Darbepoetin Trial to Improve Red Cell Mass and Neuroprotection in Preterm Infants
Phase: PHASE3
Conditions: Neurocognitive, Neuroprotective, Neonatal, Neurodevelopmental Impairment
Sponsor: NICHD Neonatal Research Network

1 
Darbepoetin Trial to Improve Red Cell Mass and 
Neuroprotection in Preterm Infants 
 
IND Number: 
100138 
 
ClinicalTrials.gov number: 
NCT03169881 
 
NRN ID number:  
NICHD-NRN-0058 
 
Lead Study Investigator: 
Robin Ohls, University of Utah 
 
Subcommittee Members:  
Kurt Schibler, Cincinnati Children’s Hospital Medical Center 
Jean Lowe, University of New Mexico 
Ed Bell, University of Iowa 
Seetha Shankaran, Wayne State University 
Abbot Laptook, Brown University 
David Carlton, Emory University 
Mariana Baserga, University of Utah 
John Flibotte, Children’s Hospital of Pennsylvania 
Abhik Das, RTI 
Andrew Bremer, NICHD 
Coordinators: Sandra Beauman and Cathy Grisby 
 
Version Date: 
2-25-19 Version 8 
 
Funding: 
NHLBI 1U01HL136318-01 (DCC 1U01HL136306-01) 
 
 

2 
Contents 
 
Section 1. 
Abstract ................................................................................................................... 3 
Section 2. 
Conflict of Interest Disclosures ............................................................................... 4 
Section 3. 
Statement of Problem ............................................................................................. 4 
3.1. 
Primary Hypothesis ..................................................................................... 4 
3.2. 
Secondary Hypotheses ................................................................................ 4 
3.3. 
Background and Rationale .......................................................................... 4 
Section 4. 
Methods ................................................................................................................ 14 
4.1. 
Study Population ....................................................................................... 14 
4.1.1. Inclusion Criteria ........................................................................... 14 
4.1.2. Exclusion Criteria........................................................................... 14 
4.2. 
Detailed Study Procedures ....................................................................... 14 
4.2.1. Screening ....................................................................................... 14 
4.2.2. Consent ......................................................................................... 14 
4.2.3. Randomization .............................................................................. 15 
4.2.4. Study Intervention and Comparison ............................................. 15 
4.2.5. Blinding/Masking .......................................................................... 18 
4.2.6. Control or Monitoring of Co-interventions................................... 18 
4.2.7. Primary Outcome .......................................................................... 18 
4.2.8. Secondary Outcomes .................................................................... 18 
4.2.9. Safety Outcomes ........................................................................... 18 
4.2.10. Compliance Monitoring ................................................................ 18 
4.2.11. Study Specimens ........................................................................... 19 
4.2.12. Post-hospital Procedures .............................................................. 19 
4.2.13. Follow-up at 24 Months ................................................................ 19 
4.2.14. Additional Follow-up Assessments ............................................... 20 
4.3. 
Potential Risks and Benefits to Subjects ................................................... 20 
Section 5. 
Analytical Plan ....................................................................................................... 21 
5.1. 
Statistical Analysis Plan ............................................................................. 21 
5.2. 
Sample Size and Power Estimates ............................................................ 22 
5.3. 
Available Population ................................................................................. 23 
5.4. 
Projected Recruitment time ..................................................................... 23 
5.5. 
Study Monitoring Plan .............................................................................. 23 
5.5.1. Reporting Adverse Events ............................................................. 23 
Section 6. 
References ............................................................................................................ 26 
 

3 
SECTION 1. 
ABSTRACT 
Study Hypothesis: Preterm infants administered weekly Darbe during the neonatal period will have 
improved neurocognitive outcome at 22-26 months compared to placebo  
 
Study Design Type: Randomized, masked, placebo-controlled trial 
 
Eligibility Criteria: Preterm infants less than or equal to 24 hours of age born 23-28 6/7 weeks gestation 
with hematocrit ≤ 60% are eligible for enrollment.  Infants with known chromosomal anomalies, known 
brain or cardiac anomalies, hemorrhagic or hemolytic disease, clinical seizures, congenital thrombotic 
disease, currently on Epo or Darbe, or systolic blood pressures >100 mm Hg while not on pressor 
support will be excluded.    
 
Study Intervention/Methods: Enrolled infants will receive weekly Darbe or placebo (sham) dosing. 
Laboratory tests will be performed on all infants while in the hospital.  Transfusions will be administered 
via protocol.  Neurodevelopmental assessment will be performed at 22-26 months.   
 
Primary Outcome: Composite cognitive score on Bayley Scale of Infant Development III.   
 
Secondary Outcomes: Hematocrit, red cell mass, donor exposures, number and volume of transfusions, 
hospital days, differences in morbidities (ROP requiring intervention, thromboses, hypertension, 
seizures, ICH, NEC requiring surgery, BPD, culture positive sepsis), moderate and severe 
neurodevelopmental impairment (NDI), death, NDI or death, and cerebral palsy at 22-26 months. The 
Data Safety and Monitoring Committee of the NRN, with approval of the Division of Blood Diseases 
and Resources of NHLBI, will oversee the study. The stopping rules will be set before the study 
begins. 
 

4 
SECTION 2. 
CONFLICT OF INTEREST DISCLOSURES 
 
Financial Conflicts of Interest of the Institutions and Investigators: None identified 
 
Plan for Managing Identified FCOIs  
 
 
SECTION 3. 
STATEMENT OF PROBLEM 
3.1. 
PRIMARY HYPOTHESIS 
 
Preterm infants administered weekly Darbe starting within 36 hours of birth and continuing to 
35 weeks gestational age will have improved neurocognitive outcome at 22-26 months (with 
deaths assigned the lowest possible score of 54), compared to placebo recipients. 
 
3.2. 
SECONDARY HYPOTHESES 
 
1. Preterm infants administered weekly Darbe during the neonatal period will have increased red 
cell mass, decreased transfusions, decreased donor exposures, and decreased volume of 
transfused red cells compared to placebo infants 
2. Preterm infants administered weekly Darbe during the neonatal period will have improved 
survival without neurodevelopmental impairment compared to placebo infants 
3. Preterm infants with peak serum Epo concentrations ≥500mU/mL will have better 
neurocognitive development than those who have peak serum Epo concentrations <500mU/mL 
4. Preterm infants administered weekly Darbe will have a decreased incidence of cerebral palsy 
compared to placebo infants 
5. Preterm infants administered weekly Darbe who receive at least 1 transfusion will have 
improved cognitive outcome compared to placebo infants who receive at least 1 transfusion  
 
3.3. 
BACKGROUND AND RATIONALE 
Advances in neonatal care have led to significant improvements in the survival of the nearly 60,000 very 
low birth weight (VLBW) infants born each year in the U.S.1,2  Improving neurodevelopmental outcomes 
for these preterm infants continues to be a major goal for neonatal care providers.  A subset of these 
infants sustain a grade 3 or 4 intraventricular hemorrhage (IVH) resulting in an increase in the incidence 
of developmental delay. Moreover, almost one third of preterm infants with normal head ultrasounds 
also develop cognitive delay.3,4  Although a variety of neuroprotective treatment strategies have been 
evaluated, no specific treatment has been identified to reduce or prevent brain injury in these most 
vulnerable preterm infants. 
A potential neuroprotective therapy involves administering erythropoiesis stimulating agents (ESAs) 
such as erythropoietin (Epo) and Darbepoetin (Darbe, a longer acting ESA). In addition to stimulating 
erythropoiesis, ESAs have been shown to be protective in the developing brain in animal models,5-9 

