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

 
 
 
 
 
 
 
 
 
 
 
A Phase I Trial of Inhaled Carbon Monoxide for the Treatment of 
Sepsis-Induced Acute Respiratory Distress Syndrome (ARDS) 
 
Protocol Version 13 
July 30, 2019 
 
NCT02425579 
 
 
 
 
 

 
 
 
 
 
A Phase I Trial of Inhaled Carbon Monoxide for the Treatment 
of Sepsis-Induced Acute Respiratory Distress Syndrome (ARDS) 
 
Protocol Version 13 
July 30, 2019 
IND 122800 
 
Sponsor: Mark A. Perrella, M.D. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
ABBREVIATIONS ................................................................................................................................... 5 
DEFINITIONS .......................................................................................................................................... 7 
PART I: STUDY SUMMARY ................................................................................................................. 9 
PART II: STUDY DESCRIPTION ....................................................................................................... 12 
1. BACKGROUND ................................................................................................................................. 12 
1.1. INTRODUCTION ......................................................................................................................................... 12 
1.2. BIOLOGICAL PROPERTIES OF CO ......................................................................................................... 12 
1.3. CO AND ANIMAL MODELS OF SEPSIS AND ALI  ............................................................................... 12 
1.4. LUNG PROTECTIVE EFFECTS OF CO  ................................................................................................... 14 
1.5. CO DELIVERY VIA AN INHALED ROUTE IS SAFE IN HUMAN SUBJECTS .................................... 14 
1.6. STEADY STATE DIFFUSING CAPACITY AND THE SAFETY OF INHALED CO  ............................ 16 
1.7. DELIVERY OF INHALED CO TO MECHANICALLY VENTILATED SUBJECTS .............................. 16 
1.8. CO DELIVERY SYSTEM TESTING IN BABOON MODEL OF PNEUMONIA .................................... 17 
1.9. CO DOSING STRATEGY USING CFK EQUATION ................................................................................ 19 
1.11. STUDY RATIONALE ................................................................................................................................ 20 
2. OBJECTIVES AND STUDY DESIGN ............................................................................................. 21 
2.1. STUDY OBJECTIVE ................................................................................................................................... 21 
2.2. HYPOTHESES ............................................................................................................................................. 21 
2.3. STUDY DESIGN .......................................................................................................................................... 21 
2.4. ACCRUAL OBJECTIVE ............................................................................................................................. 21 
2.5. STUDY PRODUCT, DOSE, ROUTE, AND REGIMEN ............................................................................ 22 
3. ENDPOINTS ....................................................................................................................................... 22 
3.1. PRIMARY ENDPOINT ................................................................................................................................ 22 
3.2. SECONDARY ENDPOINT ......................................................................................................................... 22 
3.3. OTHER SECONDARY ENDPOINTS ......................................................................................................... 22 
3.4. FOCUSED SAFETY ANALYSIS ................................................................................................................ 23 
4. STUDY POPULATION AND ENROLLMENT .............................................................................. 23 
4.1. SCREENING ................................................................................................................................................ 23 
4.2. INCLUSION CRITERIA .............................................................................................................................. 23 
4.3. EXCLUSION CRITERIA ............................................................................................................................. 24 
4.4. ENROLLMENT, RANDOMIZATION AND STUDY INITIATION TIME WINDOW ............................ 25 

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4.5. INFORMED CONSENT ............................................................................................................................... 25 
4.6. ENROLLMENT AND RANDOMIZATION ............................................................................................... 25 
5. STUDY PROCEDURES .................................................................................................................... 26 
5.1 CO OR PLACEBO STUDY PROCEDURES ............................................................................................... 26 
5.2. VENTILATOR PROCEDURES ................................................................................................................... 31 
6. DATA COLLECTION ....................................................................................................................... 31 
6.1. BACKGROUND ASSESSMENTS .............................................................................................................. 31 
6.2. BASELINE ASSESSMENTS ....................................................................................................................... 32 
6.3. ASSESSMENTS AFTER ENROLLMENT ................................................................................................. 33 
6.4. ASSESSMENTS AFTER HOSPITALIZATION ......................................................................................... 37 
6.5. ENDPOINT DETERMINATIONS ............................................................................................................... 38 
7. STATISTICAL CONSIDERATIONS AND SAFETY ASSESSMENT ........................................ 38 
7.1. STATISTICAL CONSIDERATIONS .......................................................................................................... 38 
7.2. PHASE I SAFETY ASSESSMENT ............................................................................................................. 39 
7.3. SUMMARY GUIDELINES FOR SRC AND DSMB ASSESSMENT ........................................................ 39 
8. DATA COLLECTION AND SITE MONITORING ....................................................................... 39 
8.1. DATA COLLECTION .................................................................................................................................. 39 
8.2. SITE MONITORING .................................................................................................................................... 39 
9. RISK ASSESSMENT ......................................................................................................................... 40 
9.1. RISKS OF ACTIVE STUDY DRUG ........................................................................................................... 40 
9.2. RISKS OF BLOOD DRAWS ....................................................................................................................... 40 
9.3. MINIMIZATION OF RISKS ........................................................................................................................ 40 
9.4. POTENTIAL BENEFITS ............................................................................................................................. 40 
9.5. RISKS VERSUS BENEFITS ........................................................................................................................ 41 
10. HUMAN SUBJECTS ........................................................................................................................ 41 
10.1. SELECTION OF SUBJECTS ..................................................................................................................... 41 
10.2. INFORMED CONSENT ............................................................................................................................. 42 
10.3. CONTINUING CONSENT ........................................................................................................................ 42 
10.4. IDENTIFICATION OF SURROGATES .................................................................................................... 42 
10.5. JUSTIFICATION OF SURROGATE CONSENT ..................................................................................... 43 
10.6. ADDITIONAL SAFEGUARDS FOR VULNERABLE SUBJECTS ........................................................ 43 
10.7. CONFIDENTIALITY ................................................................................................................................. 43 

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11.  ADVERSE EVENT REPORTING ................................................................................................ 44 
12. REFERENCES .................................................................................................................................. 46 
13. APPENDICES ...................................................................................................................................52 
APPENDIX A: GUIDELINES FOR EVIDENCE OF INFECTION ..................................................... 52 
APPENDIX B: CFK EQUATION AND PREDICTED COHB CURVES IN HUMANS .................... 54 
APPENDIX C: PREDICTED COHB LEVELS IN ARDS USING CFK EQUATION ........................ 57 
APPENDIX D:  DE-IDENTIFIED DATA FOR SCREENED, NON-ENROLLED SUBJECTS ......... 58  
APPENDIX E: AMBIENT CO TESTING ............................................................................................ 59 
APPENDIX F: TIME-EVENTS SCHEDULE  ..................................................................................... 85 
APPENDIX G: VENTILATOR PROCEDURES  ................................................................................. 86  
APPENDIX H: DEAD SPACE MEASUREMENT .............................................................................. 89  
APPENDIX I: GENETIC TESTING ..................................................................................................... 92 
APPENDIX J: DATA AND SAFETY MONITORING BOARD ......................................................... 93 
APPENDIX K: ADVERSE EVENTS ..................................................................................................   94 
APPENDIX L: IL682 CO-OXIMETER PROCEDURE .......................................................................  95 
APPENDIX M: NEUROCOGNITIVE DATA- CO AND IPF ............................................................ 104 
APPENDIX N: LINDE CSR - SAFETY AND TOLERABILITY OF INHALED CO ....................... 105 
APPENDIX O: CO DELIVERY SYSTEM OPERATOR’S MANUAL ............................................  340 
APPENDIX P: CO DELIVERY SYSTEM TECHNICAL INFORMATION ....................................  367 
 
 
 
 

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ABBREVIATIONS 
 
ABG = arterial blood gas 
ALI = Acute Lung Injury 
Ang-2 = Angiopoietin-2 
ARDS = Acute Respiratory Distress Syndrome 
BAL = Bronchoalveolar Lavage 
BMI = Body Mass Index 
BUN = Blood Urea Nitrogen 
BWH = Brigham and Women's Hospital 
CO = carbon monoxide 
COHb = carboxyhemoglobin 
CHF = Congestive Heart Failure 
CFK equation = Coburn-Foster-Kane equation 
CPAP = Continuous Positive Airway Pressure 
CPR = Cardiopulmonary resuscitation 
CT = Computed Tomography 
DCC = Data Coordinating Center 
DBP = Diastolic Blood Pressure 
DLCO = Diffusing capacity for carbon monoxide 
DSMB = Data Safety Monitoring Board 
FiO2 = Fraction of Inspired Oxygen 
GCS = Glasgow Coma Scale 
iCO =  inhaled carbon monoxide 
ICU = Intensive Care Unit 
IL-1! = Interleukin 1! 
IL-1Ra = IL-1 receptor antagonist 
IL-6 = Interleukin 6 
IL-8 = Interleukin 8 
IL-10 = Interleukin 10 
IL-18 = Interleukin 18 
IMV = Intermittent Mechanical Ventilation 
INR = International Normalized Ratio 
mBW = measured body weight 
MGH = Massachusetts General Hospital 
NO = nitric oxide 
OR = Odds Ratio 
PaCO2 = Partial pressure of arterial carbon dioxide 
PaO2 = Partial pressure of arterial oxygen 
PAP = Pulmonary Artery Pressure 

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PB = Barometric Pressure 
PBW = Predicted Body Weight 
PEEP = Positive End-Expiratory Pressure 
Pplat = Plateau pressure 
ppm  = parts per million 
PS = Pressure Support Ventilation 
Receptor for Advanced Glycation Endproducts = RAGE 
SRC = Scientific Review Committee 
SBP = Systolic Blood Pressure 
SBT = Spontaneous Breathing Trial 
Sequential Organ Failure Assessment= SOFA 
SpO2 = Oxygen Saturation 
SpCO = Non-invasive COHb by pulse oximetry 
VFD = Ventilator-free Days 
VA = Alveolar ventilation 
Vd/Vt = Dead space 
von Willebrand factor = vWF 
WBC = White Blood Cell 
 
 
 

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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. 
 
Drug held/hold drug: Study medication withheld for 24 hours.  
 
Drug permanently discontinued: Study medication stopped for remainder of the trial. 
 
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). 
 
Sepsis: Suspected or known infection.  
 
Study Drug: Defined as inhaled carbon monoxide at 100 ppm, 200 ppm, or dosing algorithm-specified 
dose, 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. 
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. 
 

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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. 

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Part I: Study Summary 
 
Title: A Phase I Trial of Inhaled Carbon Monoxide for the Treatment of Sepsis-Induced Acute 
Respiratory Distress Syndrome (ARDS) 
 
Objective: To assess the safety of inhaled carbon monoxide (iCO) in intubated patients with sepsis-
induced ARDS. 
 
Hypotheses:  
• Administration of inhaled CO therapy will be safe in intubated patients with sepsis-induced 
ARDS. 
 
