Intermittent Hypoxia and Caffeine in Infants Born Preterm (ICAF)
Phase: PHASE2
Conditions: Intermittent Hypoxia
Sponsor: Children's National Research Institute

  
 
INTERMITTENT HYPOXIA AND CAFFEINE IN INFANTS BORN 
PRETERM (ICAF) 
Protocol Number: R01HD089289 
National Clinical Trial (NCT) Identified Number:  NCT03321734 
Principal Investigators:  
Carl E. Hunt, MD 
Eric Eichenwald, MD     
Sponsor: NIH 
Funded by: NIH: 1R01HD089289 - 01A1 
Version Number:  4.2 
 09 September 2021 
 
 

  
 
Table of Contents 
STATEMENT OF COMPLIANCE ............................................................................................................................ 4 
1 
PROTOCOL SUMMARY .............................................................................................................................. 4 
1.1 
Synopsis ............................................................................................................................................... 4 
1.2 
Study Schema ..................................................................................................................................... 8 
1.3 
Schedule of Activities (SoA) .............................................................................................................. 9 
2 
INTRODUCTION ......................................................................................................................................... 10 
2.1 
Study Rationale ................................................................................................................................. 10 
2.2 
Background ........................................................................................................................................ 10 
2.2.1 
Known Potential Risks .................................................................................................. 12 
2.2.2 
Known Potential Benefits ............................................................................................. 13 
2.2.3 
Assessment of Potential Risks and Benefits ............................................................. 14 
3 
OBJECTIVES AND ENDPOINTS ............................................................................................................. 14 
4 
STUDY DESIGN .......................................................................................................................................... 17 
4.1 
Overall Design ................................................................................................................................... 17 
4.2 
Scientific Rationale for Study Design ............................................................................................. 17 
4.3 
Justification for Dose ........................................................................................................................ 18 
4.4 
End of Study Definition ..................................................................................................................... 18 
5 
STUDY POPULATION ................................................................................................................................ 18 
5.1 
Inclusion Criteria ............................................................................................................................... 18 
5.2 
Exclusion Criteria .............................................................................................................................. 19 
5.3 
Lifestyle Considerations ................................................................................................................... 19 
5.4 
Screen Failures ................................................................................................................................. 19 
5.5 
Strategies for Recruitment and Retention ..................................................................................... 19 
6 
STUDY INTERVENTION ............................................................................................................................ 20 
6.1 
Study Intervention(s) Administration .............................................................................................. 20 
6.1.1 
Study Intervention Description .................................................................................... 20 
6.1.2 
Dosing and Administration ........................................................................................... 20 
6.2 
Preparation/Handling/Storage/Accountability ............................................................................... 21 
6.2.1 
Acquisition and accountability ..................................................................................... 21 
6.2.2 
Dose Preparation, and Labeling .................................................................................. 21 
6.2.3 
Product Storage and Accountability ........................................................................... 22 
6.3 
Measures to Minimize Bias: Randomization and Blinding .......................................................... 23 
6.4 
Study Intervention Compliance ....................................................................................................... 23 
6.5 
Concomitant Therapy ....................................................................................................................... 24 
6.5.1 
Rescue Medicine ........................................................................................................... 24 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT DISCONTINUATION/WITHDRAWAL 25 
7.1 
Discontinuation of Study Intervention ............................................................................................ 25 
7.2 
Participant Discontinuation/Withdrawal from the Study .............................................................. 25 
7.3 
Lost to Follow-Up .............................................................................................................................. 25 
8 
STUDY ASSESSMENTS AND PROCEDURES ..................................................................................... 27 
8.1 
Efficacy Assessments ...................................................................................................................... 27 
8.1.1 
Oxygen Saturation......................................................................................................... 27 
8.1.2 
Inflammarory Biomarkers ............................................................................................. 27 
8.1.3 
Quantitative MRI Structural, Microstructural and Metabolic Biomarkers of Acute Brain Injury
 
28 
8.1.4 
Salivary caffeine levels ................................................................................................. 28 
8.1.5 
Brief Infant Sleep Questionnaire (BISQ) .................................................................... 29 
8.2 
Safety and Other Assessments ...................................................................................................... 29 

  
8.2.1 
Telephone assessment of status at home ................................................................. 29 
8.3 
Adverse Events and Serious Adverse Events .............................................................................. 29 
8.3.1 
Definition of Adverse Events (AE) .............................................................................. 29 
8.3.2 
Definition of Serious Adverse Events (SAE) ............................................................. 29 
8.3.3 
Classification of an Adverse Event ............................................................................. 30 
8.3.4 
Time period and frequency for event assessment and follow-up ........................... 31 
8.3.5 
Adverse Event Reporting ............................................................................................. 31 
8.3.6 
Serious Adverse Event Reporting ............................................................................... 32 
8.3.7 
Reporting Events to Participants ................................................................................. 32 
8.3.8 
Events of Special Interest ............................................................................................ 32 
8.3.9 
Reporting of Pregnancy ................................................................................................ 33 
8.4 
Unanticipated Problems ................................................................................................................... 33 
8.4.1 
Definition of Unanticipated Problems (UP) ................................................................ 33 
8.4.2 
Unanticipated Problem Reporting ............................................................................... 33 
9 
STATISTICAL CONSIDERATIONS .......................................................................................................... 34 
9.1 
Statistical Hypotheses ...................................................................................................................... 34 
9.2 
Sample Size Determination ............................................................................................................. 34 
9.3 
Populations for Analyses ................................................................................................................. 35 
9.4 
Statistical Analyses ........................................................................................................................... 35 
9.4.1 
General Approach ......................................................................................................... 35 
9.4.2 
Analysis of the Primary Efficacy Endpoints ............................................................... 35 
9.4.3 
Safety Analyses ............................................................................................................. 38 
10 
SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS .............................. 38 
10.1 
Regulatory, Ethical, and Study Oversight Considerations .......................................................... 38 
10.1.1 
Informed Consent Process .......................................................................................... 38 
10.1.2 
Study Discontinuation and Closure............................................................................. 39 
10.1.3 
Confidentiality and Privacy ........................................................................................... 39 
10.1.4 
Future Use of Stored Specimens and Data ............................................................... 40 
10.1.5 
Key Roles and Study Governance .............................................................................. 40 
10.1.6 
Safety Oversight ............................................................................................................ 41 
10.1.7 
Clinical Monitoring ......................................................................................................... 41 
10.1.8 
Quality Assurance and Quality Control ...................................................................... 42 
10.1.9 
Data Handling and Record Keeping ........................................................................... 42 
10.1.10 
Protocol Deviations ....................................................................................................... 42 
10.1.11 
Publication and Data Sharing Policy .......................................................................... 42 
10.1.12 
Conflict of Interest Policy .............................................................................................. 43 
10.2 
Additional Considerations ................................................................................................................ 43 
10.3 
Abbreviations ..................................................................................................................................... 44 
10.4 
Protocol Amendment History .......................................................................................................... 45 
8.1.3 Quantitative MRI Structural, Microstructural and Metabolic Biomarkers of Acute Brain Injury ... 56 
REFERENCES ......................................................................................................................................................... 57 
 
 
 

 
 
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ICAF Protocol Version 4.2: 27  September 9, 2021  
 
 
 
 
STATEMENT OF COMPLIANCE 
The trial will be carried out in accordance with International Conference on Harmonization Good 
Clinical Practice (ICH GCP) and the following:  
 
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 
46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
 
All National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are 
responsible for the conduct, management, or oversight of this study have completed Human 
Subjects Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled.  Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study.  In addition, all changes to the consent form will be IRB-approved; a 
determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent, using a previously approved consent form. 
1 
 
PROTOCOL SUMMARY 
1.1 
SYNOPSIS  
Title: 
Intermittent Hypoxia and Caffeine in Infants Born Preterm (ICAF) 
Study Description: 
 
 
 
 
 
 
 
 
 
 
 
Our proposal will address the critical question: is persisting intermittent 
hypoxia (IH) in preterm infants associated with biochemical, structural, or 
functional injury, and is this injury attenuated with extended caffeine 
treatment? We will study the effects of caffeine on IH in 220 preterm infants 
born at ≤30 weeks + 6 days gestation. Infants who are currently being 
treated with routine caffeine, and who meet eligibility criteria, will be 
enrolled between 32 weeks + 0 days and 36 weeks + 6 days PMA.  At 
enrollment, infants will be started on continuous pulse oximeter recording 
of O2 saturation and heart rate.  If, based on standard clinical criteria, the 
last dose of routine caffeine is given on or before the day the infant is 36 
weeks + 5 days PMA,   then, ideally,  on the day following, but no later than 
the third calendar day following, their last dose of routine caffeine 
treatment, infants will be randomized (110/group) to extended caffeine 
treatment or placebo. Randomized infants should begin receiving study 
drug (i.e. 5 mg/kg/of caffeine base, or equal volume of placebo) on the day 
of randomization, but no later than the third calendar day following the last 
dose of routine caffeine.  Prior to 36 weeks + 0 days PMA, study drug will 
be given once daily (i.e. 5mg/kg/day) and beginning at 36 weeks + 0 days 
PMA, study drug will be given twice daily (i.e. 10 mg/kg/day).  The last dose 
of study drug will be given at 42 weeks + 6 days PMA. Pulse oximeter 

 
 
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recordings will continue either until hospital discharge or for 1 week after 
discontinuing study drug depending on whether the infant was recruited 
at a study site at which home oximetry recording is being performed. Two 
caffeine levels will be obtained, the 1st at one week after beginning study 
drug, but no later than 14 days post-first-dose of study drug. The 2nd 
targeted at 40 weeks + 0 days PMA, but no earlier than 39 weeks+ 0 days 
and no later than the last day of study drug, whether in hospital or at home. 
Inflammatory biomarkers will be measured at study enrollment and again 
at 38 weeks + 0 days PMA plus or minus 3 days, or within 2 calendar days 
prior to hospital discharge, whichever comes first. At study sites recruiting 
into the MRI part of the study, quantitative MRI/MRS should be obtained 
between study enrollment and 3 calendar days after starting study drug and 
again at a target date of 43 weeks + 0 days, but no later than 46 weeks + 6 
days PMA. 
Objectives: 
 
Primary Objective 1: Compare the extent of IH exposure, from 
randomization through 42 weeks + 6 days PMA (within each gestational 
week and overall), in infants randomized to extended caffeine treatment to 
infants assigned to receive placebo.   
Primary Objective 2: Compare changes in a panel of inflammation-related 
cytokines and chemokines, from enrollment to the target age of 38 weeks 
+ 0 days PMA, in infants randomized to extended caffeine treatment to 
infants assigned to receive placebo.  
Primary Objective 3: Compare changes in quantitative MRI structural, 
microstructural and metabolic biomarkers of acute injury, from enrollment 
to 43-46 weeks PMA, in infants randomized to extended caffeine treatment 
to infants assigned to receive placebo.  
Secondary Objective 1: Examine the association between salivary caffeine 
concentrations and IH outcomes at the one week post randomization and 
40 weeks + 0 days PMA assessments. 
Secondary Objective 2: Determine whether caffeine effects on changes in 
inflammatory or MRI biomarkers from baseline to follow-up are mediated 
by caffeine-related reduced IH.  
 
 

 
 
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ICAF Protocol Version 4.2: 27  September 9, 2021  
 
 
 
 
Endpoints: 
Primary:  
• 
Extent of IH as measured by seconds below 90% saturation per 24 
hours of recorded oximetry data within each week PMA, and overall   
• 
Plasma concentration changes in inflammatory biomarkers 
between baseline and 38 weeks + 0 days PMA 
• 
MRI changes in microstructural measures between baseline and 
end of study (between 43 weeks + 0 days and 46 weeks + 6 days 
PMA).   
Secondary 
• 
#IH events per 24 hours of recorded oximetry data, # sec < 80% 
saturation per 24 hours of recorded oximetry data, nadir saturation 
during events, and the duration of events and area under of curve 
during events below 90% and 80% threshold saturation  
• 
Changes in inflammatory biomarkers and MRI measures of regional 
tissue volume between baseline and end of study in relation to IH 
measures 
Study Population: 
Sample size: 220 infants, 110 caffeine and 110 placebo-treated  
Gender: Male and female 
Age: Enrollment at PMA 32 weeks + 0 days - 36 weeks + 6 days 
Demographic Group: Infants born at ≤30 weeks + 6 days gestation 
General Health Status: Clinically stable, on caffeine treatment and expected 
to receive last dose of routine caffeine treatment no later than 36 weeks + 
5 days PMA 
Geographic location: NICU or step down unit at a participating hospital, with 
final weeks at home following NICU discharge  
Phase: 
Not applicable 
 
Description of 
Sites/Facilities Enrolling 
Participants: 
 
Neonatal Intensive Care Units (NICU) in  hospitals in metropolitan areas in 
the U.S. While inpatient the research will take place in the hospital. Once 
discharged, if the subject has been enrolled at a site performing MRIs,  the 
patient will return to specified outpatient locations for performance of the 
follow-up MRI.  At the end of the study,  study termination procedures will 
be performed in a manner convenient for the family, and may be performed 
in person or remotely, and if applicable  may be performed at the time of 
an MRI.  
   
Description of Study 
Intervention: 
Enrolled infants will be randomized to receive caffeine or equal volume 
placebo. The caffeine base dose will be 5 mg/kg once daily when the infant 
is at age 35 weeks + 6 days PMA or less. When the infant is at 36 weeks + 0 
days PMA or more the caffeine base dose will be 5 mg/kg twice daily, based 
on the expected increased rate of caffeine metabolism that occurs as 
infants age.  
Study Duration: 
Enrollment is scheduled to start in December, 2018 and will continue at 
least through May of 2022, with completion of follow-up and data analyses 
thereafter. 
 
 

 
 
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Participant Duration: 
The maximum time between study enrollment to study completion for an 
individual participant would be 15 weeks if enrollment was as early as 32 
weeks + 0 days and the final visit was at the latest time permitted at 46 
weeks + 6 days.   Since enrollment is permitted as late as 36 weeks + 6 days, 
and the final study visit may occur as early as 43 weeks + 6 days, a 
participant may complete the full study in as few as 7 weeks.    
 
Hospital discharge most commonly occurs between 34 and 40 weeks PMA, 
and we expect most infants to have roughly half their time in the study as 
in-patient and half as outpatient, though this may be vary widely.   
 
 
 
The end of study procedures include: return of the oximeter (if applicable), 
salivary sample and any unused study drug.  As noted above, these 
procedures will be done at the convenience of the family, and although a 
face-to-visit is preferred,  all items may be shipped back if, for any reason, 
the parents prefer not to travel to the site.    
 
 
 
 

 
 
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1.2 
STUDY SCHEMA 
Our goal is to randomize 220 infants born at a gestational age of 30 weeks + 6 days or less, who get their 
last dose of routine caffeine treatment between 32 weeks + 0 days and 36 weeks + 5 days PMA, to receive 
either extended caffeine treatment or placebo to 42 weeks + 6 days.  As illustrated below, all NICU 
admissions of infants born at 30 weeks + 6 days or less will be noted in the screening log.  Formal screening 
for eligibility will begin at 32 weeks + 0 days, with enrollment once infants meet all eligibility requirements, 
and the infant is at 36 weeks + 6 days or less PMA.  Between enrollment and randomization, baseline data 
will be collected.  Randomization will be performed, ideally,  on the day following, but no later than the 
third calendar day following, the last dose of routine caffeine, as long as randomization and dosing is by 
36 weeks + 6 days PMA.   Outcome measures will include oxygen saturation recordings, plasma biomarkers 
and brain MRI. 

