Clinical trial NCT02907593
Conditions: Bronchopulmonary Dysplasia (BPD)
Keywords: Bronchopulmonary dysplasia Pulmonary Surfactant Steroids Extremely low gestational age
Sponsor: Cynthia McEvoy
Start date: 2016-09-01; Completion date: 2018-04-01
Enrollment: 25 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
This is a phase I/II open-label study to determine the lowest, safe, effective dose of budesonide given with calfactant as the vehicle.

Detailed description:
This is a phase I/II open-label study to determine the lowest safe, effective dose of budesonide given with calfactant as the vehicle; the investigators will perform an unblinded dose escalation study. The investigators will administer four dosing levels of budesonide suspended in calfactant beginning with 0.025 mg/kg of budesonide administered to 8 extremely low gestational age newborns (ELGANs) who are intubated at 3-10 days of age. Daily doses (at the same dosage) will be administered to infants who remain intubated for a potential of 5 total doses in each patient. Subsequent groups of 8 infants each will receive 0.05 mg/kg, 0.10 mg/kg, and 0.15 mg/kg of budesonide in calfactant (up to 5 total doses in each patient). A total of up to 32 infants will be enrolled in the trial. The investigators will evaluate the clinical, laboratory and safety data from each group of treated infants to 28 days of age before moving to the next dosing level of budesonide.

Arms: arm 1: 0.025 mg/kg Budesonide in Calfactant arm 2: 0.050 mg/kg Budesonide in Calfactant arm 3: 0.10 mg/kg Budesonide in Calfactant arm 4: 0.15 mg/kg Budesonide in Calfactant
Interventions: intervention 1: Budesonide in Calfactant
Intervention descriptions: intervention 1: Budesonide in Calfactant

Protocol documents:
Prot_SAP_000.pdf: 
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Protocol Version 3.0 
 
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Steroids and Surfactant in 
Extremely Low Gestation Age 
Infants (SASSIE) 
Pilot Dose Escalation Trial 
 
 
IND Number 128102 
Principal Investigator:  Cindy McEvoy, MD, MCR 
 
Funded by the Thrasher Foundation 
 
 
 
 
 
Version 3.0 
October 16, 2017 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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TABLE OF CONTENTS 
1.Objectives ........................................................................................................................ 4 
Specific Aim 1 ................................................................................................................. 4 
Secondary Specific Aims ................................................................................................. 5 
2. Background ..................................................................................................................... 6 
2a.Epidemiology and pathogenesis ................................................................................. 6 
2b.Current therapeutic approaches .................................................................................. 6 
   2c.Pilot studies/ potential role of budesonide in surfactant ............................................. 8 
   2d.Anti-Inflammatory effects .......................................................................................... 9 
3. Study Design ................................................................................................................. 11 
4. Study Population ........................................................................................................... 11 
4a.Number of subjects ................................................................................................... 11 
4b.Inclusion and exclusion criteria ................................................................................ 11 
4c.Vulnerable populations ............................................................................................. 12 
4d.Setting ....................................................................................................................... 12 
4e Recruitment methods ................................................................................................ 13 
4f. Consent process ........................................................................................................ 13 
5. Procedures Involved...................................................................................................... 13 
5a. Study medication dosing .......................................................................................... 13 
5b. Tracheal aspirates .................................................................................................... 15 
5c. Plasma samples ........................................................................................................ 16 
5d. Clinical data collection…………………………………………………………......16 
5e. Oxygen/Flow reduction challenge test .................................................................... 17 
5f. Pulmonary function test…………………………………........................................17 
5g.  Future contact ......................................................................................................... 18 
5h.  Adverse event and protocol violation reporting ..................................................... 18 
  
6. Data and Specimens ...................................................................................................... 23 
6a. Handling of data and specimens .............................................................................. 23 
6b. Sharing of results with subjects ............................................................................... 23 
7. Data Analysis…………………………………………………………………………. 23 
7a.Primary outcome ....................................................................................................... 23 
7b.Secondary outcomes ................................................................................................. 24 
7c.Clinical parameters ................................................................................................... 25 
7d.Budesonide pharmacokinetic modeling .................................................................... 25 
7e.Clinical respiratory status at 28 days ........................................................................ 25 
7f. Safety analyses ......................................................................................................... 25 
7g. Exploratory outcomes .............................................................................................. 25 
 
8. Privacy, Confidentiality and Data Security .................................................................. 26 
9. Provisions to Monitor the Data to Ensure the Safety of Subjects ................................. 26 


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10.Risks and Benefits........................................................................................................ 26 
10a.Risks to subjects...................................................................................................... 26 
10b.Potential benefits to subjects .................................................................................. 27 
11. Drugs or Devices......................................................................................................... 28 
12. Multi-Site Coordination .............................................................................................. 28 
References ......................................................................................................................... 29 
 
Tables: 
Table 1. In vitro surface tension properties of calfactant supplemented with budesonide...8 
Table 2ab. Dosage preparation of budesonide and calfactant……………………………..14 
Table 3. Study procedure table………. …………………………………………………...22 
 
Figures: 
Figure 1. Budesonide addition to calfactant: effect on adsorption………………………... 9 
Figure 2. Budesonide affects on cytokines in human fetal lung tissue………………….....10 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1. Objectives 
The overall goal of this protocol is to determine the optimal dose and frequency of treatment 
with budesonide suspended in surfactant and administered intratracheally  to effectively and 
safely treat intubated extremely low gestational age newborn (ELGAN, <28 weeks of 
gestational age) infants at risk for bronchopulmonary dysplasia (BPD).1;2  
 
We hypothesize that an appropriate dose of budesonide delivered directly to the lung 
suspended in  the surfactant calfactant (Infasurf) will be effective and safe in improving the 
infant’s respiratory status, as demonstrated by an improvement in  respiratory assessments at 
up to 28 days of age, and decreased inflammatory response (a critical contributor to BPD in 
ventilated preterm infants).   
 
This is a dose escalation study of budesonide suspended in calfactant as the vehicle into the 
infant’s lungs. Measures of drug efficacy will include standardized clinical assessments to 28 
days of age and cytokine levels in tracheal aspirate.  Dose-dependent safety will be measured 
by monitoring adverse events potentially attributable to study treatment and budesonide 
systemic pharmacokinetics.  
 