 
Study Design: 
1. Multi-center, prospective, randomized, placebo-controlled Phase 1 clinical trial of inhaled CO 
for the treatment of sepsis-induced ARDS.  
2. In Cohorts 1 and 2, intubated patients will be randomized to receive inhaled CO or inhaled air 
placebo for up to 90 minutes daily. 
3. Treatment will continue for 5 days, until discontinuation of mechanical ventilation, or death, 
whichever comes first. 
4. In Cohort 1, patients will be randomized to iCO at 100 ppm or placebo (2:1 ratio).  
5. In Cohort 2, patients will be randomized to iCO at 200 ppm or placebo (2:1 ratio).  
6. Patients will be followed for 60 days or until discharge from the hospital to home with unassisted 
breathing, whichever occurs first. 
 
Sample Size/Interim Monitoring: 
1. A total of 20 patients will be enrolled to achieve dosing of 12 subjects meeting protocol defined 
criteria for cohort completers. Enrollment goal for cohort completers will be as follows: Cohort 
1- 6 subjects (2:1 CO:placebo); Cohort 2- 6 subjects (2:1 CO:placebo). 
2. The primary analysis will be safety. 
3. Trial progress will be monitored after each cohort by an independent Data and Safety Monitoring 
Board to determine if the study should proceed to the next cohort. The DSMB will also monitor 
trial quality and feasibility approximately every 6 months and will be available as needed on ad 
hoc basis. 
 
Inclusion Criteria: Patients with ARDS from sepsis will be enrolled as defined below.  
• Patients with sepsis are defined as those with suspected or documented infection: 
o Suspected or proven infection: Sites of infection include thorax, urinary tract, abdomen, 
skin, sinuses, central venous catheters, and central nervous system (Appendix A). 
 
All eligible patients meet the new definition of sepsis (suspected or proven infection and a SOFA " 
2) as PaO2/FiO2 ratio < 300 = 2 SOFA points1. 
• 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 infiltrates consistent with pulmonary edema on frontal chest radiograph 

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3. A need for positive pressure ventilation by an endotracheal or tracheal tube  
4. No clinical evidence of left atrial hypertension for bilateral pulmonary infiltrates. 
 
• ARDS onset is defined as the time the last of criteria 1-4 are met. ARDS must persist through 
the enrollment time window of 120 hours. 
• Infiltrates considered “consistent with pulmonary edema” include any infiltrates not fully 
explained by mass, atelectasis, or effusion or opacities known to be chronic (greater than 1 
week). Vascular redistribution, indistinct vessels, and indistinct heart borders alone are not 
considered “consistent with pulmonary edema” and thus would not count as qualifying 
opacities for this study. 
 
Exclusion Criteria: 
1. Age less than 18 years 
2. Greater than 120 hours since ARDS onset  
3. Pregnant or breast-feeding  
4. Prisoner 
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  
7. Physician refusal to allow enrollment in the trial 
8. Moribund patient not expected to survive 24 hours 
9. No arterial line/no intent to place an arterial line 
10. No intent/unwillingness to follow lung protective ventilation strategy  
11. Severe hypoxemia defined as SpO2 <95 or PaO2 <80 on FiO2 "0.8 
12. Hemoglobin < 7.5 g/dl or hemoglobin < 8 g/dl and actively bleeding  
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. Cardiopulmonary disease classified as NYHA class IV 
18. Stroke (ischemic or hemorrhagic) within the prior 3 months  
19. Diffuse alveolar hemorrhage from vasculitis 
20. Use of high frequency ventilation 
21. Participation in other interventional studies involving investigational agents 
22. Burns > 40% total body surface area  
23. Use of inhaled pulmonary vasodilator therapy (eg. NO or prostaglandins) 
 
Endpoint: The primary endpoint is safety of inhaled CO, defined by the incidence of pre-specified 
administration-associated adverse events and severe adverse events, in sepsis-induced ARDS patients.  
 

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Secondary Endpoint: The secondary endpoint is determination of the accuracy of the inhaled CO 
dosing approach in intubated patients with sepsis-induced ARDS. 
 
Other Secondary Endpoints:  
1. Mean daily Sequential Organ Failure Assessment (SOFA) score 
2. PaO2/FiO2 ratio and Oxygenation Index  
3. Lung injury score 
4. Vasopressor-free days 
5. Ventilator-free days 
6. ICU-free days at day 28 
7. Hospital-free days at day 60 
8. Hospital mortality to day 28 and 60 
9. Plasma biomarkers of inflammation (IL-6, IL-8, IL-10, IL-1Ra, IL-18, IL1!, and circulating 
mitochondrial DNA), lung epithelial injury (RAGE), endothelial injury (vWF, Ang-2), markers 
of change in other end-organ function (e.g., creatinine, liver function tests, lactate) 
 
Focused Safety Analysis: The incidence of elevation in plasma COHb " 10% measured on study days 
1-5 and pre-specified administration-associated adverse events and serious adverse events. 
 
Study Drug Dosing: All study drug doses will be administered via inhalation using a mechanical 
ventilator approved for nitric oxide (NO) delivery and the CO Delivery System. The study drug will be 
blinded to the study coordinator using identical tanks containing either CO or placebo air. The 
administering respiratory therapist and a physician study staff member will be unblinded to the treatment 
assignments. 
 
Completion of study drug administration: Study drug administration will be stopped when one of the 
following conditions is met, whichever comes first: 
1. Completion of the fifth dose of study drug   
2. Discontinuation of mechanical ventilation 
3. Death 
4. Pre-specified criteria met for permanent discontinuation of study drug (Section 5.1.9) 
 
 
 
 
 
 
 
 
 
 
 

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Part II: Study Description 
 
A Phase I Trial of Inhaled Carbon Monoxide for the Treatment of Sepsis-
Induced Acute Respiratory Distress Syndrome (ARDS) 
 
1. Background  
 
1.1. Introduction 
The 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 U.S.2,3. Despite 
decades of research and recent advances in lung protective ventilator strategies4, morbidity and mortality 
remain unacceptably high. Furthermore, no specific effective pharmacologic therapies currently exist. 
Sepsis, a clinical syndrome manifested by a systemic inflammatory response to an underlying 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.5-7, 
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 sepsis-related ARDS indicates 
a need for new treatments that target novel pathways. Carbon monoxide (CO) represents a novel 
therapeutic modality in ARDS based on data obtained in experimental models of ARDS and sepsis over 
the past decade. 
 
1.2. CO is an endogenously produced gaseous molecule with pleiotropic biological functions  
CO is made in the body by heme oxygenase-1 (HO-1), one of the few inducible molecules that can 
protect the lungs from an increased oxidant burden under circumstances of stress8. 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 in the past to exert biological functions as diverse as protection against oxidative injury9-11, 
inflammation12, and cell death13,14, inhibition of cell proliferation15, suppression of matrix production16, 
increased fibrinolysis17, as well as enhanced phagocytosis18,19 and macrophage efferocytosis20, 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 
biogenesis21, enhancement of autophagy19, and acceleration of resolution of inflammation via 
biosynthesis of specialized proresolving mediators20.  
 
1.3. Administration of inhaled CO protects against endotoxemia and lung injury in animal models 
Our laboratory has a long history of studying experimental ALI using animal models, including 
hyperoxia and endotoxin exposure, bleomycin, ischemia/reperfusion, and ventilator-induced lung injury 
(VILI). Our published studies have indicated that application of CO at low concentration can confer 
tissue protective effects in these ALI models10,11,16,22-27. In addition, we and others have demonstrated 
that CO decreases inflammation, enhances phagocytosis, and improves mortality in models of sepsis 
including endotoxemia12,28,29, hemorrhagic shock30 and cecal ligation and puncture (CLP)18,19. In 
addition, CO has been shown to have beneficial therapeutic effects in pre-clinical models of disease 
including pulmonary hypertension31-34, vascular injury35-39, and transplantation25,26,40-48 (Table 1). 
 
 

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Table 1: Pre-clinical studies using inhaled CO 
 
Model 
Year 
Species 
CO (ppm) 
Outcome 
Reference 
Lung injury (VILI, 
acid, hyperoxia, 
LPS) 
1999, 2003, 2004, 
2008, 2009, 2010 
mouse, rat, 
macaques 
10-250, 500 
Less inflammation 
& lung injury 
10,11,22-
24,49 
Bleomycin lung 
fibrosis 
2005 
mouse 
250 
Decreased lung 
hydroxyproline, 
fibronectin, collagen 
16 
Ischemia-
reperfusion (hind 
leg, lung) 
2001, 2003, 2006, 
2007, 2009 
mouse, rat 
250, 500, 1000
Less remote organ 
inflammation, less 
apoptosis, improved 
survival 
14,17,27,50-
52 
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 
25,26,40-
43,45-48,53 
Endotoxemia 
2000, 2003, 2004 
rat, mouse 
10-250 
Improved survival, 
decreased 
inflammation 
12,28,29 
Hemorrhagic 
shock 
2005 
mouse 
250 
Decreased end organ 
injury/ischemia 
30 
Cecal ligation and 
puncture 
2008, 2014 
mouse 
250 
Improved survival, 
decreased 
inflammation, 
enhanced 
phagocytosis 
18,19 
Pulmonary arterial 
hypertension 
2006 
rat, mouse 
50, 250 
Reversal of 
established PAH & 
reversal of 
remodeling 
31 
Vascular injury 
2003, 2005, 2006, 
2007 
mouse, pig 
100, 250-500 
less intimal 
hyperplasia, reduced 
thrombosis 
35-39 
Cardiopulmonary 
bypass 
2004, 2008, 2009 
pig 
250 
Less lung injury, 
decreased cardiac 
edema, apoptosis 
54-57 
Doxorubicin 
cardiomyopathy 
2007 
mouse 
500 
Improved cardiac 
function 
58,59 
Asthma 
2003 
mouse 
250-1000 
Reduced 
inflammation & 
bronchoconstriction 
16,60,61 
Cerebral malaria 
2007 
mouse 
250 
Reduced incidence 
of cerebral malaria 
62 
Hepatitis 
2003, 2007 
mouse 
100, 250, 500 
Improved survival, 
decreased apoptosis 
63,64 
Colitis, ileus 
2005 
mouse, rat, 
pig 
250 
Reduced injury & 
inflammation, 
improved motility 
31,65-67 

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Sickle cell disease 
2009 
mouse 
250 
Reduced 
leukocytosis 
68 
Collagen-induced 
arthritis 
2009 
mouse 
200 
Improved arthritis 
score, less 
inflammation 
69 
Ureteral 
obstruction 
2008 
mouse 
250 
Reduced renal 
fibrosis 
70 
 
1.4. Lung protective effects of carbon monoxide 
Numerous studies have examined the protective effects of low concentrations of carbon monoxide on 
the pulmonary parenchyma and vasculature. Inhaled CO prolongs survival and prevents tissue injury and 
epithelial cell death in rodents subjected to high oxygen stress10. CO also reduces lung cell apoptosis 
during lung ischemia-reperfusion injury in mice27 and prevents tissue injury during mechanical 
ventilation in mice by preventing alveolar-capillary barrier dysfunction and reducing inflammation22-24. 
Low concentration inhaled CO can also reverse established pulmonary hypertension in rats31 and has 
been shown to protect against endothelial apoptosis13. 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 fibrosis17. 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.   
 
