Inhaled Nitric Oxide and Neuroprotection in Premature Infants
Phase: PHASE2
Conditions: Prematurity, Bronchopulmonary Dysplasia, Intraventricular Hemorrhage, Periventricular Leukomalacia
Sponsor: University of Chicago

 
 
 
 
 
Cover Page for Protocol 
Study Title: 
Inhaled Nitric Oxide and Neuroprotection in Premature Infants 
NCT Number:  
NCT00515281 
Document Date: 
10May2017 
 

Principal Investigator:    Schreiber, Michael D. 
 
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SPECIFIC AIMS 
 
With the advances in modern neonatal intensive care medicine in the last 2o years, survival of extremely 
preterm infants weighing less than 1500g (<3 lbs, 5 oz) has risen markedly. However, with this increased 
survival has come a marked increase in the number of infants with serious neurodevelopmental disabilities: 
Premature infants with birth weights less than 1500g who survive to go home are at significant risk for serious 
neurodevelopmental problems: cognitive and motor delays, blindness, deafness, and cerebral palsy. In a recent 
randomized, placebo-controlled clinical trial, we assessed whether giving mechanically ventilated preterm 
infants inhaled nitric oxide gas (iNO) for 1 week after birth decreased the incidence of death and chronic lung 
disease. An unanticipated outcome of that study17 and a subsequent study of those infants at 2 year of age14 was 
that premature infants treated with inhaled nitric oxide (iNO) have improved neurodevelopmental outcomes 
and physical growth at 2 years corrected age, compared with placebo-treated infants14. INO therapy, therefore, 
appears to be a new treatment to protect the premature brain during development outside the womb. The 
overall goal of this application is understand the efficacy of iNO treatment in improving 
neurodevelopmental outcomes in at-risk premature infants.  
 
The improvement in neurodevelopmental outcome we reported was primarily due to a decrease in the risk 
of cognitive impairment14, measured with the Bayley scales of Infant Development2, a standardized tool for 
assessing neurodevelopmental outcome in this age group. As mentioned above, the initial hypothesis of the 
study was that iNO therapy would decrease the incidence of death and chronic lung disease. However, we 
found that the beneficial effect of iNO therapy on neurodevelopment was independent of important clinical 
factors known to affect neurodevelopmental outcome, including whether infants had chronic lung disease. 
Furthermore, the magnitude of improvement in neurodevelopmental outcome persisted after statistical 
adjustment for the presence and absence of intraventricular brain hemorrhage or periventricular leukomalacia 
(PVL, a softening of the white matter of the brain, due to reduced blood flow). Therefore, iNO therapy appears 
to be directly neuroprotective in ventilated, preterm infants. The number of children in each group having 
cerebral palsy, motor delay, blindness, or deafness did not differ between iNO- and placebo-treated infants. 
 
Advances in neonatal intensive care medicine notwithstanding, we have not successfully developed 
neuroprotective therapies for these at risk preterm infants; iNO therapy, therefore, constitutes the first 

Principal Investigator:    Schreiber, Michael D. 
 
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intervention to allow the preterm brain to develop normally outside the womb. In our previous study, we 
treated babies for one week regardless of their level of prematurity. However, we do not know what duration of 
treatment will result in maximum neuroprotection, and will give the best neurodevelopmental outcome to the 
most infants. It is likely that maximal benefit of iNO therapy will occur with longer treatment, throughout the 
preterm period. Our goal, therefore, is to determine whether iNO therapy given during throughout the 
preterm period results in further improved neurodevelopmental outcomes, and to understand the biological 
relationships between iNO treatment and neurodevelopmental outcome. To achieve this goal, we propose 
performing a randomized, placebo-controlled, double-blind study of the effect of iNO therapy with gestational 
age-determined treatment length on neurodevelopmental and growth outcomes of preterm infants. Our 
Specific Aims are: 
 
Specific Aim #1. Determine the effectiveness of iNO therapy on improving 
neurodevelopmental outcome in all O2-dependent premature infants having birth weights < 
1500g. We hypothesize that iNO therapy will improve neurodevelopmental outcome and growth at 18 
months postconceptional age compared with placebo treated infants. In a double-blind, placebo-controlled 
clinical trial, premature infants being cared for in the Intensive Care Nursery at the University of Chicago 
Medical Center requiring any level of respiratory support will be randomized to receive iNO or placebo gas. 
Treatment will continue until infants either no longer require supplemental O2 therapy or reach 33 weeks 
gestational age, whichever comes first. At 18 months postconceptional age, infants will have neurodevelopment 
assessed and secondary clinical outcomes measured. The relative risks of abnormal neurodevelopmental 
outcome in the two groups will be compared. The roles played by variables known to independently affect 
neurodevelopmental outcome, as well as variables known to mediate abnormal neurodevelopmental outcomes 
will be statistically assessed. 
 
Specific Aim #2. Assess the mechanisms by which iNO therapy alters neurodevelopmental 
outcome in preterm infants. We hypothesize that the amount of nitric oxide delivery to tissues will be 
positively correlated with neurodevelopmental outcome. Using routinely collected arterial and venous blood 
from infants in this study, measurements of nitrosylated hemoglobin and nitrite will be made. Arterial-venous 
differences will be calculated and mean differences compared across treatment groups. Arterial venous 

Principal Investigator:    Schreiber, Michael D. 
 
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differences will be regressed against neurodevelopmental outcome. Plasma levels of cyclic guanosine 
monophosphate (cGMP), the intracellular second messenger induced by nitric oxide will also be measured and 
levels regressed against neurodevelopmental outcome. 
 
