Clinical trial NCT03079102
Conditions: Heart Arrest, Out-Of-Hospital
Sponsor: Cameron Dezfulian
Start date: 2017-08-26; Completion date: 2020-06-02
Enrollment: 57 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Phase II double blind (participants and investigator) placebo controlled randomized (1:1) clinical trial of inhaled nitric oxide (iNO) 20 ppm administered over 12h beginning as soon as possible but within 4 h of return of spontaneous circulation (ROSC) from out-of-hospital cardiac arrest (OHCA). Planned enrollment is 180 subjects over 48 months at University of Pittsburgh Medical Center (UPMC) Hospitals with randomization stratified in blocks of 8. Recruitment will be performed under exception from informed consent (EFIC) to facilitate early enrollment and treatment. The study will have a pre-specified safety analysis at the mid-point (after 1 year or 60 patients whichever occurs first). Subjects will be screened by members of the University of Pittsburgh post-cardiac arrest service (PCAS), all of whom will serve as the study co-investigators, and the Research Coordinators. Notification of inclusion under EFIC will be performed as soon as possible by a member of the study team generally to a surrogate as the subjects will be comatose after OHCA.

Detailed description:
Subjects will be identified upon emergency department (ED) arrival or upon transfer from an outside facility and screened for enrollment by a PCAS physician as soon as possible. Eligible patients will have receive any required resuscitation (including central venous and arterial line placement, endotracheal intubation and hemodynamic resuscitation as needed) prior to having baseline labs and studies performed. Subjects will then be started on study drug delivered via the mechanical ventilator with a concealed canister (subject, providers and outcome assessors blind to treatment assignment). Randomization will be performed 1:1 in blocks of 8 using a random number generator. A randomization list will be prepared in advance by the director of respiratory therapy (RT) at each site and verified by a company representative. These individuals (who are not part of the study team) will be unblinded and assure that allocation to placebo and intervention is accurate. The allocation list will assign study drug canisters (by barcode) to each subject in order of study ID. Treatment assignment will be revealed after opening a sealed opaque envelope once enrollment is confirmed by a physician investigator. Study drug will then be administered by RT based on allocation. During study drug administration hourly vital signs will be obtained and methemoglobin levels for safety. Study drug will be weaned off after 12h over the course of 1h (10, 5, 4, 3, 2, 1 ppm each for 10 min) prior to discontinuation. Subjects will then receive standard post-resuscitation care with outcomes assessed by a blinded study team member (see below for outcomes).

Additional clinical variables to be collected Demographics and baseline function. Age, sex, race, maximum education level, employment status, marital status, Barthel activities of daily living (ADL) index prior to OHCA.

Arrest data. Location of OHCA, witnessed, bystander cardiopulmonary resuscitation (CPR), estimated no flow and low flow times, presenting rhythm, doses of epinephrine administered, shocks administered, recurrent arrest, date and time of ROSC.

Medical comorbidities. Diabetes, hypertension, active smoking, hyperlipidemia, chronic obstructive pulmonary disease (COPD), hypertension, drug abuse, prior myocardial infarction, prior coronary artery bypass grafting (CABG), prior coronary angiography with angioplasty or stent, congestive heart failure (EF on last echocardiogram \[ECHO\] prior to OHCA), obstructive sleep apnea, pulmonary hypertension, calculated Charlson comorbidity index (CCI).

Home medications. Statins, nitrates, anticoagulation, antiplatelet agents Hospital Interventions. Coronary angiography, percutaneous coronary intervention, CABG, mechanical ventilation hours and fraction of inspired oxygen (FiO2) from 0-24h after therapy start In-hospital medications. Alteplase, anti-epileptic medication use (valproate, phenytoin, lacosamide, levetiracetam), neurostimulants (methylphenidate, bromocriptine, modafinil, amantadine), cumulative dose of fentanyl, propofol, midazolam, cis-atracurium and vecuronium in at 24 (+/- 12 hours), 48 (+/- 12 hours), and 72 (+/- 12 hours) hours after therapy initiation

Data Storage Subjects will be assigned a study identifier (ID) upon entry and all data/samples stored using that ID. Linkage to patient identifiers will be maintained in a secure spreadsheet and will include name, date of birth and medical record number. Clinical data will be entered on case report forms (source documentation) which will be stored in a locked filing cabinet within a locked office assigned to the study team. Deidentified clinical and lab data will all be subsequently entered from the case report forms into a web based database (REDCap) to be maintained by Dr. Dezfulian's research assistant who has prior experience from other studies.

Statistical Analysis Plan Continuous data will be compared using t-tests and repeated measures ANOVA to compare between iNO and placebo groups at multiple times. Dichotomous outcomes including the primary endpoint will be compared by chi squared test. Time to awakening and 90d survival will be compared by log rank test of Kaplan-Meier survival plots. All tests will be two tailed with unadjusted p\<0.05 considered significant. In the event of a differential distribution of baseline variables strongly associated with outcome (univariate OR \>2), dichotomous outcomes will be adjusted for these baseline variables.

Arms: arm 1: 20 ppm iNO delivered via mechanical ventilator connected to the iNO ventilator delivery system (iNOvent). Drug will be started as soon as possible after return of spontaneous circulation (ROSC) but no later than 4h after ROSC. Study drug will be dosed for 12h then tapered off over 1h. arm 2: Nitrogen carrier gas delivered by identical system with similar dose/taper.
Interventions: intervention 1: Nitric Oxide intervention 2: Nitrogen
Intervention descriptions: intervention 1: An endogenous gaseous signaling molecule which stimulates soluble guanylate cyclase and may act via S-nitrosation, nitrite/nitrate or nitrated fatty acid formation. intervention 2: Nitrogen is the carrier gas (vehicle) for iNO. Subjects receiving placebo will receive equivalent doses of nitrogen.

Protocol documents:
Prot_SAP_000.pdf: 
 
CLINICAL PROTOCOL 
Includes statistical analysis plan 
 
 
 
Protocol Title: 
Inhaled Nitric Oxide (iNO) after Out-of-Hospital Cardiac 
Arrest (OHCA) 
IRB Protocol Number: 
PRO16100408 
NCT Number: 
03079102 
Version # and Date: 
V11:  July 25, 2019 
NOVEMBER 30, 2021 


1 
 
TABLE OF CONTENTS 
PROTOCOL SYNOPSIS………………………………………………………………………2 
 
1. 
STUDY OBJECTIVE, SPECIFIC AIMS, BACKGROUND AND SIGNIFICANCE 
 
1.1 OBJECTIVE…………………………………………………………………………..8 
 
1.2 SPECIFIC AIMS……………………………………………………………………...8 
 
1.3 BACKGROUND AND RATIONALE………………………….…………………….9 
 
1.4 SIGNIFICANCE………………………………………………………………………9 
 
2. 
RESEARCH DESIGN AND METHODS 
 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGN……….………………..9 
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN…………………...……………10 
2.3 STUDY DRUG(S)…………………………………………………………...............16 
 
2.4 STUDY ENDPOINTS…………………………………………………………...…..19 
 
2.5 STATISTICAL ANALYSIS…………………………………………………….…..20 
 
3. 
HUMAN SUBJECTS 
 
3.1 SUBJECT POPULATION………………………………………………………......22 
 
3.2 INCLUSION CRITERIA………………………………………………………….....22 
 
3.3 EXCLUSION CRITERIA…………………………………………………………...22 
 
4. 
IRB APPROVAL AND FDA AMMENDMENTS ………………………………...…23 
 
5. 
RECRUITMENT AND INFORMED CONSENT PROCEDURES 
5.1 RECRUITMENT METHODS………………………………………………….........24 
 
5.2 INFORMED CONSENT PROCEDURES………………………..…………………24 
 
6. 
POTENTIAL RISKS AND BENEFITS 
 
6.1 POTENTIAL RISKS………………………………………………..……………….25 
 
6.2 ALTERNATIVE TREATMENTS…………………………………….…………….25 
 
6.3 POTENTIAL BENEFITS…………………………………………………...……….25 
 
6.4 DATA SAFETY MONITORING ……..………………………………………...…..25 
 
6.5 RISK MANAGEMENT PROCEDURES…………...………………………………31 
 
7. 
COSTS AND PAYMENTS 
 
7.1 COSTS………………………………………………………...……………………..32 
 
7.2 PAYMENTS………………………………………………..………………………..33 
 
8. 
QUALIFICATIONS AND SOURCE OF SUPPORT 
 
8.1 QUALIFICATIONS OF THE INVESTIGATORS………………...………………..33 
 
8.2 SOURCE OF SUPPORT……………………………………………….……………34 
 
9. 
REFERENCES…………………………………………………………….……………35 
 
 


2 
 
PROTOCOL SYNOPSIS 
 
Protocol Title: 
Inhaled Nitric Oxide (iNO) after Out-of-Hospital Cardiac 
Arrest (OHCA) 
Protocol Number: 
PRO16100408 
NCT Number: 
03079102 
Version # and Date: 
V11:  July 25, 2019 
Clinical Phase: 
 
Phase II 
Investigational Drugs: 
Nitric Oxide (Inhaled) 
Trial Site: 
UPMC Presbyterian and Mercy Hospitals 
IND Sponsor: 
Cameron Dezfulian, MD 
Investigator: 
 
Cameron Dezfulian, MD 
SubInvestigators: 
Jon Rittenberger 
Ankur Doshi 
Bradley Molyneaux 
Lillian Emlet 
Masashi Okubo 
Melissa Repine 
Katharyn Flickinger 
Francis Pike 
Nick Krehel 
Clifton Callaway 
Alexandra Weissman 
Jonathan Elmer 
Frank Guyette 
Adam Frisch 
Kelly Sawyer 
Lori Shutter 
Sara DiFiore 
Study Monitor: 
Cameron Dezfulian, MD 
Research Facilities: 
UPMC Presbyterian and Mercy Hospitals 
Clinical Laboratories: 
UPMC 
Study Rationale: 
iNO is FDA approved for the treatment of pulmonary 
hypertension in the setting of hypoxemic respiratory failure in 
neonates. It has been used off label extensively primarily for 
the treatment of hypoxemic respiratory failure and pulmonary 
hypertension with extensive safety data available for endpoints 
as far out as 2 years from use.1-6 In these studies efficacy as 
measured by increase in oxygenation was noted with doses as 
little as 1 ppm3,4 with few cases of methemoglobinemia or 
excess NO2 especially at the doses under 40 ppm. A recent 
meta-analysis of trials of iNO to treat ischemia reperfusion 
injury in humans demonstrated efficacy in the majority of 
studies and an excellent human safety profile, but noted no 
existing human evaluation of iNO in the setting of acute brain 
injury including OHCA.7  
Effects of iNO after cardiac arrest has been studied in 4 
preclinical animal studies.8-11  These published studies 
demonstrated benefit using iNO 40 ppm started within 2h of 


3 
 
ROSC (Return of Spontaneous Circulation) which is additive to 
therapeutic hypothermia8 as well as neuroprotection without 
temperature control.9 These animal models include mouse and 
pig models of asystole and ventricular fibrillation (VF). In 
addition, iNO has been shown to protect in animal models of 
focal heart12-15 and brain16-19 injury.  This is important since 
most of the mortality and morbidity after OHCA results from 
heart and brain injury.20  
Animal models of asphyxia21 and other focal brain 
ischemia reperfusion models demonstrate NO deficiency early 
in reperfusion lasting at least 6h and benefits from iNO.16,19  
However in human brain injury from stroke22,23 or trauma24 the 
CSF (Cerebrospinal Fluid) levels of NO (Nitric Oxide) 
metabolites appear elevated by 24h with one study showing this 
effect is blocked using the iNOS (inhaled Nitric Oxide 
Synthase) inhibitor 1400W.22 Based on these reports it would 
appear a state of NO deficiency is highly likely at ROSC and 
persistent for up to 24h after ROSC.  Thus the likely 
therapeutic window is from ROSC until 24h after ROSC. 
Study Objectives: 
1. To evaluate the efficacy of inhaled nitric oxide dosed shortly 
after OHCA in reducing heart and brain injury after out-of-
hospital cardiac arrest. 
2. To examine a novel point of care method, laser speckle 
contrast imaging (LSCI) reactive hyperemia (RH), as a means 
of determining iNO responsiveness in the heterogeneous 
OHCA population to permit future screening if successful. 
3. To determine the pharmacokinetics of systemic nitric oxide 
(NO) metabolites as a marker of endogenous and exogenous 
NO bioavailability in cardiac arrest survivors treated with iNO 
or placebo.  
4. To determine which NO signaling pathways are activated by 
iNO and whether these are associated with clinical benefit. 
 