 
 
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1.3 
SCHEDULE OF ACTIVITIES (SOA) 
1.  
Study drug (caffeine base 5 mg/kg QD or placebo, weight adjusted weekly while in hospital) begins 1 day after last day of routine 
caffeine, but no later than the 3rd calendar day after last dose of routine caffeine.   Dose is increased to 5 mg/kg BID at 36 + 0; finally, 
the last dose of study drug is at 42 + 6.  Note that initial dose will be BID if enrolled at 36 + 0 or later. 
2. 
2nd caffeine level targeted at 40 + 0, (no earlier than 39 + 0 and no later than last day of study drug), whether in hospital or at home. 
3. 
1st biomarker blood sample should be obtained as close as possible to the day of randomization. Whenever possible, this blood sample 
should be timed with a clinically required blood draw. To facilitate obtaining this sample at the time of a clinical blood draw, this 
sample may be obtained at any time after consent, but no later than the 2nd calendar day after the first dose of study drug.  The 2nd 
sample is to be obtained at 38 + 0 (+ or – 3 days) or within 2 calendar days prior to hospital discharge, whichever comes first. 
4. 
1st MRI/MRS is done in hospital sometime between enrollment and 3 calendar days after starting study drug.  2nd MRI/MRS is targeted 
at 43 + 0 but should be no later than 46 + 6.  MRI visit may be combined with the end of study visit. MRI/MRS scans will only be 
performed for subjects enrolled at sites participating in the MRI/MRS portion of the study. 
5. 
Includes documentation of tracking information, and parent training including: use of oximeter, drug administration, use of drug 
administration log, salivary sample collection, communication expectations and follow-up plans. 
6. 
Weekly phone calls from study staff to parent(s) begin 1- 3 days after hospital discharge and continue through end of study follow up 
visit.  A phone follow-up form will be completed to collect clinical data, adverse events, and document family questions. 
7. 
Sleep Assessment (BISQ) done over the phone with parent at 42 weeks. 
8. 
The end of study visit, which may be done remotely, is targeted for 43 + 6 days, or one week after the last dose of study drug, 
whichever comes first, but should be completed no later than 46 + 6.  Study staff will collect the study drug log, used bottles, unused 
drug, study oximeter, and frozen salivary sample. Items may be shipped back if the parents opt for a remote visit. 
9. 
Collection of concomitant medications and adverse event monitoring and reporting are continuous throughout study. 
10. Home-oximetry will only be performed for subjects enrolled at sites participating in the home-oximetry portion of the study. 
Study 
Procedures 
≤30 weeks + 6 
days thru 36 
weeks + 6 
days 
32 weeks 
+ 0 days 
thru 36 
weeks + 6 
days 
Target: 1 
day post 
last 
routine 
caffeine 
dose (but 
window of 
up to 3 
calendar 
days 
allowed) 
1 week on 
study drug 
(up to 14 
days post 
1st dose) 
36 weeks 
+ 0 days 
PMA 
Hospital 
Discharge 
40 weeks 
+ 0 days 
PMA 
42 weeks  
+ 6 days 
PMA 
43 weeks       
+ 6 Days PMA 
Screening 
X 
  
  
  
  
  
  
  
  
Informed 
Consent 
  
X 
  
  
  
  
  
  
  
Enrollment 
  
X 
  
  
  
  
  
  
  
Baseline Data 
Collection 
  
X 
  
  
  
  
  
  
  
Pulse oximetry 
recording 
  
X10 
  
Randomization 
  
  
X 
  
  
  
  
  
  
Once daily 
study drug 
  
  
X1 
  
  
  
  
  
Twice daily 
study drug 
  
  
  
  
X1 
  
Drug log 
  
  
X 
  
Saliva for 
caffeine level 
  
  
  
X 
  
  
X2  
  
  
Biomarker 
blood sample 
  
X3 
  
  
X3 
  
  
  
MRI/MRS 
  
X4 
  
  
  
  
  
  
X4 
Pre-discharge 
checklist 
  
  
  
  
  
X5 
  
  
  
Weekly parent 
phone contact 
  
  
  
  
  
X6 
  
Infant sleep Q 
  
  
  
  
  
  
  
X7 
  
End of study 
visit 
  
  
  
  
  
  
  
X8 
Concomitant 
medications 
  
X9 
Adverse Event 
  
X9 

 
 
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2 
 
INTRODUCTION 
 
2.1 
STUDY RATIONALE  
   Caffeine treatment for apnea of prematurity and related intermittent hypoxia (IH) is almost universal, 
with treatment most commonly discontinued at 33-35 weeks PMA.   However, IH continues to occur in 
infants after routine discontinuation of caffeine, and persists after hospital discharge. This IH is not 
apparent clinically and accurate detection requires continuous high resolution pulse oximetry recordings. 
Studies confirm that the IH occurring during spontaneous breathing after stopping routine caffeine is a 
manifestation of immature breathing control. It is unknown if this persistent IH results in acute injury, 
and in particular if extending the duration of caffeine treatment attenuates or prevents this acute 
injury. This study will address 3 critical questions: 1) will extending caffeine treatment from 32-35 to 42 
weeks PMA reduce extent of IH, 2) is persisting IH associated with biochemical, structural, or functional 
indicators of acute injury, and 3) does caffeine treatment continued to 42 weeks PMA attenuate this acute 
injury? This study has the potential to (1) identify early brain injury mediated by IH and mitigated by 
caffeine at appropriate dosing, and (2) help inform optimum strategies for later definitive 
neurodevelopmental assessments based on nature of the acute brain injury. This study thus has high 
potential to justify future neurodevelopmental outcome studies that will further improve outcomes of 
infants born preterm, change clinical practice, and have significant public health benefit. 
2.2 
BACKGROUND  
Acute morbidities can contribute to adverse neurodevelopment outcomes in preterm infants born at ≤30 
weeks gestation, but neural damage occurring after resolution of acute morbidities may be more subtle 
and related to cycles of inflammation and repair in the developing brain. One possible contributor to these 
more subtle injuries is intermittent hypoxia (IH), defined as repetitive cycles of hypoxia and re-
oxygenation, which occur commonly in convalescing premature infants. Caffeine treatment can improve 
both motor and cognitive neurodevelopmental outcome in premature infants, especially at higher doses, 
but mechanisms are unclear.1-8 The Caffeine for Apnea of Prematurity (CAP) Trial in infants born preterm 
at <1250 g reported 1) shorter duration of positive pressure ventilation and reduced rate of 
bronchopulmonary dysplasia (BPD) in infants treated with caffeine during the early postnatal weeks prior 
to 34-35 weeks postmenstrual age (PMA), 2) improved motor function and reduced rates of 
developmental coordination disorder at 5 years, and 3) diffusion changes by MRI consistent with 
improved white matter microstructural development.7,9,10,11 Although potential mechanisms for this 
caffeine effect were not studied in these reports, a recent study of very preterm infants in postnatal weeks 
1-10 showed for the first time a direct link between IH and motor, cognitive and language impairment 
at 18 months corrected age (adjusted risk gradient p<0.005).12 Notably, the greatest risk gradient was at 
postnatal ages 9-10 weeks, consistent with a contributory role of later IH present after stopping routine 
caffeine treatment. These data emphasize the potential importance of recurrent episodes of IH, as 
convalescing infants approach term-equivalent age, on later cognitive, language and motor impairments.    
   Studies confirm that IH during the early postnatal weeks of life in very preterm infants may be due to 
other mechanisms, including ineffective ventilation or other acute morbidities. However, IH in 

 
 
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spontaneously breathing convalescing infants is due to ventilatory immaturity with associated respiratory 
pauses or brief apneas, and has a characteristic pattern of brief desaturation from a normoxic baseline 
followed by reoxygenation and return to normoxia.13-18 Our study will assess IH only during spontaneous 
breathing in infants after resolution of acute morbidities or need for supplemental O2, and approaching 
term-equivalent age, a time when IH has been shown by us and others to be the consequence of immature 
breathing regulation. 
   IH during spontaneous breathing related to ventilatory immaturity requires continuous high resolution 
pulse oximetry recordings for detection, and consists of brief, repetitive cycles of O2 desaturation from a 
normoxic baseline, followed by return to baseline saturations. These repetitive cycles of reoxygenation 
following each IH episode are pro-inflammatory and cause oxidative stress, free radical production, and 
release of pro-inflammatory cytokines.19,20 Studies show increased levels of inflammatory biomarkers in 
animal models of IH-associated obstructive sleep apnea (OSA) and in human subjects with OSA.19,21-25 
Although inflammatory biomarkers may be elevated in the first 2-3 postnatal weeks in very preterm 
infants who develop BPD and neurodevelopmental sequelae, it is unknown if later IH during spontaneous 
breathing in convalescing preterm infants is associated with inflammation or other biochemical, 
structural or metabolic acute injury or adverse consequences.26-31 
   We have reported that clinically unrecognized IH events are still common after discontinuing routine 
caffeine treatment, typically at 34-35 weeks PMA.18 Except for 1 study, however, the potential adverse 
consequences of IH have not been investigated in human infants.12,18,32-34 In obstructive sleep apnea, 
however, even modest amounts of chronic IH have been associated with significant neurocognitive 
morbidity.23,24  Worrisome evidence from animal models also shows that IH has significant and long lasting 
effects on multiple physiological control mechanisms20-24,35-40 and neurological outcomes.41-43 We 
hypothesize that persistent IH in spontaneously  breathing preterm infants after stopping routine caffeine 
treatment is associated with acute adverse consequences.           
   The relationship between IH, adenosine, caffeine and brain development is complex and not fully 
understood. At clinically effective doses, caffeine exerts effects in the brain by blocking adenosine (Ado) 
A1 and A2A receptors, resulting in respiratory stimulation and increased alertness, vigilance and 
arousal.44-60 Ado A1 receptor activation contributes to hypoxia-induced reduction in cerebral myelination 
and ventriculomegaly. Caffeine treatment attenuates the effects of hypoxia, presumably through 
blockade of Ado A1 receptors.45,58,59 It is thus reasonable to hypothesize that similar mechanisms may be 
active in the human preterm infant. Caffeine may thus be neuroprotective through two major 
mechanisms: 1) reducing incidence and severity of IH due to its respiratory stimulatory effects, and 2) 
reducing pre- and immature oligodendrocyte injury.  
   Brain development progresses through a highly programmed series of events.61-64 Myelination in the 
cerebral hemispheres begins to accelerate at ~30-32 weeks and continues to term and beyond, and 
disturbances in these late gestation developmental processes often result in failure of normal brain 
growth, abnormal cortical organization, impaired myelination, and connectivity, commonly observed in 
surviving preterm infants.64-75 Persisting IH thus has even greater potential for later neurodevelopmental 

 
 
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disability than the IH associated with obstructive sleep apnea.23,24,76-84 Since IH can be attenuated with 
extended caffeine, persisting IH may thus be a modifiable cause of a previously unrecognized 
additional risk for disabilities associated with preterm birth.7,18,41,42,45,55,73,85-88   
   In summary, the period from 32-35 to 42 weeks PMA is a critical time for brain development, and is 
also a time when significant IH during spontaneous breathing is present, but the adverse effects of this 
IH are unknown. As the first step in understanding acute injury from IH, we address a fundamental and 
critically important question with high potential public health benefit: does continued caffeine treatment 
after receiving the last dose of routine caffeine between 32 weeks + 0 days PMA and 36 weeks and 5 
days PMA reduce extent of IH and attenuate indicators of acute injury at 43-44 weeks PMA? We will 
assess injury in 4 domains: biochemical (inflammation), structural (MRI), functional and metabolic (MRS). 
Our proposed study thus has the potential to have major impacts on clinical practice: 1) how clinicians 
assess and interpret IH, and 2) duration of pharmacological treatment with caffeine.  This will be the first 
study in human infants to assess the effects of continuing caffeine treatment in attenuating acute injury 
indicators associated with IH.  
2.2.1 KNOWN POTENTIAL RISKS  
• 
Study drug:  Caffeine is one of the most frequently used medications in the NICU for infants born very 
preterm. At the time of enrollment, infants will be tolerating routine caffeine treatment without any 
adverse clinical effects.  However, known potential side effects of caffeine, would include: 
o Transient decreased weight gain 
o Tachycardia  
o Gastrointestinal symptoms, including feeding intolerance 
o Jitteriness   
o Hypertension 
Each of these side effects would be expected in fewer than 5% of participants. 
• 
Pulse oximeter recordings: Pulse oximetry is routinely used in the NICU and sometimes also 
indicated for home use. The study oximeter will be in addition to the comparable oximeter 
routinely used clinically. We will follow routine clinical routines for frequent site relocation of the 
oximeter sensors (probes), to decrease the risk of significant skin irritation.  
• 
Blood sampling for inflammatory biomarkers:  There will be 2 blood samples obtained, one at 
enrollment and the 2nd prior to NICU discharge, and the blood volume for each sampling will be 
<1 ml. Whenever possible, these blood samples will be obtained as part of a clinically indicated 
routine heel stick or venipuncture, thus decreasing the need to cause infant discomfort beyond 
that required for routine clinical management.  
• 
Brain MRI: All MRIs performed for this study will be done without sedation, thus eliminating the most 
important potential risk beyond that associated with a clinically-indicated brain MRI.  However, the 
time required for each study MRI will be more than that required for a routine brain MRI.   
• 
Salivary samples: The 2 salivary caffeine samples for each subject will be obtained using a 
commercially available salivary collection kit identical to that used in our recent pilot study. These 
salivary collections are easily obtained and have not been associated with any infant discomfort 
or parental concern.   
• 
Loss of confidentiality is also a potential risk. However, all demographic and clinical information will 
be entered in a secure password-protected database accessible only to the research staff at that 

 
 
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clinical site and the data coordinating center (DCC) at Boston University Medical Center. Clinical site 
research staff will enter data via web access. Identifiers that have been collected will be stored 
separately from research data. The data will be in a separate locked/password protected database 
linked only by a subject ID number. Identifiers collected for the purpose of performing follow-up will 
be stored in a separate database that will be destroyed after the study is completed. The data will be 
split as it is loaded; personal identifiers (names, addresses, telephone numbers) will be entered into 
the tracking database along with a unique numeric identifier, and other data (excluding the 
participants' names and address) will be stored in the analytic database. It will be possible to link 
information in these two databases only through a coded numeric identifier; it will not be possible to 
identify individual participants in the analytic database. The separation of names and addresses from 
personal data adds an important level of security and helps to assure that participant confidentiality 
will be maintained. Follow-up data will be processed in a similar manner. The analytic database will 
contain no identifiers and will be the exclusive source for all data analyses performed at the DCC.     
Database files will be stored at the DCC on network servers located in a data center at the Slone 
Epidemiology Center at Boston University.  Slone Epidemiology Center data center is a separate 
secure room monitored by key access.  Activity on the Slone network and servers is monitored by a 
University security team.  In addition, access to the Slone network is restricted by specific Virtual 
Private Network group access and router access control lists.  Servers are protected by software 
firewalls and enterprise antivirus software. Administrators to the servers are limited to a core group 
of individuals who oversee server health and security. Network access to the servers is protected by 
requiring a user to login to an Active Directory system using the assigned username and Kerberos 
password.  Access to directories and files are restricted by user membership to network security 
groups.  Network security group membership is granted only if a user is affiliated with a research study 
or group.  When a user is not affiliated with any research study or group, access is denied and 
removed. Backup data is taken at regular intervals.  This backup data is encrypted and stored in a 
secure off-site facility for three years.  Any request for file restoration is examined, monitored, and 
documented.  After three years, backup data is verified and certified for destruction. Any websites 
used by Slone for data collection are protected with SSL protocols—commonly referred to 
HTTPS.  These protocols provide encrypted communication and secure identification of web servers 
using certificates.  Certificates are signed by trusted certificate authority. 
• 
Recurrence of Apnea-related symptoms in placebo-treated subjects: We have not observed any 
recurrent apnea-related symptoms in preliminary studies after stopping routine caffeine.18,90  
However, if a subject has apnea-related clinical symptoms after stopping routine caffeine and starting 
study drug, nasal CPAP or nasal cannula may be used as needed at the discretion of the clinical team.  
After the last dose of study drug at 42 weeks + 6 days PMA, pulse oximeter recordings will be 
continued for 1 additional week, to 43 weeks + 6 days PMA, for those participants enrolled at sites at 
which home oximetry is included in the study. No significant alarms have occurred after stopping 
extended caffeine treatment in our pilot studies, and none are anticipated in this clinical trial since at 
this PMA the extent of IH no longer differs from healthy term infants .15,16  However, the oximeter 
alarms can be activated for this final study week if deemed necessary by the clinical site investigator.  
 