BACKGROUND 
1. Neurodevelopmental outcomes of premature infants 
 
Improved survival of premature infants has generated intense interest in the quality of life of the survivors6, 
10, 20-23.   Premature infants have under-developed lungs, brains, digestive tracts, and immune systems. 
Neurological outcomes for these infants include brain injury (intraventricular hemorrhage, periventricular 
leukomalacia), blindness, deafness, chronic lung disease, developmental and motor delays and cerebral palsy. 
The incidence of each of these adverse outcomes increases as infants are born more prematurely. Complicating 
factors, such as respiratory distress syndrome also has a negative impact on these outcomes. Lower 
socioeconomic status (SES) also has a marked negative impact, with the risk of abnormal neurodevelopmental 
outcomes being significantly increased in families with lower SES13.   
 
2. Prior interventions to improve neurodevelopmental outcome 
 
The need for effective interventions to improve neurodevelopmental outcomes for these infants has been a 
topic of concern for a long time. Randomized trials of indomethacin for closure of the ductus arteriosus was 
found to decrease the most severe form of bleeding in the brain (intraventricular hemorrhage, Grade IV)11, 12, 
but a subsequent large randomized clinical trial was able to detect no differences in survival without 
neurosensory disability at 18 months corrected age16. Similarly, a number of trials assessed whether maternal 
Phenobarbital administration could decrease brain bleeding in very preterm infants < 1000g. While initial 
reports seemed positive, more modern trials have failed to show differences, either in the rate of severe brain 
bleeding or in the incidence of adverse neurodevelopmental outcome3.  
 
3. How does iNO therapy affect neurodevelopmental outcome in preterm infants? 
 
In our previous single center, double-blind, randomized, placebo controlled study of 207 infants, we 

Principal Investigator:    Schreiber, Michael D. 
 
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demonstrated that iNO decreases the rate abnormal neurodevelopmental outcome in infants with birth weights 
<2000g receiving ventilator therapy for RDS. Our population group was predominantly African-American of 
low socioeconomic status. The original intent of this study was to assess role of iNO in decreasing death and 
chronic lung disease (a complication of prematurity and the need for mechanical ventilation). Nitric oxide was 
given at the dosage rate of 10 parts per million (ppm) for 12-24 hrs, then 5 ppm for up to 7 days, or until 
extubation. Infants were also randomized to receive either conventional ventilation or high frequency 
oscillatory ventilation (HFOV), a popular ventilatory approach believed by some to decrease chronic lung 
disease. We found no differences in the incidence of chronic lung disease and death between the groups 
receiving one of the two types of ventilator therapy. Our study concluded that nitric oxide in premature infants 
with respiratory distress syndrome decreased the incidence of chronic lung disease and death, consistent with 
our primary hypothesis. We also found that infants treated with iNO also had a markedly decreased incidence 
of the primary neurological complications of prematurity – serious bleeding in the brain (intraventricular 
hemorrhage) and strokes in the white matter of the brain (periventricular leukomalacia or PVL). 
 
We then followed these infants after discharge for two years. We were able to maintain contact with 82% of 
those in the original study, a very high follow up rate for this population. In the follow-up study we found that 
iNO treated infants had decreased abnormal neurodevelopmental outcomes compared with placebo-treated 
infants. That beneficial effect persisted after statistical adjustment of the outcome for the presence of 
independent factors known to affect neurodevelopmental outcome:  birth weight, gender, exposure to postnatal 
steroids, as well as factors that mediate abnormal neurodevelopmental outcome – intraventricular 
hemorrhage/PVL and incidence of chronic lung disease. We were able to conclude, therefore, that iNO has a 
beneficial, independent effect on neurodevelopmental outcome. We also found that iNO-treated infants grew 
better. They were only 0.5 standard deviations below the mean of full-term children, compared with more than 
1 standard deviation for placebo-treated children. 
 
There have been two other large placebo-controlled, randomized, double blind studies of iNO therapy in 
premature infants. In a multi-center trial, Kinsella and colleagues at the University of Colorado8 randomized 
793 infants at 34 weeks gestation or less requiring mechanical ventilation to receive 5 ppm of iNO for 3 weeks. 
They reported no difference in BPD in the overall cohort. However, infants weighing 1000-1250g who received 

Principal Investigator:    Schreiber, Michael D. 
 
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iNO had a significantly reduced incidence of chronic lung disease compared with the placebo-treated group. 
Most excitingly, for the entire cohort, the incidence of the combined end point of IVH, PVL and 
ventriculomegaly (all signs of brain injury) was significantly reduced in iNO treated group over the placebo-
treated group. This study provides support for our single center study that iNO therapy reduces overall risk of 
brain injury. It is also of importance to note that iNO was given for periods up to three times as long as our 
study, and without any adverse outcome. 
 
Ballard et al1 from the Children’s Hospital of Philadelphia reported on their randomized double blind 
placebo-controlled, multi-center study of 582 infants weighing < 1250g who were on mechanical ventilation or 
continuous positive airway pressure (CPAP).  In that study iNO was started later, at between 7 and 21 days of 
life. In contrast to our study, iNO was begun at a higher dose (20 ppm), then weaned first at 96 hrs of 
treatment and then weekly. Patients were treated for a minimum of 24 days. The primary outcome was survival 
without chronic lung disease. iNO was shown to decrease the incidence of survival without BPD. However, 
neurodevelopmental outcomes were not evaluated. However, this study confirms our initial study’s findings 
that iNO decreases BPD. Taken together these two studies provide confirming evidence that, in preterm 
infants, iNO therapy decreases BPD and brain injury. 
 
4. What are the mechanisms by which iNO therapy alters neurodevelopmental outcome in 
preterm infants? 
 
In studies of brain injury in animals, nitric oxide (NO) has been associated with neuroprotection, mostly 
likely through actions on the blood vessels of the brain. After inhalation, iNO crosses the alveoli (air sacs) of the 
lung and enters the circulation, where it is bound to hemoglobin. It had been believed that NO bound to 
hemoglobin was inactivated. How, then could inhaled NO be delivered to target tissues, such as the brain? New 
data indicate that hemoglobin may act as a delivery system for NO to target tissues4, 5, 24.  Neuroprotection may 
be due to actions of NO on blood vessels in the brain, with its ability to increase blood flow to injured areas of 
the brain. 
 