Study Hypothesis: 
Inhaled Nitric Oxide after OHCA will reduce heart and brain 
injury vs placebo 
Study Aims: 
1. Determine whether iNO (compared to placebo) provides 
clinical benefit when used within 4h of ROSC after OHCA. 
2. Determine which mechanistic pathways are activated through 
use of iNO therapy and whether any of these are associated with 
outcome. 
3. Determine whether iNO at 20 ppm provides a physiologic 
effect as determined by a significant reduction in tricuspid 
regurgitant velocity compared to placebo. 
4. Determine whether RH measured by LSCI predicts subjects 
who will have a physiologic and/or clinical response to iNO. 


4 
 
5. Determine whether iNO reduces oxidant stress and 
mitochondrial injury after OHCA compared to placebo. 
6. Measure plasma nitrite and S-nitrosothiol levels, as a surrogate 
of total plasma bioavailable NO, in all subjects. 
Study Design: 
Phase II double blind (participants and investigator) placebo 
controlled randomized (1:1) clinical trial of iNO 20 ppm 
administered over 12 hrs beginning as soon as possible but 
within 4 hrs of ROSC after OHCA. 
 
Planned Sample Size:  
130 subjects 
Duration of Treatment: 
12h 
Major 
Inclusion 
Criteria:  
Intubated and comatose adult (>18 yo)  
Out-of-hospital cardiac arrest (OHCA)  
Cardiac arrest within an emergency department or outpatient 
medical center will be included.  
OHCA includes EMS witnessed cardiac arrest. 
ROSC within 40 min of CPR initiation 
FOUR Brainstem score ≥ 2  
Patient must have pupil OR corneal reflex before enrollment 
 
Major Exclusion 
Criteria: 
- 
Traumatic etiology of OHCA 
- 
Prisoner 
- 
Known pregnancy  
o B-HCG screening is NOT REQUIRED for 
enrollment in women of appropriate age 
- 
Hemodynamic instability defined as: 
o > 1 recurrent arrest prior to enrollment 
o Inability to maintain mean arterial blood pressure 
(MAP) > 65     using vasopressors and inotropes 
(ie actively up titrating medications or giving 
fluid bolus) 
- 
Cerebral edema defined as: 
o Head CT grey-white ratio < 1.2 
o Fixed and dilated pupils without another 
explanation 
- 
Malignant EEG upon presentation (not required as 
screening) defined as: 
o Myoclonic status epilepticus 
o Non-convulsive status epilepticus 
o Generalized periodic epileptiform discharges  
- 
ROSC >3h from time of ED arrival (Treatment allocation 
must be within 4h so anything that will prevent this is 
reason for exclusion) 
- 
Alert and interactive patient with minimal evidence of 
neurologic injury 


5 
 
- 
Plan to extubate within 12 hours 
- 
PCAS opinion that patient will die with >95% likelihood.  
This may be based on: 
o Multiple medical comorbidities 
o Late discovery of DNR or advanced directive 
o Terminal diagnosis (other than OHCA; may have 
caused OHCA) 
o Clinical judgement based on current exam and 
data 
- 
Known intracranial hemorrhage or acute cerebral 
infarction; Head CT is NOT REQUIRED prior to 
enrollment 
- 
Patient is known to be taking PDE5 inhibitors, sGC 
stimulator, or has a known diagnosis of Chronic 
thromboembolic pulmonary hypertension (CTEPH), 
pulmonary hypertension (PAH), or erectile dysfunction 
- 
Known enrollment in another acute interventional trial 
- 
 
Study Endpoints: 
Primary   
Composite of in-hospital death, unfavorable discharge location, 
NYHA class III/IV heart failure at the time of discharge* 
*In the setting of pre-existing heart failure or prior residence in 
an unfavorable location there must be at least a 1 class decrement 
(eg NYHA III -> IV) 
 
Secondary 
Functional 
-Survival to hospital discharge, 30 and 90 days after initial 
OHCA 
-Favorable cerebral performance category (CPC) defined as 1 or 
2 at discharge, 30 and 90 days after initial OHCA  
-Favorable modified Rankin score (mRS) defined as 0-3 at 
discharge, 30 and 90 days after initial OHCA 
-Favorable discharge destination or location of residence (30 and 
90 days after OHCA) defined as home or acute inpatient 
rehabilitation 
-CPC-E at hospital discharge, 30 and 90 days after initial OHCA 
-Barthel Index of Independence in Activities of Daily Living 
(ADL) at discharge, 30 and 90 days after OHCA 
 
Neurologic 
-Estimated likelihood of good neurologic outcome (0-100%) 
assessed by an experienced (>3y) member of the PCAS team 
blinded to treatment assignment on day 0-3 post-ROSC. 
-Development of malignant EEG during hospitalization  


6 
 
-Time (in hours) to awakening from ROSC; defined as following 
commands (all values > 96h assigned 100) 
-FOUR Motor + Brainstem scores on days 1-3 (requires 30 min 
off sedation; exclude endpoint if not medically permitted to 
pause sedation or paralysis) 
-Presence or absence of upper and lower extremity 
somatosensory evoked potential N20 on comatose patients 
obtained any time after post-arrest day 1. 
- Absolute value 24h after ROSC and change in value (24h vs 
0h) of neuron specific enolase (NSE) 
-EEG burst suppression ratio and change in suppression ratio 
-EEG presence of dominant continuous background and 
reactivity to photic or tactile stimulation. 
 
Cardiovascular 
-Heart rate and mean arterial pressure (preferably off the arterial 
line) at 0-13 (hourly), 24h of study drug 
-Persistent need for vasopressors in the first 24h after ROSC; 
defined 
as 
any 
use 
of 
norepinephrine, 
epinephrine, 
phenylephrine, dopamine or vasopressin for ≥3h after study drug 
is initiated 
-Persistent need of inotropes in the first 24h after ROSC; defined 
as any use of epinephrine, dobutamine, milrinone or 
isoproterenol for ≥3h after study drug is initiated. 
-ECHO measures (absolute values at end of therapy (12h) and 
change in 12h vs 0h):  
   -tricuspid valve regurgitant velocity (TRV)  
   -cardiac output based on aortic valve velocity (AV)  
   -tricuspid annular plane systolic excursion (TAPSE) 
   -left ventricular ejection fraction (EF).  
-Absolute value at end of therapy (12h), change in value (12h vs 
0h) and peak 24h post-ROSC troponin I 
-Arterial blood gases at 0, 6, 12 and 24h after study drug 
initiation (when available) 
-Upper body central venous oxygen saturation at 0, 6, 12 and 24h 
after study drug initiation (when available) 
-Lactate at 0, 6, 12 and 24h after study drug initiation  
 
Biochemical  
-NO metabolites. Plasma nitrite, S-nitrosothiols and cyclic 
guanosine monophosphate (cGMP) levels obtained at 0, 3, 6, 9, 
12h of study drug and 24h, 48h and 72h after study drug start.  
-RBC pellet for hemoglobin (Hb) studies (retained from above) 
- MetHb at 6h (safety endpoint) 
-Platelet mitochondria. Oxygen consumption rate, respiratory 
control ratio and superoxide generation (MitoSOX) at 0 and 12h 


7 
 
of study drug when available. This data will be collected only 
when patients are enrolled from 6a-6p during weekdays (70/130 
budgeted) to assure quality. 
-Plasma for ascorbate 0, 3, 12, 24h  
-Cardiolipin at 0, 3, 12, 24h  
-After obtaining informed consent (IC) banked blood for DNA 
(pharmacogenetics) 
-LSCI coupled to 1 and 5 min forearm vaso-occlusion at 0 h and 
11 h. Endpoints will include ischemic trough, time to RH and 
RH peak, blood flow nadir 1 and 5 min after cuff release 
(respectively). 
 
 
 
 
 


8 
 
1. 
OBJECTIVE, SPECIFIC AIMS, BACKGROUND, AND SIGNIFICANCE 
 
 
1.1 OBJECTIVE 
Primary: 
 
To evaluate the efficacy of inhaled nitric oxide (iNO) dosed shortly after out-of-hospital cardiac 
arrest (OHCA) in reducing heart and brain injury and death. 
 
Secondary: 
 
- 
To evaluate the efficacy of iNO in modulating surrogate markers of heart and brain. 
- 
To evaluate the effects of iNO on platelet mitochondrial function.  
- 
To examine a novel point of care method, laser speckle contrast imaging (LSCI) reactive 
hyperemia (RH), as a means of determining iNO responsiveness in the heterogeneous 
OHCA population to permit future screening if successful. 
- 
To determine the pharmacokinetics of systemic nitric oxide (NO) metabolites as a marker 
of endogenous and exogenous NO bioavailability in cardiac arrest survivors treated with 
iNO or placebo.  
- 
To determine which NO signaling pathways are activated by iNO and whether these are 
associated with clinical benefit. 
 
 
1.2 SPECIFIC AIMS 
 
Hypothesis: iNO after OHCA will reduce heart and brain injury thus reducing death when 
compared to placebo 
 
Specific Aims: 
Primary 
Determine whether iNO compared to placebo provides clinical benefit when used within 4h of 
ROSC after OHCA. 
 
Secondary  
- 
Determine which mechanistic pathways are activated through use of iNO therapy and 
whether any of these are associated with outcome. 
- 
Determine whether iNO compared to placebo provides a physiologic effect in the heart. 
- 
Determine whether LSCI measures of endothelial function are associated with subjects 
who have a physiologic and/or clinical response to iNO (heterogeneity of treatment effect). 
- 
Determine whether iNO reduces oxidant stress and mitochondrial injury after OHCA 
compared to placebo. 
- 
Measure bioavailable NO and its downstream signals in the early post-arrest period in 
OHCA subjects treated or not treated with iNO. 
 
