Preterm Erythropoietin Neuroprotection Trial (PENUT Trial)
Phase: PHASE3
Conditions: Extreme Prematurity
Sponsor: University of Washington

 
 
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1.  PROTOCOL V6.1 (UPDATED 2019-10-14) 
 
PROTOCOL TABLE OF CONTENTS 
 
 
 
 
 
 
 
 
 
SYNOPSIS  
 3 
 
1. STUDY OBJECTIVES 
 7 
 
 
1.1   Primary Objective  
 7 
 
1.2   Secondary Objectives 
 7 
 
2. BACKGROUND 
 8  
 
 
 
2.1   Rationale  
  8 
 
2.2   Supporting Data  
10 
 
3. STUDY DESIGN  
12 
 
4. SELECTION AND ENROLLMENT OF SUBJECTS  
12 
 
 
4.1  Inclusion Criteria  
12 
 
4.2  Exclusion Criteria  
13 
 
4.3  Enrollment Procedures  
14 
 
5. STUDY INTERVENTIONS 
15 
 
 
5.1   Interventions, Administration, and Duration  
15 
 
5.2   Handling of Study Interventions 
21 
 
5.3   Concomitant Interventions  
23 
 
5.4   Adherence Assessment  
24 
 
6. CLINICAL AND LABORATORY EVALUATIONS  
25 
 
 
6.1   Schedule of Evaluations  
25 
 
6.2   Data to be Collected on Each Subject 
27 
 
 
6.3   Timing of Evaluations  
27 
 
6.4   Special Instructions and Definitions of Evaluations  
39 
 
7. MANAGEMENT OF ADVERSE EVENTS  
43 
 
8. CRITERIA FOR INTERVENTION DISCONTINUATION  
43 
 
9. STATISTICAL CONSIDERATIONS  
44 
 
 
9.1   General Design Issues  
44 
 
9.2   Outcomes  
45 
 
9.3   Meeting Recruitment Targets  
49 
 
9.4   Data Monitoring  
50 
 
9.5   Formal Interim Analysis 
55 
 

 
 
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10. DATA COLLECTION, SITE MONITORING, AND ADVERSE EVENT    
56 
  
REPORTING  
 
 
10.1   Records to be Kept  
56 
 
10.2   Role of Data Management  
57 
 
10.3   Quality Assurance  
57 
 
10.4   Adverse Event Reporting  
61 
 
11. HUMAN SUBJECTS  
62 
 
 
11.1   Institutional Review Board (IRB) Review and Informed Consent  
62 
 
11.2   Subject Confidentiality  
62 
 
11.3   Study Modification/Discontinuation  
62 
 
12.   PUBLICATION OF RESEARCH FINDINGS  
62 
 
13.   REFERENCES 
64 
 
 
 
 

 
 
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SYNOPSIS 
 
Study Title:  Preterm Epo Neuroprotection Trial (PENUT Trial) 
 
Objectives 
 
Primary Objective:  
Our primary goal is to test the hypothesis that early high dose recombinant human erythropoietin 
(Epo) treatment of preterm infants born between 24-0/7 and 27-6/7 weeks of gestation will 
improve long-term neurodevelopmental outcomes measured at 22-26 months (referred 
throughout the document also as “2 year(s)”) corrected age. Specifically, we will determine 
whether Epo decreases the combined outcome of death or neurodevelopmental impairment (NDI) 
at 22-26 months corrected age.  
 
Impairment is defined as the presence of any one of the following: cerebral palsy (CP), Bayley 
Scales of Infant and Toddler Development, 3rd Edition (Bayley-III) Motor Standard Score, 
Language or Cognitive Standard Score < 70 (severe, 2 SD below mean) or 85 (moderate, 1 SD 
below mean).1, 2 CP will be identified by standardized neurologic exam and categorized as no CP, 
diplegic CP, hemiplegic CP, or quadriplegic CP. Severity of CP will be determined using the Gross 
Motor Function Classification System (GMFCS) developed by Palisano.3, 4 
 
Secondary Objectives  
 
To determine whether there are risks to high dose Epo administration in extremely low 
gestational age neonates (ELGANs) by examining, in a blinded manner, Epo-related safety 
measures comparing infants receiving Epo with those given placebo. As part of this aim, Epo 
pharmacokinetics will be determined. 
 
To test whether Epo treatment decreases circulating inflammatory mediators and known 
biomarkers of brain injury over time. 
 
To compare brain structure (as measured by MRI) in Epo treatment and control groups at 36 
weeks post menstrual age (PMA).  MRI assessments will include documentation of 
intracranial hemorrhage (ICH), white matter injury (WMI) and hydrocephalus (HC), volume of 
total and deep gray matter, white matter and cerebellum, brain gyrification, inter-hemispheric 
distance, and tract-based spatial statistics (TBSS based on diffusion tensor imaging).  
 
As an exploratory aim, we will determine which of the above MRI measurements best predict 
neurodevelopment at 22-26 months corrected age. 
 
To determine the incidence of acute kidney injury (AKI) in this cohort of extremely low 
gestational age neonates (REPAIReD ancillary study). 
 
Study Design 
Prospective, multicenter, randomized, double blind, placebo controlled trial. 
 
 

 
 
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Figure 1 depicts an overview of the proposed trial. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Interventions and Duration 
Enrollment will occur prior to 24 hours of age. After enrollment but prior to the first dose of study 
drug, a cranial ultrasound (CUS) will be obtained to document whether an intracranial hemorrhage 
has occurred prior to dosing (while the goal will be to obtain the ultrasound prior to study drug 
dosing, a 6 hour grace period will be allowed). Baseline blood will be drawn to measure Epo 
concentration, inflammatory mediators and biomarkers of brain injury. Subjects will be 
randomized to Epo treatment or placebo by the data coordinating center (DCC). Each subject will 
be on the study (intervention + time off intervention) until completion of the follow-up visit at 22-
26 months corrected age. After the in-person 2-year visit, with parental permission, PENUT 
subjects will continue to be followed at 6-month intervals by phone. 
 
Study Drug  
Drug Dosages and Routes of Administration 
Subjects will be randomized to Epo treatment or placebo.  
 
Initially, subject will receive Epo 1000 units /kg or placebo IV every 48 ± 2 hours x 6 doses (during 
the first 2 weeks of life when physiologic vulnerability is highest).  
 
This will be followed by maintenance Epo 400 units/kg subcutaneously, or sham injection, 
Monday, Wednesday and Friday, during the period of oligodendrocyte vulnerability (to 32-6/7 
weeks). Controls will not receive any injections. Maintenance study drug doses will begin on the 
day closest to completing the high dose series. 
 
To maintain the blind in the maintenance portion of the treatment period, control infants will have 
Figure 1. Study Overview 
 

 
 
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PENUT Protocol V6.1 October 14, 2019 
 
an empty syringe dispensed from pharmacy. Infants assigned to placebo will not undergo any 
subcutaneous maintenance injections. All blinded doses will be dispensed in an opaque bag with 
tamper resistant tape. Maintenance treatments will be administered behind blinds or curtains. 
After Epo injection or sham injection (no shot), all infants will have a 2” by 2” gauze placed over 
the “injection site”, and this will be covered and wrapped with gauze that is taped to itself, so no 
adhesive touches the child’s skin. 
 
Group 1 (Epo): 
1. High dose Epo (1000 units/kg) will be administered intravenously every 48 hours x 6 
doses. Dosing will be based on birth weight. Study drug may be administered by the 
bedside nurse or a research nurse, given that the study drug appears identical. 
 
2. Maintenance Epo (400 units/kg) will be administered by subcutaneous injection three 
times weekly. Dosing will be adjusted weekly after the subject has regained birth weight. 
A nurse who is not directly involved with the care of the patient, and who will maintain 
confidentiality will administer all treatments. A cohort of charge nurses, or General Clinical 
Research Center (GCRC) nurses would be ideal for this task. 
 
 
Group 2 (Placebo): 
1. Placebo (saline) will be given intravenously every 48 hours x 6 doses. The volume of 
the saline placebo will be equivalent to the volume which would be administered if the 
subject was receiving active drug. Study drug may be administered by the bedside nurse 
or a research nurse, given that the study drug appears identical. 
   
 
2. Empty syringes (SHAM doses) will be delivered to infants in the control group however 
they will not undergo subcutaneous injections. Maintenance doses will be administered 
behind blinds or in a private room, and the “injection site” will be covered as described 
above. A nurse who is not directly involved with the care of the patient, and who will 
maintain confidentiality will administer all treatments. A cohort of charge nurses, or 
General Clinical Research Center (GCRC) nurses would be ideal for this task. 
 
Exceptional situations: 
 
If a dose of study drug is unavoidably delayed or missed, the dose should be given as soon 
as possible. This should be recorded as a study deviation. The normal schedule of 
administration should then be resumed. At least 24 hours must separate the high dose study 
drug doses. This allows the plasma Epo concentration to return to baseline. 
 
 
If severe edema (anasarca) or other circumstances prohibit the use of subcutaneous 
injection, up to three doses of Epo or saline placebo may be given intravenously using the 
normal IV protocol. In this case, pharmacy must be notified so they can provide an 
appropriate study drug formulation for IV use (saline rather than an empty syringe used for 
sham injection).  This should not be recorded as a deviation for up to 3 doses.  Subsequent 
IV doses would be considered a deviation. 
 
 
If the baby’s IV is removed prior to completing the 6 intravenous study drug doses, the dose 
can be administered subcutaneously.  This should be recorded as a study deviation. 
 
Interventions that each subject will experience: 
1. 
Cranial ultrasound prior to study drug dosing (all subjects) 
2. 
Intravenous study drug treatment (Epo or placebo) for 6 doses, day 1-11 (all subjects) 

 
 
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3.  
Epo group: Subcutaneous Epo injections three times a week until 32-6/7 weeks PMA; 
Control group: Sham injections (no shots) three times a week until 32-6/7 weeks PMA 
4.  
Iron supplementation (all infants) 
5. 
Timed blood draws totaling 2 mL during the first 2 weeks of life (see Figure 3) 
6. 
Ten timed, sequential urine collections from study entry to discharge 
7. 
Brain MRI at 36 weeks PMA (220 subjects only) 
8. 
Follow-up phone calls at 4, 8, 12, 18 months corrected age 
9. 
Follow-up visit at 22-26 months corrected age will include: Blood pressure, weight, 
(optional) blood draw (0.5 mL), urine collection, standardized neurologic exam, GMFCS 
evaluation, Bayley Scales of Infant and Toddler Development, 3rd Edition, and M-CHAT-
R questionnaire. 
10. 
Follow-up phone calls will continue at 30, 36, 42, 48, 54, 60 months corrected age. 
 
Sample Size and Population 
In order to maintain 752 (376 per arm) who survive and are followed at 22 to 26 months corrected 
age, we will enroll a total of 940 subjects. This number accounts for attrition due to death, loss to 
follow-up, and accounts for multiple births. The Vermont Oxford Network reports that 25% of 
ELBW infants are from multiple births and a variance inflation factor would therefore involve the 
expected cluster size (1.25) and the within-cluster correlation (ρ) and equal:  1 + (1.25-1) ρ. If the 
within sib-ship correlation is 0.50 then the variance inflation factor (VIF) would be 1.125 implying 
a 12.5% (n=94) increase in target sample size necessary to retain appropriate power. Due to 
withdrawal or attrition by 2 years, we anticipate losing 12.5% of the effective sample size of 752 
and will therefore increase the overall sample by an additional 94 subjects for a total enrollment 
target of n=940 subjects. 
 
Qualified infants less than 24 hours of age of both sexes and all races will be entered into the 
study if their parents or legal guardians consent.  
 
Randomization will be stratified by site, multiple births, and by gestational age: 24-0/7 to 25-6/7 
and 26-0/7 to 27-6/7. We will use block randomization within site using variable blocks of size 4, 
6, 8 and 10 subjects. Using block randomization ensures that equal numbers of subjects are 
randomized to the intervention and control arm and that the two groups are balanced at period 
enrollment intervals. For multiple births (twins, triplets) all infants will be randomized into the same 
treatment group (e.g. effective randomization of the mother).  
 
Table 1. Frequently Used Abbreviations: 
BDNF 
Brain derived neurotrophic factor 
MDI 
Mental developmental index 
BPD 
Bronchopulmonary dysplasia 
NDI 
Neurodevelopmental impairment 
CCC 
Clinical coordinating center 
NEC 
Necrotizing enterocolitis 
CP 
Cerebral palsy 
NICU 
Neonatal intensive care unit 
DCC 
Data coordinating center 
PDA      Patent ductus arteriosus 
DEHSI  
Diffuse excessive high signal intensity 
PDI 
Psychomotor developmental index 
ELGANs  
Extremely low gestational age neonates 
PMA 
Postmenstrual age 
Epo 
Erythropoietin 
PVL 
Periventricular leukomalacia 
EpoR 
Epo receptor 
ROP 
Retinopathy of prematurity 
GMFCS  
Gross motor function classification system 
SOP 
Standard operating procedure 
HC 
Hydrocephalus 
SC 
Subcutaneous 

 
 
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IV 
Intravenous 
TBSS Tract based spatial statistics 
ICH 
Intracranial hemorrhage 
VON     Vermont Oxford Network 
M-CHAT-R Modified Checklist for Autism in Toddlers 
WMI 
White matter injury 
 
 
 
1. 
STUDY OBJECTIVES 
 
1.1 
Primary Objective 
Hypothesis 1. Epo treatment from 24 hours to 32-6/7 weeks PMA of ELGANs will safely decrease 
the combined outcome of death or NDI from 40% to 30% measured at two years corrected age.  
 
Objective 1. Our primary goal is to test the hypothesis that early high dose recombinant human 
erythropoietin (Epo) treatment of preterm infants born between 24-0/7 and 27-6/7 weeks of 
gestation will improve long-term neurodevelopmental outcomes measured at 22-26 months 
corrected age.  
 
1.2   
Secondary Objectives  
Hypothesis 2. High dose Epo will be safe to administer from birth to 32-6/7 weeks of gestation in 
this population of ELGANs. 
 
Objective 2. To determine whether there are excess adverse events in Epo treated ELGANs 
compared to control infants. Three time periods will be considered: 1. Safety during the treatment 
period; 2. Safety during the initial hospitalization; and 3. Long term outcomes. Timed Epo 
concentrations will be measured to monitor for drug accumulation and safety. A Population 
Pharmacokinetic Analysis will be done using these data.  
 
Hypothesis 3. Therapeutic Epo administration during the period of oligodendrocyte vulnerability 
(24-32 weeks of gestation) will promote normal brain development by increasing brain derived 
neurotrophic factor (BDNF), decreasing circulating inflammatory mediators, and thereby 
decreasing biomarkers of brain injury. 
 
Objective 3. To test whether Epo treatment changes sequential measures of BDNF, circulating 
inflammatory mediators and known biomarkers of brain injury in preterm infants. 
 
Hypothesis 4. Epo treatment will improve brain development as assessed by MRI at 36 weeks 
PMA. 
 
Objective 4. To compare brain structure (as measured by MRI) in Epo treatment and control 
groups at 36 weeks PMA. MRI assessments will include documentation of intracranial 
hemorrhage (ICH), white matter injury (WMI) and hydrocephalus (HC), volume of total and deep 
gray matter, white matter and cerebellum, brain gyrification, inter-hemispheric distance, and tract-
based spatial statistics (TBSS) based on diffusion tensor imaging. As an exploratory aim, we will 
determine which of the above MRI measurements best predict neurodevelopment (CP, cognitive 
and motor scales) at 22-26 months corrected age. 
 
Hypothesis 5.  Epo treatment may affect kidney development and incidence of acute kidney injury. 
 

 
 
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PENUT Protocol V6.1 October 14, 2019 
 
Objective 5.  To establish the incidence of acute kidney injury in ELGANs, and to determine 
whether Epo treatment influences this. 
2. 
BACKGROUND 
2.1 
Rationale 
Study Population. ELGANS are at high risk of death or NDI. In fact, of 9,575 ELGANs born 
between 2003 and 2007 and admitted to the NICU, death or NDI occurred in 91%, 80%, 66% and 
56% of those born at 24, 25, 26 and 27 weeks gestation, respectively.5 These sobering statistics 
do not include infants that died before admission to a NICU, or those who died within 12 hours of 
admission. Major morbidities, which include CP, deafness, blindness, and mental disabilities, are 
present in 50% of surviving extremely preterm infants at school age.6-19  Long-term follow-up 
studies are now increasingly identifying behavioral dysfunctions such as attention deficit disorder 
and autism spectrum disorder.20-24 Sequelae of extreme prematurity are a tremendous burden to 
the individuals, their families, and to our health care system, accounting for nearly half of the 
health care dollars spent on newborn care. Clearly, a neuroprotective intervention that improved 
outcomes for ELGANs would be profoundly beneficial both to the individual, the family and to 
society.25, 26 We have chosen to study ELGANs born between 24-0/7 and 27-6/7 weeks of 
gestation because of their high likelihood of poor outcome, and the absence of therapeutic 
interventions to improve outcome. Because these infants are at such high risk of death or 
impairment, a strategy of prophylactic intervention is reasonable as there is great potential for 
benefit.25, 26   
 
Restrictions: Babies born at 23-0/7 to 23-6/7 will be excluded from this study because their 
mortality and morbidity is too high. At this time, best estimates of death rates are 74%, and 
unimpaired survival rates are 1%.5, 19 More mature babies are also excluded because their risk of 
poor outcomes is lower, thus prophylactic use of an unproven therapy is not indicated. 
 
Intervention Regimen. Study infants in the Epo group will receive 6 IV doses of 1000 U/kg during 
the first 2 weeks of life when physiologic vulnerability is highest. This will be followed by 
maintenance dosing of 400 U/kg SC 3 times per week during the period of oligodendrocyte 
vulnerability (to 32-6/7 wks).  
 
Dose Justification. The Epo dose, and 
duration of therapy chosen for this study is 
based upon available preclinical and 
clinical data for Epo neuroprotection.27 In 
rodent and ovine models of neonatal brain 
injury, Epo doses of 1000-5000 U/kg/dose 
result 
in 
sustained 
neuroprotection, 
improving 
both 
short 
and 
long-term 
structure and function.28-31 Higher doses 
are needed for neuroprotection due to the 
low percentage of circulating Epo that 
crosses the blood brain barrier.32 
 
Although doses as high as 3000 U/kg/dose 
are being tested in preterm infants without 
apparent adverse effects,33 preclinical data 
suggest that Epo neuroprotection has a U-shaped dosing curve, with too little or too much Epo 
Figure 2. Epo concentrations in human preterms 
compared with rat pups given 5000 U/kg s.c. or i.p.  
 
0
3
6
9
12
15
18
21
24
0
2000
4000
6000
8000
10000
12000
14000
16000
ELBW 500 U/kg i.v.
ELBW 1000 U/kg i.v.
Rats 5000 U/kg i.p.
Rats 5000 U/kg s.c.
Epo Dose
Time (h)
Erythropoietin mU/mL

 
 
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PENUT Protocol V6.1 October 14, 2019 
 
resulting in diminished efficacy.29, 34 In rats, repeated doses of 1000-5000 U/kg show optimal 
neuroprotection. To estimate how neuroprotective Epo doses in rat pups relate to human 
pharmacokinetics, plasma Epo concentrations were measured in extremely low birth weight 
infants (< 1000 g birth weight) after 500, 1000, and 2500 U/kg/dose.35 Nonlinear kinetics were 
noted, consistent with previous studies in neonates.36 In these infants, IV administration of 500 
and 1000 U/kg resulted in similar peak concentrations but faster clearance than were achieved in 
rat pups after 5000 U/kg (Figure 2). Table 2 shows a comparison of pharmacokinetic indices in 
neonatal rats and humans. Doses of 1000 U/kg Epo resulted in area under the curve (AUC) 
measurements most similar to the most protective dose in rats.29 The 500 U/kg dose fell short 
(one third to one quarter the protective AUC), while 2500 U/kg was close to three times the optimal 
dose in rats. Minimum steady-state concentrations (mean = 576 mU/ml) were produced using the 
1000 U/kg/dose.  
 