1.5. CO delivery via an inhaled route is safe in 
human subjects 
Carbon monoxide 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 literature71.  
This model has been tested and confirmed in humans 
for varying inspired CO levels and durations of 
exposure72-78 (Figure 1).  
 
Extensive data is available regarding the safety and 
tolerability of low dose inhaled CO in healthy 
volunteers76,79-84 and more recently, in subjects with 
COPD85 (Table 2). Previous studies have carefully 
measured carboxyhemoglobin levels in response to inhaled CO and demonstrated that low dose carbon 
monoxide is safe in healthy normal volunteers76,79-84. 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 potentials81. In a recent study aiming to simulate cigarette smoke inhalation, Zevin et al. 
exposed healthy volunteers to CO (1200-1500 ppm) for ten minutes, repeating every 45 minutes for 16 
hours per day for 7 days82. In this study, mean COHb levels were 5 ± 1% and no adverse events were 
reported82.  
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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Table 2: Clinical Trials of CO in Human Subjects 
Study 
CO Exposure 
COHb 
Adverse Effects 
Stewart (1970)81 
100 ppm for 8 h 
11-13% 
No adverse effects 
Peterson (1975)76 
50-200 ppm, up to 5.25 h  
1-20% 
None noted 
Hausberg (1997)79 
1000 ppm x 30 min, then 100 ppm x 30 
min  
8.3 ± 0.5% 
None reported 
Zevin (2001)82 
1500 ppm x 10 min, then every 45 min x 
16 h for 7 days 
5 ± 1% 
None reported 
Ren (2001)84 
4000 ppm until COHb ~ 10%, then 
repeated to keep COHb ~ 10% for 8 h 
9.7 ± 0.1% 
None reported 
Mayr (2005)80 
500 ppm for 1 h  
6.5-7.7% 
Mild headache in 1 
subject 
Bathorn (2007)85 
125 ppm for 2 h, 4 consecutive days  
2.1-3.4% 
2 COPD exacerbations, 
judged unrelated 
Rhodes (2009)83 
100 ppm for 1 h for 5 days 
3.3 ± 0.6% 
No adverse effects 
NCT00094406 
(IND# 70,694) 
100 ppm for 6 h 
6.5 ± 1.7% 
No adverse effects 
NCT01214187 
(IND# 109,756) 
100-200 ppm x 2 h, 2 times weekly x 12 
wks 
ongoing 
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 a CO regimen aiming to maintain a 
COHb level of 10% for 8 hours84. COHb levels ranged from 9.1 to 10.5% (mean 9.7%) and no adverse 
effects were reported84. Similar results have been published in a number of other studies, and none have 
reported adverse events76,79,80,83. Of note, baseline COHb levels of 3% have been reported in some urban 
areas81 and levels as high as 10-15% may be observed in asymptomatic chronic smokers86-88. 
 
Table 3: Effects of CO on Vital Signs and Laboratory Values 
In addition, we demonstrated 
the safety of inhaled carbon 
monoxide 
in 
healthy 
volunteers after endotoxin 
instillation 
(NCT00094406- 
IND# 
70,694). 
In 
this 
placebo-controlled study, 24 
healthy 
volunteers 
(11 
females; mean age 26.2 ± 5.2 
yrs) were randomized to 
receive room air or CO (100 
ppm) inhalation for 6 hrs starting immediately after bronchoscopic endotoxin instillation (4 ng/kg). In 
this study, subjects had a mean COHb of 6.5% ± 1.7% and CO inhalation was well tolerated and was not 
associated with any clinically significant abnormalities in vital signs, laboratory parameters, 

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neurocognitive studies (including immediate and delayed memory, attention, language and 
visuospatial/constructional function) or adverse events (Table 3).  
 
In addition to the experience in normal volunteers, a recently completed clinical trial demonstrates the 
feasibility of administering a low dose of inhaled CO to humans with chronic obstructive pulmonary 
disease (COPD) (NCT00122694)85. In this study, ex-smoking subjects with stable COPD were treated 
with inhaled CO 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 of methacholine 
responsiveness85. Finally, low dose inhaled CO has been administered to subjects with idiopathic 
pulmonary fibrosis in an ongoing multicenter phase II clinical trial with no serious adverse events 
related to CO therapy reported to date (NCT01214187- IND# 109,756). Taken together, these findings 
demonstrate that experimental administration of several different concentrations of CO is well tolerated 
and that low dose inhaled CO can be safely administered to subjects in a controlled research 
environment.  
 
1.6. 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 1900’s, Krogh and Barcroft 
developed it into a standard test procedure for both understanding alveolar membrane function as well as 
diagnosing diseases of the alveolar-capillary membrane. Up until the 1970’s, steady state diffusing 
capacity was the standard diagnostic test for pulmonary laboratories worldwide when 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 minutes89. 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 carboxyhemoglobin levels raised above 3-6% following 
the diagnostic procedure. This longstanding diagnostic procedure reinforces that inhaling a constant 
concentration of low dose carbon monoxide can be safely done without significant adverse events. 
 
1.7. Delivery of inhaled CO to mechanically ventilated 
subjects 
In order to study inhaled CO in mechanically ventilated 
subjects with ARDS in this Phase I trial, a Carbon Monoxide 
Delivery System (Figure 2) developed by 12th Man 
Technologies will be used (Appendices O and P- 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 carbon monoxide gas into the inspiratory 
path of the patient breathing circuit independent of the 
Figure 2.  CO Delivery System (12th 
Man Technologies) 

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patient’s inspiratory flow, while the patient’s respiration is supported by a ventilator. For safety reasons, 
the CO Delivery System has twin microprocessors so 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 
carbon monoxide and oxygen concentrations. 
 
The CO Injector is a core component for the delivery of CO.  The CO Injector is constructed of carbon 
monoxide 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 carbon monoxide 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. 
 
1.8. Testing of the CO Delivery System in a Baboon Model of Pneumococcal Pneumonia 
We evaluated the safety and efficacy of the CO Delivery System developed by 12th Man Technologies 
in a baboon model of S. pneumoniae pneumonia. Four juvenile, male colony-bred baboons (Papio 
cynocephalus) were intubated, sedated, and mechanically ventilated90 with a Puritan Bennett 840 
ventilator in volume-control mode. 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 CO at 200 ppm through the ventilator via the CO 
Delivery System for 60-90 minutes. After CO 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 and blood 

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gases respectively. 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.  
 
Animals met pre-specified pneumonia criteria (white blood cell count, microbiology, and 
signs/symptoms) as previously defined90. At 24-48 hours post-inoculation, animals developed fever 
(39.2 ± 0.8° C), tachycardia (HR 125 ± 15 beats/min), and tachypnea (RR 39 ± 8 breaths/min, or 25 ± 
16% above baseline). Mean arterial blood pressure was unchanged throughout the experiment. Chest 
radiographs showed bilateral lower lobe opacities. Laboratory analysis was notable for hypoxemia 
(PaO2 76 ± 7 mm Hg) and leukocytosis (17.9 ± 5 thousand/#L) with 90% neutrophils on differential. At 
168 hours, there was a late-onset thrombocytosis (669 ± 52 thousand/#L). BAL fluid (BALF) gram stain 
at 24-48 hours demonstrated numerous neutrophils with moderate gram-positive diplococci consistent 
with S. pneumoniae. S. pneumoniae was isolated from BALF at 48 hours (8.4 x 104 ± 7.1 x 104 
CFU/mL) and from blood at 24-48 hours (14 ± 10 CFU/mL); however, BALF and blood were sterile at 
168 hours. Pneumococcal urinary antigen was negative at baseline but detected in all animals at 24, 48, 
and 168 hours post-inoculation. Post-mortem examination of the lungs at 168 hours revealed minimal to 
moderate bilateral lower lobe consolidation, without pleural effusions or pleuritis. H&E stained lung 
tissue showed mild alveolar filling by mononuclear cells with and without fibrin.  
 
Following one hour of CO administration at 200 ppm, animals achieved the pre-specified desired goal 
COHb level of 6-8% (7.2 ± 0.6%). Arterial COHb levels were 1.07 ± 0.2 at baseline, and increased 
linearly to 2.2 ± 0.4%, 3.45 ± 0.1%, 4.2 ± 0.3%, 5.2 ± 0.5%, 6.2 ± 0.6%, and 7.2 ± 0.6% at 10, 20, 30, 
40, 50 and 60 minutes of CO administration, respectively (p=0.0002) (Figure 3A). One animal was 
intentionally subjected to 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 1.0 FiO2, returning to near baseline levels after 82 ± 9.5 minutes 
(Figure 3B).  
 
Using the IL 682 Co-Oximeter, we found that venous and arterial COHb levels were highly correlated 
(r=0.9904, p<0.0001). 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]). (Figure 4). This tight correlation argues that venous measurements 

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are as accurate and reliable as arterial measurements. We also found superior accuracy and precision of 
the IL 682 Co-Oximeter compared with the 
AVOXimeter 4000 Co-Oximeter. The IL 682 Co-
Oximeter had increased sensitivity to measure 
COHb, especially at low COHb levels, compared 
with 
the 
AVOXimeter 
4000 
Co-oximeter. 
Modeling these data with type II linear 
regression, we found that the regression line was 
a poor model of the data with a slope of 0.6062 
(95% CI [0.4937 – 0.7188]) and a y-intercept of -
2.233 (95% CI [-2.828 – -1.638]) (Figure 5).  
 
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 h 
time-weighted average U.S. Department of 
Labor, 
Occupational 
Safety 
& 
Health 
Administration, 
https://www.osha.gov/dts/sltc/ 
methods/inorganic/id209/id209.html). 
 
1.9. 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, 
Appendix B)71,76 to predict COHb levels 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 
by 
the 
CFK 
equation 
(r=0.9251, 
p<0.0001). However, there was superior correlation 
between measured COHb levels and COHb levels 
predicted by the CFK equation using the 20 minute 
COHb (r=0.9767, p<0.0001) (Figure 
6A). Modeling these data with linear 
regression, 
we 
found 
that 
the 
regression line was a near-perfect 
diagonal with a slope of 1.002 (95% 
CI [0.9468 – 1.057]) with a y-intercept of 0.423 (95% CI [0.056 – 0.789]) and R2=0.9878 (p<0.0001). 
Furthermore, the 20 minute COHb was highly accurate in predicting the 60 minute COHb with a 
difference between predicted and actual COHb of 0.28 ± 0.43% (95% CI [-0.4 – 0.97]) (Figure 6B). 
Using the 20 minute COHb level as input into the CFK equation, this method can be used to predict the 
60 minute COHb level with high accuracy. 
 
1.10. Programming of the CFK equation 

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The above predictions using the CFK equation were made using a computer program generated in 
MATLAB 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 minute COHb. This program was 
validated by generating the previous published curves (Figure 1) that were derived from predicted 
values using the CFK equation76 (Appendix B). In addition, we used the programmed CFK equation 
and published values for CFK variables in ARDS (DLCO, VA, Hgb, weight, FiO2) to predict COHb 
levels in ARDS patients (average and severe) for a given CO concentration and duration of exposure 
(Appendix C).  
 