In the laboratory component of this study, we propose to measure NO metabolites in the blood of our study 
babies treated with iNO or placebo. Recent data now suggest that NO, both administered as well as naturally 

Principal Investigator:    Schreiber, Michael D. 
 
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produced, is transported in blood to target tissues, where it can increase local blood flow. This has been 
documented in humans, where inhalation of NO was found to increase blood flow in the arm of exercising 
volunteers5. Furthermore, NO has been shown to be carried on the hemoglobin molecule in red blood cells – 
the hemoglobin becomes nitrosylated, carrying an NO molecule4. Thus, iNO markedly elevates levels of total 
nitrosylated hemoglobin in human volunteers24. Most importantly, there is a marked difference in the amount 
of nitrosylated hemoglobin between the arterial circulation carried to the tissues, and the venous circulation 
carried away from the tissues4. This difference indicates that inhaled NO delivered in the arterial blood is being 
removed from hemoglobin in the tissues, consistent with its effect in organ capillary beds. In addition to 
nitrosylating hemoglobin, plasma nitrite also appears to be a source of bio-available NO. Nitrite, generated 
from the reaction of NO with oxygen, can be converted back to NO by means of naturally occurring enzymes in 
the body. In fact, similar to levels of nitrosylated hemoglobin, large differences in arterial and venous nitrite 
levels have been documented in humans, and has been shown to be bioactive5. 
 
In the laboratory component of this study, we propose to measure NO metabolites in blood in children 
treated with iNO or placebo, and calculate the NO delivery to tissues using arterial and venous measurements. 
We will describe the time course of these metabolites during the course of iNO therapy, and correlate the level 
of NO metabolites with neurodevelopmental outcome. 
 
SIGNIFICANCE 
Despite much research into potential neuroprotective treatments for premature infants, the risk of abnormal 
neurodevelopmental outcome persists. Concurrently, the number of surviving preterm infants is rising because 
of advances in neonatal intensive care medicine. Inhaled nitric oxide is the only treatment shown to improve 
neurodevelopmental outcome in preterm infants. The results from the proposed study will establish whether 
iNO is effective in improving neurodevelopmental outcome for the vast majority of premature infants, i.e. those 
infants needing some respiratory support. If our hypothesis is upheld, iNO it has the potential to markedly 
impact the quality of life for these babies and their families. 
 

Principal Investigator:    Schreiber, Michael D. 
 
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RESEARCH PLAN 
Overall Experimental Plan:  The study is a single-site, randomized, double-blind, placebo-controlled trial 
of inhaled nitric oxide therapy in all preterm infants with any respiratory disease who weigh <1500g at birth. 
The trial is designed to treat these infants with nitric oxide from birth to 33 weeks gestation – a critical period 
of brain development in preterm infants. Our primary objective is to compare neurodevelopmental outcomes 
between our iNO and placebo-treated patients. We also wish to study the mechanisms underlying the 
protective effect of iNO, by assessing the relationship between nitric oxide delivery to tissues and 
neurodevelopmental outcome.  
 
The major differences between this study and our previous study are:  
 
1) Inclusion of all preterm infants requiring at least supplemental oxygen therapy. Approximately half of all 
infants weighing <1500 at birth do not require mechanical ventilation with a breathing machine, which was 
one criterion for inclusion in our previous study. However, these infants are still at risks for abnormal 
neurodevelopmental outcomes. By including all infants requiring at least supplemental oxygen by nasal 
cannula, we hope to obtain information regarding the potential beneficial effects of iNO applicable to the 
majority of at-risk preterm infants. 
 
2) Gestational age-dependent duration of iNO therapy. In our previous study, infants were treated with iNO 
or placebo for 7 days (if still on mechanical ventilation). Consequently, infants received iNO therapy during 
different periods of brain development: for example, infants of 24 weeks gestation (16 weeks early) received 
iNO during a period during brain cells were being rapidly produced, while infants of 3o week’s gestation 
received iNO after brain cells had been made and were now forming connections. In this study, our goal is to 
provide iNO during the entire time the premature brain is developing outside the womb. Therefore, infants will 
be treated from birth until 33 weeks gestation, so that less mature infants will be treated for a longer time. 
 
3) Specific design of the study to assess neurodevelopmental outcome. Our previous study had been 
designed to assess the effect of iNO treatment on the incidence of chronic lung disease and death; we assessed 
these infants’ neurodevelopmental outcomes because of our unanticipated finding of decreased brain bleeding 
and softening. This study has as its primary outcome the incidence of abnormal neurodevelopmental outcome. 
 
4) Laboratory-based Specific Aim to assess the mechanisms of the iNO-induced neuroprotection. 

Principal Investigator:    Schreiber, Michael D. 
 