2.2.2 KNOWN POTENTIAL BENEFITS  
Direct Potential Benefits:  
• 
Infants randomized to receive extended caffeine treatment may benefit directly by having less 
intermittent hypoxia and hence less inflammation and improved brain MRI findings 

 
 
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• 
Placebo-treated infants will benefit from the additional support and information provided by 
research staff, and will have the potential for benefit from information obtained from the MRI 
evaluations, which would not have been done absent enrollment in this study. Except for the 
inflammatory biomarkers and brain MRIs, placebo-treated enrollees are equivalent to “usual 
care” clinical practice in NICUs today.  
 
General Future Potential Benefits: 
• 
This study will fill several major gaps in our current knowledge: 1) The impact on 
neurodevelopmental outcome of the frequency and severity of IH in the NICU and at home until 
43 weeks PMA, assessed by continuous pulse oximeter recordings of oxygen saturation and heart 
rate, 2) the frequency and severity of IH in preterm infants receiving caffeine vs. placebo while in 
the NICU, 3) the frequency and severity of IH persisting at home after discharge (for participants 
receiving home oximetry), and 4) the efficacy of continuing caffeine treatment until 42 weeks + 6 
days PMA with regard to reducing IH and improving neurodevelopment.   
• 
Infants randomized to caffeine may benefit from improved neurodevelopmental outcome related 
to attenuated IH during the period of exposure to study drug.  
2.2.3 ASSESSMENT OF POTENTIAL RISKS AND BENEFITS  
• 
The risk: benefit ratio is favorable 
• 
Extending caffeine treatment to 42 weeks + 6 days PMA has the potential of improving brain 
structure and function as determined by MRI, and improving neurodevelopmental outcomes.  
• 
Infants randomized to placebo will be receiving what is currently the standard of care when their 
last routine caffeine dose is between 32 weeks + 0 days PMA and 36 weeks and 5 days PMA.  
• 
The potential risks are primarily related to blood sampling and the brain MRIs. As discussed above, 
however, the blood samples will be obtained as a part of clinically indicated blood sampling, and 
the MRIs will be performed without sedation.  
• 
In summary, the potential benefits exceed the potential risks of participation in this study, and 
there are no increased risks if randomized to receive placebo since placebo infants will otherwise 
be receiving the same clinical care as comparable infants not enrolled in this study.     
 
 
 
 
 
 
 
 
 
 
 
  
3 
OBJECTIVES AND ENDPOINTS 
 

 
 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
Primary 
 
 
Primary Objective 1: Compare the 
extent of IH exposure, from 
randomization through 42 weeks 
+ 6 days PMA (within each 
gestational week and overall), in 
infants randomized to extended 
caffeine treatment to infants 
assigned to receive placebo. 
Primary Objective 2: Compare 
changes in a panel of 
inflammation-related cytokines 
and chemokines, from enrollment 
to the target age of 38 weeks + 0 
days PMA, in infants randomized 
to extended caffeine treatment to 
infants assigned to receive 
placebo.  
 
Primary Objective 3: Compare 
changes in quantitative MRI 
structural, microstructural and 
metabolic biomarkers of acute 
injury, from enrollment to 43-46 
weeks PMA, in infants 
randomized to extended caffeine 
treatment to infants assigned to 
receive placebo.  
  
# of sec of IH <90% at each PMA 
week  
 
 
 
 
 
 
 
Change in plasma concentration 
of each inflammatory 
biomarker from baseline to 38 
weeks PMA. 
 
 
 
 
 
 
 
 
Difference between 
neuroimaging measures at 
baseline and at 43-46 weeks 
PMA, including diffusion tensor 
imaging (DTI) and proton 
magnetic resonance 
spectroscopy (MRS). 
# of sec of IH <90% the most 
representative measure to 
quantitate extent of IH 
 
 
 
 
 
 
Measuring biomarker 
concentrations at 38 weeks 
will be a sufficient interval for 
changes since baseline, and 
will be the interval during 
which differences in extent of 
IH will be maximal.  
 
 
 
 
Quantitative MRI is a sensitive 
modality for assessing 
structural, biochemical, and 
functional changes 
consequent to IH and extent 
of improvement with 
extended caffeine treatment. 
Assessing at study end will 
provide maximal time for 
adverse MRI changes 
consequent to IH since 
starting study drug.  
Secondary 
 
 
Secondary Objective 1: Examine 
the association between salivary 
caffeine concentrations and IH 
outcomes at the one week post 
randomization and 40 weeks + 0 
days PMA assessments. 
Secondary Objective 2: 
Determine whether changes, from 
baseline to follow-up, in 
Extent of IH (# of sec <90%) 
paired with saliva caffeine level 
during PMA week of oximetry 
recording. 
 
 
Change in plasma concentration 
for each inflammatory 
 
# of sec of IH <90% is the 
most representative measure 
of extent of IH during the 
week corresponding in time 
to the caffeine level. 
 
 
See above   

 
 
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OBJECTIVES 
ENDPOINTS 
JUSTIFICATION FOR 
ENDPOINTS 
inflammatory or MRI biomarkers 
are mediated by caffeine-related 
reduced IH. 
biomarker, from baseline to 38 
weeks PMA, in relation to IH 
measures. 
Change in each MRI parameter, 
from baseline to 43-46 weeks 
PMA in relation to IH measures. 
 
 
 

 
 
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4 
STUDY DESIGN  
4.1 
OVERALL DESIGN 
HYPOTHESES: 
Hypothesis 1: Caffeine will reduce extent of IH compared to placebo, and this caffeine effect will persist 
to 42 weeks PMA.    
Hypothesis 2: Extended caffeine treatment will be associated with less acute inflammation at 37-38 weeks 
compared to placebo, and these effects will be mediated by attenuated IH. 
Hypothesis 3: Quantitative MRI analyses in the active caffeine group, compared to the placebo group, will 
be associated with 1) greater white matter microstructural organization, 2) improved neuronal 
metabolism, and 3) greater regional brain volume, and that these effects will be mediated by attenuated 
IH. 
TYPE/DESIGN OF TRIAL: 
Randomized, placebo-controlled, double-blinded clinical trial   
METHODS TO BE USED TO MINIMIZE BIAS: 
All clinical team personnel and all research staff (except research pharmacist) will remain blinded to study 
group until study completion. Caffeine and equal volume placebo will be prepared by research pharmacist 
and will be equivalent in color and consistency. The protocol has been designed such that there should be 
no need to break the code for any individual subject. 
NUMBER OF STUDY GROUPS/ARM & STUDY INTERVENTION DURATION: 
Infants will be randomized in a 1:1 ratio to 1 of 2 treatment arms, caffeine base (Arm 1) and placebo (Arm 
2). Infants from multiple births will be cluster randomized. The duration of the intervention will be from 
randomization between 32 weeks + 0 days PMA and 36 weeks + 6 days PMA through 42 weeks + 6 days 
PMA, the age at which extent of IH no longer differs from healthy infants born at term.  
 
MULTI-SITE STUDY: 
There will be enrollment at multiple hospitals across the U.S. 
NAME OF STUDY INTERVENTION:  
Caffeine versus placebo 
 
INTERIM ANALYSIS:  None planned 
STRATIFICATION: 
A site-stratified randomly-permuted blocked randomization design will be used to ensure balance at each 
site. To ensure balance by gestational age at birth, randomization will also be stratified into 2 birth 
gestational age categories: 1) <28 weeks 2) 28 weeks + 0 days through 30 weeks + 6 days. The 
randomization scheme will be developed by the DCC using specialized software, and clinical sites will be 
provided appropriate enrollment logs and randomization procedures from the research pharmacy. 
 
SUB-STUDIES:  No ancillary studies planned at this time 
 
4.2 
SCIENTIFIC RATIONALE FOR STUDY DESIGN 

 
 
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Our pilot studies have demonstrated that IH associated with immature breathing pattern in infants born 
preterm can be attenuated with caffeine. It is unknown, however, whether persisting IH after stopping 
routine caffeine treatment in the NICU, which may only evident with continuing high resolution pulse 
oximetry, results in any long-term injury. The results in the group randomized to extended caffeine 
treatment (Arm 1) will be compared to placebo (Arm 2) to determine if extended caffeine treatment is 
superior to placebo in attenuating or eliminating the adverse effects of persisting IH on inflammation and 
brain MRI structural, microstructural, and metabolic markers of injury. 
 
4.3 
JUSTIFICATION FOR DOSE 
Infants will be started on oral caffeine base at 5 mg/kg/day or equivalent volume of placebo. At 36 weeks 
+ 0 days PMA, study drug dose will be increased to 5 mg/kg BID (total daily dose 10 mg/kg) or equivalent 
volume of placebo. The dose will be weight-adjusted weekly until NICU discharge.  The starting dose and 
dose escalation at 36 weeks + 0 days PMA reflect increasing clearance rates that have been established in 
pilot studies as being appropriate to achieve caffeine levels within the most likely therapeutic range.  All 
subjects will be on enteral medications when enrolled. 
4.4 
END OF STUDY DEFINITION 
 
Each subject will be considered to have completed the study after the planned pulse oximeter recording 
data is completed and inflammatory biomarker samples are provided, and if applicable, has completed 
the outpatient brain MRI between 43 weeks + 0 days and 46 weeks + 6 days PMA, as shown in the 
Schedule of Activities (SOA), Section 1.3. 
 
5 
STUDY POPULATION 
 
5.1 
INCLUSION CRITERIA 
INCLUSION CRITERIA: 
In order to be eligible to participate in this study, an individual must meet all of the following criteria: 
 
1. Male and female infants born preterm at ≤30 weeks + 6 days postmenstrual age 
2. Current treatment with routine caffeine.  Note infants will meet this inclusion criteria through the 
third calendar day following receiving their most recent routine caffeine dose.  
3. PMA 32 weeks + 0 days - 36 weeks + 6 days 
4. Anticipated last dose of routine caffeine will be by 36 weeks + 5 days 
5. At least 12 hours of breathing room air with no ventilatory support other than on room air nasal 
air flow therapy regardless of flow rate, or on room air and receiving nasal CPAP, and relapse 
not anticipated. 
6. Able to tolerate enteral medications  
7. It is feasible to administer the first dose of study drug no later than 36 weeks + 6 days PMA 
 
 
 
 
 

 
 
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5.2 
EXCLUSION CRITERIA 
EXCLUSION CRITERIA: 
1. Intraventricular hemorrhage Grade III-IV or cystic periventricular leukomalacia 
2. Current or prior treatment for seizures 
3. Current or prior treatment for cardiac arrhythmias 
4. Known renal or hepatic dysfunction that in the opinion of the investigator would have a clinically 
relevant impact on caffeine metabolism 
5. Major malformation, inborn error of metabolism, chromosomal abnormality 
6. Presence of a condition for which survival to discharge unlikely 
7. Social, mental health, logistical or other issues that, in the opinion of the investigator, would 
impact the ability of the family to complete the study.  
 
 
 
 
5.3 
LIFESTYLE CONSIDERATIONS 
• 
Parents and clinical care providers will be asked to avoid exposure to caffeine for subjects. 
• 
Open label treatment with a methylxanthine will be documented.  
• 
There will be no restrictions on maternal diet even if breast feeding. 
5.4 
SCREEN FAILURES 
• 
Screened and potentially eligible infants for enrollment will be screen failures if they reach 36  
weeks + 6 days PMA and are still receiving routine caffeine or, for whatever reason, cannot be 
enrolled by 36 weeks + 6 days PMA. 
• 
Enrolled infants will be screen failures, and will not be randomized, if they do not continue to 
meet eligibility criteria at the time that routine caffeine treatment is being discontinued.  
• 
Analysis will be by “intention to treat,” and all randomized subjects will remain included in their 
Arm for analysis.   
5.5 
STRATEGIES FOR RECRUITMENT AND RETENTION 
RECRUITMENT: 
• 
Potential enrollees will all be inpatients in the NICU at one of the participating hospitals.  
All inpatients in the NICU born at ≤30 weeks + 6 days postmenstrual age will be captured in the 
screening log, and beginning at 32 weeks + 0 days PMA, if they are being treated with routine 
caffeine, will be screened for potential eligibility.  Screening will be discontinued whenever 
infants meet a specified exclusion criterion for enrollment or if they reach 36 weeks + 6 days 
PMA without meeting all inclusion criteria. It is estimated based on pilot studies that 30-50% of 
all screened infants will meet 1 or more exclusion criteria or fail to meet inclusion criteria.  If an 
infant is a transfer from another facility at ≤36+6, they WILL be added to the screening log.  If 
they are a transfer from another facility at ≥37+0, they WILL NOT be added to the screening log. 
• 
The families of infants who are meeting enrollment criteria will be approached by study staff 
regarding possible enrollment.   
 

 
 
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RETENTION: 
• 
As infants are approaching the time of discharge staff will utilize the pre-discharge checklist to: 
o Obtain detailed tracking information that provides information regarding where the 
infant will be cared for post-discharge and multiple methods to contact key family 
members 
o Provide and document comprehensive training of families regarding use of the oxygen 
saturation recorder 
o Provide and document training of families regarding giving study drug to their infant and 
using the home drug log 
o Provide and document training of families regarding obtaining and storing the saliva 
sample for caffeine levels 
o Provide and document training of families related to study expectations for future 
communication (i.e. telephone contacts)  and study visits for the MRI, if necessary, and 
the end of study visit  
• 
Following discharge families will be contacted within 1 – 3 days after discharge and then at least 
weekly to support families in performing study procedures including drug administration, use of 
oximeter (if applicable), and ascertain any concerns about these procedures or the clinical status 
of the infant, including the occurrence of adverse events. 
• 
The parents of each subject will receive gift cards for a total of $100, at completion of their 
participation in the study. The purpose is to defray travel expenses for the return visit, if 
applicable, and for baby supplies, indicating our appreciation for their support of the collective 
protocol requirements. 
 
6 
STUDY INTERVENTION 
  
6.1 
STUDY INTERVENTION(S) ADMINISTRATION 
 
6.1.1 STUDY INTERVENTION DESCRIPTION 
• 
The intervention for this study is the extended treatment of infants with caffeine, for evaluation 
of the efficacy of extended caffeine in ameliorating persisting IH and its adverse consequences 
including inflammation and brain MRI structural and functional injury.  Caffeine is a standard 
treatment in preterm infants in the NICU, but currently is commonly discontinued between 32 
and 36 weeks PMA.  The FDA has granted an IND Exemption for the extended treatment with 
caffeine to 42 weeks + 6 days.  
 