1.11. Study Rationale 
The purpose of this study is to assess the safety of inhaled CO therapy in mechanically ventilated 
patients with sepsis-induced ARDS. By studying subjects with sepsis and ARDS, we have targeted 
diseases that have been well studied in animal models. Furthermore, by focusing on intubated subjects 
with sepsis-induced ARDS, we have chosen a group with higher disease burden than sepsis alone and 
thus likely to have both increased mortality and an increased opportunity for benefit, including a 
reduction in the requirement for mechanical ventilation.  
 
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 during 
sepsis91. Furthermore, early activation of mitochondrial biogenesis has been associated with improved 
survival in critically ill patients with sepsis92. CO has been shown to activate mitochondrial biogenesis83 
and may be a key mechanism by which CO protects against ARDS and organ failure during sepsis. The 
optimal dose and duration of CO therapy in sepsis-induced ARDS is unknown. The decision to examine 
five days of treatment in Phase 1 is based on trials in healthy volunteers demonstrating activation of 
skeletal muscle mitochondrial biogenesis with no toxicity after 5 days of CO exposure83.  
 
The rationale to examine a dose escalation of 100 and 200 ppm in Cohorts 1 and 2 is to demonstrate 
safety of low dose inhaled CO treatment in intubated patients with sepsis-induced ARDS. It is 
anticipated that the COHb levels in Cohorts 1 and 2 will be well below the target range of 6-8% given 
reductions in CO diffusion in patients with ARDS. However, this low dose escalation design was 
favored as an additional safety measure in this vulnerable population. The decision to target a COHb 
level of 6-8% is based on human studies that demonstrate activation of skeletal muscle biogenesis and 

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safety of COHb levels of 6-8% (Table 2)76,79,80,82-85. Given diffusion impairment in patients with ARDS, 
it is anticipated, based on CFK equation predictions (Appendix C), that patients will require doses in the 
range of 200-500 ppm to achieve COHb levels of 6-8%. Previous studies have demonstrated safety of 
CO inhalation at 500 ppm in humans80 and reduction of pulmonary neutrophilia in non-human 
primates49, therefore we do not anticipate epithelial toxicity at our maximal dose.  
 
2. Objectives and Study Design 
 
2.1. Study Objective: 
 
To assess the safety of inhaled carbon monoxide (iCO) in intubated patients with sepsis-induced ARDS. 
 
2.2. Hypotheses: 
 
• Administration of inhaled CO therapy will be safe in intubated patients with sepsis-induced 
ARDS. 
 
 
2.3. Study Design: 
 
We will perform a multi-center, prospective, randomized, placebo-controlled Phase 1 clinical trial of 
inhaled CO for the treatment of sepsis-induced ARDS. Intubated subjects with sepsis-induced ARDS 
will be randomized to inhaled CO versus inhaled air placebo for up to 90 minutes daily for a total of 5 
days in two separate cohorts. The DSMB will meet after each cohort to determine whether to proceed 
to the subsequent cohort (Section 7.3.). 
 
 
Cohorts 1-2: randomized, placebo-controlled, multi-center, dose escalation study (all sites- 
 
BWH, MGH, Duke, Cornell) 
 
 
 
 
 
 
Cohort 1: iCO at 100 ppm or placebo for up to 90 minutes daily for a total of 5 days  
 
 
Cohort 2: iCO at 200 ppm or placebo for up to 90 minutes daily for a total of 5 days  
 
 
 
2.4. Accrual Objective for Cohort Completers: 
 
 
Cohort 1: 4 subjects receiving iCO 100 ppm; 2 subjects receiving inhaled air placebo (all sites) 
 
Cohort 2: 4 subjects receiving iCO 200 ppm, 2 subjects receiving inhaled air placebo (all sites) 
 
2.4.1. Cohort Completers:  
 
All enrolled subjects that initiate study drug dosing procedures, as described in Section 5.1.6, will be 
included in the primary safety analysis. A subject will be considered a cohort completer if they meet the 
criteria in Section 5.1.10. 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 number of cohort completers identified in Section 2.4. We expect this to be an 
uncommon occurrence.  

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For cohort 1, we will enroll up to 4 additional subjects in order to achieve the accrual objective for 
cohort completers in Section 2.4.  
For cohort 2, we will enroll up to 4 additional subjects in order to achieve the accrual objective for 
cohort completers in Section 2.4.  
 
2.5. Study Product, Dose, Route, and Regimen: 
 
The study drug will be administered to mechanically ventilated subjects based on a dosing algorithm and 
using a CO delivery device tested in pre-clinical non-human primate models. All study drug doses will 
be administered via inhalation using a mechanical ventilator approved for NO delivery and the CO 
Delivery System. The study drug will be blinded to the study coordinator using identical tanks 
containing either CO or placebo air. The administering respiratory therapist and a physician study staff 
member will be unblinded to the treatment assignments. 
 
For all cohorts, each dose of iCO or inhaled air placebo will be administered to subjects on a mechanical 
ventilator via the CO delivery device for up to 90 minutes. All patients will receive one dose daily for 5 
days (as tolerated based upon safety stops built into the protocol).  
 
 
Cohort 1: Dose Escalation, Part I 
 
100 ppm iCO or placebo for up to 90 minutes (n=6, 2:1 ratio) (all sites) 
  
[STOP for DSMB REVIEW, (Section 7.3.)] 
 
 
 
 
 
Cohort 2: Dose Escalation, Part II 
 
200 ppm iCO or placebo for up to 90 minutes (n=6; 2:1 ratio) (all sites) 
 
[STOP for DSMB REVIEW, (Section 7.3.)] 
 
 
 
 
 
3. Endpoints 
 
3.1. Primary Endpoint 
 
The primary endpoint is safety of inhaled CO, defined by the incidence of pre-specified administration-
associated adverse events and severe adverse events, in sepsis-induced ARDS patients.  
 
3.2. Secondary Endpoint 
 
The secondary endpoint is determination of the accuracy of the inhaled CO dosing approach in intubated 
patients with sepsis-induced ARDS. 
 
3.3. Other Secondary Endpoints:  
1. Mean daily Sequential Organ Failure Assessment (SOFA) score 
2. PaO2/FiO2 ratio and Oxygenation Index  

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3. Lung injury score 
4. Vasopressor-free days 
5. Ventilator-free days 
6. ICU-free days at day 28 
7. Hospital-free days at day 60 
8. Hospital mortality to day 28 and 60 
9. Plasma biomarkers of inflammation (IL-6, IL-8, IL-10, IL-1Ra, IL-18, IL1!, and circulating 
mitochondrial DNA), lung epithelial injury (RAGE), endothelial injury (vWF, Ang-2), markers 
of change in other end-organ function (e.g., creatinine, liver function tests, lactate) 
 
3.4. Focused Safety Analysis: 
 
The incidence of elevation in plasma COHb " 10% measured on study days 1-5 and pre-specified 
administration-associated adverse events (Section 5.1.9.) and serious adverse events (Section 11.).  
 
4. Study Population and Enrollment 
 
4.1. Screening 
 
Study coordinators will screen intensive care units daily to identify potential subjects for enrollment. 
Permission to approach patients and/or their families will be requested from the attending physicians. 
All patients meeting the inclusion criteria will be entered into a screening log. If the patient is not 
enrolled, the screening log will include information explaining why enrollment did not occur (exclusion 
criteria, attending physician denial, patient refusal, etc. see Appendix D for a listing of the de-identified 
data to be collected on screened, non-enrolled subjects). 
 
4.2. Inclusion Criteria: 
All patients (age 18 and older) will be eligible for inclusion if they meet all of the below criteria for 
sepsis and ARDS.  
 
• Patients with sepsis are defined as those with suspected or documented infection: 
o Suspected or proven infection: Sites of infection include thorax, urinary tract, abdomen, 
skin, sinuses, central venous catheters, and central nervous system (Appendix A). 
 
All eligible patients meet the new definition of sepsis (suspected or proven infection and a SOFA " 
2) as PaO2/FiO2 ratio < 300 = 2 SOFA points1. 
 
• 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 infiltrates consistent with pulmonary edema on frontal chest radiograph 
3. A need for positive pressure ventilation by an endotracheal or tracheal tube  
4. No clinical evidence of left atrial hypertension for bilateral pulmonary infiltrates. 
 

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• ARDS onset is defined as the time the last of criteria 1-4 are met. ARDS must persist through 
the enrollment time window of 120 hours. 
 
• Infiltrates considered “consistent with pulmonary edema” include any infiltrates not fully 
explained by mass, atelectasis, or effusion or opacities known to be chronic (greater than 1 
week). Vascular redistribution, indistinct vessels, and indistinct heart borders alone are not 
considered “consistent with pulmonary edema” and thus would not count as qualifying 
opacities for this study. 
 
4.3. Exclusion Criteria: 
1. Age less than 18 years 
2. Greater than 120 hours since ARDS onset  
3. Pregnant or breast-feeding  
4. Prisoner 
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  
7. Physician refusal to allow enrollment in the trial 
8. Moribund patient not expected to survive 24 hours 
9. No arterial line/no intent to place an arterial line 
10. No intent/unwillingness to follow lung protective ventilation strategy  
11. Severe hypoxemia defined as SpO2 <95 or PaO2 <80 on FiO2 "0.8 
12. Hemoglobin < 7.5 g/dl or hemoglobin < 8 g/dl and actively bleeding  
13. Subjects who are Jehovah’s Witnesses or are otherwise unable or unwilling to receive blood 
transfusions during hospitalization 
14. Acute myocardial infarction or acute coronary syndrome 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. Cardiopulmonary disease classified as NYHA class IV 
18. Stroke (ischemic or hemorrhagic) within the prior 3 months  
19. Diffuse alveolar hemorrhage from vasculitis 
20. Use of high frequency ventilation 
21. Participation in other interventional studies involving investigational agents 
22. Burns > 40% total body surface area 
23. Use of inhaled pulmonary vasodilator therapy (eg. NO or prostaglandins) 
 
Reasons for Exclusions: 
 
Patients less than 18 years of age are excluded because the study ICUs do not admit pediatric patients.  
Patients with ARDS for more than 120 hours are excluded to evaluate more clearly the effects of CO 
early in the course of lung injury. Patients with severe hypoxemia are excluded because they do not have 

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adequate reserve to tolerate the reduction in oxygen carrying capacity. Patients with hemoglobin < 7.5 
g/dl, hemoglobin < 8 g/dl and actively bleeding, Jehovah’s witnesses, or patients otherwise unable or 
unwilling to receive blood transfusions during hospitalization are excluded because of the volume of 
blood required for monitoring during CO therapy may place these patients at greater risks from 
complications of anemia. Moribund patients and patients with large body surface area burns have a high 
incidence of adverse events and lactic acidosis that will confound the safety assessment. Pregnancy and 
recent stroke are exclusions because CO 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 different from ARDS and diffuse alveolar damage. Patients with acute 
myocardial infarction 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 congestive heart 
failure are excluded because of concerns about ventricular arrhythmias. Patients ventilated with high 
frequency ventilation are excluded because dosing of CO during this mode of ventilation is unreliable. 
Patients on inhaled pulmonary vasodilators are excluded as these inhaled medications may interfere with 
the dosing of inhaled CO. 
 