Table 2. Epo Pharmacokinetics 
 
Neonatal Rodents37 
Preterm Infants < 1000 gm35 
Epo Dose 
5000 U/kg s.c. 
5000 U/kg i.p. 
500 U/kg 
1000 U/kg 
2500 U/kg 
AUC (U*h)/L 
117,677 
140,331 
31,412 ± 2780 
81,498 ± 7067 
317,881±22941 
Cmax (U/L) 
6,224 
10,015 
8078 ± 538 
14017 ± 1293 
46467 ± 2987 
T1/2 Half-life (h) 
8.4 
6.7 
5.4 ± 0.6 
7.1 ± 0.7 
8.7 ± 1.4 
 
What constitutes clinical significance? Clinically significant studies are those that improve 
clinical practice and patient outcomes. Changes to clinical practice can occur slowly or rapidly 
depending on a number of factors: importance of the results, the size of the studies, risk of the 
therapy, and endorsement of medical associations such as the AAP. Ibrahim et al. performed a 
13-item web-based questionnaire asking neonatologists what would convince them to adopt a 
new therapy in infants < 28 weeks of gestation. The survey assumed no adverse results of  
treatment. Responses are shown in Table 3. We also have examples of changes in clinical 
practice based on published trials. Prenatal steroids reduce the risk of death or morbidity in 
preterm infants < 32 weeks 
gestation with a risk reduction of 
over 40%.38 Despite this known 
benefit, 
change 
in 
clinical 
practice was slow, and required 
endorsement from ACOG. In 
contrast, 
therapeutic 
hypothermia 
for 
neonatal 
encephalopathy has been the 
subject of intense study, with 
multicenter 
randomized 
controlled trials published in 2005-2009.39-41 A meta-analysis of these studies reporting on 767 
infants showed benefits of cooling with a significant reduction in death or NDI at 18 months: typical 
Risk Ratio, 0.81 (95% Confidence Intervals, 0.71, 0.93), P=0.002. The number needed to treat 
(NNT) ranged from 7-9 to prevent death or NDI. Cooling for neonatal encephalopathy has rapidly 
become the standard of care. Animal studies of neonatal Epo neuroprotection using appropriate 
dosing have shown improvement of 49 to 79%.27 Early clinical trials have shown safety and 
preliminary benefit. Neonatologists are comfortable using this drug for erythropoiesis. We expect 
that Epo treatment will improve NDI-free survival compared to that seen in the ELGAN and NICHD 
trials. We expect that this benefit will translate into shorter and less complicated hospital stays 
and better neurodevelopmental outcomes. This outcome of the trial would represent an important 
advance in the care of ELGANs that could change the standard of care for high-risk infants. If Epo 
treatment has no demonstrable benefit, or if adverse effects are observed, this information will 
Table 3. Survey of Neonatologists. “Would you 
change practice if a study showed treatment 
X…?” 
% 
would 
change  
N=226 
Reduced # Bayley scores < 80 by 25% 
41 
Reduced # Bayley scores < 80 by 50% 
66 
Improved Bayley scores by 10 points 
56 
Decreased CP by 25% 
50 
Decreased CP by 50% 
81 
Study size 200 per arm 
29 
Study size 400 per arm 
64 

 
 
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also be useful in the field because Epo is currently used anecdotally in many NICUs, without trial-
based evidence, for some severely ill infants. The results of the PENUT trial will be of particular 
interest to pediatricians trained in neonatal-perinatal medicine. This group of practitioners is 
largely centered in academic or medical centers and they attend several meetings each year 
where new information about treatment can be presented. The PENUT trial results will be 
presented at regional Pediatric Research Society meetings such as the Western and Eastern 
Society for Pediatric Research, and at national and international meetings including the Pediatric 
Academic Societies combined meetings, the European Society for Pediatric Research meeting, 
the American Academy of Pediatrics meetings, and at other venues such as “Hot Topics in 
Neonatology”. 
 
Clinical Impact. Mortality of extremely preterm infants has decreased,42, 43 but morbidity in 
survivors remains high due in part to increased survival of sicker infants. There remains an 
enormous burden of both medical and neurodevelopmental impairment in these children. Using 
the birth statistics for the US in 2009, we anticipate the magnitude of Epo treatment to translate 
as follows: with Epo treatment, the annual burden of death or severe NDI will decrease from 40% 
(12,226 babies) to 30% (9,170 babies, primary outcome), and that death plus moderate or severe 
NDI will decrease from 60% (18,340 babies) to 40% (12,226 babies, secondary outcome). We 
anticipate this change will derive primarily from improved neurodevelopmental outcomes. If Epo 
is proven to safely reduce combined morbidity and mortality of ELGANs, we anticipate a shift in 
neonatology practice that would improve the lives of babies and their families, and decrease the 
cost of healthcare. 
 
2.2  
Supporting Data 
Vulnerabilities of the preterm brain. ELGANs are born during the second trimester when the 
fetal brain is rapidly increasing in size, shape and complexity.44-46 Proper brain development is 
vulnerable to interruption by hypoxia-ischemia, oxidant stress, inflammation, and excitotoxicity, 
as evidenced by structural, biochemical, and cell-specific injury.47-53 Pre-oligodendrocytes, which 
emerge and mature between 24 and 32 weeks of development, are particularly susceptible to 
injury, resulting in the WMI characteristic of preterm infants.47, 54-56 Although the transition from 
fetal to early postnatal life is the period of greatest vulnerability,57 ELGANs remain at risk for brain 
injury throughout the period of oligodendrocyte development.  
 
Epo Neuroprotection. Epo has anti-inflammatory, anti-excitotoxic,58 anti-oxidant,59 and anti-
apoptotic effects on neurons and oligodendrocytes, and promotes neurogenesis and 
angiogenesis, which are essential for injury repair and normal neurodevelopment. Epo effects are 
dose-dependent, and multiple doses are more effective than single doses.28, 29, 60 Epo reduces 
neuronal loss and learning impairment following brain injury,61, 62 and even when initiated as late 
as 48-72 hours after injury, there is evidence of improved behavioral outcomes, enhanced 
neurogenesis, increased axonal sprouting, and reduced WMI.63, 64  
 
Protective effects. Perinatal inflammation (chorioamnionitis, necrotizing enterocolitis (NEC), or 
sepsis) is associated with increased risk of NDI.13, 65, 66 Microglial activation67 and increased 
cytokine expression, particularly TNF-, interleukin (IL)-6, and IL-8, have been associated with 
brain injury in preterm infants68, 69 and in animal models of neonatal brain injury.70 Epo has 
demonstrated anti-inflammatory effects, which may contribute to neuroprotection in the scenario 
of preterm birth and increased inflammatory activity.71-77  
 
White matter injury. WMI is a common brain injury affecting preterm infants.48, 52, 55 Epo 

 
 
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PENUT Protocol V6.1 October 14, 2019 
 
decreases WMI in adult and neonatal animal models of brain injury30, 63, 78-83 and maybe in 
humans.84 Mechanisms for this may be Epo protection of vulnerable preoligodendrocytes: 
functional Epo receptors are expressed by immature oligodendrocytes; Epo promotes the 
proliferation, maturation and differentiation these cells,85 and protects them from injury induced 
by interferon-γ, LPS, and hypoxic-ischemia.63, 86, 87  
Apoptosis. Neurons in the developing brain are more likely than adult neurons to undergo 
apoptosis if exposed to injurious stimuli,88, 89 and the anti-apoptotic properties of Epo may protect 
these vulnerable neurons.29, 90, 91  
 
Repair. Epo stimulates growth factors required for normal brain growth such as brain-derived 
neurotrophic factor (BDNF) and glial cell derived neurotrophic factor (GDNF).92, 93 Epo enhances 
neurogenesis,28, 60, 91, 93-96 angiogenesis, repair and plasticity, thus providing long lasting 
neuroprotective and trophic effects.60, 64, 94, 97-100  
 
Reactive Iron. Iron is highly reactive and normally sequestered by transport proteins. Unbound 
iron produces free radicals and subsequent oxidative injury. Preterm infants have measurable 
free iron, which increases after transfusions of red blood cells or during metabolic instability such 
as sepsis.101-104 In our phase I/II study of Epo administration to extremely low birth weight infants 
we observed a transient increase in reticulocytosis, indicating an increase in iron utilization.105 
Epo may contribute to neuroprotection by decreasing free iron.  
 
Molecular mechanisms of Epo neuroprotection. EpoR are present on neuron progenitor 
cells,93 neurons,106 astrocytes,85 oligodendrocytes,107 microglia,108 endothelial cells93 and 
erythrocyte progenitors. Epo has direct neuroprotective effects via EpoR binding: Epo-bound 
receptors dimerize to activate anti-apoptotic pathways via phosphorylation of JAK2, 
phosphorylation and activation of MAPK, ERK1/2, as well as the PI3K/Akt pathway and STAT5, 
which are critical in cell survival.90, 108 Epo also functions through indirect effects, increasing iron 
utilization by increasing erythropoiesis, and by decreasing inflammation73, 76 and oxidative 
injury.109, 110  
 
Translational trials of neonatal Epo neuroprotection are in progress. Epo improved 
neurodevelopmental outcomes in two trials of term neonates with hypoxic-ischemic brain 
injury.111, 112 Retrospective studies of cohorts of preterm infants that received Epo for 
erythropoiesis compared to controls show improved outcomes.113-115 Two preliminary reports of 
preterm infants treated prospectively also show benefit:  
 
1) Preterm infants 500 to 1250 g treated with Epo (400 U/kg 3x/week) from birth to 35 weeks PMA 
had an average cumulative cognitive score 10 points higher than placebo/controls (98±14 Epo 
vs. 88±14 for controls). Epo recipients also performed statistically better than controls on object 
permanence testing;116   
 
2) A phase III trial is ongoing in Switzerland to test the safety and neuroprotective efficacy of 3000 
U/kg/dose x 3 doses given in the first 3 days after birth to neonates (<1500 g, < 32 weeks PMA). 
This group has presented preliminary data in abstract form on 100 infants showing that such 
dosing is safe, and that the Epo-treated group showed less WMI by MRI;84  
 
3) Follow-up of the infants studied in our phase I/II trial35 shows that Epo treatment correlated with 
improvement of cognitive (R= .22, p < 0.05) and motor (R = .15, p < 0.05) scores.117   
 
Known and potential risks of intervention: In adults, complications of prolonged Epo treatment 
include polycythemia, seizures, hypertension, stroke, myocardial infarction, congestive heart 

 
 
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failure, tumor progression, and shortened time to death. None of these adverse effects have been 
reported in Epo-treated neonates in over 3000 patients enrolled in randomized controlled trials.118 
Epo trials in neonates for the purposes of testing its erythropoietic effect have shown it to be a 
safe drug for use in this population. There is robust data from preclinical animal work showing 
that Epo, when used at optimal doses (1000-5000 U/kg), shows short and long term improvement 
in brain injury that approximates 50-80%, and no safety issues have been discovered. Preterm 
infants administered Epo may be at increased risk of ROP and other as yet unknown 
complications. 
 
Federal oversight. This study of high dose Epo for the purposes of neuroprotection of preterm 
infants is registered with the FDA (IND # 12656). Dr. Juul, University of Washington, Seattle WA 
is the holder of the IND. The trial has been registered with clinicaltrials.gov and has been assigned 
Identifier Number NCT01378273. 
 
3. 
STUDY DESIGN 
 
Study Overview. This is a randomized, placebo controlled, double blind study of Epo 
neuroprotection in an ELGAN population. Figure 1 provides an overview of the study. 940 patients 
will be enrolled at 16-18 sites across the country in order to evaluate 752 at 22-26 months 
corrected age. Enrollment and initial treatment with study drug will occur by 24 hours after birth. 
Subjects will be randomized to either Epo treatment or placebo, and treatment will continue until 
32-6/7 weeks PMA.  Short term, intermediate and long term safety measures will be determined 
by comparing Epo-treated and control infants. Mechanisms of Epo neuroprotection and potential 
biomarkers of outcome will be sought by measuring sequential inflammatory cytokines and 
markers of brain injury. In a subset of subjects, a brain MRI will be done at 36 weeks PMA to 
determine whether Epo treatment preserves brain growth and decreases injury. After discharge 
from the hospital, phone contact will be made at 4 month intervals. Data will be collected on 
interval medical history and functional status. Face to face follow-up will occur at two years 
corrected age, at which time standardized neurodevelopmental assessments will be made. The 
primary outcome is death or severe NDI at 22-26 months corrected age, with a secondary 
outcome of death, severe or moderate NDI. 
 
4. 
SELECTION AND ENROLLMENT OF SUBJECTS 
4.1 
Inclusion Criteria 
 
NICU Inpatients between 24-0/7 and 27-6/7 weeks of gestation 
 
24 hours of age or less 
 
Arterial or venous access 
 
Parental consent 
 
When calculating gestational age, the following hierarchy will be used in order of accuracy: 
1.  Gestational age by in vitro fertilization 
2.  Gestational age by first trimester assessment (0 to 14-0/7 weeks) 
3.  Gestational age by second trimester assessment (up to 28-0/7 weeks) 
4.  Last menstrual period (LMP) 
5.  Newborn maturational assessment 
 
We estimate, based on the ELGAN study, that 62% of subjects will have the most accurate dating 

 
 
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based on #1 or #2, that 29% will have dating based on second trimester fetal ultrasound, 7% by 
menstrual dating, and only 1% by postnatal physical exam. 
 
Postnatal age will be calculated based on time and date of birth. 
 
Arterial or venous access is considered to be a functioning peripheral intravenous catheter, a 
peripherally inserted central catheter (PICC), an umbilical venous (UVC) or arterial catheter 
(UAC). One of these catheters must be available for the administration of study drug. It is 
preferable that the patient also have a catheter suitable for phlebotomy, although that is not an 
absolute entry criterion for the study. 
 
To be admitted into this study, the legal guardian of the patient must give written informed consent. 
Each site will obtain IRB approval for the study, and will have IRB approved consent forms 
available in English and a second language of their choosing. A translator will be provided for 
non-English speaking individuals as needed. If a translator is not available, the family will not be 
approached. 
 
4.2     Exclusion Criteria 
• 
Known major life-threatening anomalies (e.g. fetal diagnosis of brain, cardiac, or renal 
malformations 
• 
Known or suspected chromosomal anomalies 
• 
Severe hematologic crises such as clinically evident disseminated intravascular 
coagulopathy, twin-twin transfusion such that 1 twin is not eligible due to polycythemia or 
hydrops 
• 
Polycythemia (hematocrit > 65%) 
• 
Hydrops fetalis 
• 
Known congenital infection such as toxoplasmosis, CMV, rubella or syphilis.  
• 
Prior administration of erythropoietin to the baby 
• 
Infant is likely to die due to severity of illness or withdrawal of support is being considered 
prior to enrollment 
 
As part of standard care of an extremely preterm infant, a CBC will be obtained within the first 24 
hours of life. If the HCT is > 65%, the patient will not be eligible for the study. If the PLT count is 
< 50K, or if there are clinical signs of bleeding, a coagulation panel will be checked to determine 
whether there are signs of disseminated intravascular coagulopathy. If this condition exists, the 
patient is not eligible for the study.  
 
All patients eligible for this study will be hospitalized in the neonatal intensive care unit (NICU). 
Many concurrent illnesses are expected for this population of extremely preterm infants. These 
include, but are not limited to: ICH, periventricular leukomalacia (PVL), hydrocephalus (HC), 
respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), hypotension, patent 
ductus arteriosus (PDA), NEC, early and late onset sepsis, and retinopathy of prematurity (ROP). 
None of these will exclude a child from study entry, but all will be considered in the safety 
evaluation of Epo.  
 
4.3 Enrollment Procedures.  
 
4.3.1 Screening. Admissions to the antepartum unit and to the NICU at each site will be screened 

 
 
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at least daily for admissions that are potentially eligible for the trial. This will be done by the 
research coordinator or their designee. Once identified, the Attending health care provider will 
seek parental agreement for an investigator to meet and discuss the study. If the mother is 
interested in learning more about the study, the attending health care provider can notify the 
research coordinator or investigator so they can discuss the study with the parent and seek 
consent. When possible, and if there is IRB approval for this, consent will be obtained prior to 
delivery. Many mothers of preterm infants are admitted to an antepartum service for bed rest, 
fluids and observation. They are not acutely laboring, and the stress of imminent delivery is less 
acute. If permitted by the local IRB, these mothers will be approached during a time when they 
are able to fully evaluate the study and have opportunities for reflection and questions. 
 
4.3.2 A site screening log will be maintained to document patients screened, reasons for 
ineligibility, and reasons for nonparticipation of eligible subjects. This document will be maintained 
by the site research coordinator or their designee and will contain the following information: 
screening number, screening date, patient surname, medical record number, date of birth, 
gender, eligibility (yes/no), whether they were enrolled, and if not enrolled, the reason why. If a 
subject is enrolled, document that consent was obtained, and the study ID number. 
 
In addition, selected site screening information will be entered electronically and maintained in 
the PENUT Portal data management system. Entered data will not contain patient names or 
medical record numbers. This cumulative log will be reviewed at regular intervals to determine 
enrollment and reasons for non-enrollment.  This information will be used to consider whether 
other recruitment methods should be used.  
 
4.3.3 Consent procedures. Antenatal consent will be obtained when possible. The study 
investigator will obtain permission from the Maternal Fetal Medicine Attending to approach the 
mother to discuss the study. If consent is obtained prenatally, this will be re-confirmed at the time 
of the child’s birth if required by the local IRB. Postnatally, permission to approach the family will 
be obtained from the Attending Neonatologist. The Attending health care provider will seek 
parental agreement for an investigator to meet and discuss the study. If the parents are interested, 
the study investigator will discuss the study with family and seek consent in person. The 
consenting legal guardian will receive a copy of the consent form to review, and once signed, will 
be given a copy to keep. Ideally, if the Attending physician is also a study investigator, an alternate 
study investigator, or their designee, should obtain consent, so as to avoid the appearance of 
coercion. If this is not required by the site IRB, an investigator who is also the Attending physician 
may obtain consent. Investigators will only approach family after infant’s attending heath care 
provider gives permission and family indicates that they are interested in further information about 
the study. No alteration in care will otherwise occur. The attending neonatologist and family can 
withdraw child from study at any time. If permissible by the site IRB, phone consent may be 
obtained, but must be reaffirmed with the family when they are present. Investigator should retain 
original signed document. The investigator will obtain consent in a room which ensures the privacy 
of the family, and which is free of potential coercive influences. Consent for participation must be 
obtained before the baby is 24 hours of age. Consent for participation the continued phone 
contacts may be obtained at the 2-year in-person visit, or by phone. 
 
A certified interpreter will be provided if a family has limited or no English speaking abilities. They 
will review the consent form with the family, and interpret the verbal explanation of the study 
during the discussion between the Investigator and the family members. If individual sites have a 
large population of non-English speakers, consent forms will be translated into the appropriate 
languages. If an interpreter is not available in a timely manner, the family will not be approached. 
 

 
 
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The parents of the research participants will be given opportunity to review the study both verbally 
and in writing. They will be given opportunity to ask questions of the investigator prior to giving 
consent. A sample consent form is shown in Appendix 1. 
 
4.3.4  Randomization procedures. Site-specific study personnel associated with the PENUT 
Trial will screen and randomize patients by logging in to the PENUT Portal at www.penut-trial.org. 
Once it is confirmed that a patient meets inclusion criteria, does not have any exclusion criteria, 
and that consent and HIPAA forms have been signed, the DCC web page will generate the patient 
study ID for trial affiliated staff to provide to the site pharmacy at the time of study drug ordering. 
The site pharmacy will then look up the study ID in their binder and dispense the blinded study 
drug as requested.   
 
5. 
STUDY INTERVENTIONS 
5.1 
Interventions, Administration, and Duration 
All patients will be treated in the NICU. Each subject will be randomized to one of two groups after 
establishing eligibility and obtaining signed consent for study participation and HIPAA compliance. 
Interventions/Treatments will be as outlined in Table 4. Study drug will be administered according 
to the schedule shown in Figure 3. 
 
Cranial Ultrasound. After written consent is obtained, but before administration of study drug, a 
cranial ultrasound will be done to document whether an intracranial hemorrhage has occurred. 
Results of this examination will not affect clinical care, eligibility, or randomization for the study, 
but will be used as part of the safety analysis. Dr. Manjiri Dighe, a UW radiologist who will be 
blinded to the treatment groups and any clinical information about the child, will read the cranial 
ultrasounds. If a cranial ultrasound is indicated based on the patient's condition, it will be ordered 
by the caregiver on site and read by the site radiologist as part of clinical care. For more details 
please see the Cranial Ultrasound guidelines, Appendix 6. 
 
Phlebotomy: Subjects will have four timed blood samples drawn during the first two weeks of life 
(see Figure 3). The first blood draw may be obtained from cord blood, if available. At the two year 
follow-up visit, an additional (optional) blood sample of 0.5 mL will be drawn. 
Figure 3. 
 

 
 
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Cerebral Spinal Fluid (CSF): If a clinically-indicated spinal tap is ordered for an enrolled subject 
(e.g. to rule out meningitis), then residual CSF may be available for the PENUT study to collect.  
In that event, we request 0.15 mL CSF be collected and frozen for measurement of CSF Epo 
concentration in any infant who undergoes a diagnostic lumbar puncture during the initial 
hospitalization. If residual CSF is available for the PENUT study, then a small systemic blood 
sample (0.15 mL) will ideally also be collected within one hour of when the CSF sample is 
collected and processed to plasma as described above. These biologic specimens will be sent to 
the UW CCC lab for analysis. Diagnostic spinal taps provide a serendipitous opportunity to 
correlate circulating Epo/cytokine levels with CSF levels. If significantly elevated Epo 
concentrations are present, further analysis, either by Western blotting or MALDI TOF mass 
spectrometric measurement will be considered. If no blood can be obtained, CSF should still be 
collected and sent to the CCC. 
 
