Conditions: Neonatal Encephalopathy Birth Asphyxia
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 500
Healthy volunteers: no

Brief summary:
Hypoxic-ischemic encephalopathy (HIE) occurs when a baby gets reduced blood flow and oxygen to the brain near the time of birth. This results in death or neurologic disabilities including cerebral palsy and cognitive impairment in up to half of affected infants. This clinical trial will determine if the drug erythropoietin (Epo) added to hypothermia (usual therapy) will improve outcomes for infants suffering from HIE.

Detailed description:
Neonatal hypoxic-ischemic encephalopathy (HIE) refers to brain injury resulting from reduced blood and oxygen flow to a baby's brain near the time of birth. HIE affects up to 12,000 newborns each year in the U.S. Half of affected infants have a bad outcome including death, cerebral palsy and cognitive impairment despite receiving hypothermia, the only available treatment. Erythropoietin (Epo) is a cytokine with remarkable neuroprotective and neuroregenerative effects demonstrated in animal models of neonatal brain injury. In a phase I trial of Epo + hypothermia, the investigators found that Epo 1000 U/Kg/dose best reproduced the pharmacokinetics of neuroprotective dosing in animal models. Long term outcomes were better than expected based on entry criteria and MRI findings. A phase II trial compared 50 cooled infants randomized to receive Epo or placebo. Infants treated with hypothermia + Epo had less brain injury on early MRI, and better 12-month motor development. The investigators hypothesize that Epo given to cooled infants with moderate/severe HIE will reduce the combined primary outcome of death or neurodevelopmental impairment from 49 to 33%. This is a randomized, double-blind, placebo-controlled trial of Epo therapy in 500 infants with HIE undergoing hypothermia. Specific aims are 1) To determine if 5 doses of Epo 1000 U/kg IV reduces the rate of death, motor or cognitive deficits at 2 years; 2) To assess safety of Epo by evaluating clinical toxicity; and 3) To determine whether Epo decreases the severity of neonatal brain injury as evidenced by early MRI and circulating biomarkers of brain injury. The investigators anticipate that Epo will confer improved 2-year neurodevelopmental outcome, will be safe, and will decrease brain injury severity as determined by early biomarkers.

Arms:
arm 1: Erythropoietin 1000 U/kg IV, at about 1, 2, 3, 4, and 7 days of age (i.e., 5 doses) arm 2: Normal saline IV (equal volume), at about 1, 2, 3, 4, and 7 days of age

Interventions: intervention 1: Normal saline placebo intervention 2: Erythropoietin
Intervention descriptions: intervention 1: Equal volume of normal saline to be used as placebo intervention 2: Epogen drawn from commercially available single dose 4000U/mL vials

Protocol document:
Prot_000.pdf: 
 
 
 
 
 
 
Official Title: 
HIGH DOSE ERYTHROPOIETIN FOR ASPHYXIA AND ENCEPHALOPATHY 
 
NCT number: 
NCT02811263 
 
Document: 
Protocol version 2.11 
 
Document Date: 
November 23, 2020 
 
 
 
 
 
 
 
 


 
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HIGH DOSE ERYTHROPOIETIN FOR ASPHYXIA AND ENCEPHALOPATHY 
 
A randomized, placebo-controlled, double-masked 500 - subject clinical trial 
of erythropoietin for the treatment of 
neonatal hypoxic-ischemic encephalopathy (HIE) 
 
 
Study Chairs: 
Yvonne Wu, MD, MPH (Multi-PI CCC) 
Professor of Neurology and Pediatrics 
Division of Child Neurology 
University of California, San Francisco 
 
Sandra Juul, MD, PhD (Multi-PI CCC) 
Professor of Pediatrics 
Division of Neonatology 
University of Washington, Seattle 
 
Patrick Heagerty, PhD (PI of DCC) 
Professor and Chair of Biostatistics 
University of Washington, Seattle 
 
Funded by: 
The National Institute of Neurological Disorders and Stroke (NINDS) 
1U01NS092764-01 
5U01NS092553-02 
 
 
Sponsor of active IND: 
Yvonne Wu, MD, MPH 
IND 102,138 
 


 
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KEY STUDY PERSONNEL 
Study Chairs 
Yvonne Wu, MD, MPH (Multi-PI CCC) 
Professor of Neurology and Pediatrics 
Division of Child Neurology 
University of California, San Francisco 
Patrick Heagerty, PhD (PI of DCC) 
Professor and Chair of Biostatistics 
University of Washington, Seattle 
 
Sandra Juul, MD, PhD (Multi-PI CCC) 
Professor and Chief 
Division of Neonatology 
University of Washington, Seattle 
 
HUB Leaders 
Fernando Gonzalez, MD  
University of California San Francisco 
San Francisco, CA 
 
Dennis Mayock, MD 
University of Washington 
Seattle, WA 
Neuroimaging Core 
Jessica Wisnowski, PhD 
University of Southern California 
Los Angeles, CA  
Robert McKinstry, MD, PhD 
Washington University 
St Louis, MO 
Ashok Panigrahy, MD 
University of Pittsburgh Medical Center 
Pittsburgh, PA 
Amit Mathur, MD 
St. Louis University 
St. Louis, MO 
Stefan Bluml, PhD  
University of Southern California 
Los Angeles, CA 
Yvonne Wu, MD MPH 
University of California San Francisco 
San Francisco, CA 
Follow-Up Core 
Karl Kuban, MD  
Boston Medical Center/Boston University 
Boston, MA 
Elizabeth Rogers, MD 
University of California, San Francisco 
San Francisco, CA 
 
Jean Lowe, PhD 
University of New Mexico 
Albuquerque, New Mexico 
Michael O’Shea, MD, MPH 
University of North Carolina 
Chapel Hill, NC 
Biomarker Core 
Sandra Juul, MD, PhD  
University of Washington 
Seattle, WA 
 
An Massaro, MD  
Children’s National Medical Center 
Washington, DC 
Medical Monitor 
Michael Schreiber, MD 
University of Chicago Medicine 
Chicago, IL 
NINDS Representatives 
Adam Hartman, MD, and  
Scott Janis, MD 
NIH NINDS, Bethesda, MD 
 
 
 


 
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SUMMARY 
Study Name 
HEAL Trial: High-dose Erythropoietin for Asphyxia and Encephalopathy 
Study Drug 
Erythropoietin (Epo), Epoetin alfa, 4000 Units/mL 
Subject 
Population 
500 infants with moderate to severe HIE undergoing therapeutic hypothermia 
Objectives 
1) To determine if 5 doses of Epo 1000 U/kg (birth weight) intravenous (IV) 
reduces the rate of death or neurodevelopmental impairment (mild, moderate, or 
severe) at 24 months of age.  
2) To assess safety of Epo. 
3) To determine whether Epo decreases the severity of HIE-induced brain injury 
as evidenced by early MRI and plasma biomarkers of brain injury. 
Design 
Prospective, randomized, double-masked, placebo-controlled study  
Arms 
Treatment: Hypothermia (standard of care) plus Epo 
Placebo: Hypothermia (standard of care) plus normal saline 
Procedures 
- 
Screen all cooled infants to evaluate eligibility for HEAL 
- 
Determine severity of encephalopathy using modified Sarnat exam 
- 
Consent and randomize patients 
- 
Randomized subjects receive five doses of either study drug or placebo  
- 
First dose administered within 24+2 hours of age (Study Day 1); subsequent 
doses given at the same time of day on Study Days 2, 3, 4, and 7 
- 
Brain MRI and MR spectroscopy ideally performed between 96 and 144 
hours post-birth, as part of routine clinical care 
- 
Blood collected prior to administration of first study drug dose, and on 
Study Days 2 and 4 (3 samples total, 1.5 mL each) 
- 
Urine (2 samples) collected on Study Days 0-1, and after completion of 
rewarming on Study Days 3-4 
- 
Phone follow-up at 4, 8, 12, 18, and 24 months: parental questionnaires 
regarding intervening medical and developmental history, and 
developmental milestones (Warner Initial Developmental Evaluation of 
Adaptive and Functional Skills (WIDEA), 12, 18, and 24 months only); All 
phone interviews may be done in person if desired 
- 
24-month in-clinic evaluation includes a standardized neurologic exam, 
Bayley III, Gross Motor Function Classification System (GMFCS) evaluation, 
Child Behavior Checklist (CBCL), and growth parameters. For extenuating 
circumstances, for example, COVID-19 restrictions, the in-person elements 
of the final endpoint evaluation may be performed up to 36 months of age. 
- 
Extended contact phone calls every 6 months from 2.5 through 8 years of 
age (WIDEA at 30 and 36 months) 
Sites 
At least 15, and up to 30 centers in the United States 
 
 


 
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TABLE OF CONTENTS 
SYNOPSIS........................................................................................................................................... 8 
1 
STUDY OBJECTIVES ................................................................................................................ 9 
 
SPECIFIC AIMS ................................................................................................................................. 9 
1.1.1 
Aim 1: Efficacy ......................................................................................................................... 9 
1.1.2 
Aim 2: Safety ........................................................................................................................... 9 
1.1.3 
AIM 3: Biomarkers of Neonatal Brain Injury ......................................................................... 10 
 
ENDPOINTS ................................................................................................................................... 10 
 
OVERVIEW OF STUDY DESIGN ...................................................................................................... 10 
1.3.1 
Definition of Study Day .......................................................................................................... 10 
2 
BACKGROUND ..................................................................................................................... 13 
 
RATIONALE .................................................................................................................................... 13 
2.1.1 
Patient population ................................................................................................................. 13 
2.1.2 
Mechanisms of Epo Neuroprotection .................................................................................... 13 
2.1.3 
Pre-clinical studies of Epo in neonatal HI brain injury ........................................................... 14 
2.1.4 
Dosing regimen ..................................................................................................................... 14 
2.1.4.1 
Why multiple and late doses? ...................................................................................... 15 
2.1.4.2 
Dose selection .............................................................................................................. 15 
2.1.4.3 
Route of administration ............................................................................................... 16 
2.1.5 
Need, relevance and priority for the study ............................................................................ 16 
 
SUPPORTING DATA ....................................................................................................................... 16 
2.2.1 
Clinical studies of Epo and HIE .............................................................................................. 16 
2.2.2 
Safety ..................................................................................................................................... 17 
2.2.3 
Pilot studies ........................................................................................................................... 18 
3 
STUDY DESIGN ..................................................................................................................... 21 
4 
SELECTION AND ENROLLMENT OF SUBJECTS ......................................................................... 21 
 
INCLUSION CRITERIA ..................................................................................................................... 21 
4.1.1 
Prior therapy.......................................................................................................................... 22 
4.1.2 
Demographic characteristics ................................................................................................. 22 
 
EXCLUSION CRITERIA .................................................................................................................... 22 
 
STUDY ENROLLMENT PROCEDURES ............................................................................................. 22 
4.3.1 
Identification and recruitment .............................................................................................. 22 
4.3.2 
Screening for eligibility and monitoring of recruitment targets ........................................... 23 
4.3.3 
Consent procedures ............................................................................................................... 23 


 
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4.3.4 
Stratification, Randomization and Masking Procedures ....................................................... 23 
5 
STUDY INTERVENTIONS ........................................................................................................ 23 
 
STUDY DRUG ADMINSTRATION AND DURATION ......................................................................... 23 
 
HANDLING OF STUDY DRUG ......................................................................................................... 24 
 
CONCOMITANT AND REQUIRED INTERVENTIONS ........................................................................ 25 
 
PROHIBITED AND PRECAUTIONARY INTERVENTIONS .................................................................. 25 
 
ADHERENCE ASSESSMENT ............................................................................................................ 25 
6 
CLINICAL AND LABORATORY EVALUATIONS .......................................................................... 26 
 
SCHEDULE OF EVALUATIONS ........................................................................................................ 26 
 
TIMING OF EVALUATIONS ............................................................................................................ 29 
6.2.1 
Pre-Randomization Evaluations – Timing ............................................................................. 29 
6.2.2 
Birth Hospitalization Evaluations – Timing ........................................................................... 29 
6.2.3 
Follow-Up Evaluations – Timing ............................................................................................ 29 
6.2.4 
Final Endpoint Evaluation ...................................................................................................... 30 
6.2.5 
Future Studies ........................................................................................................................ 30 
 
INSTRUCTIONS AND DEFINITIONS OF EVALUATIONS ................................................................... 30 
6.3.1 
Informed Consent .................................................................................................................. 30 
6.3.2 
Clinical assessment ................................................................................................................ 30 
6.3.2.1 
Screening clinical assessment (<24+2 hours): .............................................................. 30 
6.3.2.2 
Demographics ............................................................................................................... 32 
6.3.2.3 
Day five clinical assessment ......................................................................................... 32 
6.3.2.4 
Newborn hearing screening ......................................................................................... 32 
6.3.3 
EEG assessments ................................................................................................................... 33 
6.3.3.1 
EEG interpretation ........................................................................................................ 33 
6.3.4 
Laboratory Evaluations ......................................................................................................... 33 
6.3.5 
Plasma and urine biomarkers; baseline Epo measurement .................................................. 33 
6.3.6 
Neuroimaging: Brain MRI/MRS ............................................................................................. 34 
6.3.7 
Phone questionnaires ............................................................................................................ 34 
6.3.8 
Neurodevelopmental examinations (24 months) .................................................................. 34 
6.3.8.1 
Standardized neurologic examination. ......................................................................... 36 
6.3.8.2 
Gross Motor Function Classification Scale (GMFCS)131 ................................................ 36 
6.3.8.3 
Bayley Scales of Infant Development III (Bayley-III) ..................................................... 37 
6.3.8.4 
Developmental history ................................................................................................. 37 
6.3.8.5 
Medical and social history ............................................................................................ 37 


 
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6.3.8.6 
Growth parameters ...................................................................................................... 38 
6.3.8.7 
Child Behavior Checklist (CBCL) .................................................................................... 38 
6.3.8.8 
Warner Initial Developmental Evaluation of Adaptive and Functional Skills (WIDEA) 38 
6.3.9 
Extended Contact Phone Calls ............................................................................................... 38 
7 
MANAGEMENT OF SERIOUS ADVERSE EVENTS AND COMPLICATIONS OF HIE ........................ 39 
 
SERIOUS ADVERSE EVENT DEFINITIONS ....................................................................................... 39 
 
SAE RELATEDNESS & SEVERITY ..................................................................................................... 40 
 
SAE AND UNEXPECTED, RELATED EVENT REPORTING .................................................................. 40 
 
ANTICIPATED MEDICAL COMPLICATIONS (ADVERSE EVENTS) IN HIE PATIENTS.......................... 44 
 
CRITERIA FOR MODIFICATION OF INTERVENTION REGIMEN ....................................................... 44 
7.5.1 
Criteria for Withholding/Stopping the Study Drug ................................................................ 44 
7.5.2 
Restarting Study Drug ........................................................................................................... 44 
 
PROTOCOL VIOLATION/ DEVIATION REPORTING ......................................................................... 44 
7.6.1 
Protocol Violations: ............................................................................................................... 44 
7.6.2 
Protocol Deviations may include the following items, among others: .................................. 45 
8 
CRITERIA OF INTERVENTION DISCONTINUATION .................................................................. 45 
9 
STATISTICAL CONSIDERATIONS ............................................................................................ 45 
 
GENERAL ....................................................................................................................................... 45 
 
OUTCOMES ................................................................................................................................... 46 
9.2.1 
Primary outcome ................................................................................................................... 46 
9.2.1.1 
Motor Deficit – Cerebral Palsy ...................................................................................... 46 
9.2.1.2 
Motor Deficit: Gross Motor Functional Classification System (GMFCS) ...................... 47 
9.2.1.3 
Cognitive Deficit: Bayley III Cognitive Score < 90 ......................................................... 47 
9.2.1.4 
Adjudication of Primary Outcome ................................................................................ 47 
9.2.2 
Secondary outcomes ............................................................................................................. 48 
 
DATA ANALYSES ............................................................................................................................ 48 
9.3.1 
Primary outcome analyses .................................................................................................... 48 
9.3.2 
Secondary Outcomes ............................................................................................................. 49 
9.3.3 
Exploratory Analyses ............................................................................................................. 49 
9.3.4 
Analyses of Safety Data ......................................................................................................... 49 
9.3.5 
Neuroimaging and circulating biomarker analyses .............................................................. 50 
9.3.6 
Compliance, Retention, and Missing Data ............................................................................ 50 
9.3.6.1 
Missing data and dropouts. .......................................................................................... 50 
 
SAMPLE SIZE AND ACCRUAL ......................................................................................................... 51 
9.4.1 
Primary Outcome Sample Size Calculations: ......................................................................... 51 


 
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9.4.2 
Sample size for efficacy secondary outcomes ....................................................................... 53 
9.4.3 
Sample size for MRI/MRS biomarkers ................................................................................... 54 
9.4.4 
Sample size for plasma biomarkers of brain injury and inflammation ................................. 54 
9.4.5 
Achieving the sample size...................................................................................................... 55 
 
DATA MONITORING ...................................................................................................................... 55 
 
DATA AND SAFETY MONITORING PLAN (DSMP) .......................................................................... 55 
10 
DATA COLLECTION, SITE MONITORING, AE REPORTING......................................................... 57 
 RECORDS TO BE KEPT ................................................................................................................... 57 
10.1.1 
Subject identifiers .............................................................................................................. 57 
10.1.2 
Documentation ................................................................................................................. 57 
 ROLE OF DATA MANAGEMENT ..................................................................................................... 58 
10.2.1 
The Clinical Site responsibilities in data collection and management .............................. 59 
10.2.1.1 
Data Collection Protocol ............................................................................................... 59 
10.2.1.2 
Description & Flow of Case Report Forms (CRFs) ......................................................... 59 
10.2.2 
Data Security and Confidentiality ..................................................................................... 59 
 QUALITY ASSURANCE.................................................................................................................... 59 
10.3.1 
Data Audit ......................................................................................................................... 60 
 ADVERSE EXPERIENCE REPORTING ............................................................................................... 60 
11 
HUMAN SUBJECTS ............................................................................................................... 60 
 INSTITUTIONAL REVIEW BOARD REVIEW AND INFORMED CONSENT ......................................... 60 
 SUBJECT CONFIDENTIALITY .......................................................................................................... 60 
 STUDY MODIFICATION/DISCONTINUATION ................................................................................. 60 
12 
PUBLICATION OF RESEARCH FINDINGS ................................................................................. 61 
13 
REFERENCES ........................................................................................................................ 62 
14 
APPENDICES ........................................................................................................................ 71 
 ACRONYMS ................................................................................................................................... 71 
 
 
 


 
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SYNOPSIS 
Hypoxic-ischemic encephalopathy (HIE) is a significant cause of neonatal encephalopathy. Although 
neonatal infection, stroke, and metabolic abnormalities can also cause NE, HIE accounts for about 60% 
of cases.1 The incidence of HIE ranges from 1-7 per 1000 births in developed countries, depending on 
the definition used, with most estimates ranging between 1-3 per 1000. An estimated 12,000 infants are 
affected each year in the US.  
Neonatal HIE accounts for 22% of annual neonatal deaths worldwide, totaling 814,000 deaths in 2008.2 
HIE is unpreventable in most cases, and therapies are limited. Hypothermia initiated within 6 hours 
improves outcome,3-7 yet despite this therapy 44-53% of infants with HIE die or suffer moderate to 
severe disabilities including cerebral palsy (CP), intellectual disability, epilepsy, and visual impairment. 
When a normally developing fetus suffers brain injury leading to lifelong neurologic disability, there is 
excruciating heartbreak for all involved. Each year, infants who develop CP as a consequence of 
neonatal HIE will impose an economic burden of $1.7 billion in their lifetime medical and non-medical 
costs in the U.S. This figure does not include the costs incurred by children with cognitive impairment, 
epilepsy, visual impairment and other adverse neurodevelopmental outcomes due to HIE. Additional 
neuroprotective therapies are urgently needed.  
The hematopoietic cytokine erythropoietin (Epo) has remarkable neuroprotective and 
neuroregenerative effects in the brain in animal models of neonatal brain injury.8-13 Over 70 pre-clinical 
studies have tested the neuroprotective effects of Epo following hypoxic-ischemic brain injury, and 
these have produced impressive histologic and functional evidence for benefit ranging from 40 to 78% 
improvement in infarct size.11,14-20 In non-human primates, Epo reduces the rate of CP and improves 
neurologic function in animals undergoing hypothermia for HIE.21  
Two clinical trials suggested that infants with HIE treated with 5-7 doses of Epo experienced improved 
neurologic outcomes compared to infants who received placebo.22,23 In a pilot phase I study, we found 
that Epo was safe, exhibited desirable pharmacokinetics, and resulted in a surprisingly low rate of 
moderate/severe disability even among infants with significant brain injury seen on MRI.24 In a phase II 
study, we found that infants randomized to receive multiple doses of Epo had less brain injury on MRI 
and better 12-month motor outcomes than those who received placebo.25 Given the compelling 
preclinical data, the suggestive findings from human trials, the favorable safety and pharmacokinetic 
data, and the unacceptable rate of adverse long-term neurologic outcomes in HIE, we will now perform 
a phase III trial to determine whether high-dose Epo improves neurodevelopmental outcomes following 
HIE.  
 
 


 
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1 Study Objectives 
We hypothesize that high-dose Epo given to cooled infants with moderate/severe HIE will reduce the 
primary outcome of death or neurodevelopmental impairment (mild, moderate or severe) at 24 months 
of age from 49% to 33% or less. We further hypothesize that neonatal Epo will be safe, will decrease 
brain injury severity on neonatal MRI, and will decrease serial inflammatory cytokines and biomarkers of 
brain injury. We expect this finding will change clinical practice. In a randomized, double-masked, 
placebo-controlled efficacy trial of 500 infants with HIE, we have three specific aims, which will be 
evaluated by the endpoints detailed below: 
- 
Aim 1: Efficacy 
- 
Aim 2: Safety 
- 
Aim 3: Biomarkers of neonatal brain injury 
 
 SPECIFIC AIMS 
1.1.1 Aim 1: Efficacy 
To determine whether Epo therapy (1000 U/kg (birth weight) given intravenously (IV) on Study 
Days 1, 2, 3, 4, and 7) reduces the composite primary outcome of death or neurodevelopmental 
impairment at 24 months of age. Since death is a competing outcome, it is critical to include it in 
the primary outcome measure.   
Neurodevelopmental impairment (mild, moderate, or severe) is defined as any of the following: 
• 
Gross Motor Function Classification System (GMFCS) level ≥1, or 
• 
GMFCS = 0 or 0.5 AND CP (any type), or 
• 
Bayley Scales of Infant and Toddler Development 3rd Edition (Bayley III) Cognitive Score <90  
Secondary analyses: we will examine the effect of Epo on CP, severity of motor impairment, Bayley 
III cognitive and language scores, epilepsy, and behavioral abnormalities. We will also evaluate how 
Epo shifts the distribution of outcomes by evaluating a 4-level outcome measure: 1) normal; 2) mild 
motor and/or cognitive impairment; 3) moderate/severe motor and/or cognitive impairment; and 
4) death.  
Overall severity will consist of the worst severity observed in either motor or cognitive outcomes. 
Severity of motor impairment is defined by type of CP and GMFCS level. Severity of cognitive 
impairment is determined by Bayley III cognitive score, where Severity is defined as:  
Severe: <70 
Moderate 70-84 
Mild: 85-89 
None: ≥90  
1.1.2 Aim 2: Safety 
To establish the safety of high dose Epo treatment in the setting of HIE and hypothermia, by 
comparing Epo-related safety measures in infants treated with Epo vs. placebo.  