Specific Aim 1/Primary Objective:  
To determine the clinical and anti-inflammatory efficacy of escalating doses of budesonide 
suspended in calfactant and administered into the lungs of ELGANs  by monitoring the infant’s 
clinical outcome up to 28 days of age, including the Respiratory Severity Score (RSS) 3 and 
measuring  tracheal aspirate cytokine levels.1;2  
 
Dose escalation will be defined as effective at the dose level in which 5 of 8 infants achieve:   
 At least 1 one of the following: 
1) Extubation within 72 hours of first dose or after < 3 doses-without re-
intubation before 28 days of age  
2) RSS on nasal continuous positive airway pressure (NCPAP) < 1.5 or on 
nasal cannula Fi02 <25% at < 2L/min at 28 days of age, persisting for at 
least 72 hours. 
3) Cumulative supplemental oxygen < 4.2 from time of enrollment to 28 days 
of age4 (see calculation below) 
4) No respiratory support at 28 days, including no supplemental oxygen by 
nasal cannula  
 AND a > 50% suppression of the tracheal aspirate interleukin-8 (IL-8) or CCL2 
(chemokine ligand 2) at 24-72 hours (or prior to extubation if occurs at <24 hours 
after dosing) after the initial dose in 5 of 8 of the infants (this may be a different 
combination of 5 patients than those who met the above criteria).   
Surfactant alone has been shown to transiently decrease RSS by 10%,5 so a sustained 
decrease in RSS would indicate budesonide effects. Data from human fetal lung explant 
studies from Dr. Phil Ballard’s laboratory6 (see section 2d below) demonstrates that 
budesonide reversibly suppresses a variety of inflammatory markers from tracheal aspirates 
and that IL-8 and CCL2 are useful biomarkers for in vivo glucocorticoid anti-inflammatory 
effects. In fetal human lung explants, 30nM Budesonide decreased IL8 and CCL2 
by ≥75% within the 24 hours of dosing. Data from a matched historical control group from the 
Trial of Late SURFactant (TOLSURF)3 will be used to evaluate the clinical response to 


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administration of calfactant alone over the first 28 days as well as changes in cytokines with 
treatment in banked tracheal aspirate samples. Secondary analysis4 of data from the 
TOLSURF study demonstrated that a cumulative supplemental oxygen (average daily FiO2- 
0.21 x number of days being monitored) of > 4.2 from day of life 5 to 28 had a 70% specificity 
for the development of BPD and/or death. As long as there are no safety concerns, dose 
escalation will be increased by one more dosing level above this defined effective dosing level 
to assess for any potential further benefits in terms of respiratory status at 28 days of age 
and/or cytokine suppression.   
 
Secondary Specific Aims/Secondary Objectives:  
1. Safety Objectives:   
Safety assessments will include the subject’s vital signs, clinical laboratory testing, morbidities 
associated with prematurity, morbidities associated with administration of  budesonide in 
calfactant, and Adverse Events (AE)s. Clinical parameters/ AEs of interest are those potentially 
consistent with elevated glucocorticoid levels and will be specifically evaluated.  A Data and 
Safety Monitoring Board (DSMB) will be established to review safety data. The incidence of 
morbidities will be compared with the incidence of these morbidities reported in the 
TOLSURF study3 due to the similarities in these populations. 
2. Serial Budesonide Levels:   
We will obtain dried blood spot samples (DBS; about 0.2 mL per sample)7 at 15 minutes, and 1 
and 4 hours after the first dose of budesonide in calfactant for budesonide levels. This is a total 
of 0.6 mL per patient or 1.2 mL/kg in a 500 gram newborn. Trough concentrations (Cmin)will 
be drawn before potential subsequent daily doses for a potential total of 7 total DBS or a total 
of 1.4 mL (2.8 mL/kg for a 500 gram baby over a five day period which does not represent an 
excessive blood loss). Analysis of the DBS and pharmacokinetic modeling are being 
performed within the Department of Pharmacology/ Toxicology at the University of Utah School 
of Medicine. Based on preliminary data in lambs,8 we could expect a Cmin of 1.9 ng/mL at 9 
hours post-dosing.  Non-compartmental and population pharmacokinetic analyses will be 
performed to establish the extent of budesonide exposure.  Commonly reported 
pharmacokinetic parameters will be calculated including:  clearance, volume of distribution, 
area under the curve (AUC), half-life, peak concentration (Cmax), and trough concentration 
(Cmin). 
3. Clinical Respiratory Outcomes:   
Clinical respiratory status (RSS and oxygen requirement) at 28 days of age will be compared 
between dosing groups and also compared to the matched historical control patients from the 
TOLSURF study.3  The individual patient’s decrease in RSS will also be correlated with their 
budesonide exposure measures by the area under the concentration time curve (AUC).  
4. Exploratory Measurements (Pulmonary Function Tests, Other Anti-Inflammatory 
Biomarkers, and Airway Microbiome Analysis) 
Pulmonary function tests will be completed on subjects born at Oregon Health and Science 
University (OHSU)  prior to the first dose of study medication, 24 hours after the first dose, 
prior to extubation and then at discharge at 34-36 weeks of post menstrual age. Passive 
respiratory compliance (Crs) and resistance (Rrs) will be measured with the single breath 
occlusion technique and functional residual capacity (FRC) with the nitrogen washout 
technique as previously described. 9-12 International criteria for testing will be followed to 
ensure testing quality.9-12  
 


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Once the dose of budesonide is identified that  effectively suppresses IL8 and CCL2 , a limited 
set of tracheal aspirates (from infants treated with that dose ) will be sent to Dr. Ambalavanan’s 
laboratory at the University of Alabama for multiplex analysis of up to 32 cytokines of interest in 
the development of BPD13 as well as airway microbiome analysis to assess potential 
modulation of the mucosal microbiome  by glucocorticoids.14  A limited set of tracheal aspirates 
will also be sent to Dr. Ambalavanan’s laboratory after treatment of the first several patients for 
pilot analysis of the 32 cytokines of interest.  
 