5 
making it possibly beneficial for very premature infants who are at risk for intraventricular hemorrhage, 
hypoxic-ischemic injury, and developmental delay.  The neuroprotective mechanisms of ESAs include 
increased neurogenesis,10 decreased neuronal susceptibility to glutamate toxicity,11,12 decreased 
neuronal apoptosis,13-17 decreased inflammation,18,19 decreased nitric oxide-mediated injury,20-22 
increased antioxidant response,23-25 decreased axonal degeneration,26 and increased protective effects 
on glia.27-29   
We evaluated hematologic and neurodevelopmental outcomes at 18 – 22 months corrected age in a 
multicenter trial of preterm infants randomized to receive Epo, Darbe, or placebo for the first 10 
postnatal weeks (Clinical Trials #NCT00334737; IND#100138).  During the hospital phase ESA treated 
infants received fewer transfusions and were exposed to fewer donors29a.  During the follow up phase 
ESA treated infants had significantly higher composite cognitive outcomes and lower rates of 
neurodevelopmental impairment.29b Hematocrits were higher in the ESA treated group, and cognitive 
scores inversely correlated with red cell transfusion volumes. Developmental assessment at 3.5-4 years 
of age continued to show improved cognitive outcomes in ESA recipients, including higher scores of 
executive function.  We previously published data30 suggesting a relationship between serum Epo 
concentrations and cognitive outcomes in ELBW infants.  Positive correlations between peak and trough 
ESA concentrations and full scale IQ remained significant at 4 years.  In addition, preliminary data from 
our laboratory show a neurotropic effect of Darbe that may exceed Epo.  Based on preliminary 
pharmacokinetic and erythrokinetic data on the administration of Darbe to preterm infants, we propose 
a randomized, masked, controlled study to evaluate effects on red cell mass and developmental 
outcomes of preterm infants receiving standard hematopoietic doses of Darbe.   
Erythropoiesis Stimulating Agents (ESAs) 
Darbepoetin alfa  
A modified version of Epo, known as darbepoetin alfa (Darbe), was developed by Amgen in 1998.33, 34 
Darbe has a longer serum half-life than Epo, allowing for increased dosage intervals to be used. Studies 
using Darbe once a week to once every three weeks (as compared to 3 doses per week of Epo) in adult 
oncology and end stage renal failure patients have demonstrated hemoglobin increases equivalent to 
that of Epo.35, 36 Side effects are similar to Epo, and the production of anti-Epo antibodies has not been 
reported.37  Some hospital formularies are moving from one ESA, Epo, to the newer long-acting ESA, 
Darbe.  Given its potential for similar efficacy with fewer injections, we considered Darbe to be a 
beneficial alternative to Epo to prevent anemia in preterm infants.  Following our in vitro evaluation of 
fetal and neonatal erythroid progenitor responsiveness to Darbe36, 38, we evaluated erythrokinetics and 
pharmacokinetics of Darbe administered to preterm infants.39-42 Compared with adults, neonates had 
more rapid clearance, suggesting that dosing in neonates would require a higher unit dose/kg and a 
shorter dosing interval than is generally used for adult patients.  Preterm infants showed a dose-
response relationship to Darbe that closely mimicked erythropoietic doses of Epo in preterm infants.41,42 
ESAs as Neuroprotectants 
The initial enthusiasm for Epo came from its effect on erythropoiesis in premature infants. Recently, in 
vitro, animal, and adult clinical studies suggest that Epo plays a potentially important role in 
angiogenesis and neurogenesis in the central nervous system.43, 44  It is now known that Epo and Epo 
receptors are present in non-hematopoietic developing neural tissue in animals as well as humans.45-47  
Similar to the mechanism leading to erythropoiesis, Epo binds to its receptor in non-hematopoietic 
tissues, activating cellular mechanisms that include cell maturation, division, and importantly, inhibition 
of apoptosis.48, 49 Studies evaluating Epo in adult and neonatal animal models report the prevention of 
hypoxic-ischemic brain injury, a decrease in infarction volume, reduced vasoconstriction, decreased 
neuronal apoptosis, and decreased neurologic deterioration in animals treated with high doses of Epo.14, 

6 
48-53 The mechanisms by which Epo is neuroprotective are under active investigation.  It has been shown 
that neuroprotection in part involves decreased expression of pro-inflammatory and anti-apoptotic 
genes of injured brain.54 Epo has protective effects both in the central nervous system and the 
peripheral nervous system.55, 56 These broad effects suggest that Epo may have multiple mechanisms of 
protection, some that affect neurons directly, and others that are likely secondary to the effects of Epo 
on other cells or systems. To date, Epo has been shown to increase neurogenesis,10 decrease neuronal 
susceptibility to glutamate toxicity,11,12 decrease neuronal apoptosis,13-17 decrease inflammation,18, 19 
decrease nitric oxide-mediated injury,20-22 increase antioxidant response,23-25 prevent axonal 
degeneration,26 and increase protective effects on glia.27-29 Epo may also provide neuroprotection by 
regulating blood flow to the brain following injury, as is suggested by Epo neuroprotection in the model 
of subarachnoid hemorrhage.57, 58 Jantzie et al showed that Epo signaling promotes critical stages of 
oligodendroglial lineage development and recovery after prenatal injury, and concluded that Epo 
treatment may be beneficial to preterm patient populations with developmental brain injury hallmarked 
by white matter injury.79 In addition, MRIs performed at 36 weeks corrected age in former preterm 
infants randomized to 3 high doses of Epo (3000 units/kg IV at 3 hours, 12-18 hours, and 36-42 hours of 
age) or placebo (Swiss high dose Epo study) revealed decreased white matter and grey matter injury in 
the Epo recipients,80 and improved fractional anisotropy.80a Developmental outcomes of the infants 
enrolled in that study remain to be determined. 
Human clinical trials of Epo for treatment of adult stroke have been performed.  Reduced stroke lesion 
size and evolutions as well as decreased serum markers of glial damage were seen in adults treated with 
Epo,59 however, in a larger RCT60, increased mortality was noted (16.4% in Epo-treated subject 
compared to 9.0% in control patients).  The FDA placed a hold on high dose Epo studies evaluating 
neuroprotective effects in 2007 to more closely evaluate initial clinical studies.  Exploratory subgroup 
analysis of the RCT revealed that patients not receiving thrombolysis most likely benefited from EPO 
during clinical recovery.  The glial markers S100B and glial fibrillary acid protein (GFAP) and the neuronal 
marker ubiquitin C-terminal hydrolase (UCH-L1) were measured by enzyme-linked immunosorbent assay 
in serum on d 1, 2, 3, 4 and 7 post-stroke. All biomarkers increased post-stroke. Overall, EPO-treated 
patients had significantly lower concentrations (area under the curve) over 7 d of observation, as 
reflected by the composite score of all three markers (Cronbach α = 0.811) and by UCH-L1. S100B and 
GFAP showed a similar tendency78.  The FDA hold on ESA studies for neuroprotection was removed in 
2008 and clinical studies were allowed to move forward.  No RCTs evaluating Epo in preterm or term 
neonates have shown a difference in mortality. 
Our group was the first to report potential neurodevelopmental improvements in Epo-treated ELBW 
infants30 (reviewed in the preliminary studies section). Two other studies have also reported improved 
outcomes in Epo-treated preterm infants.  Brown et al evaluated outcomes of 82 infants <1500 g and ≤ 
30 weeks of gestation, evaluated at 2 years after neonatal Epo treatment.61 Higher MDI scores were 
associated with higher cumulative doses of Epo, among other factors.  Neubauer and colleagues 
compared 89 Epo-treated with 57 control ELBW neonates at 10-13 years of age.62 Epo-treated neonates 
had better outcomes, with 55% of the Epo group assessed as normal compared to 39% in the control 
group (p<0.05). IQ scores were also higher in Epo treated patients (90.8 compared to 81.3 in control 
infants, p< 0.005), especially in those infants with grade III-IV IVH. 
In addition to hematopoiesis, Epo is also a vascular growth factor.  Epo may play a role in the developing 
human eye, as increased vitreal Epo concentrations have been measured that exceed serum 
concentrations.63  Retrospective analyses and meta-analyses have raised concerns about an association 
between retinopathy of prematurity (ROP) and early Epo administration.64  However, no randomized 
controlled trials (RCTs) published in peer-reviewed journals have reported an increased incidence of ROP 

7 
in the Epo-treated group, and the most recent meta-analysis of early Epo studies (starting Epo in the 
first week of life) show no statistical increase in stage 3 or greater ROP.64 Brown and colleagues 
performed a retrospective analysis of Epo dose and ROP on the same data base of infants that showed 
improved MDI, reporting an increase in ROP with increased total dose.65  In contrast, Neubauer and 
colleagues62 reported the incidence of ROP ≥stage III was 11.4% in Epo recipients versus 16.1% in the 
control group (p=NS).  In the Swiss high dose Epo study, there were no differences in any level of ROP 
between the Epo treated and placebo groups,65a nor were there differences in intracranial hemorrhage 
(any grade) or cystic PVL.  The incidence of ROP in Epo-treated infants continues to be closely evaluated.    
Darbe as a Potential Neuroprotectant 
Recent studies of ESA neuroprotection have evaluated Darbepoetin.66,67  Both Epo and Darbe cross the 
blood-brain barrier at a similar rate as albumin, suggesting that they cross the blood-brain barrier by 
way of extracellular pathways.66 In an adult animal model of ischemia, Darbe conferred behavioral and 
histological neuroprotection after focal ischemia.67  In those animal studies, the darbepoetin doses 
tested were identical to the doses used in our previous RCT (10 g/kg/dose), increasing the probability 
that preterm infants randomized to the Darbe arm are indeed receiving neuroprotective doses.  In 
addition, a study on neuroprotection in term infants with hypoxic ischemic encephalopathy treated with 
Epo showed promise in decreasing neurodevelopmental morbidity to a similar extent as that seen in 
recent cooling trials.68  Epo doses in that term infant study (300-500 units/kg every other day) were 
comparable to doses used clinically in preterm infants to stimulate erythropoiesis.   
Recent clinical studies in term infants with HIE treated with high dose Epo (500-2,500 units/kg/dose) 
showed equal efficacy at 1,000 and 2,500 units/kg/dose (NEAT Study69), and phase III trials are 
proceeding at 1000 units/kg.  Similarly, studies of prophylactic high dose Epo for neuroprotection in 
ELBW infants reported area under the curve (AUC) values at 1,000 units/kg/dose that were similar to 
neuroprotective AUC values in animal studies.70, 71  
The neurodevelopmental consequences of extreme prematurity are enormous. Unfortunately, there are 
no proven neuroprotective treatments available for these children. Developing and evaluating novel 
neuroprotective strategies is a critical need in neonatal care with tremendous public health implications. 
Converging lines of evidence from both animal and human studies demonstrate the potential for ESAs as 
neuroprotective agents.  Mechanisms of neuroprotection may include improved red cell mass, increased 
oxygen availability to tissues, and decreased adverse impact of transfusions, in addition to the biologic 
mechanisms of inhibition of apoptosis and increased cell differentiation. Although the safety and 
beneficial effect of ESAs on development is supported by preclinical and clinical studies, a large RCT 
evaluating long term neurodevelopmental effects of Darbepoetin in premature infants has not been 
performed. Our randomized, controlled investigation is powered to answer the question: Do ESAs 
improve clinically relevant developmental functions? Our primary outcome measures are well 
standardized and have functional significance. This study differs from previous studies in the following 
ways: 
1. Darbe will be compared to weekly placebo, (those in the placebo group will not receive injections; 
masking will be maintained by administering drug/placebo behind a curtain and placing a Band-Aid) 
2. Dosing will begin at least 1 day earlier on average than in any previously published study – this is 
potentially important in terms of timing for neuroprotection 
3. Correlations will be performed to better understand the relationship between Darbe administration, 
transfusions, red cell mass, serum Darbe (Epo) concentrations, and cognitive development at 2 years 
of age. 

