Clinical trial NCT02151877
Conditions: Ischemia/Reperfusion Injury After Neonatal Cardiac Surgery Inflammatory Reaction After Neonatal Cardiac Surgery
Keywords: Ischemia/reperfusion injury
Sponsor: Wake Forest University Health Sciences
Start date: 2014-07-01; Completion date: 2018-08-15
Enrollment: 24 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Around 7500 neonates born yearly in the United States have complex congenital heart disease that require surgical repair in the first few days of life. The complexity of the surgical repair requires long periods of cardiopulmonary bypass (CPB) and the use of intermittent periods of low flow or complete circulatory arrest. The immature neonatal vital organs are more prone to the complications of the cardiopulmonary bypass circulation, namely ischemia/reperfusion (I/R) injury and systemic inflammatory response. Inhaled nitric oxide (NO) is used frequently in neonates for the treatment of pulmonary hypertension, Additionally, many studies have shown that NO has an anti-inflammatory effect by reducing I/R injury and endothelial dysfunction.

The purpose of this pilot study is to assess the efficacy of NO administration via the CPB circuit in attenuating the CPB induced I/R injury and systemic inflammatory reaction in neonates undergoing repair of complex congenital heart defects. Specific goals will be to demonstrate that NO use via CPB will:

* Decrease markers of I/R injury and systemic inflammatory response.
* Decrease platelet activation leading to reduced postoperative bleeding and transfusion requirements.
* Decrease postoperative organ dysfunction, and hence decrease operative mortality and postoperative morbidity.

Twelve neonates undergoing repair of complex congenital heart defects will receive NO via the CPB circuit, for the duration of surgery. They will be compared to a control group of 12 similar patients. Serum levels of different ischemic reperfusion injury and inflammatory markers will be measured at different time points after surgery and will be correlated with different end organ function tests and clinical course in the postoperative period. The results will be compared between the two groups to try to determine the clinical benefit of NO administration through CPB circuit.

Detailed description:

Arms: arm 1: neonates receiving inhaled NO into the cardiopulmonary bypass circuit during cardiac surgery arm 2: neonates not receiving inhaled NO into the cardiopulmonary bypass
Interventions: intervention 1: Inhaled Nitric Oxide intervention 2: placebo
Intervention descriptions: intervention 1: delivering inhaled Nitric Oxide into the cardiopulmonary bypass circuit during neonatal cardiac surgery intervention 2: inhaled Nitric Oxide not delivered to the cardiopulmonary bypass circuit during neonatal cardiac surgery

Protocol documents:
Prot_SAP_000.pdf: 
PROTOCOL TITLE: Efficacy of Nitric Oxide Administration in Attenuating Ischemia / 
Reperfusion Injury During Neonatal Cardiopulmonary Bypass 
Version3_6.3.16 
 
HRP-503 Template Protocol 
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Title: Efficacy of Nitric Oxide Administration in Attenuating Ischemia / Reperfusion 
Injury During Neonatal Cardiopulmonary Bypass 
 
NCT: 02151877 
 
 
Version 3_3Jun2016 


PROTOCOL TITLE: Efficacy of Nitric Oxide Administration in Attenuating Ischemia / 
Reperfusion Injury During Neonatal Cardiopulmonary Bypass 
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Protocol Title  
Efficacy of Nitric Oxide Administration in Attenuating Ischemia / Reperfusion 
Injury During Neonatal Cardiopulmonary Bypass 
1) 
IRB Review History 
N/A 
2) 
Objectives 
 
Hypothesis: The introduction of nitric oxide (NO) directly into the cardiopulmonary 
bypass circuit, during neonatal surgical repair of congenital heart lesions, will attenuate 
ischemia reperfusion injury that can complicate the surgical procedure and postoperative 
recovery. 
 
Specific Objectives: During the Norwood procedure for hypoplastic left heart syndrome 
(HLHS) or its variants, there is around 20 to 30 minutes of ischemia to the myocardium 
between cardioplegia doses, and 20 to 30 minutes of interruption of flow to the lower 
body during reconstruction of the aortic arch. The proposed study will help to know if 
NO can decrease ischemia reperfusion injury to the myocardium, as well as to the lower 
body organs (liver, kidney, gastro-intestinal tract). 
 
Specific Aims:   
1) Evaluate whether NO delivered through the neonatal CPB circuit can decrease the 
clinical signs of ischemia/reperfusion injury and/or cardiac dysfunction as 
reflected by fluid balances, inotropic support, diuretic requirement, length of 
ventilator support, length of ICU stay, and length of hospital stay. 
 
2) Evaluate whether NO delivered through the neonatal CPB circuit can decrease 
various biochemical markers of ischemia/reperfusion injury and oxidative damage 
[cardiac troponin I, interleukens (IL), tumor necrosis factor (TNF), N-terminal 
prohormone for brain natriuretic peptide (NT-proBNP),lactate dehydrogenase 
(LDH), plasma anti-oxidant levels, plasma malondialdehyde (MDA) levels].  
 
3) Evaluate the safety of NO delivered through the neonatal CPB circuit as measured 
by adverse events during the course of the study.  
 