2. Background 
      
a. Epidemiology and pathogenesis 
Extremely low gestational age newborn (ELGAN, born at < 28weeks gestation) infants are at 
risk for BPD, a chronic lung disease that is associated with mortality, ongoing pulmonary 
morbidity, including asthma, and neurodevelopmental abnormalities.15 BPD occurs in >70% of 
ELGANs who require ventilatory support after 3 days of postnatal life.16;17  
 
BPD is the most common morbidity of ELGANs and despite significant focused research its 
incidence has remained largely unchanged for the last 20 years.15  BPD develops in 10,000-
15,000 infants in the United States per year and impacts the infant’s pulmonary and overall 
health as well as the financial and emotional well-being of the family.18 BPD is associated with 
increased respiratory diseases into childhood, recurrent hospitalizations, poorer 
neurodevelopmental outcomes, and importantly lifelong alterations in lung function.19 
 
The pathogenesis of BPD is multi-factorial including intrauterine and extrauterine inflammation 
as well as hypoxia, hyperoxia, ventilator trauma and/or infection after birth in susceptible 
infants with immature lungs.20 Inflammation is a common pathway of many factors contributing 
to the genesis and evolution of BPD. 21 Despite much research, as outlined below, there are 
few therapies that have been shown to decrease BPD. Because increased synthesis of 
proinflammatory cytokines is an early event associated with BPD, treatment to reduce 
inflammation and these cytokines has a good chance of reducing BPD.22 
 
b. Current therapeutic approaches to attempt to prevent/decrease BPD 
Randomized controlled trials (RCTs) to decrease BPD have included studies of pharmacologic 
treatments, respiratory care practices, fluid and nutritional therapies, and evidence-based 
standardized bundled practices.  Three medications have been shown to prevent / decrease 
BPD: Caffeine, vitamin A and corticosteroids given during the first week of life.23  The numbers 
needed to treat are approximately 10 (95% CI 7-16) for caffeine and 12 (95% CI 6-94) for 
vitamin A.23 Prophylactic use of systemic corticosteroids during the first week of life is no 
longer recommended because of potential harmful effects that include intestinal perforation 
and increased incidence of cerebral palsy.24  Both antenatal steroids and postnatal surfactant 
therapy decrease respiratory distress syndrome and death, but have not been shown to 
decrease BPD in survivors.20 Inhaled nitric oxide (iNO) did not decrease BPD in an individual 
patient meta-analysis of 3,298 premature infants.25  However, iNO did significantly reduce BPD 
in African Americans in a second individual participant data meta-analysis. Several 
pharmacologic trials proved ineffective in preventing BPD including: administration of 
superoxide dismutase, glutathione, cimetidine, and macrolide antibiotics.15 
 
Many RCTs of modified respiratory support in very low birth weight (VLBW) infants have been 
performed with limited success. Several large trials26-28 randomized VLBW infants to intubation 


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versus continuous positive airway pressure (CPAP) in the delivery room, but a significant 
decrease in BPD was not been clearly demonstrated. Some trials suggest that permissive 
hypercapnia may decrease BPD, but a RCT of early permissive hypercapnia in 220 patients 
was negative.29  Trials comparing different modes of ventilation have also been largely 
negative, including a meta-analysis of independent individual patient data comparing high 
frequency oscillatory versus conventional ventilation.30 Nutritional studies for BPD prevention, 
including randomization to aggressive versus conventional parenteral nutrition, early lipid 
administration, and restricted versus liberal water administration have been negative.  Studies 
using implementation science methodologies of best practices have reported decreased BPD 
in single centers. However, a study with seven intervention centers using a cluster, 
randomized, controlled design showed no difference in BPD.31 
 
Despite strong evidence implicating inflammation in BPD causation, strategies directed at 
modulating inflammation for the prevention of BPD have been limited or linked with significant 
adverse effects.  Antenatal steroid use may modulate the severity of BPD, yet direct evidence 
for preventing BPD is lacking.20 The potential role for other anti-inflammatory agents, such as 
azithromycin,32 remains unproven, and the use of selective anti-cytokine therapies remains 
unstudied in this population. Preclinical studies have implicated the regulation of nuclear factor 
kappa B (NFKB) as a key determinant of the inflammatory response; however, its role 
supporting normal lung development may limit NFKB as a potential target in the preterm 
infant.22  Mesechymal stem cell (MSC) therapy has been shown to prevent experimental BPD 
in rodents, and has potent anti-inflammatory properties in addition to its growth-promoting 
effects in the lung.  Whether MSC or conditioned media from MSC may provide a novel and 
effective anti-inflammatory strategy for the prevention of  BPD is unknown.22 
 
The use of postnatal systemic corticosteroids (primarily dexamethasone) has a long but 
controversial history of use in treating ventilator dependent infants at high risk for developing 
BPD.33-35  Postnatal use of dexamethasone became routine after the first reports of its benefits 
in the mid 1980’s and practice evolved with infants being treated prophylactically with longer 
and higher doses starting as early as day one of life.36 This practice stopped abruptly after a 
meta-analysis of controlled trials demonstrated an increased risk of cerebral palsy 
after systemic corticosteroids.24  Unfortunately no distinction was made between targeted use 
of steroids in patients at risk for BPD and indiscriminant or prophylactic use. No studies 
analyzed the pharmacokinetics of dexamethasone in a systematic manner to determine what 
exposure was related to AE’s.  The subsequent statement from the American Academy of 
Pediatrics strongly recommended against systemic postnatal steroids outside of randomized 
clinical trials and this statement in itself contributed to the early stopping of an ongoing RCT of 
postnatal dexamethasone whose primary outcome was neurodevelopmental 
outcomes.37  Several studies have now shown that coincident with the overall decreased use 
of postnatal steroids, the incidence of BPD is increasing.38Also, a meta-regression analysis 
demonstrated that if an infant’s risk of BPD is >55%, the use of postnatal systemic steroids will 
decrease the risk of poor neurodevelopmental outcome. 39 The revised AAP statement is now 
more tolerant of targeted use of systemic corticosteroids deferring to the clinician to use clinical 
judgement with regard to potential benefits and risks in ventilator dependent infants at risk for 
BPD. 40  
 
 
 