8 
Preliminary Translational Laboratory Studies 
1. Erythropoietin and Darbepoetin Increase Fetal 
Erythropoiesis in Dose Dependent Fashion: Erythroid 
progenitor cells were isolated from 12-22 week fetal liver and 
marrow, and from term (37-41 weeks) and preterm (<32 
weeks) cord blood.74  The number of burst forming units-
erythroid (BFU-E) colonies derived from fetal marrow 
progenitor cells increased significantly with both Darbe 
(p<0.01, 10 vs 50, 100, and 500 ng/mL; Figure 1) and rHuEpo (p 
<0.01, 0.05 vs 0.5, 1.0, and 2 U/mL).  Progenitors isolated from 
fetal liver and from term and preterm cord blood were 
similarly responsive.  Preterm cord blood progenitors were 
more sensitive than term progenitors to Darbe at every 
concentration (p<0.01). 
 
Figure 1. Comparison of Epo versus Darbe in growth of 
human erythroid progenitors isolated from marrow. 
2. Erythropoietin and Darbepoetin Increase Fetal Neurogenesis in Dose 
Dependent Fashion: We performed in vitro studies of Darbe and Epo on 
human fetal neuronal cell cultures.73 Cells were incubated with Epo (0-10 
units/mL) or Darbe (0-1000 ng/mL) for 7 to 10 days, and cell count and 
phenotype determined (Figure 2, right).  Pre-clinical and clinical studies 
performed by Amgen (the manufacturer of Epo and Darbe) and 
performed by clinical investigators determined the conversion to achieve 
equivalent potency is: 1 unit Epo = 100 ng Darbe.  Based on this 
comparison, neuronal cells appeared 10-fold more responsive to Darbe 
than Epo in vitro, suggesting that for neurogenesis, 1 unit Epo would be 
comparable to 10 ng Darbe (therefore 1,000 units Epo would compare to 
10 micrograms Darbe).  Both Darbe and Epo increased Epo receptor gene 
 
Figure 2. Comparison of Epo versus 
Darbe in growth of mid-gestation human 
fetal brain cells.  
expression (greater with Darbe), and increased expression of the anti-apoptotic gene Bcl.  The 
phenotype of cultured cells did not differ between Darbe and Epo; however nesting gene expression 
was four-fold greater in Darbe-cultured cells than Epo cultured cells, suggesting increased progenitors.  
 
Preliminary Clinical Studies  
1. Short term developmental outcome of ELBW infants treated with early Epo and iron: Working with 
NRN Centers, we performed a follow-up study of ELBW infants originally enrolled in the NICHD Epo 
Study73. There were no significant differences between treatment groups in weight or length, or in the 
percentage weighing <10th percentile at the time of discharge or at 18-22 months (corrected age)  
follow-up (Table 1), and no difference was found in the 
mean head circumference between groups (Table 1, right).  
However, there was an important difference in the 
incidence of microcephaly between groups. Over twice as 
many children developed microcephaly in the placebo 
group compared to those treated with Epo (32.7% vs 
14.3%). To understand this difference, longer term studies 
that include markers of neuroplasticity will be required.  
Table 1. NICHD Follow up Study 
Parameter 
Epo 
(n=51) 
Placebo 
(n=51) 
weight (kg) 
9.8±1.3 
10.0±1.5 
length (cm) 
80.2±3.7 
80.8±3.3 
HC (cm) 
47.0±2.0 
46.6±1.7 
% Wt < 10%tile* 
49.0 
50.0 
% Ln < 10%tile* 
40.8 
36.5 
% HC < 10%tile* 
14.3 
32.7 
   * National Children’s Health Study guidelines 

9 
2. Developmental outcome correlates with neonatal Epo concentration: we evaluated the relationship 
between Epo concentrations and neurodevelopmental outcome in the subgroup of infants (those from 
the University of New Mexico) enrolled in the NICHD Epo study (summarized above).30  The investigators 
responsible for neurodevelopmental evaluation were masked to the treatment groups.  Serum Epo 
concentrations were significantly different between groups during the study (Table 2).  At 18-22 months 
follow up, Epo recipients (n=7) had BSID II MDI scores of 96±11 and placebo infants (n=8) had MDI 
scores of 78±7 (p=0.15).  There were no differences between groups in anthropometric measurements. 
Two of six infants in the Epo group and 4 of 6 infants in the placebo group had some form of NDI.  Post 
hoc analysis showed that infants with peak Epo concentrations ≥500 mU/mL (Figure 3) had higher MDIs 
(100±15) than infants with Epo concentrations <500 mU/mL (77±16; p<0.05). This study was the first to 
demonstrate that elevated serum Epo concentrations to levels studied in animal models of 
neuroprotection can be achieved using subcutaneous Epo at doses of 400 units/kg 3x/week, which is 
considered a normal “hematopoietic” dose.  Based on this information, we chose Darbe doses that 
would also achieve Epo concentrations ≥500mU/mL.  
 
Table 2. Epo concentrations and developmental testing 
 
Epo (n=7)  
Placebo (n=8) 
Epo conc.  
2,023±652 mU/mL 
22±4 mU/mL 
MDI scores 
96±11 
78±7 
PDI scores 
87±13 
80±7 
Any NDI 
2/6 
4/6 
Figure 3. MDI scores at 18-22 months follow up.  MDI 
scores were higher in infants with peak Epo 
concentrations ≥500 mU/mL (right) compared to 
those with concentrations <500 mU/mL (left; p<0.05).  
 
3. Randomized, masked, dose response study of Darbepoetin in preterm infants: Following single dose 
pharmacokinetic studies of Darbe administered to VLBW infants,39, 40 we completed a randomized, 
masked, dose response study of Darbe in preterm infants ≤1,500 grams birth weight.74  Sixteen Infants 
were randomized to one of the following doses: 0, 2.5, 5.0, or 10 g/kg/dose given once a week SC for 4 
doses. We saw a clear dose response relationship in production of reticulocytes (Figure 4), with the  
greatest response occurring in infants receiving 10 
g/kg dose.  No adverse effects were reported in any 
of the infants.  The results demonstrated preliminary 
safety and efficacy, and confirmed the findings of our 
pharmacokinetic studies, in that higher doses (than 
are used in adults for erythropoiesis) resulted in a 
robust biologic response without adverse effects. A 
Darbe dose of 10 g/kg SC will generate serum Epo 
concentrations well above 500 mU/mL, based on 
single 4 g/kg SC dose achieving concentrations of 
2,000-4,000 mU/mL.39 
 
Figure 4. Absolute retic counts (ARC) in preterm infants 
treated with 0-10 g/kg doses of Darbe for 4 weeks; 
*p<0.05 versus 0 and 2.5 g/kg. 
 
4. 
Neurocognitive Outcomes at 18-22 Months are Improved in Former Preterm Infants Administered 
Darbepoetin or Erythropoietin: We evaluated infants at 18-22 months and hypothesized that those 
* 
ARC 
 (103/L) 
0
100
200
300
400
500
600
day 1
day 14
day 28
10.000
5.000
2.500
0.000
0 g/kg 
2.5 g/kg 
5.0 g/kg 
10.0 g/kg 
 
0
20
40
60
80
100
120
140
M DI
MDI 
<500          >500 
mU/mL     mU/mL 

10 
previously randomized to ESAs would have improved neurodevelopmental outcomes compared to 
placebo.76, 77  A total of 102 Infants (500-1,250 grams, <48 hours of age) were randomized in masked 
fashion to Darbe (10 g/kg, 1x/wk SC), Epo (400 units/kg, 3x/wk SC) or placebo, dosed through 35 weeks 
postmenstrual age. All infants received supplemental iron, folate, and vitamin E, and were transfused 
according to a standardized, restrictive transfusion protocol. During the treatment phase, ESA recipients 
received half the number of transfusions (1.3 versus 2.6) and were exposed to half the donors (0.7 
versus 1.4) than infants in the placebo group.  The incidence of morbidities, specifically ROP ≥ stage 3, 
either requiring surgery (2, 1, and 2 infants in Darbe, Epo, and placebo groups, respectively) or regressed 
(0, 1, and 2 infants in Darbe, Epo, and placebo groups, respectively), was similar in all three groups. 
Infants were evaluated at 18-22 months corrected age using the Bayley Scales of Infant Development 
(BSID-III). Object permanence (OP, a measure of executive function and early working memory) was 
calculated from the BSID-III. Anthropometrics and assessment of cerebral palsy (CP), blindness and 
deafness were determined.  Of the original 102 infants (946±196 grams, 27.7±1.8 weeks gestation) 
enrolled, 99 were evaluated during the hospital phase, 5 died prior to discharge (1/33 Darbe, 1/33 Epo, 
3/33 Placebo) and 14 infants were lost to follow-up (5 Darbe, 3 Epo, 6 placebo). The 80 infants evaluated 
(29 Epo, 27 Darbe, 24 placebo) were comparable between groups for age at testing, birth weight, 
gestational age, antenatal steroids, and morbidities associated with prematurity (ICH, BPD, and ROP). 
Anthropometrics did not differ among groups.  
 