3) 
Background 
 
Around 25,000-30,000 cardiac surgical operations are performed on pediatric patients 
each year, and around 7500 of them are performed on neonates. These operations are 
made possible by the use of cardiopulmonary bypass (CPB), which allows for circulatory 
and pulmonary support while the heart is being repaired. Although life-saving, the use of 
CPB is associated with a variety of adverse effects, such as systemic inflammatory 
responses, neurological insufficiencies, coagulopathies, and most importantly 
ischemia/reperfusion (I/R) injury. Adverse heart-related events after surgically induced 


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I/R continues to be a major contributor to the morbidity and mortality after pediatric 
cardiac surgery. With the absence of pulsatile flow of the cardiopulmonary bypass, the 
ischemia/reperfusion cellular injury is even more exaggerated in different vital organs, 
like the heart, lung, liver, and kidneys. This is exaggerated by episodes of circulatory 
arrest or low flow status necessitated by the complexity of the repair. This is also more 
prominent in neonates because of the complexity of the heart defects requiring longer 
time on cardiopulmonary bypass, and because of the immaturity of neonatal organ 
systems. According to the Society of Thoracic Surgeons database, most of the neonatal 
cardiac repairs belong to the Risk Adjustment for Congenital Heart Surgery (RACHS) 5 
and 6 complexity categories, with a national hospital mortality of around 20% (1). 
 
The morbidity associated with inadequate cardiac protection suggests that new strategies 
to enhance cardiac protection are needed. Recently, there has been much interest in the 
pharmacological conditioning of the heart to produce adequate protection during CPB-
associated surgically induced I/R.  More specifically, and at a cellular level, recent 
laboratory data suggests that certain agents, such as nitric oxide (NO), can protect cardiac 
cells against I/R injury by protecting the cardiac mitochondria. The mitochondria are the 
energy producing apparatus in human cells. Thus, by protecting the mitochondria, one 
can protect the cardiac cell and minimize the effects of surgically induced I/R injury 
during the repair of congenital heart defects.  
 
Ischemia/reperfusion injury and systemic inflammatory response syndrome are linked. 
After a period of ischemia, reperfusion stimulates the production of additional pro-
inflammatory mediators, in particular, reactive oxygen free radicals and cytokines by 
parenchymal, endothelial, platelet, and inflammatory cells, contributing to elevated serum 
levels of inflammatory mediators. Cell death results from coagulation necrosis and 
inflammatory mediator-induced apoptosis. Endothelial cell injury results in impairment 
of the NO-cyclic guanosine monophosphate signaling cascade and endothelial-dependent 
vasodilatation (2). Alternations in NO generation appear to underpin the interrelationship 
of endothelial function and inflammation (3). NO might play several protective roles in 
reperfusion injury (4). NO is an important regulator of apoptosis, capable of both 
inducing and inhibiting apoptosis (5). NO has been shown to inhibit platelet adhesion and 
aggregation (6) and is able to block monocyte adherence and migration (7). Furthermore, 
NO is an inhibitor of leukocyte activation, preventing neutrophil-endothelial adhesion 
and the generation of oxygen free radicals (8).   
 
The use of inhaled NO as a pulmonary vasodilator agent in neonates is well established.  
In addition to its potent vasodilatory effects in the pulmonary vasculature, inhaled NO 
has also been shown to have significant peripheral anti-inflammatory effects. The safety 
of perioperative administration of NO via the CPB circuit has been described in animals 
and human beings (9,10,11). Inhaled NO during adult cardiac surgery patients has been 
reported to reduce systemic inflammatory markers, attenuate endothelial dysfunction, and 
reduce ischemia/reperfusion injury (12,13,14). Administration of NO to adult patients 
undergoing cardiac surgery has been shown to blunt the release of markers of cardiac 
injury and decreases ventricular dysfunction during and immediately after CPB.  
 


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A more recent study in infants has also shown that the NO administration via the CPB 
circuit can reduce various markers of inflammation and reduce postoperative 
complications (15). To date, there have been no such studies relating NO administration 
via the CPB circuit and ischemia reperfusion injury in neonates undergoing surgical 
repair of congenital heart lesions. 
  
4) 
Inclusion and Exclusion Criteria 
 
Inclusion criteria: 
 
• Neonates, age 0-30 days 
• Full term, > 37 weeks gestation 
• Birth weight ≥ 2.6 kg 
 
Exclusion criteria: 
 
• Preoperative sepsis  
• Renal dysfunction, (preoperative creatinine greater than 1.0) 
• Intracranial hemorrhage (preoperative intraventricular hemorrhage more or equal 
to grade 1) 
• Known Chromosomal abnormalities and/or known genetic syndromes. (The 
Fluorescent in situ hybridization (FISH) test is routinely done on all neonates 
with cardiac disease. If the test results are not received before surgery, the 
subject will be withdrawn as indicated in protocol section 14.) 
• Prior cardiac intervention, catheter-based or surgical 
• RV wall and endocardial fibroelastosis (as determined by pre-operative 
echocardiography). 
 
6)    Study-Wide Number of Subjects 
We will enroll a total of 24 subjects with 12 subjects in the treatment group 
and 12 subjects in the control group.  
 
7) Study-Wide Recruitment Methods 
 
The study will be performed at ACH-OL. The pediatric cardiac surgery division 
performs approximately 400 surgeries each year at this site. Twenty-four neonates 
with HLHS or its variants requiring surgical repair in the first few days of life will be 
randomized into 2 groups. 
 
All newborns with diagnosis of HLHS or its variant, who undergo Norwood 
procedure and satisfy the inclusion criteria will be invited to participate in the study.  
Most of these patients are diagnosed prenatally by fetal echocardiography and are 
born at ACH-OL. Few patients with no prenatal diagnosis or who are born at an 
outside institution are transferred to ACH-OL for further management.  Parents will 


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be informed about the study by our team of Pediatric Cardiac surgeons when they see 
the child for a clinical preoperative exam. This exam typically occurs after the child is 
born and the diagnosis is confirmed by echocardiography, and before the scheduled 
surgical procedure.  
 