 
1.3. BACKGROUND and RATIONALE 


9 
 
 
 
OHCA presently results in substantial mortality (~250,000 deaths annually in the US) due to 
brain and heart injury. iNO has shown promise in reducing brain and heart injury in numerous 
animal studies of focal ischemia-reperfusion injury (IR) and in animal studies of cardiac arrest. A 
recent meta-analysis of iNO in human studies targeting IR showed promise in the majority of 
trials. iNO has been used in dozens of human clinical trials with few adverse effects compared to 
placebo thus safety is not a major concern. iNO has never been examined as a therapy following 
human brain IR or OHCA thus its efficacy in this setting remains unclear. Since the clinical 
experience with iNO suggests good safety and the preclinical/clinical data suggests potential 
benefit, this represents a promising therapy for human OHCA brain and heart IR injury and 
warrants investigation.   
 
1.4 SIGNIFICANCE 
 
OHCA carries an extremely high mortality rate resulting from brain and heart injury as well as 
substantial morbidity in long term survivors. At present, there are no alternative therapies to 
mitigate this injury. Thus iNO could save lives and/or improve long term cardiac and/or 
neurologic function therefore addressing a significant public health concern lacking any current 
therapies.  
 
2.   RESEARCH DESIGN AND METHODS 
 
2.1 CLASSIFICATION AND METHODOLOGICAL DESIGNS 
 
The iNO OHCA study is a phase II double blind (participants and investigator) placebo 
controlled randomized (1:1) clinical trial of iNO 20 ppm administered over 12h (with 1 h 
weaning off) beginning as soon as possible but within 4 h of return of spontaneous circulation 
(ROSC) after OHCA. Planned enrollment is 130 subjects over 24 months at UPMC Presbyterian 
(PUH) and Mercy (MER) with randomization stratified in blocks of 8. Recruitment will be 
performed under exception from informed consent (EFIC) to facilitate early enrollment and 
treatment. The study will have a safety monitor to assess any concerns during its conduct and a 
pre-specified safety analysis at the mid-point (after 1 year or 60 patients whichever occurs first). 
Subjects or their surrogate (as subjects will be enrolled when comatose) will be notified as soon 
as possible by a member of the research team of enrollment and can elect whether to permit 
blood banking for future genetic studies.  
 
Duration of subject participation is 90 ± 3 d of ROSC. Most data will be collected within four 
days of ROSC, which includes the 12h of study drug administration and 1h weaning of study 
drug. After the initial four days have passed follow up will be done at hospital discharge (DC; 
which includes death), day 30, and day 90 after OHCA to evaluate outcomes. 
 
Subjects will not be withdrawn from or prevented from receiving any known effective therapy 
for the purposes of participating in this research.   
 
2.2 DETAILED DESCRIPTION OF STUDY DESIGN 


10 
 
 
 
Study Visits and Procedures:   
 
 
**Please note that the 0h ECHO and LSCI may be collected up to one hour after the 
administration of the study drug to avoid issues with timing of enrollment. 
 
The majority of data, including all clinical lab data, that is collected for this study will be 
gathered during a single hospitalization. All labs will be collected within 30 minutes of their 
assigned time.  
Follow-up data that will be obtained will not require a subsequent visit, but rather just a 
phone call.  
Most of the baseline data elements can be effectively gathered retrospectively and will not be 
needed prior to starting the study drug.  A small number of data elements will indeed need to be 
captured before study drug is administered but these can be done fairly rapidly to preclude 
delays in therapy onset. The same is true for data elements in days 1-3 post-arrest (0-24 h after 
ROSC is considered day 1) 
 
Eligibility will be determined by a University of Pittsburgh Post-Cardiac Arrest Service (PCAS) 
physician, all of whom are routinely consulted in the care of this patient population and will 
serve as co-investigators on the study. Inclusion and exclusion criteria will be based on clinical 
characteristics (see attached Screening questionnaire). Due to the urgent need for intervention in 
this population, we will enroll subjects under an exception from informed consent (EFIC).  Once 
eligibility is determined, patients will be randomized using block randomization to receive iNO 
or placebo. Randomization will be stratified in blocks of 8 by center. The randomization list will 
be created prior to the beginning of the study by the Respiratory Therapy (RT) directors (Edgar 
Delgado, PUH, and Robert Bizilla, MER) and confirmed by a representative of Mallinckrodt. 
These 3 individuals who will not be involved in any portion of the conduct or analysis of the 
study will retain these lists and update them as additional subjects are enrolled using new 
canisters of study drug. This list will set an order in which canisters of iNO study drug, coded by 
bar code, will be administered at each site. Treatment assignment will be made known to the 
study team after enrollment is confirmed by means of a sealed opaque envelope which contains 
the cylinder barcode for the subject as well as the subject ID. These envelopes will be with other 
research materials at each study site.  iNO canisters will be stored in PUH Rm A-125 and Mercy 
(ED compressed gas storage).  Prior to each enrollment the next canister to be used at a site will 
be loaded onto an iNOvent delivery device and the gas calibrated permitting rapid deployment to 
the next subject enrolled at that site. Check lists will be maintained as each site documenting 
calibration and each use on a study subject. The gas canister bar code will be recorded in our 
Timepoint
Task
Pre-Enrollment
Screening for inclusion and exclusion criteria
Before Administration of Study Drug, 0h
PCAS MD Baseline, Baseline lab data, ECHO and LSCI
During Study Drug Administration, 0h-13h
Vitals, Labs, ECHO, LSCI
Retrospective Baseline Data
Demographics, OHCA data, Clinical Lab data
Day 1 Post ROSC
VS, Labs, Clinical Lab ata, PCAS MD Daily data, Interventions, Medications, SOFA
Day 2 Post ROSC
VS, Labs, Clinical Lab ata, PCAS MD Daily data, Interventions, Medications, SOFA
Day 3 Post ROSC
VS, Labs, Clinical Lab ata, PCAS MD Daily data, Interventions, Medications, SOFA
Discharge
Survival, CPC, mRS, CPC-E, Barthel ADL, NYHA Class, Location
30d Follow Up
Survival, CPC, mRS, CPC-E, Barthel ADL, NYHA Class, Location
90d Follow Up
Survival, CPC, mRS, CPC-E, Barthel ADL, NYHA Class, Location


11 
 
database with the subject ID permitting subsequent unblinding of treatment assignment. 
Canisters will be used for 4 subjects prior to return to the company and replacement with a new 
(full) canister of study drug. 
 
A clinical respiratory therapist (RT) will administer iNO at 20 ppm for 12h, with a 1h wean 
consisting of 10 min at 10 ppm, 10 min at 5 ppm, 10 min at 4 ppm, 10 min at 3 ppm, 10 min at 2 
ppm, and 10 min at 1 ppm before subject is taken off of study drug. Study drug will generally be 
connected in the emergency room or ICU. 
 
The following data elements will be collected during the course of the study at the times 
indicated. Data elements marked with an asterisk indicate variables or outcomes essential for 
conduct of the study and analysis of efficacy and/or safety. These data elements should be 
obtained in all subjects with no missing data expected. In addition to these data elements, we are 
collecting a large amount of secondary data intended for use in mechanistic studies and 
hypothesis generation.  In many cases, these data elements WILL NOT be able to be collected in 
all subjects. Such missing data will be handled as designated within an a priori statistical analysis 
plan but the presence of missing data will in no way impede our ability to meet the objectives of 
this phase 2 RCT.  
 
BASELINE DATA 
• 
*Date of OHCA 
• 
*Time of OHCA (If not available we will use Time of ROSC after subtracting estimated 
no flow and CPR time based on transfer records/physician report) 
• 
Witnessed 
• 
Bystander CPR 
• 
EMS Call Date 
• 
EMS Call Time 
• 
EMS Arrival Date 
• 
EMS Arrival Time 
• 
No flow time 
• 
CPR (low flow) time 
• 
Presenting rhythm 
• 
*Date and Time of ROSC (If not available we will use ED admission date/time from 
referring hospital record. If unavailable, stat medevac call date/time minus 30 mins. If 
unavailable, time of arrival minus 15 min) 
• 
PCAS ID 
• 
*Last name 
• 
*First name 
• 
*Date of Birth 
• 
*Medical record number 
• 
Marital status 
• 
Race/Ethnicity 
• 
*Male Sex 
• 
*Height 
• 
*Weight 
• 
      Post-resuscitation chest pain 


12 
 
• 
Charlson Comorbidity 
• 
Age Adjusted for Charlson Comorbidity Index (CCI) 
• 
*New York Heart Association (NYHA) class pre-OHCA (if no surrogate is present and 
no medical records are found on the subject this will be assumed to be NYHA class I) 
• 
Barthel ADL pre-OHCA 
• 
Education level 
• 
Private insurance 
• 
Medicare 
• 
Medicaid 
• 
Employed 
• 
Active smoker 
• 
Smoker >20 pack-years 
• 
Prior stroke 
• 
Prior Coronary artery bypass grafting (CABG) 
• 
Noninsulin-dependent diabetes mellitus (NIDDM2) type 2 
• 
Insulin-dependent diabetes mellitus (IDDM2) type 2 
• 
Diabetes mellitus (DM1) type 1 
• 
Prior myocardial infarction (MI) 
• 
Congestive Heart Failure (CHF) 
• 
Chronic Obstructive Pulmonary Disease (COPD) 
• 
Prior cardiac arrest 
• 
Dementia 
• 
Hyperlipidemia 
• 
Hypertension 
• 
Prior coronary stent 
• 
Obstructive sleep apnea 
• 
Pulmonary hypertension 
• 
*Location pre-OHCA 
• 
*Best estimate of CPC pre-OHCA (if no surrogate is present and no medical records are 
found on the subject this will be assumed to be CPC 1)  
• 
*Best estimate of mRS pre-OHCA ((if no surrogate is present and no medical records are 
found on the subject this will be assumed to be mRS 0)  
• 
Rib fracture after ROSC 
• 
Sternal fracture  
• 
Witnessed aspiration 
• 
Infection present at baseline 
• 
Infection type 
• 
Sputum color 
• 
Vent mode 
• 
Set Tidal Volume (Vt) or Inspiratory Pressure (Pinsp) 
• 
Positive end expiratory pressure (PEEP) 
• 
Fraction of inspired oxygen (FiO2) expressed as % oxygen 
• 
Peak Pressure (PIP) 
• 
Plateau Pressure (Pplat) 
• 
Chest X-Ray reading possibly consistent with pneumonia 
• 
Positive airway culture  


13 
 
 
Toxicology report 
Cause of arrest 
History of drug/alcohol use 
Current drug/alcohol use 
 
• 
ECHO RESEARCH ONLY To be performed by PCAS physician or research assistant 
trained by Marc Simon, MD, who is a cardiologist and Associate Professor of Medicine at the 
University of Pittsburgh. 
o 
Tricuspid valve regurgitant velocity 
o 
Aortic valve velocity (CO estimate) 
o 
Pulmonary valve velocity 
o 
Tricuspid annular plane systolic excursion (TAPSE)  
o 
LVEF 
• 
*LSCI RESEARCH ONLY To be performed by a research assistant trained by the PI. A 
complete description is available in section CS10.1. 
o 
Baseline blood flow and RH after 1 min vasoocclusion [resolution 1 image/s; 10 image/s 
averaging] 
o 
Baseline blood flow and RH after 5 min vasoocclusion [begin 5 min after last test]* 
• 
*Baseline labs (troponin, arterial blood gas, lactate, plasma nitrite level,) 
• 
Additional plasma labs (neuron specific enolase, platelet mitochondria, ascorbate, 
cardiolipin, SNO, cGMP, plasma banking) 
• 
Urine labs (renal failure and nitrated fatty acid measurements) 
• 
Blood for genomics (retained unless informed consent declined) 
• 
*Baseline vital signs VS (temperature, heart rate [HR], mean arterial blood pressure 
[MAP], arterial oxygen saturation [SpO2]) 
 
