mL of blood will be collected for research samples during the study from each 
study subject as follows: 
 
Sample collection  
Day 1 
Day 2 
Day 3 
Day 5 
Total 
Volume 
ABG, lactate, and COHb 
analysis 
3 mL 
3 mL 
3 mL 
 
9 mL 
4 mL EDTA tube (plasma 
and DNA)- pre and post-
treatment 
 12 mL 
12 mL 
12 mL 
 4 mL 
40 mL 
2.5 mL PAXgene tube 
(RNA)- pre and post-
treatment 
7.5 mL 
7.5 mL 
7.5 mL 
2.5 mL 
25 mL 
Primary Cell Isolation 
8 mL 
 
 
8 mL 
16 mL 
Total Volume 
30.5 mL 
22.5 mL 
22.5 mL 
14.5 mL 
90 mL 
 
Additional blood (29 mL) may be drawn during the study for research purposes as below if not drawn as 
part of usual clinical care. 
 
Sample 
collection Baseline 
Day 
1 
Day 
2 
Day 
3 
Day 
4 
Day 
5 
Day 
7 
Day 
14 
Day 
28 
Total 
Volume 
ABG, 
lactate 
2 mL 
 
 
 
 
 
 
 
 
2 mL 
CBC 
2 mL 
2 mL 
2 mL 
2 mL 
 
 
 
 
 
8 mL 
Metabolic 
panel 
3 mL 
 
 
 
 
 
 
 
 
3 mL 
Bilirubin 
 
2 mL 
2 mL 
2 mL 
2 mL 
2 mL 
2 mL 
2 mL 
2 mL 
16 mL 
Total 
Volume 
7 mL 
4 mL 
4 mL 
4 mL 
2 mL 
2 mL 
2 mL 
2 mL 
2 mL 
29 mL 
 
Samples will be sent to the central repository at BWH to be stored. Study subject ID numbers will identify 
samples during shipment and storage in the central repository. In the future, the CCC will instruct the 
repository to prepare the appropriate samples for shipment to sites for biomarker analyses. The CCC will 
not record or store unique patient identifiers (such as initials, date of birth, hospital record numbers, 
addresses, phone numbers, etc.) in the database. All data released by the CCC for studies will be linked to 
the specimen but will be de-identified. The link (key) between the de-identified database and the subject 
will be removed two years after the primary publication. Samples collected for this trial will be frozen and 

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stored in the biorepository for future research related to carbon monoxide, ARDS, sepsis, critical illness or 
other lung diseases. 
 
8.2 
SCREENING PROCEDURES 
 
No procedures will be required to determine subject eligibility. Patients will be screened based on 
laboratory values (eg. arterial blood gas analysis) and radiologic tests (eg. CXR) that are otherwise 
conducted as part of routine clinical care in the ICU. No study procedures will be initiated prior to 
obtaining written informed consent. 
 
During the screening evaluation, the subject’s medical history, vital signs, ventilator settings/mechanics, 
laboratory values, and chest X-ray will be reviewed to determine if the patient meets eligibility criteria to 
participate in the study.  
 
• 
Review medical history, vital signs, and concomitant medications 
• 
Review ventilator settings/mechanics and PaO2/FiO2  
• 
Review chest X-ray performed as part of clinical care 
• 
Review laboratory values drawn as part of clinical care including arterial blood gas (ABG), lactate, 
and troponin if available 
• 
Review electrocardiogram (EKG) performed as part of clinical care 
• 
Review urine or blood pregnancy testing if subject is female and able to become pregnant  
 
A screening log will be kept to track subjects that could potentially meet inclusion criteria. All patients 
meeting the inclusion criteria will be entered into the StudyTRAX database. If the patient is not enrolled, 
information explaining why enrollment did not occur will be entered into StudyTRAX (exclusion criteria, 
attending physician denial, patient refusal, etc. see Section 8.2.2 for a listing of the de-identified data to 
be collected on screened, non-enrolled subjects). 
If any patient meets criteria for study enrollment, the attending physician will be asked for permission to 
approach the patient or his/her surrogate for informed consent. 
8.2.1 ASSIGNING A SCREENING ID 
 
All subjects who are screened will be given a Reference ID and a Project ID. The Reference ID is 
automatically generated as a consecutive number by StudyTRAX (eg. Ref-0001, Ref-0002). The Reference 
ID carries with the subject throughout projects; it is universal to the database and does not contain any 
project-specific information.  
 
Additionally, a specific Project ID will be assigned to each subject that contains the site and a sequence 
unique to each site as follows (eg. BWH-1b-0001, MGH-1b-0001, BWH-1b-0002 ... WCM-1b-0001). Sites 
will be identified with a three-letter code as follows: BWH (Brigham and Women’s Hospital), MGH 
(Massachusetts General Hospital), Duke University Hospital (DUH), and Weill Cornell Medicine (WCM). 
The sites operate as separate projects, therefore coordinators and physicians only have access to the 
subjects at their site, unless additional privileges/permissions are granted in the database. All subjects 
who have been screened will be included in the Subject Screening Log.  
8.2.2 SCREENED, NOT ENROLLED 
 

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Subjects who are screened, but not enrolled into the study, either because they meet one or more 
exclusion criteria or decline participation, will be assigned a screening ID. The following de-identified data 
will be collected for all subjects that have been screened, but not consented/enrolled.  
 
• 
Date of intubation, qualifying PaO2/ FiO2 and ARDS onset 
• 
PaO2 
• 
SpO2 
• 
FiO2 
• 
Month of the year that patient met screening criteria (1-12) 
• 
Gender 
• 
Ethnicity 
• 
Age (if age >89, 89 will be entered for age) 
• 
Patient location (e.g. MICU, SICU, etc.) 
• 
Reason(s) patient excluded from study 
• 
If not excluded, not enrolled, why? 
• 
Lung injury category (e.g. sepsis, pneumonia) 
• 
Site of infection if lung injury category is sepsis 
 
This data will be entered into the StudyTrax database under the screening ID.  
8.3 
RANDOMIZATION PROCEDURES (DAY 0) 
8.3.1 RANDOMIZATION 
 
All ARDS criteria (for those enrolled with sepsis-induced ARDS) and pneumonia criteria in ventilated 
patients must occur within the same 24 hour period. The onset of ARDS is when the last criterion is met. 
Patients must be enrolled within 168 hours of ARDS onset or diagnosis of pneumonia in a ventilated 
patient. Information for determining when these time window criteria were met may come from either 
the site hospital or reports from a referring hospital.  
 
Following randomization, the low tidal volume protocol for mechanical ventilation must be initiated 
within two hours for those subjects with ARDS..  
 
The first treatment of study drug should be given within 24 hours of randomization. The day of 
randomization will be considered study day zero. 
 
Eligible participants will be randomized to one of the two treatment arms using a permuted block 
method146 with random block sizes. The randomization ratio will be 2:1 iCO:placebo. The randomization 
will not be stratified by any factors. The study biostatistician will verify all the necessary randomization 
information (blinding method, randomization method, block size, and number of treatment groups) in the 
StudyTRAX randomization configuration module.  
 
Site investigators will review all potential study participants with one of the CCC physician members. 
Following informed consent, the unblinded administering RT or physician or blinded study coordinator 
will randomize subjects in the database using the StudyTRAX randomization tool. The unblinded 
administering RT or physician will obtain the subject’s treatment assignment through the “View 
Treatment” button in the blinded database, a privilege granted to unblinded RTs and physicians only. CO-

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related measurements (SpCO, COHb, ambient CO) and drug-related information will be in a separate 
project within the database, which the blinded study coordinators will not have access to. This central 
review of randomization by the CCC unblinded physician will ensure that no subjects are enrolled in this 
multi-center trial during planned or unplanned study holds.  
8.3.2 ASSIGNING A RANDOMIZATION  
All subjects will be randomized through the StudyTRAX database using a permuted block method with 
random block sizes. The unblinded physicians or RTs or blinded coordinators will randomize the subject 
after enrollment. Subjects will receive a Randomization ID (RI-SiteCode-Seq) which includes a Site Code 
and Sequence starting with 0001 per site (eg. RI-BWH-0001, RI-MGH-0001, RI-DUH-0001, RI-WCM-0001).  
8.3.3 VENTILATOR PROCEDURES 
FiO2 will be increased prior to study drug administration in order to achieve a PaO2 ≥ 80 or SpO2 ≥ 95%. 
Ventilator management, including weaning, should follow the modified ARDS Network lower tidal volume 
(6 mL/kg PBW) protocol (Appendix D)13. Study drug administration will be continued in patients 
undergoing weaning from mechanical ventilation as in unless the subject is deemed ready for extubation 
by the clinical team.  
8.4 
BASELINE ASSESSMENTS 
The following baseline previous medical history and admission information will be recorded after 
informed consent has been obtained and within the 24-hour interval preceding initiation of study drug 
treatment. 
 
1. Demographic and Admission Data 
2. Pertinent Medical History and Physical Examination 
3. Height; gender, measured body weight (MBW); calculated predicted body weight (PBW). 
4. Time on ventilator prior to enrollment 
5. Type of Admission 
a. Medical 
b. Surgical scheduled 
c. Surgical unscheduled 
d. Trauma 
6. Presumed site of infection 
7. Acute or Chronic renal failure and use of dialysis 
8. Presence of the following chronic diseases: 
a. Cancer 
b. Hematological malignancy 
c. AIDS 
d. Diabetes mellitus 
e. COPD 
f. Asthma  
g. Liver cirrhosis 
h. Hypertension 
i. 
Coronary artery disease 
j. 
Congestive heart failure  

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k. Peripheral vascular disease  
l. 
Dementia 
m. Prior stroke with sequelae 
9. Survey of smoking history including:  
a. Ever smoker (> 100 cigarettes in lifetime)? 
b. If yes, current smoker?  
 