4.4. Enrollment, Randomization, and Study Initiation Time Window  
 
All ARDS criteria must occur within the same 24 hour period. The onset of ARDS is when the last 
criterion is met. Patients must be enrolled within 120 hours of ARDS onset. Information for determining 
when these time window criteria were met may come from either the site hospital or a referring hospital 
reports. Following randomization, the low tidal volume protocol for mechanical ventilation must be 
initiated within one hour.  
 
The first treatment of study drug must be given within 24 hours of randomization. The day of 
randomization will be considered study day zero. Study day 1 will be the 24 hour period following 
randomization. Following randomization, the low tidal volume protocol for mechanical ventilation must 
be initiated within one hour. 
 
4.5. Informed Consent 
 
Written informed consent will be obtained from each patient or surrogate. If available, informed consent 
will be obtained from, in order of preference: the subject’s court appointed guardian or from a health 
care proxy/person with durable power of attorney. If no such representative exists, then the subject’s 
“next of kin” will be approached, consistent with hospital policy for obtaining consent for other medical 
procedures. Informed consent will be sought from the following individuals, in the order listed: spouse; 
natural or adoptive parent; adult child; adult brother or sister; any other available adult relative related 
through blood or marriage known and documented to have made decisions for the subject in prior health 
care settings. Patients who regain decision-making capacity prior to discharge from the study hospital 
will be asked to provide written consent for ongoing participation in the study. No study procedure will 
be conducted before obtaining informed consent. If consent from a Legally Authorized Representative 
(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 
using an IRB-approved telephone script. Consent obtained by telephone must comply with all regulatory 
requirements about the process, the consent elements, and documentation of consent. 
 
4.6. Enrollment and Randomization 
 

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Site investigators will review all potential study participants with one of the Data Coordinating Center 
(DCC) physician members. Once a potential participant has completed screening and is determined to 
meet eligibility criteria, the site personnel will call or page the DCC physician to request a 
randomization number and study assignment. The DCC personnel will enter a randomization number 
and an assigned treatment from the pre-provided randomization list into the unblinded electronic 
database. The DCC will also notify the site investigator of the assigned treatment by phone. The site 
investigator or person administering the study drug will log into the database to find out the subject’s 
treatment assignment and note this in the Subject Drug Administration form. 
 
In cohorts 1 and 2, eligible participants will be randomized to one of the two treatment arms using a 
permuted block method93. Randomization ratio will be 2 (iCO):1 (placebo) for cohorts 1 and 2. A 
biostatistician will generate the randomization codes and incorporate into the REDCap randomization 
module. The site investigator will call or page an unblinded DCC physician member to request a 
randomization number and study assignment which will be obtained from the REDCap randomization 
module. 
 
The study coordinators at each site who are responsible for data entry will be blinded to the study 
treatment assignments. The respiratory therapist and a physician study staff member administering the 
study drug will conceal the gas cylinders, the CO delivery device, and measurements of COHb, SpCO, 
and ambient CO levels, to assure that the study coordinator remains blinded to the study drug 
assignment. The physician study staff member will be responsible for maintaining a separate password-
protected file with the subject identification number and CO-related measurements (SpCO, COHb, 
ambient CO) which the study coordinator will not have access to. While investigators will be unblinded 
throughout the study due to safety monitoring in the Phase 1 trial, 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 three sites. This will prevent 
investigators from knowing the treatment assignment of the fifth or sixth patient enrolled into cohort 2. 
A lead respiratory therapist and co-investigator at MGH (Dr. Hess) may become unblinded to the 
treatment assignments at other sites to assist with study drug administration procedures if necessary. 
 
As for adverse event adjudication, the Medical Monitors (Drs. Baron and Thompson) will review the 
events 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). We do not see a way around site level adjudication by the site PIs evaluating AEs with 
knowledge of the treatment assignment but do conduct a 100% audit of source documents for unreported 
AEs. 
 
Central review of randomization by the DCC physician will assure that no subjects are enrolled in this 
multicenter Phase 1 study during planned or unplanned study holds. The DCC will pause enrollment and 
notify all sites of any planned or unplanned study holds. 
 
5. Study Procedures  
 
5.1 CO or Placebo Study Procedures 
 
5.1.1. Study Drug Dose 
 
The study drug will be administered as follows: 

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• Subjects in Cohort 1 will be treated with inhaled CO at a dose of 100 ppm or placebo. (all sites) 
• Subjects in Cohort 2 will be treated with inhaled CO at a dose of 200 ppm or placebo. (all sites) 
 
Placebo will consist of medical-grade air in identical appearing gas cylinders. 
 
5.1.2. Treatment Period 
 
The study drug will be administered for up to 90 minutes daily for five 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. 
 
5.1.3. CO Delivery System  
 
Inhaled CO or placebo will be administered to mechanically ventilated subjects using a mechanical 
ventilator approved for NO delivery and the CO Delivery System developed by 12th Man Technologies 
(Figure 2, Appendices O and P). See Appendices O and P 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 7) as described in Appendices O and P. As per the 
SOP and illustrated in the schema in Figure 7, 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 a respiratory 
therapist and a physician study staff member will be 
present at the bedside during and immediately after 
administration of the study drug for any clinical 
concerns that arise. The administering respiratory 
therapist and a physician study staff member will be 
unblinded to the treatment assignments and will conceal 
the gas cylinders and CO delivery device to assure that 
the study coordinator remains blinded to the study drug 
assignment. 
 
5.1.4. CO or Placebo Cylinders 
 
The gas cylinders proposed for this clinical trial are AG 
aluminum cylinders with a CGA 500 valve and will be 
supplied by Praxair (see attached letter from Praxair 
for Chemistry data). The cylinder contains approximately 360 liters of 5000 ppm (0.5%) Carbon 
Monoxide in room air (21% oxygen) and poses no increased flammability risk. Placebo tanks will 
contain medical grade air (see attached letter from Praxair for Chemistry data). The gas cylinder’s 
nominal size is 5 inches in diameter by 17 inches tall. See Appendix P for potential leak sources that 
could release CO into the room besides through the exhalation valve. 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 

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studies using the CO Delivery System 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 the MGH at 500 ppm for over 2 hours and ambient CO levels were near zero and well 
below the OSHA PEL (Appendix E).  
 
5.1.5. CO Monitoring 
 
Subjects will have blood drawn daily for measurement of lactate as well as COHb using an IL682 Co-
Oximeter (Appendix L) prior to administration of study drug. If COHb "3% or lactate "4 mmol/L, the 
study drug will be not be given on that study day. In addition, subjects will also have baseline SpCO 
measured noninvasively using a Masimo Radical 7 (Rad-7) pulse oximeter. During and after 
administration of the study drug, carboxyhemoglobin levels will be measured invasively (COHb by 
IL682) at the following time points: 20 min, 60 min, 75 min, 90 min, and 3 hours. In addition, COHb 
will be monitored continuously throughout the treatment period with a noninvasive pulse oximeter 
(SpCO by Rad-7).  
 
The concentration of the study drug will be measured by the built-in gas monitor in the CO Delivery 
System. 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 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 carbon monoxide detectors will be calibrated every 6 months per the 
manufacturer’s instructions to ensure proper functioning. 
 
All CO monitoring will be carried out by the administering respiratory therapist and/or the physician 
study staff member and concealed from the study coordinator. The physician study staff member will be 
responsible for maintaining a separate password-protected file with the subject identification number 
and CO-related measurements (SpCO, COHb, ambient CO) which the study coordinator will not have 
access to. 
 
5.1.6. CO Dosing Algorithm Using the CFK Equation 
 
Patients in Cohorts 1 and 2 will be administered inhaled CO at 100 and 200 ppm, respectively, for up to 
90 minutes. Subjects randomized to placebo will be administered inhaled air using the identical delivery 
system and ventilator. COHb will be measured at baseline and after 20 minutes of CO treatment. The 
CFK equation will be used to predict the 90 minute COHb level. No adjustment in the CO 
concentration will be made in Cohorts 1 and 2.  
 
 
1. Arterial blood (2.5 ml) will be drawn for baseline ABG and COHb prior to CO or placebo 
administration. 
2. Subjects will be treated with placebo or inhaled CO at 100 ppm in Cohort 1 and 200 ppm in 
Cohort 2. 

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3. After 20 minutes, arterial blood will be drawn for COHb measurement.  
4. DLCO will be estimated from the sample taken at 20 minutes using the computer-
programmed CFK equation (Appendix B). 
5. The 90 minute COHb level will be predicted using the CFK equation and compared with the 
90 minute measured COHb.   
6. Arterial blood will be drawn for COHb measurements at 20 min, 60 min, 75 min, 90 min, 
and 3 hours (1.5 ml each time point). Arterial blood will be drawn for both ABG analysis and 
COHb measurement at completion of study drug (2.5 ml). 
 
On days 1-5, a total of approximately 11 ml of arterial blood will be drawn during the study drug 
administration for safety monitoring of COHb levels, ABG analysis, and measurement of DLCO. 
 
5.1.7. Daily Hold Parameters Prior to Study Drug Administration 
 
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, lactate and COHb 
levels will not be measured on days the study drug is being held. 
 
A 12 lead EKG will also be performed daily prior to study drug administration to evaluate for cardiac 
exclusion criteria ie. acute myocardial infarction or acute coronary syndrome (ACS). 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 guidelines94-
96, 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 
(0.1mV) in other contiguous chest leads or the limb leads; New or presumably new left bundle 
branch block (LBBB)94-96. 
 
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)94-96. 
 
Note, troponin (I or T) may be increased in patients with sepsis and ARDS97 in the absence of an acute 
MI or ACS from coronary artery disease. If, in the judgment of the clinical team a septic patient with 
elevated troponin levels has no other indication of an MI or ACS, the patient may still be eligible for 
enrollment. 
 

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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 <80 on FiO2 "0.8 
• Hemoglobin < 7.5 g/dl or hemoglobin < 8 g/dl and actively bleeding  
• Diffuse alveolar hemorrhage from vasculitis 
• Use of high frequency ventilation 
• Use of inhaled pulmonary vasodilator therapy (eg. NO or prostaglandins) 
 
5.1.8. Interruption of Dosing During Study Drug Administration 
 
Subjects will have arterial blood drawn for COHb measurements prior to study drug treatment and 20 
min, 40 min, 60 min, 75 min, 90 min, and 3 hours after study drug treatment. It is anticipated that the 
predicted and achieved COHb in Cohorts 1 and 2 will be well below the target range of 6-8% given 
reductions in CO diffusion in patients with ARDS. However, the following parameters will be used to 
shorten the 90 minute exposure should CO uptake be higher than anticipated: 
 
The study drug will be stopped prior to 90 minutes: 
1. If measured COHb > 7% at any time during study drug treatment. 
2. If COHb is predicted to be  > 7% prior to 90 minutes. If this occurs, study drug treatment 
will be stopped at the time the COHb is predicted to be 7% by the CFK equation and an 
arterial COHb measured at that time. 
3. If the investigator, attending physician, the patient, or their surrogate decides that the 
study drug should be discontinued. 
 