 
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1.1.3 AIM 3: Biomarkers of Neonatal Brain Injury 
Aim 3a. To determine whether early high dose Epo decreases the severity of brain injury as 
evidenced by early neonatal MRI and MR spectroscopy. 
Aim 3b. To determine whether early high dose Epo decreases the severity of brain injury as 
evidenced by serial circulating biomarkers of inflammation and brain injury. 
 ENDPOINTS 
Primary Efficacy Endpoint  
1. Death or neurodevelopmental impairment at 24 months of age, compared between the two 
groups. 
Secondary Efficacy Endpoints 
2. Comparison of CP, motor impairment, Bayley III cognitive and learning scores, epilepsy, and 
behavioral abnormalities at 24 months, compared between the two groups. 
Safety Endpoints 
3. Rates of Epo-related reported adverse events through hospital discharge, compared between 
the two groups. 
4. Rates of Epo-related reported adverse events through 24 months, compared between the two 
groups. 
Exploratory Analyses of Biomarkers of Neonatal Brain Injury 
5. Evaluation of serial circulating biomarkers of inflammation/brain injury, and MR evidence of 
brain injury obtained during the first week of life, compared between the two groups. 
6. Evaluation of predictive values of serial circulating biomarkers of inflammation/brain injury, 
and MR evidence of brain injury obtained during the first week of life, for predicting 24 -month 
neurodevelopmental outcomes. 
 OVERVIEW OF STUDY DESIGN  
1.3.1 Definition of Study Day 
To ensure study procedures are consistent across sites, the following definition will be used: 
Study Day: Study Day 1 is defined as the calendar day on which Intervention 1 (first administration 
of study drug) occurs. If the study drug is first administered on the calendar day following the day 
of birth, then the day of birth will be called Study Day 0.  
We propose a multicenter, randomized, double-masked, placebo-controlled Phase III clinical trial. We 
will enroll 500 infants at a minimum of 15, and a maximum of 30 sites. Patients will be randomized to 
receive either IV Epo 1000 U/kg/dose based on birth weight, or an equal volume of IV normal saline 
(NS). Study drug will be administered on study days 1, 2, 3, 4, and 7. All enrolled infants will receive 
standard clinical care including 72 hours of therapeutic hypothermia. The initial neonatal intervention 
and safety monitoring will begin at time of study consent (prior to 24 hours of age), and will be 
continued to hospital discharge. Follow-up evaluations will be performed at 4, 8, 12, 18, and 24 months 
via phone questionnaire, and at 24 months via in person neurodevelopmental examination.  
Primary endpoint at age 24 months ± 56 days (i.e., ± 2 months) will be determined by presence of death 
or neurodevelopmental impairment.  


 
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Blood samples will be obtained from all subjects at 3 time points: baseline, Study Day 2, and Study Day 
4. Urine samples will be obtained from all subjects at 2 time points: after consent on Study Day 0-1, and 
after completion of rewarming on Study Day 3-4. We will measure a baseline plasma Epo concentration 
prior to the administration of the first study drug dose. A subset of 200 patients identified post-hoc after 
meeting pre-defined criteria for quality of MRI/MRS data will have their samples measured for 
circulating and urinary biomarkers of brain injury and inflammation. All infants will have a brain MRI and 
MRS, ideally between 96 and 144 hours post-birth, as part of the standard clinical management of 
cooled infants. The study neuroradiologists will perform masked centralized reading of these neonatal 
brain MRI and MRS scans.  
Sample Size and Population. We will enroll 500 infants ≥ 36 weeks gestational age with moderate or 
severe HIE. Eligible subjects (≥ 36 weeks of gestation, admitted to a participating site Neonatal Intensive 
Care Unit for therapeutic hypothermia) must meet the following criteria from 1 AND 2. 
1.  Perinatal depression = at least one of the following:  
a. Apgar < 5 at 10 minutes of age, or 
b. Need for resuscitation at 10 minutes (i.e., chest compressions, or positive pressure 
respiratory support including endotracheal, mask ventilation, or CPAP), or   
c. pH < 7.00 in a cord gas (arterial or venous) or in an infant gas (arterial or venous) 
obtained at < 60 minutes of age, or 
d. Base deficit ≥ 15mmol/L in a cord gas (arterial or venous) or in an infant gas (arterial or 
venous) obtained at < 60 minutes of age 
2. Moderate/severe encephalopathy = at least 3 of 6 modified Sarnat criteria present between 1-
6 hours of age (see Table 4).3,5,26  
Assuming 10% loss of follow-up at 24 months, we will enroll 500 infants, in order to evaluate 450 infants 
(225 in each arm) at 24 months of age. We plan to apply for additional funding to allow us to follow 
these children to age 8. 
 
 


 
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Table 1. Study Procedure Chart 
Age 
<24 hours 
Entry/ Day 1* 
Study Day 
Discharge 
Months 
2 
3 
4 
5 
6 
7 
4 
8 
12 
18 
24 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomization 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Epo or placebo IV  
 
X 
X 
X 
X 
 
 
X 
 
 
 
 
 
 
Hypothermia 
X 
 
 
 
 
 
 
 
 
 
 
Blood sample 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
Urine sample 
X 
 
after 
rewarming 
 
 
 
 
 
 
 
 
 
MRI/MRS 
 
 
 
 
96-144 hours 
post-birth 
 
 
 
 
 
 
 
 
Sarnat exam 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
Phone interview 
 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
Neurodevelopmental 
exam 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
* Study Day 1 is defined as the calendar day on which Intervention 1 is administered. If the study drug 
is administered on the calendar day following the day of birth, then the day of birth will be called 
Study Day 0. 
 
Months 
Age 
30 
36 
42 
48 
54 
60 
66 
72 
78 
84 
90 
96 
Extended Contact 
phone calls 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Each of the enrolling sites has extensive experience with neonatal clinical research, performs passive or 
active hypothermia during transport, performs whole body cooling, and has an active neonatal follow-
up program.  
 
 


 
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2 BACKGROUND 
 RATIONALE 
In 2008, it is estimated that birth asphyxia (lack of oxygen and perfusion to the brain and other vital 
organs) caused 814,000 deaths worldwide, and contributed to 22% of neonatal deaths. Hypoxic-
ischemic brain injury remains a significant problem in the U.S., affecting 1-3 per 1000 births.1,27-29 
Therapeutic hypothermia has proven to be neuroprotective, but neonates who received hypothermia in 
clinical trials still experience unacceptably high rates of death (mean 28%, range 24-38); cognitive 
dysfunction (24%, range 21-25); CP (22%, range 13-28) and death or moderate/severe disability (48%, 
range 44-53).4-7,30 Other neurologic disabilities after HIE despite hypothermia include epilepsy (19%, 
range 15-24) and cortical visual impairment (6%, range 1-10). New complimentary therapies, to further 
improve outcomes, are desperately needed, and these must be tested in the context of therapeutic 
hypothermia. Epo is one such promising neuroprotective therapy, with compelling preclinical data, 
supportive phase I and II clinical trials, and a reassuring safety profile in neonates. The pharmacokinetics 
of Epo in the setting of HIE and hypothermia have been defined,31 and it is now ready for evaluation of 
safety and efficacy in a phase III trial.  
2.1.1 Patient population 
Newborn infants 36 weeks of gestation or greater, with laboratory and physical exam findings 
compatible with moderate or severe HIE who have started therapeutic hypothermia by 6 hours of 
age. Male and female infants of all races and ethnicities cared for at participating centers are 
eligible. 
2.1.2 Mechanisms of Epo Neuroprotection 
Epo, a glycoprotein originally identified for its role in erythropoiesis, has neuroprotective and 
reparative effects in the central nervous system (CNS).32-37 Epo functions by binding to its 
homodimeric cell surface receptor (Epo-R). Epo-Rs are expressed by a variety of cell types in the 
CNS,38,39 including neuronal progenitor cells,35 subsets of mature neurons,40 astrocytes,41 
oligodendrocytes,42 microglia,43 and brain endothelial cells.35 Epo and Epo-R expression in the 
brain is high during fetal development but declines rapidly after birth. In the setting of hypoxia 
ischemia, Epo-R expression in neurons, astrocytes, and microglia is rapidly upregulated. Increased 
Epo expression follows, mediated via hypoxia-mediated stabilization of neuronal transcription 
factor hypoxia-inducible factor-1α, if the insult is of sufficient duration.38,39,44 In the absence of 
Epo-Epo-R binding, cells are predisposed to apoptosis, while in the presence of Epo, cells are 
preserved.80,87 This creates an important rationale for exogenous Epo administration, given that 
brain injury can occur after brief but catastrophic insults such as placental abruption or cord 
accidents, which are insufficient to stimulate an increase in Epo synthesis.88  
Epo signaling targets several acute intracellular mechanisms important in newborn hypoxic-
ischemic brain injury.45 Epo exerts direct neuroprotective effects on neurons by activating anti-
apoptotic pathways,46-48 but also decreases inflammation,49,50 increases anti-oxidant activity,51,52 53 
and reduces excitotoxic cell injury.54 Epo protects neuronal progenitor cells from interferon-γ, 
lipopolysaccharide (LPS) and hypoxic-ischemic injury,18,55,56 protects oligodendrocytes and 
improves white matter survival assessed by MRI and pathologic analysis. 18,57,58  
In addition to acute effects, Epo promotes neurogenesis and long-term repair.59-62 In vitro, Epo 
increases the number of newly generated neuronal precursor cells and directs stem cells to 
differentiate into neurons.41,63,64 Epo also stimulates production of growth factors such as vascular 


 
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endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and glial cell derived 
neurotrophic factor (GDNF) that have important neurogenic and pro-angiogenic effects.35,65 
Overall, Epo not only decreases cell death but also enhances neurogenesis and angiogenesis in a 
number of in vitro and in vivo models of brain injury, which may be necessary for long-term 
improvement in brain histology and functional performance.17,59-62,66    
Epo signaling is mediated by binding to Epo-R and activation of several important intracellular 
signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt, Janus kinase 2 
(JAK2)/STAT5, and extracellular signal-regulated kinase (ERK)1/2 pathways. Akt limits 
inflammation,67 decreases apoptotic cell death and increases angiogenesis,68 while STAT5 plays a 
role in cell survival.69 The ERK pathway has anti-apoptotic and anti-inflammatory effects in vitro 
and is critical in neurogenesis and cell fate commitment.70,71  
2.1.3 Pre-clinical studies of Epo in neonatal HI brain injury 
Over 70 pre-clinical studies have tested the neuroprotective effects of Epo following hypoxic-
ischemic brain injury, and these have produced overwhelming histologic and functional evidence 
for benefit.10,20,72 Pre-treatment with Epo prior to hypoxic-ischemic injury is neuroprotective in 
neonatal rodents.73-78 A single dose of Epo administered immediately after hypoxia-ischemia in the 
P7 rat (comparable to human infant at term79) significantly reduces infarct volume, improves 
short-term spatial memory19 and decreases hemispheric brain loss 6 weeks after injury, with 
increased neurogenesis seen in the subventricular zone.17 Multiple doses of Epo after neonatal 
stroke reduce infarct volume in a dose-dependent manner,15 with improvement in sensorimotor 
function.16 Three doses of Epo given after neonatal brain injury result in improved histologic 
outcomes at 1 week,80 and marked preservation of neurologic function at 3 months, including 
improved learning and memory, swim speed, balance and forepaw use, as well as improved 
hemispheric and regional brain volumes.81 Epo results in decreased neuronal loss and learning 
impairment following neonatal hypoxic ischemic brain injury.11,14 When Epo therapy is initiated as 
late as 48 hours after hypoxic-ischemic event, one sees improved behavioral outcomes, enhanced 
neurogenesis and reduced white matter injury at 14 days.18,59 In a rat model of focal ischemic 
injury, we recently found that Epo 1000 U/kg starting as late as 7 days after injury leads to 
significant preservation of brain volume and improved sensorimotor function.82 This finding 
emphasizes the late neuroregenerative effects of Epo. Finally, in non-human primates, Epo 
reduces the rate of CP and improves neurologic function in animals undergoing hypothermia for 
HIE.21  
2.1.4 Dosing regimen 
We will administer 5 IV doses of Epo 1000 U/kg or NS. The first dose will be given at < 24+2 hours 
of age, and subsequent doses on study days 2, 3, 4, and 7. Our dosing regimen is justified by the 
following points, each of which is expanded upon below: 
• 
Multiple doses over 1 week produce maximal neuroprotection (see 2.1.4.1) 
• 
Epo 1000 U/kg IV produces total drug exposure (AUC) and maximum concentration levels 
that produce optimal neuroprotection in animal models (see 2.1.4.2) 
• 
Epo 1000 U/kg falls within the range of doses (500-2500 U/kg) reported in clinical studies 
to improve outcomes in neonatal HIE22,23 (see 2.2.1) 
• 
Epo 1000 U/kg IV appears well-tolerated in newborn infants (see 2.2.2) 


 
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2.1.4.1 Why multiple and late doses? 
Pre-clinical studies show that multiple doses of Epo administered over 1-2 weeks 
produce maximal neuroprotection. Whereas a single Epo dose had no long-term 
histological or behavioral effects after neonatal ischemia injury, rats receiving 3 doses 
of Epo (immediately after injury, 24 hours, and 7 days after injury) demonstrated 
improved function and regional brain volumes into adulthood.81 Further, a multiple 
dose Epo treatment protocol initiated one week following injury in the rat model was 
associated with significant behavioral improvements and preservation of brain 
volume.82 These data provide the rationale for a dose at 7 days following injury, or 
prior to discharge. Prolonged administration of Epo increases oligodendrogenesis and 
neurogenesis while decreasing astrogliosis.18 Rats given multiple doses of Epo 
received a smaller cumulative dose than did the single dose treated rats, suggesting 
that neuroprotection and repair are dependent on a prolonged course of 
administration. Multiple-dose Epo treatment protocols that are initiated between 48 
hours and 7 days after early brain injury also improve short-term sensorimotor 
performance and decrease brain injury, increasing the number of oligodendrocyte 
precursor cells, and enhancing the reorganization of white matter in both mouse and 
rat models of immature or mature brain injury.13,82,83  
2.1.4.2 Dose selection 
Dose selection was informed by our pilot Phase I and Phase II pharmacokinetic 
data.31,84 Among infants being cooled for HIE, Epo 1000 U/kg IV produced overall 
exposure (AUC) and maximum concentration levels that most closely mimic 
established neuroprotective levels from preclinical studies.31  
Sandra Juul (CCC Multi-PI) has shown that multiple doses of Epo 5000 U/kg 
administered subcutaneously (SC) or intraperitoneally (IP) afford the greatest amount 
of neuroprotection in a rodent model.80 At this dose, the mean AUC ranged from 
117,677 (SC) to 140,331 Uh/L (IP) and the mean Cmax ranged from 6,224 U/L (SC) to 
10,015 U/L (IP)85  
From our phase I study (see 2.2.3),84 we know that Epo 1000 U/kg IV, given in 
conjunction with hypothermia, produces drug exposure levels (AUC 131,054 ±17,083 
Uh/L and Cmax 13,780 ± 2,674 U/L) that most closely mimic the target 
neuroprotective levels described above. In contrast, Epo 500 U/kg produced 
insufficient plasma elevations, and doses of 2500 U/kg produced AUC and Cmax 
values that exceeded the optimal neuroprotective range by 3-fold.  
Although the upper safety limit of Epo is unknown, doses ranging from 300 to 3,000 
U/Kg/dose have been studied in randomized controlled trials of preterm and term 
neonates with no resulting safety issues. Preclinical data suggest that too much Epo 
(30,000 U/kg/dose) can lead to diminished efficacy,80 and may be harmful.86 Epo 1000 
U/kg/dose IV is a moderately high dose that establishes Epo exposure levels within 
the optimal neuroprotective range, while also minimizing risks associated with giving 
too much Epo. 
Low doses of Epo (200-400 U/kg) used to treat anemia do not raise CSF Epo 
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Epo administered systemically has been shown to increase measured Epo in the CSF 
or brain tissues of both experimental models and human infants, particularly in the 
setting of acute brain injury.22,85,87-91 In a phase II trial, we confirmed that for neonates 
with HIE receiving hypothermia, Epo 1,000 U/kg every 24 hours resulted in consistent 
achievement of target exposures associated with neuroprotection in animal models.31 
In summary, Epo 1000 U/kg/dose IV produces drug exposure levels that afford optimal 
neuroprotection. This dose is likely to minimize risks associated with giving too much 
Epo, especially in the setting of hypothermia, which may slow drug clearance (see 
2.2.3).  
2.1.4.3 Route of administration 
We will administer Epo IV. Our pilot data84 suggest that Epo 1000 U/kg given IV is safe 
and provides pharmacokinetics consistent with neuroprotective effects in animal 
models (i.e., rats and monkeys). For patients who have lost IV access, SC 
administration will be permitted. 
2.1.5 Need, relevance and priority for the study 
HIE affects up to 12,000 infants annually in the U.S., and accounts for 22% of neonatal deaths 
worldwide, totaling 814,000 deaths in 2008.2 Although hypothermia improves outcomes, 44-53% 
of affected newborns die or experience neurologic disability despite this therapy.3-7,30 New 
treatments are urgently needed. Epo has neuroprotective actions and regenerative effects that go 
beyond the effects of hypothermia alone.  
The proposed research has the potential to significantly reduce suffering from life-long neurologic 
disabilities, and to lead to enormous societal cost savings. In 2012 currency, the lifetime cost of CP 
is estimated at 1.15 million dollars per affected individual.92 Using a conservative estimate of 20% 
CP rates in infants with HIE and hypothermia,3,5,7,93 and a conservative HIE incidence of 2 per 1000, 
each year babies born with HIE introduce an economic burden that will ultimately total $1.7 billion 
in lifetime costs due to CP alone. Similar calculations using CDC cost data92 and rates of disability 
derived from hypothermia studies3,5,7 suggest that each year, HIE produces additional lifetime 
costs of $1.6 billion for intellectual disability.  If no benefits of Epo are found, the trial will 
nevertheless generate important information about clinical and laboratory antecedents of 
neurodevelopmental disabilities due to newborn brain injury, and the data will inform the next 
generation of neonatal neuroprotection trials.  
 SUPPORTING DATA 
2.2.1 Clinical studies of Epo and HIE 
To date, five phase I or II studies have suggested that Epo improves outcomes after HIE.22,23,94  
In an Egyptian study, 15 infants were given 5 daily SC doses of Epo 2500 U/kg starting at 1 day of 
age.23 Compared to placebo controls, those who received Epo had improved 
electroencephalogram (EEG) backgrounds and reduced biomarkers of oxidative stress at 2 weeks 
of age, and better neurodevelopment at 6 months of age.  
In a randomized trial performed in China, researchers treated 84 infants with HIE with Epo 300-
500 U/Kg IV every other day for two weeks. Compared to infants who received placebo, those 
who received Epo were less likely to die or have moderate to severe disability at 18 months of age 
(44% vs. 25%, P=0.02).22 No side effects of Epo therapy were seen. 


 
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The Egyptian and Chinese studies are limited by small size, inadequate masking, deviation from 
intention to treat analysis, and lack of hypothermia. There is currently insufficient evidence to 
support the clinical use of Epo in treating HIE.  
In our phase I pilot trial of Epo for HIE, neurodevelopmental outcomes were available for 22 of 24 
treated infants.24 For 22 infants who received multiple doses of Epo at 250, 500, 1000, or 2500 
U/kg/dose, mean age at last assessment was 22 months (range 8-34). There were no deaths. Eight 
(36%) had moderate to severe brain injury on neonatal MRI.  Moderate to severe disability 
occurred in only 1 child (4.5%).24 
In a phase II randomized, multi-center, double-masked, placebo controlled trial of Epo for HIE 
(NEATO, Wu PI), 50 newborns undergoing hypothermia for moderate/severe HIE were 
randomized to Epo 1000 U/kg/dose or placebo (NCT# 01913340). Infants who were randomized to 
receive Epo exhibited less brain injury on MRI and better 12-month motor outcomes than those 
who received placebo.25 Further studies are needed to definitively establish the efficacy of Epo in 
treating HIE. 
2.2.2 Safety 
Reported Epo side effects in adults with renal failure include hypertension, thrombosis, red cell 
aplasia, myocardial infarction, stroke, congestive heart failure, seizures, tumor progression and 
increased death.95-99 It is important to note that these side effects have been observed with long 
term chronic therapy, and none of these side effects have been reported in neonates.  
Based on the adult experience, potential risks of Epo are listed in the Consent form as follows: 
Rare but serious:  
• 
Increased blood pressure (reported in adults; not reported in infants) 
• 
Increased clotting (reported in adults; not reported in infants) 
• 
Increased risk of seizures (reported in adults on dialysis; not reported in infants) 
• 
Increased risk of death (reported in adults with cancer or stroke; not reported in infants) 
• 
Polycythemia (reported in adults with long term use; not reported in infants) 
There is an extensive literature of Epo therapy in preterm infants. Between 1991 and 2006, over 
2400 infants were enrolled in 30 randomized controlled trials of Epo for anemia of prematurity, 
with Epo therapy ranging from 70 to 5000 U/kg/week (35 – 750 U/kg/dose) lasting 2 weeks to 
several months in duration.100 Although a concern has been raised that chronic Epo therapy in 
infants under 32 weeks of gestation might increase the risk of retinopathy of prematurity101 and 
skin hemangiomas,102 these findings have not been reported in prospective randomized controlled 
trials in preterm or term infants. 103,104 
No adverse events have been reported in prospective studies of high-dose Epo in neonates:  
1. Phase II studies of HIE: 300-2500 U/kg Epo (N=182) (see 2.2.1)22,23,25,94,105 
2. Phase I study of HIE: up to 6 doses of Epo 250–2500 U/kg (N=24)84 (see 2.2.3)  
3. Phase III study of preterm infants (ongoing): 6 doses of 1000 U/kg followed by maintenance 
doses of 400 U/kg three times a week until 32-6/7 corrected weeks of gestation (N=941) (PI 
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4. Phase III study of HIE (ongoing): 3 doses of Epo 1000 U/kg (n=115) (Patkai PI, Neuroepo study) 
5. Phase I/II study of preterm infants: 3 daily doses 500-2,500 U/kg (N=30)107  
6. Phase III trial of preterm infants: 3 daily doses 3000 U/kg (N=30)108 
7. Phase I/II study of congenital heart disease: 3 daily doses 1000 U/kg (N=33)109  
8. Phase II study of perinatal stroke: 3 daily doses 1000 U/kg (N=25)110  
9. Phase II of preterm infants: 400 U/kg 3 times a week (N= 56)111,112 
10. Phase III of preterm infants: 3 daily doses 3000 U/kg (N=448)108,113  
Therefore, high-dose Epo appears to be safe when administered to neonates in the first 1-2 weeks 
after delivery. Epo 1000 U/kg/dose falls well within the range of high dose Epo treatment 
regimens that have been studied in a number of neonatal neuroprotection trials, none of which 
have raised concerns regarding safety. Polycythemia is a theoretical concern, but our experience 
in term HIE (see 2.2.3) and in preterm infants107 suggests that hematocrit in fact decreases over 
time because of phlebotomy losses in these critically ill infants.  
2.2.3 Pilot studies 
Phase I dose escalation and pharmacokinetic study (IND 102,138)  
We performed a phase I study of the safety and pharmacokinetics of Epo + hypothermia.84 This 
was a multicenter, open label, dose escalation study involving 5 sites (UCSF, Oakland Children’s 
Hospital, University of Washington, Children’ National Medical Center, Santa Clara Valley Medical 
Center). Patients had moderate/severe HIE with acidosis (mean cord pH = 6.84, SD 0.14), neonatal 
resuscitation > 10 minutes (88%) or 10 minute Apgar < 5 (63%). 
We tested 4 doses of Epo IV: 250 (N=3), 500 (N=6), 1000 (N=7) and 2500 U/kg/dose (N=8). 
Patients received up to 6 doses every 48 hours, with the first dose given within 24 hours of age. 
Average age at consent was 15.4 hours (SD 5.7), and average length of hospital stay was 13.5 days 
(SD 7.2). Patients received an average of 4.8 doses.  Reasons for not completing the 6-dose 
regimen include being discharged from the hospital and loss of IV access. 
Epo followed nonlinear pharmacokinetics, consistent with previous reports.85,114,115 As the dose of 
Epo increased 4 and 10-fold (from 250 to 1000 and 2500 U/kg), the overall exposure to circulating 
Epo (AUC) increased 7.1 and 17.8 times, respectively (Table 2). However, there was no excessive 
accumulation of drug following multiple doses, at any of the doses studied. Steady-state plasma 
Epo concentrations were attained by the second dose for all four dosages, and peak and trough 
concentrations were stable across doses. Plasma Epo concentrations demonstrated fairly limited 
variability across individual patients, with an average coefficient of variation of 26% for Cmax. 
Compared to premature infants given identical doses of IV Epo,107 our patients demonstrated 
about a 2-fold reduced rate of Epo elimination. Possible explanations for the slower drug 
elimination observed include hypothermia treatment, renal compromise, older gestation, hypoxia-
ischemia, or a combination of these factors. 
Epo 1000 U/kg achieved a mean AUC and Cmax levels that most closely mimic the target 
neuroprotective levels derived from pre-clinical studies (Table 2). In contrast, Epo 500 U/kg/dose 
produced insufficient plasma elevations, and doses of 2500 U/kg produced AUC and Cmax values 
that exceeded the optimal neuroprotective range by about 3-fold.  
 