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Specific Aim #1. Assess the role of iNO therapy on neurodevelopmental outcome in all oxygen-
dependent premature infants having birth weights < 1500g. 
(i) Rationale. We have previously found that brief (1 week) treatment of ventilated preterm infants reduces 
abnormal neurodevelopmental outcome. Because brain development continues throughout gestation, we 
hypothesize that iNO therapy will be protective to the preterm brain if given from birth until the risk for 
abnormal neurodevelopmental outcome is similar to that of term infants. 
(ii) Study design. We have designed a randomized, placebo controlled, double blind study of iNO therapy 
during Intensive Care Nursery stay, followed by blinded neurodevelopmental assessment at 18 months 
corrected age, a commonly used developmental endpoint.  
(iii) 
Patients. All premature infants cared for in the Intensive Care Nursery at the University of Chicago 
Medical Center, who are less than 72 hours old, weighing less than 1500 grams at birth, and requiring any 
respiratory support, will be eligible for the study. Enrolled infants are anticipated to have the same 
demographics as we have previously reported14: 52% male, 70% African-American, 45% single parent 
household. Potential subjects will be identified and informed consent sought, if possible, prior to birth, 
otherwise, within the first 3 days after birth.  
(iv) 
Exclusion criteria. Those infants with severe congenital anomalies, including major congenital heart 
disease, or major kidney, lung or brain malformations will be excluded, as will be those infants with a genetic 
syndrome. Infants judged by the physician to not be viable will be excluded from the study, as will extremely 
sick infants requiring very high ventilatory pressures (Oxygen Index ≥ 2o). This latter group is excluded 
because of a previous study by Van Meurs19 which showed that infants in this category are made worse by 
inhaled nitric oxide.   
(v) Patient randomization. Randomization will be performed as we have previously published17. To keep birth-
weight distribution comparable in the two groups, we will use five 250-gram-birth weight strata for 
randomization. To ensure equal enrollment in the two groups, infants will be randomly assigned within each 
stratum, according to a permuted block design to receive inhaled nitric oxide (INOmax®, INO Therapeutics) or 

Principal Investigator:    Schreiber, Michael D. 
 
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oxygen placebo. This strategy provides a balanced group assignment with every 4 enrollments.   
(vi) 
Patient enrollment at study entry procedures. We will enroll patients and enter them into the study as 
we have previously published17. Briefly, potential subjects will be identified, prenatally if possible, and the 
parents approached for informed consent. With consent obtained, and the infant born, admitted, and verified 
to meet inclusion criteria, a sealed envelope containing the randomized treatment assignment, appropriate 
identifying labels, etc., will be pulled and opened by the respiratory therapist. Documentation of the treatment 
assignment and patient data will be recorded by the respiratory therapist in the study book (kept in the 
respiratory therapy room), and an INOvent® brought to the bedside. The INOvent will be set to deliver either 
iNO or oxygen according to the treatment randomization and then covered to conceal the treatment group 
from the caregivers and investigators.  
(vii) 
Treatment. iNO or placebo (oxygen) will be given 5 ppm while ventilated or on continuous positive 
airway pressure (CPAP) and 10 ppm while on nasal cannula oxygen. In infants receiving iNO by nasal cannula, 
iNO concentrations are set to 10 ppm, because previous studies have shown that delivery 10 ppm by nasal 
cannula results in nasopharyngeal concentrations of about 5 ppm9. These dosages will continue until the 
infant no longer needs supplemental oxygen or reaches 33 weeks post conceptional age. All infants will have 
iNO delivery devices (INOvents) at the bedside to deliver either iNO or O2, and will be shrouded to hide from 
caregivers the nature of the gas being delivered. Only respiratory therapy and data safety monitor to know of 
gas assignments.  
(viii) 
Data Collection. Clinical data (fluids, medications, diagnoses, ventilator settings) will be collected daily 
by our research professional. 
(ix)     Sample size. In our previous study of infants with RDS17, iNO treatment cut the risk of abnormal 
neurodevelopmental outcome by half. The baseline incidence of abnormal neurodevelopmental outcome is 
40% in this age group by published NICHD data7. Therefore to see a decrease of abnormal 
neurodevelopmental outcome to 20% (half of baseline incidence) with 80% power, 82 babies are needed in 
each arm of the study, for a total of 164 babies. We have previously published an overall mortality rate for 
intubated infants < 2000g in our nursery of 23%17. Because we are including non-intubated infants, we 

Principal Investigator:    Schreiber, Michael D. 
 
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estimate a mortality rate of about 18%. Therefore, to ensure sufficient infants surviving to follow up, we 
anticipate needing to enroll approximately 215 babies over 18 months. With a historic 80% follow-up rate, we 
will need a total of 268 infants. Every year we admit approximately 175-200 infants with birth weights < 
1500g. We therefore anticipate enrollment taking as long as 18 months. Our non-consent rate historically is < 
10%.  
(x) Standards of neonatal intensive care. Because we have shown no effect of ventilatory strategy on 
neurodevelopmental outcome, there will be no external control of ventilatory modality. Because this is a 
single-site study, variability in practice exists only between our eight member group of neonatologists, with 
constant care provided by nurse practitioners, residents, and nursing staff.  
(xi)     Safety monitoring. As per our clinical routine, methemoglobin is regularly monitored with arterial blood 
gases. Although we and others have shown that iNO therapy is safely used in preterm infants, interim safety 
analyses will be performed when 50% of patients enrolled. Deaths will be compared, as will the incidence of 
severe intraventricular hemorrhage, and tests of significance made with Chi-square. The study will be stopped 
if the incidence of either index in the iNO groups is significantly higher than in placebo groups.  
(xii) 
Primary Outcome. As we have published14, we will measure abnormal neurodevelopmental outcome at 
18 months corrected age. Three disability levels will be assessed:  disability (cerebral palsy, blind in both eyes, 
or deaf in both ears); delay (score on Mental or Psychomotor Developmental Index of the Bayley Scales of 
Infant Development < 70 without a disability) or normal (without disability or delay).  
(xiii) 
Secondary Outcomes. Other neurodevelopmental outcomes to be measured include the incidences of 
disability or delay; mean cognitive (MDI) and psychomotor (PDI) function scores will be obtained and infant 
growth charted. Important clinical diagnoses known to affect the risk of adverse neurodevelopmental 
outcomes will be tracked: respiratory distress syndrome, bronchopulmonary dysplasia, necrotizing 
enterocolitis, intraventricular hemorrhage (with grade), periventricular leukomalacia, retinopathy of 
prematurity, and failed hearing screen. Finally, detailed clinical data will be tracked on a daily basis, including 
fluids, ventilatory status, blood gases, feedings, etc.  
 

Principal Investigator:    Schreiber, Michael D. 
 