• Estimate of pack years (# packs per day) x (# years smoked) 
c. If former smoker, when did the subject quit smoking? 
10. Pregnancy test (serum or urine) for women of childbearing potential  
 
The following baseline clinical parameters will also be recorded. If more than one value is available for 
this 24-hour period, the appropriate values for the APACHE II calculator will be recorded. If no values are 
available from the 24 hours prior to study drug administration, then values must be measured prior to 
initiation of study drug. A full description of the below data collection is in Section 8.1. 
Baseline Clinical Parameters 
1. Vital Signs 
2. CVP if available 
3. Electrocardiogram (EKG) 
4. Ventilator Parameters  
5. Frontal Chest Radiograph (qualifying radiograph)  
6. Lung Injury Score (LIS) 
7. APACHE II Score  
8. SOFA Score 
9. Glasgow Coma Score (GCS)  
10. Richmond Agitation Sedation Scale (RASS) 
11. ABG (qualifying arterial blood gas)  
12. SpO2 
13. Scv02 if available 
14. Complete blood count (CBC) 
15. Basic metabolic panel   
16. PT/PTT/INR if available 
17. Serum CK, AST, ALT, Albumin, Total Protein if available 
18. Bilirubin  
19. Lactate  
20. Vasopressors and Inotropes Review 
21. Fluid intake, fluid output (most recent 24 hour value) or mean hourly value for most recently 
available period 
22. Renal replacement therapy status 
23. Concomitant Medications Review 
24. Discarded bronchoalveolar lavage (BAL) fluid and plasma if available  
 
8.5 
STUDY DRUG ADMINISTRATION DAYS 1 – 3 
 
Study Day 1 will be within 24 hours of randomization. Prior to treatment on each study day, blood will be 
drawn for measurement of COHb level, complete blood count (CBC), lactate, blood gas analysis (either 
ABG or venous blood gas [VBG]), and additional laboratory values (as outlined in Section 1.3) if not 
ordered by the clinical team as part of routine ICU care. The subject’s chart will be reviewed daily for 

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interval medical events. The subject’s ventilator settings, vital signs, laboratory values, EKG, chest X-ray, 
and concomitant medications will be reviewed to determine if the subject remains eligible for treatment 
on each study day. Daily Hold Parameters are outlined in Section 6.1.6. 
 
If the subject remains eligible for treatment, the study visit will require approximately 3 hours. Treatment 
will last 90 minutes with additional safety monitoring for an additional 90 minutes immediately following 
study drug completion. Blood will be drawn at serial time point during treatment and after treatment for 
measurement of COHb levels and blood gas analysis (Table 5). 
• 
Blood collection for COHb measurements pre-treatment and after 20 minutes for estimation of 
DLCO and determination of CFK-equation determined iCO dose to achieve a COHb level of 7% 
(target 6-8%) at 90 minutes (in subjects randomized to CO) 
• 
Blood collection for COHb measurements at 40 minutes, 60 minutes, 75 minutes, 90 minutes, 
and 3 hours for safety monitoring  
• 
Arterial or venous blood collection for blood gas analysis pre-treatment and at 90 minutes 
• 
Blood collection for research labs (EDTA and PAXgene tubes) pre- and post-treatment (90 min, 3 
hours)  
• 
Urine collection for research labs pre- and post-treatment (90 min, 3 hours) 
8.5.1 REFERENCE MEASUREMENTS (DAYS 1 – 3) 
The following parameters will be measured and recorded daily on study Days 1 through 3 from 3:00-10:00 
AM using the values closest to 8:00 am on the days specified in the Schedule of Activities (Section 1.3). 
On days when the study drug is administered, the ventilator parameters will be recorded using the values 
closest to the study drug administration. Details regarding the below assessments are found in Section 
8.1. 
 
The following conditions will be ensured prior to reference measurements:  
• 
No endobronchial suctioning for 10 minutes 
• 
No invasive procedures or ventilator changes for 30 minutes 
• 
All vascular pressures will be zero-referenced to the mid-axillary line with the patient supine.  
 
Reference Measurements 
1. Vital Signs 
2. CVP if available 
3. EKG 
4. Ventilator Parameters Assessment (closest to the time of study drug administration) 
5. Frontal Chest Radiograph when available 
6. Lung Injury Score (LIS) 
7. SOFA Score 
8. GCS  
9. RASS 
10. Scv02 if available 
11. CBC 
12. Basic metabolic panel if available and requested by treating physician 
13. PT/PTT/INR if available and requested by treating physician 
14. Serum CK, AST, ALT, Albumin, Total Protein if available and requested by treating physician 
15. Bilirubin  
16. Lactate  

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17. Vasopressors and Inotropes Review 
18. Fluid intake, fluid output (most recent 24 hour value) or mean hourly value for most recently 
available period 
19. Renal replacement therapy status 
20. Concomitant Medications Review 
21. Adverse Event Monitoring 
22. Blood sample collection for research labs 
23. Urine sample collection for research labs 
24. Microbiological results when available 
25. Discarded BAL fluid and plasma if available  
8.5.2 PRE-TREATMENT ASSESSMENTS (DAY 1 – 3) 
The following assessments will be done on Days 1-3 prior to study drug administration. No treatment will 
be started before these assessments are performed and the pre-treatment administration checklist is 
completed by the study investigator. 
• 
Blood Gas Analysis (arterial or venous): Arterial or venous blood will be drawn for blood gas analysis 
using the IL GEM Premier Co-oximeter or local laboratory at each center. The blood gas will be 
assessed daily prior to starting study drug administration. The study drug will not be started until this 
result is obtained and reviewed by the study physician investigator for a potential study hold. The 
following values will be recorded from the ABG or VBG results: pH, PCO2, PO2, and base excess or 
deficit. 
• 
Hemoglobin Level: Hemoglobin will be measured and assessed daily prior to study drug 
administration. The hemoglobin level will be measured by the local laboratory at each center. The 
study drug will not be started until this result is obtained and reviewed by the study physician 
investigator for a potential study hold. 
• 
COHb Level: The baseline COHb level will be drawn and assessed by an unblinded physician 
investigator, just prior (less than 15 minutes) to starting the study drug administration. COHb will be 
measured by study investigators using an IL GEM Premier Co-oximeter at the subject’s bedside. These 
results are obtained within less than 5 minutes. The study drug will not be started until this result is 
obtained and reviewed for a potential study hold. All COHb level result printouts should be filed in the 
unblinded subject file.  
• 
Lactate Level: Lactate level will be measured and assessed daily by the local laboratory at each center 
prior to study drug administration. The study drug will not be started until this result is obtained and 
reviewed by the study physician investigator for a potential study hold. 
• 
EKG: A 12 lead EKG will be obtained and reviewed daily by the study staff physician prior to study drug 
administration to evaluate for cardiac exclusion criteria (acute MI, ACS, or unstable atrial or 
ventricular arrhythmia). The study drug will not be started until the EKG is obtained and reviewed by 
the study physician investigator for a potential study hold. The EKG will also be officially read locally 
at each site. 
• 
Calibration of IL GEM Premier Co-oximeter and COventDS: The IL GEM Premier Co-oximeter and 
COventDS will be calibrated according to the Manual of Operations prior to study drug administration. 
• 
Calculate Deadspace (Vd/Vt): Dead space fraction will be measured daily by the administering RT or 
physician prior to study drug administration using a NICO or NM3 monitor (Respironics) and the PCO2 
obtained from an arterial or venous blood gas. 

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• 
Ambient Air CO Monitoring: Ambient air CO levels will be measured before and throughout study 
drug treatment using a calibrated Dräger Pac 7000 CO detector. This will ensure that ambient levels 
are maintained within acceptable limits for occupational exposure. 
• 
Review of Reference Measurements: The unblinded study investigator will review reference 
measurements outlined in Section 8.5.1. 
• 
Review of Adverse Events: The blinded study coordinator will review events over the past 24 hours 
that may meet criteria for adverse events.  
• 
Review Daily Hold Parameters (detailed information in Section 6.1.6) prior to starting Study Drug 
administration. 
8.5.3 STUDY DRUG ADMINISTRATION AND MONITORING 
The study drug will be administered for up to 90 minutes daily for up to 3 days following randomization 
or until discontinuation of mechanical ventilation, whichever occurs first according to Section 6.1.9. See 
Sections 6.1.6, 6.1.7, and 6.1.8 for information about study drug hold, interruption, and permanent 
discontinuation criteria.  
Study Drug Dosing: 
Subjects randomized to CO will be administered iCO at 200 ppm for 20 min as a “test dose” on each 
treatment day to determine in real-time each subject’s CFK equation-determined personalized dose. 
COHb levels will be measured before and after the 20 min test dose, and these two levels entered into 
the CFK equation calculator program to estimate DLCO and compute the iCO dose to achieve a COHb level 
of 7% (target 6-8%) at 90 min. After the iCO dose is determined, subjects will be administered iCO at the 
CFK equation-determined personalized dose (200-500 ppm) but not to exceed 500 ppm or 90 min total 
(including test dose). Subjects randomized to placebo will be administered air.  
 
Monitoring during Study Drug Administration: 
Subjects will be monitored with continuous pulse oximetry, cardiac monitoring, and either intermittent 
(at least every 15 minutes) or continuous blood pressure monitoring (if an arterial line is in place) 
throughout the course of the 90-minute study drug treatment. 
 
SpCO and SpO2 will be monitored continuously using a Masimo Rad-7 non-invasive pulse oximeter, and 
recorded at the time points specified in Table 5. COHb levels will be measured using an IL GEM Premier 
Co-oximeter and recorded at the time points specified below. Arterial or venous blood will be drawn for 
blood gas analysis prior to treatment and at the completion of study drug administration (90 min or earlier 
if discontinued prior to 90 min). If study drug treatment is discontinued prior to 90 min, all of the 
scheduled 90-minute measurements will be made and recorded at the time of study drug discontinuation. 
Ambient CO levels will be monitored continuously using a Dräger Pac 7000 CO detector, and recorded at 
the time points specified below. 
  
   Table 5. CO Monitoring During Treatment 
 
 
ABG or 
VBG 
(pH, 
PCO2, 
PO2) 
FiO2 
COHb level 
(IL GEM 
Premier co-
oximeter) 
CO Dose 
Adjustment 
(CFK 
calculator)  
SpCO 
(Masimo 
Rad-7) 
SpO2 
(Masimo
Rad-7) 
Ambient 
CO Level 
(Dräger 
Pac CO 
detector) 

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Pre-
treatment 
X 
X 
X 
 
X 
X 
X 
20 min 
 
 
X 
X 
X 
X 
X 
40 min 
 
 
X 
 
X 
X 
X 
60 min 
 
 
X 
 
X 
X 
X 
75 min 
 
 
X 
 
X 
X 
X 
90 min* 
X 
X 
X 
 
X 
X 
X 
180 min 
 
 
X 
 
X 
X 
X 
  *90 min or upon completion of study product if discontinued sooner than 90 min. 
8.6 
POST-TREATMENT ASSESSMENTS 
8.6.1 STUDY DAYS 4, 5, AND 7  
If the subject remains hospitalized, the following assessments will be completed. See Section 8.1 for 
descriptions of study assessments. 
• 
Review interval medical events, ventilator settings, vital signs, and concomitant medications 
• 
Review EKG on study day 7, and other days if available 
• 
Review chest X-ray on study day 7, and other days when available 
• 
Urine and blood collection for research labs on study day 5 
• 
ABG or VBG when available  
• 
SpO2 
• 
Lung injury score (LIS) 
• 
Dead space fraction (Vd/Vt) on day 7 
• 
Obtain Glasgow Coma Score (GCS), SOFA score, and Richmond Agitation Sedation Scale (RASS) 
• 
The following lab values will be recorded if available and requested by treating physician: CBC, 
basic metabolic panel, PT/PTT/INR, Serum CK, AST, ALT, Albumin, Total Protein, and Lactate  
• 
Collection of blood for bilirubin if not drawn by the clinical team 
• 
Vasopressors and inotropes review 
• 
Fluid intake and output (most recent 24 hour value) or mean hourly value for most recently 
available period on days 4 and 5 
• 
Renal Replacement Therapy Status 
• 
Adverse event review 
• 
Microbial results when available 
• 
Discarded BAL and plasma samples when available 
8.6.2 STUDY DAY 14 
If the subject remains hospitalized, the following assessments will be completed. See Section 8.1 for 
descriptions of study assessments. 
• 
Ventilator Parameters Assessment 
• 
SOFA Score 
• 
GCS 
• 
SpO2 when available 
• 
Blood collection for total bilirubin if not drawn by the clinical team 