STUDY DRUG (0-12 h) DATA 
(Refer to Chart below) 
 
• 
*Every 3 hour vital signs VS (temperature, HR, MAP, SpO2) 
• 
Hourly monitoring of VS, SAEs, vent settings 
• Pulmonary Arterial Pressures (systolic, diastolic, mean) – often not available 
• Central Venous Pressure – often not available 
• Sv02 – often not available 
 
• 
*Plasma labs at 6 and/or* 12 h (troponin, arterial blood gas, lactate, methemoglobin, 
plasma nitrite level) 
• 
Additional plasma labs obtained variably at 3, 6, 9 and/or 12 h (neuron specific enolase, 
platelet mitochondria, ascorbate, cardiolipin, plasma banking) 
• 
Urine labs at 0, 12 and 24 h (renal failure and nitrated fatty acid measurements) 
• 
*LSCI (RESEARCH ONLY, as above)  
o 
11-12 h RH after 1 min vasoocclusion [resolution 1 image/s; 10 image/s averaging] 
o 
11-12 h RH after 5 min vasoocclusion [begin 5 min after last test] 
• 
ECHO (RESEARCH ONLY, as above (11-12 h after study drug) 
o 
Tricuspid valve regurgitant velocity 


14 
 
o 
Aortic valve velocity (CO estimate) 
o 
Pulmonary valve velocity 
o 
Tricuspid annular plane systolic excursion (TAPSE)  
o 
LVEF 
 
*Methemoglobin to be collected at 6 hours only 
 
DAY 1-3 (0-72 h post-ROSC) DATA 
 
During post-ROSC days 1-3, the following data elements will be collected by a physician 
coinvestigator/research assistant prospectively or by a research assistant retrospectively. Refer to 
chart for visual of when labs will be drawn.  
 
• 
Plasma labs measured variably between 24 and 72 h (troponin, arterial blood gas, lactate, 
plasma nitrite level, neuron specific enolase, ascorbate, cardiolipin, plasma banking) 
• 
Urine labs measured variably between 24 and 72 h (renal failure and nitrated fatty acid 
measurements) 
• 
*Full Outline of UnResponsiveness (FOUR) Score (components below) 
• 
Eyes 
• 
Motor 
• 
Brainstem 
• 
Reflex 
• 
*Glasgow Coma Score (GCS) 
• 
Eyes 
• 
Verbal 
• 
Motor 
• 
EEG interpretation 
• 
Dominant background present 
• 
Reactivity present 
• 
Seizures present 
• 
Seizure type 
• 
Burst-suppression ratio 
• 
Estimate of survival to hospital DC 
• 
Median nerve SSEP 
• 
Witnessed aspiration 
• 
New suspected infection last 24h 
• 
Infection type 
• 
Sputum color 
• 
Vent mode 
• 
Set Vt 
• 
Set PC 
• 
PEEP 
• 
FiO2 (Use % oxygen) 
• 
Peak Pressure 
• 
Plateau Pressure 
• 
CXR last 24h read may be pneumonia 
• 
Positive airway culture last 24h 


15 
 
• 
Vital Signs (VS) (highest and lowest for each) 
• 
Time to follow commands (assigned once) 
• 
SOFA (worst in prior 24h period; some components may be missing) 
• 
Respiratory (when no PaO2 is available for PaO2:FiO2 ratio, use SpO2:FiO2 ratio) 
• 
Nervous (based on GCS below) 
• 
Cardiovascular 
• 
Liver 
• 
Coagulation 
• 
Renal 
• 
Interventions 
• 
Coronary angiography 
• 
PCI 
• 
ECMO 
• 
CPR 
• 
CABG 
• 
Mechanical ventilation hours 
• 
NIPPV hours 
• 
24h FiO2 AUC 
• 
Tracheostomy 
• 
Medications (dichotomous for use not dose) 
• 
Alteplase 
• 
Anti-epileptic 
• 
Midazolam infusion 
• 
Intermittent Benzodiazapine 
• 
Propofol infusion 
• 
Intermittent Propofol 
• 
Opiate infusion 
• 
Intermittent opiate 
• 
Neuromuscular blockade infusion 
• 
Intermittent neuromuscular blockade 
• 
Neurostimulants 
• 
*Vasopressors 
• 
*Inotropes 
• 
Ketamine 
• 
Steroids (hydrocortisone/methylprednisone) 
• 
Cumulative vasopressor index (CVI; worst in prior 24h period) 
• 
*BUN (highest value in prior 24 h) 
• 
*Creatinine (highest value in prior 24 h) 
• 
Hb 
• 
WBC 
• 
platelets 
• 
PT 
• 
INR 
• 
PTT 
• 
Bilirubin 
• 
AST 


16 
 
• 
ALT 
• 
BNP 
• 
SvO2 (%) 
• 
venous PaO2 
• 
venous PaCO2 
 
Research labs will be obtained by a study coordinator within 30 minutes of the assigned 
draw time. Labs will be run either within the UPMC Clinical Labs or the research labs of Drs. 
Dezfulian or Shiva as specified.  We will also be collecting a small blood sample for an ancillary 
biomarker study for those subjects wishing to participate.  
 
The final data to be collected will be the 90 d or discharge outcomes (whichever is later).  
Information that cannot be obtained by record review will be obtained either in person, or via a 
phone call by a member of the study team. 
 
 


17 
 
 
 
TABLE 1 
 
 
 
 
Outcomes 
The following will be recorded at DC (+/- 3 days), and for follow up at day 30 (+/- 3 days) and 
day 90 (+/- 3 days) after OHCA:  
 
*Survival 
Cause of death 
date/time of death 
Vital signs
0
3
6
9
12
24
48
72
Temperature
X
X
X
X
X
X
X
X
Heart Rate
X
X
X
X
X
X
X
X
Systolic Blood Pressure
X
X
X
X
X
X
X
X
Diastolic Blood Pressure
X
X
X
X
X
X
X
X
Mean Arterial Pressure
X
X
X
X
X
X
X
X
Arterial SBP
X
X
X
X
X
X
X
X
Arterial DBP
X
X
X
X
X
X
X
X
Arterial MAP
X
X
X
X
X
X
X
X
Pulmonary artery systolic pressure
X
X
X
X
X
X
X
X
Pulmonary artery diastolicpressure
X
X
X
X
X
X
X
X
Pulmonary artery mean pressure
X
X
X
X
X
X
X
X
Central venous pressure
X
X
X
X
X
X
X
X
Cardiac output 
X
X
X
X
X
X
X
X
Vasopressors
X
X
X
X
X
X
X
X
Inotropes
X
X
X
X
X
X
X
X
Sedation
X
X
X
X
X
X
X
X
Neuromuscular blockade
X
X
X
X
X
X
X
X
Time from study drug start (hours)
Time from study drug start (hours)
Labs (highlight represents UPMC Labs)
0
3
6
9
12
24
48
72
Troponin
X
X
X
Arterial blood gas (ABG)
X
X
X
Lactate
X
X
X
Methemoglobin
X
Neuron specific enolase (NSE)
X
X
Plasma NO metabolites
X
X
X
X
X
X
X
X
Platelet mitochondria
X
X
Ascorbate
X
X
X
X
Cardiolipin
X
X
X
X
Inflammation (integrin α9β1 and 
CD11b)
X
X
X
X
Banked plasma
X
X
X
X
X
X
X
X
DNA for pharmacogenomics
X
Whole blood pellet
X
X
X
X
X
X
X
X
Urine for renal failure markers and 
NO2-cLA
X
X
X
*DNA will be collected with the first scheduled blood draw after obtaining informed consent 


18 
 
*CPC 
*mRS 
CPC-E 
Barthel ADL 
*NYHA Class 
*Location of residence 
 
 
 
 
2.3 STUDY DRUG 
2.3.1. Study Drug Preparation and Dispensing 
iNO used in this study will be sourced and formulated by INO Therapeutics, part of Mallinckrodt 
Hospital Products IP Limited. Cross reference authorization has been granted to us by 
Mallinckrodt for information on the study drug, known as INOmax under New Drug Application 
(NDA) 20-845. Placebo gas wherein nitrogen (the carrier vehicle gas) is substituted for iNO will 
also come from Mallinckrodt.  
 
Mallinckrodt will deliver gas cylinders to the Respiratory Therapy (RT) Department at UPMC 
PUH or MER.  Each cylinder will have a bar code which corresponds to its contents (iNO or 
placebo). Randomization will be done in advance in blocks of 8 at each site by the RT directors 
using the website http://www.graphpad.com/quickcalcs/randomize1.cfm. The random order list 
generated will list the order in which subjects coming in to each site are assigned to cylinders of 
study drug. In each group of 8, 4 subjects will be assigned to one placebo cylinder and 4 subjects 
will be assigned to one iNO cylinder. RT will dispense study drug upon patient enrollment  and 
the blinded research assistant (RA) will transport the cylinder/iNOvent, making note of its 
barcode to permit subsequent treatment assignment, to the patient’s bedside for connection to the 
ventilator by RT.  All patients will be on mechanical ventilation via an endotracheal tube (ETT).  
iNO is a commonly used drug within UPMC and existing protocols for its use will be followed 
in connecting the drug to the ventilator circuit. 
 
2.3.2. Drug Administration 
 
Research subjects will be comatose, so no specific instructions will be provided to them. RT will 
connect the study drug to the mechanical ventilator and the drug will be dosed via ETT for the 
entire 12 h period at 20 ppm followed by a 1 h wean. All patients will receive the same pattern of 
administration. 
 