5.1.9. Permanent Discontinuation of Study Drug Administration 
 
Permanent discontinuation of the study drug is defined as cessation of the study drug without the intent 
of restarting the study drug during the five-day treatment period.   
 
Permanent discontinuation of the study drug inhalation will occur in the following situations: 
 
• Occurrence of pre-specified administration related adverse events:  
o Acute myocardial infarction within 48 hours of study drug administration 
o Acute cerebrovascular accident (CVA) 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 patient experiences serious adverse events related to the study drug (Section 11 and 
Appendix K) 
• 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 adverse event, then appropriate 
adverse event reporting procedures will be followed (Section 11 and Appendix K). 

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• Daily baseline COHb levels greater than 3% leading to three missed drug doses. 
• Three or more missed drug administrations due to adverse events. 
 
Patients who have their study drug permanently discontinued will continue their participation in the 
study, and will be followed to determine their vital status to hospital day 60 or hospital discharge, as 
outlined in the Time-Events Schedule (Appendix F). 
 
5.1.10. Completion of 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. Five days after study drug administration   
2. Discontinuation of mechanical ventilation 
3. Completion of one or more doses of study drug 
4. Death 
 
The optimal duration of CO therapy for sepsis-induced ARDS is unknown. The decision to examine 5 
days of exposure in Phase I was based on trials in healthy volunteers showing skeletal muscle 
mitochondrial biogenesis activation with no toxicity after a 5 day exposure. Multisystem organ failure is 
the most common cause of death in patients with ARDS and dysregulated mitochondrial biogenesis has 
been described in non-survivors of sepsis and may be a key element in the development of organ 
failure92. Thus we chose a 5 day exposure in Phase I to provide more a more complete safety assessment 
for the anticipated Phase II dosing regimen. 
 
5.2. 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, will follow the modified ARDS Network lower tidal 
volume (6 ml/kg PBW) protocol (Appendix G) 4. If not already being utilized, this low tidal volume 
protocol for mechanical ventilation must be initiated within one hour of randomization. Since the time a 
patient achieves unassisted ventilation affects a secondary endpoint, VFDs, and because recent 
evidence-based consensus recommendations have identified a best practice for weaning, weaning 
strategy will also be controlled by protocol rules in accordance with these evidence-based 
recommendations. This newer weaning strategy is a simplified version of the protocolized weaning 
strategy used in prior ARDS Network studies (Appendix G). Study drug administration will be 
continued in patients undergoing weaning from mechanical ventilation as in Appendix G unless the 
subject is deemed ready for extubation by the clinical team. The study drug will not be administered on 
the day of planned extubation if the subject has passed a spontaneous awakening trial and spontaneous 
breathing trial and the clinical team has made the decision for extubation.  
 
6. Data Collection 
 
6.1. Background Assessments 
 
1. Demographic and Admission Data 
2. Pertinent Medical History and Physical Examination 

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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. Risk factors for ARDS (sepsis, aspiration, trauma, pneumonia, drug overdose, other) 
7. Acute or Chronic renal failure and use of dialysis 
8. Presence of the following chronic diseases: 
a. Metastatic 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  
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  
 
6.2. Baseline Assessments 
 
The following information will be recorded during the 24-hour interval preceding initiation of study 
drug treatment.  
 
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. 
 
1. Vital Signs: Heart rate (beats/min), systemic systolic, diastolic, and mean arterial blood 
pressure (mm Hg), body temperature (°C), and central venous pressure (CVP) if available. 
2. Electrocardiogram (EKG) 
3. APACHE II Score and SOFA Score 
4. Ventilator mode, tidal volume, FiO2 and PEEP, peak inspiratory pressure, plateau pressure, 
compliance, minute ventilation, set respiratory rate and total respiratory rate, and mean 
airway pressure. If on a pressure-cycling mode, peak pressure during inspiration will be 
assumed to be the plateau pressure. 
5. Arterial PaO2, PaCO2, pH, SpO2, and base excess (qualifying arterial blood gas), SpCO, 

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COHb, and Scv02 if available 
6. Serum CK, AST, ALT, Bilirubin, and Lactate  
7. Frontal Chest Radiograph (qualifying radiograph) – radiographic lung injury score (# of 
quadrants), barotrauma if available 
8. Vasopressors or inotropes (epinephrine, norepinephrine, phenylephrine, vasopressin, 
dopamine, dobutamine, phosphodiesterase inhibitors, including dose) 
9. Suspected or known site of infection 
10. Fluid intake, fluid output (most recent 24 hour value) or mean hourly value for most recently 
available period 
11. Serum electrolytes, glucose, albumin, and total protein 
12. Complete blood count (CBC), prothrombin time (PT), International Normalized Ratio (INR) 
13. Glasgow Coma Score  
14. Presumed site of infection, if sepsis is the etiology of ARDS. 
15. Concomitant medications: Aspirin, Angiotensin converting enzyme inhibitors (ACEIs), 
Steroids, Statins, Nitroprusside, Methylene blue, Nitrates, Neuromuscular blockade, Inhaled 
NO or prostaglandins   
16. Discarded bronchoalveolar lavage fluid and plasma will be obtained (when available) from 
the Crimson Biospecimen bank (BWH Biobank which provides discarded samples from 
consented subjects) for levels of cytokines, mediators, and protein. Plasma and BAL fluid 
will be divided into equal aliquots in specified tubes, and frozen at -800 C. 
 
6.3. Assessments after Enrollment 
 
The following data will provide the basis for assessing protocol compliance and safety as well as 
between-group differences in several efficacy variables. Data for each of the variables will be recorded 
on the days shown in the Time-Events schedule (Appendix F) or until death, discharge from the 
intensive care unit, or unassisted ventilation for 48 hours. 
 
Reference Measurements (Days 1 - 5) 
 
The following parameters will be measured and recorded daily on every day of study drug 
administration from 4:00-10:00 am using the values closest to 8:00 am on the days specified in the 
Time-Events schedule (Appendix F). The following conditions will be ensured prior to 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.  
 
1) CO or Placebo Administration- To be collected only on days when study drug is being 
administered 
a) Cohort 
b) Dose 
c) Time of administration 
d) COHb and SpCO (0, 20 min, 40 min, 60 min, 75 min, 90 min, 3h after administration) 
e) Tank label and pressures in the tank before and after dose  
f) Safety check 
g) CO ambient levels before and after administration 
2) Assisted ventilation- record daily up to day 5  
a) Mode of ventilation, Minute ventilation, Tidal volume, FiO2, PEEP, Respiratory Rate, 
Peak, plateau, and mean airway pressures 

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b) Pressure during inspiration if on a pressure targeted mode (PSV, PCV, etc). 
c) Arterial PaO2, PaCO2, pH, base excess, and SaO2; if no ABG available, SpO2 
d) Inspiratory flow rate  
e) End tidal CO2 (ETCO2)- To be collected only on days when study drug is being 
administered 
f) Dead space (as measured by NICO, see Appendix H) and alveolar ventilation- To be 
collected only on days when study drug is being administered 
3) EKG 
4) Lactate- To be collected only on days when study drug is being administered 
5) Total Bilirubin 
6) Arterial Blood Gas before and after study drug administration (according to Section 5.1.6) 
7) Fluid intake and output in the past 24 hours and diuretic administration if applicable  
8) Renal replacement therapy  
9) Metabolic panel if available and requested by treating physician 
10)  Serum CK, AST, ALT, Albumin, Total Protein if available and ordered by the treating physician 
11) Complete blood count (CBC), prothrombin time (PT), International Normalized Ratio (INR) if 
available and requested by treating physician 
12) Vital signs: Heart rate, systolic and diastolic blood pressure, body temperature, CVP 
13) SOFA score daily  
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  
14) Richmond Agitation Sedation Scale (RASS) 
15) Frontal Chest Radiograph – radiographic lung injury score (# of quadrants), barotrauma if 
available 
16) Vasopressors or inotropes (epinephrine, norepinephrine, phenylephrine, vasopressin, dopamine, 
dobutamine, phosphodiesterase inhibitors, including dose)  
17) Concomitant medications: Aspirin, Angiotensin converting enzyme inhibitors (ACEIs), Steroids, 
Statins, Nitroprusside, Methylene blue, Nitrates, Neuromuscular blockade, Inhaled NO or 
prostaglandins  (Yes/No each day).  
18) Adverse Event Monitoring (Section 11 and Appendix K) 
19) Microbiological results when available 
a) Blood cultures 
b) Urine cultures 
c) Sputum cultures 
d) BAL cultures 
e) CSF cultures if available 
f) Stool  
g) Other 
20) On every day of study drug administration, blood (4 ml) will be collected in EDTA anti-
coagulated blood samples for cytokines, mediators, and markers of inflammation. Blood will be 
collected before and after study drug administration (90 min and 3 hr). Plasma will be obtained 
and divided immediately after centrifugation into equal aliquots in specified tubes and frozen at –
80°C. Blood for DNA banking by isolation of cell pellets from whole blood (Appendix I) will 
be obtained on every study day. 
21) On days 1 (before study drug administration), 3 and 5 (90 min and 3 hr after study drug 

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administration), blood (2.5 ml) for RNA will be collected in Paxgene tubes and frozen at –80°C. 
22) Discarded bronchoalveolar lavage fluid and plasma will be obtained (when available) from the 
Crimson Biospecimen bank (BWH) on days 1-5 for levels of cytokines, mediators, and protein. 
Plasma and BAL fluid will be divided into equal aliquots in specified tubes, and frozen at -800 C. 
 
Post-Treatment Assessments 
 
Day 7  
1) Assisted ventilation 
a) Mode of ventilation, Minute ventilation, Tidal volume, FiO2, PEEP, Respiratory Rate, 
Peak, plateau, and mean airway pressures, compliance 
b) Pressure during inspiration if on a pressure targeted mode (PSV, PCV, etc). 
c) Arterial PaO2, PaCO2, pH, base excess, and SaO2; if no ABG available, SpO2 
d) Inspiratory flow rate 
2) EKG 
3) Lactate if available and requested by treating physician 
4) Total Bilirubin 
5) Arterial or venous blood gas 
6) Renal replacement therapy  
7) Metabolic panel if available and requested by treating physician 
8) Serum CK, AST, ALT, Albumin, Total Protein if available and ordered by the treating physician 
9) Complete blood count (CBC), prothrombin time (PT), International Normalized Ratio (INR) if 
available and requested by treating physician 
10) SOFA score 
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 
11) Richmond Agitation Sedation Scale (RASS) 
12) Frontal Chest Radiograph – radiographic lung injury score (# of quadrants), barotrauma if 
available 
13) Vasopressors or inotropes (epinephrine, norepinephrine, phenylephrine, vasopressin, dopamine, 
dobutamine, phosphodiesterase inhibitors, including dose)  
14) Concomitant medications: Aspirin, Angiotensin converting enzyme inhibitors (ACEIs), Steroids, 
Statins, Nitroprusside, Methylene blue, Nitrates, Neuromuscular blockade, Inhaled NO or 
prostaglandins  (Yes/No each day). 
15) Adverse Event Monitoring 
16) Discarded bronchoalveolar lavage fluid and plasma will be obtained (when available) from the 
Crimson Biospecimen bank (BWH) on days 1-5 for levels of cytokines, mediators, and protein. 
Plasma and BAL fluid will be divided into equal aliquots in specified tubes, and frozen at -80º C. 
 