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• 
ABG or VBG when available 
• 
CBC and basic metabolic panel when available 
• 
Vasopressors and Inotropes Review 
• 
Renal Replacement Therapy Status 
• 
Microbiological results when available 
8.6.3 STUDY DAY 28  
If the subject remains hospitalized, the following assessments will be completed. See Section 8.1 for 
descriptions of study assessments. 
• 
Review interval medical events 
• 
Obtain GCS and SOFA score at Day 28 or on discharge date 
• 
Collection of blood for total bilirubin if not drawn by the clinical team 
• 
CBC and basic metabolic panel if available 
• 
Microbial results when available 
• 
Vasopressors and Inotropes Review 
• 
Renal Replacement Therapy Status 
• 
Discharge, ventilator, and vasopressor status 
 
If the subject has been discharged home or to a long-term acute care facility, the subject will be contacted 
by telephone to: 
• 
Obtain vital status 
8.6.4 STUDY DAY 60 
If the subject remains hospitalized, the following assessments will be completed. See Section 8.1 for 
descriptions of study assessments. 
• 
Discharge, ventilator, and vasopressor status 
 
If the subject has been discharged home or to a long-term acute care facility, the subject will be contacted 
by telephone to: 
• 
Obtain vital status 
8.6.5 MONTH 3 AND 6 ASSESSMENTS 
Subjects will be contacted by phone at 3 and 6 months for the following assessments. See Section 8.1 for 
descriptions of study assessments: 
• 
Obtain vital status 
• 
Neurocognitive testing- Telephone versions of the Montreal Cognitive Assessment (MoCA) and 
Hayling sentence completion test 
 
8.7 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.7.1 DEFINITION OF ADVERSE EVENTS (AE) 
 

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Adverse event means any untoward medical occurrence associated with the use of an intervention in 
humans, whether or not considered intervention-related (21 CFR 312.32 (a)). 
 
8.7.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
 
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of either the 
investigator or sponsor, it results in any of the following outcomes:  
 
• 
results in death;  
• 
is life threatening (places the subject at immediate risk of death from the event as it occurred); 
• 
requires inpatient hospitalization or prolongation of existing hospitalization;  
• 
results in a persistent or significant disability/incapacity;  
• 
results in a congenital anomaly/birth defect; or  
• 
any other adverse event that, based upon appropriate medical judgment, may jeopardize the 
subject’s health and may require medical or surgical intervention to prevent one of the outcomes 
listed above. 
 
Organ failures related to ARDS or the patient’s underlying condition that are systematically captured by 
the protocol should not be reported as adverse events unless they are considered to be study related. If 
the suspected adverse reaction is unexpected, it will be classified as a serious unexpected suspected 
adverse reaction (SUSAR). 
8.7.3 DEFINITION OF ADMINISTRATION RELATED ADVERSE EVENTS 
For this trial, a subset of adverse events will be considered to be “administration related adverse 
events”. These "administration related adverse events" will by definition be considered suspected 
adverse reactions. These events are: 
• 
Acute MI* within 48 hours of study drug administration 
• 
Acute stroke** within 48 hours of study drug administration 
• 
New onset atrial or ventricular arrhythmia requiring DC cardioversion within 48 hours of study 
drug administration 
• 
Increased oxygenation requirements defined as: an increase in FiO2 of ≥ 0.2 AND increase in PEEP 
≥ 5 cm H2O within 6 hours of study drug administration 
• 
Increase in any protocol-specified measurement of COHb ≥ 10%  
• 
Increase in lactate by ≥ 2 mmol/L within 6 hours of study drug administration 
 
* Acute MI is defined below according to the Executive Group on behalf of the Joint European Society of 
Cardiology (ESC)/American College of Cardiology (ACC)/American Heart Association (AHA)/World Heart 
Federation (WHF) Task Force for the Universal Definition of Myocardial Infarction144. We will also rely 
upon subspecialty consultation to confirm the presence or absence of an acute MI. 
 
Acute myocardial infarction is defined as acute myocardial injury with clinical evidence of acute 
myocardial ischemia and with detection of a rise and/or fall of cardiac troponin values with at least 1 value 
above the 99th percentile upper reference limit and at least 1 of the following: 
• Symptoms of myocardial ischemia; 
• New ischemic ECG changes; 
• Development of pathological Q waves; 

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• Imaging evidence of new loss of viable myocardium or new regional wall motion abnormality 
in a pattern consistent with an ischemic etiology; 
• Identification of a coronary thrombus by angiography or autopsy (not for types 2 or 3 MIs). 
 
** Acute stroke is defined as an acute neurological deficit attributed to an acute focal injury of the central 
nervous system by a vascular cause, including cerebral infarction, intracerebral hemorrhage, and 
subarachnoid hemorrhage based on neuropathological, neuroimaging, and/or other clinical evidence of 
permanent injury145. We will also rely upon subspecialty consultation to confirm the presence or absence 
of an acute stroke. 
 
8.7.4 CLASSIFICATION OF AN ADVERSE EVENT 
 
8.7.4.1 SEVERITY OF EVENT 
For adverse events (AEs) not included in the protocol defined grading system, the Common Terminology 
Criteria for Adverse Events (CTCAE) Version 5.0 guidelines will be used to describe severity.  
 
• 
Grade 1 Mild – asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated.  
• 
Grade 2 Moderate – minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental activities of daily living (ADL). 
• 
Grade 3 Severe – medically significant but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling; limiting self-care ADL. 
• 
Grade 4 Life-threatening – urgent intervention indicated.  
• 
Grade 5 Death 
 
8.7.4.2 RELATIONSHIP TO STUDY INTERVENTION 
All adverse events (AEs) will have their relationship to study intervention assessed by a designated blinded 
study physician at each site. Additional AE adjudication will be provided by one of the Medical Monitors 
who will review all AEs in an unblinded fashion as is currently recommended by the FDA for SAEs 
(Guidance for Industry and Investigators Safety: Reporting Requirements for INDs and BA/BE Studies 
(Section VI.C); December 2012). The Medical Monitors at the CCC will work collaboratively with the 
reporting investigator to determine if a serious adverse event has a reasonable possibility of having been 
caused by the study drug or procedure. 
 
The degree of certainty about causality will be graded using the categories below. In a clinical trial, the 
study product must always be suspect.  
 
• 
Definitely Related – There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out.  
• 
Probably Related – There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely.  
• 
Possibly Related – There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of the trial medication). However, other 
factors may have contributed to the event (e.g., the participant’s clinical condition, other 
concomitant events).  
• 
Probably not related – A clinical event, including an abnormal laboratory test result, whose 
temporal relationship to study intervention administration makes a causal relationship 

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improbable (e.g., the event did not occur within a reasonable time after administration of the 
study intervention) and in which other drugs or chemicals or underlying disease provides plausible 
explanations (e.g., the participant’s clinical condition, other concomitant treatments). 
• 
Not Related – The AE is completely independent of study intervention administration, and/or 
evidence exists that the event is definitely related to another etiology. There must be an 
alternative, definitive etiology documented by the clinician. 
 
Administration related adverse events, as outlined in Section 8.7.3 of the protocol will always be 
considered suspected adverse reactions if they occur within protocol-defined timeframes (Section 8.7.3) 
of study drug exposure. 
 
8.7.4.3 EXPECTEDNESS  
The designated blinded study physician at each site and the Medical Monitor(s) will be responsible for 
determining whether an AE is expected or unexpected. An AE will be considered unexpected if the nature, 
severity, or frequency of the event is not consistent with the risk information previously described for the 
study product as described in the Investigator’s Brochure.  
8.7.5 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
 
Blinded study coordinators at each site will collect adverse event information daily during the period from 
enrollment to study day 7.  A blinded study physician who is designated at each site will review AE 
information collected by the blinded study coordinators and adjudicate any clinical adverse events that 
occur during the period from enrollment to study day 7. Adverse event collection begins after the patient 
or surrogate has signed informed consent and has received study drug or undergone study procedures. If 
a patient experiences an adverse event after consent, but prior to receiving study product, the event will 
NOT be collected unless the investigator feels the event may have been caused by a protocol procedure. 
The designated blinded study physician will evaluate any changes in laboratory values and physical signs 
and will determine if these changes are clinically important. All clinically important adverse events will be 
recorded in the case report form regardless of attribution to study product. After study day 7, adverse 
events are not required to be reported unless the investigator feels the events were related to either 
study product or a protocol procedure.  
 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured on the 
appropriate case report form (CRF). Information to be collected includes event description, time of onset, 
clinician’s assessment of severity, relationship to study product (assessed only by those with the training 
and authority to make a diagnosis), and time of resolution/stabilization of the event. All AEs occurring 
while on study must be documented appropriately regardless of relationship. All AEs will be followed to 
adequate resolution. 
 
Any medical condition that is present at the time that the participant is screened will be considered as 
baseline and not reported as an AE. However, if the pre-existing medical condition deteriorates at any 
time between the first study product administration and study day 7, it will be recorded as an AE. Organ 
failures related to ARDS or the patient’s underlying condition that are systematically captured by the 
protocol should not be reported as adverse events unless they are considered to be study related. 
 

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Changes in the severity of an AE will be documented to allow an assessment of the duration of the event 
at each level of severity to be performed. AEs will be followed for outcome information until resolution 
or stabilization or until 28 days (or hospital discharge whichever occurs first) after enrollment. 
 