Brain MRI: A subset of 220 subjects will undergo a brain MRI at 36 weeks PMA. Prior to scanning, 
infants will be fed and swaddled. If needed, the patient can be immobilized using a MedVac Infant 
Vacuum Immobilization Bag (contour Fabricators, Inc., Fenton, Michigan), or comparable device 
as per usual site practices for neonatal MRI scans.119 Per usual site practice, ear plugs, and/or 
ear covers [MiniMuffs® (Natus Medical Inc., San Carlos CA)] will provide noise reduction. Exams 
will be rescheduled if the child cannot be quieted sufficiently for the exam. Subjects will be 
monitored with an MRI-compatible pulse oximeter. A caregiver trained in MRI procedures and 
neonatal resuscitation will be in attendance.  
 
Table 4. Phase III Trial 
Time Line 
Procedure 
Sites 
Control 
Epo  
0-24 hours from birth 
Consent 
All 
All 
All 
Consent to 24 h of age 
Randomization 
All 
All 
All 
Consent to 24 h of age 
Cranial Ultrasound 
All 
All 
All 
Consent to 24 h of age 
Baseline blood (0.5 mL) and 
urine sample (1 -2 mL) 
All 
All 
All 
Study day 1 
Epo dose 1 IV 
All 
Placebo 
Epo 1000 U/kg 
Study day 3 
Epo dose 2 IV 
All 
Placebo 
Epo 1000 U/kg 
Study day 5 
Epo dose 3 IV 
All 
Placebo 
Epo 1000 U/kg 
Study day 3-5 
Urine sample (1-2 mL) 
All 
All 
All 
Study day 7 
Epo dose 4 IV 
All 
Placebo 
Epo 1000 U/kg 
Study day 7 
Blood draw #2 (0.5 mL) 
All 
All 
All 
Study day 8 
Begin iron supplementation per 
guidelines 
All 
All 
All 
Study day 7-9 
Urine sample (1-2 mL) 
All 
All 
All 
Study day 9 
Blood draw #3 (0.5 mL) 
All 
All 
All 

 
 
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Epo Risks: In adults, complications of prolonged Epo treatment include polycythemia, seizures, 
hypertension, stroke, myocardial infarction, congestive heart failure, tumor progression, and 
shortened time to death.  
 
None of these adverse effects have been reported in Epo-treated neonates. No prospective 
studies of neonatal Epo treatment have reported group differences in the incidence of neonatal 
morbidities, including ICH, ROP, NEC, BPD, or late onset sepsis,120 but a Cochrane review 
reported an increased risk of ROP with early treatment.121 This report was recently revised, with 
no statistical increase in ROP noted for early or late Epo administration.122 One retrospective 
report also described an increase in cutaneous hemangiomas in Epo-treated preterm infants.123  
 
In the 2 pilot studies of high dose Epo administered to preterm infants done, one at the University 
of Washington Medical Center and one in Switzerland, no increased risk of any complications 
were noted in a total of 60 treated patients, including ROP.33, 35 In the UW study, we observed 5 
cases of severe ROP in 23 surviving controls compared to 6 of 24 Epo treated infants.35 
 
Study day 9 
Epo dose 5 IV 
All 
Placebo 
Epo 1000 U/kg 
Study day 11 
Epo dose 6 IV 
All 
Placebo 
Epo 1000 U/kg 
First Monday, 
Wednesday or Friday 
following IV doses 
Begin maintenance Epo vs. 
sham injections (nothing) 
All 
No injection 
Epo 400 U/kg 
Study day 14  1 day 
Blood draw #4 (0.5 mL), Urine 
sample (1-2 mL) 
All 
All 
All 
Study week 3, 4, 6, 8, 10, 
12 
Urine samples (1-2 mL) 
All 
All 
All 
Continue study drug until 
32-6/7 weeks PMA 
Maintenance Epo or sham 
injections (no shots) 
All 
No injection 
Epo 400 U/kg 
36-0/7 to 36-6/7 weeks  
MRI 
MRI sites 
110 per arm 
110 per arm 
Discharge 
Physical exam, Urine sample  
and questionnaire 
All 
All 
All 
4 m corrected age 
Phone contact 
All 
All 
All 
8 m corrected age 
Phone contact 
All 
All 
All 
12 m corrected age 
Phone contact 
All 
All 
All 
18 m corrected age 
Phone contact 
All 
All 
All 
22-26 m corrected age 
Bayley III, Neuro exam, M-
CHAT-R 
All 
All 
All 
22-26 m corrected age 
Weight, blood pressure, urine 
collection 
(1-2 
mL), 
and 
(optional) blood draw #5 (0.5 
mL) 
All 
All 
All 
30 m chronological age 
Phone contact 
All 
All 
All 
36 m chronological age 
Phone contact 
All 
All 
All 
42 m chronological age 
Phone contact 
All 
All 
All 
48 m chronological age 
Phone contact 
All 
All 
All 
52 m chronological age 
Phone contact 
All 
All 
All 
60 m chronological age 
Phone contact 
All 
All 
All 

 
 
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Safety data from a prospective randomized controlled phase II trial of high dose Epo in 
Switzerland (ClinicalTrials.gov: NCT00413946), in which 3000 U/kg daily was given to infants 26-
0/7 to 31-6/7 weeks of gestation at 3, 12-18 and 36-42 hours after birth (N=229 treated, 214 
controls) has been reassuring.  There were no relevant differences between the groups for short-
term outcomes including mortality, ROP, IVH, sepsis, NEC, and BPD.124 Higher hematocrit was 
noted at day 7-10 in the treated group. Improved white matter was also noted at term corrected 
age by MRI.125, 126 
  
Three updated Cochrane reviews have been published reviewing the safety and efficacy of Epo 
for erythropoietic purposes,122, 127, 128 and another meta-analysis focused specifically on Epo and 
ROP.129 Conclusions from these reviews are as follows: Epo, whether used early or late, reduces 
the use of one or more RBC transfusions, and the number of RBC transfusions per infant. No 
increase in any of the complications of prematurity are noted, i.e. use of erythropoietic doses of 
Epo are safe in preterm infants. The safety of higher, neuroprotective doses has not yet been 
systematically evaluated in large population studies. 
 
Epo is a potent erythropoietic growth factor. Thus, high doses of Epo given for neuroprotective 
treatment might be expected to increase erythropoiesis, and possibly megakaryocytopoiesis. In 
neonatal rats, there is a transient increase in hematocrit following high-dose Epo,62 but in preterm 
infants, while 3 doses of high dose Epo increased reticulocytosis, they did not affect hematocrit, 
likely due to early phlebotomy losses.35 In term infants with neonatal encephalopathy, after up to 
6 doses of high dose Epo, hematocrit fell an average of 14%, reflecting phlebotomy losses in 
these sick infants.130 The effect of brief treatments of high-dose Epo on iron balance is not known. 
Prolonged Epo treatment in neonates must be accompanied by iron supplementation, and does 
improve erythropoiesis.116 This is, in fact, currently the primary indication for Epo use in neonates. 
Dosing of 400 U/kg/dose three times a week has been shown to decrease (but not eliminate) 
need for blood transfusions.131 
 
1) Safety during the high dose treatment period. Serious adverse events (SAEs) specific to 
Epo will be defined as any of the following during the treatment period:  
• 
Hypertension: subject required prolonged antihypertensive therapy (> 1 month) and/or will 
be discharged on medication 
 
Polycythemia (central hematocrit > 65%, or hematocrit increase ≥ 15% not due to red blood 
cell transfusion) 
 
Major venous or arterial thrombosis (involving a major vessel not related to an infusion line, 
requiring anticoagulation, or symptomatic such as causing superior vena cava syndrome) 
 
Serious adverse events most likely related to prematurity will include 
 
Severe pulmonary hemorrhage (severe, e.g., need for increased respiratory support, positive 
pressure. Not pink tinged secretions with suctioning). 
 
Necrotizing enterocolitis (stage 2B or 3) 
 
Severe sepsis: Blood culture-proven bacterial or fungal sepsis requiring blood pressure 
support or significant new respiratory support.  
 
Intracranial hemorrhage (grade III or IV) 
 
ROP requiring intervention (surgery or Avastin). 
 
ROP will be included as a severe adverse event, although due to the timing of screening, which 
begins at 31 weeks, it will most likely be too late to discontinue treatment. Each case of ROP will 
be reviewed in real time. Subjects will be screened for ROP as per the 2006 guidelines: for 24-27 
week gestation infants, the first screening will occur at 31 weeks PMA.132 The timing of follow-up 

 
 
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ROP exams will be determined by findings on the initial exam. 
 
Resolution of SAEs: 
 
NEC 
-When infant is able to take full feeds, or what is considered equivalent to full feeding for 
that particular infant. If the child does not make it to full feedings due to short gut, note 
the method of feeding at the time of discharge (e.g. Home TPN) 
 
 
ROP 
-When an infant is diagnosed by an ophthalmologist exam with mature vessels 
-They no longer require frequent follow up ophthalmology appointments  
 
 
IVH 
-When shunt placement has occurred.  
-When sequential cranial ultrasounds show the ventricle size is stable or getting smaller. 
 
 
Hypertension 
-When hypertensive medications are discontinued 
-If infant is discharged on antihypertensive medications  
 
 
Sepsis 
-When antibiotics are completed. 
 
 
Thrombosis 
-When anticoagulation treatment ends. 
 
 
Pulmonary Hemorrhage 
-When no more blood in tracheal secretions and with stable vent settings. 
 
Additional serious events which may be expected or unexpected include: 
 
Death 
 
Cardiac arrest 
 
Other unexpected life threatening event 
 

 
 
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The following flow diagram will be used to determine how to label an adverse event. 
Criteria for Temporarily Withholding/Stopping Study Drug and/or Supplemental Iron.  
1. Polycythemia: Central hematocrit (Hct) > 65%: Study drug should be held until Hct is < 55%. 
2. Severe sepsis: Blood culture-proven bacterial or fungal sepsis requiring blood pressure 
support or significant new respiratory support. Supplemental iron should be held until blood 
culture is negative for 72 hours. Rationale: Iron has been reported to be permissive for 
selected gram-negative bacteria and might worsen the patient's condition during sepsis. 
There is no known relationship between Epo and sepsis, so study drug does not need to 
be held. 
 3. Unexplained recurrent seizure (unrelated to ICH, PVL or other known pathology): Study 
drug should be held until seizures are well controlled by medication. Restarting study drug 
will be determined by the Medical Monitor, DSMB, and CCC. 
4. Major venous or arterial thrombosis (clot).  
a. Study drug should be held for any thrombosis that is treated with a course of 
anticoagulation. 
b. Study drug should be held for any symptomatic thrombosis involving a major vessel 
(e.g. symptoms such as superior vena cava syndrome) 
AdverseEvent
Serious
NotSerious
SeriousAdverseEvent
(SAE)
AdverseEvent
(AE)
Epo
Related
NotEpo
Related
Epo
Related
NotEpo
Related
Serious
Adverse
Reac on(SAR)
Serous
AdverseEvent
(SAE)
Adverse
Reac on(AR)
AdverseEvent
(AE)
Expected
Unexpected
SeriousAdverseReac on(SAR)
SuspectedUnexpectedSerious
AdverseReac on(SUSAR)

 
 
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5. Sustained hypertension requiring medical intervention: Study drug should be 
discontinued if blood pressure requires treatment.  When blood pressure returns to normal 
range (systolic blood pressure < 100 mmHg), study drug can be resumed. This is true even 
if patient is still being treated, but blood pressure is being controlled.   
 
In the event that study drug is discontinued in any subject, sites will encourage parents to 
continue to allow their child to participate in the follow-up evaluations. 
 
2) Safety during the initial hospitalization. Due to the inherent vulnerability of this patient 
population, the rate of comorbidity is high, particularly in the most immature infants. The most 
current week-specific data for large populations of similar infants are provided by the neonatal 
research network (9575 infants of extremely low GA (22–28 weeks),5 and by the ELGAN study 
group (1506 infants 23-27 weeks).43, 133-136 We will use standard definitions of respiratory distress 
syndrome (RDS), BPD, ICH, PVL, early-onset and late-onset sepsis, NEC, patent ductus 
arteriosus (PDA), clinically diagnosed seizures (confirmed by EEG), hypertension, and ROP.5 We 
will track the incidence of each of these disorders for all enrolled subjects using REDCap data 
entry forms.  
 
Prior to study drug dosing, study staff will check subject’s medical record for SAEs and 
criteria for temporarily withholding/stopping study drug and/or supplemental iron.   
 
3) Long term outcomes. During the phone interviews that will occur at 4, 8, 12, 18, 30, 36, 42, 
48, 54, 60 months, and at the face-to-face 2 year (22-26 months) corrected age visit, data will be 
obtained regarding interval hospitalizations, medication use, resource utilization, medical 
diagnoses, and oxygen requirement. At 12 months, parents will also be queried about their child’s 
development.  At two years corrected age, weight, blood pressure, and neurodevelopmental 
outcomes will be documented (neurologic exam, Bayley III scores, and M-CHAT-R evaluation). 
Urine and blood will also be collected at this time. 
 
5.2 
Handling of Study Interventions 
Procrit will be utilized for this study (manufactured by Amgen, distributed by Centocor Ortho 
Biotech). 
 
Study Drug Procurement by Site Research Pharmacies 
Investigational Drug Services or corresponding pharmacy services at each site will purchase 
single dose, preservative free vials of PROCRIT, Epoetin alfa, 2000 Units/mL (NDC 59676-302-
01) for use on this study.  An inventory of drug will be purchased specifically for this study. The 
invoices for the purchase of the Procrit will be retained at the site in the same manner that a study 
drug shipping receipt would be retained.  A copy of the invoice and the drug accountability logs 
must be submitted for reimbursement at approximately six month intervals by pharmacy.  
Because of the blinded nature of the PENUT Trial, the pharmacy must send this information 
directly to the CCC financial coordinator.  The study investigator(s) and coordinator(s) cannot 
review this information.   
 
Single-dose, Preservative-free Vial: Each 1 mL of solution contains 2000 Units of Epoetin alfa, 
2.5 mg Albumin (human), 5.8 mg sodium citrate, 5.8 mg sodium chloride, and 0.06 mg citric acid 
in Water for Injection, USP (pH 6.9 ± 0.3). This formulation contains no preservative. 
 
Preservative free normal saline will be used as placebo for the first 6 doses, and will be provided 
by local sites.  

 
 
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Blinding/Drug Administration. 
After enrollment, the DCC will provide randomization information to the site pharmacy. The site 
pharmacy will then dispense study drug in a closed container. Epo and placebo (saline) are the 
same in appearance, and will be labeled “study drug”. The first 6 doses of study drug will be 
administered IV push followed by 1 mL saline flush given over 2 minutes. These IV doses can be 
administered either by the bedside nurse or a research nurse since the study drug will be blinded 
by the pharmacy. 
 
Maintenance Epo will be administered by subcutaneous injection using a 27 or 30 gauge needle 
on Monday, Wednesday and Friday, beginning on the day closest to completing the high dose 
series. To maintain the blind, pharmacy will dispense “PENUT STUDY drug” in a syringe to all 
infants. Control infants will not undergo any maintenance injections. A nurse who is not directly 
involved with the care of the patient, and who will maintain confidentiality will administer 
treatments behind curtains or blinds. A cohort of charge nurses, or General Clinical Research 
Center (GCRC) nurses would be ideal for this task. After injection or sham injection, all infants 
will have the injection site covered by a 2 x 2 gauze and wrapped with a gauze bandage, which 
is secured by taping it to itself.  The time, date, and study drug intervention will be documented 
for each drug administration event. 
 
Exceptional situations: 
 
If a dose of study drug is unavoidably delayed or missed, the dose should be given as soon 
as possible. This should be recorded as a study deviation. The normal schedule of 
administration should then be resumed. At least 24 hours must separate the high dose study 
drug doses. This allows the plasma Epo concentration to return to baseline. 
 
 
If severe edema (anasarca) or other circumstances prohibit the use of subcutaneous 
injection, up to three doses of Epo or saline placebo may be given intravenously using the 
normal IV protocol. In this case, pharmacy must be notified so they can provide an 
appropriate study drug formulation for IV use (saline rather than an empty syringe used for 
sham injection).  This should not be recorded as a deviation for up to 3 doses.  Subsequent 
IV doses would be considered a deviation. 
 
 
If the baby’s IV is removed prior to completing the 6 intravenous study drug doses, the dose 
can be administered subcutaneously.  This should be recorded as a study deviation. 
 
The risks of intravenous administration of medication include infiltration, infection, and possible 
bleeding. The risks of subcutaneous injections include bruising, pain at the site of injection, and 
infection. 
 
Iron supplementation: Iron supplements (oral and intravenous preparations) will be obtained  
from each site pharmacy as per their usual practice.  
 
Iron Treatment Plan. Please see Appendix 7.  
Maintaining iron sufficiency in a growing preterm infant is important for normal brain growth. 
Because of this, we have established iron guidelines that are to be followed at all sites. When 
enteral feedings are started, a standard iron containing formula will be used if breast milk is 
unavailable. Once infants (all subjects) have an enteral intake of 60 mL/kg/d and are at least one 
week old, they will be started on enteral iron at a dose of 3 mg/kg/d total. Enteral iron will be 
increased to 6 mg/kg/d when infants achieve an enteral intake of 100 to 120 mL/kg/d.137 This is a 
well-tolerated dose that helps to prevent iron deficiency in preterm infants.137 Serum ferritin or 

 
 
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ZnPP/H ratios should be checked at 14 and 42 days, and iron adjusted accordingly. If the ferritin 
is not in the desired range at the 14 day check, please adjust the iron dose and recheck ferritin 
(or ZnPP/H) at 28 days (see figure). If subjects are not able to tolerate enteral feedings and oral 
iron supplements, they will be given maintenance iron parenterally as noted in Appendix 7. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
We estimate that less than 20% of babies will receive one parenteral dose of iron, since feedings 
are most often started by day 2 or 3, and advanced by 10 to 20 mL/kg/day. More than two 
parenteral doses will be rare. If the child is NPO for a prolonged period, parenteral iron dosing 
should be adjusted based on serum ferritin or ZnPP/H.  
 
5.3 
Concomitant Interventions 
Infants < 27-6/7 weeks of gestation require treatment for associated problems of prematurity. 
Interventions include, but are not limited to, mechanical ventilation, sedation, treatment of 
seizures, treatment of PDA either medically or surgically, treatment of sepsis (bacterial or fungal), 
transfusions with packed red blood cells or platelets, treatment of NEC either medically or 
surgically, and assessment and treatment for ROP. Preterm infants are also at risk for acute 
kidney injury and late hypertension. Data will be collected on associated conditions and 
medications used to treat them during the course of the subject enrollment. 
 
5.3.1  Required Interventions. 1) Epo/placebo treatment as designated by randomization; 2) 
cranial ultrasound within 6 hours of (preferably prior to) first study drug dose; 3) iron 
supplementation; 4) blood drawing 2.5 mL for Epo and biomarkers plus up to 1.2 mL for iron 
studies; 11 urine samples obtained at baseline and study days 3-5, 7-9, 14±1 day, then during 
weeks 3, 4, 6, 8, 10 and 12, and at the 2 year follow-up; 5) MRI at 36 weeks PMA depending on 
study site; neurodevelopmental follow-up at 22-26 months corrected age at all study sites. With 
parental permission, ongoing follow-up will be obtained by phone at 6-month intervals. 
 
 

 
 
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5.3.2  Prohibited Interventions. Use of any erythropoietic agents such as Epo or Darbepoetin 
off protocol is prohibited for subjects enrolled in this trial up to 44 weeks PMA. 
 
5.3.3  Precautionary Interventions. The attending physician or responsible family member can 
remove a subject from the study at any time. If a subject develops any SAE presumed to be 
related to study drug, study drug will be discontinued as determined by the site PI and the CCC 
PI in consultation with the Medical Monitor. 
 
5.4 
Adherence Assessment 
All medication dosing will occur in the NICU and will be documented by the nurse and the 
pharmacy. Accuracy and compliance will be assessed by measured Epo concentrations drawn 
as shown in Figure 3. 
 
A cranial ultrasound will be done before (or within 6 hours of) initial study drug dosing and will be 
logged into the PENUT Portal. 
 
Case report forms (CRF) will be completed and entered into the PENUT Portal: baseline, high 
dose and discharge. 
 
In a subset of 220 subjects, a brain MRI will occur prior to discharge at 36 weeks PMA, and will 
be documented. The research study coordinator will log phone contacts after discharge, and the 
follow-up assessments will be documented on study forms, which will be submitted electronically 
to REDCap.  
 
All contact with families after discharge will be recorded in the Follow-Up CRFs.  The follow-up 
phone calls at 4, 8, 12, and 18 months corrected age will assess medical health and functional 
condition and remind parents of need for neurodevelopmental assessment. Attendance at follow-
up clinic for the in-person 2 year corrected age assessment will be documented. 
 
Protocol Violation/Deviation Reporting.  
Violations of the study drug administration protocol require completion of a Study Deviations and 
Violations form and notification to the CCC and DCC within 7 working days. Protocol deviations 
should also be recorded on the Study Deviations and Violations form. 
 
Major protocol violations include: enrollment in light of exclusion criteria, consent obtained not in 
accordance with IRB guidelines, unblinding of study personnel, study drug administration or 
dosing error. 
 
Study deviations include: failure to administer all doses of study drug, study drug administration 
deviated from PENUT protocol, no baseline cranial ultrasound done, no baseline blood drawn, 
baseline cranial ultrasound delayed > 6 hours after 1st dose study drug and first study drug dose 
give > 26 hours of life (24 hours plus 2 hours of wiggle room). 