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(xiv) 
    Measures to ensure long-term neurodevelopmental follow-up. The very high follow-up rates we have 
previously published require intensive efforts of a number of teams following a patient’s discharge, including 
social services and the research staff. All preterm infants will be followed in our high risk follow-up clinic. This 
clinic is unusual, in that it provides routine, well-child care to its babies, as well as subspecialty care and 
neurodevelopmental care. Providing routine care provides an important incentive to parents to come to clinic, 
since they only have one appointment to make. In addition, we provide regular reminders of the role of the 
University of Chicago Medical Center in the lives of our families: patients receive holiday and birthday cards, 
and personalized invitations to our regular patient reunions. Finally, families who come for follow-up visits are 
given gifts and their travel expenses are reimbursed. Families are rewarded at a 9 month follow-up visit and at 
the 18 month follow-up visit. With this approach, our drop-out rate is less than 15% -- very low for this 
population by published reports.  
(xv) 
Assessment of neurodevelopmental outcome. As we have previously published14, at 18 months post 
conceptional age, developmental screening with Bayley exams will be performed in our High Risk Follow-up 
Clinic by Dr. Michael Msall, who will also perform neurological examinations to identify those infants with 
cerebral palsy. Other assessments will include measurements of physical growth and review of routine visual 
and hearing examinations to determine blindness or deafness. Questionnaires assessing socioeconomic status 
and self report of racial ethnicity will be administered. All examiners will be unaware of treatment regimen 
assignment. In our previous studies, our follow-up rate of >80% is very high by published standards, especially 
for an inner city community such as we serve. We anticipate that rate will continue for the current study.  
(xvi) 
    Data analysis. As we have previously published17, data will be analyzed as “Intent to Treat.”. Clinical 
and demographic variables between groups will be compared with Fisher’s exact test for categorical variables 
and Wilcoxon rank-sum tests for continuous variables. Primary outcomes will be analyzed with generalized 
linear models to obtain relative risks of primary outcome in the two groups. Effects of potential confounders 
will be adjusted for in the model: birth weight, sex, socioeconomic status, as well as for potential the 
intermediate variables: prolonged exposure to postnatal steroids, presence of chronic lung disease, severe IVH, 
or periventricular leukomalacia (PVL). Further analyses of Bayley scores, both cognitive (MDI) and 
psychomotor (PDI) will be performed. 

Principal Investigator:    Schreiber, Michael D. 
 
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(xvii) Anticipated results and critique. We anticipate that infants treated with iNO will demonstrate 
significantly decreased incidences of abnormal neurodevelopmental outcomes and will also demonstrate 
significantly increased cognitive abilities, as demonstrated by MDI scores. We further anticipate that these 
effects will persist after statistical adjustment for factors independently predisposing to abnormal 
neurodevelopmental outcome, such as male gender and gestational age, as well as adjustment for factors 
potentially mediating adverse neurodevelopmental outcome, such as chronic lung disease, intraventricular 
hemorrhage, or PVL. 
 
In our previous study, we found that some infants tolerated our standard weaning protocol from iNO 
poorly, requiring much slower weaning and therefore a longer time on very low concentration (1-2 ppm) of NO. 
One mechanism underlying this minor complication is decreases in native NO production by blood vessels of 
the lung during prolonged periods of iNO delivery. Therefore, it is conceivable that extremely preterm infants, 
who will, by design, receive iNO for up 8 or 9 weeks, exhibit intolerance to weaning off iNO. Accordingly, we 
will wean patients by 1 ppm every 12-24 hrs, the protocol which served us well in the previous study in this 
setting. 
 
Specific Aim #2. Assess the mechanisms by which iNO therapy alters neurodevelopmental 
outcome in preterm infants.  
(i) Rationale. We have previously found that iNO therapy reduces the risk of abnormal neurodevelopmental 
outcomes independently of the level of prematurity or complications of prematurity known to affect 
neurodevelopmental outcome. Therefore, it appears that iNO has direct, beneficial effects on the preterm 
brain and its response to the stress of existence outside the womb. Previously, iNO was believed to be 
inactivated in the blood vessels in the lung, by binding to hemoglobin in the blood. However, there is 
increasing evidence that hemoglobin binding does not inactivate NO permanently, but rather transports it for 
delivery to target tissues4, 5, 24.  The goal of this Specific Aim, therefore, is to assess the amount of delivered NO 
that is transported to our patients’ tissues, measure the amount of NO activity in our patients’ blood, and 
examine the relationships between these measures, the iNO concentrations administered, and the patients’ 
neurodevelopmental outcomes.  

Principal Investigator:    Schreiber, Michael D. 
 