For this trial, a subset of adverse events will be considered to be “administration related adverse 
events”. These "administration related adverse events" will by definition be considered suspected 
adverse reactions. These events are: 
• 
Acute MI within 48 hours of study drug administration 
• 
Acute stroke within 48 hours of study drug administration 
• 
New onset atrial or ventricular arrhythmia requiring DC cardioversion within 48 hours of study 
drug administration 
• 
Increased oxygenation requirements defined as: an increase in FiO2 of ≥ 0.2 AND increase in PEEP 
≥ 5 cm H2O within 6 hours of study drug administration 
• 
Increase in any protocol-specified measurement of COHb ≥ 10%  
• 
Increase in lactate by ≥ 2 mmol/L within 6 hours of study drug administration 
 
8.7.6 ADVERSE EVENT REPORTING  
 
All clinically important AEs as described in Section 8.7.5, will be recorded in the StudyTRAX database. For 
each AE, an email will be sent to the CCC, site PI, and blinded physician adjudicating AEs at each site. AEs 
will be summarized using System Organ Class and preferred terms. Tables will show by treatment group 
the number and percentage of participants experiencing an AE within a System Organ Class or within 
preferred term category by severity or by relationship to study product. For the calculations in these 
tables, a participant with multiple AEs within a category will be counted once under the maximum severity 
or the strongest recorded causal relationship to study product. Formal statistical testing comparing groups 
is not planned since interpretation of differences must rely heavily upon clinical judgment. 
 
8.7.7 SERIOUS ADVERSE EVENT REPORTING  
 
Site investigators will report all serious AND unexpected adverse events or reactions, regardless of 
relationship to the study intervention, to the CCC by entering the event information into StudyTrax within 
24 hours of becoming aware of the event. StudyTrax will automatically send an email alert to the CCC and 
DCC study leadership. The site investigator should include an assessment of whether there is a reasonable 
possibility that the study intervention caused the event. 
 
Investigators will also report all serious AND administration related adverse events (as described in 
Section 8.7.3) to the CCC by email within 24 hours of becoming aware of the event. An email will also be 
sent from the StudyTRAX database alerting the CCC to the event. The CCC will review the event and may 
inform the site to permanently discontinue study drug administration to the subject (Section 6.1.8), and 
may hold enrollment pending Executive Committee (EC) and DSMB review (Section 10.1.6). The CCC may 
also request additional information from the site investigator. 
 
If the event is characterized as unexpected and related or possibly related to the research and indicates 
that there are new or increased risks to subjects, the CCC will notify the MGB IRB within 5 working days/7 
calendar days of the date the investigator first becomes aware of the problem according to the MGB IRB 
requirements.  
 

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The CCC will report all unexpected and study-related deaths or life-threatening suspected serious 
adverse events (SUSAR) to the FDA within 7 calendar days of receipt. The CCC will report all deaths 
occurring during the study hospitalization and all serious, unexpected, and study-related adverse events 
and all administration related adverse events to the DSMB, by email, or telephone, within 7 calendar 
days of the CCC being notified of the event. A written report will be sent to the DSMB and the FDA within 
15 calendar days. Site investigators will also be sent these reports and will follow their local IRB’s policy 
for reporting.  
 
8.8 
UNANTICIPATED PROBLEMS 
 
8.8.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
An unanticipated problem involving risks to participants or others is defined as any incident, experience, 
or outcome that meets all of the following criteria: 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and 
informed consent document; and (b) the characteristics of the participant population being 
studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
8.8.2  UNANTICIPATED PROBLEM REPORTING  
Site investigators will report unanticipated problems (UPs) to the PI and CCC within 24 hours. 
Unanticipated problems will be reported to the sIRB within 5 working days/7 calendar days of the date 
the investigator first becomes aware of the problem. The UP report will include the following information: 
• 
Protocol identifying information: protocol title and number, PI’s name, and the IRB project 
number; 
• 
A detailed description of the event, incident, experience, or outcome;  
• 
An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP;  
• 
A description of any changes to the protocol or other corrective actions that have been taken or 
are proposed in response to the UP. 
 
9 
STATISTICAL CONSIDERATIONS  
 
9.1 
STATISTICAL HYPOTHESES 
 
Hypothesis: We hypothesize that low dose iCO will be safe and well-tolerated and that the CFK equation-
based iCO personalized dosing algorithm will be accurate in achieving a target COHb level of 6-8% in 
patients pneumonia (without ARDS and sepsis or with sepsis without ARDS criteria) and with sepsis-
induced ARDS at study entry.  
 

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We further hypothesize that precision-based iCO therapy will reduce the severity of pneumonia and ARDS 
and organ failure by suppressing mitochondrial dysfunction, inhibiting inflammasome activation and 
necroptosis, and accelerating resolution of inflammation.  
 
Primary Endpoints:  
1. The primary endpoint is safety of iCO, defined by the incidence of pre-specified 
administration-related AEs and SAEs.  
Administration-related SAEs are: 1) myocardial infarction; 2) stroke; 3) new onset arrhythmia 
requiring DC cardioversion; 4) worsening hypoxemia1,2; 5) COHb ≥ 10%; and 6) increase in 
lactate by ≥ 2 mmol/L. 
2. Accuracy of the CFK equation-based personalized iCO dosing algorithm to achieve a COHb 
level of 6-8%. 
 
Secondary Endpoints: 
1. Lung Injury Score (LIS) on days 1-5 and day 7 
2. PaO2/FiO2 ratio on days 1-5 and day 7 
3. Oxygenation index (OI) on days 1-5, and 7 
4. Dead space fraction (Vd/Vt) on days 1-3 and day 7 
5. Sequential Organ Failure Assessment (SOFA) scores on days 1-5, 7, 14, and 28 
6. Ventilator-free days at day 28 
7. ICU-free days at day 28 
8. Hospital-free days to day 60 
9. Hospital mortality to days 28 and 60 
10. Neurocognitive function at 3 and 6 months 
 
Exploratory Endpoints: 
1. Plasma and urine biomarkers of mitochondrial dysfunction (mtDNA) 
2. Plasma and urine biomarkers of inflammasome activation (IL-18)  
3. Plasma and urine biomarkers of necroptosis (RIPK3) 
4. Plasma Lipid mediators (LM) and specialized pro-resolving mediators (SPMs) 
 
9.2 
SAMPLE SIZE DETERMINATION 
 
As the primary endpoint of this Phase Ib trial is safety, this sample size of 36 (2:1 CO:placebo) was chosen 
based on a probability to estimate the mean difference between measured and predicted COHb levels in 
the CO group within 0.5% with 95% confidence to support the accuracy of the CFK equation.  
 
Our preliminary data from our Phase Ia trial demonstrate that the CFK equation is highly accurate at 
predicting COHb levels following a 20 minute exposure to 200 ppm iCO (r=0.916, R2=0.9204; p<0.0001). 
The mean difference between measured and predicted COHb levels was 0.1467 ± 0.2738. Using a more 
conservative estimate of standard deviation (SD)=0.6, we will have >99% probability that 95% confidence 
intervals around the mean difference between measured and predicted COHb levels will be -0.353% to 
0.647%. Our calculation includes an 8% dropout rate.  
 

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The goal of the safety evaluation for this study is to identify safety concerns associated with CO 
administration. The ability of the study to detect administration-related AEs or SAEs can be expressed by 
the true event rate above which at least one SAE would likely be observed and the true event rate below 
which no events would likely be observed. Specifically, for the CO arm of the study (n=24), there is a 90% 
probability of observing at least one AE if the true background rate of that AE is 10% and a 97% probability 
of observing at least one AE if the true background rate of that AE is 15%. For the placebo arm of the study 
(n=12), there is an 83% probability of observing at least one AE if the true background rate of that AE is 
15% and a 91% probability of observing at least one AE if the true background rate of that AE is 20%. In 
our Phase Ia trial, there were no administration-related AEs and no SAEs related to study product or 
procedures. 
 
Probabilities of observing one or more events between the groups are presented in Table 6 below for a 
range of possible true AE rates. These calculations provide a more complete picture of the sensitivity of 
this study design to identify potential safety problems with CO. 
 
Table 6. Probability of observing 1 or more events between the groups for different true AE rates 
 
        CO group  
 
 
 
   
     Placebo Group 
n 
True AE rate 
Power to 
detect ≥1 AE 
 
N 
True AE rate 
Power to 
detect ≥1 AE 
24 
0.010 
0.20 
 
12 
0.010 
0.10 
24 
0.020 
0.36 
 
12 
0.020 
0.20 
24 
0.035 
0.54 
 
12 
0.035 
0.32 
24 
0.050 
0.68 
 
12 
0.050 
0.43 
24 
0.100 
0.90 
 
12 
0.100 
0.69 
24 
0.150 
0.97 
 
12 
0.150 
0.83 
24 
0.200 
0.99 
 
12 
0.200 
0.91 
 
9.3 
POPULATIONS FOR ANALYSES 
All randomized participants will be included in the intent-to-treat (ITT) population. All randomized 
subjects who receive at least one dose of study product (CO vs. placebo) will be included in the safety 
analyses (i.e.. modified ITT). For secondary and exploratory endpoints, subjects may also be analyzed 
according to the number of study drug doses completed.   
 
9.4 
STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
We will use the Mann-Whitney U or t-test, chi-square (χ2) or Fisher’s exact tests to analyze baseline 
demographics and clinical characteristics between the treatment groups. Distribution of numerical 
outcomes will be evaluated prior to any analysis. Numerical outcomes will be compared between 
treatment arms using the Mann-Whitney U or t-test and categorical outcomes compared using either χ2 
or Fisher’s exact test. 
 
9.4.2 ANALYSIS OF THE PRIMARY SAFETY ENDPOINT(S) 
 
We hypothesize that iCO will be safe and well-tolerated and that our iCO dosing algorithm will be accurate 

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in achieving target COHb levels. 
 
We will examine all AEs including pre-specified administration-related AEs by the type of event, as well as 
by body system class, and will report proportions and 95% CI. We will compare the occurrence of specific 
AEs in each arm by the χ2 test, and the number of AEs per patient in each arm by the Wilcoxon-Mann-
Whitney test. 
 
COHb levels will be summarized descriptively reporting mean, standard deviation, median, minimum, and 
maximum at each time point, each day, by treatment arm. The mean difference between predicted and 
measured COHb levels with 95% confidence intervals will be presented for the iCO group. Repeated 
measures analysis of variance (ANOVA) will be used to assess the effects of treatment, time (over 3 days), 
and the interaction between time and treatment for 90-minute COHb levels. ANOVA will be repeated to 
assess the effects of treatment, time (over 3 days), entry criteria (ARDS/Sepsis vs. Non-ARDS/Sepsis 
Pneumonia vs. Non-ARDS/non-Sepsis Pneumonia), and two-way/three-way interactions of time, 
treatment and entry criteria. We will also compare the occurrence of COHb ≥ 10% in each arm by the χ2 
test.  
 
Accuracy of the CFK equation-based personalized iCO dosing algorithm to achieve target COHb levels will 
also be analyzed by Spearman correlation and Bland-Altman plots using measured vs. predicted COHb 
levels and modeled using linear or non-linear regression.  
 