8) 
Study Timelines 
The estimated duration needed to recruit the 24 subjects for the study is 
around 2 years. Each subject will be enrolled in the study from the time of 
surgery (Norwood procedure) until discharge. After all the subjects have been 
enrolled and the data has been collected, we anticipate that 2 to 3 months to 
finish analyzing the data and write the manuscript.  
 
9) 
Study Endpoints 
 
The primary study endpoints are to evaluate whether NO delivered through the 
neonatal CPB circuit can decrease the clinical signs of ischemia/reperfusion injury 
and/or cardiac dysfunction. Clinical parameters include: 
o fluid balance 
o diuretics requirement 
o inotropic support 
o duration of mechanical ventilation (hours) 
o ICU length of stay (LOS) 
o Hospital LOS 
o End organ failure (measured by BUN, creatinine, and liver function tests) 
 
The secondary study endpoints are to evaluate whether NO delivered through the 
neonatal CPB circuit can decrease various biochemical markers of 
ischemia/reperfusion injury and oxidative damage. Markers to be analyzed will 
include: 
o cardiac troponin I  
o N-terminal prohormone for brain natriuretic peptide (NT-proBNP) 
o plasma malondialdehyde (MDA) levels  
o superoxide dismutase 
o interleukens (IL) 
o lactate dehydrogenase (LDH) 
o procalcitonin 
o tumor necrosis factor (TNF alpha) 
o C-reactive protein (CRP) 
o Creatine phosphokinase-MB (CPK-MB) 
 
The primary safety measure is the level of methemoglobin while NO is being 
administered during cardiopulmonary bypass. The level will be checked every 


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½ hour per standard clinical practice, and NO administration will be adjusted 
accordingly, to keep the methemoglobin level below 3%.  
 
10) Procedures Involved 
 
Design 
The study will employ a double-blind placebo-controlled randomized block design. 
 
Sample 
Twenty-four neonates with HLHS or its variants requiring surgical repair in the first few 
days of life will be randomized into 2 groups. 
 
Group 1 will include 12 subjects who will receive inhaled NO via the CPB circuit for the 
duration of surgery. Group 2 will include 12 control subjects. These controls will receive 
an identical NO via CPB set-up, but will not receive NO during surgery. The subjects in 
both groups will undergo the standard Norwood procedure with a central systemic to 
pulmonary shunt or right ventricle to pulmonary artery shunt (Sano shunt). The only 
difference between groups will be the administration of NO via CPB. 
 
Standard Intra-operative protocol: 
After induction of general anesthesia using opiates (Fentanyl) and muscle relaxant, 
appropriate arterial line and central venous line access will be obtained. Intra-operative 
monitoring of arterial blood pressure, central venous pressure, systemic oxygen 
saturation, cerebral and somatic (kidney) near-infrared spectroscopy, will be done 
throughout surgery.  
 
After adequate positioning, a regular median sternotomy incision will be done. The 
thymus gland is resected for better exposure. Under full heparinization (activated clotting 
time >300sec), CPB is instituted using an arterial cannula inserted in the ascending aorta 
(or ductus arteriosus in patients with HLHS), and a venous cannula inserted in the right 
atrium or 2 separate superior and inferior vena cava cannulas.  
 
Once on CPB, the patient’s temperature is cooled down to 28 degrees Celsius. Cooling 
and warming the patient are done with pH stat strategy, during which the carbon dioxide 
level is allowed to rise in the CPB circuit to maintain a normal temperature corrected pH. 
All patients receive inhaled NO through the endotracheal tube after weaning from CPB.   
 
Research procedures 
The subjects will be randomized by the research perfusionist to either groups of the 
study. Only the perfusionist will be aware of the randomization process and NO delivery. 
The surgeon will be blinded to group assignment. Group 1 (treatment group) will receive 
NO via the CPB for the duration of surgery. Once the patient is off CPB, NO 
administration via CPB will be discontinued and switched to endotracheal tube 
administration. Group 2 (control group) will be treated with a strategy identical to that in 
the treatment group, with the exception that they will not receive NO via CPB.  


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For subjects in Group 1, NO will be delivered using the Inovent Transport delivery 
device (Ikaria, Hampton, NJ) after calibrating the machine according to the 
manufacturer’s instruction. While setting up and priming the CPB circuit, the NO 
delivery tubing will be incorporated into the oxygenator inlet gas tubing using special 
reducing connectors. NO sampling will be performed by adding a luer lock connector to 
the inlet gas tubing proximal to the oxygenator and attaching the sampling line to it.  
 
Once CPB is initiated, NO delivery will be started at 40ppm as measured by the sampling 
line, and maintained at 40ppm during the procedure unless the serum methemoglobin 
level increases above 3%. Once the patient is off CPB, NO delivery will be discontinued, 
and the subject will be managed similar to controls. 
 
Post-surgical care 
After finishing surgery, all subjects will recover in the Pediatric Surgical Heart Unit 
(PSHU) under the direct care of the pediatric cardiac intensivist and the surgical team. 
Neither physician nor nurse involved in the post-operative care will be aware of the 
subject’s randomization assignment. 
 
Data collection (see schedule of events below) 
All patients will have the following pre-operative labs drawn before surgery as part of our 
standard of care: complete blood count (CBC), complete metabolic panel, coagulation 
profile, and blood type and cross match.  
 