 
Table 3. Neurodevelopment and neurodevelopmental impairment at 18-22 months 
 
After adjustment for gender, analysis of covariance resulted in significant differences between groups 
(Table3): cognitive scores were similar for Darbe (96.2±7.3; mean±SD) and Epo (97.9±14.3) compared to 
placebo (88.7±13.5; p=0.01 vs ESA recipients; Table 4). OP was higher for the Darbe group compared to 
the Epo group (p=0.05); both Epo and Darbe groups had higher OP scores than the placebo group 
(p=0.01). None in the ESA groups had CP, compared with 5 in the placebo group (p=0.002). No 
differences between groups were noted in blindness or deafness, and there were no infants in the 
Darbe group with a cognitive score <85. Infants receiving ESAs showed improved cognitive outcomes 
compared to placebo at 18-22 months. The differences in incidence of CP (22% placebo versus 0% Darbe 
or Epo) might be explained by potential white matter neuroprotection of ESAs,80 but this hypothesis 
remains to be tested.  
 

11 
5. 
Preschool Neurocognitive Outcomes in Preterm Infants Administered ESAs: We enrolled 
children who completed the original RCT into the BRITE Study (Brain Imaging and Developmental Follow 
up of Infants Treated with Erythropoietin, NCT#01207778), and compared developmental outcomes 
with children born healthy at term (TC).  All participants were assessed using measures of full scale IQ 
(FSIQ) and general language from the Wechsler Preschool and Primary Scale of Intelligence-III, and an 
overall measure of executive function, based on tests evaluating inhibitory control and spatial working 
memory. Rates of neurodevelopmental impairment were compared across groups.  Multivariate analysis 
of variance compared children randomized to ESAs (39), placebo (14), and TC (24). FSIQ and 
performance IQ (PIQ) were significantly higher in the ESA group compared to the placebo group (Table 
4). Follow-up analyses revealed that the children receiving ESAs performed better than placebo on 
executive function tasks. ESA group’s performance was below that of TC, but results did not reach 
significance on executive function. Neurodevelopmental impairment was lower in the ESA than the 
placebo group (p=0.016). 
Table 4. Neurodevelopment in Preschool Children  
 
ESA 
Placebo 
Term 
P0 
P1 
Subjects — no. 
39 
14 
24 
 
 
Full-scale IQ 
91.49 (18.05) 
79.14 (18.53) 
102.58 (12.78) 
0.034 
0.011 
   Verbal IQ  
92.97 (17.00) 
79.07 (19.57) 
103.92 (10.55) 
0.015 
0.006 
   Performance IQ  
91.69 (18.30) 
82.86 (16.90) 
100.83 (15.20) 
0.12 
0.071 
General Language 
Comprehension 
89.82 (16.98) 
84.79 (17.11) 
101.83 (15.11) 
.35 
.006 
Executive function 
99.88 (11.65) 
91.52 (13.26) 
105.15 (7.79) 
0.035 
0.083 
   Working Memory 
100.63 (14.58) 
91.57 (17.24) 
103.92 (12.44) 
0.047 
0.35 
   Inhibition 
99.13 (15.21) 
91.53 (17.61) 
106.36 (9.95) 
0.11 
0.066 
Values represent mean standard deviation;  
P0: treated vs placebo; P1: treated vs term  
 
 
 
 
 
6. 
Neurocognitive Outcomes in Preterm Infants Administered ESAs in Relation to 
Transfusion Number and Volume: It is unclear if the improved cognitive outcomes seen in the ESA 
recipients are a result of neuroprotective effects of ESAs, improved oxygenation due to higher 
hematocrit in ESA recipients, decreased neuronal injury or morbidity due to fewer transfusions, or some 
combination of the three.  We analyzed our transfusion and cognitive outcomes data to identify 
relationships between transfusions and cognitive outcomes.  We hypothesized that cognitive scores 
would be inversely correlated with the number or volume of red cell transfusions received during the 
initial hospitalization.   
Both composite cognitive scores and 
social emotional scores were inversely 
correlated with the volume of red cell 
transfusions (Figure 5, A (left) and B 
(right)).  Moreover, there was a trend 
towards an inverse correlation 
between cognitive score and number 
of transfusions (p=0.08).  
 
Figure 5A. Cognitive score at 2 years vs 
transfusion volume  
 
R=0.26; p=0.02 
 
Figure 5B. Social Emotional score at 
2 years vs transfusion volume  
 
R=0.22; p=0.05 
 
50
60
70
80
90
100
110
120
130
cognitive
-20
0
20
40
60
80
100 120 140 160 180
volume
60
70
80
90
100
110
120
130
140
150
se
-20
0
20
40
60
80
100
120 140
160 180
volume

12 
Significant differences were 
seen in cognitive and language 
scores between ESA treated and 
placebo groups when ≥1 
transfusions were administered; 
these differences were not 
present in infants who did not 
receive transfusions (Figure 6, A 
(left) and B (right) panels). At 4 
years of age, the impact of 
transfusions was still present 
but to a lesser extent: 
differences in full scale IQ 
between non-transfused ESA 
and non-transfused placebo 
trended towards higher scores 
in the ESA treated group 
(p=0.07). 
 
 
 
Figure 6A. Cognitive scores in ESA 
treated (blue) versus placebo (red), 
untransfused versus 1 or more 
transfusions. 
 
 
 
 
 
Figure 6B. Language scores in ESA 
treated (blue) versus placebo (red), 
untransfused versus 1 or more 
transfusions. 
We speculate that transfusions negatively impact cognitive development, and that ESAs provide 
neuroprotection from these effects. 
 
7. 
Epo concentrations correlate with cognitive score at 2 and 4 years: Epo concentrations were 
measured during the hospital phase.  We hypothesized that neurodevelopmental outcomes at 2 and 4 
years of former preterm infants previously randomized to ESAs would be positively correlated with peak 
Epo concentrations.  Darbe recipients had the highest peak and trough concentrations (Figure 8, left 
panel: * p<0.05, ESA vs placebo; § p<0.05 Darbe vs Epo).  Cognitive scores significantly correlated with 
trough concentrations (Figure 8, right panel: p<0.05), and there was a trend towards correlation 
between cognitive scores and peak Epo concentrations (p=0.08).  
 
 
Figure 8.  Epo concentrations (left) and relation of Epo trough to cognitive outcome (right) at 2 years 
 
At 4 years of age, full scale IQ (Figure 9, A and B) and performance IQ (Figure 9, C and D) correlated with 
peak Epo concentrations (Figure 9, A and C), and with trough concentrations (Figure 9, B and D). 
 
 
 
 
50
60
70
80
90
100
110
120
130
cognitive
no
yes
placebo
ESA
40
50
60
70
80
90
100
110
120
130
no
yes
placebo
ESA
-100
0
100
200
300
400
500
Concentration
50
60
70
80
90 100 110 120 130
composite cognitive
No Tx 
≥1 Tx 
Cognitive Score 
P=0.76 
P=0.003 
0
200
400
600
800
1000
1200
1400
1600
Baseline
Peak
Trough
* § 
* * 
* 
Epo 
(mU/mL) 
Epo 
Darbe 
Placebo 
Baseline 
Peak 
Trough 
No Tx 
≥1 Tx 
Language Score 
P=0.62 
P=0.03 

13 
Figure 9.  Epo Concentrations and Cognitive Outcome at 4 Years 
A 
B 
C 
D 
 
 
Summary and Interpretation of Preliminary Studies 
Preliminary studies suggest that ESAs improve short term and preschool neurodevelopmental outcome 
in premature infants. Our prior work also indicates that Darbe appears safe within established dosing 
guidelines, and that the doses of Darbe administered should be adequate to achieve serum Epo 
concentrations ≥500 mU/mL. Before treatment recommendations can be made however, a well 
designed RCT is needed to evaluate the effect of ESAs on various cognitive domains. Furthermore, 
understanding the neurologic mechanisms affected by ESAs in former premature infants is necessary to 
determine how best to modify therapy to achieve optimal neurodevelopmental outcome.  The NICHD 
Neonatal Research Network has the necessary neurodevelopmental testing expertise to investigate 
potential neurologic effects in former preterm infants. 
 