 
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Table 2. Single dose pharmacokinetic parameters of 4 different Epo doses given in conjunction with 
hypothermia, in neonates with HIE. 
 
PK parameter 
250 U/kg 
(n=3) 
500 U/kg 
(n=6) 
1000 U/kg 
(n=7) 
2500 U/kg 
(n=8) 
Target level* 
AUC ([U*h]/L) 
18426 ± 
8976 
50306 ± 7426 
131,054 ± 
17,083 
328002 ± 
61945 
117,677- 
140,331 
Cmax 
(mU/mL) 
3156 ± 1615 
7046 ± 814 
13,780 ± 
2,674 
33316 ± 
7377 
6,224 - 10,015 
Cl (ml/h/kg) 
15.6 ± 6.3 
10.1 ± 1.5 
7.7 ± 0.9 
7.9 ± 1.5 
 
t ½ (h) 
7.6 ± 6.9 
7.2 ± 1.9 
15.0 ± 4.5 
18.7 ± 4.7 
 
*Target levels are AUC and Cmax levels that produce optimal neuroprotection in animal 
models. 
 
No deaths or serious adverse effects were noted in the pilot trial. No patients experienced 
polycythemia. Mean hematocrit decreased from 45.6 to 41.5% between day 1 and final day of 
testing (5-14 days). Brain MRI performed at a median of 6 (range 4-13) days of age revealed no 
intracranial hemorrhages or sinovenous thromboses. MRI was normal in 13 (54%), demonstrated 
watershed injury in nine (42%), basal ganglia injury in one (4%), and focal arterial infarction in one 
(4%). Among infants with HIE who have undergone hypothermia, normal MRI/MRS scans have 
been reported in 17-54%.116-118 Outcomes were available for 22 of 24 infants. For these 22 infants, 
mean age at last assessment was 22 months (range 8-34). There were no deaths. Eight (36%) had 
moderate to severe brain injury on neonatal MRI.  Moderate to severe disability occurred in 1 
child (4.5%), in the setting of moderate to severe basal ganglia/thalamic injury.  
 
 


 
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Figure 1. Epo pharmacokinetics in 24 infants undergoing hypothermia for HIE.  
 
Phase II Randomized Controlled Trial of Epo for HIE (NEAT-O study, IND 102,138) 
In a phase II randomized, multi-center, double-masked, placebo controlled trial of Epo for HIE 
(NEATO, Wu PI), 50 newborns undergoing hypothermia for moderate/severe HIE were 
randomized to Epo 1000 U/kg/dose or placebo (NCT# 01913340). Consent rate was 79%, and 
enrollment was completed ahead of schedule. The first study drug dose was given at mean 16 
hours of age. Follow up rate at 6 months of age was 97%. No safety concerns were raised by the 
Data Safety Monitoring Board (DSMB).  
Our phase II trial (N=50) was not designed to show efficacy; yet the results are promising.25 
Neonatal brain MRI performed at mean 5.1 (SD 2.3) days was independently reviewed by 2 
masked evaluators, and scored using the Washington University MRI scoring system.119 The MRI 
brain injury severity score was significantly lower in the Epo-treated than placebo group (4.0 vs. 
16.4, P = 0.003). Similarly, moderate/severe brain injury was less common (5% vs. 44%), and 
normal brain MRIs were more common (36% vs. 12%) in infants who received Epo vs. placebo. 
Subcortical injury (i.e., injury to the basal ganglia, thalamus or posterior limb of the internal 
capsule), a well-known predictor of adverse neurologic outcome,117,120 was significantly reduced in 
the Epo treated group (36% vs. 68%, P=0.04). At 12 months of age, we assessed development 
using the Alberta Infant Motor Scale (AIMS) and the Warner Initial Developmental Evaluation of 
Adaptive and Functional Skills (WIDEA),121,122 a questionnaire that assesses self-care, motor 
function, communication and social cognition in young infants and children. Infants who received 
Epo scored better on the AIMS score (53.2 vs. 42.8, P=0.03), and on the WIDEA motor subscore 
(28.6 vs. 23.8, P=0.05) than infants who received placebo.25  
 
 
 


 
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3 STUDY DESIGN 
Overview: We propose a multicenter, randomized, double-masked, placebo-controlled trial to test the 
hypothesis that Epo given to infants with moderate-severe HIE will safely lead to improved 
neurodevelopmental outcome at 24 months. We will enroll 500 newborns ≥ 36 weeks gestational age 
with moderate/severe HIE determined by neurologic symptoms, Apgar score, level of acidosis, and need 
for resuscitation. Study drug (Epo or placebo) will be given IV on study days 1, 2, 3, 4, and 7.  
Primary outcome will be assessed at 24 months. For extenuating circumstances, for example, COVID-19 
restrictions, the in-person elements of the final endpoint evaluation may be performed up to 36 months 
of age. The primary endpoint is the composite of death or neurodevelopmental impairment. Impairment 
(mild, moderate, or severe) is defined as of any of the following: 1) GMFCS level ≥1, or 2) GMFCS = 0 or 
0.5 AND CP (any type), or 3) Bayley III <90. Since death is a competing outcome, it is critical to include it 
in the primary outcome measure.  
Secondary analyses at age 24 months: we will assess the effect of Epo on a) presence of CP, b) severity 
of motor impairment, c) Bayley III cognitive and language scores, d) epilepsy (i.e., ≥ 2 afebrile, 
unprovoked seizures), and e) behavioral abnormalities (i.e., attention problems or aggressive behavior) 
based on the Child Behavior Checklist (CBCL) externalizing score. 
Additional exploratory analyses include the effect of Epo on sensory deficits that can result from HIE. We 
will collect 24-month data regarding the presence of cortical visual impairment diagnosed by an 
ophthalmologist, and hearing impairment requiring hearing aids. To elucidate the effect of Epo on all 
severities of impairment, we will perform a secondary analysis of the effect of Epo on the following 4-
level outcome: 1) normal, 2) mild motor and/or cognitive impairment, 3) moderate/severe motor 
and/or cognitive impairment, and 4) death. Severity of motor impairment will be determined by type of 
CP and GMFCS level. Severity of cognitive impairment will be determined by the Bayley III Cognitive 
Score. 
 
4 SELECTION AND ENROLLMENT OF SUBJECTS 
 INCLUSION CRITERIA 
1. Newborns ≥ 36 weeks gestational age 
2. Receiving active or passive whole body cooling/hypothermia since < 6 hours of age 
3. Perinatal depression based on at least one of the following: 
a. Apgar score < 5 at 10 minutes, or 
b. Need for resuscitation at 10 minutes (i.e., chest compressions, or positive pressure 
respiratory support including endotracheal,  mask ventilation, or CPAP), or  
c. pH < 7.00 in a cord gas (arterial or venous) or in an infant gas (arterial or venous) 
obtained at < 60 minutes of age, or  
d. Base deficit ≥ 15 mmol/L in a cord gas (arterial or venous) or in an infant gas (arterial or 
venous) obtained at < 60 minutes of age 
4. Moderate to severe encephalopathy, based on presence of at least 3 of 6 Sarnat criteria 
present between 1-6 hours after birth:  
a. Reduced consciousness 
b. Decreased spontaneous activity 
c. Posture 


 
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d. Tone 
e. Primitive reflexes (suck and Moro) 
f. Autonomic abnormality (pupils and respirations) 
4.1.1 Prior therapy 
All enrolled infants will have started hypothermia by 6 hours of age, which is standard of care at 
each of the study sites. Hypothermia will consist of whole body cooling at all study sites. 
4.1.2 Demographic characteristics 
Enrollment is irrespective of gender, race and ethnicity.  
 
 EXCLUSION CRITERIA 
1. Study drug unlikely to be administered within 26 hours of birth 
2. Infant has living twin (or higher order multiple) who is also being cooled. 
3. Birth weight < 1800 g (i.e., intrauterine growth restriction) 
4. Genetic or congenital condition that affects neurodevelopment or requires multiple surgeries 
(e.g., congenital viral infection, hydrops, complex congenital heart disease, severe dysmorphic 
features, etc.) 
5. Head circumference <30 cm 
6. Redirection of care is being considered due to moribund condition 
7. Patient anticipated to be unavailable for evaluation at age 2 
8. Polycythemia (hematocrit > 65.0%) 
9. Parents/legal guardians with diminished capacity or autonomy1 
10. Infant is participating or intends to participate in another interventional study during the birth 
hospitalization (note: does not include observational studies) 
11. Sentinel event and encephalopathy occurred only after birth2 
12. Unable to consent in primary language of parent(s) 
  
 STUDY ENROLLMENT PROCEDURES 
4.3.1 Identification and recruitment 
Infants ≥ 36 weeks gestational age who are undergoing hypothermia for HIE will be screened by 
the study investigators for eligibility. A study investigator will discuss the child’s condition with the 
attending physician. With the attending physician’s approval, the parents will be approached by a 
study investigator and/or study coordinator who will explain the study and obtain informed 
consent. Parents who are at an outside hospital may be given study written materials by the 
transport team, after which a study investigator may obtain consent by phone, given the time 
sensitive nature of the study. A brochure and consent form explaining the study in lay language 
will be provided to the family either in person or via fax if the parents are at an outside hospital. 
 
1 Please refer to your individual IRB requirements regarding permission to enroll an infant in the study if only one 
parent/legal guardian meets this exclusion criterion. 
2 Some infants may experience a sentinel event after birth leading to a poor blood gas before 1 hour of age, but 
have no prior signs of low Apgars, acidosis, prolonged resuscitation, or encephalopathy. These infants will be 
excluded from the study.  


 
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Once written consent is obtained (in person or by phone), the infant will be enrolled and 
randomized to receive Epo study drug or placebo. Consent rate is expected to be 80% or more.  
4.3.2 Screening for eligibility and monitoring of recruitment targets 
Data will be collected on all patients who are screened for enrollment. These data will include the 
inclusion/exclusion criteria in Sections 4.1 and 4.2 above, and hence confirm eligibility or provide 
the reasons for ineligibility, as well as reasons for nonparticipation of eligible subjects. Non-HIPAA 
related data on all screened patients will be submitted to the Data Coordinating Center (DCC), and 
summary reports will be reviewed by the site Principal Investigators (PIs) and Clinical Steering 
Committee. All sites are anticipated to enroll at least 12-20 infants a year. We will assess site 
enrollment on a monthly basis, and if enrollment at an individual site falls below expected levels 
(i.e., enrolling only 3 infants in 6 months, or 1.5 infants every 3 months), the Executive Committee 
and DSMB will evaluate performance. The multi-PI’s will give the site warning, and if 
underperformance continues, the site may be dropped from the trial.  
4.3.3 Consent procedures 
A study investigator and/or coordinator will obtain parental informed consent. For out-born 
infants whose parent(s) are not physically present, we will obtain written consent over the phone. 
That is, if the parent(s) are at an outside hospital, we will provide them with a copy of the consent 
form, either from the Transport team, or via fax. The consenting study personnel will then read 
through the consent form with both parents (when possible) over the phone, or in person if 
possible. Consent will be documented according to local and national regulations. 
4.3.4 Stratification, Randomization and Masking Procedures 
The DCC will provide each site pharmacy with a randomization look-up table that pairs each study 
ID with a treatment allocation. Randomization is stratified by site and HIE severity level. See 
6.3.2.1 for definitions of “moderate” and “severe” HIE, based on modified Sarnat score. The site 
pharmacy will then draw up the assigned study drug dose into a syringe, based on the 
randomization list. The appearance of the syringes of Epo and placebo are identical, thus the 
bedside staff remain masked. Only the research pharmacist and DCC biostatistician will be un-
masked to treatment assignment. All parents, study and clinical staff, as well as all Clinical 
Coordinating Center (CCC) members, will remain masked to treatment assignment throughout 
follow-up. To ensure balance within clinical centers, the randomization will be implemented using 
a randomly permuted blocks design to ensure approximate balanced assignments within site and 
HIE severity level. The sequence of assignments for each site and stratum will be prepared in 
advance by the DCC.  
After all final follow-up evaluations have been completed, which may be at age 8 or older if 
additional funding is obtained to follow subjects beyond 24 months of age, an effort will be made 
to inform participating families of the completion of the study, and families will be offered an 
opportunity to learn the treatment group assignment for their child. 
 
5 STUDY INTERVENTIONS 
 STUDY DRUG ADMINSTRATION AND DURATION 
Patients will be randomized to receive either IV Epo 1000 U/kg/dose (based on birth weight), or an equal 
volume of IV NS. Study drug (Epo vs. NS) will be administered on study days 1, 2, 3, 4, and 7, with the 


 
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same dose/volume used for each administration. We will administer Epoetin alpha for injection (4000 
U/mL), manufactured by Amgen Inc., prepared and used according to the package insert. Study drug will 
be infused via IV push over 1-2 minutes, followed by 1 mL normal saline flush. If an institution does not 
allow medications to be given to neonates via IV push, study drug may be infused via pump infusion 
over 5 minutes, followed by 1 mL NS flush. Potential side effects of the drug administration include 
introduction of infection, and bleeding from the line site. All subjects will be treated in a level III or IV 
neonatal intensive care unit.  The initial study drug dose should be given as soon as possible after 
randomization and no later than 24 +2 hours of age.  Each subsequent dose should be given within +/- 2 
hours of the time that the first dose was given (i.e., within a 4 hour window). This window provides 
some flexibility so that study drug does not have to be given during change of nursing shift, or while the 
infant is undergoing a procedure, for example.  Patients will not be kept in the hospital solely for 
completion the study intervention. If patients are discharged from the hospital prior to day 7, they may 
receive the final study drug dose on the day of discharge, provided that it given at least 20 hours 
following the prior dose. If the patient loses IV access prior to receiving all study drug dose(s), then the 
remaining dose(s) may be given SC. That is, placement of a new IV for the sole purpose of giving study 
drug will not be required.  
 HANDLING OF STUDY DRUG 
Each participating hospital will order Epo 4000 unit/mL, 1-ml vials, from a commercial wholesaler. Sites 
will generally purchase sufficient Epo to treat 2-4 patients randomized to the Epo arm. Each 
participating site pharmacy will be responsible for appropriately storing, accounting for and dispensing 
the study drug. Pharmacy accountability will be monitored closely. The research supply of Epo will be 
kept separate from other commercial stocks of Epo at the participating sites. Epo or placebo will be 
dispensed for IV administration as a patient-specific unit-dose syringe upon receipt of an investigator-
physician order for a research subject. Depending on the subject’s randomized treatment assignment, 
the un-masked pharmacy personnel will dispense a syringe containing the prescribed dose of Epo or an 
equivalent volume of 0.9% Sodium Chloride for Injection, USP (NS).  Both Epo and placebo syringes will 
be labeled, packaged, transported, stored and administered in an identical manner so as to maintain the 
masking. Labeling will be adapted to meet local labeling requirements, but will not un-mask the drug 
assignment. The site pharmacies shall maintain accountability logs for all research supplies of Epo. 
Accountability logs will be reviewed by the DCC. 
The sites will maintain accountability logs for the Epo inventory. These logs will include the purchase 
date (date received), lot number, expiration date, and quantity of all Epo purchased for the trial. In 
addition, every dose dispensed will also be recorded in a subject-specific accountability log. The Epo lot 
number for each dose dispensed will be recorded the inventory balance will also be noted on the 
accountability log. Individual subject drug accountability logs should be sent to the HEAL financial 
analyst on a regular basis and these logs will be reviewed by the DCC to ensure appropriate drug 
administration per the randomization log. 
Epoetin alfa purchased for the HEAL trial will be kept refrigerated, separate from the regular medication 
inventory. The refrigerator will be monitored and alarmed and will have access to emergency backup 
power. Epoetin alfa vials must be stored in a refrigerator between 35.6°F to 46.4°F (2⁰C -8⁰C). 
Epo will be labeled with stickers marked “For Investigational Use Only”. The invoices for the purchase of 
the Epoetin will be retained at the local site in the same manner that a study drug shipping receipt 
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Epo vials must be stored in the refrigerator between 36°F to 46°F (2°C to 8°C). Do not freeze vials and do 
not use a vial of Epo that has been frozen in the past. Keep vials away from direct light. Do not shake 
Epo vials. Single use vials of Epo should be used only one time. Only one dose of study drug may be 
drawn up at a time. 
If the wrong study drug is administered to a subject (i.e., Epo is given to a Control patient, or normal 
saline is given to a Treatment patient), the error should be reported in a blinded manner to the CCC as a 
protocol violation, and all future doses should be corrected to the original randomization assignment. 
The study violation must also be reported to the local IRB per institutional policy. 
 CONCOMITANT AND REQUIRED INTERVENTIONS 
Ideally, all infants enrolled in HEAL will be treated as consistently as possible across sites, so the major 
difference between subjects is their treatment with Epo vs. placebo. To this end, we have developed a 
set of recommended Clinical Guidelines regarding the treatment of infants with HIE, and sites will be 
encouraged to follow these guidelines when possible. These guidelines address 1) hypothermia therapy 
duration and goal temperature, as well as use of sedatives; 2) monitoring of laboratory tests during the 
neonatal period; 3) EEG brain monitoring; and 4) general approach to treatment of clinical and sub-
clinical seizures.  
 PROHIBITED AND PRECAUTIONARY INTERVENTIONS 
There are no prohibited and precautionary interventions for this study.  
 ADHERENCE ASSESSMENT 
Study drug will be administered by a nurse (i.e., bedside nurse, charge nurse, etc.) in the neonatal 
intensive care unit. A detailed record of study drug administration will be kept by Investigational Drug 
Services Pharmacy, and documentation of study drug administration will be completed by the nurse 
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6 CLINICAL AND LABORATORY EVALUATIONS 
 SCHEDULE OF EVALUATIONS 
Table 3. Schedule of Evaluations 
 
Evaluation 
Visit Day 
Window 
Location 
Details 
Screening 
Study Day 0 or 
1 
Before 
randomization 
NICU 
Screening Clinical Assessment 
(Apgar, resuscitation, acidosis, 
Modified Sarnat exam, 
Inclusion/Exclusion assessment) 
Therapeutic 
Hypothermia Begins 
Study Day 0 or 
1 
Begin by <6 
hours post-birth 
NICU 
Begins prior to administration of 
study drug and at <6 hours of age, 
continues for 72 hours, per 
standard of care. 
Consent & 
Enrollment 
Study Day 0 or 
1 
Prior to 24 + 2 
hours post-birth 
NICU 
Informed Consent 
Randomization 
Study Day 0 or 
1 
Prior to 24 + 2 
hours post-birth 
Pharmacy 
HEAL Portal 
Blood sample (pre-
intervention) 
Study Day 0 or 
1 
Before 
Intervention 1 
NICU 
1.5 mL blood sample 
Urine sample 1 
Study Day 0 or 
1 
1st void after 
consent 
NICU 
1-2 mL urine sample, ideally 
obtained prior to study drug 
administration (note: urine may be 
collected after study drug 
administration if not available 
earlier, and in this case, does not 
require a Protocol Deviation) 
Intervention 1 (study 
drug)* 
Study Day 1 
Prior to 24 + 2 
hours post-birth 
NICU 
Target time is as soon as possible 
Intervention 2 (study 
drug) 
Study Day 2 
24 ± 2 hours 
following 
Intervention 1 
NICU 
Target time is same time of day as 
Intervention 1 
Blood sample 2 
Study Day 2 
24 ± 2 hours 
following 
Intervention 1 
NICU 
1.5 mL blood sample. Target time is 
same time of day as Intervention 2 
Intervention 3 (study 
drug) 
Study Day 3 
48 ± 2 hours 
following 
Intervention 1 
NICU 
Target time is same time of day as 
Intervention 1 
Urine sample 2 
Study Day 3-4 
After rewarming 
completed, and 
within 24 hours 
after rewarming 
NICU 
1-2 mL urine sample 


 
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Evaluation 
Visit Day 
Window 
Location 
Details 
Intervention 4 (study 
drug) 
Study Day 4 
72 ± 2 hours 
following 
Intervention 1 
NICU 
Target time is same time of day as 
Intervention 1 
Blood sample 3 
Study Day 4 
72 ± 2 hours 
following 
Intervention 1 
NICU 
1.5 mL blood sample. Target time is 
same time of day as Intervention 4 
Brain MRI/MRS 
(routine clinical care) 
Study Day 4-5 
Ideally between 
96-144 hours 
post-birth 
NICU 
After rewarming, and ideally after 
Intervention 4 has been 
administered 
Clinical assessment 
Study Day 5 
Anytime this 
calendar day 
NICU 
Modified Sarnat exam 
Intervention 5 (study 
drug) 
Study Day 7 
144 ± 2 hours 
following 
Intervention 1 
NICU 
Target time is same time of day as 
Intervention 1 
EEG Summary 
Study Days 1-7 
N/A 
NICU 
Upload EEG reports 
Clinical Blood Test 
Summary 
Study Days 1-7 
N/A 
NICU 
Record renal function, CBC, liver 
function labs, as collected per site’s 
clinical standard of care. 
Follow-up (month 4) 
Month 4 
Day of month 
(±7 days) 
Phone 
Contact info, intervening medical 
and developmental history, assess 
adverse events 
Follow-up (month 8) 
Month 8 
Day of month 
(±7 days) 
Phone 
Contact info, intervening medical 
and developmental history, assess 
adverse events 
Follow-up (month 12) 
Month 12 
Day of month 
(±28 days) 
Phone 
Contact info, intervening medical 
and developmental history, WIDEA, 
assess adverse events 
Follow-up (month 18) 
Month 18 
Day of month 
(±28 days) 
Phone 
Contact info, intervening medical 
and developmental history, WIDEA, 
assess adverse events 
Follow-up (month 24) 
Month 24 
Day of month 
(±56 days) 
Phone 
(may also 
be done 
in clinic) 
Contact info, intervening medical 
and developmental history, WIDEA, 
assess adverse events 
Follow-up (month 24) 
Month 24 
Day of month 
(±56 days, may 
extend up to 36 
months of age in 
extenuating 
circumstances, 
e.g., COVID-19) 
Clinic 
Standard Neurologic Exam (Kuban), 
Bayley III, GMFCS Evaluation, Child 
Behavior Checklist (CBCL), growth 
parameters, assess adverse events 


 
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Evaluation 
Visit Day 
Window 
Location 
Details 
Extended Contact 
Calls 
Months 30, 36, 
42, 48, 54, 60, 
66, 72, 78, 84, 
90, 96 
Day of month 
(±56 days) 
Phone 
Confirm contact information, 
collect basic information about how 
child is doing. WIDEA at 30 and 36 
months 
Note: failure to complete, or 
completing these Extended Contact 
calls outside of window does NOT 
constitute a study deviation. 
* Study Day 1 is defined as the calendar day on which Intervention 1 is administered. If the study drug is 
administered on the calendar day following the day of birth, then the day of birth will be called Study Day 0. 
 