9.4.3 ANALYSIS OF THE SECONDARY ENDPOINT(S) 
 
We also hypothesize that iCO will reduce the severity of ARDS as measured by lung injury score (LIS), 
PaO2/FiO2, oxygenation index (OI), and dead space fraction (Vd/Vt). These endpoints will be analyzed 
using repeated measures analysis of covariance (ANCOVA) to assess the effects of treatment, time, and 
the interaction between time and treatment, while incorporating between-patient variation in baseline 
measurements and important covariates including smoking, age, sex, and severity of ARDS. Age and sex 
will be considered as important biological variables in all analyses.  
 
Interaction contrasts will include polynomial trends across time, focusing on a linear trend suggesting 
improvement and a quadratic trend suggesting that improvement is leveling off. As part of our analysis, 
we will also include the interaction between treatment and  entry criteria (ARDS/Sepsis vs. Non-
ARDS/Sepsis Pneumonia vs. Non-ARDS/non-Sepsis Pneumonia)in the model.  Because the study is not 
powered for the interaction between treatment and entry criteria, that test will be exploratory. We will 
also perform post-hoc analyses within each criteria group to generate hypotheses for future study. 
 
With 24 subjects in the CO group and 12 subjects in the placebo arm, we will have 80% power (α=0.05) to 
detect a 0.95 difference in LIS between treatment arms, assuming a conservative SD of 0.8 (Table 7). We 
will also have 80% power to detect a 78.6 mm Hg difference in PaO2/FiO2, a 12.0 difference in OI, and a 
0.14 difference in Vd/Vt between treatment arms, assuming SDs of 66, 10.1, and 0.12 respectively. 
 
Table 7. Minimal detectable difference at 80% power. 
 
Endpoint 
SD 
Detectable Difference 
(α= 0.05) 
LIS 
0.8 
0.95 
PaO2/FiO2 
66 
78.6 

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Endpoint 
SD 
Detectable Difference 
(α= 0.05) 
OI 
10.1 
12.0 
Vd/Vt 
0.12 
0.14 
 
Ventilator-free days (VFDs), hospital- and ICU-free days, and Sequential Organ Failure Assessment (SOFA) 
scores will each be analyzed separately using ANCOVA with treatment arm as the main effect. Given our 
modest sample size, we will only be powered to see large changes. 
 
Results from the Hayling Sentence Completion Test and Montreal Cognitive Assessment (MoCA) testing 
will be compared between CO and placebo groups using the Mann-Whitney U test or t-test (if normally 
distributed). If significantly different between the groups, scores from the Hayling Sentence Completion 
Test and MoCA testing may be incorporated into exploratory multiple regression analyses. 
 
Mortality will be analyzed using Cox regression models for time-to-death with treatment arm as the main 
predictor, and with important covariates including smoking, age, and sex in the model. Hazard ratio (HR) 
and 95% CI for CO relative to placebo will be reported, along with Kaplan-Meier plots and estimated 
proportions surviving at 28, 60, and 180 days.  
 
To address potential bias from unobserved study outcomes due to missed visits, premature withdrawal, 
or death and to ensure robust data analyses, we will work closely with Dr. Dale Needham at Johns Hopkins 
University, who has extensive expertise with long-term outcomes assessment after acute respiratory 
failure and critical illness. Dr. Needham will provide his expertise and assist with statistical methods 
developed by his Outcomes After Critical Illness and Surgery (OACIS) Group to address missing data and 
assess long-term outcomes that may be “truncated due to death”. We will use the statistical software 
application developed by his group using R and Shiny to impute missing data among survivors given the 
high mortality in ARDS. We will also employ other statistical approaches used by his OACIS Group to assess 
long-term outcomes in critically ill patients. These include a composite endpoint approach using both 
functional outcome and survival, as well as other statistical methods (e.g. survivors only analyses and 
survivor average causal effect [SACE]).  
9.4.4 ANALYSIS OF EXPLORATORY ENDPOINTS (BIOMARKERS) 
 
We will compare biological outcomes between treatment arms using the Mann-Whitney U or t-test for 
numerical endpoints and either χ2 or Fisher’s exact test for categorical outcomes. Lipid mediators (LM) 
and specialized pro-resolving mediators (SPM) levels will be analyzed using principal component analysis 
(PCA). 
 
We hypothesize that treatment with iCO will reduce plasma levels of mitochondrial DNA (mtDNA), IL-18, 
and receptor-interacting serine/threonine-protein kinase 3 (RIPK3). Circulating IL-18, mtDNA, and RIPK3 
levels will be log transformed before analysis. Based on our prior work, the SD of the log-IL-18 is 
approximately 0.7, approximately 1.0 for mtDNA, and approximately 2.0 for RIPK3. With 12 subjects in 
the placebo arm and 24 in the iCO arm, we will have 80% power to detect a difference in IL-18 levels of 
0.834 log, which corresponds to an approximate 2-fold difference in plasma IL-18. We will have 80% power 
to detect a difference in mtDNA of 1.190 log, which corresponds to an approximate 3-fold difference in 
the circulating mtDNA copy number between the iCO and placebo groups.  
 

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We will also have 80% power to detect a difference in RIPK3 of 2.38 log, which corresponds to an 
approximate 8 10-fold difference in the circulating RIPK3 levels between the iCO and placebo groups. 
While the magnitude of the putative CO effect on these 3 biomarkers is unknown, we found a 3-fold 
difference in IL-18 between sepsis patients with and without ARDS and a 4-fold difference in mtDNA 
between sepsis/ARDS patients and controls. Similarly, we found a 7-fold difference in median RIP3K levels 
between patients with organ failure and shock compared with ICU patients without organ failure or shock. 
Therefore detecting differences of these magnitudes is feasible in this patient population. Based on our 
preliminary data, we will also have 80% power (α=0.05) to detect a difference in a number of LM and SPM 
including PGD2, PGE2, PGF2α, TXB2, RvD2, and Mar1 (Table 8).  
 
 
 
        
Table 8. Minimal detectable difference at 80% power. 
 
 
Mediator 
Standard 
Deviation 
Detectable 
Difference 
(pg/mL) 
Pro- 
inflammatory LM 
LTB4 
17.95 
21.4 
PGD2 
0.68 
0.81 
PGE2 
1.68 
2.00 
PGF2α 
1.15 
1.37 
TXB2 
12.38 
14.75 
Pro-resolving 
SPM 
RVD2 
12.18 
14.5 
Mar1 
15.29 
18.2 
RvE1 
10.54 
12.55 
 
We will assess correlations between biomarkers and clinical and lung physiology outcomes including LIS, 
SOFA, VFDs, as well as 28 and 60 day hospital mortality. We will use multivariable linear regression as the 
primary model with LIS as the primary outcome variable. Biomarker levels tend to exhibit right 
skewedness and large outliers, so data will likely require log transformation. Additional predictor variables 
(such as demographic variables and comorbidities) will be specified a priori and interactions between 
those variables and biomarkers will be included in the model with adjustment for age and sex as important 
biological variables. To evaluate the predictive ability of biomarkers for binary outcomes, we will use two 
methods. We will use c-statistics obtained from a logistic regression model to evaluate the effect of 
biomarker measurements on increasing the area under the receiver operating characteristic (ROC) curves. 
In addition, we will test if including measured biomarkers to models including important covariates results 
in an improvement in the net reclassification index as we have done previously for mtDNA.  
 
9.4.5 SAFETY ANALYSES 
 
The primary endpoint is safety of iCO, defined by the incidence of pre-specified administration-related 
AEs and SAEs. Administration-related SAEs are: 1) myocardial infarction; 2) stroke; 3) new onset 
arrhythmia requiring DC cardioversion; 4) worsening hypoxemia1,2; 5) COHb ≥ 10%; and 6) increase in 
lactate by ≥ 2 mmol/L. The primary endpoint is also accuracy of the CFK equation-based personalized iCO 
dosing algorithm to achieve a COHb level of 6-8%. 
 
We will examine all AEs including pre-specified administration-related AEs by the type of event, as well as 
by body system class, and will report proportions and 95% CI. We will compare the occurrence of specific 
AEs in each arm by the χ2 test, and the number of AEs per patient in each arm by the Wilcoxon-Mann-
Whitney test. 

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For AEs not included in the protocol defined grading system, the CTCAE Version 5.0 guidelines will be used 
to describe severity. The CTCAE term, start and stop date, severity, relationship, expectedness, and 
outcome will be reported for all AEs. A separate table will report the incidence of pre-specified 
administration-related AEs per arm. 
 
COHb levels will be summarized descriptively reporting mean, standard deviation, median, minimum, and 
maximum at each time point, each day, by treatment arm. The mean difference between predicted and 
measured COHb levels with 95% confidence intervals will be presented for the iCO group. Repeated 
measures analysis of variance (ANOVA) will be used to assess the effects of treatment, time (over 3 days), 
and the interaction between time and treatment for 90-minute COHb levels. ANOVA will be repeated to 
assess the effects of treatment, time (over 3 days), entry criteria (ARDS/Sepsis vs. Non-ARDS/Sepsis 
Pneumonia vs. Non-ARDS/non-Sepsis Pneumonia), and two-way/three-way interactions of time, 
treatment and entry criteria. We will also compare the occurrence of COHb ≥ 10% in each arm by the χ2 
test.  
 
Accuracy of the CFK equation-based personalized iCO dosing algorithm to achieve target COHb levels will 
also be analyzed by Spearman correlation and Bland-Altman plots using measured vs. predicted COHb 
levels and modeled using linear or non-linear regression.  
 
9.4.6 PLANNED INTERIM ANALYSES  
 
Safety monitoring will be conducted on a regular basis by the EC and DSMB. The EC will independently 
review the safety data in a blinded fashion at regular intervals and make recommendations to the DSMB 
and NHLBI. The DSMB will meet at least semiannually or after enrollment of 12 subjects, whichever is 
sooner, to assess safety data on each arm of the study in an unblinded fashion. In addition, all related 
SAEs will be reported to the DSMB in an expedited fashion and the DSMB would have the option after 
every SAE report to request an ad hoc meeting if safety concerns arise. At this ad hoc meeting, the CCC 
would update the tabulations of all AEs and administration related adverse reactions for Board review. 
Recommendations by the EC and/or DSMB to amend the protocol or add additional subjects will be 
reviewed by the sIRB prior to implementation. The EC and/or DSMB may halt enrollment in the study at 
any time during the trial. 
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 
 
10.1.1.1 CONSENT/ASSENT AND OTHER INFORMATIONAL DOCUMENTS PROVIDED TO PARTICIPANTS 
 
Federal regulations 45 CFR 46.111(a)(5) require that informed consent will be sought from each 
prospective subject or the subject’s legally authorized representative (LAR). Consent forms describing in 
detail the study intervention, study procedures, and risks are given to the participant/LAR and written 
documentation of informed consent is required prior to starting intervention/administering study 
intervention.   
 