 
 
 
Peak Epo Concentration 
P=0.017 
Full 
Scale 
IQ 
Trough Epo Concentration 
P=0.032 
Full 
Scale 
 IQ 
PIQ 
P=0.039 
Trough Epo Concentration 
Peak Epo Concentration 
P=0.008 
PIQ 
 

14 
SECTION 4. 
METHODS 
4.1. 
STUDY POPULATION 
This is a randomized, masked, placebo controlled clinical study in which enrolled infants will receive 
weekly Darbe or placebo (sham) dosing.  
 
4.1.1. Inclusion Criteria 
• 
23 0/7-28 6/7 weeks gestation  
• 
≤24 hours postnatal age   
 
4.1.2. Exclusion Criteria 
• 
Hematocrit > 60% 
• 
Infants with known congenital or chromosomal anomalies, including congenital heart disease 
and known brain anomalies  
• 
Hemorrhagic or hemolytic disease  
• 
EEG- confirmed seizures  
• 
Congenital thrombotic disease  
• 
Systolic blood pressures >100 mm Hg while not on pressor support 
• 
Receiving Epo or Darbe clinically, or planning to receive Epo or Darbe during hospitalization 
• 
Infants in whom no aggressive therapy is planned  
• 
Family will NOT be available for follow-up at 22-26 months  
• 
Infant unlikely to survive 
• 
Infant enrolled in a conflicting trial 
 
4.2. 
DETAILED STUDY PROCEDURES 
 
4.2.1. Screening 
Pregnant women will be screened for potential delivery between the GA of 23 0/7 – 28 6/7 weeks.  In 
addition, admissions to the NICU (inborn and outborn) with GA 23 0/7 – 28 6/7 weeks will be screened 
for inclusion/exclusion criteria. 
 
4.2.2. Consent  
Parents of infants with GA 23 0/7 – 28 6/7 weeks and who are not known to have any exclusion criteria 
may be approached for consent before delivery if allowed by the local IRB.  This will enable collection of 
cord blood at delivery.  Parents/guardians of infants who meet inclusion criteria and who do not have 
any exclusion criteria may also be approached for consent after delivery but must be randomized within 
24 hours. 
Consenting should include informing families that cord blood collected either at delivery or from stored 
samples, peak and trough samples, and scavenged samples will be sent to the DREAM Lab at UNM to be 
used for Epo (Darbe measures as Epo) concentrations (and antibody assay if needed).  In addition, 
various lab studies are included as part of the study design.  For families that wish to participate in the 
study but do not want research labs drawn (and for those sites where research labs exceed maximum 
research blood draw limits [4.0 ml]), only serum ferritin (to adjust iron dosing) and peak Epo 
concentration will be obtained (1.5 mL total blood volume).  Information from clinically-obtained 

15 
CBCs/hematology labs will be collected.  Serum or plasma remaining after clinical labs are run will be 
collected and stored (frozen at -80 degrees centigrade) during the study to measure random Epo 
concentrations, and in the event that antibody assays are run. Deidentified Epo concentration data will 
be sent to the University of Iowa for pharmacokinetic analyses by Dr. An.   
 
4.2.3. Randomization  
After parental consent is obtained, infants will be randomized through the Data Center at RTI in a 
permutated block design using a web based electronic data capture (EDC) system. Randomized subjects 
will be stratified by center and within each center by gestation (<26 weeks, 26-28 and 6/7 weeks). 
Multiple gestation infants will be randomized to the same treatment arm.    
 
4.2.4. Study Intervention and Comparison  
Study Drug:  Infants will be randomized to one of two groups: Darbepoetin 10 g/kg/once every week 
(IV or SC), or Placebo (equal volume normal saline for IV administration, or sham dosing) once a week. 
Where normal saline for placebo is not available, dextrose 5% in water for IV administration may be 
used. If the study drug is given IV, it will be given slow IV push. If the study drug is given SC, the study 
drug will be brought to the bedside in a masked container, and injections will be shielded behind screens 
and out of earshot from caregivers and parents.  An adhesive bandage or gauze will be placed over the 
true and sham injection sites, or gauze held until no evidence of an injection is visible.  For those infants 
with IV access, IV administration of Darbe or placebo (in equal volume) is allowed. Infants will be treated 
until 35 completed weeks gestation, discharge, or transfer to another hospital.  The first dose of study 
medicine will be administered as soon as possible, at the latest by 36 hours of age. 
 
Supplementation:  Infants will receive parenteral iron dextran or iron sucrose, 3 mg/kg once a week (at 
the end of the first week) while they are receiving <60 mL/kg/day in enteral feedings.  The parenteral 
iron will be added to TPN solution and run over a 24 hour period to ensure slow delivery, or run over 4 
hours separate from TPN.  Infants will receive oral iron (5 mg/kg/day elemental iron) when receiving 80-
120 mL/kg/day of enteral feedings.  Doses of all supplements will be recorded.  Infants will receive iron 
dosing regardless of receiving a PRBC transfusion.  Infants will receive study drug regardless of iron 
dosing (red cell production will continue whether iron stores are adequate or not, but production is 
more effective if iron stores are adequate). Additionally, Infants will continue to receive iron dosing 
regardless of whether study drug is withheld (iron is adjusted per algorithm below).  
 
Iron dose monitoring and adjustment: Monitoring for iron overload/insufficiency will occur during the 
study around days 14 and 42.  Additional labs will be sent for ferritin in those cases where ferritins fall 
below 50 or above 400.  Iron supplementation will be adjusted as follows:  
Ferritin <50 ng/mL: parenteral iron 4 mg/kg/week; OR oral iron 8 mg/kg/day, repeat ferritin in 2 weeks  
Ferritin 50-300: parenteral iron 3/mg/kg/week; OR oral iron 5 mg/kg/day 
Ferritin 301-400 ng/mL: parenteral iron 2 mg/kg/week; OR oral iron 4 mg/kg/day 
Ferritin >400 ng/mL: hold all iron, repeat ferritin in 2 weeks 
 
Iron doses are similar to the doses and dosing schedule of ELBW infants enrolled in the original NICHD 
Epo Study, and the recent ESA Study.  Ferritin concentrations in the ESA Study were used to adjust iron 
dosing.  A greater number of infants in the placebo group had their iron dose decreased at day 42 due to 
ferritin >400 (p=0.045) and a greater number of infants had their iron dose increased at day 14 
(p=0.024) and day 42 (p<0.001) in the treated groups.  There were no differences in incidence of late 
onset sepsis among groups.  A summary of iron dosing is shown in Table 5. 

16 
Table 5. Ferritin concentrations and iron dosing 
 
Darbe 
Epo 
Placebo 
P= 
Ferritin Day 14  
121 [63, 184] 
104 [58, 197] 
204 [114, 348] 
0.003 
Ferritin Day 42 
50 [27, 77] 
61 [33, 99] 
127 [67, 216] 
0.002 
Number of IV iron doses 
1.7±1.3 
1.6±1.5 
1.9±2.3 
0.317 
oral iron started (days of age) 
15.5±8.5 
12.9±8.4 
14.5±7.7 
0.205 
Ferritin <50 day 14 
7 
5 
2 
0.024 
Ferritin <50 day 42 
12 
13 
2 
<0.001 
Ferritin >400 day 14 
1 
1 
5 
0.118 
Ferritin > 400 day 42 
3 
0 
5 
0.045 
Ferritin reported as median [Q1, Q3] 
Laboratory Studies:   
The following labs will be obtained on all enrolled infants: (1) CBC with reticulocyte count prior to study 
drug and at day 14 and 42 (0.5 mL/sample); (2) Epo concentration prior to study drug and any time the 
day of the third dose (trough), and 12±2 hours after the third dose if given SC, or just after the dose if 
given IV (peak; 0.5 mL/sample); (3) ferritin concentration on study day 14 and 42 (0.5 mL/sample).  In 
addition, biomarkers of brain injury and recovery will be run on collected samples (see below).  Blood 
draws prior to first study drug administration can be obtained from the umbilical cord/placenta prior to 
start of intervention after consent is obtained, or from stored cord blood if this is site practice (1.0 mL). 
If a retic count can be obtained on the infant’s admission CBC, then that CBC can serve as the baseline 
CBC. Clinically drawn measures of erythropoiesis (e.g., hemoglobin, CBC, retic, total and direct bilirubin) 
performed over the course of the study will be recorded. For those sites where this exceeds maximum 
research blood draw limits, only ferritin (to adjust iron dosing) and peak Epo will be drawn. In addition 
to the timed samples, serum/plasma samples will be collected from clinically drawn labs (scavenged 
samples) to measure random Epo concentrations, in order to perform population pharmacokinetics.  
Remaining serum or plasma from the above samples will be collected, frozen and shipped to the 
University of New Mexico.  These samples will be stored until 6 months following the last dose of the 
last enrolled infant, then destroyed if not used for anti-Darbe antibody analysis. Epo concentration data 
will be sent to Dr. An at the University of Iowa for pharmacokinetic analyses. 
 