On the day of surgery, the following research related markers will be drawn in the 
operating room after the patient is under anesthesia and after insertion of arterial and 
central venous access (Time 0): cardiac troponin I, NT pro-BNP, plasma (MDA) level, 
superoxide dismutase, IL-6, IL-8, lactate dehydrogenase (LDH), procalcitonin, TNF 
alpha, CRP and CPK-MB. These markers will be rechecked at the end of CPB and before 
modified ultrafiltration (Time 1), after modified ultrafiltration (Time 2), 12 hours (+/- one 
hour) after CPB (Time 3) and 24 hours (+/- one hour) after CPB (in the PSHU) (Time 4). 
The amount of blood needed for each research blood draw will be 2 ml for a total of 10 
ml of blood to be drawn for research purposes.  
 
All patients will have an echocardiogram, chest radiography and the following blood tests 
drawn in the PSHU on a daily basis that includes: complete blood count (CBC), complete 
metabolic panel with kidney function and liver function tests, and coagulation profile. 
These tests are conducted as part of our standard of care.  
 
All standard of care laboratory tests will be performed and billed in accordance with 
usual hospital procedures. All research only labs will be shipped in batch to Immunetech 
labs and processed according to the appropriate procedures. 
 
Clinical data that will be collected will include: CPB time and aortic cross clamp time, 
fluid requirements, diuretic requirement inotropic support, ventilator hours, need to leave 
sternum open, ICU LOS and total hospital LOS, transfusions, blood and coagulation 


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factors, total amount of chest tube drainage, post-operative complications like bleeding, 
infection or seizures, renal dysfunction, arrhythmias, and post-operative need for 
extracorporeal membrane oxygenation (ECMO) support.  
 
Schedule of Events 
 
 
Screening  
 
 
 
 
 
 
End 
of 
Study 
Post 
study 
follow-
up 
Visit description 
Preop 
Time  
0  
(Preop) 
Time 
1 
(end 
of 
CPB 
and 
before 
MUF) 
Time 
2 (end 
of 
CPB, 
after 
MUF) 
Time 
3  (12 
hrs +/- 
one 
hour 
 Post 
CPB) 
Time 
4 (24 
hrs +/- 
one 
hour 
Post 
CPB) 
Time 
5 
Day 2 
Postop 
Time 
6 
Day 3 
Postop 
Time 
7 
D/C 
date 
Time 
8 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Demographic 
information 
X 
 
 
 
 
 
 
 
 
 
Concomitant 
medications 
X 
 
 
 
 
 
 
 
X 
X 
Laboratory tests 
 
 
 
 
 
 
 
 
 
 
Cardiac Troponin I 
 
X 
X 
X 
X 
X 
 
 
 
 
NT pro-BNP 
 
X 
X 
X 
X 
X 
 
 
 
 
Plasma MDA  
 
X 
X 
X 
X 
X 
 
 
 
 
Superoxide 
Dismutase 
 
X 
X 
X 
X 
X 
 
 
 
 
IL-6 
 
X 
X 
X 
X 
X 
 
 
 
 
IL-8 
 
X 
X 
X 
X 
X 
 
 
 
 
LDH 
 
X 
X 
X 
X 
X 
 
 
 
 
Procalcitonin 
 
X 
X 
X 
X 
X 
 
 
 
 
TNF alpha 
 
X 
X 
X 
X 
X 
 
 
 
 
CRP 
 
X 
X 
X 
X 
X 
 
 
 
 
CPK-MB 
 
X 
X 
X 
X 
X 
 
 
 
 
BUN and Cr 
 
SOC 
 
 
SOC 
 
SOC 
SOC 
 
 
Liver function tests 
 
SOC 
 
 
SOC 
 
SOC 
SOC 
 
 
Diagnostic tests 
 
 
 
 
 
 
 
 
 
 
CXR 
SOC 
 
 
 
SOC 
SOC 
SOC 
SOC 
 
 
Echocardiogram 
SOC 
 
 
 
X 
SOC 
SOC 
 
SOC 
 
Clinical course 
 
 
 
 
 
 
 
 
 
 
Fluid balance 
 
 
 
 
X 
X 
X 
X 
 
 
Diuretic 
requirement 
 
 
 
 
X 
X 
X 
X 
 
 
Inotropic support 
 
 
 
 
X 
X 
X 
X 
 
 
Ventilator Hours 
 
 
 
 
X 
X 
X 
X 
 
 
ICU LOS 
 
 
 
 
 
 
 
 
X 
 
Hospital LOS 
 
 
 
 
 
 
 
 
X 
 


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Reperfusion Injury During Neonatal Cardiopulmonary Bypass 
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Safety Adverse 
Event Reporting 
 
 
X 
 
X 
X 
X 
X 
X 
X 
Complications 
 
 
 
 
 
 
 
 
X 
X 
Mortality 
 
 
 
 
 
 
 
 
X 
X 
SOC=standard of care, MUF = modified ultrafiltration 
 
11) Data and Specimen Banking 
The data will be collected and saved on a password-protected Excel data sheet 
which will be stored on the password-protected computer of principal 
investigator. 
 
12) Data Management 
Sample Size Estimation 
Sample size estimation was calculated using PASS 12 (15) based on the 
number of subjects needed to compare the difference in fluid balance as 
measured by the total amount of fluid administered in the first 24 and 48 
hours for the two groups. Power analysis was run based on published data 
that showed a difference 130 mL between similar group assignments. (14) 
A sample size of 12 achieves 83% power to detect a mean difference of 75 
mL with an estimated standard deviation of 60 mL and with a significance 
level (alpha) of 0.05 using a two sample t-test.  
Data Management 
Standard of care lab values and clinical indicators as identified in protocol 
Section 9 will be recorded in the electronic medical record (EMR) per 
routine hospital procedure. Lab values of markers being collected for 
research only will be received in a report directly from the labs. 
Investigators will extract study data from the EMR or lab results onto a 
source document for data collection and record into an electronic 
spreadsheet. 
Statistical Analysis Plan 
Data analysis will include measures of central tendency for all 
demographic and outcome variables. The primary endpoint of clinical 
indicators, including fluid balance, inotropic support, ventilator hours, and 
LOS will be compared between groups using a Repeated Measures 
ANOVA with post hoc comparisons, Student’s t-test or chi-square as 
appropriate. A p-value less than, or equal to, 0.05 will be regarded 
statistically significant. 
Secondary outcomes to be compared between groups include the 
biochemical markers. Analysis will be done using Student’s t-test and a p-
value less than, or equal to, 0.05 will be regarded statistically significant.  