2.3.3. Dose Selection 
 
A number of studies have reviewed the safety of iNO in adults primarily in the setting of acute 
respiratory distress syndrome (ARDS), A recent meta-analysis found improved oxygenation but 
also greater acute kidney injury (AKI) resulting from iNO use.25 When this was examined by 
dose, AKI only resulted in those patients receiving higher cumulative doses of iNO (≥40 ppm for 
≥48h).26 Our study will limit iNO to 20 ppm for 12h hence AKI is not expected though we will 
track BUN and creatinine on all subjects. A separate meta-analysis examining effects of iNO in 


19 
 
acute hypoxemic respiratory failure in adults and children found no associated adverse events.27 
A study of iNO used in transport identified no adverse events associated with the drug.28 
Although iNO significantly increases methemoglobin (metHb) levels, the increase (0.3%) is 
clinically insignificant and not associated with toxicity 29 which is uncommon until levels of 20-
30% (we will consider > 10% a significant and unexpected adverse event). The best predictor of 
significant metHb >4% (as < 0-3% is considered within normal range) is cumulative iNO dose of 
>2000 ppm * h.30 In our study subjects will receive only 250 ppm * h. We will monitor 
methemoglobin as part of our study at 6h. Although iNO lowers pulmonary blood pressure it has 
not been linked to reduction in systemic arterial blood pressure. We will monitor blood pressure 
effects closely in our study given the inherent instability of cardiac arrest subjects. 
Effects of iNO after cardiac arrest has been studied in 4 preclinical animal studies.8-11  
These published studies demonstrated benefit using iNO 40 ppm started within 2h of ROSC 
which is additive to therapeutic hypothermia8 as well as neuroprotection without temperature 
control.9 These animal models include mouse and pig models of asystole and ventricular 
fibrillation (VF). In addition iNO has been shown to protect in animal models of focal heart12-15 
and brain16-19 injury.  This is important since most of the mortality and morbidity after OHCA 
results from heart and brain injury.20  
Animal models of asphyxia21 and other focal brain ischemia reperfusion models 
demonstrate NO deficiency early in reperfusion lasting at least 6h and benefits from iNO.16,19   
 
Information from prior clinical trials of iNO are available by cross reference in the Mallinckrodt 
NDA 20-845 (letter authorizing cross reference is included with application).  
 
iNO is FDA approved for the treatment of pulmonary hypertension in the setting of hypoxemic 
respiratory failure in neonates. It has been used off label extensively; primarily for the treatment 
of hypoxemic respiratory failure and pulmonary hypertension.  From these published studies, 
extensive safety data is available for a variety of endpoints with up to 2 year follow up.1-6 In 
these studies efficacy as measured by increase in oxygenation was noted with doses as little as 1 
ppm3,4 with rare cases of methemoglobinemia or excess NO2 especially at the doses under 40 
ppm. A recent meta-analysis of trials of iNO to treat ischemia reperfusion injury in humans 
demonstrated efficacy in the majority of studies and an excellent human safety profile, but noted 
no existing human evaluation of iNO in the setting of acute brain injury including OHCA.7  
 
In human brain injury from stroke22,23 or trauma24 the CSF levels of NO metabolites appear 
elevated by 24h with one study showing this effect is blocked using the inducible NO synthase 
inhibitor 1400W.22 Based on these reports it would appear a state of NO deficiency is highly 
likely at ROSC and persistent for up to 24h after ROSC.  Thus the likely therapeutic window is 
from ROSC until 24h after ROSC.  
 
Most studies that have demonstrated efficacy of iNO in animal models of CA or other IR injury 
have dosed the drug early (during CPR or at the time of ROSC). Only one study did a formal 
time course.31 In this report iNO started 2h after CA improved neurologic function/injury and 
survival but 6h later did not. Thus the therapeutic window is between CPR onset and <6h after 
ROSC. One study showed efficacy at 1h after ROSC.9 We selected a midpoint of 4h from ROSC 
as our latest time to initiate therapy but again stress that most data support earlier dosing either at 
the time of ROSC or during CPR. Thus our goal in designing a trial with recruitment under EFIC 


20 
 
is to enroll and dose patients as soon as possible after ROSC.  
 
Our patients will generally achieve ROSC in the field (pre-hopsital). Most patients at PUH, our 
larger center, arrive as transports already 1-3h post-ROSC upon arrival. Thus at the time the 
subjects arrive in our center we will be under substantial time pressure to get them on study drug 
even to achieve the goal of dosing prior 4h of ROSC. Furthermore, many of these patients are 
flown into our center or transported by ambulance with sirens activated, thus arriving 
without a family member from whom to obtain consent.  
 
 
 
 
2.3.4. Treatment Period 
 
RT will administer iNO at 20 ppm for 12h, with a 1h wean consisting of 10 min at 10 ppm, 10 
min at 5 ppm, 10 min at 4 ppm, 10 min at 3 ppm, 10 min at 2 ppm, and 10 min at 1 ppm before 
subject is taken off of study drug.  
 
2.3.5. Breaking the Blind 
 
The safety monitor can request unblinding in the case of any serious unanticipated adverse 
event (SUAE) based on clinical judgement and will unblind whenever there is a MetHb level > 
10% associated with any adverse event. The safety monitor will review clinical data from the study 
consisting of VS, vasopressor and inotrope use, labs and outcomes, at least every 4 weeks. In the 
event of a SUAE, the safety monitor will review the record within 48 h breaking the blind to 
determine whether this is a drug associated SUAE.  
The Data Safety Monitoring Board (DSMB) will meet prior to the commencement of the 
trial and every 6 months. All data will be reviewed at 12 months or 60 subjects for safety. The 
DSMB and safety monitor are authorized to unblind at any time during the conduct of the study if 
they have safety concerns. The investigators will remain blind up until the point that the DSMB 
decides to halt the trial or trial conclusion. 
 
2.3.6. Medication Compliance 
 
Since subjects are comatose and will be given study drug by endotracheal tube (ETT) over 12h 
compliance will be directly observed and verified by study team member in the research record. 
Thus, we anticipate 100% compliance unless study drug is stopped by a surrogate upon notification 
or for safety reasons both of which are unanticipated events. 
 
2.3.7. Medication Storage and Accountability 
 
Please refer to package insert of INOmax Nitric Oxide gas from INO Therapeutics/ Mallinckrodt 
for information on storage and accountability (cross reference authorized to NDA 20-845). Once 
delivered gas cylinders will be secured in a temperature controlled area within the respiratory 
therapy department. Gas cylinders (one blinded placebo and one blinded iNO) will be mounted 
on iNOvent delivery devices which will be stored within the emergency department to permit 


21 
 
rapid deployment. The contents of iNO after each use (in psi) will be documented and the 
cylinders will undergo high flow calibrations monthly and low flow calibrations weekly.  Logs 
will be maintained on clipboards connected to the iNOvent.  
 
2.3.8 Concomitant Medications 
 
The following agents will not be permitted during study drug dosing: PDE5 inhibitors (sildenafil, 
vardenafil, tadalafil), sGC stimulators (riociguat), other nitrates (nitroglycerin, nitroprusside, 
isosorbide mono- and di-nitrate).  PDE5 inhibitor and sGC stimulator use is cause for exclusion 
and these drug in general are seldom used within our population. We will likewise exclude subjects 
known to have the diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH), 
pulmonary arterial hypertension (PAH) and erectile dysfunction which are the primary indications 
for PDE5 inhibitor and sGC stimulator use. 
 
 
2.3.9 Rescue Medications 
 
Hemodynamics will be stabilized using standard inotropic and vasopressor agents as needed in the 
ICU. There is no specific reversal agent for iNO. Methemoglobinemia can be reversed with 
treatment using methylene blue.  
 
2.4 STUDY ENDPOINTS 
Primary   
Composite of in-hospital death, unfavorable discharge location, NYHA class III/IV heart failure 
at the time of discharge* 
*In the setting of pre-existing heart failure or prior residence in an unfavorable location there must 
be at least a 1 class decrement (eg NYHA III -> IV) 
 
Secondary 
Functional 
-Survival to hospital discharge, 30 and 90 days after initial OHCA 
-Favorable cerebral performance category (CPC) defined as 1 or 2 at discharge, 30 and 90 days 
after initial OHCA  
-Favorable modified Rankin score (mRS) defined as 0-3 at discharge, 30 and 90 days after initial 
OHCA 
-Favorable discharge destination or location of residence (30 and 90 days after OHCA) defined as 
home or acute inpatient rehabilitation 
-CPC-E at hospital discharge, 30 and 90 days after initial OHCA 
-Barthel Index of Independence in Activities of Daily Living (ADL) at discharge, 30 and 90 days 
after OHCA 
 
Neurologic 
-Estimated likelihood of good neurologic outcome (0-100%) assessed by an experienced (>3y) 
member of the PCAS team blinded to treatment assignment on day 3-5 post-ROSC. 
-Development of malignant EEG during hospitalization  


22 
 
-Time (in hours) to awakening from ROSC; defined as following commands (all values > 96h 
assigned 100) 
-FOUR Motor + Brainstem scores on days 1-3 (requires 30 min off sedation; exclude endpoint if 
not medically permitted to pause sedation or paralysis) 
-Presence or absence of upper and lower extremity somatosensory evoked potential N20 on 
comatose patients obtained any time after post-arrest day 1. 
- Absolute value 24h after ROSC and change in value (24h vs 0h) of neuron specific enolase (NSE) 
-EEG burst suppression ratio and change in suppression ratio 
-EEG presence of dominant continuous background and reactivity to photic or tactile stimulation. 
 
Cardiovascular 
-Heart rate and mean arterial pressure (preferably off the arterial line) at 0-12 (hourly), 24h of 
study drug 
-Persistent need for vasopressors in the first 24h after ROSC; defined as any use of norepinephrine, 
epinephrine, phenylephrine, dopamine or vasopressin for ≥3h after study drug is initiated 
-Persistent need of inotropes in the first 24h after ROSC; defined as any use of epinephrine, 
dobutamine, milrinone or isoproterenol for ≥3h after study drug is initiated. 
-ECHO measures (absolute values at end of therapy (12h) and change in 12h vs 0h):  
   -tricuspid valve regurgitant velocity (TRV)  
   -cardiac output based on aortic valve velocity (AV)  
   -tricuspid annular plane systolic excursion (TAPSE) 
   -left ventricular ejection fraction (EF).  
-Absolute value at end of therapy (12h), change in value (12h vs 0h) and peak 24h post-ROSC 
troponin I 
-Arterial blood gases at 0, 6, 12 and 24h after study drug initiation (when available) 
-Upper body central venous oxygen saturation at 0, 6, 12 and 24h after study drug initiation (when 
available) 
-Lactate at 0, 6, 12 and 24h after study drug initiation  
 
Biochemical  
-NO metabolites. Plasma nitrite, S-nitrosothiols and cyclic guanosine monophosphate (cGMP) 
levels obtained at 0, 3, 6, 9, 12h of study drug and 12h after cessation.  
-RBC pellet for hemoglobin (Hb) studies (retained from above) 
- MetHb at 6h (safety endpoint) 
-Platelet mitochondria. Oxygen consumption rate, respiratory control ratio and superoxide 
generation (MitoSOX) at 0 and 12h of study drug when available. This data will be collected only 
when patients are enrolled from 6a-6p on weekdays (70/130 budgeted) to assure quality. 
-Plasma for ascorbate 0, 3, 12, 24h  
-Cardiolipin at 0, 3, 12, 24h  
-After obtaining separate informed consent (IC) banked blood for DNA (pharmacogenetics) 
-LSCI coupled to 1 and 5 min forearm vaso-occlusion at 0 h and 11 h. Endpoints will include 
ischemic trough, time to RH and RH peak, blood flow nadir 1 and 5 min after cuff release 
(respectively). 
 
2.5 STATISTICAL ANALYSIS 
 
2.5.1 Sample Size Determination 


23 
 
Sample size calculations will be based on the primary composite endpoint and ascertained via a 
two sample test of proportions. The control rate proportion was estimated to be approximately 
80-85% and we have > 80% power to detect reduction in the composite endpoint of 
approximately 23% with 60 per arm and a type II error rate of 5%, and 65 per arm assuming 
minimal dropout in the range of 5%. Since our estimates of dropout are extremely conservative 
(our reported dropout in EFIC studies is <1%)32 we are powered to detect changes in our primary 
composite endpoint as low as 21% if we actually observe little to no dropout. The secondary 
efficacy endpoint of iNO responsiveness is overpowered to detect a decrease in PA pressure in 
the region of 20-25 mm Hg with the planned enrollment. 
 