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(ii) Study Design. Measurements of nitrosylated hemoglobin from arterial and venous blood samples will be 
made on each patient at defined points during the hospitalization: in the first 48 hrs following initiation of 
study gas (iNO or placebo), after 1 week of treatment, after 2 weeks of treatment. In infants who are treated 
longer than 2 weeks (anticipated to be all infants born before 31 weeks gestation, since they will likely still 
require some respiratory support), weekly measurements of venous nitrosylated hemoglobin will be made. 
Cyclic GMP will be measured once, after 1 week of therapy. For each patient, arterial and venous values will be 
compared, to identify the amount of NO that is delivered to tissues, and these values plotted over time. Mean 
delivered amounts will be compared across treatment groups, and individual values correlated with 
neurodevelopmental outcome. Cyclic GMP levels, a measure of nitric oxide activity, will be averaged within 
groups and treatment groups compared. 
(iii) 
Source of blood. This protocol does not require for blood draws outside the regular scope of practice. 
Samples will be taken from regularly scheduled arterial and venous draws, and leftovers from these samples 
used. 
(iv)     Measurement of nitrosylated hemoglobin. The S-nitrosylated hemoglobin will be measured using 
chemiluminescence, a technique we have used previously in the measurement of  inhaled NO in tracheal air15. 
Analysis will be performed according to published methods5. Briefly, leftover blood from routine blood draws 
(arterial and venous) will be placed into EDTA collection tubes and taken to the lab, and centrifuged at 750xg 
for 5 min. Aliquots of plasma will be taken and stored at -8oº for assays of nitrite and cGMP (see below). The 
red blood cell pellet will be removed, washed, flash-frozen on dry ice and stored at -80º C in a laboratory 
freezer. At the time of assay, the pellet will be rapidly thawed and pretreated with a 100-fold excess of KCN 
and K3Fe(CN)6, to remove No from heme while preserving the S-nitrosothiol bond. After pretreatment (30 
min), 500 µl will be passed through a Sephadex G25 column to remove contaminating nitrite and small 
nitrosothiols. Samples will then be reacted with I-3 to release NO from S-nitroso-hemoglobin, and assayed by 
chemiluminescence. Results will be calibrated against S-NO-glutathione standards. 
(v)     Measurement of plasma nitrite. Plasma nitrite will be measured using a commercially available kit 
(World Precision Instruments, Sarasota, FL) and a spectrophotometer, available in the laboratory. A 
colorimetric reaction is generated, and the absorbance is read at 540 nm. 

Principal Investigator:    Schreiber, Michael D. 
 
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(vi)    Measurement of cGMP. Guanosine 3’-5’ cyclic monophosphate will be measured in plasma using a 
commercially available competitive enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). The detection 
limit of this assay is about 6 pmol/ml, but acylation of the standards and samples will increase the sensitivity 
by 10-fold. Plasma proteins will be precipitated with ethanol and removed by centrifugation. The supernatant 
will be dried under nitrogen and re-suspended in diluted sample buffer. The assay is performed in 96-well 
format and after 18 hrs incubation at 4º C; Ellman’s reagent is reconstituted and added to the wells. 
Absorbance is read 90 min later at 405 nm. Results will be calibrated against standards assayed 
simultaneously. 
(vii) 
Data Analysis. Arterial-venous differences in S-nitroso-hemoglobin and plasma nitrite levels will be 
calculated on a patient-by-patient basis. Mean levels within groups will be compared across groups, and 
statistical significance tested with ANOVA18. Individual values of A-V differences will be compared with 
neurodevelopmental outcome on a patient-by-patient basis and assessed with regression techniques. Similar 
analyses will be performed with cGMP values. 
 
EXPECTED IMPACT OF THE PROJECT.  
Although there is great interest in the neonatal community about our prior finding of improved 
neurodevelopmental outcome and growth among premature infants treated with nitric oxide (see attached 
newspaper articles), additional corroborative data from a study specifically designed to examine the effect of 
nitric oxide therapy on neurodevelopmental outcomes is required to change current recommendations for 
neonatal care. If supportive, this specifically designed project would provide that corroborative data, such that 
we and other authorities could recommend that routine care of premature infants include nitric oxide 
treatment for improved neurodevelopmental outcomes. Consequently, this project would be expected to 
provide a marked improvement in neurodevelopmental outcome for the thousands of preterm babies born in 
the US every year. 
 

Principal Investigator:    Schreiber, Michael D. 
 
Page   16    
 
PLANS FOR ACKNOWLEDGING FOUNDATION SUPPORT FOR THE PROJECT.  
All papers reporting data from this study will specifically acknowledge the support of the Gerber Foundation. 
The text will say: “Funded by a grant from the Gerber Foundation.” Similarly, all talks, abstracts, and posters 
will contain specific acknowledgement. 
 
 
 

Principal Investigator:    Schreiber, Michael D. 
 
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Time
Project Stage
Activity
Year 1 month 1
Begin patient enrollment
randomize to NO or placebo
Begin assessment of NO delivery and metabolism during 
hospitalization
measurement of arterial and 
venous nitrite and nitrosylated 
hemoglobin
year 1 month 3
anticipate 1st discharges to home -                                       
assess complications of prematurity potentially affecting 
neurodevelopmental outcome
Routine head ultrasounds 
Routine head MRI at discharge 
Classification of chronic lung 
disease
year 1 month 9
Interim safety analysis
Comparison of death rates and 
rates of Grade IV IVH between 
groups
Year 2  month 7
End patient enrollment
Year 2 month 7
Begin assessment of 18 month neurodevelopmental outcome
Bayley scales             
Neurologic exam      
Assessment of CP status 
Assessment of Blindness 
Assessment of hearing
Year 3 month 12
End 18 month neurodevelopmental outcome assessment
Appendices 
A. Project schedule/Timeline of events 
 
B. Collaborations/Linkages. 
 
An ongoing collaboration will be made with the University of Chicago Neonatal Follow-up Clinic in which 
our infants will be seen for routine follow-up and neurodevelopmental assessment. Particularly important, will 
be our collaborations with Susan Plesha, our neurodevelopmental assessment specialist, who performs the 
Bayley developmental assessments on all our babies, as well as with our social work staff. Our social work 
staff’s involvement with our subjects’ families has been shown to be very helpful in keeping contact with them, 
and ensuring our very high follow-up rates in this economically depressed population. 
 

Principal Investigator:    Schreiber, Michael D. 
 
Page   18    
 
C. Budget 

Principal Investigator:    Schreiber, Michael D. 
 
Page   19    
 
D. Informed Consent Documentation 
THE UNIVERSITY OF CHICAGO  
The Division of the Biological Sciences. The University of Chicago Hospitals 
 
CONSENT BY SUBJECT FOR PARTICIPATION IN RESEARCH PROTOCOL 
 
Protocol Number: ____________ 
 
Patient Name:                                                    
 
 
 
 
 
 
 
 
 
 
Hospital Number:  
 
Title of Protocol:  
NITRIC OXIDE AND NEUROPROTECTION (PREMATURE INFANTS)  
 
Doctor(s) Directing Research:  
Michael D. Schreiber, M.D.   
 