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The other indicators, number of adverse events (as determined by 
methemoglobin) will also be described and reported as indicated in 
protocol Section 13. Differences in adverse event rates will be evaluated 
using descriptive and inferential statistics as appropriate. 
Data Storage 
All information gathered during this study will be kept confidential. 
Subjects will be assigned case numbers and data collection forms that 
including the patient name will only be seen by the research team. All data 
collection forms will be placed in a confidential folder within the principal 
investigator’s locked office. Tabulation of data will be stored in a 
password protected file within a password protected computer. The results 
of this study may be published in scientific journals or be presented at 
professional meetings, but no individuals will be identified. The collection 
and handling of data will be in complete accordance with the HIPPA 
regulations. The research-related records for this study may be inspected 
by a federal regulatory agency and the Institutional Review Board. 
Data collected for this study will be maintained for three years after the 
investigation is complete and the FDA is notified.  
13) Provisions to Monitor the Data to Ensure the Safety of Subjects 
MONITORING PLAN 
The individuals responsible for data safety and monitoring will be Dr. 
Elzein (PI). Quality control will include regular data verification and 
protocol compliance checks by Dr. Elzein. 
COLLECTION AND REPORTING OF SAEs AND AEs 
Throughout the study, Dr. Elzein and the study research coordinator will 
monitor study participants for adverse events.  Dr. Elzein will review all 
adverse events (AEs) and serious adverse events (SAEs) individually and in 
aggregate on a biweekly basis.  Dr. Elzein will report all AEs and SAEs to 
the Advocate Health Care IRB according to the AE reporting guidelines.  
For this study, the following standard AE definitions will be used: 
Adverse event:  Any unfavorable and unintended sign (including an 
abnormal laboratory finding), symptom or disease, that did not exist at the 
time of the subject’s entry into the study and that is temporally associated 
with the use of the study medication, regardless of whether it is considered 
related to the study medication.   
Suspected adverse reaction: A suspected adverse reaction means any 
adverse event for which there is a reasonable possibility that the drug caused 
the adverse event. For the purposes of IND safety reporting, ‘reasonable 
possibility’ means there is evidence to suggest a causal relationship between 
the drug and the adverse event. A suspected adverse reaction implies a lesser 
degree of certainty about causality than an adverse reaction. 


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Adverse Reaction: An adverse reaction means any adverse event caused by 
a drug. Adverse reactions are a subset of all suspected adverse reactions 
where there is reason to conclude that the drug caused the event.  
Unexpected Adverse Event:  An adverse event or suspected adverse 
reaction is considered “unexpected” if it is not consistent with the risk 
information described in this plan.    
The following list of medically important unexpected events is intended to 
serve as a guideline for determining which condition may be considered 
reportable. The list is not intended to be exhaustive: 
• Renal failure  
• Delay of chest closure (i.e. > 7 days) 
• Surgical wound from study related surgery necessitating 
debridement 
• Sepsis (i.e. confirmed by positive laboratory culture) 
• Delayed extubation (i.e. intubation period lasting > 3 weeks) 
 
Serious Adverse Event:  A significant hazard or side effect, regardless of 
the Investigator’s opinion on the relationship to treatment group.  SAEs will 
be recorded and reported using the INO Therapeutics/Ikaria SAE form (see 
Attachment A). A serious adverse event is any untoward medical occurrence 
that at any dose: 
• 
Results in death 
• 
Is life-threatening (defined in this case as an event in which the 
subject was at risk of death at the time of the event/reaction; it does 
not refer to an event/reaction which hypothetically might have 
caused death if it were more severe) 
• 
Requiring prolongation of existing hospitalization (i.e., > 4 weeks 
which is a typical hospitalization time for this population) 
• 
Results in persistent or significant disability/incapacity (e.g., stroke, 
seizure) 
• 
Required intervention to prevent permanent impairment/damage 
(e.g., re-intubation, ECMO, additional surgical intervention) 
AEs will be graded by the investigator according to the following scale:  
Mild:  An event that is easily tolerated by the subject, causing minimal 
discomfort and not interfering with everyday activities.  This includes 
transient laboratory test alterations. 