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c. Pilot studies/potential role of budesonide in surfactant 
Given the limited number of therapies shown to effectively decrease BPD and the importance 
of inflammation in its pathogenesis, it is critical to find a method to administer glucocorticoids 
that will target their anti-inflammatory effects to the lung and minimize systemic effects. 
Budesonide is a glucocorticoid with strong local anti-inflammatory effects and limited transfer 
to the systemic circulation when given intratracheally with surfactant as the vehicle. Two 
published randomized trials 1;2  of about 380 patients have reported that intratracheal 
installation of budesonide combined with surfactant as the vehicle for distribution into the lungs 
significantly improved respiratory status and decreased the incidence of BPD or death in 
preterm infants without adverse side effects. Both studies reported no differences in the 
incidence of neuromotor dysfunction, mental or psychomotor developmental indexes, or 
physical growth parameters at follow-up at 2-3 years of age.1;2;41 The most recent study was 
accompanied by an editorial by highly respected neonatologists discussing the great promise 
of this therapy to prevent BPD.42 
 
The premise of using surfactant as a vehicle to deliver budesonide is based on surfactant’s 
ability to generate a convection flow that can be used to deliver medications to the lung 
periphery.43  Recent in vitro studies in Dr. Ballard’s (co-investigator on present study) lab have 
shown supplementation of calfactant with budesonide did not alter either surface tension 
properties6 (Table 1) and improved adsorption to the air-liquid interface.44 (Figure 1) This is 
similar to findings from Yeh et al2 who demonstrated that Survanta (100 mg or 4 mL/kg) mixed 
with budesonide (0.25 mg or 1 mL/kg) did not affect the biophysical and chemical properties of 
the surfactant. 
 
Table 1. In vitro surface tension properties of calfactant supplemented with budesonide 
Time (h) 
STmina (mN/m) 
Time to STmina (sec) 
0 
0.3 ± 0.7 
10.5 ± 1.6 
24 
1.6 ± 0.3* 
12.7 ± 3.3 
48 
1.2 ± 0.5* 
12.6 ± 2.4 
All values are within the normal range for unsupplemented Infasurf.;aSTmin = minimum surface tension ; Values 
represent means ± SD (n=6 for time 0 and n=12 for 24 and 48 h exposure at room temperature).*p<0.01 vs time 0 
 
 
Figure 1 . Budesonide addition to calfactant:effect on adsorption.  
A.Representative PL dose-response experiment for calfactant 
supplemented with budesonide 0-5% showing higher Max but 
similar ½Max with 5% budesonide (mean±SEM for triplicates).  B. 
Budesonide dose-response curve for Max film formation at 1.5 µg 
PL.  Data are mean± SEM for 4 experiments. The multimodal 
curve suggests a threshold beneficial effect between 1 and 5% 
budesonide.6   
 
d. Anti-Inflammatory Effects of Budesonide in Human 
Fetal Lung Explants  Dr. Ballard’s laboratory has 
completed a study of budesonide responses in fetal lung 
explants.6  Explants of second trimester human fetal lung 
were cultured with or without dexamethasone, budesonide, 
and in the presence and absence of calfactant. Tissue and 
media were collected for LC/MS/MS, microscopy, assay of 
IL-8 and CCL2 cytokines by Western blotting and ELISA, 
and RNA analysis.  Budesonide at 30 nM suppressed 


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medium levels of both cytokines by up to 80% at 12 hours and suppression was reversible on 
removal of steroid 
(Figure 2). Results for cytokine suppression were similar for budesonide suspended in 
calfactant. In dose-response experiments, half-maximal suppression occurred at 0.04±0.02 
and 0.05±0.02 nM for IL-8 and CCL2, respectively, consistent with the reported high potency of 
budesonide in other systems. Using RNAseq analysis, he identified 230 genes, including IL-8 
and CCL2, that were decreased by >50% with a False Discovery Rate of <0.05 (data not 
shown due to space limitation), indicating that budesonide down-regulates a network of 
inflammatory mediators in lung tissue acting at the level of transcription. In studies using 
LC/MS/MS of explant tissue and culture medium,levels of the major budesonide metabolite 
(16α-hydroxyprednisolone) was <5% of the budesonide level.  Thus, most metabolism of 
budesonide in vivo occurs in non-pulmonary tissues, 
primarily the liver. 
Figure 2: Chemokine release from human fetal lung explants 
cultured with budesonide or dexamethasone is maximally decreased 
by 24 hours and reversed by 72 hours. (A) Representative CCL2 
Western blot on explant culture media ± 30 nM budesonide or 30 
nM dexamethasone collected every 24 hours (CCL2 N.D. in tissue, 
IL-8 N.D. by Western in media or tissue).  (B) IL-8 and CCL2 levels 
in explant culture media as measured by ELISA reduced by up to 
90% of control levels within 12 – 16 hours and 8 – 12 hours 
respectively (n=4, 30 nM budesonide). (C,D) Washout experiments 
exposed human fetal lung explants to 3 nM budesonide, 3 nM 
dexamethasone, or control media for 4 hours, followed by a saline 
rinse and replacement with steroid- and serum-free control media 
changed every 24 hours out to 96 hours.  Levels of (C) CCL2 and 
(D) IL-8 were measured by ELISA in media collected every 24 
hours.  Explants cultured in budesonide or dexamethasone but not 
washed out were included for comparison.  For CCL2, the 
differences between washout levels compared to control levels were 
statistically significant at all time points while IL-8 levels were 
significantly different at 48 and 72 hours.  Dashed line indicates 
control levels.  (wo = washout).6 
 
In preparation for this planned dose escalation trial, 
investigators from our group recently conducted a 
pharmacokinetic investigation of a combination 
budesonide and surfactant mixture that was instilled into 
the lungs of 7 preterm lambs.8 All of the animals 
received 0.25 mg/kg budesonide with surfactant as the 
vehicle, which is the same dose used in the above trials 
by Yeh et al.1;2  Plasma and tissue samples were 
obtained and assayed to quantify budesonide, 16α-hydroxyprednisolone, and budesonide 
palmitate concentrations using LC/MS/MS. Peak plasma concentrations were found to be 
inversely correlated with the oxygenation index (correlation coefficient of -0.75). Plasma 
budesonide concentrations were extremely low (~10% of expected) for two lambs that had 
high oxygenation indices and were excluded from further analyses. For the remaining 5 
preterm lambs, a non-compartmental analysis demonstrated a mean AUCinf of 148.8 (SD ± 
28.2) ng*hr/mL, a half-life of 4.8 ± 1.8 hrs, and a peak concentration of 46.2 ± 17.7 ng/mL. 
Using population pharmacokinetic methods, a one-compartment model with an exponential 
residual error model and first-order absorption adequately described the data.8 The apparent 
clearance and volume of distribution were estimated at 6.3 L/hr and 29.1 L, respectively. 