 

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Day 14 
 
1) Assisted ventilation 
a) 
Mode of ventilation, Minute ventilation, Tidal volume, FiO2, PEEP, Respiratory Rate, 
Peak, plateau, and mean airway pressures, compliance 
b) Pressure during inspiration if on a pressure targeted mode (PSV, PCV, etc). 
c) 
Arterial PaO2, PaCO2, pH, base excess, and SaO2; if no ABG available, SpO2 
d) Inspiratory flow rate  
2) SOFA score 
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 
3) Arterial blood gas 
4) Vasopressors or inotropes (epinephrine, norepinephrine, phenylephrine, vasopressin, dopamine, 
dobutamine, phosphodiesterase inhibitors, including dose) 
5) Renal replacement therapy 
6) Adverse Event Monitoring if remains in ICU 
 
Day 28 and 60 
Vital Status  
Glasgow Coma Scale at Day 28 or on discharge date 
Adverse Event Monitoring if remains in ICU 
 
Day 60 
Vital Status  
 
A total of approximately 100 ml of blood will be collected during the study from each study subject as 
follows: 
 
Before and during study drug administration: 
 
 
Sample collection  
Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
4 ml EDTA Vacutainer Tube   
X 
 X 
 X 
X  
X 
2.5 ml Paxgene Tube  
X 
 
 
  
 
5.5 ml for ABG and COHb Analysis 
X 
X 
X 
X 
X 
 
 
 After study drug administration (90 min and 3 hours): 
 

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Sample collection  
Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
4 ml EDTA Vacutainer Tube- 2 tubes 
X 
 X 
 X 
X  
X 
2.5 ml Paxgene Tube- 2 tubes 
 
 
 X 
 
X 
 
In the case of a study drug hold, blood will only be drawn once and the tubes drawn will be the tubes as 
indicated in the table below. As blood will only be drawn once on study drug hold days, this will 
decrease the volume of blood drawn for those subjects.  
 
Sample collection  
Day 1 
Day 2 
Day 3 
Day 4 
Day 5 
4 ml EDTA Vacutainer Tube- 2 tubes 
X 
 X 
 X 
X  
X 
2.5 ml Paxgene Tube- 2 tubes 
X 
 
 X 
 
X 
 
Samples will be sent to a central repository to be stored (as described below). Central repository 
accession numbers will identify samples during shipment and storage in the central repository. In the 
future, the data coordinating center (DCC) will instruct the repository to prepare the appropriate samples 
for shipment. The key relating the subject study ID number to the new specimen accession number will 
be kept at the DCC in a restricted access electronic file. The DCC 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 DCC for studies will be linked to the specimen but will be de-
identified. The link (key) between the de-identified database and the patient will be removed two years 
after the primary publication. Samples collected for this trial will be frozen and stored at a biorepository 
for future research. 
 
6.4. Assessments after Hospitalization 
 
Vital status will be collected at 28 and 60 days through telephone interviews with patients. Surrogates 
will be contacted in the case that patients cannot be reached. In addition, we will verify duration of 
survival for patients 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). We will use each patient’s social 
security number (SSN) for an exact NDI match. We will collect contact information for the patient and 
alternative contact information on up to 3 individuals. This information and the SSN will be collected on 
paper at the time of consent, and forwarded via secure fax to the DCC. Contact information and SSN 
will be maintained on paper and will not appear in the DCC database.  
 
Long term cognitive function will not be assessed in this Phase 1 study due to multiple confounding 
factors. Cognitive impairment is highly prevalent among survivors of critical illness98-103. In addition, 
neurocognitive deficits observed at three months after hospitalization persist at 12 months following 
critical illness in the majority of patients103. In survivors of ARDS, cognitive impairment has been 
observed in approximately 30-55% of patients following one year of hospitalization98,102. Similarly, 
moderate to severe cognitive impairment has been observed in survivors of severe sepsis up to one year 
following hospitalization99.  
 
Furthermore, several studies have demonstrated the safety of CO, including lack of adverse 
neurocognitive effects, at levels of COHb <10%, which exceed the levels anticipated in our study. 
Neurocognitive effects of CO have been extensively evaluated in previous human studies81,104, our 
endotoxin study in healthy volunteers (NCT00094406), and our recent trial of CO treatment in IPF 
patients (NCT01214187). Stewart et al. demonstrated no impairment in performance testing in healthy 

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humans exposed to CO at 100 ppm for 8 hours with COHb levels of 11-13%81. In addition, we 
demonstrated lack of adverse neurocognitive effects in healthy volunteers exposed to CO at 100 ppm for 
6 hours with COHb 6.5% ± 1.7%. In addition, a recent study by Linde Gas Therapeutics assessed the 
safety of inhaled CO in 32 healthy subjects. CO was well tolerated with no significant neurocognitive 
effects observed in subjects with COHb levels of 2-8.8% (Appendix N). Furthermore, we recently 
evaluated neurocognitive function in subjects enrolled in our CO IPF trial using the Montreal Cognitive 
Assessment tool. Subjects with IPF receiving biweekly treatment with inhaled CO at 100-200 ppm for 2 
hours demonstrated no impairment in neurocognitive function after 2.5 weeks of follow-up (Appendix 
M). In addition to these studies, a recent review of the literature suggests that neurocognitive effects are 
only seen once COHb rises above 15-20% 104. 
 
In addition, this Phase 1 dose-finding and safety trial is not powered to see differences in outcomes. 
Should this treatment proceed to a Phase 2 study, it will be powered to allow for a direct assessment of 
the incremental risk, if any, of carbon monoxide inhalation on cognitive function in patients with sepsis 
and ARDS.   
 
6.5. Endpoint Determinations 
 
1. Vital status at 60 days until discharged home on unassisted breathing 
2. Time of initiation of unassisted breathing (assuming a patient achieves 48 consecutive hours of 
unassisted breathing) 
3. Need for re-instituting assisted or mechanical ventilation after achieving 48 consecutive hours of 
unassisted breathing 
4. Need for, timing, and duration of dialysis 
5. Vital status 48 hours after initiation of unassisted breathing 
6. ICU length of stay in calendar days including ICU days after readmission to ICU 
7. Hospital length of stay in calendar days and discharge disposition (home, other facility, with or 
without assisted ventilation) 
8. Administration associated adverse events (Section 5.1.9.) 
9. All adverse events 
 
7. Statistical Considerations and Safety Assessment 
 
7.1. Statistical Considerations 
 
For this phase I safety trial, any patient who is randomized and receives a portion of any dose of 
treatment will be included in the primary safety analysis. Baseline demographics and clinical 
characteristics will be summarized descriptively overall and by group. The primary endpoint of this 
Phase I trial is safety of inhaled CO, defined by the incidence of pre-specified CO-administration 
associated adverse events and severe adverse events. All safety data and on-study vital signs will be 
summarized descriptively for each treatment group. Although the study is not powered to demonstrate 
significant differences in secondary endpoints, numerical secondary outcomes will be compared 
between two groups using either Student’s t tests or Mann-Whitney U tests. Categorical secondary 
outcomes will be compared using either chi-square ($2) test or Fisher’s exact test. All statistical analyses 
will be performed using SAS v 9.4 or newer versions (Cary, NC) or equivalent statistical packages. 
 
For secondary and exploratory endpoints, analyses may be limited to subjects who have met the criteria 

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for study drug completion as per Section 5.1.10. Subjects who have not met the criteria for study drug 
completion (as per Section 5.1.10) may be analyzed separately.  
 
For secondary and exploratory endpoints, subjects may also be analyzed according to number of study 
drug doses completed. 
 
7.2. Phase I Safety Assessment 
 
The Scientific Review Committee (SRC, Appendix J) will independently review the safety data within 
14 days after the last enrolled subject in each cohort has completed the study drug, and make 
recommendations to the DSMB. In addition, site investigators will notify the DCC of any severe 
unexpected adverse event (Appendix K) within 24 hours of becoming aware of the event. The DCC will 
review all reported events and report all serious, unexpected, and study-related adverse events and all 
“administration related adverse events” to the SRC for review and input by email or telephone prior to 
submission to the DSMB within 7 calendar days of the DCC being notified of the event. The decision 
about whether the frequency of adverse events is too high will not have formal evaluation criteria. The 
DSMB will be provided with summary statistics of baseline and on-study vital signs and laboratory 
values as well as tabulations of all the study endpoints. 
 
7.3. Summary Guidelines for SRC and DSMB Assessment 
 
The SRC and DSMB will meet following completion of subjects within each cohort. The DSMB will 
perform a safety evaluation and make the decision whether to proceed with the next cohort. The DSMB 
will make a recommendation to either: proceed to the next cohort; add additional subjects to a 
given cohort; or terminate the study. Recommendations by the SRC or DSMB to add additional 
subjects to a cohort will be reviewed by the Institutional Review Board (IRB) of each study hospital 
prior to enrollment of additional subjects. The SRC and DSMB may halt enrollment in the study at 
any time during the trial. 
 
7.3.1. Decision to Proceed to Cohort 2 
 
A decision to proceed to Cohort 2 using 200 ppm of inhaled CO will be made by the DSMB after review 
of safety data obtained up to 14 days from the last dose in the last patient in Cohort 1.  
 
8. Data Collection and Site Monitoring  
 
8.1. Data Collection 
 
Study coordinators will collect data and enter it directly into the web-based data entry system managed 
by the Data Coordinating Center or record on paper data forms. Data will be transferred to the DCC on a 
prescribed basis through a web-based data entry program. 
 
8.2. Site Monitoring 
 
Site visits will be performed on a regular basis by the DCC, to ensure that all regulatory requirements 
are met and to monitor the quality of the data collected. Records of Institutional Review Board 
approvals and patients’ charts will be examined on a spot check basis to evaluate the accuracy of the 
data entered into the database 

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9. Risk Assessment 
 
9.1. Risks of Active Study Drug 
 
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 drug administration and COHb and lactate levels will be 
carefully monitored as outlined in the Study Protocol. There may be other risks of inhaled CO in patients 
with ARDS that are currently unknown. Subjects will be monitored closely throughout their 
participation in the trial. 
 
9.2. Risks of blood draws 
 
All patients will have blood drawn for research purposes. Most blood will be drawn through indwelling 
catheters. Risks of drawing blood percutaneously are uncommon and include bleeding and bruising. 
 