 
 
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6. 
CLINICAL AND LABORATORY EVALUATIONS 
6.1   
Schedule of Evaluations 
Table 5. Evaluation 
Screening 
Pre-Entry 
(Prior to 24 hrs) 
Entry 
1-7 day 
7-10 days 
12-15 days 
28-42nd days 
32 weeks PMA 
34-36 weeks PMA 
Discharge 
4 months CA 
8 months CA 
12 months CA 
18 months CA 
24-26 months CA 
30 months  
36 months 
42 months 
48 months 
52 months 
60 months 
Screen 
admissions 
to 
antepartum unit and NICU 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Documentation of Gestational 
Age 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed Consent 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Maternal 
Demographics 
and 
History 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Screening Cranial ultrasounds 
 
 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Blood Pressure  
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
X 
 
 
 
 
 
 
Urine collection 
 
 
 
X 
X 
X 
X
X 
X
X 
X 
X 
X 
 
 
 
 
X 
 
 
 
 
 
 
Epo concentration 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adherence Assessments 
 
 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Stored Plasma for biomarkers 
 
 
 
X 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ferritin or ZnPP/H 
 
 
 
 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRI (subset of 220 subjects) 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Hematology 
(CBC/transfusions/phlebotomy 
loss) as available from clinical 
care 
 
 
 
X 
 
 
X 
X 
X 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Liver/kidney Function Tests (as 
clinically available) 
 
 
 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 

 
 
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Complete Physical Exam 
including weight, head 
circumference and length 
 
 
 
X 
 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Telephone contact to review 
current status 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
Comprehensive 
Neurodevelopmental 
Assessment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 

Page 27 of 75 
PENUT Protocol V6 October 14, 2019 
 
6.2 
Data to be collected on Each Subject 
Gestational age, birth weight, maternal history, placenta histology (intrauterine infection, insufficiency), Apgar 
scores at 1, 5 and 10 minutes, will be documented in addition to measures of maternal education, SES, and 
other factors known to affect cognitive development (e.g. drug/alcohol exposure). NINDS common language will 
be used when possible. A cranial ultrasound will be obtained prior to study drug dosing. 
 
6.3   
Timing of Evaluations 
Data collection for hematology, chemistry, liver and kidney function will be as clinically available. These tests are 
done routinely in all extremely preterm infants as part of usual care, and will not be required as part of the study 
protocol. The research nurse or designee will collect these results from the medical record. 
 
Cranial ultrasounds at baseline, 7-10 days and at 36 weeks PMA (30 days of age or later is acceptable) will be 
sent to UW CCC, as will all brain MRIs. Analysis will be done using the most severe lesion noted. 
 
6.3.1  Pre-Randomization Evaluations 
These evaluations occur prior to the subject receiving any study interventions.  
 
Screening  
If appropriate, families will be approached for study entry while on the antepartum service, if delivery seems 
reasonably imminent. All NICU admissions will also be screened daily for eligible patients. 
 
Pre-Entry 
After birth, consent must be obtained by 24 hours of age to be eligible for study participation.  The time 
window for initiation of study intervention from birth is 24 hours. 
 
6.3.2  On-Study/On-Intervention Evaluations 
After consent is obtained, a cranial ultrasound must be obtained prior to, or within 6 hours, of the first study drug 
dose. Results of the cranial ultrasound will not affect randomization or clinical care. These data will be used for 
later analysis of outcomes only.  
 
Physical exam will be done at study entry, after the first 6 doses of intravenous study drug, and at discharge. 
Complete blood counts (CBC) drawn as part of clinical care will be recorded, as will laboratory evaluations of 
renal and hepatic function. 
 
Blood draws: Blood draws are timed according to Study drug dosing (see Figure 3). All subjects will have 5 
blood draws (0.5 mL volume each) as part of this study. This blood will be used to determine plasma Epo 
concentrations, biomarkers of brain injury, and the effect of Epo on systemic inflammatory response. The cell 
pellets will be frozen and saved for future studies that are beyond the scope of this study.  A final (optional) blood 
draw will be obtained at the 2-year follow-up visit.  This blood will be evaluated for renal function for the purposes 
of identifying markers of kidney injury at follow-up. 
 
Collection Schedule  
1. Day 0: Baseline - prior to first study drug dose (may use cord blood) 
2. Day 7: 30 min after the 4th study drug dose (peak) 
3. Day 9: 30 min prior to the 5th study drug dose (trough) 
4. Day 14 ± 1 day: Denote the time of draw on sample relative to drug dosing 
5. 22-26 months corrected age 
 
Blood draws will be synchronized with the clinically needed blood samples when possible. Epo measurements 
will be done in all subjects, and cytokine measurements (Aim 3) will be done in the subset of 220 subjects (110 
per arm) who will also be undergoing an MRI at 36 weeks PMA.  
 

 
 
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Following serum ferritin (0.4 mL) or ZnPP/H (0.1 mL) to assess iron status is strongly recommended beginning 
at 14 days. If these lab values are outside the normal range, it is recommended that iron supplementation be 
adjusted, and repeat labs (ferritin or ZnPP/H) be followed more closely (at 2-week intervals). This would increase 
the total blood volume drawn for study purposes by 0.2 (if ZnPP/H is assessed) to 0.8 additional mL of blood (if 
ferritin is assessed). These volumes (Maximum of 2.8 mL) are within the IRB recommendations for infants of this 
weight: “Existing guidelines for blood sample volume limits (ranging from 1–5% of total blood volume within 24 
hours and up to 10% of total blood volume over 8 weeks) are consistent with the limited evidence available on 
“minimal risk” to children”.138 Given that the blood volume of premature infants ranges from 80 to 85 mL/kg, their 
total blood volume would range from 40 mL (80 x 0.5 Kg) to 85 mL (85 x 1 Kg). The maximum amount of blood 
to be drawn for study purposes is 2 + 0.8 mL = 2.8 mL, which is within the guidelines. 
 
Blood draw precautions: If there is a venous or arterial catheter in place appropriate for blood drawing, this will 
be used preferentially. If no access is available, standard neonatal phlebotomy techniques will be used.  
 
Urine collections 
Rationale: Little is known about acute kidney injury (AKI) in extremely preterm infants. Serum creatinine (SCr) 
and urine output are not ideal markers to define AKI, especially in neonates. Even in adults, the increase in SCr 
is known to be a relatively late occurrence. The use of SCr in neonates is complicated by the fact that in the first 
few days of life, it reflects maternal SCr. 
 
Urine biomarkers of cell injury/repair are felt to have great potential. However, before these novel biomarkers 
can be used clinically, we must evaluate their ability to predict a SCr rise, as well as short and long-term clinical 
outcomes. The goal of collecting detailed information about renal function in these infants is ultimately to improve 
the ability to diagnose AKI, and to better understand the epidemiology and pathophysiology of AKI in extremely 
low gestational age neonates. BUN and creatinine are commonly checked as part of routine care of extreme 
preterm infants. When obtained for clinical indications, these values will be recorded. 
 
Neonatal AKI has been classically defined by oliguria/anuria and/or a persistent SCr elevation. We will use 
categorical definitions to make the diagnosis of AKI, similar to definitions published by KDIGO (2012) 
http://www.kdigo.org and the AKIN groups (2007). Jetton, et al., proposed a modification of the adult Acute 
Kidney Injury Network Criteria (AKIN) definition of AKI as a standardized definition of AKI for neonates based on 
a rise in SCr from a previously documented low, rather than on an absolute SCr threshold. 
 
Stage 0: 
No change or rise < 0.3 mg/dL 
Stage 1: 
Increase in SCr 0.3 mg/dL or a SCr 150–200% from previous trough value 
Stage 2: 
Increase in SCr 201–300% from previous trough value 
Stage 3: 
Increase in SCr > 300% from previous trough value or 2.5 mg/dL or need for dialysis 
 
Urine will be obtained at timed intervals as shown in Figure 4. With the exception of the initial urine, which will 
be collected at the time of enrollment, if the date of urine collection falls on a Saturday or Sunday, the urine will 
be collected on the closest weekday.  Do not delay study drug dosing if no urine is available at enrollment, but 
still obtain first urine available.  Urine samples are requested at the time of enrollment, on day 3 to 5, day 7 to 9, 
with a preference for day 7 if that is possible, day 14 ± 1 day, and then during week 3, 4, 6, 8, 10 and 12. If 
possible, pick one day of the week to collect urines, and do this consistently. This is to minimize the work for the 
site coordinator as much as possible.  

 
 
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The urine samples will be banked for future analyses of biomarkers (neutrophil gelatinase associated lipocalin 
(NGAL), KIM-1, osteopontin (OPN), cystatin C (CysC), uromodulin (UMOD), epithelial growth factor (EGF) and 
urine β2-Microglobulin (β2MG).  There are increasing data in older children as to the validity of such markers for 
diagnosis of AKI, but very little data in preterm neonates.  Additionally, prior to including these markers as part 
of the neonatal AKI definition, more knowledge is needed on other clinical factors which may affect urinary 
concentrations of these biomarkers independent of renal function (e.g. inflammation, NEC or other neonatal 
specific conditions).  Advantages to eventually including novel renal injury biomarkers in a future definition of 
neonatal AKI are that they would ideally represent actual renal tubular injury versus the solely functional markers 
of AKI currently used. 
 
Urine will be collected by placing cotton balls in the infant’s diaper with a diaper change.  (If needed, a bag can 
be used to collect urine instead of cotton balls.)  At the next diaper change, these cotton balls will be placed in 
an empty 10 mL syringe and the plunger will be used to squeeze the urine out into collection tubes.  The collection 
tubes will be labeled with the infant’s study ID number, and date. The urine will then be frozen and sent in batches 
to the University of Washington for further processing and analysis. 
 
Brain Imaging in Premature Infants.  Please see Appendix 6. 
The review titled: “Practice parameter: neuroimaging of the neonate: report of the Quality Standards 
Subcommittee of the American Academy of Neurology and the Practice Committee of the Child Neurology 
Society” published in 2002 gave recommendations for the timing and grading of ultrasound finings.139 Cranial 
ultrasounds are recommended at day 7-10 and at 36-0/7 to 36-6/7 weeks PMA or prior to discharge if this occurs 
earlier.  Our study guidelines are modified from these recommendations in that an additional research cranial 
ultrasound will be done on day 1, prior to (or within 6 hours of) the first study drug administration.  While our 
guidelines request a late cranial ultrasound or MRI be done at 36 weeks PMA, we will accept a CUS done after 
30 days of age.  
 
Magnetic Resonance Imaging.  Please see Appendix 2 for pulse sequences. 
MRI’s will be obtained on 110 subjects from each study arm at 36 weeks PMA (220 total). All scans will be done 
on a 3T Siemens or 3T Philips scanner using an optimized protocol determined by the Imaging committee. These 
scanners are available at the University of Florida in Gainesville, Florida Hospital for Children in Orlando, 
Methodist Children’s Hospital in San Antonio, University of Minnesota in Minneapolis, Wake Forest School of 
Medicine in Winston-Salem, Johns Hopkins in Baltimore, University of Washington in Seattle and Maria Fareri 
Children’s Hospital in Valhalla.  (Please see Imaging SOP.) 
 
Figure 4.  
 

 
 
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Automated Developing Tissue Segmentation: Analyses will be based on automated processing steps 
developed specifically for analysis of early developing brain anatomy, as part of separate on-going R01 projects 
on fetal and premature neonatal brain image analysis, directed by Colin Studholme, PhD (R01 NS 061957 and 
R01 NS 055064). Validated automated techniques will segment gray matter, myelinated, and un-myelinated 
white matter using 3D T1W MRI data. These make use of age-specific spatial statistical priors synthesized from 
a computational spatio-temporal atlas140, 141 that models age-related variations in tissue volume, tissue 
probability, and MRI contrast for an automated expectation maximization based labeling scheme, illustrated in 
Figure 5.  
 
Following tissue segmentation, anatomical regions will be automatically labeled (Figure 6) using fine scale 
unbiased spatial normalization to form an unbiased minimum deformation group average anatomy. 
 
Spatial Normalization for population based analyses. Accurate spatial normalization of developing anatomies 
allows population-based mapping of structural differences in subjects. For structural image analysis we will use 
template-free, symmetric, group wise, spatial normalization of anatomies to form an unbiased average anatomy 
from all scans being studied. This will make use of accurate, automated developing brain tissue segmentation 
techniques to align only boundaries present in each subject's anatomy and exclude focal pathological variations 
in tissue contrast such as blood clots that may be present in some anatomies and potentially induce incorrect 
mappings between brain structures. 
 
Evaluation of Global and Regional Tissue Growth (GRTG) summary measures. We will first use the 
automated tissue segmentations to evaluate summary measures for global and regional growth rates in each 
subject for conventional statistical analyses. Gray matter, myelinated white matter and unmyelinated white matter 
will be estimated by integrating tissue label probabilities over the whole brain and within a set of pre-defined 
Figure 5. Preliminary results using a specialized template driven expectation maximization (EM) scheme 
(left) to label T1W MRI data of a premature neonate (right) with developed and developing tissues (cyan-
myelinated white matter boundary, blue-unmyelinated white matter boundary, red-CSF boundary 
Figure 6: Left: Example delineation of the preterm cerebellum to create a set of 10 
reference MRI scans with delineated anatomy. Right: 3D Rendering illustrating initial 
gyral marking protocols and techniques applied to a pair of MRI studies of a single infant 
acquired at 32 and 38 wks PMA.  
 

 
 
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brain regions. Each MRI will be automatically labeled with an optimized patch-based brain labeling technique142 
based on a subset of manually delineated MRI scans that are aligned to form a statistical model of the labeling. 
The labeling approach combines multi-atlas based methods with patch-based techniques to optimally assign 
region labels to each new individual MRI scan. The resulting label maps combined with tissue segmentations 
provide summary measures that can be analyzed using linear modeling described in more detail by the DCC. 
 
Evaluation of patterns of tissue growth using Tensor-Based Morphometry (TBM) of Structural MRI. We 
will use TBM to look for spatially localized biomarkers of abnormal brain tissue growth across the population 
using techniques developed and validated in fetal brain imaging.44 The local volume changes induced by the 
mapping from the group average to each individual brain will be used to characterize the developing brain 
anatomies. These will be evaluated from the Jacobian determinant of the deformation at each voxel.143 We will 
apply general linear modeling to analyze these maps to account for age and other covariates together with main 
outcome measures on a voxel-by-voxel level, generating regression coefficients and associated t-statistics at 
every voxel in the average anatomies by spatial parametric mapping.144 Correction for multiple comparisons will 
be achieved using an efficient parallel implementation of non-parametric permutation testing.145 This analysis 
permits a spatial hypothesis free exploration of how local tissue growth rates are related to outcome and 
treatment variables across the study population.  
 
Evaluation of Cortical Folding using Surface Curvature Mapping (SCM). We will use the automated 
segmentations of cortical tissue boundaries to evaluate the local and global state of neonatal brain folding to 
look for perturbations in cortical development, using techniques our group has previously used in both fetuses 
and premature neonates.146, 147 Each cortical surface will be tessellated into a locally topologically-correct 
triangular mesh148 and a set of normalized curvature measures will be evaluated at each point on the surface to 
provide direct quantitative measures of cortical evolution. Each subject’s surface folding map will be transformed 
into the unbiased population average anatomy using the deformations described above for TBM analysis. These 
are mapped onto a common tessellated average surface146 to allow statistical parametric mapping using a vertex-
by-vertex general linear model. As with TBM, this generates a map of regression coefficients and t-statistics 
(here on a surface of vertex locations), which is corrected using an efficient parallel implementation of non-
parametric permutation testing,145 again permitting a spatial, hypothesis-free exploration of how local and global 
cortical folding is related to outcome and treatment variables across the study population. Figure 6 shows an 
example of changes in surface folding over time in the developing fetus. 
 
MRI Analysis. All MRI images will be sent to UW CCC for analysis, where they will be will be converted to 
DICOM format and stored on the DICOM server.  Todd Richards, MD will write customized software to prepare 
data for analysis at the UW CCC site such as reformatting, resorting, and extraction of diffusion b-values b-
vectors for tensor calculations. He has already written b-values, b-vector extraction code for the Philips Acheiva 
scanners but will need to customize code for the Siemens scanners. Standardized phantoms (The Phantom 
Laboratory, Incorporated) will be used at each site to establish data integrity for quality control of MR data. 
 
Qualitative image analysis will be performed by the clinical neuroradiologist at each participating site so the 
information is available to the subject’s care team. For study purposes, each MRI will be evaluated by Drs. 
Dennis Shaw and Manjiri Dighe, who will be blinded to the treatment group, and all clinical data. Qualitative 
evaluation will be done using the recently published MR Imaging Assessment Tool created by the Inder group 
to define brain abnormalities in very preterm infants at term corrected age.149 This scoring system defines 
abnormalities of white matter, gray matter, and includes measures of hemorrhage, and brain loss. The table 
below is excerpted from this manuscript. 
 

 
 
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Quantitative analysis. We will use a range of highly sensitive automated techniques to quantitatively assess 
brain development at 36 weeks PMA using MRI analysis tools developed as part of on-going projects in the 
biomedical image computing group at UW (http://depts.washington.edu/bicg) led by Dr. Studholme. We will 
examine global and regional summary measures of tissue volume,150 cortical folding147 and microscopic water 
diffusion properties from each subject scan to examine whether the state of overall brain growth at 36 weeks 
PMA has been modified by the administration of Epo. We will then explore the possible presence of focal 
differences in growth patterns that may be related to specific functional deficits. This will use group wise spatial 
normalization to examine the spatial pattern of tissue volume increase,44 and water diffusion, together with 
patterns of cortical surface folding.146 
 
DTI Analysis: The methodology detailed below is currently used by our research group at the University of 
Washington’s Diagnostic Imaging Sciences Center (DISC).  
 
Preprocessing. DTI quantification will be preceded by head motion and eddy current correction using affine 
registration 
to 
a 
reference 
volume151 
with 
FDT 
(FMRIB’s 
Diffusion 
Toolbox; 
http://www.fmrib.ox.ac.uk/fsl/fdt/index.html). Using the field maps, B0-field inhomogeneity induced geometric 
distortion in the eddy current-corrected images will be corrected with PRELUDE (phase Region Expanding 
Labeller for Unwrapping Discrete Estimates)152 and FUGUE (FMRIB's Utility for Geometrically Unwarping EPIs; 
http://www.fmrib.ox.ac.uk.offcampus.lib.washington.edu/fsl/fugue/).  FSL software DTIFIT will be used to fit the 
diffusion tensor model at each voxel in order to calculate DTI Eigen vectors, Eigen values, fractional anisotropy, 
axial diffusivity, radial diffusivity, and mean diffusivity (http://www.fmrib.ox.ac.uk/fsl/fdt/fdt_dtifit.html). 
 
Development of 36 Week Infant Head Model for MRI. We will use software (MINC) 
developed 
at 
the 
Montreal 
Neurological 
Institute 
(http://www.bic.mni.mcgill.ca/ServicesSoftware/MINC) in order to co-register and 
combine 50 MRI structural brain images of normal infants at 36 weeks in order to make 
a head model to be used in DTI and VBM group comparisons Dr. Richards has 
experience using this software at I-LABS in collaboration with Dr. Patricia Kuhl where 
he used MINC to make a head model for 6 month old infant brain using 43 brains. 
 
Evaluation of white matter integrity using DTI. Group differences in fractional 
anisotropy (FA), axial diffusivity, radial diffusivity, and mean diffusivity (similar to ADC) 
will be determined using TBSS (Tract-Based Spatial Statistics).153 TBSS was 
Figure 7. TBSS  

 
 
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PENUT Protocol V6.1 October 14, 2019 
 
developed to conduct voxel wise analysis of multi-subject diffusion data utilizing improved non-linear registration 
techniques. An example of a 6 month old child studied at the University of Washington is shown in Figure 7. 
Data processing will be conducted according to the standard method detailed in the TBSS instruction manual 
(http://www.fmrib.ox.ac.uk/fsl/tbss/index.html). We plan to use Randomise, a permutation method, to test for 
between-group differences in these DTI measures. Correction for multiple comparisons will be done using whole 
brain cluster-based thresholding method:  voxel height, p < 0.01; cluster extent, p < 0.05. Our group is well 
published in this area of diffusion imaging, DTI and DTI analysis.154-157 Statistical comparison for TBSS will be 
performed using FSL-software called Randomise (http://www.fmrib.ox.ac.uk/fsl/randomise/index.html). This 
program will be used to perform a non-parametric voxel by voxel ANOVA (and correlations with clinical score) 
with multiple comparison correction using the Threshold-Free Cluster Enhancement option. Randomise is a 
permutation program enabling modeling and inference using standard General Linear Model design matrix setup 
using cluster-based tests. For more detail on permutation testing in neuroimaging see Nichols and Holmes.145 
The multiple comparison correction was performed based on cluster statistical characteristics such as extent of 
cluster size. Dr. Richards has written software to create the design matrix that will be used for both the ANOVA 
and clinical score correlations in software Randomise. 
 