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The investigator is responsible for ensuring that the patient/LAR understands the risks and benefits of 
participating in the study, and answering any questions the patient/LAR may have throughout the study 
and sharing any new information in a timely manner that may be relevant to the patient’s willingness to 
continue his or her participation in the trial. All study participants or their surrogates will be informed of 
the objectives of the study and the potential risks. The informed consent document will be used to explain 
the risks and benefits of study participation to the patient in simple terms before the patient is entered 
into the study, and to document that the patient is satisfied with his or her understanding of the risks and 
benefits of participating in the study and desires to participate in the study. The investigator is responsible 
for ensuring that informed consent is given by each patient or legal representative. This includes obtaining 
the appropriate signatures and dates on the informed consent document prior to the performance of any 
protocol procedures and prior to the administration of study agent. 
 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
 
A licensed physician investigator will obtain written informed consent after a detailed review of the 
consent form with the patient, or more likely, the LAR of the potential study participant. The majority of 
the patients approached for participation in this study will have limitations of decision-making abilities 
due to their critical illness or need for sedating medications. Hence, most patients will not be able to 
provide informed consent. Accordingly, informed consent will be sought from the potential subject’s LAR. 
 
The physician investigator will be responsible for ensuring that the subject or LAR understands the risks 
and benefits of participating in the study, and answering any questions the subject or LAR may have 
throughout the study and sharing in a timely manner any new information that may be relevant to the 
subject’s willingness to continue his or her participation in the trial. All study participants or their 
surrogates will be informed of the objectives of the study and the potential risks.  
 
The informed consent document will be used to explain the risks and benefits of study participation to 
the patient or LAR in simple terms before the patient is entered into the study, and to document that the 
patient or LAR is satisfied with his or her understanding of the risks and benefits of participating in the 
study and desires to participate in the study. The investigator is responsible for ensuring that informed 
consent is given by each patient or legal representative. This includes obtaining the appropriate signatures 
and dates on the informed consent document prior to the performance of any protocol procedures and 
prior to the administration of the study agent. 
 
The subject or LAR will be given ample opportunity to read the consent form and ask questions during the 
initial consent visit and afterwards prior to the signing of the consent form. The subject or LAR will be 
allowed to discuss the study with anyone they choose before making a decision. The physician investigator 
obtaining informed consent will explain to the subject or LAR that questions can be addressed at any point 
throughout the trial and the PI’s contact information will be made available during the consent visit for 
any questions that arise prior to or throughout the study. The subject or LAR will be given a signed copy 
of the informed consent form, which will include the site PI contact information and the subject or LAR 
will be encouraged to call with any questions. 
 
For subjects for whom consent was initially obtained from a surrogate, but who subsequently regain 
decision-making capacity while in hospital, we will obtain formal consent for continuing participation, 
inclusive of continuance of data acquisition. The initial consent form signed by the surrogate will reflect 
that such continuing consent will be obtained when possible.  
 

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The following categories of surrogates (listed in general order of preference) may provide written 
informed consent on behalf of potential subjects incapable of providing informed consent themselves, 
however the exact order will be dependent on the sIRB and local IRB requirements at each site when 
relying on an sIRB.  
• 
court appointed guardian with specific authority to consent to participation in research or 
authority to make health care decisions for a class of diagnostic and therapeutic decisions 
inclusive of the proposed research;  
• 
health care proxy/person with durable power of attorney with specific authority for making health 
care decisions inclusive of the proposed research; or  
• 
spouse, adult child, or other close family member who knows the subject well and has been 
involved in their care.  
 
Subjects and/or surrogates will be given a written copy of the consent form, as well as ample time to read 
and understand the written material. The surrogate will be asked to consider the potential subject’s own 
views prior to providing surrogate consent. An investigator will be available to answer any additional 
questions. Subjects and/or their surrogates can withdraw consent at any time.  
 
If consent from a LAR or surrogate cannot be obtained in person on behalf of a subject with impaired 
decision-making capacity, a licensed physician investigator may call the subject’s LAR to perform consent 
by phone according in accordance with the sIRB and each site’s IRB policy. Consent obtained by telephone 
must comply with all regulatory requirements about the process, the consent elements, and 
documentation of consent. 
 
 
For subjects for whom consent was initially obtained from a surrogate, but who subsequently regain 
decision-making capacity while in the hospital, we will obtain formal consent for continuing participation, 
inclusive of continuance of data acquisition. The initial consent form signed by the surrogate will reflect 
that such continuing consent will be obtained when possible.  
 
After the patient or his/her legal representative has consented to the study, a copy of the signed informed 
consent form will also be included in his or her medical record. A note will be included in the medical chart 
to make other staff aware that the patient was enrolled in the study. The pager number of the PI or Co-
investigator will be included in the note to allow the clinical staff to contact the research team at any time.   
 
Remote Consent During COVID-19 Pandemic 
 
Due to infection control measures and hospital visitor policy restrictions during the COVID-19 pandemic, 
it may not be possible for physician investigators to obtain wet consent from the subject/LAR in person. 
If unable to obtain wet consent from the subject’s LAR in person, the physician investigator will obtain 
consent remotely via telephone call in accordance with the sIRB and each site’s IRB policy and FDA 
guidance on the conduct of clinical trials during the COVID-19 pandemic. The consent form will be 
provided to the LAR via secure email or other electronic means including REDCap, Adobe Sign, DocuSign 
and asked to electronically sign the document, scan or take a picture of the consent signature page, and 
then return electronically to the research team, in accordance with the sIRB and each relying site’s 
institutional policies. 
 
 
The physician investigator will electronically sign or print and sign their own copy of the paper consent 
form and document the consent process in a note to file or informed consent process checklist. 
Documentation will include that the consent form was provided to the LAR, consent was obtained, the 

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method used to obtain consent, that the LAR signed a separate copy of the consent form, and the 
date/time of consent. A compiled copy of the consent including the document with the physician 
investigator’s signature and the LAR’s signature consent document will be retained in the study record 
and a copy of the compiled consent form will be uploaded to subject’s medical record. Consent obtained 
electronically must comply with all regulatory requirements about the process, the consent elements, and 
documentation of consent. 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause. Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping    
 
Written notification, documenting the reason for study suspension or termination, will be provided by the 
suspending or terminating party to study participants, investigators, NIH, the Investigational New Drug 
(IND) sponsor and regulatory authorities. If the study is prematurely terminated or suspended, the 
Principal Investigator (PI) will promptly inform study participants, the sIRB and site IRBs as required, and 
sponsor and will provide the reason(s) for the termination or suspension. Study participants will be 
contacted, as applicable, and be informed of changes to study visit schedule. 
 
If the trial is suspended, all enrollments and study product administration will cease, but other study 
procedures will continue. The EC and DSMB will convene for a conference call within seven days of the 
suspension to determine whether the suspension can be lifted or permanent discontinuation of study 
product administration is appropriate. Lifting the suspension requires the unanimous approval of all 
members of the EC and DSMB. 
 
10.1.3 CONFIDENTIALITY AND PRIVACY  
 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, 
and the sponsor(s) and their interventions. This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants. Therefore, the 
study protocol, documentation, data, and all other information generated will be held in strict confidence. 
No information concerning the study or the data will be released to any unauthorized third party without 
prior written approval of the sponsor. All research activities will be conducted in as private a setting as 
possible. 
 
The study monitor, other authorized representatives of the sponsor, representatives of the IRB, or 
regulatory agencies may inspect all documents and records required to be maintained by the investigator, 
including but not limited to, medical records (office, clinic, or hospital) and pharmacy records for the 
participants in this study. The clinical study site will permit access to such records. 
 
The study participant’s contact information will be securely stored at each clinical site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location for as 
long a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements. 
 
All subjects or surrogates must provide written informed consent and signed HIPAA authorization prior to 
the performance of any screening or main study procedures.  

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Subject confidentiality will be protected throughout the study and no subject-identifying information will 
be released to anyone outside the IRB-approved study. Confidentiality will be secured through several 
mechanisms. To maintain confidentiality, all research specimens, data collection forms, and study reports 
will be identified only by a coded number. The coded subject identification number will be automatically 
generated as a consecutive number by the StudyTRAX database and only IRB-approved study staff will 
have access to the codes.  
 
Investigators and study staff at each site will retain a Subject Code Key linking the subject ID number to 
the subject’s identifying information. The Subject Code Key will always be kept secure and stored 
electronically on each site’s approved storage approved for Confidential Data. All paper records linking 
the subject to their study ID code will be kept in a locked cabinet within a locked office. Any study forms 
and paper records containing personal identifier information (e.g., address, phone number) will be stored 
securely in a locked cabinet within a locked office at each study site. 
 
Clinical data abstracted from the medical record will be entered into a study-specific database in 
StudyTRAX, a secure, web-based, data management tool which is US FDA Title 21 CFR Part 11 compliant. 
StudyTRAX hosted projects are fully HIPAA compliant and ensure access control, audit control, data 
integrity, user authentication, and transmission security. The StudyTRAX database will only be accessible 
to the IRB-approved study staff. Study participants will be identified only by unique study identification 
numbers. Other than dates related to an individual (e.g., Date of Birth, Date of Informed Consent, 
admission date, test date, discharge date, death date, etc.) and ages over 89 (as applicable), no patient 
HIPAA identifiers will be entered or inputted into the StudyTRAX EDC system database. Each participating 
site will have access to only its own site’s data, with the exception of the study monitors, CCC, and DCC 
who will have access to data entered from all sites for preparation of study-related reports (DSMB, 
adverse event reporting), as well as for analysis of the study data/endpoints. 
 
Research data will be maintained by and accessible only to IRB-approved study staff and investigators. 
Any paper records including informed consent forms, contact information etc., and/or physical data will 
be stored securely in a locked file cabinet in a locked office in a secure area at each site. Any electronic 
data stored by each site (e.g., screening log, patient name, MRN, contact information) will utilize only site-
approved methods for Confidential Data. Access to data will be role based and minimum necessary access 
will be provisioned. Only de-identified data will be used in any data analysis.  
 
Access to subject data and information at the study sites, including biological samples, will be restricted 
to IRB-approved authorized personnel. All study data analyses will be done with coded numbers only. 
Subjects will not be identified by name in any reports or publications, nor will the data be presented in 
such a way that the identity of individual subjects can be inferred. Clinical information will not be released 
without the written permission of the subject, except as necessary for monitoring by the CCC, DCC, sIRB, 
NHLBI, FDA or other authorized federal agencies. All data released by the DCC for studies including genetic 
studies will be de-identified. Should subjects or surrogates revoke their consent for genetic testing, the 
clinical sites will notify the DCC and all samples collected for genetic analysis for that subject will be 
destroyed. Confirmation of destruction of samples will be forwarded from the DCC to the clinical site. 
 
Investigators will obtain consent to use social security numbers (SSN) to determine vital status of 
participants lost to follow-up. Contact information and the subject’s SSN will be collected on paper at the 
time of consent. Contact information and SSN will be maintained on paper locally and will not appear in 
the study database. 