Table 6. Laboratory Evaluation 
 
Day 1 
(before study drug) 
Day 14 
(dose 3) 
Day 42 
(dose 7) 
CBC and retic count 
X 
X 
X 
Epo concentration 
X 
Peak and 
trough 
 
Ferritin 
 
X 
X 
 
Measuring factors involved in brain injury and recovery: 
We will use Meso Scale Discovery (MSD) technology to measure markers of brain injury and recovery 
using the same serum/plasma samples on which Epo concentrations will be measured.  No additional 
sample volume is required.  The following inflammatory markers and growth factors will be considered 
for measurement: Brain-Derived Neutrotrophic Factor (BDNF), Interferon-gamma (IFN-γ),81 Interleukin 

17 
(IL)-1 β,82 IL-6, IL-8, IL-10, tumor necrosis factor-α (TNF-α), transforming growth factor (TGF)-β, matrix 
metalloproteinase (MMP)-2 and MMP-9,83 macrophage inflammatory protein-1α (MIP-1α), MIP-1β84, 
monocyte chemotactic protein-1 (MCP-1)84  and tissue inhibitor of metalloproteinase (TIMP)-1.83 
Markers of neurotoxicity and brain injury will include: S100B, glial fibrillary acidic protein (GFAP), neuron 
specific enolase (NSE), Activin A, and Ubiquitin C-terminal hydrolase-L1 (UCH-L1).85-87  
Not all of the above factors may prove to be informative.  We have specifically left off a final list of 
biomarkers to measure, in order to choose the most informative subset and appropriately manage costs 
of the MSD assays.  The above markers will be measured in a similar population of preterm infants 
enrolled in the PENUT trial, and we will use those results to inform the final selection of biomarkers to 
measure. 
All donors, transfusions and transfusion volumes will be recorded, and transfusions will be administered 
according to a transfusion protocol (below). Information regarding delayed cord clamping or cord 
milking will be collected on each infant enrolled, and centers will be encouraged to perform delayed 
cord clamping or cord milking.   
Prior to discharge, the following data will be collected: length, weight and head circumference; clinical 
outcomes (days of ventilation, hospital days, incidence of BPD [oxygen at 36 weeks], all stages of ROP 
and treatments for ROP, incidence of NEC ≥Bells stage 2, any ICH, PDA and treatments of PDA). 
The following safety data will be collected through 7 days past the last study dose or to conclusion: 
thromboses, seizures receiving treatment, hypertension receiving treatment, culture positive sepsis, soft 
tissue infections at the injection site. 
Results of clinically ordered head ultrasound or other head imaging studies, hearing and eye screening 
will be recorded.  
Transfusion Protocol:  
Infants in the study will be administered transfusions according to the following protocol.  Transfusions 
may be administered under urgent conditions when the attending clinician feels withholding a RBC 
transfusion would adversely affect the infant.  Transfusions may be considered depending on the infant 
reaching the following hemoglobin or hematocrit levels, but are not mandatory.  The transfusion volume 
will be 20 mL/kg for all transfusions administered. 
 
Table 7. Transfusion Protocol 
Hct ≤32% 
Hgb <11 gms/dL 
MAP > 8 cm H
2O (CV)  MAP > 14 (HFOV) AND FiO
2 ≥0.40 
Hct <27% 
Hgb <9 gms/dL 
Any mechanical ventilation OR non-invasive ventilation and FiO2 ≥0.40; undergoing surgery 
Hct ≤24% 
Hgb ≤8 gms/dL 
On respiratory support (≥ 1 LPM and ≥ 0.35 FiO2) 
AND ONE of: 
two or more consecutive 
episodes of tachycardia 
(heart rate >180) or 
tachypnea (RR >60) 
Increase in FiO2 by 
≥0.2 
≥2 episodes of apnea and/or 
bradycardia above baseline 
Hct ≤21%  
Hgb ≤7 gms/dL 
Asymptomatic AND: 
Absolute Retic Count < 100,000 cells/µL (<2%) (if clinically available) 
 

18 
F. Research Design 
 
4.2.5. Blinding/Masking 
Primary providers and bedside caregivers will be blinded to randomization group.  If the dose will be 
administered IV and can be masked, the study drug will be administered slow IV push by the bedside 
nurse. If the dose will be administered subcutaneously, the study drug will be brought to the bedside in 
a closed container, and injections will be shielded behind screens and out of earshot from caregivers and 
parents.  An adhesive bandage (or 2x2 gauze) will be placed over the true and sham injection sites and 
either taped or held in place until no evidence of the injection is visible.  The parents, medical providers, 
data collection staff and neurodevelopmental follow up personnel will be masked to the treatment arm. 
 
4.2.6. Control or Monitoring of Co-interventions 
Transfusions may be administered outside the protocol under urgent conditions when the attending 
clinician feels withholding a RBC transfusion would adversely affect the infant. Epo or open label 
Darbe should not be administered during any enrolled infant’s hospitalization. 
 
4.2.7. Primary Outcome 
The primary outcome is the Bayley III cognitive score. Deaths will be assigned a score of 54. The trial will 
analyze all enrolled infants as above, and will randomize infants 23 0/7-28 6/7 weeks GA into two groups 
(Darbe and placebo) in a 1:1 ratio. The primary hypothesis will evaluate whether infants receiving Darbe 
have higher cognitive scores at 22-26 months follow up, using a two-tailed test with 5% type 1 error and 
90% power. 
 
Study Limitations: This study will have sufficient statistical power to detect a meaningful difference (½ 
standard deviation or 7.5 points) in BSID III Cognitive scale score at 22-26 months. Loss to follow-up is a 
risk in any prospective study, however the excellent follow up rates of NRN sites provide the best chance 
at performing outcome studies. 
 
4.2.8. Secondary Outcomes  
Secondary outcomes that will be followed include: hematocrit, red cell mass, number and volume of 
transfusions, donor exposures, hospital days, and differences in morbidities (thromboses, hypertension, 
seizures, ICH, NEC requiring surgery, BPD, ROP requiring intervention, culture positive sepsis); NDI, 
death, NDI or death, and CP at 22-26 months. 
 
4.2.9. Safety Outcomes 
Monitored safety events are listed below.  
• 
Death 
• 
Major vessel thromboses 
• 
Receiving treatment for EEG-confirmed seizures 
• 
Receiving treatment for hypertension 
• 
Culture positive sepsis 
• 
Soft tissue infection at the injection site 
 
4.2.10. Compliance Monitoring  
Strategies to improve or monitor adherence to the study protocol will include the following: 
• Monthly recruitment reports of infants screened and enrolled (accrual figures) 

19 
• Monthly reports detailing data received at the Data Center, data consistency, missing data, 
performance measures and adherence to the study protocol (with appropriate measures taken to 
preserve the blinding of study personnel and investigators) 
• Periodic reports for the DSMC detailing data quality, protocol adherence, participant withdrawals, 
adverse events, losses to follow-up and study futility/efficacy  
• Supplementary blinded reports requested by the study investigators or sub-committee that do not 
disclose allocation group specific outcomes (primary, secondary or any safety outcomes).    
The aforementioned reports will be generated by the NRN Data Coordinating Center at RTI 
International. 
Additionally the following data will be collected to monitor adherence to the protocol: missed dose; 
missed iron dosing >7 days, doses given more than 2 days before or after a scheduled dose; study drug 
held per protocol, permanently stopping study drug per protocol; physician request to stop study drug; 
parental withdrawal from study with or without continued data collection. 
 
4.2.11. Study Specimens 
The following labs will be obtained.  Research CBC/retic counts and all but ferritins and peak Epo 
concentrations will be optional for families that wish to participate in the study but do not want 
research labs drawn or for those sites where research labs exceed maximum research blood draw limits:  
(1) Ferritin concentration around study day 14 and 42 (0.5 mL/sample)  
(2) CBC with reticulocyte count prior to study drug and around day 14 and 42 (0.5 mL/sample)   
(3) Epo concentration and biomarkers prior to initial dose of study drug and before and after the 
third dose (trough can be obtained any time during the day prior to dosing; please 
draw in conjunction with clinical labs if possible; peak should be drawn 12±2 hours 
after SC dosing, or  within 30 minutes of IV dosing (0.5 ml/sample)  
Blood draws prior to the initial dose of Darbe/placebo can be obtained from the umbilical cord/placenta 
at birth (1.0 mL).    All clinically drawn measures of erythropoiesis (e.g., hemoglobin, CBC, total and 
direct bilirubin) will be recorded and hematocrit values used to determine red cell mass (Hct x weight x 
85 mL/kg).  As mentioned above, additional serum/plasma samples will be collected from clinically 
drawn labs (up to 3 scavenged samples drawn 4-72 hours after a study dose and collected within 7 days 
of blood draw) to measure random Epo concentrations/biomarkers, in order to perform population 
pharmacokinetics, and to store in the event anti-Darbe antibody assays are run.  Remaining serum or 
plasma will be collected, frozen and shipped to the University of New Mexico.  These samples will be 
stored until 6 months following the last dose of the last enrolled infant, then destroyed if not used for 
anti-Darbe antibody analysis or for Epo concentration measurement or biomarkers measurement. 
 