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Budesonide and budesonide palmitate were unable to be detected in the brain. This finding 
suggests that intratracheal instillation results in local pulmonary deposition, with no evidence of 
budesonide accumulation in the central nervous system. Overall, these results revealed that 
peak plasma budesonide concentrations are inversely correlated with the oxygenation index 
and that lung-specific delivery avoids budesonide accumulation in the brain.8  
 
In the initial study by Yeh et al.,1 10 of the preterm infants treated intratracheally with 0.25 
mg/kg budesonide mixed with surfactant had venous blood samples collected at 30 min, 1, 2, 
4, and 8 hrs post-dosing to permit a pharmacokinetic analysis. The mean budesonide 
concentration observed at 30 mins was 20.9 (SD ± 14.4) ng/mL. The mean budesonide 
concentration measured at 8 hrs was 9.9 ± 8.2 ng/mL. Over the first 8 hrs following dosing, the 
mean AUC was calculated to be 115.7 ng*hr/mL. Of note, there was one neonate that had 
budesonide concentrations measured that were an order of magnitude lower than those 
observed among his/her peers, which may reflect insufficient lung recruitment. Yeh et al.2 also 
demonstrated significant decreases in IL-8 in tracheal aspirates at 12 hour, 3-5 days, and 7-8 
days after treatment. 
 
A dose escalation study is needed to provide critical data about the most efficacious and safest 
dose for reducing BPD in premature infants, hence the design of a multicenter RCT to evaluate 
this potential therapy for BPD. 
 
 
3. Study Design 
This is a phase I/II open-label study to determine the lowest, safe, effective dose of 
budesonide given with calfactant as the vehicle; we will perform an unblinded dose escalation 
study. We will administer four dosing levels of budesonide suspended in calfactant beginning 
with 0.025 mg/kg of budesonide (10% of the dose used in the studies by Yeh et al.1;2) 
administered to 8 ELGANs who are intubated at 3-14 days of age. Daily doses (at the same 
dosage) will be administered to infants who remain intubated for a potential of 5 total doses in 
each patient.  Subsequent groups of 8 infants each will receive 0.05 mg/kg, 0.10 mg/kg, and 
0.15 mg/kg or 0.20 mg/kg of budesonide in calfactant. Each patient will receive up to 5 total 
doses, for a total of up to 32 infants in the entire trial.  We will evaluate the clinical, laboratory 
and safety data from each group of treated infants to 28 days of age before moving to the next 
dosing level of budesonide.  
 
Control Group 
Case-matched premature infants who received calfactant alone in the Trial of Late 
SURFactant (TOLSURF)3 will serve the comparison group using banked samples of tracheal 
aspirates.  
 
4. Study Population 
 
a. Number of Subjects 
The potential study sample size is 32 total patients with 8 patients per dosing level.  Each site 
will enroll approximately 2-3 subjects per dosing level, though there is no minimum/maximum 
number of subjects per site.   
 


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b. Inclusion and Exclusion Criteria 
 
Inclusion Criteria 
a.  230/7 to 276/7 weeks, inclusive, of gestational age based on center’s best estimate of 
due date  (using  earliest obstetrical ultrasound, last menstrual period, examination, and 
other pertinent available information) 
b. Day of life 3-14 from the date and time of delivery, with the date of birth being DOL 0 
c. Intubated and mechanically ventilated and do not anticipate extubation in next 24 
hours 
 
 
Exclusion Criteria 
a. Serious congenital malformations or chromosomal abnormality (as described in the 
manual of operating procedures) 
b. Likely to be extubated in next 24 hours 
c. Clinically unstable (see criteria below) 
d. Infants who have received systemic steroids prior to dosing with study medication.  
e. Infants who have received Indocin, Ibuprofen, or acetaminophen ≤ 96 hours prior to 
enrollment window ending 
 
Definition of Clinically Unstable Infant: 
1. Active Pneumothorax with chest tube 
2. Active Pulmonary Hemorrhage 
3. Uncontrolled hypotension requiring more than 20 mcg/kg/min dopamine, 2 inotropic 
agents, or > 30 mL/kg of fluid boluses 
4. Acute NEC – less than 24 hours from diagnosis or surgery or isolated intestinal 
perforation 
5. Untreated culture positive sepsis (<6h) 
6. RSS >14 
7. Uncontrolled hyperglycemia 
8. Clinical team feels infant would not tolerate dosing procedure 
 
Note: If patient becomes unstable during the period of dosing administration, study drug 
administration will be discontinued.  If patient stabilizes again within 14 days of life, dosing may 
be continued, up to 5 total doses per subject. 
 
If a subject is consented into the study but NOT dosed (due to instability, extubation, 
withdrawal of consent, or any reason), another subject may be consented to replace the 
subject that did not get dosed. If a patient gets treated for a PDA in the midst of SASSIE study 
dosing, further SASSIE doses will be discontinued and patient may be replaced for study 
purposes. 
 
Data collected on the pre-screening log and during the screening visit in the event of a screen 
failure will be retained for separate analysis. 
 
c. Vulnerable Populations 
Neonates will be included in this research.  The inclusion of neonates is important to the 
research because they are at risk for BPD and associated morbidities.  Parent or guardian 


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permission will be obtained before the child will be enrolled in the study, as per respective IRB 
guidelines.     
 
d. Setting 
The study is an open label dose escalation trial being conducted at 4 clinical locations:  
Oregon Health and Science University in Portland Oregon (PI:  Cindy McEvoy, MD), University 
of Florida in Jacksonville, Florida and Wolfson Children’s Hospital in Jacksonville, (PI:  Mark 
Hudak, MD), Florida Hospital for Children in Orlando, Florida (PI:  Rajan Wadhawan, MD) and 
University of California in San Francisco, California (PI:  Roberta Keller, MD).  Each PI will be 
responsible for the conduct of research at their institution.  Each institution will submit to their 
respective IRB.     
 