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Certificate of Confidentiality  
To further protect the privacy of study participants, this research is protected by a Certificate of 
Confidentiality from the National Institutes of Health (NIH). This certificate protects identifiable research 
information from forced disclosure. It allows the investigator and others who have access to research 
records to refuse to disclose identifying information on research participation in any civil, criminal, 
administrative, legislative, or other proceeding, whether at the federal, state, or local level. By protecting 
researchers and institutions from being compelled to disclose information that would identify research 
participants, Certificates of Confidentiality help achieve the research objectives and promote participation 
in studies by helping assure confidentiality and privacy to participants. 
 
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA  
 
With the participant’s approval and as approved by local IRBs, coded biological samples will be stored at 
a central repository located at BWH, along with coded study data, for use by other researchers including 
those outside of the study. These samples could be used for research related to carbon monoxide, ARDS, 
critical illness or other lung diseases. The central repository at BWH will also be provided with a code that 
will allow linking the biological specimens with the data from each participant, maintaining the blinding 
of the identity of the participant. Only the originating clinical trial site will be able to link coded samples 
or data to specific individuals. 
 
Samples may also be stored in the central repository at BWH for future genetic research related to carbon 
monoxide, ARDS, critical illness or other lung diseases. Specific consent for future genetic research will be 
required.  
 
During the conduct of the study, an individual participant can choose to withdraw consent to have 
biological specimens stored for future research. However, withdrawal of consent with regards to 
biosample storage may not be possible after the study is completed.  
 
Datasets and associated documentation from the clinical trial will be submitted to the NHLBI data 
repository Biological Specimen and Data Repository Information Coordinating Center (BioLINCC). The 
datasets will be prepared in accordance with the NHLBI Policy for Data Sharing from Clinical Trials and 
Epidemiological Studies, and in accordance with the Guidelines for NHLBI Data Set Preparation. In addition 
to the clinical data from the trial, biomarker results will also be deposited in BioLINCC. The DCC will 
prepare the datasets in a manner that protects the privacy of study participants and will document the 
methods used to prepare the datasets to protect participant privacy before submission to the NHLBI data 
repository. To maintain participant confidentiality, the DCC will remove obvious identifiers and de-identify 
protected information by re-coding data in order to mask subject IDs and center identifiers. 
 
10.1.5 KEY ROLES AND STUDY GOVERNANCE 
 
Principal Investigator 
Rebecca Baron, MD 
Brigham and Women’s Hospital  
75 Francis Street, Boston, MA 
Tel: 617-525-6642 
Email: rbaron@bwh.harvard.edu 
 

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Medical Monitor 
Medical Monitor 
B. Taylor Thompson, MD 
Crystal North, MD 
Massachusetts General Hospital 
Massachusetts General Hospital 
55 Fruit Street, Boston, MA 
55 Fruit Street, Boston, MA 
Tel: 617-643-2411 
Tel: 617-726-1721 
E-mail: tthompson1@mgh.harvard.edu 
Email: cnorth@mgh.harvard.edu 
 
Medical Monitors 
The unblinded Medical Monitors will oversee regulatory reporting for the trial. They will review all 
potential study participants with site investigators and will be available for site questions that arise 
regarding safety and eligibility throughout the study. The Medical Monitors will review all AEs and prepare 
reports for all pre-specified administration-related AEs and serious treatment-related AEs for expedited 
DSMB review. They will also prepare IND safety reports for all serious, unexpected, suspected adverse 
reactions (SUSARs) for expedited DSMB and FDA review. 
Clinical Coordinating Center 
The CCC will serve as the overall coordinating center for all study sites and will be directed by  the Medical 
Monitors. The CCC will be responsible for the oversight of the conduct of the Phase Ib safety trial including 
design and implementation of the protocol, comprehensive training of site personnel, communication and 
coordination among all sites, AE reporting, and report generation for the DSMB, FDA, sIRB, and NHLBI in 
conjunction with the DCC. The CCC will train clinical trial personnel and serve as the central site to maintain 
the necessary materials to conduct the trial including standard operating procedures (SOPs), electronic 
Case Report Forms (CRFs), training documents, Site Performance Plans, Recruitment and Retention Plans, 
the Investigator’s Brochure (IB), AE Reporting Guidelines, the DSMB Charter, the Manual of Operations, 
CO administration procedures, and long term neurocognitive outcomes assessment.  
Data Coordinating Center 
The DCC will oversee the overall data management and biostatistical support for the study, and will be 
directed by Dr. Karla Ballman at WCM. In conjunction with the CCC, the DCC will be responsible for the 
statistical design of the trial, randomization, data collection and monitoring, quality control and 
completeness of study data, interim monitoring of the data, biostatistical support and data analysis, and 
report generation for the DSMB, FDA, sIRB, and NHLBI. The DCC will oversee the Data Completeness and 
Quality Monitoring Reporting Plan, and will ensure completeness of subject assessment and data 
collection, complete data cleaning, and perform primary and secondary data analyses to ensure timely 
publication and dissemination of the study results. In conjunction with the CCC, the DCC will ensure that 
the study results are reported to ClinicalTrials.gov within 12 months of the primary completion date and 
will implement the data sharing plan, including submission of study data to BioLINCC. Together, the DCC 
and CCC will work closely together to ensure the successful completion of the Phase Ib clinical trial and 
timely dissemination of its results. 
Executive Committee 
The Executive Committee (EC) will be comprised of Dr. Choi, Dr. Baron, CCC Directors, IND Sponsor, and 
the DCC study statistician. Dr. Choi and Dr. Baron will serve as heads of the overall EC. They will lead 
videoconferences to provide the structure to facilitate rapid decision-making and contingency planning 
for study implementation and execution.  The EC will also ensure rapid communication among the 
leadership group, with administrative support from the CCC Project Managers for study-related clinical 

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and scientific issues, as well as operational issues that may arise. A major role of the EC will be to 
independently review the safety data in a blinded fashion at regular intervals and make recommendations 
to the DSMB and NHLBI. 
Steering Committee 
The PIs Dr. Choi and Dr. Baron will assume primary responsibility for the overall administration of the 
project, providing scientific, administrative, and fiscal oversight. The Steering Committee will include the 
site PIs from each enrolling center. They will work closely with the EC to provide formal guidance to the 
project, with interactive input from the Steering Committee. The Steering Committee (SC) will provide 
support, advice, and guidance for the study.  
 
10.1.6 SAFETY OVERSIGHT 
 
Safety oversight will be under the direction of a Data and Safety Monitoring Board (DSMB) composed of 
individuals with the appropriate expertise. Per NHLBI Policy for Data and Safety Monitoring of Extramural 
Clinical Studies, NHLBI may suggest DSMB members for this trial. Members of the DSMB should be 
independent from the study conduct and free of conflict of interest, or measures should be in place to 
minimize perceived conflict of interest. The DSMB will meet at least semiannually or after enrollment of 
12 subjects, whichever is sooner, and on as needed basis to assess safety data on each arm of the study. 
In addition, all related SAEs will be reported to the DSMB in an expedited fashion and the DSMB would 
have the option after every SAE report to request an ad hoc meeting if safety concerns arise. At this ad 
hoc meeting, the CCC would update the tabulations of all AEs and administration related adverse reactions 
for Board review. The DMSB will operate under the rules of an approved charter that will be written and 
reviewed at the organizational meeting of the DSMB. At this time, each data element that the DSMB needs 
to assess will be clearly defined. DSMB meetings may be held by conference call or face-to-face meeting. 
 
The DSMB serves in an advisory capacity to the IND Sponsor. They will review the study protocol, monitor 
all aspects of the study (e.g., recruitment, adverse events, protocol adherence, data quality, attrition, 
demographic and baseline characteristics) and recommend protocol modifications, including early study 
termination. All proposed changes to the study protocol will be reviewed by the DSMB and the single IRB. 
 
Data and Safety Monitoring Board (DSMB) 
The principal role of the DSMB is to regularly monitor data from this trial, review and assess the 
performance of its operations, and make recommendations, as appropriate, to the Study PI, IND Sponsor, 
and Project PI with respect to: 
 
• 
Benefit/risk ratio of procedures and participant burden 
• 
Selection, recruitment, and retention of participants 
• 
Adherence to protocol requirements  
• 
Completeness, quality, and analysis of measurements  
• 
Amendments to the study protocol and consent forms 
• 
Performance of individual centers and core labs 
• 
Participant safety 
• 
Notification of and referral for adverse events  
 
The Study PI and the CCC physician members will be responsible for the preparation of DSMB reports. 
Recommendations to end, modify, or continue the trial will be prepared by the DSMB executive secretary 

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for review by the Study PI and IND Sponsor. The Study PI and IND Sponsor will act on recommendations 
expeditiously in consultations with the Executive Committee. The executive secretary of the DSMB will be 
responsible for preparing the minutes for each meeting or conference call. DSMB recommendations will 
be distributed to all study investigators. 
 
Single IRB 
This study will use a single a sIRB to conduct the ethical review required for the protection of human 
subjects in compliance with the NIH Policy on the Use of a sIRB for Multi-Site Research. The Mass General 
Brigham (MGB) IRB will serve as the sIRB of record for this multi-center study and will facilitate efficient, 
effective, and consistent review of the proposed research. The MGB IRB provides ethical and scientific 
review and continuing oversight for the human subjects research at MGH and BWH. 
 
10.1.7 CLINICAL MONITORING 
Clinical site monitoring is conducted to ensure that the rights and well-being of trial participants are 
protected, that the reported trial data are accurate, complete, and verifiable, and that the conduct of the 
trial is in compliance with the currently approved protocol/amendment(s), with International Conference 
on Harmonisation Good Clinical Practice (ICH GCP), and with applicable regulatory requirement(s). 
• 
External site monitoring visits will be performed remotely or in-person by the DCC monitors. 
• 
On-site or remote monitoring will occur prior to the initiation of the site, after the first participant 
is enrolled, and yearly thereafter (and more frequently as needed) to review regulatory 
documents and monitor medical records against the study database. More frequent on-site or 
remote visits will occur if there is a problem at the site or a new satellite or subcontracted site is 
added. Emphasis will be placed on the process used to consent participants, compliance with 
regulatory requirements and the study protocol, study drug handling, and values of key 
endpoints. 
• 
Records of IRB approvals and subjects’ charts will be examined on a spot check basis by study 
monitors.  
• 
Source documents will be reviewed for targeted source data verification related to study 
endpoints, safety and other key data variables. 
• 
Site PIs will be provided copies of monitoring reports within 14 business days of the monitoring 
visit.   
• 
Each clinical trial site will provide direct access to all source data/documents and reports for the 
purpose of monitoring and auditing by the DCC on behalf of the IND Sponsor, and inspection by 
local and regulatory authorities. 
 