 
6.1.2 DOSING AND ADMINISTRATION 
• 
The dose of caffeine base will be the same caffeine dose per kg as determined in our recent pilot 
study, both the QD starting dose and the BID dose at 36 weeks + 0 days PMA to adjust for 
increasing rates of metabolism.  
• 
The caffeine doses used have been shown in our pilot study to be sufficient to achieve and 
maintain effective systemic concentrations for significant attenuation of IH 
• 
The equal volume placebo will be identical in appearance to the caffeine  

 
 
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• 
Due to the relatively long half-life of caffeine, timing of doses may have some variance to best 
accommodate routine care and feeding schedules. The time of the 1st dose will be approximately 
24 hours after the last routine clinical dose, and the subsequent QD doses will be given at the 
same approximate time each day, as consistent with daily care schedules. Timing of the BID doses 
starting at 36 weeks + 0 days PMA will be determined as consistent with daily care schedules, and 
will then be continued at the same times every day.  After discharge, timing of the doses may 
initially be modified to be most consistent with home care schedules, and then repeated at the 
same approximate time(s) each day.  
• 
All study drug will be given prior to a feeding.    
• 
Study drug will be continued from the 1st dose, which will be given sometime between 32 weeks 
+ 0 days and 36 weeks + 6 days PMA, through the last scheduled dose at 42 weeks + 6 days PMA. 
• 
Study drug will be administered by clinical care personnel in the same manner as for routine 
caffeine use. Clinical care and/or research staff will teach parents how to administer so that 
administration at home will be comparable to that in the NICU. 
• 
For home use, families will be instructed how to draw up the proper dose from the study drug 
vials supplied by research staff. 
• 
Missed QD doses will be given as soon as possible after the missed dose, but that dose will be 
eliminated (and details recorded) if missed for more than 12 hours. For BID doses, missed doses 
will be given as soon as possible after the missed dose, but that dose will be eliminated (and 
details recorded) if missed for more than 6 hours.  
 
6.2 
PREPARATION/HANDLING/STORAGE/ACCOUNTABILITY 
 
6.2.1 ACQUISITION AND ACCOUNTABILITY 
Each clinical research site is responsible for procuring the required supplies needed to prepare blinded 
doses for both inpatient and outpatient doses. See Pharmacy Manual for specific compounding 
ingredients and instructions. 
• 
Instructions will be provided to the parents/caregivers to administer the blinded oral doses just 
prior to the morning feeding and just prior to a late afternoon or early evening feed (breast or 
bottle). A diary will be provided for recording all administered doses.  
• 
All used bottles and unused study solution will be returned to the research pharmacy (IDS) at 
study end. 
6.2.2 DOSE PREPARATION, AND LABELING 
Inpatient Doses:  Caffeine base or Placebo  
Caffeine base:  IDS pharmacies will prepare a bulk oral solution containing caffeine base (anhydrous) 10 
mg/mL, according to the instructions in the Pharmacy Manual.  A bulk solution can be prepared 
individually for a patient or can be used for multiple patients (preferred) who are assigned/randomized 
to receive active treatment. 
A 24 hour supply is prepared from the bulk solution and dispensed in a suitable-sized unit-dose, oral 
syringe. All oral syringes prepared and administered in the inpatient setting are to be labeled in a blinded 
fashion, using specific departmental procedures for labeling of blinded syringes and reflecting blinded 

 
 
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study drug information on the medication administration record (e.g. eMAR).  All syringes must be labeled 
as “For Investigational Use Only” (or alternative site specific verbiage to identify a study medication).  
Syringes can be prepared as long as 4 days prior to administration (BUD 4 days) and should be stored at 
room temperature. 
Example:  Caffeine base 10 mg/1 mL or Placebo; Caffeine base 10 mg or placebo (1 mL)       
 
Placebo:  Research pharmacies will prepare a bulk oral solution according to instructions in the pharmacy 
manual.  A bulk solution can be prepared individually for a patient or can be used for multiple patients 
(preferred) who are assigned/randomized to receive placebo treatment. 
A 24 hour supply is prepared from the bulk solution and dispensed in a suitable-sized unit-dose, oral 
syringe. All oral syringes prepared and administered in the inpatient setting are to be labeled in a blinded 
fashion, using specific departmental procedures for labeling of blinded syringes and reflecting blinded 
study drug information on a medication administration record (e.g. eMAR). All syringes must be labeled 
as “For Investigational Use Only” (or alternative site specific verbiage to identify a study medication). 
Syringes can be prepared as long as 4 days prior to administration (BUD 4 days) and should be stored at 
room temperature. 
Example:  Caffeine base 10 mg/1 mL or Placebo; Caffeine base 10 mg or placebo (1 mL) 
 
Outpatient Doses: 
Doses intended for outpatient administration, starting at time of discharge and continuing until 42 weeks 
+ 6 days PMA, are prepared by the IDS pharmacy at each clinical site and issued as a bulk solution 
containing a sufficient quantity of either the active drug (caffeine base) or placebo to last until 42 weeks 
+ 6 days (BUD 90 days).   
6.2.3 PRODUCT STORAGE AND ACCOUNTABILITY 
IDS pharmacies are temperature controlled and have access limited to pharmacy personnel only.  
Compounding and accountability records are maintained by the IDS pharmacy to reflect drug preparation 
and dispensing for patients receiving both the active and placebo solutions, prepared for both inpatient 
and outpatient treatment. All supplies used to prepare both the active and placebo oral solutions are to 
be stored in the IDS/research pharmacy, in accordance with temperature parameters indicated on the 
product label. 
Sites may use/develop their own accountability forms; specific information to be documented on the drug 
accountability record includes: patient initials, patient study ID number, individual dose (mg per mL), lot 
number and beyond use date of the prepared solution.  For outpatient dosing, the quantity dispensed 
(mLs), the dose and the dispensing date must also be recorded.  The use of an electronic record (e.g., 
Vestigo) is acceptable as long as the required information is captured, including patient-returned 
medication. 

 
 
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Any remaining doses of study drug not administered at home are to be returned by the family member 
during the end-of-study outpatient visit if applicable, or sent back, and eventually returned to the IDS 
pharmacy for on-site destruction following reconciliation and accountability of the unused doses.  Used 
bottles are to be returned to the IDS pharmacy; if remaining solution is present, it is to be measured and 
recorded as a return.  Empty bottles are to be recorded as zero mL.  Once the information for the returned 
bottle has been recorded, the solution and bottle can be disposed of as non-hazardous pharmaceutical 
waste, according to institutional procedures. 
6.3 
MEASURES TO MINIMIZE BIAS: RANDOMIZATION AND BLINDING 
Randomization: 
• 
Ideally one day following, but up to the third calendar day following the last routine caffeine 
treatment dose, enrolled infants will be randomized in a 1:1 ratio to 1 of 2 treatment arms, 
caffeine base or placebo.  
• 
Infants from multiple births will be cluster randomized.   
• 
A site-stratified randomly-permuted blocked randomization design will be used to ensure balance 
at each site.  
• 
To ensure balance by gestational age at birth, randomization will also be stratified into 2 birth 
gestational age categories: 1) <28 weeks and 2) 28 weeks + 0 days through 30 weeks + 6 days 
gestation.  
• 
A randomization scheme will be developed by the DCC using specialized software, and clinical 
sites will be provided appropriate enrollment logs and blinded randomization procedures. 
 
Blinding:  
• 
Treatment assignments will be blinded until study completion for all study personnel, except for 
the clinical site research pharmacy dispensing study drug.  
• 
The study pulse oximeter will not have visual displays or alarms for respiration or heart rate 
activated during the study period ending at 43 weeks + 6 days. The IH which is the focus of study 
typically does not result in overt apnea or bradycardia, and therefore we do not anticipate a need 
for unblinding based on oximeter results 
• 
Any requests for unblinding will be adjudicated by one of the study Principal Investigators, and 
can only be authorized by them. 
• 
Two salivary caffeine concentrations are being obtained from all subjects, but the results of the 
caffeine concentrations will be blinded until all infants have completed the study protocol. 
• 
Potential caffeine side effects, such as mild tachycardia, irritability or feeding difficulties are 
frequent in both caffeine-treated non-treated infants. If such concerns arise, however, at the 
discretion of the clinical care team, study drug may be held for the days necessary, and noted in 
study records, but would generally not be a reason to request unblinding. 
• 
If apnea-related symptoms requiring treatment occur after starting study drug, the clinical care 
team may institute treatment as needed until cessation of these symptoms, and similarly, this 
also should not require unblinding.  
6.4 
STUDY INTERVENTION COMPLIANCE 
• 
Approximately the 1st half of study duration will be in the later weeks of the NICU admission. 
During this time, compliance with protocol requirements will be closely monitored by research 
staff and documented in the database. 

 
 
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• 
To maximize compliance with study protocol during the several weeks at home, families will be 
instructed in all aspects of protocol adherence while in the NICU, and reinforced just prior to 
discharge. 
• 
Once discharged, research staff will contact each family within the first 2-3 days home to address 
parental concerns or questions, and encourage compliance with study procedures.  Staff will re-
contact each family at least weekly thereafter to support families and encourage full home 
compliance with protocol requirements.  
• 
Two salivary caffeine concentrations will be obtained in all subjects.  Although results will not be 
available until the end of the study, the results will be useful in analyses to assess adherence to 
the administration of study drug, and understand the relationship between caffeine 
concentrations and study safety and efficacy outcomes. 
6.5 
CONCOMITANT THERAPY 
Uniform treatment guidelines for routine treatment caffeine have been developed for the study and 
agreed to by sites.  The Clinical Guidelines for Routine Caffeine Use in NICU (Prior to enrollment in ICAF) 
include: For infants with birth weight < 1250 grams, initiate caffeine treatment within the first 3 days after 
birth in all infants.  If on CPAP only:  initiate caffeine treatment within the first 24 hours. If approaching 
extubation in first 3 days after birth prior to any caffeine treatment, initiate caffeine treatment 6 to 8 
hours prior to extubation.  For infants with birth weight ≥1250 grams, if on CPAP or on no respiratory 
support, initiate caffeine treatment if the infant experiences >6 apnea episodes requiring stimulation. If 
the infant requires mechanical ventilation, consider starting caffeine treatment prior to extubation.  The 
initial caffeine dose is a 20 mg/kg bolus dose of caffeine citrate (or equivalent caffeine base) followed by 
5-10 mg/kg/day maintenance dose, beginning 24 hours after the loading dose.  Levels are not required to 
monitor for dosage adjustment. Stop routine caffeine treatment once the infant is free of clinically 
significant episodes of apnea for 5-7 days off of any positive pressure (CPAP or NC > 1L) or at 34 weeks 
PMA, whichever comes first.  If the maintenance dose is less than 5 mg/kg, infants may be allowed to 
“outgrow” their dose of caffeine and the dose should be stopped as noted above (i.e. no clinically 
significant episode for 5-7 days off any positive pressure (CPAP or NC > 1L) or 34 weeks PMA, whichever 
comes first.) Following randomization, open label caffeine use is discouraged, however, there are no 
prohibited therapies and sites will manage all participants based on their best clinical judgment, with 
guidance as needed by the an ICAF Principal Investigator.   
 
6.5.1 RESCUE MEDICINE 
As noted above, rescue therapy with open-label caffeine therapy is discouraged.   Recurrent apnea-
related symptoms have not occurred in preliminary studies after stopping routine caffeine. 18,90 
However, in the event that an infant has apnea-related clinical symptoms after stopping routine caffeine 
and starting study drug, study drug should be continued, and if judged to be needed by the clinical team, 
nasal CPAP or nasal cannula is the preferred additional therapy.   If the clinical team ultimately decides 
that open-label caffeine must be used, we suggest the following:  1) Hold study drug; 2) begin open-
label maintenance caffeine citrate at 5-10 mg/kg/day on the same day; 3) as soon as open-label caffeine 
is no longer deemed necessary, stop open-label caffeine and restart study drug at the next regularly 
scheduled time for study drug; 4) there should be no need to unblind the treatment group, since 
published studies have suggested that it is safe to give the suggested dose of open-label caffeine to 
either treatment group.3 If the attending physician thinks unblinding may be needed they should contact 
the clinical site principal investigators. 

 
 
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Pulse oximeter recordings will continue through hospital discharge, or at home through 1 week 
following discontinuation of study drug, for infants recruited at sites participating in home oximeter 
recording. The PMA at which study drug is scheduled to end is well after the clinical team judged it safe 
to stop routine caffeine treatment and at a PMA at which prior studies have shown that the extent of IH 
no longer differs from healthy infants born at term. However, for sites continuing oximeter recordings 
at home, the oximeter alarms may be activated for this final study week if deemed necessary by the 
clinical site principal investigators. 
 
7 
STUDY INTERVENTION DISCONTINUATION AND PARTICIPANT 
DISCONTINUATION/WITHDRAWAL 
 
7.1 
DISCONTINUATION OF STUDY INTERVENTION 
The clinical site principal investigators may discontinue the participant’s activity without the participant’s 
consent if either of these criteria are met: 
• 
A participant consistently fails to comply with study procedures 
• 
A participant’s safety or health may be compromised by further participation 
 
7.2 
PARTICIPANT DISCONTINUATION/WITHDRAWAL FROM THE STUDY 
Each study participant may withdraw consent at any time during the study without penalty. In the event 
a participant is discontinued and thus no longer receiving the study drug, the participant will be asked to 
continue with follow-up assessment as described below, and not considered withdrawn from the study. 
If the participant refuses further follow-up, then the participant will be considered to be withdrawn. 
Counseling about the participant's health will be provided if the participant decides to discontinue 
participation in the study. Medical advice regarding what is in the best interest of the participant will be 
provided.  
All data collected up to the time of withdrawal will be reported. The End of Study eCRF will be completed, 
with the reason for withdrawal specified. 
7.3 
LOST TO FOLLOW-UP 
• 
A participant will be considered lost to follow-up if he or she fails to return for the end-of-study 
visit between 43 weeks + 0 days and 46 weeks and 6 days PMA and is unable to be contacted by 
the study site staff to (1) reschedule the final visit for the brain MRI (if applicable) to be completed 
by 46 weeks + 6 days, and (2) return all study equipment and supplies within the next 4 weeks. 
• 
If the subject does not return for the final evaluation, the site will attempt to contact the family 
and reschedule the missed visit within the next 1-4 weeks, counsel the participant on the 
importance of maintaining the assigned visit schedule, and ascertain if the family wishes to and/or 
should continue in the study. 
• 
Before a participant is deemed lost to follow-up, the clinical site investigator or designee will make 
every effort to regain contact with the participant by multiple means or by contacting designated 

 
 
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alternative contacts. These contact attempts will be documented in the participant’s medical 
record or study file.  
• 
Should the participant continue to be unreachable, the participant will be considered to have 
withdrawn from the study with a primary reason of lost to follow-up. 
 
 

 
 
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8 
STUDY ASSESSMENTS AND PROCEDURES 
 
8.1 
EFFICACY ASSESSMENTS  
8.1.1 OXYGEN SATURATION  
Continuous recording of O2 saturation using the study pulse oximeter is planned for all infants through 
hospital discharge, and for the sub-group of infants recruited at hospitals participating in home oximetry 
recording ,will continue  through 43 weeks + 6 days PMA (i.e. one week following the last dose of study 
drug).  The study oximeter will be the Masimo RAD-97, set at the minimum possible averaging time of 2-
4 sec and a sampling rate of 1 sec (high resolution).  The oximeter will be preset in the sleep lab mode, 
hence with no “probe off,” “low battery,” or physiologic visual displays or alarms.  The internal data 
storage capacity of the RAD-97 oximeter is sufficient for 28 days of continuous data collection. 
Inpatient Phase: During the inpatient phase, the RAD-97 will be a 2nd oximeter, in addition to the one used 
for standard monitoring in the NICU.  Continuous oximeter recordings will be obtained whenever in the 
NICU.   Data will be downloaded from the monitor, and archived by the DCC, at least every 4 weeks while 
in the NICU and at discharge. 
Please note:  Some sites may discontinue oximeter recordings at hospital discharge. 
Outpatient Phase:  At sites participating in the home oximetry portion of the study, prior to discharge, 
the family will be trained in the use of the RAD-97, and will be asked to use the oximeter during all sleep 
times and quiet awake periods until 43 weeks + 6 days PMA.  At home, therefore, we expect at least 8 – 
12 hours of use per day, so the memory capacity of the RAD-97 should be sufficient to complete the 
outpatient phase.   
Following discharge, the family will be contacted within the first 2 – 3 days, and then at least weekly to 
provide support in the use of the study monitor and to support other aspects of the study protocol.  As 
part of their support of the use the monitor at home, study site staff will assess if the use pattern is such 
that the oximeter memory might be close to capacity, and determine if it is necessary to schedule an 
additional data download. 
8.1.2 INFLAMMARORY BIOMARKERS 
An EDTA blood sample (0.6 – 0.8 ml) to measure a panel of inflammatory biomarkers will be obtained at 
enrollment prior to randomization, and again at 38 weeks + 0 days PMA or NICU discharge, whichever 
comes first. The blood will be centrifuged and the resultant plasma aliquoted into tubes and immediately 
frozen (-80 °C). The intent is to freeze a plasma volume of at least 0.3 ml. As specified by later shipping 
instructions, the frozen samples will be batched and shipped on dry ice overnight using shipping labels 
that will be provided.  
 