e. Recruitment Methods 
Subjects will be recruited from eligible inpatients within the Neonatal Intensive Care Units at 
the 4 sites listed above.  To facilitate timely enrollment in the trial, infants < 28 0/7 weeks 
gestational age will be screened at age 1-3 days of age by the study physicians, research 
personnel, and/or nurses for eligibility.  A pre-screening log will be kept. Records of potential 
candidates for the study will then be reviewed by one of the study investigators and/or study 
coordinator, and the child’s condition discussed with the attending physician. If eligible, the 
infant’s parents will be approached by study personnel to potentially obtain informed consent. 
This will occur in preterm infants preferably within the first 3 days of life with the expectation 
that if the infant meets criteria between 3 and 14 days of age, he/she will be enrolled at that 
time. Subjects will not be reimbursed for participation in the study. 
 
f. Consent Process 
Written informed consent will be obtained from each subject’s parent/legal guardian prior to 
any study procedures taking place. Once an eligible infant is identified, individuals authorized 
to obtain written informed consent (PI, co-investigators, and study staff specifically designated 
by the PI) will begin the informed consent discussion with the subject’s parent(s)/legal 
guardian.  The informed consent process will take place in the NICU or in a quiet, private area.   
 
The information provided in the consent will cover the elements in the CFR Part 50.25 and be 
approved by the site Institutional Review Board (IRB). This includes the investigational nature 
and objective of the trial; the procedures and treatments involved and their attendant risks, 
discomforts, and benefits; and the potential alternative therapies, alternative to not participate 
and right to withdraw without penalty, all of which will be explained to the parents in detail. All 
of the parent/legal guardian’s questions will be answered before signing the consent form.  If 
the parent/legal guardian wishes to take the consent form home to consult with other family 
members or health care providers, or to allow more time for consideration, they will be allowed 
to do so.  A copy of the signed consent form will be given to the parent/legal guardian. 
 
The informed consent process will be an ongoing active process of sharing information 
between the investigator and the parent/legal guardian(s).  If a protocol change requires a 
change to the consent form, parents/legal guardian(s) will be notified in a timely manner and 
the new informed consent form will be signed.   
 
Assent will not be obtained as the subjects in this study are neonates. 
 


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Permission may be obtained from legally authorized representatives, described as an 
individual who is authorized under applicable state or local law to consent on behalf of a child 
to general medical care when general medical care includes participation in research.   
 
5. Procedures Involved 
 
a. Study medication dosing  
Subjects will be dosed with budesonide given in calfactant as per the dose escalation plan.  
There are four dosing levels:  0.025 mg/kg, 0.050 mg/kg, 0.10 mg/kg, and 0.15 mg/kg or 0.20 
mg/kg of budesonide given with calfactant as the vehicle.  The calfactant dose is as 
recommended by the manufacturer for newborn therapy (3 ml/kg). The final product will be 
compounded according to the following formula: 
 
Table 2a.  Dosage Preparation of Budesonide and Calfactant (3 mLvial) for 1 kg infant 
Bud Dose 
(mg/kg) 
Bud Aliquot (ml/3 ml 
Calfactant) 
Final Volume 
(ml) 
Mixture Dose (ml/kg) 
0.025 
0.05 
3.05 
3.05 
0.05 
0.1 
3.1 
3.1 
0.10 
0.2 
3.2 
3.2 
0.15 
0.3 
3.3 
3.3 
0.20 
0.4 
3.4 
3.4 
 
Table 2b.  Dosage Preparation of Budesonide and Calfactant (6 mL vial) for 1 kg infant 
Bud Dose 
(mg/kg) 
Bud Aliquot (ml Budesonide /6 
ml Calfactant) 
Final Volume 
(ml) 
Mixture Dose (ml/kg) 
0.025 
0.10 
6.10 
3.05 
0.05 
0.2 
6.2 
3.1 
0.10 
0.4 
6.4 
3.2 
0.15 
0.6 
6.6 
3.3 
0.20 
0.8 
6.8 
3.4 
 
Budesonide (Pulmicort Respule 1.0 mg/2 ml); Calfactant 35 mg/ml, 3 ml/kg dose. 
Add indicated aliquot of budesonide to 3 ml or 6 mL of calfactant, gently mix, pull mixture into 
syringe at dose/kg infant weight as shown.  Note:  dosing level 4 will be either 0.15 mg/kg or 
0.20 mg/kg based on effectiveness of previous doses. 
 
The research pharmacy at the respective institutions will prepare the combination of the 
appropriate dose of budesonide (Pulmicort nebulizing suspension, Astra Zeneca, Sweden) 
suspended in calfactant (Infasurf ONY INC) as per the dose escalation protocol.  Under sterile 
conditions, the appropriate amount of budesonide will be added to the 3 or 6 ml vial of 
calfactant to provide the desired drug concentration. The contents will be mixed by gentle 
inversion of the syringe (vial) to avoid excessive bubble formation.  Because both budesonide 
and calfactant are suspensions of lipophilic substances, effective mixing is expected to occur 
rapidly. The budesonide/ calfactant mixture will be prepared in the research pharmacy shortly 
before it is sent to the NICU for instillation. If there is an unexpected delay in instillation, the 
vial will be stored at 5°C for no more than 24 hours.    
 


14 
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The study medication will be administered using standard procedures and precautions as for 
newborn treatment.  Study drug is administered via skilled personnel with care taken to assure 
adequate lung inflation during the instillation based on our experience with preterm lambs.8 
Ventilator adjustment guidelines will be provided to address the response to any significant 
changes in oxygenation, ventilation, heart rate or chest wall movement during or after the 
procedure. Vital signs, saturations, respiratory severity score and ventilator settings will be 
recorded prior to and at 60 and 120 minutes after each study procedure. Subsequent 
ventilatory management is at the discretion of the clinical medical team, with a common 
ventilator strategy to maintain adequate lung inflation and minimize over distention and oxygen 
exposure.  Up to 5 total doses will be given at daily intervals, with dosing no sooner than 18 
hours post last dose. 
    
If the infant remains intubated 18 to 24 hours after the 1st study dosing with no plan for 
extubation in next 24-48 hours, a second dose of the same dosage of budesonide will be 
given. Up to 5 total doses may be given daily if the infant remains intubated. Retreatment 
intervals may change after analysis of data from the first 8 patients in terms of respiratory 
status and cytokine suppression after dosing. If the infant is considered clinically unstable by 
the physicians at the time when a dose should be given, but later stabilizes during the study 
eligibility period (up to 14 DOL), the infant will continue with study drug dosing as per the 
dosing schedule outlined above, and is still eligible for 5 total study doses after study dosing 
resumes. 
 