 
 
 
2.5.2 Study Conduct Analysis 
Continuous data will be summarized and compared using t-tests or a non-parametric equivalent 
should the normality assumption appear tenuous. Generalized linear mixed models, which 
account for non-independence of repeated assessments and endpoint structure, will be used to 
compare between iNO and placebo groups at multiple times. Analysis of the composite endpoint 
will be ascertained using an unadjusted two sample test of proportions with a type II error rate of 
5%. In the event of imbalance of potential confounders after randomization an adjusted analysis 
will be performed using logistic regression with those unbalanced variables and treatment as the 
main effect of interest. Other secondary dichotomous outcomes will be summarized and 
compared by chi squared tests and appropriate exact methods should sample sizes be small. Time 
to event endpoints will be summarized by Kaplan Meir curves and compared using log rank 
tests. All tests will be two tailed with unadjusted p<0.05 considered significant and performed 
using SAS 9.4. 
 
 
 
 
 
2.5.3 Efficacy Analysis 
Our primary outcome is intended to prove efficacy. Improvement in any of the secondary clinical 
outcomes being assessed would be considered preliminary evidence of efficacy requiring a 
subsequent larger trial for proof. Since iNO’s effects on pulmonary vasodilation are best known, 
we will use this as our primary physiologic surrogate measure of iNO efficacy and will be 
compared using unadjusted two-sample tests or adjusted regression based methods.  
 
 
2.5.4 Safety Analysis 
Section 6.4.5 outlines all potential SAE’s and defines a SUAE as any SAE that occurs in the 
setting of MetHb > 10% which may thus be attributed to the study drug. All listed safety 
outcomes will be tabulated and summarized using basic descriptive statistics and tested using 
small sample exact methods. 
 
2.5.5 Handling Missing Data 
Since our primary endpoint is ascertained at hospital discharge we expect no missing data though 
all analyses will be performed assuming a missing at random mechanism. No data imputation 
will be performed though standard sensitivity analyses, using standard missing data techniques 
may be used. 
 
2.5.6 Data Management 


24 
 
Subjects will be assigned a study ID upon entry and all data/samples stored using that ID.  
Linkage to patient identifiers will be maintained in a secure spreadsheet and will include name, 
DOB and MRN. De-identified clinical and lab data will all be stored in a web based database to 
be maintained by Dr. Dezfulian’s research assistant who has prior experience from other studies. 
All data will be stored and managed with adherence to all relevant laws and HIPAA guidelines to 
ensure the safety and privacy of patient data.  
 
 
3.  HUMAN SUBJECTS 
 
3.1 SUBJECT POPULATION 
3.1.1 Inclusion of Women and Minorities 
Women and Minorities will be included in the subject population 
 
3.1.2 Inclusion of Children 
Children will not be included in the subject population 
 
3.1.3 Inclusion of Prisoners 
Prisoners will not be included in the subject population 
 
3.2 INCLUSION CRITERIA 
 
Inclusion criteria do not change any subgroups. 
 
Intubated and comatose adult (>18 yo)  
Out-of-hospital cardiac arrest (OHCA)  
Cardiac arrest within an emergency department or outpatient medical center will be included.  
OHCA includes EMS witnessed cardiac arrest. 
ROSC within 40 min of CPR initiation 
FOUR Brainstem score ≥ 2  
Patient must have pupil OR corneal reflex before enrollment 
 
 
3.3 EXCLUSION CRITERIA 
 
Exclusion criteria do not change any subgroups. 
 
- 
Traumatic etiology of OHCA 
- 
Prisoner 
- 
Known pregnancy  
o B-HCG screening is NOT REQUIRED for enrollment in women of appropriate age 
- 
Hemodynamic instability defined as: 
o > 1 recurrent arrest prior to enrollment 
o Inability to maintain mean arterial blood pressure (MAP) > 65 using vasopressors 
and inotropes (ie actively up titrating medications or giving fluid bolus) 
- 
Cerebral edema defined as: 


25 
 
o Head CT grey-white ratio < 1.2 
o Fixed and dilated pupils without another explanation 
- 
Malignant EEG upon presentation (not required as screening) defined as: 
o Myoclonic status epilepticus 
o Non-convulsive status epilepticus 
o Generalized periodic epileptiform discharges  
- 
ROSC >3h from time of ED arrival (Treatment allocation must be within 4h so anything 
that will prevent this is reason for exclusion) 
- 
Alert and interactive patient with minimal evidence of neurologic injury 
- 
Plan to extubate within 12 hours 
- 
PCAS opinion that patient will die with >95% likelihood.  This may be based on: 
o Multiple medical comorbidities 
o Late discovery of DNR or advanced directive 
o Terminal diagnosis (other than OHCA; may have caused OHCA) 
o Clinical judgement based on current exam and data 
- 
Known intracranial hemorrhage or acute cerebral infarction; Head CT is NOT REQUIRED 
prior to enrollment 
- 
Patient is known to be taking PDE5 inhibitors, sGC stimulator, or has a known diagnosis of 
Chronic thromboembolic pulmonary hypertension (CTEPH), pulmonary hypertension 
(PAH), or erectile dysfunction 
- 
Known enrollment in another acute interventional trial 
 
4.   IRB APPROVAL AND FDA AMENDMENTS 
 
The Investigator will obtain, from the University of Pittsburgh Institutional Review 
Board (IRB), prospective approval of the clinical protocol and corresponding informed 
consent form(s); modifications to the clinical protocol and corresponding informed 
consent forms, and advertisements (i.e., directed at potential research subjects) for study 
recruitment.    
 
The only circumstance in which a deviation from the current IRB-approved clinical 
protocol/consent form(s) may be initiated in the absence of prospective IRB approval is 
to eliminate an apparent immediate hazard to the research subject(s).  In such 
circumstances, the Investigator will promptly notify the University of Pittsburgh IRB of 
the deviation. The Investigator should also notify the sponsor of this event.   
 
The University of Pittsburgh IRB operates in compliance with FDA regulations at 21 
CFR Parts 50 and 21 CFR 56, and in conformance with applicable International 
Conference on Harmonization (ICH) Guidelines on Good Clinical Practice (CGP).  
 
In the event that the University of Pittsburgh IRB requires, as a condition of approval, 
substantial changes to a clinical protocol submitted under an FDA-accepted IND 
application, or in the event of the Investigator’s decision to modify the previously 
accepted clinical protocol: 
 
 


26 
 
• The Sponsor will submit (i.e., in advance of implementing the change) a Protocol 
Amendment to the IND describing any change to a Phase 2 protocol that significantly 
affects the safety of subjects, the scope of the investigation, or the scientific quality of 
the study.  Examples of Phase 2 clinical protocol changes requiring the submission of 
a Protocol Amendment include: 
 
o Any increase in drug dosage or duration of exposure of individual subjects to the 
investigational drug beyond that described in the current protocol, or any 
significant increase in the number of subjects under study. 
 
o Any significant change in the design of the protocol (such as the addition or 
deletion of a control group). 
 
o The addition of a new test or procedure that is intended to improve monitoring 
for, or reduce the risk of, a side effect or adverse event; or the dropping of a test 
intended to monitor the safety of the investigational drug. 
 
5. RECRUITMENT AND INFORMED CONSENT PROCEDURES 
5.1 RECRUITMENT METHODS:  
PCAS physicians are generally alerted to incoming resuscitated cardiac arrest patients either 
through a med call page (to accept the patient in transfer) or a page from emergency medical 
services stating the patient is en route to the emergency room (ER). PCAS physicians will review 
details of the patient's cardiac arrest to determine eligibility in consultation with a study 
coordinator. The ultimate designation of whether a patient is eligible will require a physical 
exam to determine neurologic function and hemodynamic stability upon ER presentation. If a 
patient is deemed eligible, they will be enrolled into the trial.  We are requesting a waiver of 
informed consent to both review medical records to determine eligibility, and to enroll patients in 
the trial. 
 
 
5.2 INFORMED CONSENT PROCEDURES  
 
Since the study population is comatose (part of inclusion criteria), their surrogates are often 
difficult to locate or emotionally not able to provide consent and the therapy must be provided 
rapidly, recruitment will be done under EFIC.  Subjects will be enrolled if they meet the 
eligibility criteria unless they have opted out of the study previously. Surrogates (the legally 
authorized healthcare representative [LAR]) will be informed that subject is enrolled in a trial at 
an appropriate time, based on the judgement of the study team, by either study coordinator or 
PCAS MD. If family chooses to withdraw consent, subject will be weaned off study drug. If the 
subject awakens, he/she will be informed of their participation in the study and that their records 
were reviewed. Banking of blood for genetic studies requires a separate informed consent.  The 
genetic sample will not be drawn until consent for the procedure has been obtained. In the event 
that an LAR is identified but is unable/unwilling to come into the hospital for an in-person 
discussion, we are requesting the option to have a physician investigator discuss the study and go 
through the consent over the phone.  We would then email a copy of the consent to the LAR and 
request that he/she respond that they are agreeable for the patient to participate in the genomic 


27 
 
portion of the trial, or indicate that they are not agreeable.  We will have two people independent 
of the study, usually clinical nurses or physicians, witness the process. We would then draft a 
Note to File to keep in the subject's study folder outlining exactly what happened, along with a 
copy of the entire email discussion.   
 
In the event that the LAR is unable/unwilling to come into the hospital and does not have access 
to email, we are requesting a waiver to document written informed consent for the notification of 
study enrollment only.  The physician investigator will discuss the study and will obtain 
permission for continued participation.  A Note to File will be placed in the subject’s record 
documenting the discussion and the LAR’s response. 
 
 
6.  POTENTIAL RISKS AND BENEFITS 
 
 
6.1 POTENTIAL RISKS 
 
6.1.1 Risk of Experimental Drug Intervention 
The known adverse events due to iNO are reviewed in the package insert and in section 2.3.3 
above. Extensive safety data is available from published studies of iNO in critically ill patients 
with up to 2 year follow up.1-6 In these studies rare cases of MetHb or excess nitrogen dioxide 
[NO2], an airway irritant, are reported.  These cases occur at doses ≥40 ppm. The INOVent 
delivery system provides constant monitoring of NO2 levels and we will monitor MetHb to avoid 
toxicity. iNO may cause rebound hypoxia after prolonged use but this generally requires over 
24h of use. We plan a 1h wean off the study drug to minimize this risk.  Other NO donors (not 
iNO) can reduce blood pressure but this has not been noted in human or animal studies of iNO at 
the doses we plan to use. Acute kidney injury (AKI) has been noted in some clinical trials of iNO 
but again only with higher doses than we propose to use. We will nonetheless monitor BUN and 
creatinine as markers of renal function. Patients receiving iNO may experience increasing 
capillary wedge pressure and worsening heart failure. We will monitor oxygen requirement 
(FiO2) and saturation (SpO2) hourly during study drug administration and the PI or a physician 
coI will be notified of an increase of >20% in the patients’ oxygen delivery (mechanical 
ventilator FiO2) or a drop of > 50 mm Hg or 25% reduction (whichever is lesser) in the PaO2 if 
the patient is on FiO2 > 0.80. This data along with any change in chest X ray (generally obtained 
daily in these patients) and hemodynamics collected hourly and hourly fluid intake and output 
will also be reviewed for each patient by the safety monitor as described above (2.3.5). 
 
6.1.2 Risk of Study Procedures 
Blood will be obtained from pre-existing indwelling catheters required as part of patient care 
therefore there is no additional risk from phlebotomy. Likewise urine will be collected from 
existing foley catheters. The total whole blood being collected is 125 ml. This represents 2.5% of 
the average human blood volume and is not expected to result in any significant added stress to 
the subjects. 
 
6.2 ALTERNATIVE TREATMENTS 
 


28 
 
No present FDA approved therapies to provide cardiac or neuroprotection after OHCA. 
 