As part of the overall QA effort, we will examine various measures of study implementation across sites. In 
particular, recruitment, retention, data completeness, and measurement precision will be tabulated and 
compared across sites and will be included in our web-based reports. QA efforts and site visits will be focused 
on any sites that show evidence of problems. 
 
Inflammatory Mediators. To test whether Epo treatment decreases serial measures of circulating inflammatory 
mediators and biomarkers of brain injury.  
 
Circulating Proteins:  
We will use Meso Scale Discovery (MSD) technology to measure the following inflammatory markers and growth 
factors: BDNF, Interferon-gamma (IFN-γ),82 Interleukin (IL)-1 β,158 IL-6, IL-8, IL-10, tumor necrosis factor-α (TNF-
α), transforming growth factor (TGF)-β, matrix metalloproteinase (MMP)-2 and MMP-9,159 macrophage 
inflammatory protein-1α (MIP-1α), MIP-1β136, monocyte chemotactic protein-1 (MCP-1)136  and tissue inhibitor of 
metalloproteinase (TIMP)-1.159 
 
Markers of neurotoxicity and brain injury will include: S100B, glial fibrillary acidic protein (GFAP), neuron 
specific enolase (NSE), Activin A, and Ubiquitin C-terminal hydrolase-L1 (UCH-L1).160-162  
 
A number of circulating proteins have been evaluated as potential biomarkers of brain injury. Promising 
biomarkers include the GFAP, Ubiquitin C-terminal hydrolase-L1 (UCH-L1), myelin basic protein (MBP), S100B, 
Activin A, and neuron-specific enolase (NSE), although the sensitivity, specificity and timing of elevation after 
injury in preterm infants have yet to be determined. All of these proteins, with varying levels of specificity, are 
elevated after brain injury in studies of adult or neonates. Due to the immaturity of myelination, MBP is unlikely 
to be useful in preterm infants. 
 
GFAP is a specific marker of differentiated astrocytes and increased circulating concentrations are detectable 
within hours and peak at up to four days after an ischemic stroke.160-163 Astrocyte foot processes make up the 
sub-endothelial component of the blood-brain barrier (BBB), so elevations of GFAP can also reflect loss of BBB 
integrity. UCHL1 expression is highly specific to neurons and to cells of the neuroendocrine system. Elevations 
have been demonstrated in term infants with HIE. S100B belongs to a family of calcium handling proteins and is 
localized predominately in glial cells in brain. It is thought to function in neurite extension, inhibition of PKC-
mediated phosphorylation, axonal proliferation, and inhibition of microtubule assembly. NSE is a glycolytic 
enzyme localized primarily to neurons. However NSE is also expressed in platelets and erythrocytes, so 
elevations may not be specific to brain injury. GFAP has not been studied in the context of preterm brain injury, 
but a direct comparison of GFAP, S100B, and NSE showed that GFAP was a superior biomarker of brain injury 
in the setting of traumatic brain injury.164  

 
 
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Activin A is a member of the transforming growth factor-β superfamily and is expressed by neurons. It is a trophic 
factor that regulates differentiation and proliferation of neurons.165  In a cohort of 53 infants, Florio, et al., showed 
that high concentrations of Activin A (> 0.8 mcg/L) in blood samples drawn during their first hour of life was highly 
correlated with ICH in preterm infants < 32 weeks gestation (100% sensitivity and 93% specificity, with positive 
predictive value of 79%).166, 167 Activin A is also increased in term newborns with moderate or severe asphyxia 
suggesting that activin is released after neuronal injury.168 
 
Population Epo Pharmacokinetic Analysis. We will measure timed Epo concentrations to monitor for 
accumulation and safety, and to confirm Epo dosing. Plasma Epo concentrations will be measured in duplicate 
using Meso Scale Discovery (MSD) technology at the UW Laboratory Core. Batched samples will be run every 
12-18 months. A population pharmacokinetic analysis will be performed using sparse plasma Epo concentration 
data. NONMEM software (Icon Development Solutions, Ellicott City, MD) will be used to determine 
compartmental population pharmacokinetic parameters (i.e., clearance and volume). The base structural model 
will be implemented as established in the population pharmacokinetic model developed from the intensive 
pharmacokinetic data of our pilot study.35 The random-effect model will be updated using the concentration data 
from this study. A covariate analysis will also be performed in a standard step-wise forward addition followed by 
backward elimination method to determine the influence of clinical factors (e.g. gestational age, birth weight, 
serum creatinine, concomitant medications) on pharmacokinetic parameters. Based on the final population 
model, post-hoc Bayesian individual pharmacokinetic parameters will then be estimated for each participant. 
Pharmacodynamic relationships will be determined between Epo pharmacokinetics and the clinical and 
biochemical markers measured during the investigation. 
 
Blood (0.5 mL/sample, 2.5 mL total) will be obtained through an indwelling umbilical arterial or venous catheter 
when possible. Samples will be spun for 10 min at 1000 g; plasma and cellular components will be frozen in 
separate labeled tubes at -70°C to -80°C. Samples will be labeled with a code provided by the DCC to maintain 
blinding. All samples will be sent to Dr. Juul’s lab at the University of Washington. Each site will be sent premade 
packages containing the instructions, data sheets, sample labels, blood collection tubes and mailing labels with 
which to process the blood samples (See Laboratory SOP).  
 
Neurodevelopmental Follow-Up: High-risk follow-up for ELGANs is part of standard care at all sites. Routine 
visits generally occur at 4 months, 8 months, 12 months, 18 months and two years corrected age. The timing of 
visits may vary depending on the child’s need for intervention and site-specific protocols. If subjects are not 
scheduled at any one of these time points for routine high risk follow-up, parents/guardians will be contacted by 
telephone at 4, 8, 12, 18, 30, 36, 42, 48, 54, and 60 months corrected age. The 30 minute phone contact will 
include an update of the subject’s medical problems, medications, use of services such as physical therapy. The 
timing of these call can be found in Table 5 and Appendix 3, “Follow-up manual,” for further details.   
 
The primary outcome will be neurodevelopment at 22-26 months corrected age. All personnel involved in the 
neurodevelopmental assessments will be blind to study treatment. Assessment at 22-26 months corrected age 
will include: 
 
 
Bayley III Scales of Infant and Toddler Development 
 
Standardized neurological examination based on ELGAN Neurological Exam Study protocol (Appendix 
3) 
 
The Gross Motor Function Classification System (GMFCS)  
 
Modified Checklist for Autism in Toddlers (M-CHAT-R) (Appendix 3) 
 
Bayley Scales of Infant Development (BSID) is the standard test used to evaluate early neurodevelopmental 
outcomes of preterm infants.13, 19, 169, 170 The second edition Bayley Scales, used from 1969 to 2005, was 
modestly predictive of cognitive function at school age with MDI < 70 showing a positive predictive value of 0.19 

 
 
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– 0.8 for identification of children with an IQ < 70 at school age.171-175 The cognitive portion of the Bayley test 
was revised in 2005176, 177 (Bayley III Scales of Infant Development): the Mental Development index (MDI) 
reported in the Bayley II was a composite of cognitive, expressive and receptive language; in the revised Bayley 
III, individual scores for cognitive, expressive and receptive language are reported in addition to an assessment 
of social-emotional, and adaptive behavior. As experience has accumulated, it has become apparent that 
children score higher on the Bayley III compared to the Bayley II.1, 177-183 Thus a cognitive score of 85 on the 
Bayley III likely corresponds to a Bayley II score of 70. Given that children assessed with the Bayley III are just 
now reaching school age, the value of the third edition of the Bayley Scales for prediction of cognitive impairment 
at school age and beyond is still under evaluation.177 In order to ensure that the outcomes of this study are 
clinically important, we have identified two important stepped outcomes: the primary outcome is very stringent, 
and uses a cut off of 2 standard deviations below the mean for cognitive or motor scales (< 70). The secondary 
outcome uses a cut off of one standard deviation below the mean for these criteria (< 85), which will still have a 
significant impact on the child, family, and healthcare system.  
 
Inter-Rater Reliability Training:  
Bayley Scales of Infant and Toddler Development, 3rd edition. Individuals performing the BSID-III assessments 
for this study will be required to be certified after attending a formal Training & Orientation Workshop 6 months 
before testing as organized by Drs. Michael O’Shea and Jean Lowe (run in tandem with a neurological training 
session). The certification process is comprised of two parts. First, each candidate Infant Assessor will review 
the test publisher’s “Enhanced Administration DVD,” which will be employed in this RCT as the “gold standard” 
for administration and scoring of the Bayley III. Second, the candidate Infant Assessor will administer the Bayley 
to healthy full-term infants who are 24 months old. Video-recordings will be made of these administrations. The 
candidate Assessor will score each of his/her pilot administrations. A copy of the candidate’s test protocol will 
be sent to the Study Psychologist (Jean Lowe, PhD) at the coordinating center, along with a copy of the video 
recording. The Study Psychologist will use these materials to assess the candidate’s ability to (a) engage the 
infant in the Bayley activities, (b) administer and score each Bayley item, and (c) compile all summary scores. 
Infant Assessors will be required to meet the calibration standard for certification: 88% score concordance on 
items correctly administered. The Study Psychologist will provide prompt item-specific feedback, so that the 
candidate can make any needed modifications. Once Infant Assessors have begun to see enrolled patients, we 
will ask each Assessor to record one Bayley administration after the first 10 patients, or after the first 6 months, 
and then semi-annually. These video-recordings will also be reviewed by the Study Psychologist to guard against 
“calibration drift” over time. Training for Bayley assessment staff will include a semi-annual assessment to ensure 
continued reliable and valid assessment data. For non-English speaking infants, we will use an interpreter who 
interprets each of the Assessor’s requests to the child.  
 
Neurological exam. To standardize the quality of data regarding neurological exams, examiners will attend a 
one-day workshop at 4 convenient hub locations in the U.S., view a training video, and then classify neurological 
findings illustrated on an assessment video.184 Inter-observer variability assessments will be done to determine 
agreement with gold standard responses. Annotated feedback will be given to examiners regarding items that 
had a less than 85% correct rate, and, based on experience in the ELGAN study, we expect agreement rate to 
rise to over 90%.43, 184 To minimize expectation bias, examiners will be blinded to the child’s medical history and 
brain-imaging studies. Findings from the neurological exam will serve as the basis for an algorithm that classifies 
infants into one of four groups: no CP, diparetic CP, hemiparetic CP, and quadriparetic CP.185 These 
categorizations correlate highly with long-term neurodevelopmental outcomes. We will use the neurologic exam 
forms created for the NINDS-funded ELGAN study (Appendix 3). In addition, CP severity will be determined 
using the Gross Motor Function Classification System (GMFCS) developed by Palisano.3, 4, 186, 187 A GMFCS 
score of 2 or greater will be considered severe CP, given that these children will not be able to walk independently 
at age 2. 
 
 
b) Standardized Diagnosis of Cerebral Palsy and Quantification of Motor Delay  
The presence and sub-type of CP will be determined using the broadly accepted and standardized ELGAN 

 
 
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Neurological Exam scoring system and CD based video teaching system.184, 185 This software and training 
program developed by the PENUT collaborator Dr. Karl Kuban provides a distributable method of neurologic 
testing of subjects in a formalized, systemized method that is highly reproducible.184 CP severity will be 
determined using the Gross Motor Function Classification System (GMFCS).4, 186, 187 
 
Rationale for diagnosing sub-types of CP: 
Although all forms of CP will be tabulated as part of the primary outcome, relationships between CP sub-types 
and cognitive development vary by the brain region affected and so we will classify CP into three sub-categories:  
- Spastic Quadriparesis: has a higher association with structural injury and quadriparetic children are 9-times 
more likely than diparetic children to be severely impaired and 5-times more likely than diparetic to be 
microcephalic. They are also twice as likely to have MDI < 70 and a high rate of positive M-CHAT-R scores vs. 
diparetic and children without CP. 
 
- Spastic Hemiparesis: represent an intermediate level of injury where 53% of this form of CP have Bayley < 70 
vs. 75% for quadriparesis and only 34% for diparesis; consistent with a more common association with unilateral 
injury and focal lesions like middle cerebral artery stroke, that spare fibers closest to the ventricle in control of 
lower extremities. 
 
- Spastic Diparesis: Diparesis is more commonly associated with symmetrical white matter injury close to the 
ventricle presumable due to injury of leg fibers of the pyramidal system that are closest to ventricles.  
 
Gross Motor Function Classification System: The Gross Motor Function Classification System (GMFCS) 
system developed by Palisano, et al. focuses on children's functional achievements rather than on their 
limitations. It places emphasis on the child’s routine performance (not necessarily their best capacity) in the 
home or community setting.  
 
The GMFCS system uses descriptions defining 5 levels of function that represent a broad ordinal scale where 
the distance between levels is not considered equal. Distinctions between levels are based on the need for 
assistive technology including mobility devices (such as walkers and wheeled mobility), and to a much lesser 
extent the quality of movements. Levels between 3 and 5 (the highest possible score) indicate progressively 
more serious limitations of gross motor function and that child is severely handicapped.3 Table 6 shows the 
classification relevant to our study population. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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Table 6. Summary of the Gross Motor Classification System for Cerebral Palsy. Adapted from Palisano et 
al., 1997. 
 
 
 
The 2-year assessment will provide a window into early language development and early gross- and fine-motor 
development. We plan to submit further grants for long-term follow-up at 5 years of age, which correlates better 
with ultimate function.188 
 
Modified Checklist for Autism in Toddlers (M-CHAT-R). Parent questionnaire (Appendix 3) will be administered 
to the mother/caretaker at the 2 year corrected age follow-up visit. This instrument is validated for screening 
toddlers between 16 and 30 months of age, to assess risk for autism spectrum disorders (ASD).  
 
“The primary goal of the M-CHAT-R was to maximize sensitivity, meaning to detect as many cases of ASD as 
possible. Therefore, there is a high false positive rate, meaning that not all children who score at risk for ASD 
will be diagnosed with ASD. To address this, we have developed a structured follow-up interview for use in 
conjunction with the M-CHAT-R; it is available at www.firstsigns.org.” Users should be aware that even with the 
follow-up questions, a significant number of the children who fail the M-CHAT-R will not be diagnosed with an 
ASD; however, these children are at risk for other developmental disorders or delays, and therefore, evaluation 
is warranted for any child who fails the screening. 
 
The M-CHAT-R can be scored in less than two minutes. We will use the scoring template available at 
www.firstsigns.org. Children who fail more than 3 items total or 2 critical items (particularly if these scores remain 
elevated after the follow-up interview) will be referred for diagnostic evaluation by a specialist trained to evaluate 
ASD in very young children.  
 
As the field of Neonatology has matured, more nuanced follow-up of NICU graduates has become available. It 
is now clear that preterm babies have a higher risk than do term infants for psychological and behavioral 
problems, with autism spectrum disorders and attention deficit problems identified specifically.189-193  We will 
therefore screen all subjects with the M-CHAT-R parental questionnaire at the 2 year corrected age follow-up 
visit.194 We anticipate positive screening will be higher in this ELGAN population than in term infants,43 and if, as 
indicated in several MRI studies,46, 193, 195-201 MRI imaging of brain structures correlates to psychological and 
behavioral function, we anticipate that Epo-treated neonates will have better preserved brain structure, and 
therefore, function. 
 
 

 
 
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6.3.3 Subject Retention. Additional contact to increase the likelihood of follow-up participation will include 
sending birthday cards and appointment reminders. Follow-up rates of > 90% have been achieved in most of 
our study sites, using these methods. An offset for parents' travel/work loss expenses ($100) will be provided at 
the completion of the face-to-face follow-up visit at 2 years corrected age. An additional $100 per visit is included 
in the budget to provide for unusually high travel expenses when needed. We estimate this will be needed for 
15% of subjects. 
 
6.3.4  Intervention Discontinuation Evaluations 
If a subject is removed from study treatment for any reason (parent decision, Attending physician choice, or 
because of SAE such as a symptomatic clot requiring anticoagulation), the reason for discontinuation will be 
recorded. Since this is an intention-to-treat study design, any subjects who discontinue intervention will continue 
to be followed and evaluated on study. Like all study subjects, these families will have a discharge questionnaire 
filled out, and will be contacted by phone at 4, 8, 12 and 18 months. They will be sent birthday cards for their 
baby. A small monetary incentive will be given to each family at the completion of the 2 year follow-up visit. For 
those individuals that must travel over 2.5 hours for the follow-up visit, overnight accommodations may be 
offered.   
 
6.3.5  On Study/Off-Intervention Evaluations 
The MRI should be done between 36-0/7 and 36-6/7 weeks of PMA. This will be done in a subset of 220 patients 
at study sites that have a 3T Siemens or 3T Philips magnet available.  
 
6.3.6  Final On-Study Evaluations 
Study drug treatment will end on the Monday, Wednesday or Friday closest to 32-6/7 weeks PMA, and each 
subject will be followed for 2 years, until their neurodevelopmental assessment at 22-26 months corrected age. 
 
At the 2 year corrected age follow-up visits, documentation of the standardized neurologic exam, and results of 
the Bayley III exam will be recorded. 
 
DOCUMENTATION AND RECORD RETENTION 
Documentation. Each site must provide the UW CCC lead PENUT study coordinator  with the following 
documents prior to study initiation. A copy of these documents must be maintained in the site investigator’s study 
files.  
 
IRB approved informed consent form - All IRB approvals and correspondence (including approved revisions, 
protocol, advertisements, etc.) - Copies of all correspondence pertaining to the study (excluding any budgetary 
matters) - Copies of all serious adverse events submitted to the IRB - Copy of all safety reports. 
 
The UW DCC co-supports an installation of REDCap, software specifically designed for electronic data capture 
that we have used successfully in other multi-site studies. REDCap features include differentiated user roles and 
privileges, password and user authentication security, electronic signatures, SSL encryption, and comprehensive 
auditing to record and monitor access and data changes (http://www.project-redcap.org/software.php). REDCap 
will serve as the architectural backbone for all data captured prospectively, with all data linked by study subject 
ID. 
 
Record Retention. The clinical site is responsible for maintaining all records (i.e., case report forms, original 
data, screening logs, signed informed consent forms, correspondence, etc.) until notified, in writing, by the UW 
CCC, that these records are no longer needed. The Investigator must notify the UW CCC lead study coordinator 
if the site or records are relocated, if the investigator leaves the institution, etc., and a new address for the records 
must be provided. 
 
6.3.7  Off-Study Requirements 
When the subject has completed the 2 year corrected age follow-up visit, there are no further requirements for 

 
 
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study participation. Participation in any further follow-up will require new consent. If consent is given, phone 
follow-up will continue at 6 month intervals up to a maximum age of 5 years. At that point, we hope to enroll 
willing participants in a follow-up study that will follow the children up to 8 years of age. 
 
6.4   
Special Instructions and Definitions of Evaluations 
Screen admissions to antepartum and NICU: Screening for eligible subjects will be done daily. For antepartum 
admissions, this will involve determining whether any admissions within the past 24 hours are likely to deliver 
imminently, and if so, whether they are within the gestational age criteria for the study. If both these criteria are 
satisfied, the Attending Perinatologist will be asked whether this patient might be an appropriate study candidate, 
and if so, whether it is permissible to approach the mother to discuss the study.  
 
For neonatal admissions to the NICU, screening will involve determining the infant’s time of birth and gestational 
age. If they qualify for the study, the Attending Neonatologist will be asked whether they meet inclusion (but not 
exclusion) criteria. If the baby meets criteria, the Attending will be asked whether it is permissible to approach 
the family to discuss the study. Once it is confirmed that the family is willing to hear about the study, they will be 
approached for informed consent. 
 
A screening log will be completed for all screened patients. 
 
6.4.1  Informed Consent 
Antenatal consent will be obtained when feasible. Prenatally, the study investigator will obtain permission from 
the Maternal Fetal Medicine Attending to approach the mother to discuss the study. Postnatally, permission to 
approach the family will be obtained from the Attending Neonatologist. The Attending physician will seek parental 
agreement for an investigator to meet and discuss the study. If the parents are interested, the study investigator 
will discuss the study with family and seek consent in person. The consenting legal guardian will receive a copy 
of the consent form to review, and once signed, will be given a copy to keep. Ideally, if the Attending physician 
is also a study investigator, an alternate study investigator, or their designee, should obtain consent, so as to 
avoid the appearance of coercion. If this is not required by the site IRB, an investigator who is also the Attending 
physician may obtain consent. Investigators will only approach family after infant’s attending heath care provider 
gives permission and family indicates that they are interested in further information about the study. No alteration 
in care will otherwise occur. The attending neonatologist and family can withdraw child from study at any time. If 
permissible by the site IRB, phone consent may be obtained, but must be reaffirmed with the family when they 
are present. Investigator should retain original signed document. Consent will be obtained by the Investigator in 
a room which ensures the privacy of the family, and which is free of potential coercive influences. Consent for 
participation must be obtained before the baby is 24 hours old. 
 
If a family has limited or no English speaking abilities, a certified interpreter will be provided. They will review the 
consent form with the family, and interpret the verbal explanation of the study during the discussion between the 
Investigator and the family members. If individual sites have a large population of non-English speakers, consent 
forms will be translated into the appropriate languages. If an interpreter is not available in a timely manner, the 
family will not be approached. 
 