Patients who are extubated within 24 hours of a study dosing will not receive subsequent study 
medication dosing. However, if an infant is re-intubated up to 14 days of life, they can restart 
study dosing again at the next appropriate dose and receive a total of 5 doses. Study sampling 
will occur according to the study procedure chart (see chart below on subsequent pages). 
 
If the attending physician deems that the infant would benefit from intravenous or oral steroids 
during the 5 days of study dosing, the following must be followed: the site PI should be notified 
promptly, and the site PI, in turn, will report this development to the study PI, Dr. McEvoy 
within 72 hours. All further dosing with the study dosing protocol will be discontinued; data and 
sample collection and AE reporting on the infants should continue as per study protocol. 
 
If an eligible patient is treated with Indocin, ibuprofen, or acetaminophen, enrollment for the 
infant will be delayed by 96 hours after the last dose. If the attending physician feels that the 
infant requires Indocin, ibuprofen, or acetaminophen for the treatment of a clinically significant 
PDA (as agreed upon via the clinical guidelines) during the 5 days of dosing, study medication 
administration will cease. An attempt will be made to replace the infant in the study with 
another for purposes of the study. In addition there should be an attempt to avoid the use of 
Indocin or Ibuprofen until > 48 hours after last study treatment dosing 
 
Study dosing will be stopped if there are any safety concerns deemed significant or potentially 
significant by the Medical Monitor.  The Medical Monitor will review SAEs and any other 
concerns/questions reported by the sites in detail and will consult with the DSMB, site PIs, 
and/or study PI as needed.   
 
If the first 3 dose levels do not meet the criteria for effectiveness as outlined in Specific Aim 1, the final 
dose level (level 4) will be increased from 0.15mg/kg to 0.2mg/kg.  If dose level 3 does meet criteria for 
effectiveness, the final dose level (level 4) will remain at 0.15mg/kg. 


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b. Tracheal Aspirate 
Lung fluid from the infants breathing tube will be collected and tested for markers of 
inflammation. Tracheal aspirate (TA) samples are collected before each of the maximum of 
five (5) doses of budesonide: calfactant. TA’s will also be drawn 12 ± 2 hours (10-14 hours) 
after the first dose and 24 ± 2 (22-26 hours) and 48 ± 2 hours (46-50 hours) after the fifth dose 
(if infant is still intubated). If extubation is planned sooner than 24 hours after any dose, a TA 
sample should be obtained during routine airway preparation procedures just prior to 
extubation. A maximum of 8 TA’s can be collected if an infant receives all 5 doses of study 
drug and remains intubated more than 24 hours after the fifth dose.  
 
Each tracheal aspirate sample will be centrifuged within 20 minutes of collection to provide a 
cell pellet and a supernatant fraction. After freezing and batch shipment to UCSF, the 
supernatant is centrifuged and assayed for total protein and for selected cytokines using 
ELISA. The TA cell pellet is used for quantitation of DNA to determine total cell number. All 
tracheal aspirate samples are labeled by study ID number and date/time, without other 
identifiers. Analyses of data from study samples as related to clinical parameters is performed 
by P Ballard with assistance of project manager Jeanette Asselin.  
 
Further exploratory analyses will be done as follows. Once the dose of budesonide is identified 
that  effectively suppresses IL8 and CCL2 , a limited set of TAs (from infants treated with that 
dose )will be sent to Dr. Ambalavanan’s laboratory at the University of Alabama for multiplex 
analysis of up to 32 cytokines of interest in the development of BPD13 as well as airway 
microbiome analysis to assess potential modulation of the mucosal microbiome  by 
glucocorticoids.14  Again, all tracheal aspirate samples are labeled by study ID number and 
date/time, without other identifiers. 
 
c. Plasma Samples 
To minimize sample collection volumes, dried blood spot (DBS) samples (about 0.2 ml per 
sample) will be obtained for the measurement of budesonide levels via the central umbilical 
line in place for clinical care or by heel stick. We are collaborating with investigators at the 
Center for Human Toxicology who have experience with DBS sampling7 and will be performing 
the DBS analysis for this study.  Filter paper will be labeled with the patient’s study 
identification number, date and time of collection. Filter paper will be handled by the edges to 
prevent cross contamination. Using a previously published assay for budesonide by 
LC/MS/MS using a Thermo LCQ Advantage Max ion trap instrument equipped with a Finnigan 
Surveyor LC pump, Surveyor Autosamples, and a universal Ion Max source,8 DBS levels for 
budesonide are linear across the range from 0.5 to100 ng/mL, and the LLOQ was 2 ng/mL.   
 
If the infant has a central line, samples of blood will be drawn to test for budesonide levels at 
15 minutes, 1 hour, and 4 hours after initial dosing for a total of 3 samples.  If the infant does 
not have a central line, 2 blood levels will be drawn at 1 and 4 hours and we will attempt to 
time them with another blood draw being done for clinical care whenever possible. Trough 
levels will be done prior to any further doses for a potential total of 7 DBS or a total of 1.4 mL 
(2.8 mL/kg for a 500 gram baby over a five day period which does not represent an excessive 
blood loss).  
 
d. Clinical Data Collection 


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Clinical data that will be collected includes patient demographics, vital signs including blood 
pressure measurements, laboratory data obtained for clinical care during calfactant: 
budesonide dosing including complete blood counts, glucose, and electrolytes and detailed 
respiratory support through 28 days of age, and 36±1 week and 40 ±1 week postmenstrual 
age (PMA), medications, significant co-morbidities, and status at discharge.  See MOP for 
more details regarding data collection.  The respiratory data includes the type of respiratory 
support, FiO2, and ventilator settings, which will be used to calculate daily respiratory severity 
scores (RSS = MAP X FiO2). Other respiratory outcomes through 28 days of age include the 
length of ventilatory support, duration of oxygen supplementation, data on medication 
administration, including use of steroids, vitamin A, caffeine, antibiotics and indomethacin. We 
will collect vital signs, saturations, RSS, and ventilator settings prior to, at 60 minutes and 120 
minutes after each dose.  Survival at discharge without BPD at 36 and 40 weeks and date of 
discharge will also be collected. Data may be entered directly onto CRFs. Please see Table 3 
below for procedures done during the study. 
 
 
e. Oxygen/Flow Reduction Challenge Test 
Oxygen/Flow Reduction Challenge Test is an assessment used to determine whether an infant 
who is receiving a small amount of supplemental oxygen or flow support at 36 weeks corrected 
age requires supplementation. This is an important component in determining whether an 
infant has BPD. Study patients who remain hospitalized at 36 (±1) weeks PMA and are stable 
on a small amount of supplemental oxygen or supplemental flow via nasal cannula are eligible 
to complete the oxygen/flow reduction challenge test. If the infant remains hospitalized at 40 
(±1) weeks PMA and meets the same criteria, the test will be completed then as well. The 
O2/Flow Challenge Worksheet will be used when completing this test. For infants no longer in 
the study hospital, a member of the study team will contact the referral center and/or parents to 
obtain information about infant’s respiratory support at 36 and 40 weeks of PMA.  
 