6.3 POTENTIAL BENEFITS 
The risks listed here are minimal in comparison to the benefits which may be gained by reducing 
life ending brain and heart injury. 
 
 
6.4 DATA SAFETY MONITORING  
 
6.4.1 Data Safety Monitoring Board 
 
A Data and Safety Monitoring Board (DSMB) will be created to review this study. 
 
6.4.2 Data Safety Monitoring Plan 
 
Data Monitoring Plan. The proposed study will use the FDA definition of adverse events (AE) and 
serious adverse events (SAE). Any SAE, which is unexpected and related to study intervention, 
will be reported immediately to the IRB and will be followed by an additional letter detailing the 
nature of the SAE. In the event that a participant either withdraws from the study or the 
investigators decide to discontinue a participant due to a SAE, the participant will be monitored 
by the co-PIs until (a) a resolution is reached (e.g., the problem has resolved or stabilized with no 
further change expected), (b) the SAE is determined to be clearly unrelated to the study 
intervention, or (c) the SAE results in death. Outcomes of SAEs will be regularly reported to the 
IRB and the sponsor. A summary of the SAEs that occurred during the previous year will be 
included in the annual progress report as well as in the annual IRB renewal. 
 
Monitoring of safety and data quality in the proposed study will be the responsibility of all 
personnel on the project, with primary responsibility and supervision by the Investigator.  The 
Institutional Review Board will approve the Notification / Statement of Informed Consent (IC) for 
the study and provide institutional oversight of data and safety issues. The study protocol will be 
approved prior to recruiting or obtaining consent from any participants. Moreover, the study will 
be reviewed at a minimum of annual basis (or more frequently as deemed necessary) by the IRB 
committee. Participants will be enrolled under EFIC and the LAR (and subject assuming 
awakening) will be provided notification of inclusion in the study and provided the option to 
withdraw IC. LAR/subject will separately be requested to provide IC for retention of blood for 
genomics. To ensure participant safety, once participants are enrolled in the study, study staff will 
immediately report all adverse and serious adverse events to one of the Investigators. The 
Investigator will, per standardized procedures, report them to the IRB for their review. These 
events should also be communicated to the sponsor of the IND. With regard to monitoring of data 
quality and protected health information, all required personnel proposed for this project will have 
the required human subjects and confidentiality training, which includes information about 
maintaining data integrity and security. Confidentiality will be guarded using established 
procedures such as storing data in locked cabinets within locked offices or locked data rooms, 
coding by study identification numbers rather than any personally identifying information to avoid 
revealing the identity of subjects, and aggregating data across participants. The key linking names 
and study identification numbers will be kept separately from the data sets with limited access by 


29 
 
study personnel (RAs and principal investigator only). Only study personnel will have access to 
the data sets on protected servers. Oversight of all aspects of data management will occur with the 
Investigator. 
 
After initial approval and at periodic intervals (at least every 6 months; to be determined by the 
committee) during the course of the study, the DSMB responsibilities are to: 
 
1. 
Review the research protocol, informed consent documents and plans for data and safety 
monitoring; 
2. 
Evaluate the progress of the study, including periodic assessments of data quality and 
timeliness, participant recruitment, accrual and retention, participant risk versus benefit, adverse 
events, unanticipated problems, performance of the trial sites, and other factors that can affect 
study outcome; 
3. 
Consider factors external to the study when relevant information becomes available, such 
as scientific or therapeutic developments that may have an impact on the safety of the participants 
or the ethics of the study; 
4. 
Review clinical center performance, make recommendations and assist in the resolution of 
problems reported by the PI; 
5. 
Protect the safety of the study participants; 
6. 
Report on the safety and progress of the study; 
7. 
Make recommendations to the PI, and if required, to the funding agency concerning 
continuation, termination or other modifications of the study based on the observed beneficial or 
adverse effects of the treatment under study; 
8. 
Monitor the confidentiality of the study data and the results of monitoring;  
9. 
Assist the PI by commenting on any problems with study conduct, enrollment, sample size 
and/or data collection. 
 
The DSMB will include experts in critical care medicine, pharmacology, and biostatistics.  
Members will consist of persons independent of the investigators who have no financial, scientific, 
or other conflict of interest with the study.  Written documentation attesting to absence of conflict 
of interest will be required. 
 
The University of Pittsburgh Office of Clinical Research, Health Sciences will provide the 
logistical management and support of the DSMB.  A safety officer (chairperson) will be identified 
at the first meeting.  This person will be the contact person for serious adverse event reporting.  
Procedures for this will be discussed at the first meeting. 
 
The first meeting will take place before initiation of the study to discuss the protocol, approve the 
commencement of the study, and to establish guidelines to monitor the study.  The follow-up 
meeting frequency of the DSMB will be determined during the first meeting.  An emergency 
meeting of the DSMB will be called at any time by the Chairperson should questions of patient 
safety arise.   
 
The DSMB will meet prior to the beginning of enrollment and every 6 months until study 
completion (planned at 2 years). A planned interim analysis will be conducted at 1 year or after 
60/130 subjects is enrolled, whichever occurs first. 


30 
 
 
 
6.4.3 Parameters to be Monitored 
The following progress will be monitored throughout the course of the research to ensure the safety 
of subjects as well as the integrity and confidentiality of their data. 
 
• An evaluation of the progress of the research study, including subject recruitment and 
retention, and an assessment of the timeliness and quality of the data. 
 
• A review of collected data (including adverse events, unanticipated problems, and 
subject withdrawals) to determine whether there is a change to the anticipated benefit-
to-risk assessment of study participation and whether the study should continue as 
originally designed, should be changed, or should be terminated. 
 
• An assessment of external factors or relevant information (eg. pertinent scientific 
literature reports or therapeutic development, results of related studies) that may have 
an impact on the safety and study participants or the ethics of the research study. 
 
• A review of study procedures designed to protect the privacy of the research subjects 
and the confidentiality of their research data. 
 
The severity of adverse changes in physical signs or symptoms will be classified as follows: 
 
• Grade 1 (Mild): asymptomatic or mild symptoms; clinical or diagnostic observation only; 
intervention not indicated. 
 
• Grade 2 (Moderate): minimal, local or noninvasive intervention indicated; limiting age-
appropriate ADL (Activities of Daily Living). 
 
• Grade 3 (Severe): medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self care 
ADL. 
 
• Grade 4 (Life-threatening): consequences; urgent intervention indicated. 
 
• Grade 5 (Death): event is a direct cause of death. 
 
6.4.4 Frequency of Monitoring 
The Investigator will review subject safety data as it is generated. The Investigator, sub-
investigators, and the research staff will meet on a monthly interval to re-evaluate study 
goals, subject recruitment, data coding and retention, documentation and identification of 
adverse events, complaints and confidentiality of subjects. There will be an evaluation of the 
progress of the research study, including assessments of data quality, time lines, participant 
recruitment, accrual, and retention. The Investigator will also review the outcome and 
adverse event data to determine whether there is any change to the anticipated benefit-to-risk 
ratio of study participation and whether the study should continue as originally designed or 


31 
 
should it be re-evaluated and changed.  
 
Independently the safety monitor will be review clinical data from the study consisting of 
subject VS, vasopressor and inotrope use and labs at baseline and during the first 24 h of 
enrollment (which includes study drug dosing) and final outcomes.  This review will occur  
at least every 4 weeks. In the event of a SUAE, the safety monitor will review the record 
within 48 h, breaking the blind to determine whether this is a drug associated SUAE 
 
 
 
6.4.5 Reportable Adverse Events 
This is a high risk population in which numerous serious adverse events are expected. 
Indeed, all subjects will require mechanical ventilation, with >50% of subjects expected to 
require >50% FiO2 and/or vasopressors during the first 24 h based on our prior data33,34 
and expected mortality is 70-80%35 as we are excluding non-comatose subjects. Thus 
numerous serious adverse events (SAE) are anticipated during the conduct of this study 
with a complete list provided below. In this context we define a SUAE as any SAE that 
occurs in the setting of MetHb > 10% which may thus be attributed to the study drug.  
 
Expected SAE (with severity grade indicated) 
• Non-central nervous system thrombosis, particularly in vessels accessed for endovascular 
devices, assessed as symptomatic venous or arterial thrombus documented 
radiographically or ultrasonographically.  (Grade 3) 
• Sepsis assessed as the presence of microbiologically proven, clinically proven, or 
suspected infection; presence of Systemic Inflammatory Response Syndrome (SIRS); and 
development of at least one organ dysfunction within the preceding 24 hours. Diagnosis 
of SIRS will require the fulfillment of at least two of the following criteria (Grade 4):  
o Hyperthermia (≥ 38°C) (which will only be evident after active temperature 
control);  
o Tachycardia (≥ 90 bpm);  
o Tachypnea (≥ 20 breaths/min) or an arterial pCO2 ≤ 4.3 kPa (32 mmHg) or 
mechanical ventilation;  
o White blood cell count ≥ 12,000/μL or ≤ 4,000/μL or left shift in differential 
white blood cell count ≥ 10%.  
• Expected organ dysfunctions after OHCA (which may accompany SIRS in sepsis as 
defined above or separately) will be defined as:  
o Acute encephalopathy not explained by psychotropic medication (or attributable 
to the cardiac arrest) [Grade 3];  
o Thrombocytopenia ≤100.000/μL or drop in platelet count > 30% within 24 hours 
not explained by hemorrhage (Grade 3);  
o Arterial hypoxemia with an arterial pO2 < 8 kPa (60 mmHg) when breathing 
room air or an oxygenation index (paO2/FiO2 ≤ 200 mmHg) not explained by 
presence of a pulmonary or cardiac disease (Grade 4);  
o Arterial hypotension with a systolic blood pressure ≤ 90 mmHg or mean arterial 
blood pressure ≤65 mmHg for at least one hour despite adequate fluid loading not 
explained by other causes of shock (Grade 4);  


32 
 
o Renal dysfunction with urine output ≤ 0.5 mL/kg/h for at least one hour despite 
adequate fluid loading or serum creatinine above 200 µmol/L (2.3 mg/dL) [Grade 
3];  
o Metabolic acidosis with base deficit ≥ 5.0 mmol/L or serum lactate ≥ 1.5 fold 
above local reference range (2.4 mmol/L at UPMC) [Grade 3]. 
• Pneumonitis assessed as the presence of air bronchograms on any chest x-ray within one 
week of the index cardiac arrest. (Grade 3) 
• Since many of these comatose OHCA subjects will receive therapeutic hypothermia, 
anticipated SAE include:  
o Electrolyte abnormalities: defined as the presence of any of the following from 
induction of hypothermia until 72 hours (all Grade 3):  
 Hypokalemia assessed as serum potassium concentration < 3.5 mmol/L. 
 Hyperglycemia assessed as serum glucose > 240 mg/dL. 
 Hypophosphatemia assessed as serum phosphate concentration < 0.8 
mmol/L. 
 Hypocalcemia assessed as serum ionized calcium < 2.2 mmol/L. 
o Bleeding (overt bleeding with Hb drop > 3 g/dL or transfusion; cardiac 
tamponade; bleeding requiring surgical intervention for control or intravenous 
vasoactive agents; intracranial hemorrhage; intraocular bleed compromising 
vision; operation for vascular closure for the purpose of controlling bleeding; fatal 
bleeding) [Grade 3] 
o Thrombosis (Grade 3) 
o Infection (Grade 3) 
o Fluid shifts defined as net fluid intake/output exceeding 3 L positive or negative 
in a 24h period (Grade 3) 
• Pulmonary Edema: We shall monitor the incidence of pulmonary edema in each 
treatment duration group and assess whether there is a significant difference between 
groups. It will be defined as the presence of congestive heart failure on a hospital-based 
chest x-ray (first emergency department or ICU chest x-ray).  OHCA is more common in 
patients with cardiomegaly, and this radiographic sign alone will not be a criterion for 
diagnosis of acute heart failure. (Grade 3) 
• Other common post-OHCA SAE (Grade 3 unless specified) include pneumonia, cerebral 
bleeding, stroke, seizures, bleeding requiring transfusion or surgical intervention, rearrest 
(Grade 5), rib fractures, sternal fractures, internal thoracic or abdominal injuries as well 
as any other major medical or surgical outcomes will be recorded as noted in the hospital 
discharge summary. 
 