The parents of the research participants will be given opportunity to review the study both verbally and in writing. 
The will be given opportunity to ask questions of the investigator prior to giving consent. 
 
If there are changes in the protocol or safety information that require consent forms to be updated, they will be 
sent through the IRB process for approval. When entering a patient into the PENUT Portal electronic database, 
documentation of a signed consent form as well as a signed HIPAA form is required prior to randomization.  
 
A model informed consent form is included as Appendix 1.   

 
 
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6.4.2  Documentation of Gestational Age will be done according to the following hierarchy, and the method by 
which gestational age was determined will be logged on the enrollment form: 
1. 
Gestational age by in vitro fertilization if available. 
2. 
Gestational age by first trimester assessment (0-14-0/7 weeks) 
3. 
Gestational age by second trimester assessment (up to 28-0/7 weeks) 
4. 
Last menstrual period (LMP) 
5. 
Newborn maturational assessment 
 
Documentation of intracranial hemorrhage. Screening Cranial Ultrasounds will be obtained as per the 
guidelines. Only the first ultrasound is done for study purposes. The subsequent two ultrasounds (or MRI at 36 
weeks) will be done as part of routine clinical care. The Imaging Guidelines delineate how pathology will be 
documented and graded. 
 
Documentation of clinical findings during the initial hospitalization will be done in REDCap after chart extraction 
after the first 6 doses of study drug have been administered, and within one month of discharge. 
 
Documentation of neurodevelopmental follow-up will be at the time of the 2 year corrected age follow-up visit. A 
standardized neurologic exam and Bayley III exam will be done. The M-CHAT-R questionnaire will also be 
administered at this time. 
 
6.4.3 Maternal demographics and history will be obtained and documented on the demographics and maternal 
history forms after consent is obtained for participation in the study. 
 
6.4.4  Treatment history. Not applicable since subjects are newborns 
 
6.4.5  Concomitant treatments while the subject is in the NICU will be documented. 
 
6.4.6  Study Intervention Modifications. Study drug and/or iron supplementation will be held or stopped for 
the following criteria:  
 
Polycythemia: Central hematocrit (Hct) > 65%: Study drug should be held until Hct is < 55%. 
 
Severe sepsis: Blood culture-proven bacterial or fungal sepsis requiring blood pressure support or new 
respiratory support. Supplemental parenteral iron should be held until blood culture is negative for 72 hours. 
Rationale: Iron has been reported to be permissive for selected gram-negative bacteria and might worsen 
the patient's condition during sepsis. There is no known relationship between Epo and sepsis, so study 
drug does not need to be held. 
 
Unexplained recurrent seizures (unrelated to ICH, PVL or other known pathology): Study drug should be 
held until seizures are well controlled by medication. Restarting study drug will be determined by the Medical 
Monitor, DSMB, and CCC. 
 
Major venous or arterial thrombosis (clot).  
 
Study drug should be held for any thrombosis that is treated with anticoagulation, and this should be reported 
to the CCC PI as an SAE. 
 
Study drug should be held for any symptomatic thrombosis involving a major vessel (e.g. symptoms such 
as superior vena cava syndrome) 
 
Sustained hypertension requiring medical intervention: Study drug should be discontinued if blood 
pressure requires treatment.  When blood pressure returns to normal range (systolic blood pressure < 100 
mmHg), study drug can be resumed. This is true even if patient is still being treated, but blood pressure is 
being controlled.   
 
If the subject requires prolonged antihypertensive therapy (> 1 month) and/or will be discharged on 
medication this is considered an SAE. Medications used to treat hypertension will be recorded.  Any work 
up done to investigate causes of hypertension will be recorded.   

 
 
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6.4.7  Clinical Assessments. 
Clinical safety parameters 
1) Complete physical exam will be done on study entry, after the first 6 doses of study drug (day 12-15) and at 
discharge. Growth parameters (head circumference, height and weight) will be obtained at these times. The 
presence of microcephaly (OFC < 10th percentile) or relative microcephaly (discrepancy of > 50 percentile 
between weight, length and head circumference) will be recorded. 
 
2) Vital signs and blood pressure. All ELGANs are monitored continuously for heart rate and arterial saturation 
(pulse oximetry), with either continuous or intermittent blood pressure readings as part of routine care in the 
NICU. The high and low blood pressure will be recorded for the first 14 days of the study, and then BP 
measurements will be recorded at 6 set time points through discharge.   
 
3) Hematologic data. Complete blood count (CBC) including hematocrit, white blood cell count, absolute 
neutrophil count, platelet counts and blood smear are obtained routinely in extremely preterm neonates as part 
of their care. This is done to evaluate for infection, and to evaluate the need for transfusion (ELGANs receive an 
average of 4 transfusions during their first month of life). Results of these tests will be recorded weekly, as 
available, through 36-6/7 weeks PMA or hospital discharge. The number and volume of blood transfusions, 
donor exposures, and phlebotomized blood volume will be recorded for the entire hospital stay. Ferritin or 
ZnPP/H will be followed to monitor iron status. While important for all preterm newborns, this is strongly 
recommended for infants receiving parenteral iron. Iron supplementation will be modified based on these results 
(See Appendix 7 Iron Guidelines). 
 
4) Renal function. Daily weights, and fluid intake and output will be recorded as available in the chart. BUN and 
creatinine will be recorded as available in the chart. If renal ultrasounds are done for clinical indications, the 
results from these studies will be recorded. Ten urine samples will be collected non-invasively at timed intervals 
from the time of enrollment until discharge. 
 
5) Liver function. Conjugated bilirubin will be recorded weekly as available, through 36-6/7 weeks PMA. These 
data are checked routinely on all infants requiring parenteral nutrition support, and as part of routine assessment 
of nutritional status.   
 
6) Respiratory data. Respiratory complications of prematurity will be documented, including respiratory distress 
syndrome, pulmonary hemorrhage, pneumothorax, pneumonia, duration of mechanical ventilation (> 1 day, > 1 
week), and oxygen use at 28 days and BPD at 36 weeks PMA. 
 
7) Complications of extreme prematurity defined as follows will be recorded:  
 
Bronchopulmonary dysplasia (BPD). BPD will be defined at 36 ± 1 weeks corrected gestational age. Infants 
requiring nasal cannula oxygen, CPAP/high flow nasal cannula or mechanical ventilation will be considered 
mild, moderate or severe BPD, respectively.  
 
 
Necrotizing Enterocolitis (NEC). Bell's staging criteria will be used to define NEC. All surgeries for NEC, 
and for strictures or bowel obstructions occurring as sequelae of NEC, will be recorded. NEC stage II or III 
is considered an SAE. 
 
 
Retinopathy of prematurity (ROP). All infants will be followed using the screening recommendations 
published in 2006.202 The international classification of ROP will be used.203 Severe ROP requiring 
intervention is considered an SAE 
 
 
Intracranial Hemorrhage (ICH), white matter injury (WMI) or hydrocephalus (HC). A cranial ultrasound will 
be done prior to the first study drug dose as part of the study. Brain imaging will also be done as part of 

 
 
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routine care at 7-10 days and 36 weeks PMA (30 days of age or later is acceptable). The presence, location 
and extent of any intracranial bleeding, hydrocephalus or periventricular echolucencies/densities will be 
documented. Grade III and IV bleeds will be considered an SAE. 
 
 
Clinical seizures. If clinical seizures are suspected, an EEG will be done to confirm the diagnosis at the 
Attending physician’s discretion. All such results will be recorded.  
 
 
Sustained hypertension requiring medical intervention: Study drug should be discontinued if blood 
pressure requires treatment.  When blood pressure returns to normal range (systolic blood pressure < 
100 mmHg), study drug can be resumed. This is true even if patient is still being treated, but blood 
pressure is being controlled. 
 
 
If the subject requires prolonged antihypertensive therapy (> 1 month) and/or will be discharged on 
medication this is considered an SAE. Medications used to treat hypertension will be recorded.  Any work 
up done to investigate causes of hypertension will be recorded.   
 
 
Patent ductus arteriosus that is treated with either medical or surgical intervention. The indications for 
treatment will be recorded (ECHO parameters and/or clinical symptoms). Age at treatment will be recorded. 
 
 
Sepsis, confirmed by positive blood culture, or presumed (clinical symptoms present and antibiotic 
treatment instituted for 7-10 days despite negative cultures). Culture proven sepsis associated with shock 
or significant respiratory deterioration is an indication to hold parenteral iron (not study drug). 
 
 
Renal (acute or chronic renal failure) and hepatic complications (cholestasis, hepatitis). 
 
 
Thyroid function (TSH and free T4) will be collected as available. 
 
 
Mortality. The timing and circumstances of any deaths in this study population will be recorded and 
reviewed by the DSMB. 
 
Data on medication administration, including use of steroids, methylxanthines, antibiotics, diuretics, 
vasopressors, antihypertensive agents, sedatives, anticoagulants, anticonvulsants, thyroid treatment, and 
indomethacin/ibuprofen will be collected. 
 
8) Blood samples. Blood (0.5 mL/sample) will be obtained through an indwelling umbilical arterial or venous 
catheter when possible. If no access is available, all efforts will be made to combine the blood draw with clinically 
indicated phlebotomy times. The timing of blood draws is shown in Figure 3. An additional (optional) blood 
sample will be drawn at the 2 year follow-up visit. 
 
The CCC will provide sample collection packs to each site. These will contain green top tubes for blood collection, 
a microfuge tube for plasma collection, and labels for subject identification and date. Samples will be spun, and 
plasma and cell pellet frozen at -70°C to -80°C. Batched samples from each site will be sent to Dr. Juul’s lab at 
the University of Washington via FedEx.  
 
Biomarkers. Plasma from these samples will be used to investigate the effect of Epo on inflammatory mediators 
and growth factors, as well as Epo pharmacokinetics. 
 
Sample storage. Samples will be spun on site to separate plasma from cells. Plasma and cells will be stored in 
separate, labeled containers at -70ºC to -80°C. They will be sent to the UW in batched quantities, after every 4-
5 subjects. The stored cells for each subject will be kept for later study of problems related to prematurity that 
are beyond the scope of this study. 

 
 
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Urine samples will be banked at the University of Washington for future analyses of biomarkers, (neutrophil 
gelatinase associated lipocalin (NGAL), osteopontin (OPN), cystatin C (CysC), uromodulin (UMOD), epithelial 
growth factor (EGF) and urine β2-Microglobulin (β2MG)).   
9) MRI will be done at selected sites at 36-0/7 to 36-6/7 weeks PMA using a predefined protocol. If an MRI is 
needed for clinical indications during this time frame, and if the appropriate protocol is utilized, it may be used 
for the purposes of the study 
 
10) Neurodevelopmental assessment will be completed on each subject at 22-26 months corrected age: 
 
 
Bayley III Scales of Infant Development: Composite Language and Composite Motor Scale in 5 domains - 
cognitive language (receptive, expressive), motor (fine and gross), social-emotional, and adaptive.176  
 
 
Modified Checklist for Autism in Toddlers (M-CHAT-R) (Appendix 3) 
 
 
Standardized neurological examination  (Appendix 3) 
 
 
The Gross Motor Function Classification System (GMFCS)  
 
6.4.8 Epo pharmacokinetics. Epo concentrations will be obtained at four time points as outlined in Figure 3, 
section 5.1. Population Pharmacokinetic Analysis will be done, and Epo concentrations will also be used to 
document study adherence for subjects assigned to the Epo (and control) arms. Plasma for inflammatory 
mediators and biomarkers of brain injury will be obtained at the same blood draw at baseline and study days 7 
and 14. 
 
7.0 
MANAGEMENT of ADVERSE EVENTS 
 
All complications will be treated in the site NICUs. 
 
8.0 
CRITERIA FOR INTERVENTION DISCONTINUATION 
 
The attending neonatologist or parent may withdraw the infant study subject at any time for any reason.  The 
reason for such withdrawal will be recorded.  The study interventions will be discontinued in any infant subject 
who suffers a SAE due to Epo administration.  The research coordinator and study investigator will evaluate all 
subjects on an ongoing basis for evidence of Epo SAE occurrence. 
  
Criteria for Temporarily Withholding/Stopping Study Drug and/or Supplemental Iron.  
1. Polycythemia: Central hematocrit (Hct) > 65%: Study drug should be held until Hct is < 55%. 
2. Severe sepsis: Blood culture-proven bacterial or fungal sepsis requiring blood pressure support or 
significant new respiratory support. Supplemental iron should be held until blood culture is negative for 72 
hours. Rationale: Iron has been reported to be permissive for selected gram-negative bacteria and might 
worsen the patient's condition during sepsis. There is no known relationship between Epo and sepsis, 
so study drug does not need to be held. 
3. Unexplained recurrent seizures (unrelated to ICH, PVL or other known pathology): Study drug should be 
held until seizures are well controlled by medication. Restarting study drug will be determined by the Medical 
Monitor, DSMB, and CCC. 
4. Major venous or arterial thrombosis (clot).  
Study drug should be held for any thrombosis that is treated with a course of anticoagulation. 

 
 
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Study drug should be held for any symptomatic thrombosis involving a major vessel (e.g. symptoms such 
as superior vena cava syndrome). 
5.  Sustained hypertension requiring medical intervention: Study drug should be discontinued if blood 
pressure requires treatment.  When blood pressure returns to normal range (systolic blood pressure < 100 
mmHg), study drug can be resumed. This is true even if patient is still being treated, but blood pressure is 
being controlled.   
 
All necessary medical interventions will be available in the event of serious adverse events stemming from a 
subjects involvement in research. All serious adverse events will be monitored closely until resolution, and they 
will be recorded and reported to the local IRB (as required) as well as the CCC PI (Dr. Juul) who will then notify 
the Medical Monitor as well as the DSMB and NINDS (via Peter Gilbert). In the event that a subject is withdrawn 
from the study for any reason, all efforts will be made to encourage parents to allow their child to continue to 
participate in follow-up visits. 
 
 
9.0 
STATISTICAL CONSIDERATIONS 
9.1   
General Design Issues 
Hypothesis: Epo treatment from 24 hours to 32-6/7 weeks postmenstrual age (PMA) of Extremely Low 
Gestational Age Neonates (ELGANs) will safely decrease the combined outcome of death or neurologic 
impairment from 40% to 30% measured at two years of age.  
 
Design:  We will enroll 940/2 = 470 subjects in each of two treatment groups:  Epo-treated (from 24 hours of age 
to 32-6/7 weeks PMA) vs. placebo control. 
 
Randomization sequences will be created centrally by the DCC. We will use block randomization within site 
using variable blocks of size 4, 6, 8 and 10 subjects. Using block randomization ensures that equal numbers of 
subjects are randomized to the intervention and control arm and that the two groups are balanced at period 
enrollment intervals. For multiple births (twins, triplets) all infants will be randomized to the same treatment group 
(e.g. effective randomization of the mother).  
 
Randomization will be stratified on site, gestational age category (24-25 weeks, 26-27 weeks), and on the 
number of babies inborn for a given pregnancy (one, two, three or more). Randomization sequences will be 
provided to the research pharmacy at each site through a study binder.  The binder will contain the complete set 
of study IDs and associated randomized assignments and will be utilized as a look-up table over the course of 
the study and for each patient’s protocol administration of the study drug. Patient study IDs will utilize the 
following format: <3 digit site code>-XXX-YY, where XXX is the mother ID and YY is the baby ID within mother 
(01, 02, 03… etc.). Study IDs within the binder will be listed by site and mother ID.  Singletons, twins and 
triplets are stratified by gestational age as shown in Table 7.  For example, twin infants from mother 201 at 
site UOW will have study IDs of UOW-201-01 and UOW-201-02. Using Table 7.1 to look up the mother ID UOW-
201, these two infants will be assigned to receive Epo over the course of the study protocol. 
 
Table 7. Randomization and stratification 
 
24-0/7 to 25-6/7 
26-0/7 to 27-6/7 
Singletons 
100-YY 
400-YY 
Twins 
200-YY 
500-YY 
Triplets 
300-YY 
600-YY 
 

 
 
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Table 7.1. Example study drug look-up table in Pharmacy study binder. 
Singleton, 24-25 weeks gestation 
Twins, 24-25 weeks gestation 
Triplets, 24-25 weeks gestation 
ID 
Baby 
Treatment 
ID 
Baby 
Treatment 
ID 
Baby 
Treatment 
UOW-101 
-01 
Epo 
UOW-201 
-01 
Epo 
UOW-301 
-01 
Placebo 
UOW-102 
-01 
Placebo 
 
-02 
 
 
-02 
 
UOW-103 
-01 
Epo 
 
 
 
 
-03 
 
UOW-104 
-01 
Placebo 
UOW-202 
-01 
Placebo 
UOW-302 
-01 
Epo 
UOW-105 
-01 
Placebo 
 
-02 
 
 
-02 
 
UOW-106 
-01 
Epo 
 
 
 
 
-03 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Singleton, 26-27 weeks gestation 
Twins, 26-27 weeks gestation 
Triplets, 26-27 weeks gestation 
ID 
Baby 
Treatment 
ID 
Baby 
Treatment 
ID 
Baby 
Treatment 
UOW-401 
-01 
Epo 
UOW-501 
-01 
Epo 
UOW-601 
-01 
Placebo 
UOW-402 
-01 
Placebo 
 
-02 
 
 
-02 
 
UOW-403 
-01 
Epo 
 
 
 
 
-03 
 
UOW-404 
-01 
Placebo 
UOW-502 
-01 
Placebo 
UOW-602 
-01 
Epo 
UOW-405 
-01 
Placebo 
 
-02 
 
 
-02 
 
UOW-406 
-01 
Epo 
 
 
 
 
-03 
 
 
 
 
 
 
 
 
 
 
 
A modified intent-to-treat (mITT) approach204 will be used, with all randomized infants who receive the first dose 
of study treatment to be included in the analysis. All pre-specified hypotheses will be tested using a two-sided 
type I error of 0.05 with no formal adjustment for multiple comparisons unless otherwise specified (such as with 
safety outcomes). Secondary analyses that focus on separate hypotheses will not require correction for multiple 
comparisons, but those analyses that use multivariate measures such as multiple brain image parameters would 
be corrected for multiple comparisons using standard methods. 
 
Given that we anticipate enrollment of multiple births we require that all analyses properly account for the within-
sibship correlation of outcomes. We will use Generalized Estimating Equations (GEE), which is a versatile 
regression approach for the analysis of discrete and continuous outcomes.205 Use of “robust” standard errors will 
provide valid statistical inference and fully account for the clustering of data.  
 
9.2  Outcomes 
Primary outcome variable: The primary outcome is the composite outcome of death or neurodevelopmental 
impairment at 22-26 months corrected age. Neurodevelopmental impairment (NDI) is defined as the presence 
of any one of the following: CP, Bayley III cognitive or motor scale < 70. There is a known inflation of scores from 
the Bayley II to III1, 178, 179 and we will therefore also consider a threshold of < 85 for secondary analysis. Subjects 
will be stratified by gestational age (24-0/7 to 25-6/7 and 26-0/7 to 27-6/7), number of babies in the pregnancy, 
and by study site. Table 8 shows how CP will be categorized based on features present on standardized 
neurologic exam. 
 
 
 

 
 
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Table 8. Motor outcome - 4 level classification 
GMFCS 
 
0 
0.5 
1 
2 
3 
4 
5 
No CP 
None 
None 
Mild 
Moderate 
Severe 
Severe 
Severe 
HP or DP 
Mild 
Mild 
Moderate 
Moderate 
Severe 
Severe 
Severe 
QP 
Moderate* 
Moderate* 
Severe* 
Severe 
Severe 
Severe 
Severe 
* It is unlikely that a child with quadriparetic CP will have a GMFCS of 0-1. However, this scenario is 
possible in cases of bilateral hemiparesis in which arms are more affected than legs. In such cases, the 
bilateral nature of the deficit, and the significant neurologic abnormalities that are noted on a 
standardized neurologic examination, warrant a designation of moderate/severe neurodevelopmental 
impairment. 
QP: quadriplegic; HP: hemiplegic; DP: diplegic 
 
 
Primary Analysis: The primary analysis will be a test of equality of the rate of the primary outcome (death or 
NDI) across the two randomized investigational groups. Specifically, we will use a GEE Wald test based on 
logistic regression, with stratification by recruitment center, multiples in gestation and gestational age. We will 
perform intent to treat analysis and expect minimal non-compliance due to the nature of the intervention in 
relation to in-patient care. For the primary endpoint we expect uniform and complete ascertainment of death but 
may not evaluate all subjects for developmental impairment. We plan to perform a primary analysis based on 
complete cases and will exclude those subjects for whom vital status is known (alive) but NDI cannot be 
assessed. Sensitivity analysis will use multiple imputation to evaluate the potential impact of any missing data. 
Secondary analysis will be for quantitative measures of brain volume, and for these endpoints an unadjusted t-
test provides inference regarding the mean response across the treatment groups. We will adjust all secondary 
outcome analyses for recruitment site using regression methods. 
 
Power and Sample Size for Primary Outcome:  In order to determine the necessary sample size for efficacy 
evaluation, we need to formulate assumptions for the primary outcome rate in the treated and untreated groups. 
 