 


 
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 TIMING OF EVALUATIONS 
6.2.1 Pre-Randomization Evaluations – Timing 
Screening <24+2 hours post-birth: Screening and pre-entry clinical assessment may occur 
concurrently. Screening does not involve procedures that are not part of routine patient care. 
Patients should be screened and evaluated as soon as possible after birth. The Sarnat examination 
should be performed and recorded between 1-6 hours of age, preferably prior to administration 
of any sedative drugs. When performed by an outside physician or an individual not trained in the 
HEAL Sarnat exam, the Sarnat exam should be confirmed by a HEAL study physician (see Section 
6.3.2.1). 
Consent and Randomization (<24 hours): Consent must occur by 24+2 hours of age (i.e., post-
birth). Study entry (i.e. randomization) may occur at any time up to 24+2 hours of age, as long as 
the first dose of study drug is administered by 24+2 hours (ideally ≤ 24 hours) of age.  
Clinical Assessment (<24 hours): For subjects successfully screened, consented, and randomized, a 
baseline blood sample will be collected prior to giving the first dose of study drug. The 1.5 mL 
blood sample must be collected within the first 24+2 hours of age, which is the latest time point at 
which drug administration can be begin. A 1-2 mL urine sample will be collected from the first void 
after consent is obtained.  The urine does not need to be collected prior to giving the first dose of 
study drug. When possible, EEG monitoring will also begin by 24+2 hours of age, as will laboratory 
monitoring according to the HEAL Clinical Guidelines and institutional guidelines for care of 
critically ill newborns. We will request placental pathology reports, as available. To facilitate 
obtaining this data, clinical pathologic examination of the placenta should be performed when 
possible. 
6.2.2 Birth Hospitalization Evaluations – Timing 
Blood and urine samples. During the same four-hour window permitted for delivery of study drug 
on Study Day 2, a 1.5 mL blood sample will be collected for biomarker analyses (Section 6.3.5). A 
final blood (1.5 mL) sample for biomarker studies will be performed during the same four-hour 
window permitted for delivery of study drug on Study Day 4. A urine sample (1-2 mL) will also be 
collected after rewarming, on Study Day 3-4. 
EEG or aEEG. EEG or aEEG will be performed during the first 3 days, consistent with HEAL Clinical 
Guidelines whenever possible. Continuous EEG monitoring is encouraged, as recommended by the 
American Clinical Neurophysiology Society in their 2011 published guidelines for care of neonates 
with HIE 123. 
MRI/MRS. The patient will undergo a clinical head MRI/MRS, ideally 96-144 hours post-birth per 
clinical standard of care. If a subject is transitioning to comfort care and has not received any head 
imaging (head ultrasound (HUS) or MRI/MRS), it is recommended to obtain a HUS if possible. 
Modified Sarnat. A modified clinical Sarnat exam will be repeated, ideally by a physician who has 
been trained on the HEAL Sarnat exam, when possible, and recorded on Study Day 5. 
6.2.3 Follow-Up Evaluations – Timing  
The following evaluations will be performed on all surviving subjects who are consented and 
randomized, regardless of whether they completed the study intervention. Note that subjects who 
are discharged from the hospital prior to completing the study intervention will still be followed, 
with all efforts made to retain such subjects in the study for purposes of collecting outcome data. 


 
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Interim phone evaluations. Follow-up phone contact will be carried out at 4 months ± 7 days, 8 
months ± 7 days, 12 months ± 28 days, 18 months ± 28 days of age, and 24 months ± 56 days of 
age. The 24-month phone call may be performed in the clinic concurrent with the 24-month visit. 
At each phone interview, contact information will be confirmed and updated as appropriate, and 
information about intervening medical and developmental history will be sought. The WIDEA 
parental questionnaire will also be administered at 12, 18, and 24 months of age.  
6.2.4 Final Endpoint Evaluation 
24-month neurodevelopmental examination. Formalized testing (Standardized neurologic 
examination, Bayley III, and GMFCS) will be done in person at 24 months ± 56 days of age (i.e., ± 2 
months). For extenuating circumstances, for example, COVID-19 restrictions, the in-person 
elements of the final endpoint evaluation may be performed up to 36 months of age. 
6.2.5 Future Studies 
We plan to apply for further funding that will allow us to continue to follow study subjects through 
age 8. 
 INSTRUCTIONS AND DEFINITIONS OF EVALUATIONS 
6.3.1 Informed Consent 
All newborn infants admitted to the intensive care nurseries at participating sites will be screened 
for eligibility by the study physicians and/or study coordinators on a daily basis. A study 
investigator or research coordinator (available by pager or cell phone) will be contacted regarding 
potential candidates. The parents or legal guardians will first be notified of the study by the 
treating physician. If they indicate an interest in learning more, then a study investigator or 
coordinator will approach the parents, describe the study, answer questions, and provide a study 
brochure and consent form. Infants must be consented, randomized, and receive study drug by 24 
+ 2 hours of age. In the event that an out-born infant does not have a parent present, we will 
attempt to obtain written consent over the phone when permitted. When an infant with HIE at an 
outside hospital is identified for transport to the study hospital, a study investigator and/or 
coordinator will be notified immediately of the impending transfer, so that necessary steps can be 
taken to achieve enrollment in the required timeframe. When possible, both parents will review, 
discuss, and sign the consent form with a study investigator and/or coordinator. At least one 
parent with legal authority to do so will be asked to sign the consent form and HIPAA form. In 
some cases, the form may be faxed, scanned and emailed, or texted as a photo to the investigator 
and/or coordinator. The signed Consent form will need to be provided per the enrolling hospital’s 
requirements. A copy of the signed consent and HIPAA forms will be given to the family. A copy of 
the signed consent form will be placed in the medical record, as well as kept in the study records. 
The original signed consent form will be kept by the local site PI. HIPAA language may be included 
in the consent form as directed by local authorities. 
6.3.2 Clinical assessment 
Each of the clinical characteristics listed below will be recorded on the Case Report Forms (CRFs) 
at the time points indicated. 
6.3.2.1 Screening clinical assessment (<24+2 hours):  
1. Apgar scores at 5 and 10 minutes 


 
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2. Resuscitation: presence of endotracheal ventilation, mask ventilation, CPAP, or 
chest compressions at 10 minutes after delivery. 
3. Acidosis: pH and base deficit in a cord, arterial, or venous blood sample taken 
before 60 minutes of age. 
4. Sarnat Exam Procedures. The modified Sarnat Exam should be performed between 
1-6 hours of age prior to administering sedative medications, by a neonatologist, 
neurologist, pediatrician or neonatal nurse practitioner. The qualifying Sarnat exam 
should be the worst exam before sedative medications are administered to the infant. 
The exam cannot be evaluated in an infant who is under the influence of paralytic 
medications.  
A Sarnat Screening Worksheet (1 page, hard copy) must be filled out for all enrolled 
infants. We will record the age of the infant (in hours) at the time of the qualifying 
exam. 
The examiner should be trained in the HEAL Sarnat examination whenever possible. If 
the examiner has not undergone HEAL Sarnat training, the exam should be confirmed 
by a HEAL physician by 24 hours of age, by at least one of the following mechanisms: 
1) The HEAL research physician performed the qualifying exam; 2) HEAL research 
physician discussed the findings with the outside physician/transport team member 
who performed the exam; or 3) HEAL research physician corroborated the exam 
findings with a treating physician who spoke with the Sarnat examiner.   
The Sarnat Screening Sheet must be signed by the HEAL Physician who confirmed the 
exam. It is permissible for signature to occur after the subject has been enrolled, 
however, whenever possible, the Sarnat should be verbally confirmed prior to 
enrollment. 
If the qualifying Sarnat exam is not done prior to receiving sedating medications, 
and/or cannot be confirmed by a HEAL physician by 24+2 hours of age, the infant 
could still qualify for the study.  However, this will be noted on the Sarnat Screening 
Sheet. If a HEAL physician determines that an enrolled infant did not actually meet 
Sarnat criteria for eligibility, and the infant has already been enrolled, then this should 
be reported as a protocol violation. 
All items on the Sarnat Screening Sheet must also be entered into the HEAL Portal in 
order for the infant to be randomized and enrolled into the study.   
The Sarnat Screening Sheet must be kept on file at each enrolling site, along with the 
infant's signed consent form.  The Sarnat Screening Sheet will be used as a source 
document during monitoring visits, to ensure accuracy of data entry into the HEAL 
Portal. 
5. Sarnat Exam Findings (Table 4).3,5,26 The HEAL study will incorporate a Modified 
Sarnat Exam to determine the degree of encephalopathy. “Severe” encephalopathy 
refers to the presence of more symptoms classified in the severe category than in the 
moderate category. If signs are equally distributed between the two categories, then 
designation of severity is based on level of consciousness.3 Degree of encephalopathy 


 
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will be calculated within the HEAL Portal, based on the exam findings that are entered 
into the screening form. 
 
Table 4. Modified Sarnat Encephalopathy3,26 
Category 
Normal 
Mild 
Abnormality 
Moderate 
Abnormality 
Severe Abnormality 
Unable to 
assess? 
1. Level of 
consciousness 
Normal 
Hyperalert or 
irritable 
Lethargic or poorly 
responsive 
Minimal or no 
responsiveness 
N/A 
2. Spontaneous 
activity 
Normal 
Slightly 
decreased 
Decreased 
Absent 
N/A 
3. Posture 
Normal 
Mild distal 
flexion 
Distal flexion, 
complete 
extension 
Decerebrate 
N/A 
4. Tone 
Normal 
Hypertonic 
Hypotonic 
Flaccid 
N/A 
5. Primitive reflexes 
 
 
 
 
 
a. Suck 
Normal 
N/A 
Weak or bite 
Absent 
Unable to 
assess 
b. Moro 
Normal 
Low threshold 
to elicit 
Weak or 
incomplete 
Absent 
Unable to 
assess 
6.  Autonomic  
 
 
 
 
 
a. Pupils 
Normal 
N/A 
Constricted 
Dilated and either 
fixed or sluggishly 
reactive; asymmetric 
Unable to 
assess 
b. Respiration 
Normal 
N/A 
Periodic Breathing 
Intubated and 
ventilated 
N/A 
 
6.3.2.2 Demographics  
Demographic information: maternal and paternal race and ethnicity; maternal 
education will be collected prior to discharge. Variables from the NINDS Common 
Data Elements will be used.  
6.3.2.3 Day five clinical assessment 
Modified Sarnat score (Table 4). At Study Day 5, the infant will undergo another Sarnat 
examination.  Unlike the initial Sarnat exam, the Sarnat scoring sheet does not need to 
be retained for this follow-up exam. Exam findings will be entered into the HEAL 
Portal. The exam should be conducted (or confirmed) by a Physician who has been 
trained on the HEAL Sarnat exam, when possible. 
6.3.2.4 Newborn hearing screening 
If performed, results from the newborn hearing screening will be recorded.  
 
 


 
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6.3.3 EEG assessments  
Brain monitoring via conventional video-EEG should start as soon as possible after NICU admission 
(see Clinical Guidelines).  For sites where video-EEG is not available 24/7, amplitude-integrated 
EEG (aEEG) monitoring should be started as soon as possible.  If patient develops suspected 
clinical or aEEG seizures, conventional video-EEG should be initiated and continued until the 
patient has been free from all seizures for 24 hours. Sites that perform continuous EEG (cEEG) 
throughout hypothermia should save de-identified cEEG data for analysis in future ancillary 
studies.    
6.3.3.1 EEG interpretation  
EEG interpretation will be based on the clinical EEG report.  
The following data will be recorded on the CRFs: 
• 
Electrographic seizures or status epilepticus 
• 
Clinical seizures  
6.3.4 Laboratory Evaluations 
Standard of care for infants with HIE includes intermittent monitoring for glucose instability, renal 
and hepatic injury. Other labs such as complete blood counts, thyroid function, and cardiac 
function may also be obtained if clinically indicated. We will record a subset of these results to 
determine the presence or absence of common complications of HIE. HEAL guidelines have 
recommended time windows for these laboratory studies, and these timings are consistent with 
existing institutional protocols at HEAL enrolling sites.   
6.3.5 Plasma and urine biomarkers; baseline Epo measurement  
Three blood samples (1.5 mL each) will be collected in EDTA tubes from each enrolled patient over 
the course of their hospital stay to be used to study circulating biomarkers of inflammation and 
brain injury. The first blood sample (collected prior to the administration of study drug) will also 
be used to measure a baseline Epo level. A baseline Epo level may be helpful to determine the 
chronicity of hypoxic-ischemic stress in utero, a potential confounder of the relationship between 
Epo therapy and 2-year outcome. Additional samples are collected on Study Days 2 and 4. 
Samples may be refrigerated for up to 4 hours prior to processing and freezing. As soon as 
possible after drawing, samples should be spun for 8 min at 2000 G, plasma removed into a 
separate container, and plasma and pellet stored in labeled tubes at -70°C or below. RNALater will 
be added to one pellet to preserve RNA for future studies. Please refer to the Laboratory SOP for 
further details. Plasma Epo concentrations will be measured in duplicate using Meso Scale 
Discovery (MSD) technology at the UW Laboratory Core.  
We will assay blood samples for circulating biomarkers of brain injury and inflammation in a 
subset of infants (N = 200; 100 treated and 100 control infants with balanced numbers of 
moderate and severe HIE. These subjects will all have MRI scans that have met pre-defined 
acceptance criteria). We will assay putative biomarkers of brain injury severity in neonates 
including: S100B, glial fibrillary acidic protein (GFAP), Tau, neuron-specific enolase (NSE), and 
ubiquitin C-terminal hydrolase-L1 (UCH-L1). Given the role of inflammation in HIE, we will also 
measure the following inflammatory markers: interleukin (IL)-1beta (β), IL-6, IL-8, and tumor 
necrosis factor-alpha (TNF-α). All samples will be run in duplicate using MSD technology in the UW 
core laboratory. This combination of factors will allow us to evaluate the subject’s inflammatory 


 
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state over time, provide insight as to the timing and severity of brain injury, and potentially, 
provide insight as to why some individuals might respond more optimally to Epo.124-127  
Residual plasma and cell pellets will be banked (with parental consent) and used for future 
mechanistic studies such as epigenetic or microRNA associations with certain outcomes. Two 
urine samples (1-2 mL each) will be banked for future studies to better understand consequences 
of HIE and/or Epo therapy (separate funding).    
6.3.6 Neuroimaging: Brain MRI/MRS 
At each of the participating sites, patients will be imaged on a 3T MRI, ideally between 96-144 
hours post-birth. This timing is consistent with the American Academy of Neurology practice 
parameter which suggested imaging before 8 days of age.128 In cooled infants, the MRI/MRS is 
most sensitive and specific if performed at < 8 days of age.117 Since therapeutic hypothermia ends 
at 4 days of age (study day 3-4), we anticipate that brain imaging can be performed after 
completion of therapeutic hypothermia and between 96-144 hours post-birth in study patients, as 
part of routine clinical care. All study sites have agreed to follow a specific study protocol including 
collection of high quality MR spectroscopy data (see below); to demonstrate excellent signal to 
noise ratio; and to have a MRI physicist and/or neuroradiologist oversee the study procedures. 
Infants may receive more than one brain MRI, and some may also receive a spine MRI. All brain 
and spine MRI studies will be anonymized and transmitted electronically in a secure fashion to the 
Neuroimaging Core.   
Any patients discharged prior to 96-144 hours post-birth may have their MRI performed prior to 
discharge, even if this timing is outside of the pre-specified study window. Similarly, if a subject is 
unable to have an MRI done during the desired window due to clinical instability such as being on 
ECMO, the MRI should be done as soon as is clinically appropriate. Out-of-window MRIs will not 
be considered protocol deviations.  
All sites will perform the following set of sequences: A) 3D T1-weighted (isotropic 1x1x1 
acquisition); B) Axial T2 weighted (1x1x2 with TE >=120); C) Diffusion tensor imaging (DTI) (25-32 
directions with b value of 1000 s/mm2; isotropic 2x2x2); and D) single voxel PRESS magnetic 
resonance spectroscopy (MRS), TE 35ms, with 1 voxel in the left thalamus/basal ganglia, and 1 
voxel in the left parietal white matter. In addition, one long TE spectrum (TE=144 ms) will be 
acquired from the identical left thalamus/basal ganglia ROI. The Neuroimaging Core will work with 
sites to ensure quality and consistency of MRI and MRS data optimized for each model of MRI, will 
perform centralized interpretation of these neonatal brain scans, and will process the MR 
spectroscopy data and provide masked interpretation of these data. 
6.3.7 Phone questionnaires 
Interim data collection will occur via telephone contact at 4, 8, 12, 18, and 24 months of age. The 
phone call interviews may also be done in person if the patient is being seen in the clinic during 
any of these times. During these follow-ups, an interval medical and developmental history 
questionnaire will be administered.  At 12, 18, and 24 months, the WIDEA, a standardized parental 
questionnaire regarding infant development will be included in the assessment.  
6.3.8 Neurodevelopmental examinations (24 months) 
At 24 months ± 56 days (i.e., ± 2 months), we will perform the following in-person evaluations to 
determine the primary and secondary outcomes. For extenuating circumstances, for example, 
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up to 36 months of age. This section provides a brief description of each of these evaluations. For 
a description of the primary outcome itself, see Section 9.2.1.   
To ensure that complete primary endpoint data can be obtained in as many subjects as possible, it 
is strongly recommended that the 24-month neurodevelopmental examinations be performed in 
the following order: 
1. Bayley III exam 
a. Cognitive  
b. Language 
c. Motor 
2. Standardized neurologic exam 
3. GMFCS 
The follow-up examination may be performed by the following individuals, provided they have 
received the appropriate training through HEAL. The preferred order is as follows: 
- 
Pediatric neurologist 
- 
Developmental pediatrician 
- 
Other pediatrician involved in infant follow-up 
- 
PNP involved in infant follow-up 
- 
Pediatric physical therapist (who is trained in the HEAL standardized assessments) 
Instructions for subjects with delayed in-person 24-month visit: 
- 
During the 24 month (±56 day) window, collect the following information by phone: 
o 24-month phone interview 
o WIDEA 
o GMFCS (by phone) 
o CBC-L (performed by phone) 
- 
The in-person visit should be performed as close to the 24 month (±56 day) window as possible, 
and before 36 months of age.  
o Bayley III exam 
o Standardized neurologic exam, including growth parameters 
o GMFCS 
o CBC-L (repeat only if the prior CBC-L was performed more than 4 months prior) 
o WIDEA (repeat only if the prior WIDEA was performed more than 4 months prior) 
Primary outcome measurement: 
a) Standardized neurologic examination, based on the examination used in the ELGAN study 
protocol129,130  
b) GMFCS131 
c) Bayley III cognitive score132 
Secondary outcomes:  
a) Developmental history 
b) Medical history including seizure history 


 
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c) Bayley III cognitive and language score 
d) CBCL externalizing score 
6.3.8.1 Standardized neurologic examination.  
The presence and type of CP will be determined using the broadly accepted and 
standardized examination described and used in the ELGAN study (the examination 
will be housed on our HEAL portal).129 This software and training program developed 
by our co-investigator Dr. Karl Kuban provides a method of neurologic testing of 
subjects in a formalized, systemized method that is highly reproducible. This 
examination is designed specifically to determine the presence and classification of CP 
in a 2-year old child. The follow-up examiners at each study site will undergo in-person 
training and certification. To standardize the quality of data regarding neurological 
exams, examiners will attend a half-day workshop at the annual PAS meeting or at a 
central location (e.g., Minnesota or Missouri) or virtual location, view a training video, 
and then classify neurological findings illustrated on an assessment video.133 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 and 
PENUT studies, we expect agreement rate to rise to over 90%.133,134 Training will entail 
participation in a training session with Dr. Kuban, followed by the review of a training 
video, and submission of a set of answers regarding scoring of the neurologic exam 
that will be used to certify the examiner.130 A re-certification process will be 
performed every 18 months to ensure that primary outcome examiners remain 
adequately trained throughout the duration of the study. The neurologic examination 
will produce one of the following neurologic diagnoses: no CP, diparetic CP, 
hemiparetic CP, or quadriparetic CP.135  These categorizations correlate highly with 
long-term neurodevelopmental outcomes. 
6.3.8.2 Gross Motor Function Classification Scale (GMFCS)131  
This classification scale 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 
defines 5 levels of function that represent an ordinal scale where the distance 
between levels is not considered equal. A brief summary of the GMFCS score at age 24 
months is as follows:   
Descriptions of each level for ages 12 to 24 months 
Level 0 = Walks ten steps independently and has symmetric gait. 
Level 0.5 = Walks ten steps independently but does not have symmetric gait. 
Level 1 = Infants move in and out of sitting and floor sit with both hands free to 
manipulate objects. Infants creep or crawl on hands and knees, pull to stand and take 
steps holding onto furniture. Some may creep or bottom shuffle, but are able to 
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Level 2 = Infants maintain floor sitting but may need to use their hands for support to 
maintain balance. Infants creep on their stomach or crawl on hands and knees. Infants 
may pull to stand and take steps holding onto furniture. 
Level 3 = Infants maintain floor sitting when the low back is supported. Infants roll and 
creep forward on their stomachs. 
Level 4 = Infants have head control but trunk support is required for floor sitting. 
Infants can roll to supine and may roll to prone. 
Level 5 = Physical impairments limit voluntary control of movements. Infants are 
unable to maintain antigravity head and trunk postures in prone and sitting. Infants 
require adult assistance to roll. 
(Modified from Palisano et al, Med Child Neurol, 1997; 39:214-233. Algorithm adapted 
by Rosenbaum P and Saigal S for TIPP Trial, and utilized in the NICHD Neonatal 
Research Network under the direction of Betty Vohr). 
To determine the level of gross motor function of the infant, follow the algorithm 
shown on the Gross Motor Function Work Sheet. The algorithm starts with normal 
function and progresses to increasing levels of functional limitations. 
6.3.8.3 Bayley Scales of Infant Development III (Bayley-III) 
The Bayley III is the most widely used developmental measure to determine 
developmental delay in high-risk infants.132,136 Since 2005, the Bayley III has been 
adopted for standard use for the vast majority of follow up programs as well as 
neonatal clinical trials including the hypothermia trials.3-5,7 We will measure Bayley III 
cognitive, motor, and language scores. Only the cognitive score will be part of the 
primary outcome measure, and as such, this portion of the Bayley III exam should be 
performed first. We will not use the social-emotional and adaptive behavioral subtests 
of the Bayley III. All individuals who will perform the Bayley exam will undergo a 
certification process to ensure reliability of test results. This process will include 
review of a “gold standard” Bayley III assessment performed by a neuropsychologist, 
and sending in a videotape of the HEAL examiner performing a Bayley evaluation of a 
2-year-old along with the completed test booklet. Dr. Lowe will evaluate this tape and 
the associated test booklet for completeness and correctness. Only individuals 
certified by Dr. Lowe on behalf of HEAL will administer the test.  
6.3.8.4 Developmental history 
We will interview the parents regarding the age of developmental milestones 
including rolling over, sitting, standing, babbling, speaking, and performing pincer 
grasp.  
6.3.8.5 Medical and social history 
General information will be gathered from families, including: 
a) Primary language spoken at home 
b) Maternal (or primary caregiver) education 
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c) Presence of afebrile seizures 
d) Use of supplemental oxygen, mechanical ventilation, or tracheostomy 
e) Use of gastrostomy or nasogastric tube feedings; ability to self-feed; ability to 
eat solids 
f) Hospitalizations 
g) Utilization of rehabilitation services (OT, PT, or ST) 
h) Maternal (or primary caregiver) employment 
i) 
Vision or auditory issues 
j) 
Medications, including seizure medications 
6.3.8.6 Growth parameters 
At 2 years, we will measure head circumference, length, and weight. 
6.3.8.7 Child Behavior Checklist (CBCL) 
At 2 years, we will administer the CBCL, a parental questionnaire that includes 99 
items that describe specific kinds of behavioral, emotional, and social problems. We 
will specifically evaluate the externalizing score for evidence of attention problems 
and aggressive behavior. This checklist will be scored centrally.  
6.3.8.8 Warner Initial Developmental Evaluation of Adaptive and Functional Skills (WIDEA) 
The WIDEA is a questionnaire developed and standardized to assess the 4 functional 
domains of self-care, motor function, communication, and social cognition in young 
children. Normative data are available for this instrument through 36 months of age. 
Dr. Elizabeth Rogers (follow-up PI) will provide an interactive WIDEA video training 
session and certification for study personnel who will administer this parental 
questionnaire.121,122  
 
6.3.9 Extended Contact Phone Calls 
At the 24-month study visit, or by phone after the visit has been completed, the parent/guardian 
should be approached for consent for participating in Extended Contact phone calls. If consent is 
obtained, these calls should be performed at the following intervals: 
• 
30 months (±56 days) 
• 
36 months (±56 days) 
• 
42 months (±56 days) 
• 
48 months (±56 days) 
• 
54 months (±56 days) 
• 
60 months (±56 days) 
• 
66 months (±56 days) 
• 
72 months (±56 days) 
• 
78 months (±56 days) 
• 
84 months (±56 days) 
• 
90 months (±56 days) 


 
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• 
96 months (±56 days) 
These phone calls will be used to maintain relationships with participating families, ensure 
updated contact information is on file, and to gather basic information about how the child is 
doing. The WIDEA may be performed at the 30- and 36-month calls. Data may be used to support 
future applications for follow-up studies on this population.  
Failure to complete these follow-up calls, or performing these follow-up calls outside of the 
requested windows, does not constitute a study deviation. 
Any deaths identified during these Extended Contact phone calls must be reported as SAEs. 
 