 
 
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Bulk analyses of inflammatory biomarker plasma concentrations will be performed at Children’s National 
Medical Center using a commercially available ELISA multi-spot assay to measure inflammation-related 
proteins from different functional categories of cytokines and chemokines, including growth factors, and 
adhesion molecules, IFN-alpha, IL-6, Gro/CXCL1, IL-1β, IL-4-6, IL-6 receptor, IL-8, IL-10, IL-13, ICAM-1, 
myeloperoxidase, CRP, MCP, IGFBP-1, MIP-1a, RANTES, and TNFa. Analytes that show strong trends or 
significance with this assay may be further analyzed with individual ELISA assays, to confirm the original 
result. 
 
8.1.3 QUANTITATIVE 
MRI 
STRUCTURAL, 
MICROSTRUCTURAL 
AND 
METABOLIC 
BIOMARKERS OF ACUTE BRAIN INJURY 
Our goal is to obtain baseline and follow-up brain MRI in approximately half of the study participants (55 
subjects in each study arm).   For additional information, refer to MRI Manual. 
 
At sites recruiting infants into the MRI portion of the study, infants will be quieted by swaddling and 
feeding before the MRI study. O2 saturation, heart rate and temperature will be monitored and recorded 
throughout the scan. All infants will be protected from MRI scanner noise by the use of ear wax, and mini 
muffs (Natus MiniMuffs Noise Attenuators). MRI acquisition protocols will be standardized and field-
tested across all sites in Y1 Q1-3, and MRI calibration studies will be performed to ensure that the MRI 
scanner properties and parameter settings during the acquisition phase are correct. Conventional MRI 
studies will be reviewed in a standardized fashion by a pediatric neuroradiologist at each site blinded to 
study randomization.  
 
All MRI data sets will be processed at the Advanced Pediatric Brain Imaging Research Laboratory (DBRL) 
at Children’s National Medical Center. All quantitative MRI outcome measures are continuous and will be 
performed by a single investigator masked to randomization. Abnormalities of brain development, 
maturation, the presence of focal destructive ischemic or hemorrhagic lesions, will be documented. 
 
8.1.4 SALIVARY CAFFEINE LEVELS 
Two salivary samples will be obtained for later analysis of caffeine concentrations, the 1st in the NICU 1 
week after starting study drug, and the 2nd, which may be either in the hospital or at home by parents, at 
about 40 weeks + 0 days PMA.  All samples will be pre-prandial. Salivary volumes of 200-300 uL will be 
collected using commercially available kits (Salimetrics, State College, PA) and frozen at -20o C at home 
and then at -20° C or colder after return to the clinical site research team for bulk analysis at the end of 
the study. Parents will return the home salivary sample at the same time as their scheduled return to the 
clinical site at study end. As specified by later shipping instructions, the frozen salivary samples will be 
batched and shipped on dry ice overnight with the plasma samples using shipping labels that will be 
provided.  
Salivary analyses will be performed by the Biomedical Research Lab at Walter Reed National Military 
Medical Center. Sample preparation and chromatography will utilize a method validated in our 
preliminary study.89,90  Chromatography will be performed using a Waters Alliance 2695 Separations 
Module equipped with a Waters 2996 Photodiode Array detector and Waters Symmetry column. 

 
 
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8.1.5 BRIEF INFANT SLEEP QUESTIONNAIRE (BISQ) 
The Brief Infant Sleep Questionnaire (BISQ) is a parent-reported questionnaire on infant/toddler sleep 
over the prior one week.  It includes 13 items in three categories (sleep duration, night wakings, and 
method of falling asleep).  The BISQ will be completed by phone during the weekly phone call that is 
scheduled between 42 weeks 0 days and 42 weeks 6 days, or in person, at study termination if this occurs 
prior to 42 weeks + 0 days.  
 
 
8.2 
SAFETY AND OTHER ASSESSMENTS 
8.2.1 TELEPHONE ASSESSMENT OF STATUS AT HOME 
Within 1 – 3 days following hospital discharge to home, and at least weekly thereafter, study site staff will 
contact families by phone and complete a phone contact log.  The phone contact log will document: 
• 
Family questions or concerns 
• 
Assessment of completion of the drug log and issues related to administration or supply of study 
drug 
• 
Assessment of extent of use of study oximeter (if applicable) and any questions, problems or 
other concerns related to study drug 
• 
Discussion of upcoming study procedures  
• 
Assessment for occurrence of adverse events 
• 
Determination of use of concomitant medications 
8.3 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
 
8.3.1 DEFINITION OF ADVERSE EVENTS (AE) 
• 
Adverse event means any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 (a)). 
• 
Since there will be multiple participating clinical sites, centralized safety oversight will be 
coordinated by the DCC in conjunction with the ICAF principal investigators. 
• 
The study does not involve an investigational new drug, but is extending the duration of caffeine 
treatment beyond the customary age of discontinuing caffeine treatment in the NICU. The FDA 
has deemed the use of caffeine in this study as consistent with prior FDA approval, and has 
therefore granted an IND Exemption.   
• 
There is no risk in randomizing to placebo; absent enrollment in this study, infants would not be 
treated with caffeine. 
8.3.2 DEFINITION OF SERIOUS ADVERSE EVENTS (SAE)  
An adverse event (AE) or suspected adverse reaction is considered "serious" if, in the view of either the 
investigator or sponsor, it results in any of the following outcomes: death, a life-threatening adverse 
event, inpatient hospitalization or prolongation of existing hospitalization, or a persistent or significant 
incapacity or substantial disruption of the ability to conduct normal life functions. Important medical 
events that may not result in death, be life-threatening, or require hospitalization may be considered 

 
 
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serious when, based upon appropriate medical judgment, they may jeopardize the participant and may 
require medical or surgical intervention to prevent one of the outcomes listed in this definition.    
Note that for the purpose of this study, delaying discharge of an infant related to achieving a specified ‘spell-free’ 
number of days shall NOT be considered a ‘prolongation of existing hospitalization’ for the purpose of categorizing 
an event as a serious adverse event. 
 
 
 
 
8.3.3 CLASSIFICATION OF AN ADVERSE EVENT 
8.3.3.1 SEVERITY OF EVENT 
The following guidelines will be used to describe severity.  
 
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
• 
Severe – Events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment. Severe events are usually potentially life-threatening or 
incapacitating.  Of note, the term “severe” does not necessarily equate to “serious”. 
8.3.3.2 RELATIONSHIP TO STUDY INTERVENTION 
All adverse events (AEs) will have their relationship to study intervention assessed by the clinical site 
principal investigators based on temporal relationship and clinical judgment. The degree of certainty 
about causality will be graded using the categories below:  
• 
Definitely Related – There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event occurs in a plausible time relationship to 
study intervention administration and cannot be explained by concurrent disease or other drugs 
or chemicals.  
• 
Probably Related – There is evidence to suggest a causal relationship, and the influence of other 
factors is unlikely. The clinical event occurs within a reasonable time after administration of the 
study intervention, is unlikely to be attributed to concurrent disease or other drugs or chemicals. 
• 
Potentially Related – There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of the trial medication). However, other 
factors may have contributed to the event (e.g., the participant’s clinical condition, other 
concomitant events). An AE may rate as “possibly related” soon after discovery, but it can be 
flagged as requiring more information and later be upgraded to “probably related” or “definitely 
related”, as appropriate. 
• 
Unlikely to be related – A clinical event whose temporal relationship to study intervention 
administration makes a causal relationship improbable and other drugs or underlying disease 
provides plausible explanations (e.g., the participant’s clinical condition, other concomitant 
treatments). 
• 
Not Related – The AE is completely independent of study intervention administration, and/or 
evidence exists that the event is definitely related to another etiology. The alternative, definitive 
etiology must be documented by the clinical care team. 
8.3.3.3 EXPECTEDNESS  

 
 
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• 
The clinical site principal investigators will be responsible for determining whether an adverse 
event (AE) is expected or unexpected.  An AE will be considered unexpected if the nature, severity, 
or frequency of the event is not consistent with the well-established risk information previously 
described for caffeine. 
 
 
• 
Expected AE for caffeine treatment can include tachycardia, feeding intolerance, jitteriness. 
However, no significant AE are expected since all enrollees will have previously tolerated routine 
caffeine treatment at systemic concentrations similar to those achieved in this study.   
 
 
8.3.4 TIME PERIOD AND FREQUENCY FOR EVENT ASSESSMENT AND FOLLOW-UP 
• 
The occurrence of an adverse event (AE) or serious adverse event (SAE) may come to the attention 
of clinical care personnel as part of routine NICU care, or by research staff or parents. 
• 
All AEs including local and systemic reactions not meeting the criteria for SAEs will be captured on 
the appropriate case report form (CRF). Information to be collected includes event description, 
time of onset, clinician’s assessment of severity, relationship to study product (assessed only by 
those with the training and authority to make a diagnosis), and time of resolution/stabilization of 
the event.  
• 
All AEs occurring while on study must be documented appropriately regardless of relationship. All 
AEs will be followed to adequate resolution. 
• 
Any medical condition that is present at the time that the participant is enrolled will be considered 
as baseline and not reported as an AE. However, if the study participant’s condition deteriorates 
at any time during the study, it will be recorded as an AE.  
• 
Changes in the severity of an AE will be documented to allow an assessment of the duration of 
the event at each level of severity to be performed. AEs characterized as intermittent require 
documentation of onset and duration of each episode.  
• 
Designated study site research staff will record all reportable events occurring in the NICU  
starting  from when informed consent is obtained until the last day of study participation (for non-        
serious AEs) or 30 days (for SAEs) after the last day of study participation.  NOTE: Last day of study 
participation may be any of the following: 
o end-of-study visit 
o follow-up MRI visit 
o Lost-to-follow-up determination date 
• 
After discharge home, research staff will query parents at least weekly about the occurrence of 
AE/SAEs since the last contact.  Events will be followed for outcome information until resolution 
or stabilization. 
 
 
 
8.3.5 ADVERSE EVENT REPORTING  
AEs and SAEs will be documented in the source records and recorded on the eCRFs using accepted medical terms 
and/or the diagnoses that accurately characterize the event. When a diagnosis is known, the AE term recorded on 
the eCRF will be the diagnosis rather than a constellation of symptoms. The clinical site principal investigators or 

 
 
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their designee will assess all AEs for seriousness, relationship to investigational product, severity, expectedness, and 
other possible etiologies. When an event has not resolved by study closure, it will be documented on the AE eCRF 
as “ongoing”. 
The timeframe for the collection of AEs and SAEs is noted above in section 8.3.4. 
 
 
 
 
 
 
8.3.6 SERIOUS ADVERSE EVENT REPORTING  
All SAEs must be reported promptly (within 24 hours) to the study DCC, whether or not the event is 
considered related to study product. Further, the clinical site principal investigators should comply with 
relevant clinical study site IRB requirements on reporting SAEs. 
Clinical site principal investigators must submit additional information as soon as it is available on the SAE 
report form. The study DCC will report unexpected SAEs associated with the use of the drug as required.  
Clinical site principal investigators must follow all relevant regulatory requirements as well as specific 
policy regarding the timely reporting of SAEs to the study DCC and the clinical study site  IRB.  
Reporting to the study DCC does not fulfill the clinical site principal investigator’s duty to report all 
unanticipated problems involving risk to human subjects or others to the IRB. The clinical site principal 
investigators will notify the clinical study site IRB and ICAF principal investigators.  
• 
Any AE considered serious by the clinical site principal investigator or an ICAF principal 
investigator, or which meets the definition of an SAE included in Section 8.3.2, Definition of 
Serious Adverse Events must be submitted on an SAE form to the Data Coordinating Center (DCC).   
• 
The DSMB will receive expedited notification of all SAEs, even if not thought to be related to study 
intervention.   
• 
All serious adverse events (SAEs) will be followed until satisfactory resolution or until the clinical 
site principal investigator deems the event to be chronic or the participant is stable. Other 
supporting documentation of the event may be requested by the Data Coordinating Center (DCC) 
and should be provided as soon as possible. 
 
8.3.7 REPORTING EVENTS TO PARTICIPANTS  
Not Applicable 
 
 
8.3.8 EVENTS OF SPECIAL INTEREST  
• 
Although rare, infants born at ≤30 weeks gestation and discharged home are at risk for an 
apparent life-threatening event (ALTE, or brief resolved unexpected event, BRUE) or for sudden 
unexpected infant death (SUID) 

 
 
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• 
Infants enrolled in this study are at no greater risk, and perhaps at even less risk, for such events 
than infants not enrolled   
 
8.3.9 REPORTING OF PREGNANCY  
Not applicable  
 
8.4 
UNANTICIPATED PROBLEMS 
 
8.4.1 DEFINITION OF UNANTICIPATED PROBLEMS (UP) 
Federal regulations (45 CFR Part 46/32 CFR 219) require that unanticipated problems involving risks to subjects or 
others be promptly reported to the IRB. These events encompass a broader category of events than SAEs and may 
include issues such as problems with loss of control of subject data or the investigational product; adverse 
psychological reactions; or breach of confidentiality. Risks to others (e.g., program personnel) must also be reported.   
 
The Office for Human Research Protections (OHRP) considers unanticipated problems involving risks to 
participants or others to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
 
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
participant population being studied; 
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
 
 
8.4.2  UNANTICIPATED PROBLEM REPORTING  
The clinical site principal investigators will report unanticipated problems (UPs) occurring from the time 
the informed consent form is obtained through 30 days after the last day of study participation to the 
clinical study site Institutional Review Board (IRB) and to the Data Coordinating Center (DCC)/ICAF 
principal investigators The UP report will include the following information: 
• 
Protocol identifying information: protocol title and number, Clinical site principal investigator’s 
name, and the IRB project number; 
• 
A detailed description of the event, incident, experience, or outcome;  
• 
An explanation of the basis for determining that the event, incident, experience, or outcome 
represents an UP;  
• 
A description of any changes to the protocol or other corrective actions that have been taken or 
are proposed in response to the UP. 
 
To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline:   
 
• 
UPs will be reported to the clinical study site IRB and to the DCC and PI/Co-PI within  1 business 
day  of the investigator becoming aware of the event.  

 
 
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All UPs should be reported to appropriate institutional officials (as required by an institution’s written 
reporting procedures), the supporting agency head (or designee), and the Office for Human Research 
Protections (OHRP) promptly after the clinical study site IRB’s receipt of the report of the problem from 
the clinical site principal investigators. 
 