The primary outcome measure is the rate of death or severe neurodevelopmental impairment (NDI). Using data 
from two sources, we can compute the expected rates of death or NDI for the neonates that we will enroll. The 
Vermont Oxford Network 2008 Follow-up Report206 evaluated the disability status of infants born in 2008 only, 
and the combined 2004-2008 cohorts. Follow-up status was determined at age 18-24 months and information 
regarding death and NDI is provided for subgroups of children based on their gestational age. Therefore, we can 
use these data to forecast expected trial results for our eligible subjects (24-27 weeks gestational age).  
 
The VON reports on severe NDI and specifically states that components of severe disability include: cerebral 
palsy, or a Bayley score less than 70 or too severely delayed for Bayley testing.206  
 
In addition, data from Gargus et al. (2009)19  provides follow-up information at 18-22 months for approximately 
3,800 neonates born between 24 and 27 weeks gestational age, based on babies born between 1998 and 2001. 
  
We have combined the VON and Gargus (2009) data in order to determine the anticipated characteristics of our 
proposed trial. While the VON data is contemporary, it has relatively low follow-up for two-year outcomes 
(approximately 50%) and therefore may be biased toward more easily followed or more favorable subjects. Table 
9 displays our best estimates of gestational age specific and overall rates of death and NDI. 

 
 
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Our 
assumed 
death rates of 
32, 19, 13 and 9 
percent 
are 
exactly 
those 
presented in the 
VON 2008 report, 
and are lower than 
the rates reported 
in Gargus (2009) 
where 
among 
neonates 
born 
between 1998 and 
2001, GA-specific 
death rates of 
45, 25, 18, and 
15 percent were observed. Using our combined data assumptions, we expect an overall death rate of 
approximately 18%, which is approximately the rate observed in VON (17%), and is lower than the 26% death 
rate observed for Gargus (2009).  
 
In addition, Gargus (2009) report GA-specific rates of NDI as 21, 25, 23, and 19 percent. The VON 2008 report 
estimates NDI rates as 22, 21, 17, and 16 percent respectively. We have combined VON and Gargus (2009) 
data and further assumed that NDI rates are decreasing with increasing gestational age to obtain our NDI 
estimates. Using these data we expect an overall NDI rate of 22%, which equals the overall rate observed in the 
VON 2008 report and in Gargus (2009). 
 
In order to estimate the overall rate observed among treated neonates, we have assumed that there will be no 
effect of treatment on death, but that Epo will lead to a decrease in the rate of NDI. If we assume a multiplicative 
reduction in the NDI rate of 0.45 then we expect a treated NDI rate of 12 percent and an overall rate of death+NDI 
of 30.4% as compared to the control rate of 40.4%. Therefore, in order to obtain a target sample size we assume:  
an overall control rate of 40%, and an overall treated rate of 30% corresponding to an overall treatment rate ratio 
of 0.75. 
 
Using the control and treated rates of 40% and 30% respectively leads to a sample size of 376 evaluated subjects 
per arm or a total evaluated sample size of 752 subjects. 
 
Efficacy Trial Statistical Analysis Plan – Secondary Outcomes 
Secondary Aims and Outcomes:  
 
To compare safety measures between infants receiving Epo and placebo to determine whether there are 
risks associated with Epo administration. 
 
To compare moderate impairment (< 85 on Bayley III cognitive or motor score, or CP). 
 
To compare neuroimaging outcomes across the two treatment groups. 
 
To assess whether treatment effects vary by gender.  
 
To evaluate whether individual biomarkers measured through 36 weeks are predictive of 2-year outcomes, 
and whether a derived multivariate combination of markers has predictive performance greater than 
individual marker performance.  
 
Analysis for Secondary Aims and Outcomes:  
 
Safety: The primary safety outcomes are the serious adverse events and adverse events listed in the CRFs. 
Analysis will compare the proportion of subjects with an AE across the two treatment groups and will use a 2-
Table 9. Combined Vermont Oxford Network (VON) and Gargus (2009) data showing death 
rates and severe neurodevelopmental impairment (NDI) rates for different gestational ages 
(GA). 
 
Gestational 
age 
(weeks) 
Fraction of 
enrolled 
 
Death 
 
NDI 
 
Total 
(Death+NDI) 
Expected 
Treated 
(Death + NDI*0.45) 
24 
0.25 
32 
25.0 
54.4 
44.8 
25 
0.25 
19 
24.2 
40.1 
32.3 
26 
0.25 
13 
21.2 
30.4 
24.7 
27 
0.25 
9 
18.1 
24.5 
19.0 
 
Overall 
 
 
18.2 
 
22.1 
 
40.4 
 
30.4 
 

 
 
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sample test of proportions. Inference for the (5) individual SAE outcomes will use a Bonferroni correction for 
multiple comparisons. 
  
Power: The expected prevalence of SAEs range from less than 1% for polycythemia, to 18% for death. In order 
to characterize power we show the effect sizes for which we have 80% power based on having n=376 subjects 
evaluated in each group:  
 
Table 10. 
Baseline (control) rate 
Alternative rate for 80% power 
Using alpha=0.05 
 
Using alpha=0.01 
5% 
11% (RR = 2.2) 
13% (RR = 2.6) 
10% 
18% (RR = 1.8) 
19% (RR = 1.9) 
15% 
24% (RR = 1.6 
25% (RR = 1.7) 
20% 
30% (RR = 1.5) 
31% (RR = 1.5) 
 
 
 
 
 
 
Secondary Efficacy Outcome: Our key secondary long-term outcome is the rate of death or NDI using severe or 
moderate impairment defined by a Bayley less than 85. In order to estimate power for the secondary outcome 
we assume the same overall rates for death and severe NDI as above, and then assume a 20% rate for moderate 
impairment. Similar to the primary outcome, we assume that the death rate is unchanged by Epo and that the 
rate of severe NDI is reduced from 22% to 12%. We also assume that the moderate NDI group would have an 
effect similar to the severe group where the 20% rate would be reduced by a factor of 0.45 to  
 
 
 
 
 
 
 
 
 
11% but that among the 10% of severe subjects impacted by Epo, half of these would only move from severe to 
moderate impairment. Therefore we assume: 
 
Power: Using these assumptions and the target evaluated of n=752 leads to greater than 95% power to detect 
a difference on the secondary outcome. 
 
Imaging Outcomes:  The neuroimaging outcomes are: Myelinated white matter volume; Total gray matter 
volume; and White matter integrity (TBSS FA corpus callosum). We will conduct a single MANOVA test using 
the multivariate outcome and comparing treated and control subjects. The mean and standard deviation will also 
be calculated (by treatment group) for each individual measure. Note that MR measures will be obtained on a 
subset of infants (110 per treatment group). 
 
Given the a priori hypothesis that treatment effect may differ according to gender we will conduct a single 
subgroup analysis that assesses treatment effects separately for males and for females. Subgroup specific 
treatment effects will be computed and inference will be based on a single Gender-by-Treatment test for 
interaction using logistic regression. 
 
Power: Multiple MRI variables will be collected and analyzed in this study. To provide a basic sample size for a 
treatment effect we selected one common basic measure of structural injury that has been identified in the 
literature for the premature neonates that we hypothesize will be preserved: The size of the cerebellum tissue 
when compared to the whole head volume. We used a comparable set of T1 weighted brain scans of 22 
Control 
Treated  
Death  
 
 
18% 
 
18% 
 
 
Severe NDI 
 
 
22% 
 
12% 
 
 
Moderate NDI 
 
20% 
 
16% = 11% remain mod + 5% from severe 
 
Total  
 
 
60% 
 
46% 

 
 
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premature babies aged between 34 and 36 PMA, with a representative range of brain injuries present on MRI 
and applied the proposed segmentation methods to the data. We estimated the ratio of the cerebellum to whole 
brain size as our marker across this group and used the mean and variance of these measures on which to base 
our power calculation for a treatment effect. Assuming age matched groups in the treated and untreated groups, 
from this data we estimate that 110 subjects will be required to see a 5% increase in the cerebellum size. The 
recognition of cerebellar atrophy in extremely preterm infants has only recently come to light with the increased 
use of MRI.193, 207-212 Cerebellar atrophy has been linked to cognitive as well as motor deficits.209, 213, 214 In 
summary, we will focus on four primary measures that are established to be predictive of 2-year 
neurodevelopmental status and therefore can be used to establish support for the hypothesis of long-term benefit 
of Epo treatment. The neuroimaging biomarkers are: 1) myelinated white matter volume; 2) total gray matter 
volume; 3) cerebellar volume and 4) white matter integrity assessed by TBSS (using FA corpus callosum).  
 
Biomarker Analysis:  We will consider two main classes of potential predictors of 2-year status:  neuroimaging 
measures and inflammatory markers. Interest is in the prognostic potential of individual and/or combined 
biomarker measurements. Given that the primary outcome is a binary measure (NDI), we will evaluate the 
predictive potential of individual quantitative measures using ROC curves showing the full potential of sensitivity 
and specificity across marker cut points. We will compute ROC curves for the (4) primary neuroimaging 
measures, and separately for individual inflammatory markers. Only 220 subjects will have data on the 
inflammatory markers, and these will be the same subjects identified for MR measures. We will derive two 
multivariate predictive models:  using the inflammatory markers; and using the MR measures. We will use AIC 
and 10-fold cross-validation to develop and validate predictive models. A final multivariate model will combine 
markers from both MR and inflammatory measures, and 10-fold cross-validation will permit inference in the 
incremental value of adding markers in combination by comparing ROC curves and associated area under the 
ROC curve (AUC). 
 
9.3   
Meeting Recruitment Targets 
We have chosen sites for this trial that can be expected to enroll a minimum of 24 infants/year in the study. We 
plan to recruit at 18 sites, several of which have participated in the NO CLD and TOLSURF studies. Based on 
our experience to date with these studies, we anticipate that the participating sites will meet their enrollment 
targets and that subject accrual will follow the projected timeline. We have provided very conservative estimates 
of enrollment, recognizing that some sites will do better, and some worse than predicted. Accordingly, we will 
assess site enrollment every 6 months after initiation of the study. Should enrollment at an individual site fall 
below 6 in a 6 month period, the UW CCC PI and Executive Committee will give warning and evaluate whether 
it is appropriate to drop the site from the trial. If no patients are entered during a 6 month period the Executive 
Committee will recommend that the site be dropped and a new site recruited. If a single site appears to be 
enrolling more than 20% of the total infants enrolled in the trial we will temporarily limit enrollment at that site. 
 
In order to obtain the target number of evaluated subjects we will recruit from 18 centers. Our current centers 
are listed below in Table 11, with numbers estimated based on the previous 2 years of admissions. If these 18 
sites are insufficient to maintain an average of 204 subjects enrolled per 6 month period, additional sites will be 
added. This will be monitored closely. 
 
Table 11. Subject Availability at Each Enrolling Site 
 
 
 
 
Site 
# admitted  
< 27-6/7 
wks 
# 
eligible 
# 
survive 
% 
enrolled 
% 
followed 
# Total    
(2.5 years) 
Beth Israel Deaconess Hospital, Boston 
47 
41 
40 
0.5 
93 
47 
Children's Hospital of the Univ of Illinois 
57 
50 
42 
0.5 
90 
47 
Children's Hospital of Minnesota, St Paul 
55 
41 
39 
0.5 
85 
41 

 
 
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Children's Hospital of Minnesota, Minneapolis 
99 
80 
70 
0.5 
96 
84 
University of Minnesota Children's Hospital 
52 
48 
46 
0.5 
100 
46 
Maria Fareri Children's Hospital, New York 
110 
88 
72 
0.5 
90 
81 
Methodist Children's Hospital, San Antonio 
64 
56 
51 
0.5 
80 
51 
Prentice Women's Hospital, Chicago 
87 
83 
70 
0.5 
93 
81 
University of Florida, Gainesville 
100 
85 
68 
0.5 
85 
72 
Florida Hospital for Children, Orlando 
75 
57 
56 
0.5 
80 
56 
University of Louisville, Kentucky 
94 
86 
71 
0.5 
90 
80 
Univ of New Mexico Children's Hospital 
50 
25 
22 
0.5 
100 
28 
University of Arkansas, Little Rock 
120 
100 
80 
0.5 
90 
90 
University of Utah, Salt Lake City 
68 
54 
49 
0.5 
90 
55 
University of Washington, Seattle 
87 
69 
58 
0.5 
85 
62 
Wake Forest School of Medicine, Winston-Salem 
120 
100 
89 
0.5 
92 
102 
Johns Hopkins Medical Center, Baltimore 
87 
80 
70 
0.5 
90 
63** 
South Miami Hospital, Miami 
47 
42 
40 
0.5 
90 
36*** 
Hopkins joined study in Feb 2015; South Miami joined study in June 2015 
**Over 1.8 years; ***Over 1.5 years 
 
In both the phase I/II Epo trial of extremely low birth weight infants and the NEAT trial (Epo neuroprotection of 
term infants with hypoxic ischemic encephalopathy), consent was obtained in over 90% of approached parents. 
This extremely high consent rate reflects the concern parents have about their children’s neurodevelopment, 
and their willingness to participate in research that might result in improved outcome. 
 
Site performance will be tracked with both enrollment and follow-up tables. Examples are shown below. 
 
EXAMPLE ENROLLMENT AND FOLLOW-UP TABLES 
 
Shell Table 1. Open Report: Cumulative and Current Report Period Study Recruitment. 
 
Current Report Period  
Cumulative 
Site 
Screened Eligible 
Enrolled 
Screened Eligible 
Enrolled 
Site-Name-1 
 
 
 
 
 
 
… 
 
 
 
 
 
 
Site-Name-18 
 
 
 
 
 
 
Total 
 
 
 
 
 
 
 
Shell Table 2. Open Report: Follow-up rates by site, n / N (%). 
Site 
4 
month 
8 
month 
12 
month 
18 
month 
24 
month 
30 
month 
36 
month 
42 
month 
48 
month 
54 
month 
60 
month 
Site-Name-1 
 
 
 
 
 
 
 
 
 
 
 
… 
 
 
 
 
 
 
 
 
 
 
 
Site-Name-
18 
 
 
 
 
 
 
 
 
 
 
 
Total 
 
 
 
 
 
 
 
 
 
 
 
 
9.4   
Data Monitoring 
Per NIH Guidelines (1998), this Data and Safety Monitoring Plan (DSMP) defines the oversight and monitoring 

 
 
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activities that will ensure and maintain both the safety of participants and the scientific integrity and validity of 
the trial data. The plan also describes the procedures for adverse event reporting and detailed guidelines for 
recommendations related to trial continuation. 
 
PENUT is a multi-center randomized placebo-controlled phase III trial to determine whether recombinant human 
erythropoietin (Epo) will safely improve the long-term neurodevelopmental outcome of preterm infants 24-0/7 to 
27-6/7 weeks of gestation. The PENUT study will randomize n=940 subjects to receive either Epo or placebo. 
 
The DSMB will monitor the rates of these comorbidities at 6 month intervals, comparing the rates in treated and 
control infants. Expected rates will be based on the published literature. For example, in the Network trial, 93% 
had RDS, 46% PDA, 36% had any ICH with 16% severe, 11% NEC, 36% late-onset sepsis, and 12% ROP. In 
the ELGAN study, 21% died by 2 years of age, 24% of all children had an ICH, 12% had moderate/severe 
ventriculomegaly, and 24% had an ultrasound lesions (echodense or lucent).134 
 
Safety Analysis: For each SAE we will tabulate the event rate by treatment group, and then compare rates 
using Fisher’s exact test. The DCC will prepare monthly summaries of reported SAEs for review by the Medical 
Monitor, the CCC PI and NINDS. 
 
9.4.1  Data and Safety Monitoring Board (DSMB) 
Review of the PENUT trial’s study performance and safety outcomes will be conducted by the Data and Safety 
Monitoring Board (DSMB) as required by the NIH for multi-site clinical investigations. The DSMB consists of 5 
members with a designated chair (Ronnie Guillet), and safety officer (Jack Widness).  Committee membership 
includes expertise in both biostatistics and bioethics, and should have clinical expertise appropriate for the 
intervention and target population. The DSMB is expected to meet two times (every 6 months) per year to review 
study performance and safety outcomes in Open and Closed Reports, and to review study enrollment reports 
quarterly. Ad hoc sessions may be scheduled as required should a serious adverse event need to be reviewed 
by the group. 
  
Aggregate safety and efficacy data and study performance monitoring data will be presented during the open 
sessions of DSMB meetings. Blinded safety and efficacy data will be presented by treatment arm during the 
closed sessions. Review by the DSMB provides assurances that the trial can continue without jeopardizing 
patient safety. The DSMB is also responsible for protecting the confidentiality of the trial data and for monitoring 
the quality of both the data and study implementation procedures.   
 
The DCC will work closely with the CCC in developing and implementing a comprehensive system for safety 
monitoring. Safety monitoring includes the systematic review of safety data for trends that may impact patient 
safety. The processing, reviewing, and reporting of adverse events (AEs) and serious adverse events (SAEs) 
are also part of this process. The infants in this study are hospitalized at study entry, and the duration of their 
hospitalization is highly variable and not predictable at birth. It ranges from as short as three months to more 
than a year.  
 
Data and Safety Monitoring Schedule:  Our target enrollment is n=940 which is expected to accrue during the 
first 2.5 years of the trial. Therefore, we will enroll approximately 200 subjects every 6 months. Our planned 
DSMB safety analyses will occur every 6 months after trial initiation.  
 
9.4.2 Monitoring Guidelines 
Based on findings following review of study data by the DSMB, the Board may recommend: continuation of the 
trial, termination of the trial, or modifications to the protocol (e.g. adding new measurements for safety monitoring, 
discontinuing high risk subjects, extending the trial in time, increasing the trial sample). Decision guidelines in 
the PENUT trial are based on group differences in adverse event rates as explained below, and on whether SAE 
rates exceed pre-determined thresholds.  
 

 
 
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9.4.2.1 Performance Monitoring  
Performance monitoring will be an ongoing activity performed by the study principal investigator and statistician, 
and status reports will be reviewed by the DSMB during their regular meetings. Procedural reviews to address 
protocol compliance with respect to subject recruitment and eligibility, retention and follow-up, randomization 
and blinding and quality of data will be conducted and monthly reports generated. Any protocol violation that 
affects patient safety will be reported to the DSMB immediately. 
 
Performance data will be reviewed in aggregate and by site. It is expected that: 
 
The response rate for determination of the primary outcome (e.g. 24 month Bayley) will be no less than 
80%; 
 
 
Missing interviews will be no greater than 15% at the 4, 8, 12 and 24 month follow-ups; 
 
 
The overall enrollment rate will not drop below the expected rate (34 subjects per month for 24 months) 
by more than 25%. 
 
Data will be entered into a central trial management system utilizing a REDCap database hosted at the DCC.  
Data will be entered into fields with automated validation and logical check built in.  Data will be double entered 
on the outcome measures of Bayley III and CP assessment by standardized neurologic exam.  Compliance will 
be assessed based on the weekly conference calls and data submitted to the DCC on a weekly basis.  If it is 
determined by the study PI that either 1) study protocol is not being followed, or 2) that reporting is inadequate 
at any site, further action will be taken to address these issues.  These actions may include additional in-person 
site visits if appropriate or additional educational/problem-solving sessions by phone or in person regarding the 
study protocol.  
 
9.4.2.2 Safety Monitoring:  
The research coordinator at each site will monitor each subject daily for the presence of any complications until 
discharge. Serious adverse events will be brought to the attention of the CCC PI (Dr. Juul) who will report them 
to the Medical Monitor (Jack Widness), DSMB and NINDS (via Peter Gilbert).  Sites will notify their local IRBs of 
SAEs per site institutional requirements. An independent Medical Monitor will review all cases of serious adverse 
events. A potential risk that is unique to preterm infants is the risk of ROP.121 In the published studies of preterm 
neonates receiving potentially neuroprotective doses of Epo, no difference has been noted between treatment 
and control groups.33, 35, 114, 115 
 
As part of the Data and Safety Monitoring Plan (DSMP) we will perform continuous and interim analysis of 
accruing safety data. We have defined potentially treatment (Epo) related serious adverse events (SAEs) that 
will be monitored throughout the course of the study. Specifically, for SAEs, we will compare absolute rates to 
expected rates based on published data for similar newborns, and will seek careful DSMB review and guidance 
when observed rates exceed pre-specified thresholds. In addition, at planned interim analysis we will formally 
compare the event rates across the two treatment groups using appropriate small sample methods such as 
Fisher’s exact test.  
 
9.4.2.3  Data and Safety Monitoring Schedule.  
See section 9.4.3.1, Table 14. 
 
9.4.2.4  Treatment Efficacy Monitoring 
Treatment efficacy will be monitored by the DSMB, but recognizing that 24 month efficacy outcomes will not be 
obtained until after enrollment is complete (or nearly complete).  Therefore, although formal interim analyses will 
be conducted using group-sequential boundaries there may not be compelling reasons to terminate the trial on 
the basis of accruing efficacy data. Efficacy measures will be provided in a blinded fashion to the DSMB with 
treatment groups labeled “A” and “B” as well as a z-score assessment of the evidence for the difference between 

 
 
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treatment arms at 24 months.  An O’Brien-Fleming interim stopping boundary will be provided to the DSMB to 
help assess the magnitude of the z-score.   
 