7 
MANAGEMENT OF SERIOUS ADVERSE EVENTS AND COMPLICATIONS 
OF HIE 
The study will be monitored by a NINDS-appointed DSMB with independent experts in the areas of 
pediatric neurology, neonatology and biostatistics. The study will be monitored for safety by the DSMB 
with reviews for enrollment, follow-up, data quality, overall study conduct, participant safety, and 
significant adverse event rates. Meeting frequency and conduct will be detailed separately by the DSMB. 
For this NINDS grant, the DSMB will follow the guidance presented in the NINDS Guidelines for Data and 
Safety Monitoring: 
http://www.ninds.nih.gov/research/clinical_research/policies/data_safety_monitoring.htm 
A neonatologist who is independent of the study will serve as the Medical Monitor, and will monitor all 
serious adverse events concurrently. The Medical Monitor will be masked to treatment allocation.  
 SERIOUS ADVERSE EVENT DEFINITIONS 
In the HEAL study, the following events, occurring within 30 days of study drug dosing, will be 
reported as Serious Adverse Events (SAEs). Death occurring any time during the neonatal or 
follow-up period will always be reported as an SAE.  
 
Systemic hypertension 
o Definition: If blood pressure is elevated enough to require antihypertensive 
therapy 
 
Polycythemia 
o Definition: Central hematocrit (Hct) > 65.0%, as measured by a central lab on 2 
consecutive free-flowing arterial or venous samples. 
(Note: High Hct values measured by point of care tests are not sufficient for 
meeting polycythemia criteria for purposes of this study) 
 
Disseminated intravascular coagulation (DIC) 
o Clinical bleeding or oozing requiring transfusion of blood products (e.g., FFP, 
cryoprecipitate or platelets) 
(Note: Laboratory findings may include elevated INR, PTT, PT, or D-dimers, or low 
PLT or fibrinogen) 
 
Major venous or arterial thrombosis (clot): Definition = any of the following 3: 
o Any thrombosis that is treated with a course of anticoagulation 
o Any venous or arterial thrombosis involving a major vessel not related to a central 
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o Any symptomatic thrombosis involving a major vessel (e.g., symptoms such as 
superior vena cava syndrome) 
 
Pulmonary hypertension 
o Elevated pulmonary vascular resistance treated with inhaled nitric oxide or ECMO 
therapy 
 
Intracranial hemorrhage 
o Intraparenchymal or intraventricular blood visualized by HUS or MRI T1 or T2 
sequences 
 
Cardiopulmonary arrest 
o Clinical code event requiring chest compressions or epinephrine bolus from which 
the infant recovers (does not lead to death), that is not secondary to endotracheal 
tube (ETT) obstruction or other mechanical issue 
 
Other unexpected life-threatening event 
o Unexpected for HIE, or unexpected based on the Epo drug profile 
 
Death 
The definition for an SAE in our population of HIE newborns is consistent with 21 CFR 312.21 
(2005).  
 SAE RELATEDNESS & SEVERITY 
For all Serious Adverse Events (SAE, as noted above), the site investigator will use their best medical 
judgment and indicate whether the SAE is related to the research (i.e., related to any research 
procedure including but not limited to the main intervention). The investigator will assign the SAE to one 
of the four categories below: 
 
Definitely related: The SAE is clearly related to the intervention 
 
Probably related: The SAE is likely related to the intervention 
 
Possibly related: The SAE may be related to the intervention 
 
Unlikely: The SAE is doubtfully related to the intervention 
 
Not related: The SAE is clearly NOT related to the intervention 
The investigator will also provide an SAE severity: 
 
Mild: asymptomatic or mild symptoms; clinical or diagnostic observations only; no intervention 
indicated 
 
Moderate: minimal, local, or noninvasive intervention indicated 
 
Severe: Severe or medically significant, but not immediately life-threatening; hospitalization or 
prolongation of hospitalization indicated; disabling 
 
Life-threatening: Life-threatening consequences; urgent intervention indicated 
 SAE AND UNEXPECTED, RELATED EVENT REPORTING 
All SAEs or events that are unexpected and thought to be related to study drug will require expedited 
reporting to the CCC. These events must be reported to the CCC PIs (Wu/Juul) within 72 hours of the site 
becoming aware of the event. Full documentation of the event is required within 7 days of becoming 
aware of the event. For in-hospital SAEs occurring during the neonatal period, it is expected that the site 
will be made immediately aware of the event. During follow-up, patients will be followed by phone (at 4, 
8, 12, and 18 months of age) and in person (at 24 months of age). During these follow-up encounters, a 
questionnaire will be administered to cover medical complications following discharge from the 


 
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neonatal intensive care unit. All deaths, SAEs occurring within 30 days of study drug dosing, and events 
unexpected and thought to be related to study drug occurring after hospital discharge must also be 
reported to the CCC PIs within 72 hours of the site becoming aware of the event. 
 


 
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Following notification from the site, the CCC will ensure reporting per Table 5 and Figure 2 below.  
 
Table 5. SAE and Unanticipated Related Drug Effects Reporting Timelines 
Group 
responsible 
for doing 
notification 
Group 
Notified 
Required Notification Timelines for: 
SAEs 
Unanticipated Related Drug Effects 
Site 
CCC PIs 
 
Notification within 72 hours of 
site becoming aware of event; 
SAE CRF completed by 7 days* 
 
Notification within 72 hours of 
site becoming aware of event; 
SAE CRF completed by 7 days* 
Site 
Site IRB 
 
Per site IRB requirements 
 
Per site IRB requirements 
CCC 
(Wu/Juul) 
Medical 
Monitor 
 
Provide SAE report within 7 days 
of site becoming aware of event 
 
Provide SAE report within 7 days 
of site becoming aware of event 
CCC (Wu) 
FDA 
 
Death or life-threatening SAES 
that are unexpected and Epo-
related: no later than 7 
calendar days of sponsor’s (CCC) 
initial receipt of the information 
 
Other SAEs that are unexpected 
and Epo-related: as soon as 
possible, but no later than 15 
calendar days of sponsor (CCC) 
determining the event qualifies 
for reporting 
 
N/A, unless serious (see SAE 
column) 
Medical 
Monitor 
DSMB 
 
Epo-related SAEs: within 15 
calendar days after sponsor’s 
initial receipt of the information 
 
Within 15 calendar days after 
sponsor’s initial receipt of the 
information 
*In some instances, the CCC and/or Medical Monitor may require more expeditious completion of the 
SAE form 
 
 
 


 
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Figure 2. SAE and Unanticipated Related Drug Effects Reporting Timelines 
 


 
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 ANTICIPATED MEDICAL COMPLICATIONS (ADVERSE EVENTS) IN HIE PATIENTS 
Critically ill infants with HIE commonly experience multiple medical complications. Medical 
complications of HIE patients will be recorded on case report forms, entered into the portal, and 
analyzed to determine if they occur in different frequencies between the treatment and control groups. 
 CRITERIA FOR MODIFICATION OF INTERVENTION REGIMEN  
7.5.1 Criteria for Withholding/Stopping the Study Drug  
- 
Central HCT > 65.0% 
- 
Systemic hypertension requiring treatment as determined by the clinical team.137,138  
7.5.2 Restarting Study Drug 
- 
Study drug will be restarted when central HCT < 60.0% 
- 
Study drug will be restarted when blood pressure is within the normal range for age for 48 
hours, or normotensive on a stable dose of anti-hypertensives. 
o If the study drug is held on Study Days 1-4, one held dose may be made up, as 
long as it is given >20 hours before and >20 hours after any other study drug 
doses, and before the Study Day 7 dose window. 
o If the study drug is held on Study Day 7, the held dose may be made up any time 
though Study Day 10.  
o Only one held dose may be made up, in total. 
- 
Note that any per-protocol blood samples should be drawn at the protocol-specified 
times. Blood sample times are not adjusted in cases where study drug is held.  
Open Label Use of Epo. Open label use of Epo is not permitted in enrolled infants during the initial 
neonatal hospitalization. It is very unlikely that open label Epo use will pose a problem, since 
standard treatment for acute anemia in term infants is red blood cell transfusion, and Epo is rarely 
(if ever) used in this clinical setting. 
If study drug is discontinued in any subject, parents will be encouraged to continue to allow their 
child to participate in the follow-up evaluations. 
 PROTOCOL VIOLATION/ DEVIATION REPORTING  
Protocol violations require completion of a Protocol Violation Form and notification to the CCC and DCC 
within 3 working days. Protocol deviations require completion of a Protocol Deviation form, and the CCC 
and DCC should be notified within 7 days of occurrence.  
7.6.1 Protocol Violations: 
- 
Un-masking of study personnel 
- 
Enrollment despite meeting exclusion criteria 
- 
Enrollment despite not meeting inclusion criteria 
- 
Consent not obtained in accordance with IRB guidelines 
- 
Study drug administration or dosing error (incorrect study drug administered)  
- 
Wrong study drug dose administered (>40 units, and >10% off from specified dose [1000 
units/kg birthweight]) 
- 
Study drug administered when it should have been withheld 


 
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- 
Open label Epo administered 
7.6.2 Protocol Deviations may include the following items, among others: 
- 
Hypothermia performed not in accordance with site’s hypothermia protocol (Examples 
include equipment malfunction, or early discontinuation due to reclassification of a 
patient as mild severity (i.e., even if a site has an early exit protocol, this early exit should 
be reported as a protocol deviation). Reasons for early discontinuation consistent with a 
site’s cooling protocol are not considered protocol deviations (e.g., stopping cooling for 
ECMO, etc.)) 
- 
First study drug dose given after 26 hours of life 
- 
Study drug dose is given outside of prescribed time window 
- 
Study drug dose not given 
- 
Wrong study drug dose administered (>40 units, but ≤10% off from specified dose [1000 
units/kg]) 
- 
Blood samples drawn outside of the accepted time windows 
- 
Blood collection missed 
- 
Blood sample not in accordance with requirements per SOP (processing error, shipping 
error, other) 
- 
Urine collected outside of the accepted time windows 
- 
Urine collection missed 
- 
Urine sample not processed appropriately 
- 
Lost urine or blood sample 
- 
Day 5 Sarnat not done 
- 
Day 5 Sarnat performed out of window 
- 
Follow-up phone contact missed 
- 
Follow-up performed out of window 
- 
24-month visit not performed before 30 months of age 
- 
24-month visit not performed before 36 months of age 
 
8 CRITERIA OF INTERVENTION DISCONTINUATION 
If the patient experiences any SAE that might be considered related to study drug, the site investigator 
should notify the HEAL CCC PI (Drs. Wu/Juul) immediately, and hold study drug until the event can be 
evaluated by the Medical Safety Monitor. The Medical Monitor will decide whether to continue therapy 
thereafter. If the remaining doses of study drug are held, all efforts will be made to maintain the 
subject’s participation in follow-up activities at 4, 8, 12, 18, and 24 months of age. The site always 
maintains the ability to hold study drug or intervene on behalf of the subject for safety reasons. 
 
9 STATISTICAL CONSIDERATIONS 
 GENERAL  
This study is a randomized parallel group double-masked, placebo controlled trial in neonates with HIE. 
Eligible neonates will be enrolled and treated with either Epo or normal saline over 7 days and followed 
for a fixed period of 24 months (±56 days) for survival and neurological outcomesThe follow-up period 
was chosen to provide meaningful motor and cognitive outcomes, since CP and motor and cognitive 


 
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deficits are more reliably diagnosed at 2 years of age. Enrolled children will be randomized, stratified by 
study site and severity of HIE, with equal likelihood to receive Epo or saline. 
Analyses will be based on a modified Intention to Treat (mITT) approach. In this approach, all 
randomized neonates who received at least one dose of study drug will be included in the analyses. 
Neonates whose parents withdrew consent after randomization and before the first dose will be 
excluded from the mITT. Since the first dose is within 24 hours of birth and within at most a few hours 
after consent and randomization, we do not expect more than a very small number of withdrawals. 
Preliminary analyses will compare baseline characteristics to explore whether there are any imbalances 
that occurred at randomization. These analyses will include comparing demographics and the Sarnat 
score at study entry.  
 
 OUTCOMES 
9.2.1 Primary outcome 
Primary outcome is the composite of death or neurodevelopmental impairment.   
Neurodevelopmental impairment (mild, moderate, or severe) is defined as any of the following: 
• 
GMFCS level ≥1, or 
• 
GMFCS = 0 or 0.5 AND CP (any type), or 
• 
Bayley III Cognitive Score <90  
Since death is a competing outcome, it is critical to include it in the primary outcome measure. We 
will use standardized, validated neurological and developmental assessments: 
• 
CP diagnosed by Standardized Neurological Examination113,114 
• 
Bayley III Cognitive Score 
• 
GMFCS131 
9.2.1.1 Motor Deficit – Cerebral Palsy  
CP will be determined by a Standardized Neurologic Examination129,130 conducted 
under the direction of Dr. Karl Kuban (Co-I), using the systematized exam and video-
based certification system created for the NINDS-funded ELGAN and PENUT 
studies.129,130 This training program provides a formal method of neurologic testing 
that is highly reproducible. The exam was created specifically to determine the 
presence and classification of CP (i.e., quadriplegic-QP, hemiplegic-HP and diplegic-DP) 
at age 2. To standardize the quality of neurologic examination findings, two follow-up 
examiners at each site will participate in a training session, review training videos, and 
submit a set of independently scored examinations that will be used for certification, 
as is being done currently in the PENUT trial.130 A re-certification process will be 
performed at least every 18 months to ensure that primary outcome examiners 
remain adequately trained throughout the study. 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 < 85% 
correct rate. Based on experience in the ELGAN study, we expect agreement rates to 
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9.2.1.2 Motor Deficit: Gross Motor Functional Classification System (GMFCS) 
The GMFCS is a well-accepted and validated tool that is used widely to classify motor 
functional outcomes. Distinctions between levels of motor function are based 
primarily on functional ability. The GMFCS was used to determine motor outcome in 
the NINDS-funded Beneficial Effects of Antenatal Magnesium Sulfate (BEAM) trial, as 
well as in 6 of 7 hypothermia trials for HIE. We will apply the BEAM trial algorithm and 
definitions when assigning GMFCS scores. Level 0 = normal gait; Level 0.5 
=asymmetric gait; Level 1 = walks independently with abnormal gait or requires ankle-
foot orthosis; Level 2 = cruises, pulls to stand, sits hands free; Level 3 = sits propped 
on hands only, rolls both ways; Level 4 = sits when supported in lower trunk, has head 
control, can roll to supine; Level 5 = no head or trunk control, no rolling, little or no 
voluntary movement. These definitions are modified from Palisano et al, Med Child 
Neurol, 1997; 39:214-233. Algorithm adapted by Rosenbaum P and Saigal S for TIPP 
Trial, and utilized in the NICHD Neonatal Research Network under the direction of 
Betty Vohr. 
9.2.1.3 Cognitive Deficit: Bayley III Cognitive Score < 90  
Bayley III Cognitive Score112 is a standard test used to evaluate early cognitive 
outcomes in high-risk infants. Severity of cognitive deficit is defined by number of 
standard deviations (SD) below the mean (i.e., severe, moderate, and mild deficit = -3, 
-2 and -1 SDs below the mean). Since the Bayley III yields higher cognitive scores than 
the previous version of the Mental Development Index on the Bayley II,136,139-141 we 
have defined cut-offs on the Bayley III that are 5-15 points shifted to the right 
compared to the Bayley II. We consider any Bayley III Cognitive Score < 90 as 
abnormal, with mild, moderate, and severe ranges as defined in Table 7. These 
definitions are consistent with findings in a contemporary cohort of infants with HIE 
who underwent hypothermia,141 and also consistent with cut-offs used in the PENUT 
trial. Each site will undergo certification by reviewing a “gold standard” Bayley III 
assessment performed by our research neuropsychologist. Each site psychologist will 
then record a Bayley III assessment of a 2-year-old child, and submit the test booklet 
and video recording for review by the research psychologist for feedback. HEAL CCC 
leadership has experience in performing multicenter Bayley III certification in other 
NIH funded studies (e.g. PENUT, TOLSURF), and will use the infrastructure in place for 
these studies. Only Bayley III examiners who have undergone this training and 
certification will be allowed to perform the 2-year primary endpoint evaluation. 
9.2.1.4 Adjudication of Primary Outcome 
There may be cases of only a phone follow-up rather than an in-person visit, or 
otherwise partial data. The CCC will create an Outcomes Adjudication Committee, 
who will be masked to the treatment assignment of the child. The Committee will 
receive all available data on the long-term outcome of these toddlers and will assess 
whether they can definitively assign a primary outcome level and what it is, or they do 
not have sufficient information to make that determination and therefore the value 
will be imputed. This model was successfully previously used in the NO CLD clinical 
trial long-term outcome.142 


 
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9.2.2 Secondary outcomes  
Overall severity will consist of the worst severity observed in either motor or cognitive outcomes. 
Severity of motor impairment will be determined by type of CP and GMFCS level (Table 6). 
Severity of cognitive impairment will be determined by Bayley III Cognitive Score (Table 7).  
At age 2, we will assess the effect of Epo on secondary outcomes: a) presence of CP, b) severity of 
motor impairment, c) Bayley III cognitive and language scores, d) epilepsy (i.e., ≥ 2 afebrile, 
unprovoked seizures), and e) behavioral abnormalities (i.e., attention problems or aggressive 
behavior) based on the CBCL externalizing score.143 To explore the effect of Epo on sensory deficits 
that can result from HIE, we will collect 2-year data regarding hearing impairment requiring 
hearing aids, and if available, information about the presence of cortical visual impairment 
diagnosed by an ophthalmologist. To elucidate the effect of Epo on all severities of impairment, 
we will analyze the effect of Epo on the following 4-level outcome: 1) normal, 2) mild motor 
and/or cognitive impairment, 3) moderate/severe motor and/or cognitive impairment, and 4) 
death.  
 
Table 6. 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 
 
Table 7. Cognitive outcome - 4 level classification 
Cognitive Deficit 
Bayley II MDI score 
(Hypothermia trials) 
Bayley III Cognitive score 
(PENUT, HEAL) 
Severe (> 3 SD) 
≤ 55 
≤ 70 
Moderate (2-3 SD) 
> 55 and ≤ 70 
70-84 
Mild (1-2 SD) 
> 70 and ≤ 85 
85-89 
None 
> 85 
≥ 90 
 DATA ANALYSES 
9.3.1 Primary outcome analyses 
The primary analysis will be a test of equality of the rate of the primary outcome (death or 
neurodevelopmental impairment (NDI)) across the two randomized investigational groups. 
Specifically, we will use a likelihood ratio test based on logistic regression, with stratification by 
recruitment center and HIE severity. We will perform Intention to Treat analysis and expect 
minimal non-compliance due to the nature of the intervention in relation to in-patient care. For 


 
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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 consider an ordered categorical two-year 
status measure (death/severe or moderate impairment/mild impairment/normal), and analysis 
will be based on generalized Wilcoxon tests or regression models for ordered categorical 
outcomes such as the proportional odds model. Secondary quantitative measures include MRI-
based injury score using the Washington University Standardized Scoring System. For these 
endpoints, a stratified t-test provides inference regarding the mean response across the treatment 
groups. We will adjust all secondary outcome analyses for recruitment site and HIE severity using 
regression methods. 
9.3.2 Secondary Outcomes 
Our key secondary long-term outcome is an ordered categorical 24-month status measure that 
classifies subjects as: dead; moderate or severe impairment; mild impairment; and normal. Use of 
this measure allows a detailed assessment of potential shifts in the distribution of outcomes 
toward improved status associated with treatment. Statistical analysis of this outcome will use a 
generalization of the Wilcoxon test that controls for recruitment site and HIE severity. Regression 
models for ordered categorical outcomes can also be used to provide adjusted treatment effect 
estimates.  
9.3.3 Exploratory Analyses 
Biomarker Prognostic Analysis. We will consider two main classes of potential predictors of 24-
month 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. We will derive two multivariate 
predictive models: using the inflammatory markers and using the MRI and MRS 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). Evaluation of whether 
treatment modifies the prognostic potential of biomarkers can be conducted by testing for the 
interaction between treatment status and individual biomarkers in predictive models for 2-year 
outcomes. 
9.3.4 Analyses of Safety Data  
Clinical safety data includes SAEs and clinical laboratory markers both from the hospitalization and 
study intervention period and from the long-term follow-up period. Safety event rates will be 
tabulated by study group and compared separately for each SAE using a multivariable logistic 
regression model, with adjustment for randomized treatment group, and randomization 
stratification factors of clinical recruitment site and HIE severity (moderate/severe). We will 
additionally create a per-patient aggregate count of SAEs and evaluate the rates between groups 
using a Poisson regression model with robust standard errors and adjustment for randomization 


 
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stratification factors. For any safety events that occur infrequently (<2%), we will use Fisher's 
exact test to compare the rates between treatment groups.  Because these are critically ill 
newborns, we anticipate that the majority of safety events will occur during the initial inpatient 
and treatment period. As a secondary analysis, we will therefore summarize and analyze safety 
events that occur during the initial hospitalization and post-discharge time 
periods separately. Statistical significance is defined conservatively at the alpha=0.05 level with no 
correction for multiple outcomes. 
Laboratory tests of organ injury are measured at baseline and at age 2-3 days as part of routine 
care. We will compare laboratory test data measured between study day 2 and 3 between 
treatment groups using analysis of covariance (ANCOVA) regression model, with adjustment for 
treatment group, the laboratory value measured at baseline, and the time between laboratory 
measurements. We will use a similar analytic approach to compare vital signs and growth 
parameters between treatment groups.   
9.3.5 Neuroimaging and circulating biomarker analyses 
Circulating biomarkers of inflammation and brain injury.  Inflammation is thought to play an 
important role in HIE and CP.144-147 Biomarkers of inflammation that we measure include: 
interleukin (IL)-1β, IL-6 and IL-8 and TNF-α. Putative biomarkers of brain injury in neonatal HIE 
include: glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), S100B, 
Tau, and neuron-specific enolase (NSE). We will select a random subset of 200 subjects (100 
treated and 100 controls with each group, for example, including both moderate and severe HIE) 
to measure circulating biomarkers of inflammation and brain injury. We will collect 3 plasma 
samples from each infant at the following time points based on hour of age: < 24 hours; Study Day 
2; Study Day 4. Our analysis will focus on time-specific comparisons of the mean biomarker 
measure across treatment groups using appropriate regression methods while controlling for site 
and HIE severity. In addition, we will conduct longitudinal analysis using linear mixed models148 
that permit an omnibus test across all four measurement times, and allow inference on 
differential rates of change across treatment groups.  
9.3.6 Compliance, Retention, and Missing Data 
Adherence and retention. Our major analyses are based on the modified ITT principle. We do not 
anticipate that non-adherence will be a major issue since the treatment is directly observed during 
a short in-hospital time frame. We will assess non-compliance, with particular focus on study 
treatment dosing and timing. If there are more than minimal issues, that will justify quantifying 
and characterizing non-adherence and doing a per-protocol analysis.  
In addition, site selection included having a committed neonatology follow-up program, and we 
expect >90% retention.  
9.3.6.1 Missing data and dropouts.  
Prevention: We will strive to sustain excellent participant involvement throughout the 
study and we have achieved 90%+ follow-up rates in numerous prior studies. The UW 
DCC will generate automated nightly reports, available to staff at the study sites, 
identifying these fields with a request to discuss and prevent further missingness. 
Important data elements will be prospectively monitored to examine patterns of 
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Handling: We will conduct a missing data analysis to describe and characterize 
enrolled participants who do not provide further response due to attrition or dropout. 
We will use inverse probability weighting in secondary analysis for each response 
regression model (e.g. Generalized Estimating Equations) to inflate the weights of 
cases that are under-represented in the analysis due to selective attrition and/or non-
participation. We will also conduct sensitivity analyses using 10-fold multiple 
imputation to assess the robustness of the results when missing data are imputed. 
Statistical uncertainty: Given the type of missing data we expect in the proposed 
study, missing completely at random (MCAR) or missing at random (MAR), both 
methods we propose to utilize for missing data properly account for statistical 
uncertainty due to missingness and will provide accurate confidence interval 
coverage.  
Tracking and reporting: The study web-based portal identifies when patients enter the 
follow-up interview window and when interviews are complete. Reasons for dropout 
will be systematically documented in the study database. In final manuscripts and 
analyses, the number of non-responders will be enumerated by study arm according 
to CONSORT guidelines.  
Sensitivity analysis: We will assess the sensitivity of inferences made from missing 
data methods first by using the two previously described methods for dealing with 
missing data, and secondly by imputing missing data under both pessimistic and 
optimistic scenarios to provide bounds on the statistical uncertainty. The 
characteristics of non-responders will be summarized in our final report and we will 
present the sensitivity of the treatment effect due to missing data. 
 