 
 
 
9 
STATISTICAL CONSIDERATIONS  
 
9.1 
STATISTICAL HYPOTHESES 
Primary Objective 1: Compare the extent of IH exposure, from randomization through 42 weeks + 6 
days PMA (within each gestational week and overall), in infants randomized to extended caffeine 
treatment to infants assigned to receive placebo. We hypothesize that caffeine will reduce the extent of 
IH compared to placebo, and that this caffeine effect will persist through 42 weeks PMA.    
 
Primary Objective 2: Compare changes in a panel of inflammation-related cytokines and chemokines, 
from enrollment to the target age of 38 weeks + 0 days PMA, in infants randomized to extended caffeine 
treatment to infants assigned to receive placebo. We hypothesize that continuing caffeine treatment will 
be associated with less inflammation compared to placebo. 
 
Primary Objective 3: Compare changes in quantitative MRI structural, microstructural and metabolic 
biomarkers of acute injury, from enrollment to 43-46 weeks PMA, in infants randomized to extended 
caffeine treatment to infants assigned to receive placebo. We hypothesize that continued caffeine 
treatment compared to placebo will be associated with 1) greater white matter microstructural 
organization, 2) improved neuronal metabolism, and 3) greater regional brain volume. 
Secondary Objective 1: Examine the association between salivary caffeine concentrations and IH 
outcomes at the one week post randomization and 40 weeks + 0 days PMA assessments. We 
hypothesize that those with higher salivary caffeine concentrations will have less severe IH outcomes. 
Secondary Objective 2: Determine whether caffeine effects on changes in inflammatory or MRI 
biomarkers from baseline to follow-up are mediated by caffeine-related reduced IH.  We hypothesize that 
caffeine effects on changes in inflammatory or MRI biomarkers will be at least partially mediated by 
reduced IH.  
9.2 
SAMPLE SIZE DETERMINATION 

 
 
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Sample Size: We will randomize 220 infants, 110 in each treatment group, with ≥100 infants/group 
available for final analyses. Sample size considerations focus on PMA week-specific differences between 
the extended caffeine vs. placebo infants for our primary outcome of #sec/day < 90% saturation from 36 
to 42 weeks PMA. Preliminary data showed effect sizes at 37 and 38 weeks PMA of 0.87 and 0.73.  With 
the decline in IH with increasing age, we expect that 1) the caffeine effect may decline and 2) moderate 
effects will be needed to achieve a meaningful reduction in IH to 42 weeks PMA.  Therefore, we consider 
an effect size of 0.50 to represent a meaningful detectable difference between groups.  Final samples of 
n=100 per group gives 82% power to detect this effect, testing at the overall 2-tailed .05 level with a 
familywise Type I error correction adjusting for multiple testing across weeks. Secondary outcomes of #IH 
events and #sec < 80% saturation/day showed effect sizes at 38 weeks PMA of 0.73 and 0.46 in our pilot 
data.  
 
 
9.3 
POPULATIONS FOR ANALYSES 
The intention-to-treat (ITT) population will consist of all randomized subjects.   
 
The modified intention-to-treat (mITT) population will include all subjects who took at least one dose of 
study drug and who have data available for efficacy outcomes.   
 
The safety population will consist of all randomized subjects who receive a dose of study drug. 
 
 
9.4 
STATISTICAL ANALYSES 
 
9.4.1 GENERAL APPROACH 
Statistical significance will be declared at the two-sided p <0.05 level; when appropriate, correction for 
multiple comparisons will be employed.  For analyses of primary and secondary objectives, we will adjust 
for demographic and clinical differences.  When employed, tests for interaction will at the 0.1 alpha level.  
Results will be presented through measures of effect and 95% confidence intervals. If lost to follow-up 
exceeds the 10% anticipated under sample size considerations, multiple imputation on the intent-to-treat 
population will be used for sensitivity analyses that account for missing data. All computations will be 
performed using the latest available version of SAS®. 
 
Demographic variables and subject characteristics will be summarized descriptively by treatment 
assignment and overall. Continuous parameters will be summarized using descriptive statistics (N, mean, 
median, standard deviation, minimum and maximum value). Categorical demographic parameters, such 
as gender, will be summarized as a percentage of the population. 
9.4.2 ANALYSIS OF THE PRIMARY EFFICACY ENDPOINTS  
Primary Objective 1: Compare the extent of IH exposure, from randomization through 42 weeks + 6 
days PMA (within each gestational week and overall), in infants randomized to extended caffeine 
treatment to infants assigned to receive placebo. 
Analyses for this objective will be performed on the mITT population and compare those randomized to 
extended caffeine vs. placebo.  To be included in IH analyses, a subject must have had at least 10 hours of 
acceptable quality oximetry data recorded within at least one PMA week. A single overall baseline SaO2 

 
 
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will be determined for each infant, confirmed in our pilot study to be ≥95% for the PMA weeks being 
studied. An IH event is defined as SaO2 decrease ≥10% (event threshold) and lasting ≥5 sec; the event ends 
when SaO2 increases above the event threshold. For sec of SaO2 below threshold, all sec below the 
threshold are included. We will compare extended caffeine vs. placebo infants on measures of IH, with # 
of sec <90% saturation/day our primary outcome. Our pilot data showed that # of sec <90% optimally 
discriminates between infants on vs. not on extended caffeine, with high correlation between #sec < 90% 
saturation/day and multiple other measures to quantify IH which will be secondary outcomes, including 
#IH events/day, # sec < 80% saturation/day, nadir saturation during events, duration of events 
(Spearman’s correlations r >0.90, and AOC during events below 90% and 80% threshold saturation 
(r=0.83).90 Preliminary data showed that these outcomes follow a log-normal distribution, and data will 
be log-transformed for analysis; non-parametric procedures will be considered as an alternative analytic 
approach if warranted by the distribution of the IH outcomes. Exploratory analyses will also compare 
caffeine vs. placebo on heart rate data. As a check on randomization, preliminary analyses will compare 
the two study groups on key characteristics including gestational age, birth weight,  infant sex, morbidity 
(e.g., necrotizing enterocolitis, mild BPD), and severity of illness measures; variables found to differ 
meaningfully between groups will be controlled for in primary analyses through multiple linear regression 
models. Primary analyses will determine how long extended caffeine provides an advantage over placebo 
by testing PMA week-specific differences between study groups through multiple regression models 
controlling for key covariates using Holm’s family-wise error adjustment to account for multiple testing 
across PMA weeks.   
Primary Objective 2: Compare changes in a panel of inflammation-related cytokines and chemokines, 
from enrollment to the target age of 38 weeks + 0 days PMA, in infants randomized to extended caffeine 
treatment to infants assigned to receive placebo.  
Analyses for this objective will be performed on the mITT population and compare those randomized to 
extended caffeine vs. placebo.  To be included in the analyses a subject must have had a baseline/follow-
up pair of blood samples for which at least one biomarker was measured on both samples.  Analysis will 
focus on change in inflammatory biomarker levels over the study period.  Preliminary analyses will 
examine the distribution of biomarkers, and data will be transformed (e.g., logged) as appropriate for 
analysis. Two approaches will be taken for the primary analyses, comparing changes in biomarkers over 
the study period in extended caffeine vs. placebo infants.  First, we will conduct separate analyses of each 
inflammatory biomarker using linear regression models to compare study groups at study completion, 
controlling for baseline levels as well as key covariates including morbidities that might be associated with 
inflammation, using the Benjamini and Hochberg method to account for multiple testing by controlling 
the false discovery rate.  Second, we will examine the structure of the biomarker data using data reduction 
procedures of factor analyses (identifying latent variables reflected by the set of biomarkers) and latent 
profile analysis (identifying subgroups of infants with similar patterns of biomarker profiles). Extended 
caffeine vs. placebo infants would then be compared on the summary measures of inflammation 
biomarkers resulting from these analyses.  

 
 
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Primary Objective 3: Compare changes in quantitative MRI structural, microstructural and metabolic 
biomarkers of acute injury, from enrollment to 43-46 weeks PMA, in infants randomized to extended 
caffeine treatment to infants assigned to receive placebo.  
Analyses for this objective will be performed on the mITT population and compare those randomized to 
extended caffeine vs. placebo.  To be included in the analyses a subject must have had a baseline/follow-
up pair of MRI studies completed from which at least one MRI endpoint could be measured on both 
assessments. 
Primary neuroimaging outcome measures will include microstructural measures derived from our DTI 
analyses (FA/MD) and proton MRS.  Secondary outcomes will include regional tissue volumes. Individual 
end-of-study MRI parameters will be compared through analysis of covariance multiple linear regression 
models controlling for the MRI parameter at baseline along with key covariates including indicators of 
comorbidities potentially associated with brain development, using the Benjamini and Hochberg method 
to account for multiple testing by controlling the false discovery rate.  .  
Secondary Objective 1: Examine the association between salivary caffeine concentrations and IH 
outcomes at the one week post randomization and 40 weeks + 0 days PMA assessments. 
Analyses will examine the association between salivary caffeine levels and IH outcomes, both at the end 
of the inpatient stay and at study completion. Caffeine levels at home will also be used to verify protocol 
adherence. Secondary analyses will also include relationship between individual neonatal morbidities and 
extent of IH in caffeine and placebo groups. Exploratory analyses will model the trends in IH parameters 
over time in the extended caffeine and placebo groups using mixed-effects linear regression models for 
longitudinal data.  Should missing data become a problem, multiple imputation methods will be used to 
account for potential bias in the complete case analysis.  
Secondary Objective 2:  Determine whether changes, from baseline to follow-up, in inflammatory or 
MRI biomarkers are mediated by caffeine-related reduced IH. 
 
We will also examine IH levels during the study period as a mediator of the extended caffeine effect.  Initial 
analyses will examine associations between IH parameters and inflammatory biomarkers, to determine 
which IH measures are most strongly correlated with inflammation at study completion. Linear structural 
equation mediational models will be used to determine whether any associations between extended 
caffeine treatment and changes in inflammatory biomarkers can be explained by IH parameters during 
the study period. The relationship between neonatal morbidities, Inflammation, and response to caffeine 
will also be assessed. 
Similarly we will also examine associations between changes in MRI parameters and levels of IH during 
the study period through correlation and linear regression models with the goal of determining IH 
parameters most strongly associated with brain injury. Structural equation mediational models will 
determine whether any associations between extended caffeine and MRI changes can be explained by 
the level of IH activity during the study. 
 

 
 
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9.4.3 SAFETY ANALYSES 
Analyses of safety outcomes will be performed on the safety population consisting of all randomized 
subjects who receive a dose of study drug.  Descriptive data on safety outcomes will be provided by 
treatment group. 
 
10 SUPPORTING DOCUMENTATION AND OPERATIONAL CONSIDERATIONS 
 
10.1 REGULATORY, ETHICAL, AND STUDY OVERSIGHT CONSIDERATIONS 
 
10.1.1 INFORMED CONSENT PROCESS 
• 
In collaboration with the clinical care team, research staff will screen all NICU patients born at ≤30 
weeks gestation and receiving routine caffeine treatment for study eligibility 
• 
Clinical care staff will inform parents of this ongoing research study and inform research staff of 
families desiring additional study information 
• 
Parents will not be approached for formal consent before the infant is 32 weeks + 0 days PMA. 
 
 
 
10.1.1.1 CONSENT/ASSENT 
AND 
OTHER 
INFORMATIONAL 
DOCUMENTS 
PROVIDED 
TO 
PARTICIPANTS 
• 
All informational documents and consent material to be provided to parents of potential study 
subjects will be submitted to study site IRB’s and utilized in accordance with procedures approved 
by the IRB. 
 
 
 
10.1.1.2 CONSENT PROCEDURES AND DOCUMENTATION 
Informed consent is a process that is initiated prior to parental agreement to participate in the 
study and continues throughout the duration of the study. The Consent Form will be Institutional 
Review Board (IRB)-approved and the parent(s) will be asked to read and review the document. 
The investigator, or their designee, will explain the research study to the parent and answer any 
questions that may arise. A verbal explanation will be provided in terms suited to the parent’s 
comprehension of the purposes, procedures, and potential risks of the study and of their rights 
as research participants.  Parents will have the opportunity to carefully review the consent form 
and ask questions prior to signing, and will have the opportunity to discuss the study with family 
or surrogates or to think about it prior to agreeing to participate. A parent will sign the informed 
consent document prior to any procedures being done specifically for the study. Parents will be 
informed that participation is voluntary and that they may withdraw from the study at any time, 
without prejudice. A copy of the informed consent document will be given to the parents for their 
records. The informed consent process will be conducted and documented in the source 
document (including the date), and the form signed, before the subject undergoes any study-
specific procedures. The rights and welfare of the subjects will be protected by emphasizing to 

 
 
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parents that the quality of medical care will not be adversely affected if they decline to participate 
in this study. 
• 
As may be deemed necessary by the clinical site, the Consent Form will be available in Spanish as 
well as in English, and a Spanish translator will be available whenever needed. 
10.1.2 STUDY DISCONTINUATION AND CLOSURE 
This study may be temporarily suspended or prematurely terminated if there is sufficient reasonable 
cause.  If the study is prematurely terminated or suspended, the clinical site principal investigators will 
promptly inform study participants, the clinical study site Institutional Review Board (IRB), and will provide 
the reason(s) for the termination or suspension.   
  
Circumstances that may warrant termination or suspension include, but are not limited to: 
• 
Determination of unexpected, significant, or unacceptable risk to participants 
• 
Demonstration of efficacy that would warrant stopping    
• 
Insufficient compliance to protocol requirements 
• 
Data that are not sufficiently complete and/or evaluable 
• 
Determination that the primary endpoint has been met 
• 
Determination of futility 
 
 
 
10.1.3 CONFIDENTIALITY AND PRIVACY  
Information about study subjects will be kept confidential and managed according to the requirements of 
the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a 
signed subject authorization informing the subject of the following:  
• 
What protected health information (PHI) will be collected from subjects in this study 
• 
Who will have access to that information and why 
• 
Who will use or disclose that information 
• 
The rights of a research subject to revoke their authorization for use of their PHI.  
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, 
retains the ability to use all information collected prior to the revocation of subject authorization.  For 
subjects that have revoked authorization to collect or use PHI, attempts should be made to obtain 
permission to collect at least vital status (i.e. that the subject is alive) at the end of their scheduled study 
period. 
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, 
and the sponsor(s) and their interventions. This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants. Therefore, the 
study protocol, documentation, data, and all other information generated will be held in strict confidence. 

 
 
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No information concerning the study or the data will be released to any unauthorized third party without 
prior written approval of the sponsor.  
 
All research activities will be conducted in as private a setting as possible. 
 
Representatives of the clinical site Institutional Review Board (IRB) or DSMB may inspect all documents 
and records required to be maintained by the clinical site principal investigators, including but not limited 
to, medical records (office, clinic, or hospital) and pharmacy records for the participants in this study. The 
clinical study site will permit access to such records. 
 
The study participant’s contact information will be securely stored at each clinical study site for internal 
use during the study. At the end of the study, all records will continue to be kept in a secure location for 
as long a period as dictated by the clinical site IRB, Institutional policies, or sponsor requirements. 
 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will 
be transmitted to and stored at the Data Coordinating Center. This will not include the participant’s 
contact or identifying information. Rather, individual participants and their research data will be identified 
by a unique study identification number. The study data entry and study management systems used by 
clinical study sites and by Data Coordinating Center research staff will be secured and password protected. 
At the end of the study, all study databases will be de-identified and archived at the Data Coordinating 
Center. 
 
 
10.1.4 FUTURE USE OF STORED SPECIMENS AND DATA  
See Section 10.1.3, Confidentiality and Privacy and Section 10.1.9, Data Handling and Record Keeping, 
for further information on future use of study records. 
 