9.4.3  Scheduled Reporting 
One month prior to each DSMB review, the Data Coordinating Center (DCC) will summarize monthly 
administrative reports that describe study progress including subject accrual by site, demographics, and the 
sites’ adherence to inclusion/exclusion criteria and other protocol requirements, including retention rates at each 
follow-up point. These reports are prepared monthly and reviewed internally by the study research team for 
ongoing quality control and are also presented to the DSMB and NIH as requested. The DCC will also produce 
safety reports that list adverse events, serious adverse events, deaths, and disease or treatment specific events 
by site and in aggregate to the DSMB. 
 
With each review the DSMB will approve the study and protocol as is, recommend protocol changes in the 
interest of patient safety or stop the study based on overwhelming evidence of treatment benefit or safety 
concerns.  The DSMB will provide the recommendation in written minutes provided to the NIH.  If the NIH concurs 
with these recommendations, they will be forwarded to the principal investigator.  The recommendations will 
include any suggested changes in the proposed timing of future DSMB reviews. The review may result in an 
amendment to the protocol, which must be approved by the IRB, the NIH and the sponsor.  
 
The DSMB report will begin with a brief narrative section that describes the status of the study, progress or 
findings to-date, issues, and the procedures that produced the report (e.g., data obtained by a specific date). 
The report will provide a study description along with a current organization chart, current timetable and study 
schedule as well as a list of study clinical and administrative centers. Data will be presented that describe the 
administrative status of the study including recruitment and forms handling. Study data reports describe 
demographic and baseline clinical characteristics and provide a safety assessment. Tables will be provided by 
site as well as for the whole study population. AE/SAE rates for each group will be presented.  
 
Following each DSMB meeting, the NINDS will send the study’s principal investigator a letter confirming that the 
DSMB met, reviewed all accumulated study data, and made a recommendation that the study continue as 
planned (or, possibly, to modify the protocol).  The principal investigator will forward a copy of this letter to each 
of the clinical investigators who, in turn, are responsible for forwarding it to their local IRB.  
 
9.4.3.1  Serious Adverse Event Reporting 
We divide potential serious adverse events (SAEs) by the severity:  Below we detail the defined SAEs with 
associated expected event rates and thresholds that would trigger DSMB review. Most of the SAEs would occur 
during the Epo treatment period or during hospitalization and therefore would be immediately recorded.  
 

 
 
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Adverse Event (AE):  In addition to the monitoring of SAEs, we will also measure and compare rates of adverse 
events across the two treatment arms. Given the high-risk study population a number of adverse events are 
expected, and the key monitoring function will be to assess whether evidence is accruing that suggests a 
differential adverse event rate associated with treatment. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Estimates here are based on three primary sources:  Stoll et al. (2010); O’Shea et al. (2009) ELGAN study; and 
The Vermont Oxford Network (VON) 2008 Follow-up Report (2011).5, 43, 206  
 
Table 14. Cumulative number of subjects evaluated for safety events and for the long-term efficacy 
outcome.  
Table 12. Serious Adverse Events 
 
 
Serious Adverse Event (SAE) Potentially Epo related 
Expected Rate* 
Threshold 
Hypertension 
20% 
25% 
Polycythemia (hematocrit > 65%) 
Rare < 1% 
2% 
Major venous or arterial thrombosis (clot) not associated 
with a central line 
Rare < 5% 
10% 
Other unexpected life threatening event  
Rare < 5% 
10% 
Serious Adverse Event (SAE) Prematurity related 
Expected Rate* 
Threshold 
Pulmonary Hemorrhage (Severe) 
7% 
15% 
NEC (Stage 2b or 3) 
12% 
25% 
Sepsis (severe) 
33% 
50% 
Intracranial hemorrhage (grade III or IV) 
16% 
25% 
Retinopathy of Prematurity (Severe) 
7% 
14% 
Other Serious Adverse Event: Expected or unexpected 
 
 
Death 
Approx. 18% 
30% 
Cardiac arrest 
Rare < 1% 
2% 
Other life threatening event 
Rare < 1% 
2% 
Table 13. Adverse Events 
 
Adverse Event (AE) 
Expected Rate 
Respiratory distress syndrome (RDS) 
93% 
Patent ductus arteriosus (PDA) 
46% 
Bronchopulmonary dysplasia  (BPD) 
42% 
Central line-related thrombosis (clot) 
40% 
Intracranial hemorrhage (ICH) 
36%total 
16% (grade III & IV) 
Necrotizing enterocolitis (NEC) 
11% 
Periventricular leukomalacia (PVL) 
4% 
Hydrocephalus 
2% 
Clinical Seizures 
6%-13% 
ROP (stages 1-2) 
59% 

 
 
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Month 
6 
12 
18 
24 
30 
36 
42 
48 
54 
60 
Safety (3 month) 
125 
375 
625 
875 
940 
 
 
 
 
 
Efficacy (24 month) 
0 
0 
0 
0 
125 
375 
625 
875 
940 
 
 
Safety evaluation will be based on all available follow-up but we expect the majority of SAEs and AEs to occur 
during hospitalization and therefore within the first three months study follow-up. SAE’s and AE’s will be 
considered by gestational age category, given that complication rates of infants 24 and 25 weeks of gestation 
are expected to be higher than those at 26 and 27 weeks of gestation. 
 
SAEs and Threshold for Action: For each SAE listed in 
Table 9 we have established expected rates of SAEs for 
the target study population, and thresholds for each 
treatment arm at which careful evaluation would be 
required by DSMB review. The DSMB would be asked to 
review all information associated with the line listing for 
the SAEs and then make a decision as to whether the 
study should be modified, continued or stopped. In Figure 
8 we show the operating characteristics for an SAE that 
has an expected (normal) rate of 5%, and a threshold of 
10% for review. If the true SAE rate is 1.5 times the 
threshold (e.g. 15%) then with 100 or more subjects there 
is a greater than 80% probability (power) that the 
observed rate of greater than the threshold would occur 
and trigger DSMB review. However, if the true SAE rate 
was only 1.25 times greater than the threshold then 150 
or more subjects would be needed to have 80% power. 
Although a total of 5 SAEs are monitored for their 
absolute rate, we will not use any multiple comparison 
correction for continuous monitoring of safety leading to DSMB review. However, formal interim analysis 
comparing safety across the two study groups will account for both multiple comparisons due to multiple 
individual safety outcomes, and for multiple interim analyses. 
 
SAE analysis uses a doubling of rates to trigger consideration of DSMB action irrespective of statistical 
significance at interim analysis, yet formal monitoring will be conducted every 6 months to evaluate the strength 
of statistical evidence for a difference across groups. Therefore, our proposed interim analyses will evaluate the 
accruing data using both formal statistical comparison in addition to consideration of absolute rates and is thus 
more conservative than use of only one criterion. Finally, we plan to conduct interim analyses that will also report 
on the specific SAEs and AEs listed in Tables 10 and 11, and will provide p-values comparing treatment groups 
for these safety outcomes in addition to the SAE outcomes. A medical safety officer not involved in the study will 
review all SAEs, and the DSMB (appointed by NINDS) will decide when they want to review each type of SAE 
or AE. We recommend that each case of ROP be reviewed as an SAE in real time.  
9.5 Formal Interim Analysis 
Our primary objective for interim analysis is to allow careful and continuing analysis of safety outcomes. 
Specifically, we propose to conduct formal statistical analysis and inference for each SAE and AE at three interim 
and one final analysis time. We will continue to analyze all AE events that have occurred during follow-up, but 
focus interim analysis on those events that occur within the first three months of follow-up since this is the time 
period in which the major treatment related events would be expected. We will conduct formal safety evaluation 
at 6, 12, 18, and 36 months following the start of enrollment. As shown above in Table 11, we expect to have 3-
month data available on 125 subjects at the first safety analysis. We plan to monitor death and will control the 
overall significance level using O’Brien-Fleming boundaries (net alpha=0.05 significance, accounting for three 
Figure 8: Probability (power) of exceeding established 
thresholds for safety outcomes. Here we consider an 
expected event rate of 5% with a threshold of 10% used to 
trigger DSMB action. 
 
0
50
100
150
200
250
300
0.0
0.2
0.4
0.6
0.8
1.0
N (number evaluated)
probability (power)
rate = 1.50 x threshold
rate = 1.25 x threshold
rate = 1.10 x threshold
rate = null value (5%)

 
 
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interim and one final analysis).215 For the other 4 SAEs, we will again use sequential monitoring boundaries to 
control the outcome-specific family-wise error rate (using a Bonferroni corrected alpha of 0.05/5 = 0.01). 
 
We have 10 AE outcomes and will display both a standard O’Brien-Fleming sequential monitoring guideline 
based on alpha=0.05, and a Bonferroni corrected sequential guideline using a significance of 0.05/9 = 0.0056 to 
correct for multiple outcomes. 
 
The CCC PI will be responsible for notifying the Medical Monitor of any safety events, and the Medical Monitor 
will notify the DSMB if indicated. All events will be reported to the DSMB in semi-annual reports. 
10.0   DATA COLLECTION, SITE MONITORING, AND ADVERSE EVENT 
REPORTING 
10.1   Records to Be Kept 
All patient data will be identified by a Study Identification Number (SID). The link between the SID and the patient 
name/medical record number will be maintained in a locked file available only to the site investigator. This link 
will be destroyed after study completion, data analysis and publication of results.  Retained medical record 
information will include maternal education, SES, other factors known to affect cognitive development (e.g. drug. 
alcohol exposure), date of birth, birth weight, medical treatments, complications, length of hospitalization, 
outcome measures including neurodevelopmental examination results, Bayley III test results, and neuroimages. 
All retained data will be coded with a SID so no direct link will be available to others besides the site investigator. 
 
Data Storage and Security 
Access to research data collected by the PENUT project will be restricted to study team members at each site 
and UW DCC personnel. The clinical recruitment sites will maintain a secure electronic database (e.g. Access, 
MySQL, REDCap, etc.) that links the SID generated by the PENUT Portal to study subject contact information. 
The clinical recruitment sites are responsible for scheduling patient follow-up visits, phone calls, and collection 
of survey data. The database will be stored on a secure electronic server with user name and passwords log in 
for individual users and will be backed up nightly. 
 
The REDCap servers are virtual machines (VMs) located on UW DCC hardware in a secure server room. This 
server room meets the technical requirements for HIPAA compliance and hosts other servers containing PHI.  
 
Storage for all study data is backed by 2 dedicated Network Appliance FAS2050 storage appliances. The filer 
provides highly fault tolerant storage using large RAID volumes, on-line hot-spare drives, and built-in, proprietary 
'snapshot' file system technology that automatically creates hourly, daily, and weekly on-line backups of modified 
files. Each filer provides approximately 2.6 terabytes (2.6TB) of usable storage space.  
 
The VM Operating System will be kept fully patched and firewalled in accordance with UW Medicine Information 
Security Policy SEC05.04, which can be found online at http://security.uwmedicine.org/policies.     
 
The UW DCC maintains a Linux-based infrastructure server that uses and manages a central department 
storage server.  A Silicon Mechanics SM-1272A rack-mount server installed in the UW DCC server room rack 
and an HP dc5750 desktop PC are installed as so-called “logical firewalls”, such that that all PCs and operate 
on a private logical network. 
 
10.2   Role of Data Management 
10.2.1  Each clinical site will be responsible for data collection on enrolled subjects and data entry into the 
REDCap system. Once the study is complete as described in 10.1, each clinical site will be responsible for 
destruction of links and any retained clinical information. 

 
 
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10.2.2  It will be the responsibility of the Data Coordinating Center (DCC) to maintain confidentiality of all the 
submitted data by appropriate data encryption methods and password protection entry to database.  Please refer 
to the DCC Manual of Procedures for more detailed information on data handling. 
 
10.3   Quality Assurance 
Each clinical site will need local IRB approval of the study protocol prior to enrollment. Each clinical site will be 
visited by Dr. Sandra Juul and the UW CCC lead study coordinator or her designee, prior to initiation of subject 
enrollment.  This visit will focus on protocol review, data collection and entry and maintenance of quality control.  
In addition, the UW CCC lead study coordinator or her designee will visit each clinical site on a regular basis, at 
least once a year to assure protocol compliance and quality data entry.  These visits will include review of all 
pertinent records, maintenance of study and pharmacy regulatory documents, and review/resolve any data 
accuracy concerns.  Each site will be required to make all study documents and pertinent records available for 
inspection by the research coordinator or other monitoring authorities.  
 
Data Quality Assurance. We will monitor the accuracy of data entry by the sites both internally and externally. 
We will review study data on arrival for completeness. We will then subject each submitted data set to a set of 
preliminary checks to search for values that are out-of-range or otherwise inappropriate.  
 
External monitoring will consist of regular monitoring visits to every site while actively enrolling. Initial monitoring 
visits will take place prior to subject enrollment, and then every six to 12 months. Using the Patient Monitoring 
Report (example below), a subset of all data points in the CRFs will be compared with the medical record for 
25% of enrolled subjects. Any outstanding data queries will be resolved with the research coordinator at the time 
of the visit. After each study site visit a report will be prepared and copies sent to the Study File, the study PI (S. 
Juul), the site PI, and the site coordinator. 
 
 

 
 
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10.4   Serious Adverse Event Reporting 
Since reporting rules vary by institution, by IRB, and by government agency (NIH), the following statements are 
a conservative guide to reporting serious adverse events for this trial and may be further refined with DSMB 
guidance. 
 
When an SAE occurs, the PI of the CCC (S. Juul) will be notified by the site within 24-72 hours per protocol (see 
Figure 9). She, or her designee, is responsible for reporting the SAE to the Medical Monitor (Jack Widness), the 
DSMB and NINDS (via Peter Gilbert).  The immediate reports will be followed promptly by detailed, written 
reports within 3-7 days per protocol.  SAE reporting will also follow the requirements of the local IRB and the 
FDA.  SAEs and/or laboratory abnormalities identified in the protocol as critical to participant safety must be 
reported. All SAEs experienced by participants during the study time frame specified in the protocol (from the 
time of study drug administration to discharge) are to be reported.  A death, found at any time point, including 
follow-up, should be reported.  The DCC will provide monthly summaries of all SAEs to the CCC PI, DCC PI, 
Medical Monitor, and Peter Gilbert (NINDS). The Medical Monitor is responsible for notifying the FDA when 
indicated.  
Figure 9 shows the flow diagram of how adverse events will be reported. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Unexpected adverse events which are serious, but not life threatening, and have a causal relation to the 
research, (unexpected in this context means not mentioned in the informed consent) must be reported to the PI 
of the CCC (S. Juul) within 72 hours per protocol (see Figure 9).  She, or her designee, is responsible for notifying 
the Medical Monitor, DSMB and NINDS within 7 days.  The local IRB should be notified as per local regulations 
Figure 9. SAE Flow Diagram 
 
 

 
 
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by the Site. The DSMB may call an emergency meeting, if necessary. 
 
11.0   HUMAN SUBJECTS 
11.1   Institutional Review Board (IRB) Review and Informed Consent 
This protocol and the informed consent document and any subsequent modifications will be reviewed and 
approved by the IRB or ethics committee responsible for oversight of the study.  A signed consent form will be 
obtained from the subject’s parent, legal guardian, or person with power of attorney.  The consent form will 
describe the purpose of the study, the procedures to be followed, and the risks and benefits of participation.  A 
copy of the consent form will be given to the subject, parent, or legal guardian, and this fact will be documented 
in the subject’s record. A model informed consent form is included in Appendix 1. 
 
11.2   Subject Confidentiality 
The UW DCC will support an https secured web page (https://www.penut-trial.org) that provides a centralized 
location for information about the Preterm Epo Neuroprotection (PENUT) Trial for patients, providers, 
investigators, and institutional agencies. The web page will also contain a link to the PENUT Portal 
https://www.penut-trial.org/portal where all research personnel will log in with individual user names and 
passwords to securely perform study data management activities. 
 
The PENUT Portal controls the assignment of sequentially generated Study Identification Numbers (SID) for all 
patients screened under the PENUT project. All patient contact information will be linked to the SID within 
databases hosted at the clinical recruitment sites and in a REDCap database at the DCC that stored separately 
from all other study or clinical data.   
 
All laboratory specimens, evaluation forms, reports, video recordings, and other records that leave the site will 
be identified only by the SID to maintain subject confidentiality.  All records will be kept in a locked file cabinet at 
each site.  All computer entry and networking programs will be done using SIDs only.  Clinical information will 
not be released without written permission of the subject, except as necessary for monitoring by IRB, the FDA, 
the NINDS, the OHRP, the sponsor, or the sponsor’s designee. 
 
Upon conclusion of the study, subjects’ families or legally authorized representative may be notified of their 
child’s study arm assignment. This information will be generated by the DCC, and given to sites to distribute in 
a confidential way appropriate to their IRB’s guidelines.  
 
Certain aspects of the subject's medical history and demographics will be collected for this study. Loss of privacy 
may lead to problems with insurability or social stigmatization. However, all data will be collected by study 
personnel with due attention to patient privacy. A Certificate of Confidentiality from the United States Department 
of Health and Human Services (DHHS) will be obtained by the CCC PI for all sites, to protect the subjects' 
confidential information. With this Certificate, study investigators cannot be forced (for example by court order or 
subpoena) to disclose information that may identify subject, subject’s parent, legal guardian, or person with 
power of attorney any federal, state, local, civil, legislative, administrative, or other proceedings. The researchers 
will use the Certificate to resist any demands for information that would identify any specific individual, except to 
prevent serious harm to anyone. 
 
11.3    Study Modification/Discontinuation 
The study may be modified or discontinued at any time by the DSMB, IRB, the NINDS, the sponsor, the OHRP, 
the FDA, or other government agencies as part of their duties to ensure that research subjects are protected. 
 

 
 
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12.0   PUBLICATION OF RESEARCH FINDINGS 
A Publications and Presentations Policy document has been developed to describe processes for defining study 
publications and presentations, for assigning authors in accord with JAMA criteria, and for reviewing publications 
prior to submission.  (Please see Appendix 4). A proposal for a manuscript will be initiated by submitting a 
structured summary proposal that includes an analysis plan. All proposals will be reviewed by the Publications 
and Presentations and Executive Committees. Similarly, all abstracts, presentations, and publications must also 
be approved by the Publications and Presentations and Executive Committees, and it will be the responsibility 
of the DCC to ensure that the process is transparent and timely.  An Excel spreadsheet or the study website will 
include a searchable list of all analysis proposals and will track their status toward publication. 
 
We expect that Epo treatment will improve NDI-free survival compared to that seen in the ELGAN and NICHD 
trials. We expect that this benefit will translate into shorter and less complicated hospital stays and better 
neurodevelopmental outcomes. This outcome of the trial would represent an important advance in the care of 
ELGANs that could change the standard of care for high-risk infants. If Epo treatment has no demonstrable 
benefit, or if adverse effects are observed, this information will also be useful in the field because Epo is currently 
used anecdotally in many NICUs, without trial-based evidence, for some severely ill infants. The results of the 
PENUT trial will be of particular interest to pediatricians trained in neonatal-perinatal medicine. This group of 
practitioners is largely centered in academic or medical centers and they attend several meetings each year 
where new information about treatment can be presented. The PENUT trial results will be presented at regional 
Pediatric Research Society meetings such as the Western and Eastern Society for Pediatric Research, and at 
national and international meetings including the Pediatric Academic Societies combined meetings, the 
European Society for Pediatric Research meeting, the American Academy of Pediatrics meetings, and at other 
venues such as “Hot Topics in Neonatology”.  
 

 
 
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13.0  REFERENCES 
 
1. 
Moore T, Johnson S, Haider S, Hennessy E, Marlow N. The Bayley-III cognitive and language scales: how do scores 
relate to the Bayley II? Arch Dis Child. 2011;96(Suppl 1):A1–A100. doi: 10.1136/adc.2011.212563.85. 
2. 
Rouse DJ, Hirtz DG, Thom E, Varner MW, Spong CY, Mercer BM, et al. A randomized, controlled trial of magnesium 
sulfate for the prevention of cerebral palsy. N Engl J Med. 2008;359(9):895-905. Epub 2008/08/30. doi: 
10.1056/NEJMoa0801187. PubMed PMID: 18753646; PubMed Central PMCID: PMC2803083. 
3. 
Palisano R, Rosenbaum P, Walter S, Russell D, Wood E, Galuppi B. Development and reliability of a system to classify 
gross motor function in children with cerebral palsy. Dev Med Child Neurol. 1997;39(4):214-23. Epub 1997/04/01. 
PubMed PMID: 9183258. 
4. 
Palisano RJ, Rosenbaum P, Bartlett D, Livingston MH. Content validity of the expanded and revised Gross Motor 
Function Classification System. Dev Med Child Neurol. 2008;50(10):744-50. Epub 2008/10/07. doi: 10.1111/j.1469-
8749.2008.03089.x. PubMed PMID: 18834387. 
5. 
Stoll BJ, Hansen NI, Bell EF, Shankaran S, Laptook AR, Walsh MC, et al. Neonatal outcomes of extremely preterm 
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