10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL 
 
Each clinical site will perform internal quality management of study conduct, data and biological specimen 
collection, documentation and completion. Each site will operate under a comprehensive Quality 
Management Plan, which incorporates internal Quality Control (QC) activities prior to data entry and 
regular internal Quality Assurance (QA) review of subjects' charts. 
 
QC procedures will be implemented beginning with the data entry system and data QC checks that will be 
run on the database will be generated. Any missing data or data anomalies will be communicated to the 
site(s) for clarification/resolution. 
 

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10.1.9 DATA HANDLING AND RECORD KEEPING  
 
10.1.9.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES  
 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
investigator. The investigator is responsible for ensuring the accuracy, completeness, legibility, and 
timeliness of the data reported. All source documents should be completed in a neat, legible manner to 
ensure accurate interpretation of data.   
 
Source data are all information, original records of clinical findings, observations, or other activities in a 
clinical trial necessary for the reconstruction and evaluation of the trial. Electronic source data are data 
initially recorded in electronic form.  
 
Study visit worksheets will be provided for use as source document worksheets for recording data for 
each participant enrolled in the study. Data should be entered in the electronic case report form (eCRF) 
in StudyTRAX as directed on the source document worksheets. Data recorded in the eCRF derived from 
source documents should be consistent with the data recorded on the source documents.  
 
Clinical data (including AEs), concomitant medications, and expected adverse reactions data) and clinical 
laboratory data will be entered into StudyTRAX, a 21 CFR Part 11-compliant data capture system provided 
by the DCC. The data system includes password protection and internal quality checks, such as automatic 
range checks, to identify data that appear inconsistent, incomplete, or inaccurate. Clinical data will be 
entered directly from the source documents. 
The study Manual of Operations will provide additional detail on data collection procedures, including 
source documentation, eCRF completion guidelines, data handling procedures and procedures for data 
monitoring and quality control that ensure these data are accurate, consistent, complete and reliable and 
in accordance with ICH 36.   
 
10.1.9.2 STUDY RECORDS RETENTION  
Study documents should be retained for at least seven years after study completion. No records will be 
destroyed without the written consent of the IND Sponsor. It is the responsibility of the IND Sponsor to 
inform the site investigators when these documents no longer need to be retained. 
 
10.1.10 PROTOCOL DEVIATIONS  
 
A protocol deviation is any noncompliance with the clinical trial protocol, International Conference on 
Harmonisation Good Clinical Practice (ICH GCP) requirements. The noncompliance may be either on the 
part of the participant, the investigator, or the study site staff. As a result of deviations, corrective actions 
are to be developed by the site and implemented promptly.  
 
It is the responsibility of the site investigator to use continuous vigilance to identify and report deviations 
in a timely manner. All deviations must be addressed in study source documents and will be reported to 
the DCC. Major protocol deviations, such as enrollment violations or any deviation that involves risks to 
participants or others, will be reported to the DCC within 4 business days of awareness. The CCC Project 
Manager will be responsible for reporting these to the single IRB within 5 business days. Minor deviations 
will be reported to the DCC as part of the site monitoring visits. 
 

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Sites should also follow their local IRB’s policy on reporting protocol deviations for studies relying on a 
single IRB. The site investigator is responsible for knowing and adhering to the sIRB and local site IRB 
requirements.  
 
10.1.11 PUBLICATION AND DATA SHARING POLICY 
 
Results from this study will be reported according to guidelines established by the NIH Public Access 
Policy, which ensures that the public has access to the published results of NIH funded research. The final 
peer-reviewed journal manuscripts will be submitted to the digital archive PubMed Central upon 
acceptance for publication. 
 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-Funded 
Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. As 
such, this trial will be registered at ClinicalTrials.gov, and results information from this trial will be 
submitted to ClinicalTrials.gov. In addition, every attempt will be made to publish results in peer-reviewed 
journals. All biomarker data generated in this study will be shared with the scientific community following 
the appropriate guidelines for data deposition. 
 
10.1.12 
CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence, such as by any outside party, is 
critical. Therefore, any actual conflict of interest of persons who have a role in the design, conduct, 
analysis, publication, or any aspect of this trial will be disclosed and managed. Furthermore, persons who 
have a perceived conflict of interest will be required to have such conflicts managed in a way that is 
appropriate to their participation in the design and conduct of this trial. All persons involved in study are 
expected to take the appropriate actions to ensure that they are in compliance with the financial 
disclosure requirements of their respective institutions. 
 
 
10.2 ABBREVIATIONS 
 
ABG 
Arterial Blood Gas 
AE 
Adverse Event 
ALI 
Acute Lung Injury 
ARDS 
Acute Respiratory Distress Syndrome 
BAL 
Bronchoalveolar Lavage 
BUN 
Blood Urea Nitrogen 
BWH 
Brigham and Women's Hospital 
CFK equation 
Coburn-Foster-Kane equation 
CFR 
Code of Federal Regulations 
CHF 
Congestive Heart Failure 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
CO 
Carbon Monoxide 
COHb 
Carboxyhemoglobin 
CONSORT 
Consolidated Standards of Reporting Trials 
CPAP 
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CRF 
Case Report Form 
DBP 
Diastolic Blood Pressure 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DLCO 
Diffusing capacity of the Lung for Carbon Monoxide 
DSMB 
Data and Safety Monitoring Board 
eCRF 
Electronic Case Report Forms 
EKG 
Electrocardiogram 
EC 
Executive Committee 
FDA 
Food and Drug Administration 
FiO2 
Fraction of Inspired Oxygen 
GCP 
Good Clinical Practice 
GCS 
Glasgow Coma Scale 
HIPAA 
Health Insurance Portability and Accountability Act  
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonisation  
iCO 
Inhaled Carbon Monoxide 
ICU 
Intensive Care Unit 
IDE 
Investigational Device Exemption 
IMV 
Intermittent Mechanical Ventilation 
IND 
Investigational New Drug Application 
INR 
International Normalized Ratio 
IRB 
Institutional Review Board 
ITT 
Intention-To-Treat 
LAR 
Legally Authorized Representative 
LIS 
Lung Injury Score 
LM 
Lipid Mediator 
MedDRA 
Medical Dictionary for Regulatory Activities 
mBW 
Measured Body Weight 
mtDNA 
Mitochondrial DNA 
MGH 
Massachusetts General Hospital 
MOP 
Manual of Procedures 
NCT 
National Clinical Trial 
NIH  
National Institutes of Health 
NO 
Nitric Oxide 
NYHA 
New York Heart Association 
OR 
Odds Ratio 
ORP 
Office of Research Protections 
PaCO2 
Partial pressure of arterial carbon dioxide 
PaO2 
Partial pressure of arterial oxygen 
PBMC 
Peripheral Blood Mononuclear Cell 
PEEP 
Positive End-Expiratory Pressure 
PI 
Principal Investigator 
Pplat 
Plateau pressure 
ppm 
Parts per million 
PS 
Pressure Support Ventilation 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 

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SBP 
Systolic Blood Pressure 
SBT 
Spontaneous Breathing Trial 
sIRB 
Single Institutional Review Board 
SOA 
Schedule of Activities 
SOC 
System Organ Class 
SOFA 
Sequential Organ Failure Assessment 
SOP 
Standard Operating Procedure 
SpCO 
Non-invasive COHb by pulse oximetry 
SPM 
Specialized Pro-resolving Mediator 
UAB 
Unassisted Breathing 
UP 
Unanticipated Problem 
US 
United States 
VBG 
Venous Blood Gas 
VFD 
Ventilator-free Days 
VA 
Alveolar ventilation 
Vd/Vt 
Dead space 
vWF 
von Willebrand factor 
WBC 
White Blood Cell 
 
 

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10.3 PROTOCOL AMENDMENT HISTORY 
 
Version 
Date 
Description of Change  
Brief Rationale 
2.0 
5/31/2022 
Synopsis: Two additional sites 
(Washington University, and New 
York-Presbyterian Brooklyn 
Methodist Hospital have been added 
to the protocol. 
Washington University and New York-
Presbyterian Brooklyn Methodist 
Hospital will be participating as sites 
in the trial. 
2.0 
5/31/2022 
6.1.8, 8.7.3: Clarification of criteria 
used to define acute MI and acute 
stroke. 
The DSMB requested clarification of 
how these administration-related AEs 
would be defined. 
2.0 
5/31/2022 
6.3, 8.3.1: The language regarding 
stratification by site was revised.  
An error was noted in the protocol. 
The randomization is not stratified by 
site or any other factor 
2.0 
5/31/2022 
Cover, 6.3, 10.1.5: The PI was 
changed from Dr. Laura Fredenburgh 
to Dr. Rebecca Baron. Dr. Baron was 
removed as Medical Monitor. 
Dr. Rebecca Baron is the new PI of the 
study and will no longer serve as 
Medical Monitor. Dr. Taylor 
Thompson and Dr. Crystal North will 
serve as Medical Monitors. 
2.0 
5/31/2022 
8.7.5: Clarification of who will collect 
AE information and assess their 
relationship to the study drug. 
The DSMB requested clarification of 
who would be monitoring AEs and 
their relationship to the study team. 
2.0 
5/31/2022 
8.7.7: Clarification that investigators 
will report AEs by entering the event 
information into the Studytrax 
database. 
StudyTrax will automatically send an 
email alert to the CCC and DCC study 
leadership.  
2.0 
5/31/2022 
9.2, 9.4.2, 9.4.5: Revision of the 
sample size determination section to 
clarify the power calculations. 
Revision of language and Table 6 
regarding the power to detect AEs 
based on the true AE rate and sample 
size. 
The prior language was incorrect and 
an error was noted in the probability 
calculations. 
 
2.0 
5/31/2022 
9.4.3, 9.4.4: Revision of language, 
Tables 7 and 8 regarding the 
detectable differences in secondary 
and exploratory endpoints with our 
sample size. 
An error was noted in these 
calculations. 
 
2.0 
5/31/2022 
9.4.6, 10.1.6: Revision of the DSMB 
meeting frequency and SAE reporting 
to the DSMB. 
The DSMB requests meeting twice 
per year or after 12 subjects are 
enrolled, whichever occurs sooner, to 
review the safety data in an 
unblinded fashion. All related SAEs 
will be reported to the DSMB in an 
expedited fashion and the DSMB 
would have the option after every 
SAE report to request an ad hoc 
meeting if safety concerns arise. 
 
 

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Wilson JG, Liu KD, Zhuo H, et al. Mesenchymal stem (stromal) cells for treatment of ards: A phase 
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Ranieri VM, Rubenfeld GD, Thompson BT, et al. Acute respiratory distress syndrome: The berlin 
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Singer M, Deutschman CS, Seymour CW, et al. The third international consensus definitions for 
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Brealey D, Brand M, Hargreaves I, et al. Association between mitochondrial dysfunction and 
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Carre JE, Orban JC, Re L, et al. Survival in critical illness is associated with early activation of 
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