Data collected for this study will be analyzed and stored at Boston University Medical Center (Data 
Coordinating Center (DCC)). After the study is completed, the de-identified, archived data will be 
transmitted to and stored at the Slone Epidemiology Center at Boston University, for use by other 
researchers including those outside of the study. Permission to transmit data to the Slone Epidemiology 
Center at Boston University will be included in the informed consent.  
 
During the conduct of the study, an individual participant can choose to withdraw consent to have 
biological specimens stored for future research. However, withdrawal of consent with regard to 
biosample storage may not be possible after the study is completed.  
 
When the study is completed, access to study data and/or samples will be provided through the DCC. 
 
 
10.1.5 KEY ROLES AND STUDY GOVERNANCE 
Principal Investigator 
Carl E. Hunt, MD 
Professor of Pediatrics 

 
 
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Children’s National Medical Center/Children’s 
Research Institute  
111 Michigan Avenue, NW  Washington, DC 20010 
301-767-5514  
carl.hunt@usuhs.edu 
chunt@childrensnational.org 
 
Co-Principal Investigator 
Eric Eichenwald, MD  
Chief, Division of Pediatrics 
Perelman School of Medicine 
University of Pennsylvania                                              
Children’s Hospital of Philadelphia                                
3401 Civic Center Blvd., Philadelphia PA  19104 
215-452-6951 
617-966-7024 (cell) 
Eichenwald@email.chop.edu 
Study Leadership 
• 
This is a Multiple PI grant application, with Drs. Hunt and Eichenwald as Co-PIs.  
• 
There will be a Steering Committee, chaired in rotating years by Drs. Hunt and Eichenwald. Other 
members will be Drs. McEntire (Project Director), Corwin (DCC), Heeren, Limperopoulos, Poets, 
Darnall and Revenis and a representative from each clinical site. This group will be responsible for 
coordinating and managing all phases of this study.  
• 
Executive Committee (EC): Drs. Hunt and Eichenwald (Co-PI), Dr. McEntire (Project Director), and 
Dr. Corwin (DCC) will provide overall study oversight and coordination, and resolution of any site-
specific issues.  
10.1.6 SAFETY OVERSIGHT 
Safety oversight will be under the direction of a Data and Safety Monitoring Board (DSMB) composed of 
individuals with the appropriate expertise, including content and statistical expertise. Members of the 
DSMB should be independent from the study conduct and free of conflict of interest, or measures should 
be in place to minimize perceived conflict of interest.  The DSMB will meet at least semiannually to assess 
safety and efficacy data on each arm of the study. The DMSB will operate under the rules of an approved 
charter that will be written and reviewed at the organizational meeting of the DSMB. At this time, each 
data element that the DSMB needs to assess will be clearly defined. The DSMB will provide its input to 
the executive committee and to NICHD staff. 
10.1.7 CLINICAL MONITORING 
There will be no formal on site clinical monitoring of data. All study data will be managed at the data 
coordinating center (DCC). Each subject will be assigned a unique study identifier code. Data entered by 
sites will be via a web-based portal and will flow directly into the main study Microsoft Access database. 
Standard quality control and cleaning procedures will be applied to ensure that accurate data entry has 
occurred. All study data will be stored on secure password-protected computer servers that are backed 
up automatically daily, and maintained by the Information Systems staff. The database will be 
programmed to produce weekly reports tracking enrollment and follow-up completion and other periodic 

 
 
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reports as required.  Virtual meetings between DCC and site investigators will be held frequently to 
monitor enrollment and track overall study conduct and any difficulties encountered. 
10.1.8 QUALITY ASSURANCE AND QUALITY CONTROL 
See Section 10.1.7, Clinical Monitoring. 
 
10.1.9 DATA HANDLING AND RECORD KEEPING  
See Section 10.1.7, Clinical Monitoring 
10.1.9.1 STUDY RECORDS RETENTION  
Study documents will be retained for a minimum of 2 years since the formal discontinuation of clinical 
development of the study intervention. These documents may be retained for a longer period, however, 
if required by local regulations.  
10.1.10 
PROTOCOL DEVIATIONS  
A protocol deviation is any noncompliance with the clinical trial protocol, International Conference on 
Harmonization Good Clinical Practice (ICH GCP), or Manual of Procedures (MOP) requirements. The 
noncompliance may be either on the part of the participant, the clinical site principal investigators, or the 
study site staff. As a result of deviations, corrective actions are to be developed by the site and 
implemented promptly.  
 
These practices are consistent with ICH GCP:  
• 
4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3  
• 
5.1 Quality Assurance and Quality Control, section 5.1.1  
• 
5.20 Noncompliance, sections 5.20.1, and 5.20.2.  
 
No protocol deviations will be approved. However, it will be the responsibility of the clinical site principal 
investigators to use continuous vigilance in order to identify and report deviations within 5-10 working 
days of identification of the protocol deviation, or within 5-10 working days of the scheduled protocol-
required activity.  All deviations must be addressed in study source documents, reported to the clinical 
site PI, DCC, and NICHD Program Official.  Protocol deviations must be sent to the reviewing Institutional 
Review Board (IRB) per their policies. The site investigator is responsible for knowing and adhering to the 
reviewing IRB requirements. Further details about the handling of protocol deviations will be included in 
the MOP. 
 
 
 
10.1.11 
PUBLICATION AND DATA SHARING POLICY 
 
• 
The Executive Committee will be responsible for developing publication procedures and resolving 
authorship issues.  
• 
This study will be conducted in accordance with the National Institutes of Health (NIH) Public 
Access Policy, which ensures that the public has access to the published results of NIH funded 
research. It requires scientists to submit final peer-reviewed journal manuscripts that arise from 
NIH funds to the digital archive PubMed Central upon acceptance for publication. 
• 
This study will comply with the NIH Data Sharing Policy and Policy on the Dissemination of NIH-
Funded Clinical Trial Information and the Clinical Trials Registration and Results Information 

 
 
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Submission rule. As such, this trial has been registered at ClinicalTrials.gov (NCT03321734), and 
results from this trial will be submitted to ClinicalTrials.gov. In addition, every attempt will be 
made to publish results in peer-reviewed journals.   
• 
Data from this study may be requested from other researchers after the completion of the 
primary endpoint by contacting the DCC.  
10.1.12 
CONFLICT OF INTEREST POLICY 
The independence of this study from any actual or perceived influence is critical.  Therefore, any actual 
conflict of interest of persons who have a role in the design, conduct, analysis, publication, or any aspect 
of this trial will be disclosed and managed. Furthermore, persons who have a perceived conflict of interest 
will be required to have such conflicts managed in a way that is appropriate to their participation in the 
design and conduct of this trial.  The study leadership in conjunction with NICHD has established policies 
and procedures for all study group members to disclose all conflicts of interest and will establish a 
mechanism for the management of all reported dualities of interest. 
10.2 ADDITIONAL CONSIDERATIONS 
None 
 
 

 
 
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10.3 ABBREVIATIONS 
 
AE 
Adverse Event 
ANCOVA 
Analysis of Covariance 
CFR 
Code of Federal Regulations 
CMP 
Clinical Monitoring Plan 
COC 
Certificate of Confidentiality 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
DCC 
Data Coordinating Center 
DHHS 
Department of Health and Human Services 
DSMB 
Data Safety Monitoring Board 
EC 
Ethics Committee 
eCRF 
Electronic Case Report Forms 
FA 
Fractional Anisotropy 
HIPAA 
Health Insurance Portability and Accountability Act  
IB 
Investigator’s Brochure 
ICH 
International Conference on Harmonization  
ICMJE 
International Committee of Medical Journal Editors 
IH 
Intermittent Hypoxia 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
ITT 
Intention-To-Treat 
LSMEANS 
Least-squares Means 
MD 
Mean Diffusivity 
MOP 
Manual of Procedures 
MRI/MRS 
Magnetic Resonance Imaging / Magnetic Resonance Spectroscopy 
MSDS 
Material Safety Data Sheet 
NCT 
National Clinical Trial 
ICU 
Neonatal Intensive Care Unit 
NIH  
National Institutes of Health 
OHRP 
Office for Human Research Protections 
PI 
Principal Investigator 
PMA 
Postmenstrual age 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SOA 
Schedule of Activities 
SOP 
Standard Operating Procedure 
TDD 
Total Daily Dose  
UP 
Unanticipated Problem 
US 
United States 
 
 

 
 
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10.4 PROTOCOL AMENDMENT HISTORY 
 
Changes in Protocol version 2.0, dated 24 NOV 2018 compared to the preliminary 
protocol version 1.1, dated 12 JUN 2018 
Location of Change 
Description of Change 
Brief Rationale 
Section 1.1 
Enrollment start date 
changed to December, 
2018 
Estimated approval date for Version 2.0 
Section 1.2 Study 
Changed randomization 
from 32+1 to 32+0 
It is possible to do randomization on same  day as enrollment 
Section 5.2 
Changed wording on 
exclusion #2 and #3 from 
“history of or current 
treatment for…” to 
“current or prior treatment 
for…” 
Clarify wording to show the exclusions are if the infant was 
treated for the conditions, not just diagnosed with the 
conditions. 
Section 6.1.2 
Deleted “simple syrup” in 
multiple sentences. Same 
deletion in other sections 
as needed. 
Simple syrup not necessary component of study drug; cherry 
syrup sufficient. 
Section 6.2.1 
For specific components 
of compounded study 
drug, refer to Pharmacy 
Manual 
Pharmacy Manual the appropriate location for this detailed 
information 
Section 6.2.2 
Stock study drug bottles 
can be stored, 
refrigerated, for up to 30 
days instead of 90 days 
As recommended by Investigational Drug Service, according 
to USP guidelines 
Sections  6.5.1, 7.1,  
8.3.3.2, 8.3.3.3 , 8.3.6, 
8.4.2, 10.1.2, 10.1.3, 
10.1.10    
Changed “study PI, 
clinical site PI, primary 
care provider ” to 
“clinical site principal 
investigators”  throughout 
document, and “PI” to 
“ICAF PI” 
Make wording consistent for “clinical site PI” and “ICAF PI “ 
throughout document 
Section 8.1.4 
Salivary Caffeine Levels, 
changed to “20 degrees or 
colder, -80 C is okay, if 
more convenient to store 
in same freezer as blood 
samples” 
Saliva samples must be frozen at 20 degrees or colder to 
prevent evaporation. 
Section 8.3.4 
Last day of study 
participation may be 
defined as “end of study 
visit, follow up MRI, or 
lost-to-follow-up 
determination date” 
Clarify definition of last day of study participation 
Section 8.3.5 
Adverse Event Reporting 
for AEs and SAES from 
time informed consent is 
signed through 30 days 
Clarify timeframe for collection of AEs and SAEs 

 
 
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after the last day of study 
participation 
Section 8.4.2    
Unanticipated problems 
need to be reported from 
when informed consent is 
signed through 30 days 
after the last day of study 
participation 
Clarify timeframe for reporting unanticipated problems 
Changes in Protocol version 3.0, dated 08 AUG 2019 compared to version 2.0, dated 24 NOV 2018 
Location of Change 
Description of Change 
Brief Rationale 
Section 1.1 
Two caffeine levels will 
be obtained, the 1st at one 
week after beginning 
study drug, ADDED: but 
no later than 14 days post-
first-dose 
Added window of 14 days post-first-dose to assist with 
protocol compliance 
Section 1.1 
Inflammatory biomarkers 
will be measured at study 
enrollment and again at 
38+0 ADDED: plus or 
minus 3 days 
Added window of + or – 3 days to assist with protocol 
compliance 
Section 1.3 
Under heading “1 week 
on study drug” ADDED: 
“up to 14 days post 1st 
dose” 
Added window of 14 days post-first-dose to assist with study 
compliance 
Section 1.3 
Under footnote 3, 
ADDED: “+ or – 3 days” 
window to 2nd sample 
collection time 
Added window of + or – 3 days to assist with protocol 
compliance 
 
 

 
 
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Section 5.1 
Inclusion Criteria #5 
changed from “Breathing 
room air with no 
ventilatory support” to 
“Breathing room air with 
no ventilatory support for 
≥ 12 hours and not 
expected to relapse (Note 
that infants on room air 
with ≤ 2.0 L/min nasal air 
flow therapy will NOT be 
considered to be on 
ventilatory support).” 
 
To specify the duration of time needed of ventilatory support 
to satisfy this criterion, and to define what should be 
considered ventilatory support in a manner that is consistent 
with typical clinical management. 
Section 6.2.2 
Removed “containing 
dilute oral cherry 
solution” 
This detail is no longer correct and was meant to be removed 
from the protocol with the updated information now included 
in detail in the Pharmacy Manual.   
Section 6.2.2 
Examples changed “20 
mg/1mL” to 10 mg/1 mL 
Current caffeine formulation, which has been updated in the 
Pharmacy Manual, is 10 mg/mL. 
Section 6.2.2 
Deleted “refrigerated”, 
and replaced with “at 
room temperature” 
This detail is no longer correct and was corrected to reflect the 
instruction to store the current formulation at room 
temperature.   
Section 6.2.2 
Deleted “provided a 2nd 
bottle…” and replaced 
with “a sufficient quantity 
of either the active drug 
(caffeine base) or placebo 
to last until 42 weeks + 6 
days (BUD 90 days).” 
This detail is no longer correct and was corrected to reflect the 
90 day BUD for the current formulation.   
 
 

 
 
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Section 8.1.4 
Replaced Prior Text: 
“Two salivary samples 
will be obtained for later 
analysis of caffeine 
concentrations, the 1st in 
the NICU 1 week after 
starting study drug, and 
the 2nd at home by 
parents at about 40 
weeks + 0 days PMA.  
All samples will pre-
prandial. Salivary 
volumes of 200-300 uL 
will be collected using 
commercially available 
kits (Salimetrics, State 
College, PA) and frozen 
at -20o C at home and 
then at  -80° C after 
return to the clinical site 
research team  in the 
same freezer as the 
plasma samples) for bulk 
analysis at the end of the 
study. 
Revised Text:   “Two 
salivary samples will be 
obtained for later 
analysis of caffeine 
concentrations, the 1st in 
the NICU 1 week after 
starting study drug, and 
the 2nd, which may be 
either in the hospital or at 
home by parents, at 
about 40 weeks + 0 days 
PMA.  All samples will 
be pre-prandial. Salivary 
volumes of 200-300 uL 
will be collected using 
commercially available 
kits (Salimetrics, State 
College, PA) and frozen 
at -20o C at home and 
then at -20° C or colder 
after return to the clinical 
site research team for 
bulk analysis at the end 
of the study.”  
 
Text revised to clarify that 2nd salivary sample may be 
either in the hospital or at home and the freezer 
temperature is permitted to be -20 degrees or colder 
hospital freezer. 
Sections 8.3.2 and 8.3.3 
Removed first sentence 
in section 8.3.3 and 
added it as the last 
sentence in 8.2.2 to fix 
Correction of typographical error. 

 
 
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formatting pagination 
error. 
Section 8.3.5 
Timeframe for the 
collection of AEs and 
SAEs…” has been 
changed to reference the 
timeframe which was 
previously provided in 
Section 8.3.4., which 
states, “…AEs through 
the last day of study 
participation, and SAEs 
until 30 days after the 
last day of study 
participation.” 
Section 8.3.4 correctly stated the policy, and there was no 
need to repeat it in section 8.3.5, which was also not 
worded consistently. 
   
Section 8.3.8 
Changed from “(ALTE, 
or brief resolved 
unexpected event, 
BRUE) or for sudden 
unexpected infant death 
(SIDS)” to “(ALTE, or 
brief resolved 
unexpected event, 
BRUE) or for sudden 
unexpected infant death 
(SUID)”     
 
Correction of acronym for Sudden Unexpected Infant 
Death 
CHANGES FROM 3.0 to 4.0 
Location of Change 
Description of Change 
Brief Rationale 
1.1 Study Description: 
5th sentence: 
CHANGED FROM: 
If, based on standard 
clinical criteria, the last 
dose of routine caffeine 
is given on or before the 
day the infant is 36 
weeks + 5 days PMA, 
then on the day 
following their last dose 
of routine caffeine 
treatment, infants will 
be randomized 
(110/group) to extended 
caffeine treatment or 
placebo.  
 