 SAMPLE SIZE AND ACCRUAL 
9.4.1 Primary Outcome Sample Size Calculations:  
Our proposed HEAL sites report an overall mortality rate of 14%. Using three large sites that 
participated in the phase II study (UCSF, Wash U, CNMC) we can also estimate the rates of 
neurodevelopmental impairment: death = 14%; moderate-severe impairment = 18%; mild 
impairment = 17%; and normal = 51%. Therefore, we anticipate a control primary outcome rate 
of 49% (death or NDI).  
Non-human data informing treatment effect size: A recent study with nonhuman primates 
(Macaca nemestrina) compared animals experiencing 15-18 minutes of umbilical cord occlusion 
that were then treated with either saline (n=14), therapeutic hypothermia (n=9), or therapeutic 
hypothermia and multiple doses of Epo.21 Among animals treated with saline 8/14 = 57% died or 
had NDI. Among animals treated with hypothermia (HT) alone 7/9 = 78% died or had NDI, while 
among animals treated with hypothermia and Epo (HT+Epo) only 5/12 = 42% were observed to die 
or have NDI. Results from Figure 2 of Traudt et al.149 (2013) show the number of animals in each 
outcome category by treatment group. These data suggest a risk ratio of 0.53 (95% CI: 0.25, 1.14) 
comparing HT+Epo to HT alone, and a risk ratio of 0.73 (95% CI: 0.32, 1.64) comparing HT+Epo 
versus saline. Therefore, animal data support an Epo effect that optimistically corresponds to a 


 
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50% reduction, and that is conservatively associated with a 27% reduction in the primary outcome 
rate.  
Phase I data informing treatment outcome rates: Given that the primary outcome is based on a 
22-26 month assessment we can rely on a recently completed long-term follow-up from a phase I 
study84 in which 24 cooled infants were given multiple doses of Epo ranging from 250U/kg to 
2500U/kg24. At 22-26 months of age n=22 subjects were followed, and 0/22 subjects died, 1/22 
had moderate-severe NDI, and 6/22 had mild NDI. This study suggests an overall primary 
outcome rate of 7/22 = 31.8% (exact confidence interval = 14%-55%) for our planned intervention 
group. We recognize that the dosing of Epo was not optimized in the phase I trial, and our 
proposed study will use multiple doses of 1000U/kg for all subjects which has been shown to yield 
plasma concentrations observed to be neuroprotective in animal studies. 
Phase II data informing treatment outcome rates: In our phase II NEATO trial, a total of n=50 
subjects were randomized to Epo (n=24) or placebo (n=26).  We find substantial differences in the 
MRI injury severity distribution with 95% of Epo treated subjects having no injury or mild injury as 
compared to only 56% of control subjects. Using a recently submitted study from Trivedi, et al., we 
can then link the MRI injury severity category to expected Bayley III Cognitive scores at 24 months. 
Using our NEATO data and Trivedi’s data we calculate a predicted mean (SD) of 97.9 (11.7) among 
Epo treated subjects, and a mean (SD) of 91.1 (15.2) among controls. The predicted 6.8-point 
mean difference for Bayley III Cognitive scores is consistent with our observed 6.3-point difference 
in mean WIDEA scores at 6 months among NEATO subjects. Predicted Bayley III Cognitive 
distributions lead to an expected 25.1% of subjects with a cognitive score of < 90 among Epo 
treated subjects, and 47.1% among controls. Incorporating expected death rates of 12% and 14% 
respectively for Epo treated and controls leads to an expected primary outcome rate of 34.1% 
among treated and 54.5% among controls. We acknowledge that our primary outcome is based 
on both Bayley III Cognitive scores, and clinical assessments of CP and GMFCS level, but expect 
Bayley status to be the major case indicator. 
Table 8. Distribution of outcomes in previous studies.  
 
NEATO 
Epo (n=22) 
NEATO 
Placebo (n=25) 
Bayley III 
Cognitive*** 
*** Trivedi et 
al. (submitted) 
Injury Severity 
 
 
Mean 
Std. Dev 
  None (0) 
8 (36%) 
3 (12%) 
96 
(8) 
  Mild (1-11) 
13 (59%) 
11(44%) 
100 
(13) 
  Moderate (12-32) 
1 (5%) 
6 (24%) 
85 
(18) 
  Severe (>32) 
0 (0%) 
5 (20%) 
76 
(19) 
Predicted Bayley III 
Mean (S.D) 
 
97.9 (11.7) 
 
91.1(15.2) 
 
 
Predicted %<90 
25.1% 
47.1% 
 
 
Predicted death/NDI 
34.1% 
54.5% 
 
 
 


 
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In addition, Cheong et al. (2012)150 evaluated the correlation between MRI findings obtained 
within 10 days of birth and 2-year clinical status outcomes for participants in the ICE trial (Infant 
Cooling Evaluation). Specifically, basal ganglia and thalamus (BGT) injuries were classified as 
abnormal if moderate/severe abnormalities were noted on T1- and T2-weighted images. In our 
phase II trial, we find only 4.5% (1/22) among Epo treated subjects have BGT injury as compared 
to 20% (5/25) among control subjects. Cheong et al. (2012) estimates that the probability of 
death/NDI at two years is 88% (PPV) for BGT abnormal subjects as compared to 32% (1-NPV) for 
BGT normal subjects. Applying these rates to our phase II BGT results yields expected death/NDI 
rates of 34.5% among Epo treated, and 43.2% among controls. For the ICE trial, the overall 
death/NDI rate for ICE hypothermia subjects was 51%, which is approximately the same as our 
expected control rate. 
Therefore, based on animal data, phase I data, and projections from phase II data we expect 
primary outcome rates from 31-35% among Epo treated subjects with a protective relative risk of 
0.65 to 0.71. 
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 Epo 
treated and control groups. The primary outcome measure is the composite rate of death or NDI, 
and current cohort studies suggest that the primary outcome occurs among 49% of infants treated 
with hypothermia alone (standard of care). Based on human data presented in Rogers et al.24 
(2014), animal studies including Traudt et al.149, and the NEATO phase II data we assume that 33% 
of treated infants will die or have NDI, corresponding to a relative risk of 0.67. Assuming an 
intervention rate of 33% yields greater than 90% power, while we have 88% power for an 
alternative of 34%. In order to compute power, we assume a 90% follow-up rate with n=225/250 
subjects evaluated in each arm. 
Table 9. Power analysis for the primary outcome assuming n=500 patients randomized and 10% loss to 
follow-up. 
Control 
Intervention 
Relative Risk 
Power 
49% 
32% 
0.65 
95% 
49% 
33% 
0.67 
92% 
49% 
34% 
0.69 
88% 
49% 
35% 
0.71 
83% 
 
9.4.2 Sample size for efficacy secondary outcomes 
Our key secondary long-term outcome is an ordered categorical 24-month status measure that 
classifies subjects as: dead; moderate or severe impairment; mild impairment; and normal. Use of 
this measure allows a detailed assessment of potential shifts in the distribution of outcomes 
toward improved status associated with treatment. Statistical analysis of this outcome will use a 
generalization of the Wilcoxon test that controls for recruitment site and HIE severity. Regression 
models for ordered categorical outcomes can also be used to provide adjusted treatment effect 
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To consider power we have used 2013 data from three large sites participating in the current 
phase II trial (UCSF, Wash U, CNMC) to estimate the distribution of outcomes within the control 
group. We calculate power for the stratified Wilcoxon test assuming a set of alternatives that are 
consistent with the assumption of a 33% rate of death or impairment (Scenarios 1 and 2) or a 32% 
rate (Scenarios 3 and 4) associated with intervention that was used to power the primary analysis. 
We assume a small effect on the death rate, and compute power for alternative shifts in the 
distribution of outcomes. For example, in Scenario 1 we assume no reduction in the death rate, 
and an 8% reduction in the moderate/severe category (relative risk = 0.55), and an 8% reduction 
in the rate of mild impairment (relative risk = 0.50) which yields 81% power using a two-sided 
alpha=0.05 test. Scenario 3 considers a larger reduction in the rate of moderate/severe and a 
smaller reduction in the mild category and yields 90% power. Scenarios 3 and 4 consider a 32% 
overall rate of impairment or death and yield 90% or greater power. Therefore, our study is 
adequately powered to detect modest but clinically important shifts in the outcome distribution. 
Table 10. Power analysis for a four-level outcome measure with n=500 patients randomized and 
assuming 10% loss to follow-up. 
 
Neurodevelopmental Impairment 
 
 
Normal 
Mild 
Moderate/Severe 
Mortality 
Power 
Placebo Arm 
51% 
17% 
18% 
14% 
-- 
Epo Arm 
 
Scenario 1 
67% 
9% 
10% 
14% 
81% 
Scenario 2 
67% 
11% 
10% 
12% 
86% 
Scenario 3 
68% 
9% 
9% 
14% 
86% 
Scenario 4 
68% 
11% 
9% 
12% 
92% 
 
Given the a priori hypothesis that treatment effect may differ according to gender or HIE severity 
we will conduct a pair of subgroup analyses that assesses treatment effects separately for males 
and for females, and separately for moderate and severe HIE. Subgroup specific treatment effects 
will be computed and inference will be based on a single Covariate-by-Treatment test for 
interaction using logistic regression. 
9.4.3 Sample size for MRI/MRS biomarkers 
For imaging measures, we will have data for all subjects. Assuming a 10% missing data rate we 
have 80% power to detect a difference in the mean of secondary outcomes across treatment 
groups of 0.26 SDs. For the inflammation and brain injury biomarkers, we will have a total of 200 
subjects and have 80% power to detect a mean difference across the treatment groups of 0.4 SDs. 
9.4.4 Sample size for plasma biomarkers of brain injury and inflammation 
A sample size of 91 neonates per group would have been required to detect a 0.5 SD difference on 
the log scale with a single observation per neonate with 80% power while controlling for a Type I 
error of 0.0125 (4 markers, Bonferroni). However, since we have 4 samples per neonate, a sample 


 
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size of 100 neonates per treatment group (with 400 samples per group, but correlated) will be 
more than sufficient to attain >80% power. 
9.4.5 Achieving the sample size 
The plan is to enroll and randomize 500 patients over a 36-month period. The 17 sites that were 
included in the original study proposal cooled a total of 530 HIE infants in the year 2014. From the 
NEATO phase II randomized controlled trial study, we estimate that at least 40-45% of these 
infants would be eligible and will consent for a phase III trial, yielding at least 16 enrolled infants 
per month. With a further conservative estimate of 14 per month allowing for slower enrollment 
during start-up, we can enroll 500 patients in 36 months.  
 DATA MONITORING 
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. Using the 
Patient Monitoring Report, a subset of all data points in the CRFs will be compared with the medical 
record for 20-25% of enrolled subjects. Any outstanding data queries will be resolved with the research 
coordinator at the time of the site-monitoring visit. After each study site monitoring visit, a report will 
be prepared and copies sent to the Study File, the study PIs (Y. Wu and S. Juul), the site PI, and the site 
coordinator. The quality and completeness of other deliverables (blood samples, MRIs) will be 
monitored. 
 DATA AND SAFETY MONITORING PLAN (DSMP) 
The DSMB will review the accruing data to: 1) ensure that the study is adequately enrolling; 2) to ensure 
that there are no serious safety concerns; and 3) to assess whether the study efficacy appears 
overwhelming. The DSMB will be assigned by NINDS. The research coordinator at each site will monitor 
each subject weekly for the presence of any complications. Serious adverse events will be brought to the 
attention of the DSMB, and if appropriate, the IRB, in writing. An independent medical monitor will 
review all cases of serious adverse events.  
As part of this 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. The DCC PI will remain masked to assignment while the study staff 
statistician will not.  
The primary outcome of the study is a composite endpoint of mortality or NDI at 24 months of age. 
Therefore, monitoring the primary outcome for treatment efficacy or futility is challenging. Based on 
enrollment plans, the majority of patients will have been randomized by the time NDI is assessed at 24 
months for any participants, and therefore we do not expect to be able to conduct a first interim 
analysis prior to the completion of enrollment. We expect to have the primary outcome evaluated on 
the first quarter of subjects (n=125) after 34 months of recruitment at which point we expect to have 
randomized n=450 of the total n=500 subjects (90% enrollment completed). Therefore, any actions that 
a DSMB might take to prevent subsequent patients from receiving an ineffective treatment (futility) or 
to make available a useful treatment (efficacy) will not have a direct impact on patients participating in 


 
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this study. As a surrogate for long-term 24-month clinical efficacy on NDI, one alternative would be to 
monitor directly early MRI results as intermediate outcomes or surrogates. However, a large study 
published by Cheong et al.,150 found that early MRI measurements had poor sensitivity (27-60%) for 
accurately predicting death or NDI at 2 years. Therefore, we do not recommend using early MRI 
measures as a surrogate for the long-term outcome for monitoring treatment efficacy. Given that we 
are not conducting interim analysis directly on the primary outcome measure for efficacy or futility, we 
do not make adjustments to sample size or statistical power to account for interim alpha spending. 
Our primary objective for interim analyses is therefore to allow for careful and continued monitoring of 
mortality and safety outcomes. We propose to conduct formal statistical analysis and inference for 
mortality at three interim and one final analysis time. We will continue to present mortality and SAE 
data from all available follow-up data, but we expect most treatment-related safety events to occur 
within the first three months of follow-up. We will conduct formal safety evaluation at 6, 12, 18, and 24 
months following the start of enrollment, where approximately 25, 50, 75, and 100% of the study cohort 
will have been randomized and followed for at least 3 months. As part of each interim analysis, we plan 
to monitor mortality as a primary safety endpoint and will control the overall significance level using 
O’Brien-Fleming boundaries (net alpha=0.05 significance, accounting for three interim and one final 
analysis). The DSMB will also monitor all other SAEs utilizing the same O’Brien-Fleming sequential 
monitoring boundaries without further adjustment for multiple comparisons, but allowing for flexibility 
to continue the study if the O’Brien-Fleming boundary is reached on a secondary SAE endpoint. 
Table 11. O’Brien-Fleming Monitoring Boundaries 
Enrollment 
Monitoring p-value: Death 
25% 
0.000014734 
50% 
0.0030359 
75% 
0.016248 
100% 
0.030701 
 
EXAMPLE SAFETY MONITORING TABLE 
Shell Table 1. Closed Report: Serious adverse event (SAE) incidence rate (number of events/number at 
risk) and threshold for action. 
 
Serious Adverse Events 
Group 
A 
Group 
B 
Expected 
(Range) 
Threshold 
for Action 
p-value* 
Systemic hypertension 
 
 
0.2-3.0151-153 
6% 
 
Polycythemia 
 
 
1.0-5.0%154 
10% 
 
Disseminated intravascular coagulation 
(DIC) 
 
 
11.0-45.0%3-7 
50% 
 
Major venous or arterial thrombosis 
 
 
0-2.0%4,6,155 
8% 
 
Pulmonary hypertension 
 
 
6.0-
22.0%3,4,7,156 
30% 
 
Intracranial hemorrhage 
 
 
7.9-31.3%6,7 
37% 
 
Cardiopulmonary arrest 
 
 
0.002-
0.13%157 
5% 
 
Other unexpected life-threatening event 
 
 
1.0%4 
5% 
 


 
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Death 
 
 
8.1-35.1%3-
7,155,158,159 
40% 
 
* P-value calculated for the rate ratio comparing treatment Group A to Group B. 
In addition, we will provide a table comparing complications of HIE between the groups. 
 
Data Safety Monitoring Schedule: Our target enrollment is n=500 which is expected to accrue during 
the first 4.0 years of the trial. Therefore, we will enroll approximately 83 subjects every 6 months. Our 
planned DSMB safety analyses will occur every 6 months after trial initiation. At the first safety 
evaluation, we expect to have 83 subjects enrolled, but would only have discharge outcomes on the first 
60 subjects. Each subsequent 6-month period would increase the number of babies by 83 leading to the 
following cumulative number of subjects available for analysis. Safety evaluation will be based on all 
available follow-up but we expect the majority of SAEs and AEs to occur during the neonatal 
hospitalization and therefore within one month of age (2-4 weeks) in the majority of cases. 
Table 12. Cumulative number of subjects evaluated for safety events and for the long-term efficacy outcome 
Month  
6 
12 
18 
24 
30 
36 
42 
48 
54 
60 
66 
Safety (1 mo)  
60 
143 
226 
309 
392 
475 
500 
 
 
 
 
Efficacy (24 mo)  
0 
0 
0 
0 
60 
143 
226 
309 
392 
475 
500 
 
10 DATA COLLECTION, SITE MONITORING, AE REPORTING 
 RECORDS TO BE KEPT 
All data will be entered into CRFs in the HEAL Portal that are developed specifically for the study, using a 
REDCap platform. These forms will include screening, enrollment and follow-up data for infants. The 
infant’s parent or legal guardian must read, understand, and sign an IRB approved informed consent 
form. Additionally, it will be recorded in the infant’s medical record that the infant is involved in this 
study. The Investigator will retain the original signed consent form in a secured location. Investigators 
must provide all information required by the protocol on the CRFs in the HEAL Portal provided for the 
study.  
At the DCC, all data received is identified by a unique subject ID. No subject identifiers are collected 
other than date of birth 
10.1.1 Subject identifiers 
Only the clinical site will have the Screening Log that maps the ID number, infant initials and 
randomization number to the subject’s name and contact information at their site. At the clinical 
sites, all subject data are maintained in locked file cabinet or other secure locations with limited 
access by researchers and staff. Laboratory samples will be identified by a study ID number. A list 
of each sites’ subjects will be kept separately in password protected or locked files within that site 
investigator’s office.  
10.1.2 Documentation 
Each site must provide the CCC at the University of California, San Francisco with the following 
documents prior to study initiation. A copy of these documents must also be maintained 
throughout the study in the investigator’s study files. 
• 
IRB approved informed consent form 


 
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• 
Completed 1572 form, signed and dated CVs, proof of current CITI training or equivalent 
(Human Subjects Training), Good Clinical Practice certification, financial conflict of 
interest forms, and proof of current medical licensure for all investigators 
• 
All IRB approvals and correspondence (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 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 
CCC, that these records are no longer needed. The Investigator must notify CCC project director if 
the site or records are relocated, if the investigator leaves the institution, etc. and a new address 
for the records must be provided. 
 ROLE OF DATA MANAGEMENT 
The DCC will be at University of Washington, Seattle WA. The DCC will create an https secured web-
based portal specially designed for the purposes of this study. The study portal will provide a highly-
structured repository to store and process study data from e-CRFs, as well as to protect its integrity and 
confidentiality. This tool will assist the CCC by providing efficient protocol management and study 
enrollment and retention oversight. Access to the study portal will be granted by the DCC with 
differential access rights based on role. Users at a given site will only have access to data on study 
participants at their own site. 
The DCC is responsible for:  
• 
Providing tools for data collection, management and monitoring for the clinical sites  
• 
Supporting a web page and portal and providing training for research staff 
• 
Providing and managing secure log-in access to the study portal 
• 
Biostatistics and analysis support 
The DCC also has the responsibility to:  
• 
Generate and distribute masked randomization assignments to clinical site pharmacies 
• 
Ensure study drug accountability at all sites 
• 
Provide data summaries and statistical analyses for the study  
• 
Receive information about SAEs  
• 
Generate semi-annual data and safety monitoring reports for independent assessment by the 
study DSMB 
 
The DCC will facilitate user access by sponsoring UW NetIDs for each individual involved with the HEAL 
project. For each clinical recruitment site, the DCC will provide up to two initial training sessions to 


 
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familiarize research staff and investigators with the web-based data management portal. As research 
staff turnover during the course of the study, data management training videos and a FAQ page will be 
accessible on the portal home page in order to ensure that new staff are facile with the system.  
10.2.1 The Clinical Site responsibilities in data collection and management 
10.2.1.1 
Data Collection Protocol 
• 
For screening and enrollment, the site will access the REDCap study portal via 
the web, provide screening information about the patient (Screening ID will 
be generated) and if eligible for randomization, receive the study participant 
number (Study ID).  
• 
Study-related data, as outlined in the schedule of visits and evaluations above, 
will be entered online by site personnel directly into the CRFs within the 
REDCap study portal.  
10.2.1.2 
Description & Flow of Case Report Forms (CRFs) 
• 
Copies (PDF versions) of all CRFs are printable from the study portal. 
• 
CRFs can be printed and used to capture data, and later entered into the 
REDCap study portal. 
10.2.2 Data Security and Confidentiality 
The DCC employs procedural and technical controls to ensure the security, integrity and 
confidentiality of subject data that are in compliance with established regulations and standards 
for Information Technology Security. While only limited identifiable data will be collected by the 
DCC (date of birth, dates of service), this server room meets the technical requirements for HIPAA 
compliance and hosts other servers containing PHI. Multiple levels of data security are in place 
designed to prevent unauthorized access and limit authorized access to the computer systems and 
prevent data corruption and loss. These include firewall and network intrusion detection devices, 
malware protections, account and system security features, as well as written policies and 
procedures. 
 QUALITY ASSURANCE 
We will monitor the accuracy of data entry by the sites both internally and externally. For internal 
monitoring, completed online CRFs (in the HEAL Portal) are reviewed on a regular basis, and issues are 
clarified as necessary with site coordinators who act as liaison between the sites and the DCC as needed.  
PI’s and Study Coordinators from the CCC will perform regular monitoring visits to every site while 
actively enrolling. Initial monitoring visits will be targeted for after the first 6-8 patients have completed 
data collection at a given site. A subset of all data points in the online CRFs will be compared with the 
medical record. Any outstanding data queries will attempt to be resolved with the research coordinator 
at the time of the visit. After each study site monitoring visit, a report will be prepared and copies sent 
to the Study File, the DCC, the study PIs, the site PI, the site coordinator and the CCC Study Coordinators. 
As part of the overall quality assurance (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. 