4.2.12. Post-hospital Procedures 
Post-hospital procedures will include tracking for 22-26 month neurodevelopmental follow-up, including 
phone follow up following discharge per site practice. Ancillary studies may be added that include MR 
Imaging at some sites. 
 
4.2.13. Follow-up at 24 Months 
Neurodevelopmental evaluation will be performed by certified follow up examiners at 22-26 months 
corrected age.  Evaluations will consist of the following: BSID III, neurologic evaluation, anthropometric 
measurements, and assessment of severity of neurodevelopmental impairments (NDI). For the purposes 
of this study, severe, moderate or mild NDI will be defined as follows (Table 8): severe NDI will be 
defined by any of the following: a BSID III cognitive score < 70, Gross Motor Functional (GMF) Level of 3-

20 
5, blindness (<20/200 vision) or profound hearing loss (inability to understand commands despite 
amplification); moderate NDI will be defined as a BSID III cognitive score 70-84 and either a GMF level of 
2 or a hearing deficit requiring amplification to understand commands or unilateral blindness; mild NDI 
will be defined by a cognitive score 70-84, or a cognitive score ≥ 85 and any of the following: presence of 
a GMF level 1 or hearing loss not requiring amplification.  Normal (no NDI) will be defined by a cognitive 
score ≥ 85 and absence of any neurosensory deficits.  
 
Table 8. Definition of neurodevelopmental impairment (NDI) 
 
Normal 
Mild 
Moderate 
Severe 
Cognitive 
≥85 
70-84 or ≥85 
and any below 
70-84 
<70 
(GMFCS) 
 
1 
2 
3-5 
Vision 
No apparent 
problem 
No apparent 
problem 
Limited with correction/ 
unilateral blind 
Bilateral blind 
(cannot correct) 
Hearing 
No apparent 
problem 
Hearing loss 
not requiring 
amplification 
Moderate, can hear and follow 
directions with amplification 
No functional hearing with 
amplification 
 
4.2.14. Additional Follow-up Assessments 
Estimated mortality for initial hospitalization is 25%.  Every effort will be made to maintain contact with 
the remaining families of enrolled infants so that appropriate follow-up assessments can be made.  
Estimated follow up rates are 90%.   
 
4.3. 
POTENTIAL RISKS AND BENEFITS TO SUBJECTS 
 
Side effects are minimal and uncommon in adults receiving Darbe, and are similar to side effects of 
recombinant Epo, which are hypertension (10%), pain (5%), rash (1%), and rarely, thromboses (≤1%) or 
seizures (≤1%).  Three infants with seizures and two infants with transient hypertension were identified 
in the Darbe group in the ESA Study (p>0.2 versus placebo group for both side effects).  At present there 
are no other published reports of neonates receiving long term Darbe.  There are no long term adverse 
effects reported of neonates receiving Epo.62  
Preliminary analyses performed by Amgen reported no anti-Darbe antibodies in any of the infants 
receiving Darbe in our previous study.29a Pure red cell aplasia (PRCA) has been reported in adults who 
developed antibodies against Epo or Darbe.  The incidence of PRCA in adults receiving Darbe is 
extremely low (14 cases/100,000 patient years81), less than half that seen in adults receiving Epo, and 
there have been fewer than 20 reported cases in the literature.  Cases of PRCA after Epo dosing dropped 
dramatically in 2006 following identification of a specific antigenic single use syringe/stopper.  No cases 
of PRCA have been reported in neonates. 
The risks of drawing blood from a vein or giving a subcutaneous shot include discomfort at the site of 
needle insertion (5%), possible bruising and swelling around the insertion site (5%), skin irritation from 
the bandaid, and, rarely, an infection (less than 1%).  This procedure may be uncomfortable due to local 
pain at the time of the needle puncture and during the time blood is drawn.  There may be other risks to 
this study which are not yet known.  Of the 33 infants we have monitored who have received more than 
one dose of Darbe, no side effects have been noted. 
Potential benefits of Darbe include increased BSID III cognitive scores, improved executive function, 
decreased transfusions and decreased donor exposures.   

21 
SECTION 5. 
ANALYTICAL PLAN 
5.1. 
STATISTICAL ANALYSIS PLAN 
All analyses will be performed on an intent-to-treat basis, with the exception that survivors who are lost 
to follow up (estimated to be less than 10% in each treatment group) will be excluded from analysis of 
outcomes evaluated at 22-26 months. The primary outcome is BSID III composite cognitive score. The 
primary analysis (and all analyses examining outcomes by treatment) will be adjusted for the 
stratification variables of gestation and center. Since our primary outcome is continuous, we will use 
general linear mixed models (GLMM) to estimate the adjusted mean difference in BSID III cognitive 
scaled scores between the two treatment groups; the model will include a random effect for familial 
clustering. The p value used to assess statistical significance will depend on the alpha spent on interim 
efficacy analyses (see section 5.5.1.2). If no interim efficacy analyses are conducted, a p value less than 
0.05 in favor of the treatment effect will be considered as a statistically significant evidence of treatment 
benefit. If interim analyses for efficacy are conducted, the p value will be reduced by the alpha spent on 
those analyses.  
Comparisons of secondary outcomes between groups will be considered descriptive, and not formal 
tests of hypotheses. For continuous secondary outcomes measured serially over time, including 
hematocrit, CEV, platelet count, and absolute reticulocyte count we will use longitudinal GLMM 
accounting for the lack of independence between repeated measured on the same participant to obtain 
estimates of the values over time in each treatment group and adjusted mean differences between the 
two groups. Because the timing of these measures will vary by infant, days since treatment initiation will 
be a continuous covariate in the longitudinal models, and quadratic and cubic effects for time may be 
included depending on the outcome. Interactions between the time effects and treatment group will be 
included to assess whether the outcome measures have different trajectories of change over time in the 
two groups. Continuous outcomes measured at one time point, such as length of hospital stay, will be 
analyzed using similar GLMM models that are not longitudinal. 
For categorical outcomes including NDI, death, and other morbidities, and for count outcomes such as 
number of transfusions and donor exposures, we will use robust Poisson regression implemented in a 
generalized estimating equations (GEE) model to adjust for familial clustering, and with fixed effects for 
center and gestational age group, to obtain adjusted relative risk estimates for the treatment effect.  
We will examine hematocrit and red cell mass, also known as circulating erythrocyte volume (CEV) and 
calculated as hematocrit (%) x estimated blood volume (85 mL/kg) x weight (kg). While hematocrit may 
remain the same or even decrease slightly, preterm infants are constantly growing, and therefore the 
total number of red cells and CEV increases over time. Each of these outcomes will be measured at 
multiple points in time, and the repeated measures general linear mixed models to be used in analysis 
will accommodate variations in the timing of assessments between infants. Analyses examining these 
outcomes by treatment group will be adjusted for the stratification variables of gestation (<26 weeks, 26 
to 28 completed weeks) and center by their inclusion as fixed effects in the statistical models. General 
linear mixed models (GLMM) will also include a random effect to account for familial clustering due to 
the randomization of multiple births to the same treatment group.   
For outcomes measured serially over time, including hematocrit, CEV, platelet count, absolute 
reticulocyte count, and transfusion volume, we will use longitudinal GLMM accounting for the lack of 
independence between repeated measures on the same participant to obtain estimates of the values 
over time in each treatment group and adjusted mean differences between the two groups. Because the 
timing of blood draws and transfusions will vary by infant, days since treatment initiation will be a 
continuous covariate in the longitudinal models, and quadratic and cubic effects for time may be 

22 
included depending on the outcome. Interactions between the time effects and treatment group will be 
included to assess whether the outcome measures have different trajectories of change over time in the 
two groups.  
 
5.2. 
SAMPLE SIZE AND POWER ESTIMATES 
The table below presents a range of sample size estimates for each arm of the two-arm Darbe study for 
different underlying assumptions about the study.  Key assumptions for this study that are incorporated 
into each of the sample sizes calculated in the table are: (1) multiples will be randomized to the same 
arm; (2) 75% of the infants will survive on both arms with survival equal on the two arms and infants 
who do not survive will have an imputed BSID score of 54; (3) an additional 10% of infants will be lost 
prior to the follow-up and will be excluded from the analysis under the assumption that the data are 
missing at random; (4) Composite cognitive scores for survivors on the two arms will have mean values 
in the range of 85 and 95 with the true standard deviation will be in the range of 10 to 15 among 
survivors.  Under these assumptions, the standard deviation across the mixture of survivors and non-
survivors with scores imputed at 54 was found to be in the range of 17 to 20 if the underlying standard 
deviation among survivors was 10 and between 19 and 22 if the underlying standard deviation among 
survivors was 15, so three values over the range of 17 to 22 were considered.  The adjustment for 
multiples was estimated to be in the range of 12% to 15%. Note that the final sample sizes incorporate 
both the multiple randomization effect and the loss to follow-up percentages.  Sample sizes were 
computed for both 80% power and 90% power.  
 