 
CHANGED TO: 
If, based on standard 
clinical criteria, the last 
dose of routine caffeine 
is given on or before the 
day the infant is 36 
To allow a 3-day window from last day of clinical 
caffeine to randomization and dosing. 

 
 
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weeks + 5 days PMA,   
then, ideally,  on the day 
following, but no later 
than the third calendar 
day following, their last 
dose of routine caffeine 
treatment, infants will be 
randomized (110/group) 
to extended caffeine 
treatment or placebo. 
 
 
1.1 SYNOPSIS Study 
Population 
Remove “one of 8” 
participating hospitals 
We continue to increase the number of participating 
hospitals to help increase enrollment. 
1.1 SYNOPSIS Description 
of Sites/Facilities 
Remove “8” (hospitals) 
and “six” (metropolitan 
areas)  
We continue to increase the number of participating 
hospitals to help increase enrollment. 
1.1 SYNOPSIS Study 
Duration 
Change sentence from 
“Enrollment is scheduled 
to start in December, 
2018 and will continue 
through the end of 2021, 
with completion of 
follow-up and data 
analyses extending 
through May of 2022” 
TO:.”Enrollment is 
scheduled to start in 
December, 2018 and will 
continue at least through 
May, 2022, with 
completion of follow-up 
and data analyses 
thereafter.” 
To allow time for adequate enrollment which has 
been delayed partially due to COVID impacts. 
1.2 Study Schema 
2nd-to-last sentence 
CHANGED FROM: 
Randomization will be 
performed within 24 
hours following the last 
dose of routine caffeine, 
as long as randomization 
is by 36 weeks + 6 days 
PMA. 
 
 CHANGED TO:: 
“Randomization will be 
performed, ideally,  on 
the day following, but no 
later than the third 
calendar day following, 
the last dose of routine 
caffeine, as long as 
randomization and 
To allow a 3-day window after the last dose of routine 
caffeine for randomization and dosing. 

 
 
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dosing is by 36 weeks + 
6 days PMA.    
4.1 STUDY DESIGN 
Overall Design, Multi-Site 
Study 
Substitute “multiple” 
for “8”, remove “6” , 
delete all cities and 
states listed. 
We continue to increase the number of participating 
hospitals to help increase enrollment. 
5.1 STUDY 
POPULATION: Inclusion 
Criteria #2 
Added Sentence to end 
of criteria #2:. “ Note 
infants will meet this 
inclusion criteria through 
the third calendar day 
following receiving their 
most recent routine 
caffeine dose.” 
To allow for a window of 3 days after last dose of 
clinical caffeine to be considered “on caffeine”, 
therefore meeting the eligibility requirement.  
5.1 STUDY 
POPULATION: Inclusion 
Criteria #5 
Criterion #5 changed 
from: “Breathing room 
air with no ventilatory 
support for ≥ 12 hours 
and not expected to 
relapse (Note that infants 
on room air nasal air 
flow therapy, will NOT 
be considered to be on 
ventilatory support). “  
 
CHANGED TO: At 
least 12 hours of 
breathing room air with 
no ventilatory support 
other than on room air 
nasal air flow therapy 
regardless of flow rate, 
or on room air and 
receiving nasal CPAP, 
and relapse not 
anticipated. 
 
 
  
To increase eligibility to now include infants on 
respiratory support to include nasal air flow therapy 
of any flow rate, or nasal CPAP providing they are 
breathing ROOM AIR with NO SUPPLEMENTAL 
OXYGEN. 
 
Also to be more consistent with evolving changes in 
clinical practice. 
5.5 Recruitment 
2nd bullet, last sentence, 
ADDED:” If an infant is 
a transfer from another 
facility at ≤36+6, they 
WILL be added to the 
screening log.  If they are 
a transfer from another 
facility at ≥37+0, they 
WILL NOT be added to 
the screening log.” 
 
To explain screening log protocol for infants 
transferred from other facilities. 
5.5 Recruitment 
3rd bullet, 2nd sentence 
deleted: 
“Based on pilot studies, 
it is estimated that 
Estimation of pilot study’s consent rate to this study’s 
participation is an inaccurate comparison because of 
COVID’s influence on ability of staff to 
approach/consent  families. 

 
 
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ICAF Protocol Version 4.2: 27  September 9, 2021  
 
 
 
 
about 30-35% of 
families approached for 
consent will consent to 
participate.” 
   
8.1.2 Inflammatory 
Biomarkers 
2nd paragraph: Remove 
“40 plex V Plex” and 
“(MesoScale 
Diagnostics, Rockville 
MD) 
Changes made to reflect newly available  assay 
methods. 
10.1.1.1 
CHANGED FROM: The 
Consent Form describing 
in 
detail 
the 
study 
intervention, 
study 
procedures, and risks will 
be given to the parents of 
each 
eligible 
subject. 
Written documentation 
of informed consent will 
be obtained prior to 
enrolling each infant 
 
CHANGED TO: 
All 
informational 
documents and consent 
material to be provided 
to parents of potential 
study subjects will be 
submitted to study site 
IRB’s and utilized in 
accordance 
with 
procedures approved by 
the IRB. 
 
DELETED: The Consent 
Form is being submitted 
with this protocol 
 
To allow for various consenting methods approved by 
site-specific  IRBs 
10.1.1.2 
Added: ór their 
designee” to: “the 
investigator will explain 
the research study to 
the parent…”  
 
Removed “written” 
from consent 
To allow for investigator-designated study team 
members to explain the study to potential 
participants. 
 
 
 
 
Consent, written or otherwise,  may be obtained per 
IRB requirements. 
10.1.5 Key Roles and Study 
Governance: Study 
Leadership 
2nd bullet: CHANGE 
FROM: There will be a 
Steering 
Committee, 
chaired in rotating years 
by 
Drs. 
Hunt 
and 
To condense listings of committees, and to align 
meetings with current standards of virtual meetings. 

 
 
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Eichenwald. 
Other 
members will be Drs. 
McEntire 
(Project 
Director), Corwin (DCC), 
and 
Darnall, 
Dobson, 
Fort, Frantz, Hartman, 
Heeren, Limperopoulos, 
Poets, Revenis, Rhein, 
and Stark. This group will 
be 
responsible 
for 
coordinating 
and 
managing all phases of 
this study. They will meet 
at 
funding 
onset 
to 
establish responsibilities 
and timelines, complete 
the manual of operations, 
and then meet biweekly 
in Quarter (Q) 1-2 of Year 
1, and monthly thereafter. 
The committee will meet 
face-to-face initially and 
once/year; 
all 
other 
meetings will be virtual. 
Y1 will be used to 
complete 
all 
project 
materials, 
train 
site 
personnel, 
and 
obtain 
IRB 
approvals. 
CHANGE TO: 
There will be a Steering 
Committee, chaired in 
rotating years by Drs. 
Hunt and Eichenwald. 
Other members will be 
Drs. McEntire (Project 
Director), Corwin (DCC), 
Heeren, Limperopoulos, 
Poets, 
Darnall 
and 
Revenis 
and 
a 
representative from each 
clinical site. This group 
will be responsible for 
coordinating 
and 
managing all phases of 
this study 
 
10.1.5 Key Roles and Study 
Governance: Study 
Leadership 
3rdbullet:Change 
FROM: 
Executive 
Committee (EC): Drs. 
Hunt 
and 
Eichenwald 
(Co-PI), Dr. McEntire 
(Project Director), and 
Dr. Corwin (DCC) will 
To condense listings of committees, and to align 
meetings with current standards of virtual meetings. 

 
 
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provide 
overall 
study 
oversight 
and 
coordination, 
and 
resolution of any site-
specific issues. The EC 
will 
meet 
meet 
in 
conjunction 
with 
SC 
meetings, and will also 
meet virtually as needed, 
biweekly. CHANGE TO: 
Executive 
Committee 
(EC): Drs. Hunt and 
Eichenwald (Co-PI), Dr. 
McEntire 
(Project 
Director), and Dr. Corwin 
(DCC) 
will 
provide 
overall study oversight 
and coordination, and 
resolution of any site-
specific issues. The EC 
will meet meet virtually 
as needed, generally on a 
biweekly basis. 
 
 
10.1.11 
2nd bullet: 
Added 
“PubMed 
Central” after “digital 
archive…” 
To specify where the final peer-reviewed journal 
manuscripts will be published. 
CHANGES FROM PROTOCOL VERSION 4.1 to 4.2 
1.1 Synopsis 
Added: Pulse oximeter 
will continue either until 
hospital discharge or for 
1 
week 
after 
discontinuing 
study 
drug, 
depending 
on 
whether the infant was 
recruited at a study site 
at which home oximetry 
recording 
is 
being 
performed. 
Since home oximetry is challenging for parents, and a 
potential deterrent to enrollment, especially in the 
pandemic environment, we have determined that it 
was appropriate to offer sites the option of not 
participating in the home oximetry, and that overall 
enrollment would enhance our ability to achieve study 
goals, even if the home oximetry data were available 
on only a subset of the infants. 
1.1 Schedule of Activities 
Added footnotes 10 and 
11 noting that Home-
Oximetry and MRIs 
performed for subjects 
enrolled at sites 
participating in  the 
home oximetry and MRI 
portions of the study, 
respectively. 
 
To clarify the MRI procedure language related to 
participation being only in a subset of subjects. 
2.2.1 Known Potential 
Risks, 7th bullet 
Added: 
for 
those 
participants enrolled at 
sites at 
which 
home 
Since home oximetry is challenging for parents, and a 
potential deterrent to enrollment, especially in the 
pandemic environment, we felt it was appropriate to 

 
 
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oximetry is included in 
the study. 
offer sites the option of not participating in the home 
oximetry, and that overall enrollment would enhance 
our ability to achieve study goals, even if the home 
oximetry data were available on only a subset of the 
infants. 
2.2.2 Known Potential 
Benefits: (General future 
potential benefits to fill 
major gaps in our current 
knowledge) 
Added: “(for participants 
receiving 
home 
oximetry),” to benefit #3) 
the 
frequency 
and 
severity of IH persisting 
at home after discharge  
Since home oximetry is challenging for parents, and a 
potential deterrent to enrollment, especially in the 
pandemic environment, we felt it was appropriate to 
offer sites the option of not participating in the home 
oximetry, and that overall enrollment would enhance 
our ability to achieve study goals, even if the home 
oximetry data were available on only a subset of the 
infants. 
4.4. End of Study Definition 
Added the BOLDED 
words:  Each subject 
will be considered to 
have completed the study 
after the planned pulse 
oximeter recording data 
is completed and 
inflammatory biomarker 
samples are provided, 
and if applicable, has 
completed the outpatient 
brain MRI between 43 
weeks + 0 days and 46 
weeks + 6 days PMA, as 
shown in the Schedule of 
Activities (SOA), 
Section 1.3. 
 
Added language to clarify the MRI procedure and 
oximetry procedure related to participation being for 
only a subset of subjects. 
5.5 Recruitment and 
Retention: (retention) 
Added “if applicable” to 
the section on contacting 
families 
on 
use 
of 
oximeter. 
Added language to clarify the home-oximetry 
procedure related to participation being only for a 
subset of subjects 
6.5.1 Rescue Medicine 
Added: Pulse oximeter 
recordings will continue 
through hospital 
discharge, or at home 
through 1 week 
following 
discontinuation of study 
drug, for infants 
recruited at sites 
participating in home 
oximeter recording. The 
PMA at which study 
drug is scheduled to end 
is well after the clinical 
team judged it safe to 
stop routine caffeine 
treatment and at a PMA 
at which prior studies 
have shown that the 
Added language to clarify the home-oximetry 
procedure related to participation being only for a 
subset of subjects. 

 
 
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extent of IH no longer 
differs from healthy 
infants born at term. 
However, for sites 
continuing oximeter 
recordings at home, the 
oximeter alarms may be 
activated for this final 
study week if deemed 
necessary by the clinical 
site principal 
investigators. 
 
8.1.1 Oxygen Saturation 
Removed 
and 
added 
language: 
Continuous 
recording of O2 saturation 
using the study pulse 
oximeter is planned for 
all 
infants 
through 
hospital discharge, and 
for the sub-group of 
infants 
recruited 
at 
hospitals participating in 
home oximetry recording 
,will continue  through 43 
weeks + 6 days PMA (i.e. 
one week following the 
last dose of study drug).   
Removed and added language to clarify the home-
oximetry procedure related to participation being 
only for a subset of subjects. 
8.1.1 Oxygen Saturation: 
Outpatient Phase 
Added: 
At 
sites, 
participating in the home 
oximetry portion of the 
study, prior 
Added language to clarify the home-oximetry 
procedure related to participation being only for a 
subset of subjects. 
8.1.3 
QUANTITATIVE 
MRI 
STRUCTURAL, 
MICROSTRUCTURAL 
AND 
METABOLIC 
BIOMARKERS 
OF 
ACUTE BRAIN INJURY 
 
Added: 
“approximately 
half 
of 
the 
study 
participants” to our goal 
is to obtain baseline and 
follow-up MRI pairs in: 
To clarify the percentage of participating subjects on 
whom we aim to obtain MRI pairs. 
8.1.3 Quantitative MRI 
Structural, Microstructural 
and Metabolic Biomarkers of 
Acute Brain Injury 
Added: 
“At 
sites 
recruiting infants into the 
MRI 
portion 
of 
the 
study,” 
Added language to clarify the MRI procedure related 
to participation being for only a subset of subjects. 
1.1 Synopsis 
OBJECTIVES: “Phase” 
Deleted “phase 3”, added 
“not applicable” 
Our protocol does not fit the definition of a Phase 3, or 
Phase 2, study 
1.1 Synopsis 
OBJECTIVES, Description 
of Sites/Facilities Enrolling 
Participants 
After “once discharged”, 
Added “if the subject has 
been enrolled at a site 
performing MRIs 
To clarify the MRI procedure related to participation 
being for only a subset of participants. 
1.1 Synopsis 
OBJECTIVES, Description 
of Sites/Facilities Enrolling 
Participants 
Added “At the end of the 
study, study termination 
procedures 
will 
be 
performed in a manner 
To clarify the end of study procedures, may be done 
remotely for the convenience of the family. 

 
 
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convenient for the family, 
and may be performed in 
person or remotely, and if 
applicable 
may 
be 
performed at the time of 
an MRI. 
6.2.3 
Added “if applicable, or 
sent back” 
To clarify that for the convenience of the family, study 
drug may be shipped back in lieu of an in-person visit. 
1.1 Participant Duration 
Added “The end of 
study 
procedures 
include: return of the 
oximeter 
(if 
applicable), 
salivary sample 
and 
any unused study drug.  
As noted above, these 
procedures will done at 
the convenience of the 
family, and although a 
face-to-visit 
is 
preferred,  all items 
may be shipped back if, 
for any reason, the 
parents prefer not to 
travel to the site. 
To clarify that the end-of-study procedures may be done 
in person or remotely for the family’s convenience. 
1.3 Schedule of Activities, 
footnote 8 
Added, “which may be 
done remotely” (referring 
to the end of study visit), 
and added, “Items may be 
shipped 
back 
if 
the 
parents opt for a remote 
visit. 
To clarify that end-of-study procedures may be done 
remotely for the parents’ convenience. 
5.5 Retention 
3rd bullet:  Added “if 
applicable” 
to 
“return 
visit”. 
To clarify that end-of-study procedures may be done 
remotely for the parents’ convenience. 
 
 
 
 
 
 
 
 
 
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