9.3. Minimization of Risks 
 
Federal regulations at 45 CFR 46.111(a) (1) requires that risks to subjects are minimized by using 
procedures which are consistent with sound research design. There are several elements of study design 
in the present protocol that meets this human subject protection requirement. First, several of the 
exclusion criteria prohibit participation of patients who might be at increased risk from the effects of CO 
as outlined in Section 4.3.). These include individuals with severe hypoxemia, acute myocardial 
infarction or acute coronary syndrome within 90 days, angina pectoris with activities of daily living, 
cardiopulmonary disease (NYHA class IV), CVA within 3 months, as well as women who are pregnant 
or breast feeding. 
 
Second, to limit risk of toxicity during drug administration, we will: 
 
1. Monitor inhaled CO concentrations via the CO analyzer in the CO Delivery System 
during drug administration.  
2. Perform serial bedside measurements of arterial COHb at 20, 40, 60, 75, and 90 minutes 
during study drug administration as well as 3 hours after treatment.  
3. Measure SpCO at the same time points throughout the study.  
4. Use the CFK equation to predict increases in COHb for a given study drug concentration 
and exposure duration.  
 
Third, there are provisions in the protocol for daily hold parameters, interruption of dosing, and 
permanent discontinuation of the study drug. Fourth, we will monitor for adverse effects by monitoring 
baseline COHb and lactate prior to each dose. We will not administer the study drug if COHb "3% or 
lactate "4 mmol/L. 
 
Finally, the SRC and DSMB will meet after completion of each cohort for safety evaluation before 
proceeding to the next cohort.  
 
9.4. Potential Benefits 
 

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Study subjects may or may not receive any direct benefits from their participation in this study. Potential 
benefits from the administration of CO include decreased requirement for ventilatory support, decreased 
days spent in the ICU, increased organ failure free days, and enhanced survival. Finally, there are 
potential benefits to society since the discovery of agents that can reduce the substantial mortality and 
morbidity of ARDS would enhance the health of society.  
 
9.5. Risks versus 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.” Based on the preceding assessment of risks and potential benefits, the risks to 
subjects are reasonable in relation to anticipated benefits.   
 
Blood draws: The risks associated with this common clinical practice are small, however, the knowledge 
gained in furthering our understanding of the pathophysiology and potentially leading to better and 
targeted therapy may be substantial. 
 
CO Treatment: Although there is a risk of toxicity associated with inhalation of high concentrations of 
CO, low dose inhaled CO has been shown to be protective in animal models and safe in humans. Data 
from animal studies demonstrate that inhaled CO has beneficial effects on outcomes in sepsis and ALI. 
There is potential for benefit to society and individual patients should treatment prove to be of benefit 
for future patients with ARDS. 
 
10. Human Subjects 
 
Each study participant or a legally authorized representative must sign and date an informed consent 
form. Institutional review board approval will be required before any subject is entered into the study. 
10.1. Selection of Subjects 
 
10.1.1. Equitable Selection of Subjects 
 
Federal regulations at 45 CFR 46(a)(3) require the equitable selection of subjects. The ICUs will be 
screened to determine if any patient meets the inclusion and exclusion criteria. Data that have been 
collected as part of the routine management of the subject will be reviewed to determine eligibility. No 
protocol-specific tests or procedures will be performed as part of the screening process. If any subjects 
meet criteria for study enrollment, then the attending physician will be asked for permission to approach 
the patient or his/her surrogate for informed consent. Justifications of exclusion criteria are given in 
Section 4.3. These exclusion criteria neither unjustly exclude classes of individuals from participation in 
the research nor unjustly include classes of individuals from participation in the research. Hence, the 
recruitment of subjects conforms to the principle of distributive justice. 
 
10.1.2. Justification of Including Vulnerable Subjects 
 
The present research aims to investigate the safety of a type of treatment for patients with sepsis-induced 
ARDS. Due to the nature of these illnesses, the vast majority of these patients will have impaired 
decision-making capabilities. This study cannot be conducted if enrollment is limited to only those 
subjects with decision-making capacity. Potential benefits to participation in this study are increased 
survival and VFDs. 

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10.2. Informed Consent 
 
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. The investigator is responsible for 
ensuring that the patient understands the risks and benefits of participating in the study, and answering 
any questions the patient 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. 
 
10.3. Continuing Consent 
 
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. 
 
10.4. Identification of Surrogates 
 
Many of the patients approached for participation in this research protocol will have limitations of 
decision-making abilities due to their critical illness. Hence, most patients will not be able to provide 
informed consent. Accordingly, informed consent will be sought from the potential subject’s legally 
authorized representative. Regarding proxy consent, the existing federal research regulations (‘the 
Common Rule’) state at 45 CFR 46.116 that “no investigator may involve a human being as a subject in 
research…unless the investigator has obtained the legally effective informed consent of the subject or 
the subject’s legally authorized representative”; and defines at 45 CFR 46 102 (c) a legally authorized 
representative (LAR) as “an individual or judicial or other body authorized under applicable law to 
consent on behalf of a prospective subject to the subject’s participation in the procedures(s) involved in 
the research.” OHRP defined examples of “applicable law” as being state statutes, regulations, case law, 
or formal opinion of a State Attorney General that addresses the issue of surrogate consent to medical 
procedures. Such “applicable law” could then be considered as empowering the surrogate to provide 
consent for subject participation in the research. Interpretation of “applicable law” is therefore state 
specific and hence, will be left to the discretion of the individual IRBs of the respective clinical centers 
involved in the study. 
 
According to a previous President’s Bioethics Committee (National Bioethics Advisory Committee), an 
investigator should accept as an LAR…a relative or friend of the potential subject who is recognized as 
an LAR for purposes of clinical decision making under the law of the state where the research takes 
place105. Finally, OHRP has opined in their determination letters that a surrogate could serve as a LAR 
for research decision making if such an individual is authorized under applicable state law to provide 
consent for the “procedures” involved in the research study106. 
 

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10.5. Justification of Surrogate Consent 
 
According to the Belmont Report, respect for persons incorporates at least two ethical convictions; first, 
that individuals should be treated as autonomous agents, and second, that persons with diminished 
autonomy are entitled to protection. One method that serves to protect subjects is restrictions on the 
participation of subjects in research that presents more than minimal risks. Commentators and Research 
Ethics Commission have held the view that it is permissible to include incapable subjects in research 
that involves more than minimal risk as long as there is the potential for beneficial effects and if the 
research presents a balance of risks and expected direct benefits similar to that available in the clinical 
setting107. Several U.S. task forces have deemed it is permissible to include incapable subjects in 
research. For example, the American College of Physicians’ document allows surrogates to consent to 
research involving incapable subjects only “if the net additional risks of participation are not 
substantially greater than the risks of standard treatment108.” Finally, the National Bioethics Advisory 
Committee (NBAC) stated that an IRB may approve a protocol that presents greater than minimal risk 
but offers the prospect of direct medical benefits to the subject, provided that…the potential subject’s 
LAR gives permission…”105. 
 
10.6. Additional Safeguards for Vulnerable Subjects 
 
The present research will involve subjects who might be vulnerable to coercion or undue influence. As 
required in 45CFR46.111(b), we recommend that additional safeguards be included to protect the rights 
and welfare of these subjects. Such safeguards might include, but are not limited to: a) assessment of the 
potential subject’s capacity to provide informed consent, b) requirement for subject’s assent, c) the 
availability of the LAR to monitor the subject’s subsequent participation and withdrawal from the study, 
and d) augmented consent processes. The specific nature of the additional safeguards will be left to the 
discretion of the individual IRBs. 
 
10.7. Confidentiality 
 
All subjects or surrogates must provide written informed consent and signed HIPAA authorization prior 
to the performance of any screening or main study procedures. Federal regulations at 45 CFR 46 111 (a) 
(7) requires that when appropriate, there are adequate provisions to protect the privacy of subjects and to 
maintain the confidentiality of data. Subject confidentiality will be protected throughout the study and 
no subject-identifying information will be released to anyone outside the study. Confidentiality will be 
secured through several mechanisms. To maintain confidentiality, all laboratory specimens, evaluation 
forms, and reports will be identified only by a coded number. The coded number will be generated at 
random by a computer, and only the study investigators will have access to the codes. All records will be 
kept in a locked, password protected computer. All computer entry and networking programs will be 
done with coded numbers only. Any study forms and paper records containing personal identifier 
information (e.g., address, phone number) will be kept secured and locked at each clinical center. No 
personal identifiers will be placed on biological samples and other documents forwarded to central labs. 
All paper case report forms will be maintained in a locked cabinet inside a locked office. No personal 
identifiers, such as name, address, or social security number will be entered into the study database. Any 
subject-specific data reported to any study committees will only be identified by subject ID number. 
Access to all subject data and information at the clinical centers, including biological samples, will be 
restricted to authorized personnel. Finally, 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 

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identifiers. Clinical information will not be released without the written permission of the patient, except 
as necessary for monitoring by the National Heart, Lung, and Blood Institute, the Federal Drug 
Administration or other authorized Federal Agencies, local IRBs, and the Data Coordinating Center. 
 
11.  Adverse Event Reporting 
 
Investigators will determine daily if any clinical adverse events occur during the period from enrollment 
to ICU discharge as described in Appendix K. An adverse event is any untoward medical occurrence 
associated with the use of a drug, whether or not considered drug related. The investigator 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 drug. 
 
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, as outlined in Appendix K.  These events are: 
 
• New onset atrial or ventricular arrhythmias requiring DC cardioversion within 48 hours of 
study drug administration 
• Myocardial infarction within 48 hours of study drug administration 
• CVA within 48 hours of study drug administration 
• Increase in O2 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 lactate by 2 mmol/L within 6 hours of study drug administration 
• Increase in any protocol specified measurement of COHb  " 10%  
 
Investigators will report all serious AND unexpected adverse events or reactions, as defined in 
Appendix K, as well as serious AND “administration related adverse events” as described above, to the 
DCC by phone, fax or email within 24 hours of becoming aware of the event. The DCC will review the 
event and may inform the site to permanently discontinue study drug administration to the subject 
(Section 5.1.9.), and may hold enrollment pending SRC and DSMB review (Section 7.3.). The local 
Institutional Review Boards will also be notified according to local requirements. The investigator will 
then submit a detailed written report to the DCC and the Institutional Review Board no later than 5 
calendar days after the investigator discovers the event.  
 
The DCC will report all unexpected and study-related deaths or life-threatening suspected serious 
adverse events to the FDA within 7 days. The DCC 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 DCC being 
notified of the event. A written report will be sent to the DSMB and the FDA within 15 calendar days, 
and these reports will be sent to investigators for submission to their respective Institutional Review 
Boards. The DSMB will also review all adverse events during scheduled interim analyses. The DCC 
will distribute the written summary of the DSMB’s periodic review of adverse events to investigators for 
submission to their respective Institutional Review Boards in accordance with NIH guidelines.  
 
Investigators must also report Unanticipated Problems, regardless of severity, associated with the study 
drug or study procedures within 24 hours. An unanticipated problem is defined as follows: 

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Unanticipated Problem (UP): any incident, experience, or outcome that meets all of the following 
criteria: 
• Unexpected, in terms of nature, severity, or frequency, given the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and 
informed consent document; and the characteristics of the subject population being studied; 
• Related or possibly related to participation in the research, in this guidance document, 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; 
• Suggests that the research places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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