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Moderate:  An event that is sufficiently discomforting to interfere with 
normal everyday activities.  This includes laboratory test alterations 
indicating injury, but without long-term risk. 
Severe:  An event that prevents normal everyday activities. If 
prolongation of hospitalization is greater than 4 weeks, required for 
treatment it becomes an SAE.  (An AE that is severe should not be 
confused with a SAE.  Severity is a category utilized for rating the 
intensity of an event; and both AEs and SAEs can be assessed as mild, 
moderate or severe.) 
However, relating the intensity of clinical AEs to functional daily life and 
everyday activities may not be assessable in children in the PSHU.  
Therefore, if not possible to assess, the intensity of clinical AEs will be 
assessed by the investigator as per his/her best judgment. 
The study will use the following AE attribution scale:  
Unrelated:  The AE is clearly related to other factors such as the 
subject’s clinical state, therapeutic interventions, or concomitant drugs 
administered to the subject (i.e., another cause of the event is most 
plausible and/or a clinically plausible temporal sequence is inconsistent 
with the onset of the event).   
Unlikely related:  The event was most likely produced by other factors 
such as the subject’s clinical state, therapeutic interventions or 
concomitant drugs administered to the subject, and does not follow a 
known response pattern to the study drug. 
Possibly related:  The event follows a reasonable temporal sequence 
from the time of drug administration and/or follows a known response 
pattern to the study drug; but it could have been produced by other 
factors such as the subject’s clinical state, therapeutic interventions, or 
concomitant drugs administered to the subject. 
Probably related:  The event follows a reasonable temporal sequence 
from the time of drug administration and follows a known response 
pattern to the study drug; and it cannot be reasonably explained by other 
factors such as the subject’s clinical state, therapeutic interventions, or 
concomitant drugs administered to the subject. 
AEs will be monitored throughout the time that a subject is in the trial 
(hospital length of stay). AEs will be assessed through a review of the hospital 
chart on a weekly basis and with a physical examination of the subject, if 
indicated.   
REPORTING OF ADVERSE EVENTS  
Events determined by the PI to involve injury or to be unexpected problems involving 
risk and probably related to the study drug will be reported by the PI to the IRB 
within 5 days per HRP 214. Adverse events that are determined by the PI not to 


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involve injury or not a related risk of the use of NO during the Norwood surgery, will 
be reported per IRB policy at the time of continuing review.  
 
An IND safety report will be reported to the FDA by the PI when an event meet all 
three of the definitions for suspected adverse reaction, serious and unexpected. All 
FDA reporting will be consistent with 21 CFR 312.32 (c). For each reportable event, 
the information to be recorded in the source document includes the nature, date, 
intensity, duration, attribution (relationship to study drug), action taken and outcome 
of the event. 
• Unexpected serious suspected adverse reactions will be reported to FDA as 
soon as possible but no later than within 15 calendar days following the PI 
initial receipt of the information. 
• Unexpected fatal or life-threatening suspected adverse reactions will be 
reported to FDA as soon as possible but no later than 7 calendar days 
following the PI initial receipt of the information.  
 
 
DATA ANALYSIS PLANS AND INTERRIM REPORTS 
An independent Data Safety Monitoring Board (DSMB) will perform a 
statistical review of the study data and will have access to fully unblinded* 
patient data if necessary, to ensure patient safety. The DSMB will be 
comprised of 3 individuals who are not involved in the conduct of the study, 
representing pediatric cardiology or pediatric intensive care, perfusion and 
research. The DSMB will be empowered to recommend, based on safety 
considerations, modifications to the protocol or early termination of the study.  
The board will function independently of the investigators.  AEs and SAEs 
will be monitored by the DSMB in an unblinded* manner during the study 
after half of the subjects (N=12, 6 in each group) are enrolled. Subsequent 
safety reports will be completed after each set of 6 subjects. 
*A the randomization code will be kept strictly confidential.  It will be 
provided to the DSMB by the perfusionists (Vincenzo Rizzo, Cindy Urbas) to 
ensure patient safety. 
  
The interim analyses will be performed to evaluate differences in AE and SAE 
rates between the two treatment groups, and to evaluate the primary endpoint 
(fluid balance, morbidity and mortality). The DSMB will prepare a report 
after each review which will be given to the PI, and, in turn, this report will be 
submitted by Dr. Elzein to the IRB. 
The DSMB will be comprised of the following individuals:  
• 
Denise Angst, PhD, RN – Chair of DSMB, Director, Advocate Center for 
Pediatric Research  
• Christine Steffensen, PharmD, MA - Clinical Coordinator, Pediatrics 
and Pediatric Critical Care, Advocate Children’s Hospital-Oak Lawn 


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• Luis E. Torero, MD – Pediatric Pulmonologist, Torero Pulmonary 
Specialists, S.C. 
14) Withdrawal of Subjects 
The FISH test to test for chromosomal abnormality is done routinely on all 
neonates with cardiac disease. If the FISH test result is abnormal after the 
Norwood procedure is performed, indicating genetic syndromes, the subject 
will be withdrawn from the study. 
It may be possible that RV wall and endocardial fibroelastosis may not be 
evident on the pre-operative echo but that it would be identified during the 
operation. If this condition is identified during the operation, the subject will 
be withdrawn at that time, randomization will be unblended and the subject 
will be treated as if they are not in the study (e.g. will not have extra study 
blood drawn). 
At any time, the attending physician can withdraw a patient from this study if 
they feel it is warranted based on clinical condition.  Parents will be notified 
by the attending physician. The parents have the right to withdraw their child 
from the study at any time. If the parent decides to withdraw their child from 
the study, they will be asked if medical record collection can continue until 
the end of hospitalization. Documentation of the conversation and their 
decision will be entered into the research chart and medical record.  
 
15) Risks to Subjects 
We have been using inhaled NO during and after surgery at our center for 
over 8 years. The direct risk of using NO in the CPB circuit during surgery is 
related to elevated methemoglobin serum level. An elevated methemoglobin 
level is usually treated effectively by decreasing the rate of NO 
administration. All surgical staff and team members are familiar with NO use.  
NO administration via the cardiopulmonary bypass circuit will be monitored 
by checking frequent methemoglobin levels. Per our standard practice, this 
level is checked with every arterial blood gas which is usually performed 
every 30 minutes while the patient is on cardiopulmonary bypass. If the 
methemoglobin level increases to 3%, NO administration will be decreased 
until methemoglobin falls below this level. No other research-related risks are 
anticipated.  
After the surgery, subjects will be monitored for any adverse events 
throughout the course of their hospitalization as outlined in safety monitoring 
plan protocol (Section 13). Appropriate medical care will be provided for any 
adverse events. 
 