Phone: 773-702-6210 
 
 
 
 
              
 
 
Jeremy D. Marks, Ph.D. M.D.  
 
 
 
  
         
 
 
 
Michael Msall, M.D. 
 
 
 
 
         
 
 
 
 
You are being asked to have your baby participate in a research study. The doctors at The University of Chicago 
Hospitals and The Division of Biological Sciences study the nature of disease and attempt to develop improved methods 
of diagnosis and treatment. In order to decide whether or not you want to be part of this research study, you should 
understand enough about its risks and benefits to make an informed judgment. This process is known as informed consent. 
This consent form gives detailed information about the research study, which will be discussed with you. Once you 
understand the study, you will be asked to sign this form if you wish to participate. 
 
I.  NATURE AND DURATION OF PROCEDURE (S):    
 
Your premature baby has an illness, which results from the immaturity of the lung to exchange adequate amounts of 
oxygen into your baby's blood. Some medical investigators have found that the breathing a gas, called nitric oxide, causes 
more blood to travel through the lungs, and results in more oxygen in the blood. Nitric oxide, which occurs normally in 
the body, relaxes blood vessels. By using higher than normal nitric oxide concentrations, the blood vessels in the lung 
may relax further and improve oxygenation. We have used nitric oxide to successfully treat preterm newborn babies who 
need to be on a breathing machine, and have found that babies treated with nitric oxide not only have fewer complications 
of their lung disease, but may have improved neurodevelopmental outcomes. This study is specifically designed to 
determine whether nitric oxide improves neurodevelopmental outcomes in premature babies with some difficulty 
breathing, whether or not they need mechanical ventilation.  
 
If your baby is assigned to receive inhaled nitric oxide, the amount of gas will be carefully controlled and adjusted to 
the amount that best improves the function of your baby's lungs. Your baby will remain on inhaled nitric oxide until s/he 
reaches 33 weeks of gestation or no longer needs supplemental oxygen. 
 
After being discharged from the hospital, your baby will be seen in our High Risk Follow-up Clinic at 9 and 18 
months of corrected age. He or she will be examined and will have developmental testing done by our developmental 
pediatrician. No invasive testing will be performed.  
 
II. POTENTIAL RISKS AND BENEFITS:   Based on the limited human and animal studies, in conditions similar to 
those encountered by your baby, no serious side effects of inhaled nitric oxide, in the concentrations delivered, have been 
demonstrated. Although not reported in humans, high concentrations of nitric oxide theoretically could cause bleeding 
problems. In addition, high concentrations of nitrogen dioxide, a by-product of nitric oxide, could cause lung injury. The 
precise concentration of nitrogen dioxide is continuously monitored and maintained within accepted guidelines. As is the 
case with any new drug, unknown side effects could occur. These potential risks are small compared to the risks of current 
conventional therapy. Determining which treatment will be most effective with the least side effects is the purpose of this 
study. The results will not to be known until after the study is completed and the data have been analyzed. Because your 
baby will receive pain medication and/or sedatives, no discomfort from this study should be experienced. Your baby's 
records will be kept as confidential as is possible within the law. 
 
FINANCIAL CONSIDERATIONS   Study gas and equipment will be provided free of charge. There will be no additional 
costs to you or your insurance carrier related to this study. Follow-up examinations will be performed at no additional cost 
to the family. Travel and meal allowances will be provided. 

Principal Investigator:    Schreiber, Michael D. 
 
Page   20    
 
 
BENEFITS OF PARTICIPATION   The potential benefits are that the treatment your baby receives may prove more 
effective than the currently available treatments and may prove to be helpful in preventing or ameliorating acute 
pulmonary injury which leads to the development of chronic lung disease. 
 
III. POSSIBLE ALTERNATIVES:   The alternative is to continue standard treatment using oxygen, mechanical 
ventilation, and other medications. Such currently used standard therapy is moderately successful in many cases.  
 
AGREEMENT TO CONSENT 
 
The research project and the treatment procedures associated with it have been fully explained to me. All 
experimental procedures have been identified and no guarantee has been given about the possible results. I have had the 
opportunity to ask questions concerning any and all aspects of the project and any procedures involved. I am aware that 
participation is voluntary and that I may withdraw my consent at any time. I am aware that my decision not to participate 
or to withdraw will not restrict my access to health care services normally available at The University of Chicago 
Hospitals. Confidentiality of records concerning my involvement in this project will be maintained in an appropriate 
manner. Records of study participants will be identified by code numbers. Results will be compiled as aggregate data. 
When required by law, the records of this research may be reviewed by applicable government agencies including the 
Federal Food and Drug Administration. The study sponsor may receive records of this research but will have no access to 
the names of the patients. 
 
I understand that in the event of physical injury resulting from this research, The University of Chicago Hospitals will 
provide me with free emergency care, if such care is necessary. I also understand that if I wish, The University of Chicago 
Hospitals will provide non-emergency care, but the Medical Center assumes no responsibility to pay for such care or to 
provide me with financial compensation. 
 
I, the undersigned, hereby consent to participate as a subject in the above described research project conducted at The 
University of Chicago Hospitals. I have received a copy of this consent form for my records. I understand that if I have 
any questions concerning this research, I can contact the doctor(s) listed above. If I have questions concerning my rights 
in connection with the research, I can contact the Institutional Review Board, at 773-702-1472. 
 
After reading the entire consent form, if you have no further questions above giving consent, please sign where 
indicated. 
 
 
Doctor:  
                
 
 
                     
 
      
 
 
 
 
Signature of Parent or Guardian 
 
 
Witness:                                                             
 
 
 
Date: __/__/__ Time: ______________ 
 
 
 
 
 
 
 
                           
 
 
 
  
 
 
 
 

Principal Investigator:    Schreiber, Michael D. 
 