6.4.6 Adverse Events Reporting Timeline 
 
All SUAE associated with the use of the study drug (ie in the setting of MetHb > 10% or discovered 
to be drug associated after unblinding by the safety monitor and considered potentially resulting 
from the study drug by the DSMB) will be reported to the IRB within 24 hours of discovery of the 
incident with subsequent follow-up submission of a detailed written report. 
 
The FDA will be notified by telephone or facsimile transmission of a human SUAE no later than 
7 calendar days after receiving the respective human adverse event information, followed by the 


33 
 
subsequent submission of a written IND Safety Report. 
 
Serious and unexpected adverse events associated with the use of the study drug or procedures will 
be reported to the IRB with subsequent follow-up submission of a detailed written report in 
accordance with the respective policies and procedures of the IRB. Written IND Safety Reports 
will be submitted to the FDA as soon as possible and, in no event, later than 15 calendar days 
following the investigator-sponsor’s receipt of the respective adverse event information. 
 
A summary report of the findings will be prepared and submitted to the regulatory agencies. 
 
 
6.4.7 Withdrawal of Subjects and Stopping Criteria: 
 
 
Subjects withdrawn from the study due to concern for SAE will be followed for all clinical 
outcomes but no further lab or imaging data will be collected. These subjects will not be replaced 
within the study. Subjects will not be withdrawn after enrollment except if requested by the 
subject or surrogate after notification. In this event all data and specimens required for study 
outcomes obtained up to that time and clinical outcomes to DC will be retained.  
 
Discontinuation of the Clinical Trial:  
This study can be stopped only by the DSMB.  In such a case we will complete collection of 
clinical outcomes in all enrolled subjects (up to 90 d after OHCA).  Subjects will not be notified 
of early study termination. 
 
6.5 RISKS MANAGEMENT PROCEDURES 
 
6.5.1 Protection Against Risks   
 
General Risks of Study Protocol and Procedures 
All research interventions/activities will be conducted in private patient care areas. The collection 
of sensitive information about subjects is limited to the amount necessary to achieve the aims of 
the research, so that no unneeded sensitive information is being collected. Specifically we will 
only obtain 4 elements of protected health information (PHI) which could identify the subjects.  
These elements are the subject name (first, middle initial, last), date of birth and UPMC medical 
record number. These data elements will be stored in a password protected Microsoft Excel 
spreadsheet which links the PHI to the subject identifier (ID).  The document will be stored on a 
UPMC cloud server drive accessible only to the principal investigator (PI) and data manager. This 
folder lies behind the UPMC firewall and hence has all security provisions attributable to the 
electronic medical record associated with it. 
 
Those data elements which will be obtained by direct observation or physical examination of 
subjects will initially be recorded on a Case Report Form (CRF) which will be linked to the subject 
using the subject ID only. The Investigator will review, approve and sign/date each completed 
CRF; the Investigator’s signature serving as attestation of the Investigator’s responsibility for 
ensuring that all clinical and laboratory data entered on the CRF are complete, accurate and 
authentic. These data elements will subsequently be transferred by a research assistant to the 


34 
 
electronic database described below. The completed CRF will be stored in a locked filing cabinet 
within the locked office of the PI. 
   
All staff will sign confidentiality statements. Access to the database will be limited to the data 
manager and staff under the supervision of the Investigator. 
 
Specimens will be only be referenced by subject ID as described above. These specimens will be 
stored safely in the custody of the Investigator responsible for the individual assays. The 
Investigators will limit future access to any remaining sample to only those investigators with 
prior IRB approval for their studies. 
 
All staff involved in this study are properly credentialed and instructed in the areas of testing, 
confidentiality, and safety.  
 
The Investigator will retain the data for the entire period of this study and will retain the 
specified records and reports for up to two years after the marketing application is approved for 
the investigational drug; or, if a marketing application is not submitted or approved for the 
investigational drug, until two years after investigations under the IND have been discontinued 
and the FDA so notified. The Investigator may continue to use and disclose subjects’ de-
identified information for the purpose of this study for a minimum of seven years after final 
reporting or publication of the study. If the subject and/or legal representative decide to withdraw 
or be withdrawn from study participation, they may request that the study data and samples be 
destroyed. Subject names or other directly identifiable information will not appear on any 
reports, publications, or other disclosures of clinical study outcomes. 
 
6.5.2 Protection Against Potential Risks of Experimental Intervention 
 
The study drug (iNO) and administration system (INOVent) are already in clinical use within the 
UPMC system in the ICUs where subjects will receive care.  Existing protocols for administration 
will be followed by trained RTs. The investigators are all qualified to perform the elements of data 
acquisition whether by exam, observation or phlebotomy as described below (8.1). All members 
of the study team will have received required training on protection of human research participants 
(CITI Good Clinical Practice Module) and all have prior clinical trial experience. During study 
drug administration the patient’s VS, need for vasopressor/inotropic medications, ventilator 
settings, fluid balance and labs will be reviewed by a research assistant present throughout 
administration who will report any adverse events to a physician member of the PCAS team (on 
call 24/7). The PCAS co-investigators are empowered to stop study drug based on their discretion 
as is the safety monitor who can be consulted if needed.  The safety monitor will review data on 
VS, need for vasopressor/inotropic medications, ventilator settings, fluid balance and labs at least 
every 4 weeks and data reviewed at least every 6 months by the DSMB. 
 
7.  COSTS AND PAYMENTS 
 
7.1 COSTS 
 


35 
 
No additional cost to patients or insurers for study drug or labs and data collected as part of this 
study. 
 
7.2 PAYMENTS 
 
No compensation for study participation. 
 
 
8.  QUALIFICATIONS AND SOURCE OF SUPPORT 
 
 
8.1 QUALIFICATIONS OF THE INVESTIGATORS 
 
Cameron Dezfulian, MD, is an Assistant Professor of Critical Care Medicine and Clinical and 
Translational Sciences. He is an experienced researcher in cardiac arrest heart and brain injury, 
post-resuscitation care, nitric oxide and free radical biology and mitochondrial function. He is a 
Post Cardiac Arrest Service Physician at UPMC Presbyterian-Shadyside and routinely cares for 
the patient population being studied in this project. He also has the scientific background and lab 
resources as part of the Safar Center for Resuscitation Research to perform the studies planned. 
 
Jon Rittenberger, MD, MS is an Assistant Professor of Emergency Medicine and an experienced 
researcher in acute neurological emergencies. He is a Post Cardiac Arrest Service Physician at 
UPMC Presbyterian-Shadyside and routinely cares for cardiac arrest patients as part of his 
clinical duties. 
 
Clifton W. Callaway, MD, PhD - Professor and Vice-Chair in Emergency Medicine, Consultant 
on Post-cardiac arrest care. Dr. Callaway has career focus on cardiac resuscitation, acute brain 
injury, and also cares for cardiac arrest patients as part of his normal clinical duties. 
 
Dr. Elmer, Dr. Guyette, Dr. Frisch, Dr. Weissman, Dr. Molyneaux, Dr. Shutter, Dr. Sawyer, Dr. 
Emlet and Dr. Doshi are all attending physicians in Emergency/Criticial Care Medicine. They 
routinely care for cardiac arrest patients as part of their clinical duties, and have taken part in 
several research projects within the department. 
 
Dr. Okubo is an emergency medicine physician who is presently performing a clinical and 
research fellowship in the area of cardiac arrest resuscitation and thus also has expertise with this 
patient population. 
 
Ms. Flickinger, MS, is an exercise physiologist and research specialist in the Applied Physiology 
Lab. Ms. Repine is also a research specialist in the Applied Physiology Lab. Both specialists 
have at least 3 years of experience in the human physiology lab and are trained in IV placement 
and obtaining blood draws. They both have experience in physiologic monitoring and 
thermoregulation. 
 
Sara DiFiore is the study coordinator for the Department of Emergency Medicine. She has 
several years experience in the regulatory field and has participated in many of the department's 


36 
 
clinical trials. 
 
Nick Krehel is a research assistant employed by Dr. Dezfulian. He previously worked in the 
laboratory of Dr. Dezfulian where he participated in several translational projects. He is adept at 
processing blood samples, a number of basic lab techniques (such as measure of NO metabolites 
being used in this study) and has been trained to conduct clinical research by Dr. Dezfulian. He 
will gather much of the data used in this study. 
 
Dylan Burbee is a new research assistant employed by Dr. Dezfulian. He previously worked in a 
University of Pittsburgh laboratory where some of his duties included processing blood samples 
and a number of basic lab techniques. He was trained to perform ECHO and LSCI as well as 
how to collect clinical data for this study and has become the administrator of the REDCap 
database. 
 
 
8.2 SOURCES OF SUPPORT 
 
Investigator initiated research award from Mallinckrodt Pharmaceuticals. 
 
 


37 
 
 
9.  REFERENCES 
1. 
Ballard PL, Truog WE, Merrill JD, et al. Plasma biomarkers of oxidative stress: 
relationship to lung disease and inhaled nitric oxide therapy in premature infants. 
Pediatrics. 2008;121(3):555-561. 
2. 
Dellinger RP, Trzeciak SW, Criner GJ, et al. Association between inhaled nitric oxide 
treatment and long-term pulmonary function in survivors of acute respiratory distress 
syndrome. Critical care (London, England). 2012;16(2):R36. 
3. 
Dellinger RP, Zimmerman JL, Taylor RW, et al. Effects of inhaled nitric oxide in patients 
with acute respiratory distress syndrome: results of a randomized phase II trial. Inhaled 
Nitric Oxide in ARDS Study Group. Critical care medicine. 1998;26(1):15-23. 
4. 
Lundin S, Mang H, Smithies M, Stenqvist O, Frostell C. Inhalation of nitric oxide in 
acute lung injury: results of a European multicentre study. The European Study Group of 
Inhaled Nitric Oxide. Intensive care medicine. 1999;25(9):911-919. 
5. 
Patrianakos-Hoobler AI, Marks JD, Msall ME, Huo D, Schreiber MD. Safety and 
efficacy of inhaled nitric oxide treatment for premature infants with respiratory distress 
syndrome: follow-up evaluation at early school age. Acta Paediatr. 2011;100(4):524-528. 
6. 
Walsh MC, Hibbs AM, Martin CR, et al. Two-year neurodevelopmental outcomes of 
ventilated preterm infants treated with inhaled nitric oxide. The Journal of pediatrics. 
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