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16) Potential Benefits to Subjects 
There are no guaranteed benefits to any subjects in this study.  The results of 
this study will be used to enhance our understanding of the effectiveness of 
NO to potentially decrease ischemia reperfusion injury that can complicate the 
surgical procedure and postoperative recovery and may benefit future patients. 
Potential benefits to the subject could include decreased ischemia/reperfusion 
injury and systemic inflammatory reaction that is commonly seen after 
neonatal cardiac surgery. This will be reflected by improved post-operative 
myocardial function and preserved end organ function (kidney, lung, liver), 
shorter intensive care unit and hospital stay and improved mortality and 
morbidity.  
 
17) Vulnerable Populations 
 
This research involves children as a vulnerable population.  Informed 
consent/parental permission will be assured by offering parents multiple opportunities 
to ask and have their questions answered about the study before enrollment and 
throughout the study time period. Informed consent/parental permission 
documentation will be completed as discussed in Section 30. Subjects will be too 
young to provide assent as they will all be neonates.  
 
18) Multi-Site Research 
The study will be performed exclusively at ACH-OL. 
 
19) Community-Based Participatory Research 
Not applicable 
 
20) Sharing of Results with Subjects 
The results of some tests routinely performed as standard of care (including 
lab values) may be shared with the family by the physician involved in the 
care. No further sharing of research-only procedures or data will be provided 
to subjects or their families.  
 
21) Setting 
The research will be conducted at ACH-OL with the research interaction 
taking place in the operating room and post-surgical data collection occurring 
while subjects are cared for in the PSHU. 
 


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22) Resources Available 
The Norwood procedure has been performed at our institution for over 20 
years. Our operative mortality varies between 5 and 10%; the national 
operative mortality for the Norwood procedure is around 16%. In the past, we 
performed between 20 and 30 Norwood procedure per year. During 2013, we 
performed around 15 procedures with zero mortality. Our estimated period to 
recruit the 24 subjects for the study is 2 years (taking into consideration 
parents who decline participation). 
Dr. Michel Ilbawi and I (Chawki Elzein) are the surgeons who will be 
performing these surgeries. Dr. Ilbawi has over 20 years experience and Dr. 
Elzein has approximately 10 years’ experience with the Norwood procedure 
and the postoperative care of patients. The PSHU is covered 24 hours a day, 
seven days a week, with a team of experienced pediatric cardiac intensivists 
and experienced nursing staff. Dr. Andrew VanBergen, chief of cardiac 
critical care, has approximately 7 years’ experience with postoperative care of 
Norwood procedure patients. The perfusionists run the cardiopulmonary 
bypass machine during the Norwood procedure. They have more than 10 
years experience with extracorporeal circulation during complex pediatric 
cardiac operations. There is a back-up ECMO (extracorporeal membrane 
oxygenation) team available in case of sudden postoperative cardiorespiratory 
decompensation.  In addition, Vivian Cui and Robert Metzger, Pediatric Echo 
cardiographers will be performing the echocardiography on the subjects.   
 
 
23) Prior Approvals 
Not applicable 
 
24) Recruitment Methods 
 
See protocol Section 7.   
 
25) Local Number of Subjects 
See protocol Section 6. 
 
26) Confidentiality 
All information gathered during this study will be kept confidential.  Data collection 
forms and the database that include the patient name and other PHI will be seen only 
by the principal investigator and research team. All information and data gathered in 
this study will be placed in a password protected computer with restricted access by 


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only the investigative team. The results of this study may be published in scientific 
journals or be presented at professional meetings, but no individuals will be 
identified. The research-related records for this study may be inspected by a federal 
regulatory agency and the Institutional Review Board.   
 
Data will be stored on Advocate’s secure computer network which will only be 
accessible by the study investigators. 
 
27) Provisions to Protect the Privacy Interests of Subjects 
See protocol Section 12 and 26. 
 
28) Compensation for Research-Related Injury 
No compensation will be provided for research-related injury. No funds have 
been set aside by Advocate Health Care as compensation for injury or any 
associated costs.  
 
29) Economic Burden to Subjects 
The patient is not responsible for any extra costs related to the research. The 
cost of NO used in CPB during surgery, and the cost of the serum 
ischemia/reperfusion and inflammatory markers, will be covered by IKARIA 
Inc.  
 
30) Consent Process 
Written parental permission/informed consent will be obtained from each child’s 
parent or legal guardian prior to surgery (See Attachment B- Parental Permission 
form and Attachment C – HIPPA Authorization Form).  We will follow procedures 
outlined in SOP HRP-090 to obtain informed consent/parental permission. 
The neonate remains in the hospital from birth until the Norwood procedure is 
performed.  Parents will be informed about the study by our team of Pediatric Cardiac 
surgeons when they see the child for a clinical preoperative exam.  This exam 
typically occurs after the child is born and the diagnosis is confirmed by 
echocardiography, and before the scheduled surgical procedure.  
The informed consent/parental permission process will take place at the same 
time as obtaining consent for surgery. The consent for surgery is usually 
obtained 1-2 days before surgery date. Only the study investigators (Chawki 
Elzein, Andrew VanBergen) will be involved in obtaining the research 
consent.  Approximately 15 to 30 minutes will be assigned for discussing the 
research study and potential related risks to the subject.  The person obtaining 
the research consent will clearly distinguish its difference from the surgical 