Effect Size 
Standard 
Deviation 
Multiple 
Effect 
Sample Size Per 
Arm 
Survivor 
Aggregate 
80% 
Power 
90% 
Power 
7.5 
5.625 
17 
12% 
179 
238 
7.5 
5.625 
17 
15% 
241 
322 
7.5 
5.625 
19.5 
12% 
234 
313 
7.5 
5.625 
19.5 
15% 
241 
322 
7.5 
5.625 
22 
12% 
299 
397 
7.5 
5.625 
22 
15% 
307 
408 
10 
7.5 
17 
12% 
101 
134 
10 
7.5 
17 
15% 
104 
138 
10 
7.5 
19.5 
12% 
134 
178 
10 
7.5 
19.5 
15% 
137 
183 
10 
7.5 
22 
12% 
169 
225 
10 
7.5 
22 
15% 
174 
231 
 
In our previous multicenter study comparing cognitive outcomes at 18 to 22 months, we used 
information presented in our preliminary data section that showed a difference of 15±15 MDI points 
among survivors between the two groups.  The differences in our current Darbe study are 8±12 points 
on the BSID composite cognitive score among survivors despite the relatively small sample size. We 
anticipate that the proposed trial will find a difference at least that large between survivors randomized 
to receive Darbe compared to those randomized to placebo, but the study is conservatively powered to 
detect a difference of 7.5 points. When non-survivors with assigned scores of 54 are included, the 

23 
overall expected difference between the treatment groups becomes 5.625 points.  Using a conservative 
estimate of differences in BSID III cognitive score of 5.625±19.5 points between Darbe recipients and 
controls, with 90% power and an  of 0.05, estimating a survival rate of 75% and an additional loss at 
follow up of 10%, and assuming multiples would be assigned to the same treatment arm, and assigning a 
score of 54 to non-survivors, a total of 322 (rounded to 325) infants would need to be enrolled in each 
arm of the study, for a total of 650 infants. 
 
5.3. 
AVAILABLE POPULATION 
The number of eligible NRN admissions is approximately 525 infants per year.   
 
5.4. 
PROJECTED RECRUITMENT TIME 
Based on a consent rate of 50%, enrollment for this study should be completed within 24-30 months of 
IRB approval at all sites, and all follow up completed by 48-60 months.  Compatibility with other IND-
associated studies will need to be determined, and could impact enrollment and length of the study. 
 
5.5. 
STUDY MONITORING PLAN 
 
5.5.1. Reporting Adverse Events 
The trial will use NRN standard definitions for adverse events (AEs) and serious adverse events (SAEs), 
and collect the data on such events using specialized forms for this purpose. All SAEs that are 
unexpected and/or at least possibly related to the study intervention will be reported to NICHD and the 
NRN Data Center at RTI within 24 hours, along with a completed Medwatch form. All such events that 
are marked as possibly related to the intervention will be forwarded to the Chair of the independent 
NRN DSMC for any further action.  The independently appointed DSMC makes recommendations to the 
Director of NICHD and is entrusted with the following charges:  
(i) 
Review the research protocol, review model informed consent documents, and plans for 
data and safety monitoring, including all proposed revisions;  
(ii) 
Review methodology used to help maintain the confidentiality of the study data and the 
results of monitoring by reviewing procedures put in place by investigators to ensure 
confidentiality; 
(iii) 
 Monitor study design, procedures and events that will maximize the safety of the study 
participants and minimize the risks; 
(iv) 
Evaluate the progress of the study, including periodic assessments of data quality and 
timeliness, participant recruitment, accrual and retention, participant risk versus benefit, 
performance of the study site(s), and other factors that may affect study outcome; 
(v) 
Consider factors external to the study when relevant information becomes available, such 
as scientific or therapeutic developments that may have an impact on the safety of the 
participants or the ethics of the studies; 
(vi) 
Review serious adverse event documentation and safety reports and make 
recommendations regarding protection of the safety of the study participants. 
5.5.2    Data Monitoring Plan and Stopping Rules 
Data Management: Data will be collected using the stated forms, then entered into a centralized web 
based NRN Data Management System developed and maintained by the NRN Data Center at RTI. The 
data entry software will perform range checks and consistency checks and errors will be corrected on 
the spot. The Data Center will perform further consistency checks as well as more sophisticated machine 
checks designed to detect consistencies across forms. Edit errors detected at the Data Center will be 

24 
sent electronically back to the study sites for reconciliation. The final analysis database will be in the SAS 
format. 
 
Performance Monitoring: Standard reports will be generated from the study database by the Data 
Center. These include:  
• Monthly recruitment reports that provide the number of infants screened and enrolled  
• Monthly reports detailing data received at the Data Center, missing data, adherence to study 
protocols and a variety of performance measures  
• Periodic reports for the DSMC that detail data quality and protocol adherence, as well as 
adverse events, efficacy data and withdrawals or losses to follow-up  
• Ad Hoc reports requested by the study investigators that do not disclose treatment group 
specific outcomes information (primary, secondary or any safety outcomes).  
 
Stopping Rules: Information from clinical labs (CBC and reticulocyte count) are collected to monitor 
effects of study drugs and procedures.  Criteria are in place for holding and/or stopping the study drug.  
Criteria for holding the study drug include:  
1. Starting with the third dose, the study drug will be held if the known hematocrit on the day of 
dosing is ≥50% (excluding baseline labs, and not due to a blood transfusion) and will continue 
to be held until the hematocrit is <50%. 
2. If hypertension occurs during the study period in which a clinical decision is made to initiate 
antihypertensive treatment, the study drug will be held until hypertension resolves with 
treatment.   
3. All local or systemic skin reactions to the drug will be recorded, including soft tissue infections    
directly related to the injection site.  If any reaction to the study medicine occurs which causes 
a systemic reaction (hypotension, systemic skin reaction, respiratory compromise) the study 
drug will be held. 
 
Criteria for permanently stopping the study drug: 
1. If EEG-confirmed seizures not controlled by anticonvulsant medication occur during the study 
period, the study drug will be stopped. 
2. If hypertension recurs following antihypertensive treatment and resumption of the study 
drug, the study drug will be stopped. 
3. If any major vascular thrombosis is documented, the study drug will be stopped. 
 
5.5.2.1 
Safety 
Since the primary outcome is available only at 22-26 months follow up, safety will be the primary focus 
of interim monitoring for this trial.  The interim safety analysis will compare the incidence of 
serious adverse events (SAEs) across the placebo and Darbe groups. The DSMC will conduct pre-
specified formal safety looks at the interim data after the first 20 patients enrolled have reached 35 
completed weeks gestation, discharge, or transfer to another hospital, and subsequently, after 25%, 
50% and 75% of the enrolled patients have reached the same milestone. Pocock stopping bounds will be 
used as stopping rules for safety, based on the 4 planned interim safety looks at the data.  Thus, at each 
interim safety look, a p value less than 0.0158 from comparing the incidence of SAEs across groups, 
may be used by the DSMC as evidence of significant harm from the study intervention. SAEs are 
defined as any adverse event (defined in section 4.2.9) that results in any of the following.  
a. Death of infant. 
b. Prolonged hospitalization of infant. 
c. Persistent or significant disability/incapacity of the infant. 

25 
d. Required medical or surgical intervention to prevent any of a through c above. 
e. Is considered life-threatening if no medical intervention is provided. 
 
The above analysis will be conducted using robust Poisson regression implemented in a generalized 
estimating equations (GEE) model to adjust for familial clustering, and adjusting for gestation age group 
and center to obtain the p value for comparison with the cut-off of 0.0158. Given that there are 15 
centers in the NRN, adjustment for center may not be computationally feasible for the first few interim 
analyses, in which case GLMM with center as a random effect may be attempted if adjustment for 
center is deemed crucial for inference.  
 
5.5.2.2 
Efficacy 
Since the primary outcome is only available at 22-26 months follow up, and the projected enrollment 
period for this study is less than 3 years, interim efficacy monitoring for this trial may become moot if 
the trial finishes enrollment before substantial primary outcome data is accrued. However, in the event 
that recruitment is slow, there may be some interim efficacy data available for the DSMC to review. 
Interim efficacy looks will be performed once 25% of the enrolled infants, regardless of survival, would 
reach 2 years corrected age, and every 25% thereafter (provided enrollment/intervention is not already 
complete). To control the overall Type I error rate, a Lan-DeMets alpha spending function with an 
O’Brien Fleming-type stopping bound will be used. The above analysis will be conducted using GLMM for 
the primary outcome, adjusting for gestation age group and center, with a random effect for familial 
clustering, to obtain the p value for comparison with the appropriate stopping boundary.  
 
5.5.2.3 
Futility 
Just like interim efficacy, interim futility analyses will be limited in usefulness because the primary 
outcome is obtained at 22-26 months follow up. Thus, it is expected that vast majority of the subjects 
may be enrolled before the primary outcome can be determined in the first enrolled survivors.  
Interim futility looks will thus be performed on the primary outcome once 25% of the enrolled infants, 
regardless of survival, reach 2 years corrected age and every 25% thereafter. Specifically, the conditional 
power to detect a statistically significant treatment benefit will be calculated at each of these looks 
based on the accrued data and assuming the hypothesized treatment benefit for the unobserved data. 
The DSMC can recommend stopping further enrollment for futility if, at any of these interim looks, the 
conditional power for treatment benefit is less than 0.2. 
 

26 
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