Page   21    
 
E. CV’s of Principal Investigators 
 

Principal Investigator:    Schreiber, Michael D. 
 
Page   22    
 
F. References 
 
1. Ballard RA, Truog WE, Cnaan A, et al. (2006) Inhaled Nitric Oxide in Preterm Infants Undergoing 
Mechanical Ventilation. N Engl J Med 355:343-353 
2. Bayley N (1993) Bayley scales of infant development (2nd ed.) San Antonio, TX:Harcourt Brace. 
3. Crowther C, Henderson-Smart D. (2003) Phenobarbital prior to preterm birth for preventing neonatal 
periventricular haemorrhage. Cochrane Database of Systematic Reviews Issue 3:Art. No.: CD000164 
4. Gladwin MT, Ognibene FP, Pannell LK, et al. (2000) Relative role of heme nitrosylation and beta-cysteine 93 
nitrosation in the transport and metabolism of nitric oxide by hemoglobin in the human circulation. 
Proc Natl Acad Sci U S A 97:9943-8 
5. Gladwin MT, Shelhamer JH, Schechter AN, et al. (2000) Role of circulating nitrite and S-nitrosohemoglobin 
in the regulation of regional blood flow in humans. Proc Natl Acad Sci U S A 97:11482-7 
6. Hack M, Wilson-Costello D, Friedman H, Taylor GH, Schluchter M, Fanaroff AA. (2000) Neurodevelopment 
and predictors of outcomes of children with birth weights of less than 1000 g: 1992-1995. Arch Pediatr 
Adolesc Med 154:725-31 
7. Hintz SR, Kendrick DE, Vohr BR, Kenneth Poole W, Higgins RD, For The NICHD Neonatal Research N. 
(2006) Gender differences in neurodevelopmental outcomes among extremely preterm, extremely-low-
birthweight infants. Acta Paediatr 95:1239-48 
8. Kinsella JP, Cutter GR, Walsh WF, et al. (2006) Early Inhaled Nitric Oxide Therapy in Premature Newborns 
with Respiratory Failure. N Engl J Med 355:354-364 
9. Kinsella JP, Parker TA, Ivy DD, Abman SH. (2003) Noninvasive delivery of inhaled nitric oxide therapy for 
late pulmonary hypertension in newborn infants with congential diaphragmatic hernia. The Journal of 
Pediatrics 142:397-401 
10. Laptook AR, O'Shea TM, Shankaran S, Bhaskar B. (2005) Adverse neurodevelopmental outcomes among 
extremely low birth weight infants with a normal head ultrasound: prevalence and antecedents. 
Pediatrics 115:673-80 
11. Ment LR, Ehrenkranz RA, Duncan CC, et al. (1994) Low-Dose Indomethacin and Prevention of 
Intraventricular Hemorrhage: A Multicenter Randomized Trial. Pediatrics 93:543-550 

Principal Investigator:    Schreiber, Michael D. 
 
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12. Ment LR, Oh W, Ehrenkranz RA, et al. (1994) Low-dose indomethacin therapy and extension of 
intraventricular hemorrhage: A multicenter randomized trial. The Journal of Pediatrics 124:951-955 
13. Ment LR, Vohr B, Allan W, et al. (2003) Change in Cognitive Function Over Time in Very Low-Birth-Weight 
Infants. JAMA 289:705-711 
14. Mestan KL, Marks JD, Hecox K, Huo D, Schreiber MD. (2005) Neurodevelopmental outcomes of 
premature infants treated with inhaled nitric oxide. New England Journal of Medicine 353:23-32 
15. Roberts JD, Fineman JR, Morin FC, et al. (1997) Inhaled Nitric Oxide and Persistent Pulmonary 
Hypertension of the Newborn. N Engl J Med 336:605-610 
16. Schmidt B, Davis P, Moddemann D, et al. (2001) Long-Term Effects of Indomethacin Prophylaxis in 
Extremely-Low-Birth-Weight Infants. N Engl J Med 344:1966-1972 
17. Schreiber MD, Gin-Mestan K, Marks JD, Huo D, Lee G, Srisuparp P. (2003) Inhaled nitric oxide in 
premature infants with the respiratory distress syndrome. New England Journal of Medicine 
349:2099-2107 
18. Snedecor GW (1934) Analysis of Variance and Covariance Ames, Iowa:Iowa State University Press. 
19. Van Meurs KP, Wright LL, Ehrenkranz RA, et al. (2005) Inhaled Nitric Oxide for Premature Infants with 
Severe Respiratory Failure. N Engl J Med 353:13-22 
20. Vohr BR, Wright LL, Dusick AM, et al. (2000) Neurodevelopmental and functional outcomes of extremely 
low birth weight infants in the National Institute of Child Health and Human Development Neonatal 
Research Network, 1993-1994. Pediatrics 105:1216-26 
21. Wilson-Costello D, Borawski E, Friedman H, Redline R, Fanaroff AA, Hack M. (1998) Perinatal correlates of 
cerebral palsy and other neurologic impairment among very low birth weight children. Pediatrics 
102:315-22 
22. Wilson-Costello D, Friedman H, Minich N, Fanaroff AA, Hack M. (2005) Improved survival rates with 
increased neurodevelopmental disability for extremely low birth weight infants in the 1990s. Pediatrics 
115:997-1003 
23. Wilson-Costello D, Friedman H, Minich N, et al. (2007) Improved neurodevelopmental outcomes for 
extremely low birth weight infants in 2000-2002. Pediatrics 119:37-45 

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24. Xu X, Cho M, Spencer NY, et al. (2003) Measurements of nitric oxide on the heme iron and beta-93 thiol of 
human hemoglobin during cycles of oxygenation and deoxygenation. Proc Natl Acad Sci U S A 
100:11303-8