 
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The DCC will use a web- and email-based reminder system to identify when patients enter a follow-up 
evaluation window and when evaluations are complete. Data managers from the DCC will assess data 
quality by running validation checks on a regular basis. Data fields are verified against documented 
expectations using suitable legal ranges, field requirements, reports, and other data consistency checks. 
Any discrepancies are reported back to the clinical sites for further review and correction. The reporting 
page of the study portal will contain a window-based reporting system that checks for the timely entry 
of data within the anticipated length of neonatal hospitalization (7-20 days) and the schedule of follow-
up visits 
10.3.1 Data Audit 
After all subject data have been submitted and data queries addressed, the database will be 
considered ready to lock.  
 ADVERSE EXPERIENCE REPORTING  
See Table 5 above for the flow chart reflecting AE and SAE reporting. 
 
11 HUMAN SUBJECTS 
 INSTITUTIONAL REVIEW BOARD REVIEW AND INFORMED CONSENT 
This protocol, the informed consent document, and any subsequent modifications will be reviewed and 
approved by the IRBs responsible for oversight of the study at each study site. Any substantial changes 
in protocol must be approved by the DSMB. The consent form will describe the purpose of the study, 
the procedures to be followed, Certificate of Confidentiality language, and the risks and benefits of 
participation. A list of key elements to be included in the consent will be prepared by the CCC, reviewed 
by the DCC, and reviewed and ultimately approved by the DSMB. The approved consent at UCSF will be 
used as a template for preparation of the consent at the sites. A copy of the signed consent form will be 
given to the parent or legal guardian, a copy placed in the patient’s chart, and the original signed 
consent will be stored by the site coordinator. 
 SUBJECT CONFIDENTIALITY 
All laboratory specimens, evaluation forms, reports, and other records that leave the site will be 
identified only by the Study Identification Number (Subject Study ID), as well as date and time for 
laboratory specimens, to maintain subject confidentiality. All records will be kept in a locked file cabinet. 
All computer entry and networking programs will be performed using Subject Study ID only. Clinical 
information will not be released without written permission of the subject, except as necessary for 
monitoring by the IRB, the FDA, the NINDS, and by the CCC and DCC. 
 STUDY MODIFICATION/DISCONTINUATION 
The study may be modified or discontinued at any time by a local IRB, the NINDS, the DSMB, or the FDA 
as part of their duties to ensure that research subjects are protected. Any changes to the protocol 
require a written protocol amendment that must be approved by the Executive and Steering 
Committees and by the DSMB prior to implementation. Amendments that affect patient eligibility, study 
protocol, or consent changes require additional approval by the IRB at each site. These amendments, 
should they be required, will become a part of the protocol and maintained by the Investigator as part 
of the study documentation. For amendments affecting only administrative aspects of the study that do 
not require formal IRB approval, the IRB at each of the sites must be informed of such changes. Other 


 
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changes in the study conduct are not permitted. Any unforeseen changes must be recorded in the 
clinical study report.  
 
12 PUBLICATION OF RESEARCH FINDINGS 
Publication of the results of this trial will be governed by the policies and procedures developed by the 
Executive Committee. Any presentation, abstract, or manuscript will be made available for review by the 
NINDS prior to submission. 


 
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Douglas-Escobar M, Yang C, Bennett J, et al. A pilot study of novel biomarkers in neonates with 
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classifying cerebral palsy in young children. J Pediatr. 2008;153(4):466-472. 
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neurologic examinations of 1-year-old children in a multicenter epidemiologic study. J Child 
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Kuban KC, Allred EN, O'Shea M, Paneth N, Pagano M, Leviton A. An algorithm for identifying and 
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therapy in the era of Bayley-III. J Perinatol. 2014. 
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preterm infants treated with inhaled nitric oxide. J Pediatr. 2010;156(4):556-561 e551. 
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risk for cerebral palsy in term and near-term infants. Ann Neurol. 2009;66(5):663-670. 
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150. 
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analysis of trial data. BMJ. 2010;340:c363. 
 
 
 


 
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14 Appendices 
 ACRONYMS 
AE 
Adverse Event 
aEEG 
Amplitude-integrated EEG 
AIMS 
Alberta Infant Motor Scale 
ANCOVA 
Analysis of Covariance 
AUC 
Area Under the Curve 
Bayley III 
Bayley Scales of Infant and Toddler Development, 3rd Edition 
BDNF 
Brain-Derived Neurotrophic Factor 
BEAM 
Beneficial Effects of Antenatal Magnesium Sulfate 
BGT 
Basal Ganglia and Thalamus 
CBCL 
Child Behavior Checklist 
CCC 
Clinical Coordinating Center 
cEEG 
Continuous EEG 
CNS 
Central Nervous System 
CONSORT 
Consolidated Standards of Reporting Trials 
CP 
Cerebral Palsy 
CPAP 
Continuous Positive Airway Pressure 
CRF 
Case Report Form 
CSF 
Cerebrospinal Fluid 
DCC 
Data Coordinating Center 
DIC 
Disseminated Intravascular Coagulation 
DSMB 
Data Safety Monitoring Board 
DSMP 
Data Safety Monitoring Plan 
DTI 
Diffusion Tensor Imaging 
ECMO 
Extracorporeal Membrane Oxygenation 
EEG 
Electroencephalogram 
ELGAN 
Extremely Low Gestational Age Newborns 
Epo 
Erythropoietin 
Epo-R 
Erythropoietin Receptor 
ERK1 
Extracellular Signal-Related Kinase 


 
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ETT 
Endotracheal Tube 
FDA 
Food and Drug Administration 
GDNF 
Glial cell derived neurotrophic factor 
GFAP 
Glial Fibrillary Acidic Protein 
GMFCS 
Gross Motor Function Classification System 
HCT 
Hematocrit 
HIE 
Hypoxic-Ischemic Encephalopathy 
HIPAA 
Health Insurance Portability and Accountability Act 
HT 
Hypothermia 
HUS 
Head Ultrasound 
ICE Trial 
Infant Cooling Evaluation Trial 
IL 
Interleukin 
IND 
Investigational New Drug 
IP 
Intraperitoneal 
IRB 
Institutional Review Board 
IV 
Intravenous 
JAK2 
Janus Kinase 2 
MAR 
Missing at Random 
mITT 
Modified Intent to Treat 
MCAR 
Missing Completely at Random 
MRI 
Magnetic Resonance Imaging 
MRS 
Magnetic Resonance Spectroscopy 
MSD 
Meso Scale Discovery 
NDI 
Neurodevelopmental Impairment 
NEAT 
Neonatal Erythropoietin in Asphyxiated Term Newborns 
NEATO 
Neonatal Erythropoietin and Therapeutic Hypothermia Outcomes in Newborn 
Brain Injury 
NINDS 
National Institute of Neurological Disorders and Stroke 
NS 
Normal Saline 
NSE 
Neuron-Specific Encolase 
OT 
Occupational Therapy 


 
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PENUT 
Preterm Erythropoietin Neuroprotection Trial 
PI 
Principal Investigator 
PI3K 
Phosphatidylinositol-3 kinase 
PT 
Physical Therapy 
QA 
Quality Assurance 
SAE 
Serious Adverse Event 
SC 
Subcutaneous 
SD 
Standard Deviation 
SOP 
Standard Operating Procedure 
ST 
Speech Therapy 
STAT5 
Signal Transducer and Activator of Transcription 5 
TNF-α 
Tumor Necrosis Factor-α 
TOLSURF 
Trial of Late Surfactant 
UCH-L1 
Ubiquitin C-terminal Hydrolase-L1 
VEGF 
Vascular Endothelial Growth Factor 
WIDEA 
Warner Initial Developmental Evaluation of Adaptive and Functional Skills 
 

SAP_001.pdf: 
 
 
 
 
 
 
Official Title: 
HIGH DOSE ERYTHROPOIETIN FOR ASPHYXIA AND ENCEPHALOPATHY 
 
NCT number: 
NCT02811263 
 
Document: 
Statistical Analysis Plan (SAP) version 2.0 
 
Document Date: 
February 15, 2022 
 
 
 
 
 
 
 
 


 
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A randomized, placebo-controlled, double-masked 500 - subject clinical trial 
of erythropoietin for the treatment of 
neonatal hypoxic-ischemic encephalopathy (HIE) 
 
Statistical Analysis Plan 
 
 
 
 


 
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TABLE OF CONTENTS 
1. 
General Design Considerations ............................................................................................................ 4 
1.1 
Hypothesis ............................................................................................................................................... 4 
1.2 
Design ...................................................................................................................................................... 4 
1.3 
Randomization ........................................................................................................................................ 4 
1.4 
Analysis .................................................................................................................................................... 4 
2. 
Primary Outcome ................................................................................................................................ 4 
2.1 
Motor Deficit - Cerebral Palsy (CP) ......................................................................................................... 5 
2.2 
Motor Deficit – Gross Motor Functional Classification System (GMFCS) ............................................... 5 
2.3 
Cognitive Deficit – Bayley III .................................................................................................................... 5 
2.4 
Adjudication of Primary Outcome .......................................................................................................... 6 
3. 
Secondary Outcomes ........................................................................................................................... 6 
4. 
Exploratory Outcomes ......................................................................................................................... 7 
4.1 
Ordinal Exploratory Outcome ................................................................................................................. 7 
4.2 
Circulating Biomarkers ............................................................................................................................ 7 
4.3 
Neuroimaging Outcomes ........................................................................................................................ 7 
4.4 
Hearing and Cortical Visual Impairment ................................................................................................. 8 
5. 
Data Analyses ...................................................................................................................................... 8 
5.1 
Primary Outcome .................................................................................................................................... 8 
5.2 
Secondary Outcomes .............................................................................................................................. 8 
5.3 
Exploratory Outcomes............................................................................................................................. 8 
5.4 
Exploratory Prognostic Biomarkers ......................................................................................................... 9 
5.5 
Safety Data .............................................................................................................................................. 9 
5.6 
Laboratory Data ...................................................................................................................................... 9 
5.7 
Circulating Biomarkers ............................................................................................................................ 9 
5.8 
Neuroimaging Outcomes ...................................................................................................................... 10 
6. 
Compliance, Retention, and Missing Data .......................................................................................... 10 
6.1 
Adherence and Retention ..................................................................................................................... 10 
6.2 
Missing Data and Dropouts ................................................................................................................... 10 
6.3 
Handling ................................................................................................................................................ 10 
6.4 
Statistical Uncertainty ........................................................................................................................... 10 
6.5 
Sensitivity Analysis ................................................................................................................................ 10 
7. 
Sample Size and Accrual .................................................................................................................... 11 
7.1 
Primary Outcome Sample Size Calculations .......................................................................................... 11 
7.2 
Sample size for efficacy secondary outcomes ...................................................................................... 13 


 
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7.3 
Sample size for MRI/MRS biomarkers ................................................................................................... 13 
7.4 
Sample size for plasma biomarkers of brain injury and inflammation ................................................. 14 
7.5 
Achieving the sample size ..................................................................................................................... 14 
8. 
Data Monitoring ................................................................................................................................ 14 
9. 
Data and Safety Monitoring Plan (DSMP) ........................................................................................... 14 
10. References ........................................................................................................................................ 16 
 
 


 
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1. 
General Design Considerations 
1.1 Hypothesis 
Epo given to infants with moderate-severe HIE will safely lead to improved neurodevelopmental 
outcome at 24 months. 
1.2 Design 
This study is a randomized parallel group double-masked, placebo-controlled trial in neonates with HIE. 
We will enroll 500/2 = 250 subjects in each of two treatment groups:  Epo-treated vs. placebo control. 
Eligible neonates will be enrolled and treated with either Epo or normal saline (on study day 1,2,3,4, and 
7) and followed for a fixed period of 24 months (±56 days) for survival and neurological outcomes. For 
extenuating circumstances, for example, COVID-19 restrictions, the in-person elements of the final 
endpoint evaluation may be performed up to 36 months of age. The follow-up period was chosen to 
provide meaningful motor and cognitive outcomes, since CP and motor and cognitive deficits are more 
reliably diagnosed at 2 years of age. Enrolled children will be randomized, stratified by study site and 
severity of HIE, with equal likelihood to receive Epo or saline. 
1.3 Randomization 
Randomization sequences will be created centrally by the Data Coordinating Center (DCC). The DCC will 
provide each site pharmacy with a randomization look-up table that pairs each study ID with a treatment 
allocation. Randomization is stratified by site and HIE severity level (“moderate” vs. “severe” HIE) based 
on the modified Sarnat score. The site pharmacy will then draw up the assigned study drug dose into a 
syringe, based on the randomization list. The appearance of the syringes of Epo and placebo are 
identical, thus the bedside staff remain masked. Only the research pharmacist and DCC biostatistician 
will be un-masked to treatment assignment. All parents, study and clinical staff, as well as all Clinical 
Coordinating Center (CCC) members, will remain masked to treatment assignment throughout follow-up. 
To ensure balance within clinical centers, the randomization will be implemented using a randomly 
permuted blocks design to ensure approximate balanced assignments within site and HIE severity level. 
The sequence of assignments for each site and stratum will be prepared in advance by the DCC. 
1.4 Analysis 
Analyses will be based on a modified Intention to Treat (mITT) approach. In this approach, all 
randomized neonates who received at least one dose of study drug will be included in the analyses. 
Neonates whose parents withdrew consent after randomization and before the first dose will be 
excluded from the mITT. 
 
2. 
Primary Outcome 
We used standardized, validated neurological and developmental assessments at 24 months:  
• 
CP diagnosed by Standardized Neurological Examination1,2 
• 
Bayley III Cognitive Score  
• 
GMFCS3 
The primary outcome is the composite of death or any neurodevelopmental impairment. 
Neurodevelopmental impairment (mild, moderate, or severe) is defined as any of the following:  
• 
GMFCS level ≥1, or  


 
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• 
GMFCS = 0 or 0.5 AND CP (any type), or  
• 
Bayley III Cognitive Score < 90 
Due to COVID-19 restrictions, the Bayley III, Neuro examination, and GMFCS data collection windows 
were extended from their original window of 22-26 months of age up to 36 months of age. The Bayley III 
is normed by age through 36-months, and CP status is not anticipated to change between 22 and 36 
months of age. Therefore, this window extension does not affect the validity of the primary outcome 
measure. 
2.1 Motor Deficit - Cerebral Palsy (CP) 
Cerebral palsy (CP) will be determined by a Standardized Neurologic Examination1,2 conducted under the 
direction of Dr. Karl Kuban (Co-I), using the systematized exam and video-based certification system 
created for the NINDS-funded ELGAN and PENUT studies.1,2 This training program provides a formal 
method of neurologic testing that is highly reproducible. The exam was created specifically to determine 
the presence and classification of CP (i.e., quadriplegic-QP, hemiplegic-HP and diplegic-DP) at age 2. To 
standardize the quality of neurologic examination findings, two follow-up examiners at each site will 
participate in a training session, review training videos, and submit a set of independently scored 
examinations that will be used for certification, as was done in the PENUT trial.2 A re-certification 
process will be performed at least every 18 months to ensure that primary outcome examiners remain 
adequately trained throughout the study. 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 < 85% correct rate. Based on experience in the ELGAN study, we expect 
agreement rates to exceed 90%. 
2.2 Motor Deficit – Gross Motor Functional Classification System (GMFCS) 
Gross Motor Functional Classification System (GMFCS) The GMFCS is a well-accepted and validated tool 
that is used widely to classify motor functional outcomes. Distinctions between levels of motor function 
are based primarily on functional ability. The GMFCS was used to determine motor outcome in the 
NINDS-funded Beneficial Effects of Antenatal Magnesium Sulfate (BEAM) trial, as well as in 6 of 7 
hypothermia trials for HIE. We will apply the BEAM trial algorithm and definitions when assigning GMFCS 
scores. Level 0 = normal gait; Level 0.5 =asymmetric gait; Level 1 = walks independently with abnormal 
gait or requires anklefoot orthosis; Level 2 = cruises, pulls to stand, sits hands free; Level 3 = sits propped 
on hands only, rolls both ways; Level 4 = sits when supported in lower trunk, has head control, can roll to 
supine; Level 5 = no head or trunk control, no rolling, little or no voluntary movement. These definitions 
are modified from the Palisano3 algorithm adapted by Rosenbaum P and Saigal S for TIPP Trial, and 
utilized in the NICHD Neonatal Research Network under the direction of Betty Vohr. 
2.3 Cognitive Deficit – Bayley III 
Bayley III Cognitive Score < 90 Bayley III Cognitive Score4 is a standard test used to evaluate early 
cognitive outcomes in high-risk infants. Severity of cognitive deficit is defined by number of standard 
deviations (SD) below the mean (i.e., severe, moderate, and mild deficit = -3, -2 and -1 SDs below the 
mean). Since the Bayley III yields higher cognitive scores than the previous version of the Mental 
Development Index on the Bayley II,5-8 we have defined cut-offs on the Bayley III that are 5-15 points 
shifted to the right compared to the Bayley II. We consider any Bayley III Cognitive Score < 90 as 
abnormal, with mild, moderate, and severe ranges as defined in Table 2. These definitions are consistent 
with findings in a contemporary cohort of infants with HIE who underwent hypothermia,7 and also 
consistent with cut-offs used in the PENUT trial. Each site will undergo certification by reviewing a “gold 
standard” Bayley III assessment performed by our research neuropsychologist. Each site psychologist will 


 
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then record a Bayley III assessment of a 2-year-old child and submit the test booklet and video recording 
for review by the research psychologist for feedback. HEAL CCC leadership has experience in performing 
multicenter Bayley III certification in other NIH funded studies (e.g., PENUT, TOLSURF), and will use the 
infrastructure in place for these studies. Only Bayley III examiners who have undergone this training and 
certification will be allowed to perform the 2-year primary endpoint evaluation. 
2.4 Adjudication of Primary Outcome 
There may be cases of only a phone follow-up rather than an in-person visit, or otherwise partial data. 
The CCC will create an Outcomes Adjudication Committee, who will be masked to the treatment 
assignment of the child. The Committee will receive all available data on the long-term outcome of these 
toddlers and will assess whether they can definitively assign a primary outcome level and what it is, or 
they do not have sufficient information to make that determination and therefore the value will be 
imputed. This model was successfully previously used in the NO CLD clinical trial long-term outcome. 
 
3. 
Secondary Outcomes 
At age 2, we will assess the effect of Epo on secondary outcomes: a) presence of CP, b) severity of motor 
impairment, c) Bayley III cognitive and language scores, d) epilepsy (i.e., ≥ 2 afebrile, unprovoked 
seizures), and e) behavioral abnormalities (i.e., attention problems or aggressive behavior) based on the 
CBCL externalizing score.9  
Severity of motor impairment will be determined by type of CP and GMFCS level (Table 1). 
Table 1. 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 
 
Severity of cognitive impairment will be determined by Bayley III Cognitive Score (Table 2). 
Table 2. Cognitive outcome – 4 level classification. 
Cognitive Deficit 
Bayley II MDI score 
(Hypothermia trials) 
Bayley III Cognitive score 
(PENUT, HEAL) 
Severe (> 3 SD) 
≤ 55 
≤ 70 
Moderate (2-3 SD) 
> 55 and ≤ 70 
70-84 
Mild (1-2 SD) 
> 70 and ≤ 85 
85-89 
None 
> 85 
≥ 90 
 


 
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4. 
Exploratory Outcomes 
4.1 Ordinal Exploratory Outcome 
 To elucidate the effect of Epo on all severities of impairment, we will analyze the effect of Epo on the 
following 4-level outcome: 1) normal, 2) mild motor and/or cognitive impairment, 3) moderate/severe 
motor and/or cognitive impairment, and 4) death. Use of this measure allows a detailed assessment of 
potential shifts in the distribution of outcomes toward improved status associated with treatment. 
4.2 Circulating Biomarkers 
Inflammation is thought to play an important role in HIE and CP.10-13 Biomarkers of inflammation that we 
measure include: interleukin (IL)-1β, IL-6 and IL-8 and TNF-α. Putative biomarkers of brain injury in 
neonatal HIE include: glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), 
S100B, Tau, and neuron-specific enolase (NSE). 
4.3 Neuroimaging Outcomes 
The HEAL neuroimaging outcome measure is the total MRI injury score based on a previously validated 
MRI scoring system for HIE.14 This score indicates the extent of signal abnormality (i.e., 0=none, 1 ≤25%, 
2=25%–50%, 3 ≥50%) for each of the deep grey nuclei, PLIC, cerebral cortex, white matter and 
cerebellum, as well as brainstem (scored on a 0–2 scale) separately for each sequence. ‘Signal 
abnormality’ is defined qualitatively as either abnormally low or high signal on T1w and T2w images or 
areas of restricted diffusion on Trace-weighted or ADC images calculated from the DTI sequence. We do 
not employ a quantitative threshold for ADC, consistent with the previously validated scoring system.14,15 
Areas with high ADC are not scored as injury for the DTI images, which is designed to measure acute 
injury; however, correspondent areas of low and/or high signal on the T1w and/or T2w images are 
scored. 
For each ROI, injury is scored qualitatively for each sequence on a 4-point scale based on the extent of 
signal abnormality (0=none, 1 = <25%, 2=25%–50%, 3 ≥50%), except for the brainstem which is scored on 
a 3-point scale (0=none; 1=focal; 2=multifocal/widespread). 
To compute the MRI injury score, each scan is reviewed independently by two of three experienced 
readers (AMM, JLW, and RCM), who are blinded to the infant’s clinical course, treatment assignment and 
MRS findings. After the primary review, each scan undergoes final consensus review during which any 
discrepancies are reviewed by all three reviewers and resolved by consensus. The individual and 
consensus scores are then classified as none (total=0), mild (1–11), moderate (12–32) or severe (33–
138), in accordance with the previously validated MRI injury classification.14 Inter-rater reliability 
between the three independent readers will be determined for both the total score and for the 
categorical classification (none, mild, moderate, severe). For the total score, we will use a general linear 
mixed effects model to estimate the intraclass correlation coefficient (ICC).16 For the categorical 
classification, we will use kappa.17  
Secondary MRI measures include the MRI injury severity classification discussed above as well as 
classification of injury pattern and acuity. Injury pattern is classified as: (1) normal MRI (defined as no 
evidence of injury); (2) central (injury to the BGT±perirolandic cortex); (3) peripheral (injury to the 
parasagittal cortex and/or WM, i.e, ‘borderzone/watershed distribution); (4) global (injury to BGT+ 
total or near total involvement of cortex±underlying WM); (5) punctate WM Lesions (discrete foci of 
injury typically 1–10 mm in size localised to the periventricular WM or centrum semiovale); (6) arterial 
ischaemic stroke (infarct localised to the vascular territory of the middle, anterior or posterior cerebral 
arteries); (7) other focal lesions (includes venous infarcts, contusions, and unilateral lesions to the BGT, 


 
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cortex or white matter not classified elsewhere) and (8) atypical/not otherwise specified (with additional 
text field for describing the lesion). The scoring system allows for multiple patterns to be coded, which 
will allow us to determine the frequency of individual patterns as well as the co-occurrence across 
patterns. 
4.4 Hearing and Cortical Visual Impairment 
To explore the effect of Epo on sensory deficits that can result from HIE, we will collect 2-year data 
regarding hearing impairment requiring hearing aids, and if available, information about the presence of 
cortical visual impairment diagnosed by an ophthalmologist. 
5. 
Data Analyses 
5.1 Primary Outcome 
The primary analysis will be a test of equality of the rate of the primary outcome (death or 
neurodevelopmental impairment (NDI)) across the two randomized investigational groups. Specifically, 
we will use a likelihood ratio test based on logistic regression, with stratification by recruitment center 
and HIE severity. We will perform modified Intention to Treat (mITT) 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 consider an ordered categorical two-year status measure (death/severe or moderate 
impairment/mild impairment/normal), and analysis will be based on generalized Wilcoxon tests. 
5.1.1 Subgroups 
Given the a priori hypothesis that treatment effect may differ according to gender or HIE severity 
we will conduct a pair of subgroup analyses that assesses treatment effects separately for males 
and for females, and separately for moderate and severe HIE. Subgroup specific treatment effects 
will be computed and inference will be based on a single covariate-by-treatment test for 
interaction using logistic regression. 
5.2 Secondary Outcomes 
Generalized linear regression models will be used for binary and continuous secondary outcomes, 
adjusted for recruitment site and HIE severity.  
 