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consent.  If the parents want to think about the research study, they will have 
ample time to decide whether to enroll or not.  All questions will be answered 
prior to obtaining consent.   
Non-English Speaking Subjects 
There is a potential that a neonate with Spanish speaking parents will be 
invited to participate in this study. In this case, the Spanish short form will be 
used for consent with the English consent presented orally in Spanish with the 
assistance of an interpreter (we have computer Skype based interpreters for 
over 10 languages are available at ACH-OL). In addition, one of the sub-
investigators, Andrew VanBergen, MD, speaks fluent Spanish and may be 
available to present the study to any Spanish speaking patients. It is 
anticipated that this process may be used for 1-2 patients only. 
Subjects who are not yet adults (infants, children, teenagers) 
All patients are neonates with HLHS or its variants. The Norwood procedure 
is usually performed at 4 to 7 days old. The consent form will be obtained 
from at least one parent before any study procedures are performed, though 
both parents will be encouraged to discuss the child’s participation. The 
mother will have to sign the consent form if the parents are not married. If 
there are social issues and DCFS is involved in consenting for the patient, the 
patient will not be enrolled. 
31) Process to Document Consent in Writing 
We will be following procedures outlined in SOP HRP-091. The consent for 
the research study will be documented together with the consent for surgery as 
a note in the medical record of the patient.  
 
32) Drugs or Devices 
Inhaled NO is already available and has been used at ACH-OL for over 7 
years. This study will be the first time that NO will be administered via the 
cardiopulmonary bypass circuit at our institution but it has been used as part 
of the cardiopulmonary bypass circuit in children at another institution. (14) 
An investigator-initiated IND application was submitted to the FDA. 
Registration at clinicaltrials.gov has been submitted and is under review.   
 
33) References 
1- Society of Thoracic Surgery Database. 
2- Cooper WA, Duarte IG, Thourami VH, Nakamura M, Wang NP, Brown 
WM III, et al. Hypothermic circulatory arrest causes multisystem vascular 
endothelial dysfunction and apoptosis. Ann Thorac Surg, 2000;69:696-703. 


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3- Chello M, Mastroroberto P, Perticone F, Celi V, Colonna A. Nitric oxide 
modulation if neutrophil-endothelium interaction: difference between arterial 
and venous coronary bypass graft. J Am Coll Cardiol. 1998;31:823-6. 
4- Gianetti J, Del Sarto P, Bevilacqua S, Vassalle C, De Filippis R, Kacila M, 
et al. Supplemental nitric oxide and its effect on myocardial injury and 
function in patients undergoing cardiac surgery with extracorporeal 
circulation. J Thrac Cardiovasc Surg. 2004;127:44-50. 
5- Uchiyama T, Otani H, Okada T, Ninomiya H, Kido M, Imamura H, et al. 
Nitric oxide indices caspase-dependent apoptosis and necrosis in neonatal rat 
cardiomyocytes. J Mol Cell Cardiol. 2002;34:1049-61. 
6- Sawicki G, SalasE, Murat J, Miszta-Lane H, Radomski MW Release of 
gelatinase a during platelet activation mediates aggregation.. Nature. 
1997;386:616-9. 
7- Comini L, Bachetti T, Agnoletti L, Gaia G, Curello S, Milanesi B, et al. 
Induction of functional inducible nitric oxide synthase in monocytes of 
patients with congestive heart failure: links with tumor necrosis factor –alpha. 
Eur Heart J. 1999;20:1503-13. 
8- Van Dervort AL, Yan L, Madara PJ, Cobb JP, Wesley RA, Corriveau CC, 
et al. Nitric oxide regulated endotoxin-induced TNF-alpha production by 
human neutrophils. J Immunol. 1994;152:4102-9.  
9- Palatianos GM, Paziouros K, Vassili MI. Effects of exogenous nitric oxide 
during cardiopulmonary bypass on lung postperfusion histology. ASAIO J, 
2005 Jul-Aug:51(4):398-403. 
10- Zhou Jing, Wu Xiao-dong, Lin Ke. Blood Hibernation: a novel strategy to 
inhibit systemic inflammation and coagulation induced by cardiopulmonary 
bypass. Chinese Medical Journal 2010; 123(13):1741-1747. 
11- Lowson SM, Hassan HM, Rich GF The effect of nitric oxide on platelets 
when delivered to the cardiopulmonary bypass circuit.. Anesthesia and 
Analgesia 1999 Dec; 89(6):1360-5. 
12- Vinten-Johansen J, Sato H, Zhao ZQ. The role of nitric oxide and NO-
donor agents in myocardial protection from surgical ischemic-reperfusion 
injury. Int Jour Cardiology 1995 July; 50(3): 273-81.  
13- Lefer AM. Attenuation of myocardial ischemia-reperfusion injury with 
nitric oxide replacement therapy. Ann Thor Surg 1995 Sept; 60(3):847-51. 
14- Checchia PA, Bronicki RA, Muenzer JT, Dixon D.Nitric oxide delivery 
during cardiopulmonary bypass reduces postoperative morbidity in children- a 
randomized trial. J Thorac cardiovasc Surg 2013 Sept;146(3): 530-6. 
15- Hintze, J. PASS 12. Kaysville, Utah: NCSS, LLC; 2013. 
 
Attachments 


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Attachment A – INO Therapeutics / Ikaria Serious Adverse Event Form 
Attachment B- Parental Permission form 
Attachment C – HIPPA Authorization Form 
Attachment D – Eligibility Checklist 
Attachment E – Data Collection Form 
Attachment F – FDA letter of receipt