5.3 Exploratory Outcomes  
Generalized linear regression models will be used for exploratory binary and continuous outcomes, 
adjusted for recruitment site and HIE severity. Regression models for ordered categorical outcomes 
(proportional odds models) will be used to provide treatment effect estimates adjusted for recruitment 
site and HIE severity. Quantitative measures include MRI-based injury score using the Washington 
University Standardized Scoring System. For these endpoints, differences and 95% confidence intervals 
in median brain MRI injury scores between groups will be assessed using bootstrapped resampling with 
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5.4 Exploratory Prognostic Biomarkers  
We will consider two main classes of potential predictors of 24-month 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. We will derive two 
multivariate predictive models: using the inflammatory markers and using the MRI and MRS 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). Evaluation of whether treatment modifies 
the prognostic potential of biomarkers can be conducted by testing for the interaction between 
treatment status and individual biomarkers in predictive models for 2-year outcomes. 
5.5 Safety Data 
Clinical safety data includes serious adverse events (SAEs) and clinical laboratory markers both from the 
hospitalization and study intervention period and from the long-term follow-up period. Because these 
are critically ill newborns, we anticipate that most safety events will occur during the initial inpatient and 
treatment period. Safety event rates will be tabulated by study group and compared separately for each 
SAE using a multivariable logistic regression model, with adjustment for randomized treatment group, 
and randomization stratification factors of clinical recruitment site and HIE severity (moderate/severe). 
We will additionally create a per-patient aggregate count of SAEs and evaluate the rates between groups 
using a Poisson regression model with robust standard errors and adjustment for randomization 
stratification factors. For any safety events that occur infrequently (<2%), we will use Fisher's exact test 
to compare the rates between treatment groups. All safety events identified post-birth will be included 
in the primary analysis, while the secondary analyses will evaluate safety events occurring post-
treatment with the first study drug dose as well as those events assessed as being possibly related to the 
study drug. Statistical significance is defined conservatively at the alpha=0.05 level with no correction for 
multiple outcomes. 
5.6 Laboratory Data 
Laboratory tests of organ injury are measured at baseline and at age 2-3 days as part of routine care. We 
will compare laboratory test data measured between study day 2 and 3 between treatment groups using 
analysis of covariance (ANCOVA) regression model, with adjustment for treatment group, the laboratory 
value measured at baseline, and the time between laboratory measurements. We will use a similar 
analytic approach to compare vital signs and growth parameters between treatment groups. 
5.7 Circulating Biomarkers 
Inflammation is thought to play an important role in HIE and CP.10-13 Biomarkers of inflammation that we 
measure include: interleukin (IL)-1β, IL-6 and IL-8 and TNF-α. Putative biomarkers of brain injury in 
neonatal HIE include: glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), 
S100B, Tau, and neuron-specific enolase (NSE). We will select a random subset of 200 subjects (100 
treated and 100 controls with each group, for example, including both moderate and severe HIE) to 
measure circulating biomarkers of inflammation and brain injury. We will collect 3 plasma samples from 
each infant at the following time points based on hour of age: < 24 hours; Study Day 2; Study Day 4. Our 
analysis will focus on time-specific comparisons of the mean biomarker measure across treatment 


 
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groups using appropriate regression methods while controlling for site and HIE severity. In addition, we 
will conduct longitudinal analysis using linear mixed models18 that permit an omnibus test across all four 
measurement times and allow inference on differential rates of change across treatment groups. 
5.8 Neuroimaging Outcomes 
The primary neuroimaging outcome measures are: (1) the total injury score (derived from the 
HEAL/Wash U MRI scoring system above) and (2) the ratio of lactate/NAA determined for the left 
thalamus and parietal white matter from the quantitative MRS data. For the MRI injury score and the 
lactate/NAA ratio, we will use linear regression to compare Epo treated patients to controls while 
adjusting for site and HIE severity since these are factors used to stratify the randomization. If we have 
missing data due to either early patient death or other factors (e.g., family declines MRI or infant is 
unable to complete all the sequences), we will estimate the resultant sampling bias by comparing our 
final neuroimaging sample to the overall HEAL sample with regard to patient demographics and primary 
outcome data. 
6. 
Compliance, Retention, and Missing Data 
6.1 Adherence and Retention 
Our major analyses are based on the modified ITT principle. We do not anticipate that non-adherence 
will be a major issue since the treatment is directly observed during a short in-hospital time frame. We 
will assess non-compliance, with particular focus on study treatment dosing and timing. If there are 
more than minimal issues, that will justify quantifying and characterizing non-adherence and doing a 
per-protocol analysis. In addition, site selection included having a committed neonatology follow-up 
program, and we expect >90% retention. 
6.2 Missing Data and Dropouts 
We will strive to sustain excellent participant involvement throughout the study and we have achieved 
90%+ follow-up rates in numerous prior studies. The UW DCC will generate automated nightly reports, 
available to staff at the study sites, identifying these fields with a request to discuss and prevent further 
missingness. Important data elements will be prospectively monitored to examine patterns of 
missingness. 
6.3 Handling 
In final manuscripts and analyses, the number of non-responders will be enumerated by study arm 
according to CONSORT guidelines. We will conduct a missing data analysis to describe and characterize 
enrolled participants who do not provide further response due to attrition or dropout. We will also 
conduct sensitivity analyses using 10-fold multiple imputation to assess the robustness of the results 
when missing data are imputed. 
6.4 Statistical Uncertainty 
Given the type of missing data we expect in the proposed study, missing completely at random (MCAR) 
or missing at random (MAR), both methods we propose to utilize for missing data properly account for 
statistical uncertainty due to missingness and will provide accurate confidence interval coverage. 
6.5 Sensitivity Analysis 
We will assess the sensitivity of inferences made from missing data methods first by using the two 
previously described methods for dealing with missing data, and secondly by imputing missing data 
under both pessimistic and optimistic scenarios to provide bounds on the statistical uncertainty. The 


 
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characteristics of non-responders will be summarized in our final report where we will present the 
sensitivity of the treatment effect due to missing data. 
 
7. 
Sample Size and Accrual 
7.1 Primary Outcome Sample Size Calculations 
Our proposed HEAL sites report an overall mortality rate of 14%. Using three large sites that participated 
in the phase II study (UCSF, Wash U, CNMC) we can also estimate the rates of neurodevelopmental 
impairment: death = 14%; moderate-severe impairment = 18%; mild impairment = 17%; and normal = 
51%. Therefore, we anticipate a control primary outcome rate of 49% (death or NDI). 
7.1.1 Non-human Data Informing Treatment Effect Size 
A recent study with nonhuman primates (Macaca nemestrina) compared animals experiencing 15-
18 minutes of umbilical cord occlusion that were then treated with either saline (n=14), 
therapeutic hypothermia (n=9), or therapeutic hypothermia and multiple doses of Epo.19 Among 
animals treated with saline 8/14 = 57% died or had NDI. Among animals treated with hypothermia 
(HT) alone 7/9 = 78% died or had NDI, while among animals treated with hypothermia and Epo 
(HT+Epo) only 5/12 = 42% were observed to die or have NDI. Results from Figure 2 of Traudt et 
al.20 show the number of animals in each outcome category by treatment group. These data 
suggest a risk ratio of 0.53 (95% CI: 0.25, 1.14) comparing HT+Epo to HT alone, and a risk ratio of 
0.73 (95% CI: 0.32, 1.64) comparing HT+Epo versus saline. Therefore, animal data support an Epo 
effect that optimistically corresponds to a 50% reduction, and that is conservatively associated 
with a 27% reduction in the primary outcome rate. 
7.1.2 Phase I Data Informing Treatment Outcome Rates 
Given that the primary outcome is based on a 22-26 month assessment (and for extenuating 
circumstances such as COVID-19 restrictions, up to 36 months of age) we can rely on a recently 
completed long-term follow-up from a phase I study21 in which 24 cooled infants were given 
multiple doses of Epo ranging from 250U/kg to 2500U/kg24. At 22-26 months of age n=22 subjects 
were followed, and 0/22 subjects died, 1/22 had moderate-severe NDI, and 6/22 had mild NDI. 
This study suggests an overall primary outcome rate of 7/22 = 31.8% (exact confidence interval = 
14%-55%) for our planned intervention group. We recognize that the dosing of Epo was not 
optimized in the phase I trial, and our proposed study will use multiple doses of 1000U/kg for all 
subjects which has been shown to yield plasma concentrations observed to be neuroprotective in 
animal studies. 
Table 3. Distribution of outcomes in previous studies 
 
NEATO 
Epo (n=22) 
NEATO 
Placebo (n=25) 
Bayley III 
Cognitive 
Trivedi et 
al. (submitted) 
Injury Severity 
 
 
Mean 
Std. Dev. 
  None (0) 
8 (36%) 
3 (12%) 
96 
(8) 
  Mild (1-11) 
13 (59%) 
11(44%) 
100 
(13) 
  Moderate (12-32) 
1 (5%) 
6 (24%) 
85 
(18) 
  Severe (>32) 
0 (0%) 
5 (20%) 
76 
(19) 
Predicted Bayley III (Mean (SD)) 
97.9 (11.7) 
91.1(15.2) 
 
 
Predicted %<90 
25.1% 
47.1% 
 
 
Predicted death/NDI 
34.1% 
54.5% 
 
 


 
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7.1.3 Phase II Data Informing Treatment Outcome Rates 
In our phase II NEATO trial, a total of n=50 subjects were randomized to Epo (n=24) or placebo 
(n=26). We find substantial differences in the MRI injury severity distribution with 95% of Epo 
treated subjects having no injury or mild injury as compared to only 56% of control subjects. Using 
a recently submitted study from Trivedi, et al., we can then link the MRI injury severity category to 
expected Bayley III Cognitive scores at 24 months. Using our NEATO data and Trivedi’s data we 
calculate a predicted mean (SD) of 97.9 (11.7) among Epo treated subjects, and a mean (SD) of 
91.1 (15.2) among controls. The predicted 6.8-point mean difference for Bayley III Cognitive scores 
is consistent with our observed 6.3-point difference in mean WIDEA scores at 6 months among 
NEATO subjects. Predicted Bayley III Cognitive distributions lead to an expected 25.1% of subjects 
with a cognitive score of < 90 among Epo treated subjects, and 47.1% among controls. 
Incorporating expected death rates of 12% and 14% respectively for Epo treated and controls leads 
to an expected primary outcome rate of 34.1% among treated and 54.5% among controls (Table 
3). We acknowledge that our primary outcome is based on both Bayley III Cognitive scores, and 
clinical assessments of CP and GMFCS level, but expect Bayley status to be the major case 
indicator. 
In addition, Cheong et al.22 evaluated the correlation between MRI findings obtained within 10 
days of birth and 2-year clinical status outcomes for participants in the ICE trial (Infant Cooling 
Evaluation). Specifically, basal ganglia and thalamus (BGT) injuries were classified as abnormal if 
moderate/severe abnormalities were noted on T1- and T2-weighted images. In our phase II trial, 
we find only 4.5% (1/22) among Epo treated subjects have BGT injury as compared to 20% (5/25) 
among control subjects. Cheong et al. estimates that the probability of death/NDI at two years is 
88% (PPV) for BGT abnormal subjects as compared to 32% (1-NPV) for BGT normal subjects. 
Applying these rates to our phase II BGT results yields expected death/NDI rates of 34.5% among 
Epo treated, and 43.2% among controls. For the ICE trial, the overall death/NDI rate for ICE 
hypothermia subjects was 51%, which is approximately the same as our expected control rate. 
Therefore, based on animal data, phase I data, and projections from phase II data we expect 
primary outcome rates from 31-35% among Epo treated subjects with a protective relative risk of 
0.65 to 0.71. 
7.1.4 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 Epo treated and control groups. The primary 
outcome measure is the composite rate of death or NDI, and current cohort studies suggest that 
the primary outcome occurs among 49% of infants treated with hypothermia alone (standard of 
care). Based on human data presented in Rogers et al.23 (2014), animal studies including Traudt et 
al.20, and the NEATO phase II data we assume that 33% of treated infants will die or have NDI, 
corresponding to a relative risk of 0.67. Assuming an intervention rate of 33% yields greater than 
90% power, while we have 88% power for an alternative of 34% (Table 4). In order to compute 
power, we assume a 90% follow-up rate with n=225/250 subjects evaluated in each arm. 
Table 4. Power analysis for the primary outcome assuming n=500 patients randomized and 10% 
loss to follow-up 
Control 
Intervention 
Relative Risk 
Power 
49% 
32% 
0.65 
95% 
49% 
33% 
0.67 
92% 


 
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49% 
34% 
0.69 
88% 
49% 
35% 
0.71 
83% 
 
7.2 Sample size for efficacy secondary outcomes 
Our key secondary long-term outcome is an ordered categorical 24-month status measure that classifies 
subjects as: dead; moderate or severe impairment; mild impairment; and normal. Use of this measure 
allows a detailed assessment of potential shifts in the distribution of outcomes toward improved status 
associated with treatment. Statistical analysis of this outcome will use a generalization of the Wilcoxon 
test that controls for recruitment site and HIE severity. Regression models for ordered categorical 
outcomes can also be used to provide adjusted treatment effect estimates.  
To consider power we have used 2013 data from three large sites participating in the phase II trial (UCSF, 
Wash U, CNMC) to estimate the distribution of outcomes within the control group. We calculate power 
for the stratified Wilcoxon test assuming a set of alternatives that are consistent with the assumption of 
a 33% rate of death or impairment (Scenarios 1 and 2, Table 5) or a 32% rate (Scenarios 3 and 4, Table 5) 
associated with intervention that was used to power the primary analysis. We assume a small effect on 
the death rate, and compute power for alternative shifts in the distribution of outcomes. For example, in 
Scenario 1 we assume no reduction in the death rate, and an 8% reduction in the moderate/severe 
category (relative risk = 0.55), and an 8% reduction in the rate of mild impairment (relative risk = 0.50) 
which yields 81% power using a two-sided alpha=0.05 test. Scenario 3 considers a larger reduction in the 
rate of moderate/severe and a smaller reduction in the mild category and yields 90% power. Scenarios 3 
and 4 consider a 32% overall rate of impairment or death and yield 90% or greater power. Therefore, our 
study is adequately powered to detect modest but clinically important shifts in the outcome distribution. 
Table 5. Power analysis for a four-level outcome measure with n=500 patients randomized and 
assuming 10% loss to follow-up 
 
Neurodevelopmental Impairment 
Mortality 
Power 
Normal 
Mild 
Moderate/Severe 
Placebo Arm 
51% 
17% 
18% 
14% 
-- 
Epo Arm 
 
Scenario 1 
67% 
9% 
10% 
14% 
81% 
Scenario 2 
67% 
11% 
10% 
12% 
86% 
Scenario 3 
68% 
9% 
9% 
14% 
86% 
Scenario 4 
68% 
11% 
9% 
12% 
92% 
 
Given the a priori hypothesis that treatment effect may differ according to gender or HIE severity we will 
conduct a pair of subgroup analyses that assesses treatment effects separately for males and for 
females, and separately for moderate and severe HIE. Subgroup specific treatment effects will be 
computed and inference will be based on a single Covariate-by-Treatment test for interaction using 
logistic regression. 
7.3 Sample size for MRI/MRS biomarkers 
For imaging measures, we will have data for all subjects. Assuming a 10% missing data rate we have 80% 
power to detect a difference in the mean of secondary outcomes across treatment groups of 0.26 SDs. 
For the inflammation and brain injury biomarkers, we will have a total of 200 subjects and have 80% 
power to detect a mean difference across the treatment groups of 0.4 SDs. 


 
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7.4 Sample size for plasma biomarkers of brain injury and inflammation 
A sample size of 91 neonates per group would have been required to detect a 0.5 SD difference on the 
log scale with a single observation per neonate with 80% power while controlling for a Type I error of 
0.0125 (4 markers, Bonferroni). However, since we have 4 samples per neonate, a sample size of 100 
neonates per treatment group (with correlated 400 samples per group) will be more than sufficient to 
attain >80% power. 
7.5 Achieving the sample size 
The plan is to enroll and randomize 500 patients over a 36-month period. The 17 sites that were included 
in the original study proposal cooled a total of 530 HIE infants in the year 2014. From the NEATO phase II 
randomized controlled trial study, we estimate that at least 40-45% of these infants would be eligible 
and will consent for a phase III trial, yielding at least 16 enrolled infants per month. With a further 
conservative estimate of 14 per month allowing for slower enrollment during start-up, we can enroll 500 
patients in 36 months.  
 
8. 
Data Monitoring 
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. Using the Patient Monitoring Report, a 
subset of all data points in the CRFs will be compared with the medical record for 20-25% of enrolled subjects. 
Any outstanding data queries will be resolved with the research coordinator at the time of the site-monitoring 
visit. After each study site monitoring visit, a report will be prepared and copies sent to the Study File, the 
study PIs (Y. Wu and S. Juul), the site PI, and the site coordinator. The quality and completeness of other 
deliverables (blood samples, MRIs) will be monitored. 
 
9. 
Data and Safety Monitoring Plan (DSMP) 
An NINDS-appointed independent Data and Safety Monitoring Board (DSMB) will review the accruing data to: 
1) ensure that the study is adequately enrolling; 2) to ensure that there are no serious safety concerns; and 3) 
to assess whether the study efficacy appears overwhelming. The DSMB will be assigned by NINDS. The 
research coordinator at each site will monitor each subject weekly for the presence of any complications. 
Serious adverse events will be brought to the attention of the DSMB, and if appropriate, the IRB, in writing. An 
independent medical monitor will review all cases of serious adverse events.  
As part of this 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. The following SAEs will be prospectively reviewed by the DSMB:  
• 
Systemic hypertension requiring treatment  
• 
Polycythemia (hematocrit > 65%) 
• 
Major venous or arterial thrombosis (clot) 
• 
Disseminated Intravascular Coagulation (DIC) 
• 
Pulmonary Hypertension 
• 
Intracranial Hemorrhage 


 
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• 
Cardiopulmonary Arrest 
• 
Death 
• 
Other unexpected life-threatening event (unexpected for HIE or Epo drug profile) 
 
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. The DCC PI will remain masked to 
assignment while the study staff statistician will not.  
The primary outcome of the study is a composite endpoint of mortality or NDI at 24 months of age. Therefore, 
monitoring the primary outcome for treatment efficacy or futility is challenging. Based on enrollment plans, 
most patients will have been randomized by the time NDI is assessed at 24 months for any participants, and 
therefore we do not expect to be able to conduct a first interim analysis prior to the completion of enrollment. 
We expect to have the primary outcome evaluated on the first quarter of subjects (n=125) after 34 months of 
recruitment, at which point we expect to have randomized n=450 of the total n=500 subjects (90% enrollment 
completed). Therefore, any actions that a DSMB might take to prevent subsequent patients from receiving an 
ineffective treatment (futility) or to make available a useful treatment (efficacy) will not have a direct impact 
on patients participating in this study. As a surrogate for long-term 24-month clinical efficacy on NDI, one 
alternative would be to monitor directly early MRI results as intermediate outcomes or surrogates. However, a 
large study published by Cheong et al.,22 found that early MRI measurements had poor sensitivity (27-60%) for 
accurately predicting death or NDI at 2 years. Therefore, we do not recommend using early MRI measures as a 
surrogate for the long-term outcome for monitoring treatment efficacy. Given that we are not conducting 
interim analysis directly on the primary outcome measure for efficacy or futility, we do not adjust sample size 
or statistical power to account for interim alpha spending. 
Our primary objective for interim analyses is therefore to allow for careful and continued monitoring of 
mortality and safety outcomes. We propose to conduct formal statistical analysis and inference for mortality at 
three interim and one final analysis time. We will continue to present mortality and SAE data from all available 
follow-up data, but we expect most treatment-related safety events to occur within the first three months of 
follow-up. We will conduct formal safety evaluation at 6, 12, 18, and 24 months following the start of 
enrollment, where approximately 25, 50, 75, and 100% of the study cohort will have been randomized and 
followed for at least 3 months. As part of each interim analysis, we plan to monitor mortality as a primary 
safety endpoint and will control the overall significance level using O’Brien-Fleming boundaries (net alpha=0.05 
significance, accounting for three interim and one final analysis). The DSMB will also monitor all other SAEs 
utilizing the same O’Brien-Fleming sequential monitoring boundaries (Table 6) without further adjustment for 
multiple comparisons but allowing for flexibility to continue the study if the O’Brien-Fleming boundary is 
reached on a secondary SAE endpoint. 
Table 6. O’Brien-Fleming Monitoring Boundaries 
Enrollment 
Monitoring p-value: Death 
25% 
0.000014734 
50% 
0.0030359 
75% 
0.016248 
100% 
0.030701 
 
Data Safety Monitoring Schedule: Our target enrollment is n=500 which is expected to accrue during the first 
4.0 years of the trial. Therefore, we will enroll approximately 83 subjects every 6 months. Our planned DSMB 
safety analyses will occur every 6 months after trial initiation. At the first safety evaluation, we expect to have 
83 subjects enrolled, but would only have discharge outcomes on the first 60 subjects (Table 7). Each 
subsequent 6-month period would increase the number of babies by 83 leading to the following cumulative 


 
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number of subjects available for analysis. Safety evaluation will be based on all available follow-up, but we 
expect most SAEs to occur during the neonatal hospitalization and therefore within one month of age (2-4 
weeks) in most cases. 
Table 7. Cumulative number of subjects evaluated for safety events and for the long-term efficacy 
outcome. 
Month  
6 
12 
18 
24 
30 
36 
42 
48 
54 
60 
66 
Safety (1 mo) 
60 
143 
226 
309 
392 
475 
500 
 
 
 
 
Efficacy (24 mo)  
0 
0 
0 
0 
60 
143 
226 
309 
392 
475 
500 
 
 
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