Prior to starting the test, patients will be evaluated by a physician to determine stability and 
also to collect baseline data using a pulse oximeter. The pulse oximeter collects baseline data 
from the infant on their current oxygen and nasal cannula liter flow/minute support. Data is 
collected for 5 minutes, with the time and oxygen saturation recorded at every minute. If 
oxygen saturations recorded are ≥90% after 5 minutes, the infant may complete the oxygen 
reduction phase. 
 
The oxygen/flow reduction challenge test will follow the current NIH recommendations for BPD. 
First, the FiO2 will be weaned by 0.20 every 5 minutes until either: 1) The infant is in room air 
(FiO2 0.21) and off nasal cannula with adequate saturations OR 2) The infant fails the 
oxygen/flow challenge exam with saturations ˂ 90% for 5 continuous minutes OR saturation 
˂80% for 15 seconds. Once the patient is placed in room air off all support, the infant will be 
monitored and data collected for one hour or until the criteria for failure is met.  
 
An alternative weaning schedule can also be used if the supplemental oxygen is being 
delivered by nasal cannula without blender/medical air. The flow will be weaned by 50% every 
10 minutes until the flow if ≤ 0.15 lpm, then the cannula will be gently removed from the nares. 
The wean will be discontinued if the infant fails the oxygen/flow challenge exam with 
saturations ˂ 90% for 5 continuous minutes OR saturation ˂80% for 15 seconds. Again, once 


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the patient is placed in room air off all support, the infant will be monitored and data collected 
for one hour or until the criteria for failure is met.  
 
Possible test results include: 1) Pass: All saturations ≥90% for one hour; 2) Fail: Saturation ˂ 
90% for 5 continuous minutes or saturation ˂80% for 15 seconds. At the conclusion of the 
oxygen/flow challenge the infant should be returned to their previous level of supplemental 
oxygen. The clinical care team will be notified of the results of the challenge test.   
 
 
f. Pulmonary Function Testing in Patients Enrolled at Oregon Health & Science 
University 
 
Non-invasive pulmonary function tests (PFTs) have been used to quantify the response of the 
newborn infant to a number of medications including antenatal10-12 and postnatal 
corticosteroids,9;45 and these physiologic measurement have been shown to correlate with the 
infant’s clinical course.9;11  Neonatal PFTs are regularly performed at OHSU and in the present 
study, PFTs will be done at OHSU prior to the first dose of study medication, 24 hours after the 
first dose, prior to extubation and then at discharge at 34-36 weeks of postmenstrual age. 
International criteria for testing acceptance will be followed as outlined below.9;11;12 
 
PFTs will be measured with a computerized infant pulmonary function cart (SensorMedics 
2600, SensorMedics Inc., Yorba Linda, CA and the Master Screen BabyBody, Jaeger/Viasys). 
Passive respiratory compliance (Crs) and resistance (Rrs) will be obtained with the single-
breath occlusion technique12 and functional residual capacity (FRC) with the nitrogen washout 
method.10;11 In intubated patients including those requiring surfactant, testing will be performed 
by connecting the infant’s endotracheal tube into the system via a three-way valve that also 
connected to the ventilator. In non-intubated patients, a face mask will be used.   
 
For the single-breath occlusion technique, the airway will be briefly occluded at end inspiration 
until an airway pressure plateau is observed and the Hering Breuer reflex invoked. The linear 
portion of the passive flow-volume curve will be identified, and a regression line drawn to 
obtain the best fit. From the intercepts on the flow and volume axes, respiratory system 
compliance and resistance will be calculated. Acceptance criteria will include: 1) stable end 
expiratory baseline; 2) plateau pressure lasting >100 msec; 3) plateau pressure varying by < ± 
0.125 cm H2O; 4) acceptable flow-volume curve by visual inspection, with linear data segment 
identified; 5) at least ten breaths accepted with a coefficient of variation of <20%.10-12 
 
For the nitrogen washout technique, calibration will be done with 2 known volumes, and a 
calibration line will be constructed for the system at the specific flow rate. The calibration curve 
will then be used to correlate the nitrogen washed out to the infant’s FRC. The system will be 
corrected for dead space present and corrected the FRC to body temperature, pressure, and 
water-saturated conditions. Total FRC will be related to body weight. Acceptance criteria will 
include: 1) infant supine and quietly asleep; 2) test initiated at end expiration; 3) no evidence of 
leak on tracing of the washout; 4) consistent tracings; 5) at least 3 measurements with a 
coefficient of variation of < 10%.10-12 
 
g. Future Contact 


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We may contact subjects in the future if there are things we need to discuss or ask regarding 
this study or for future study opportunities, if they have agreed on the consent form to this 
future contact.  We will also review the subject’s medical records in the future to look at 
behavior and breathing outcomes up to 24 months of age.  We may also review and obtain  
other health information which may be important for the study.  We may obtain this information 
so that we can be sure to have all of the important information we need for the study.   
 
 
 
 
h. Adverse Event and Protocol Violation Reporting 
 
All AE’s reported or observed from the time the patient is consented into the study through the 
end of study participation (28 days) will be documented/reported.   
 
Definitions 
 
Adverse Event:  An Adverse Event (AE) is any untoward medical occurrence in a clinical 
investigation subject administered a pharmaceutical product and that does not necessarily 
have a causal relationship with this treatment.  An AE can therefore be any unfavorable and 
unintended sign (including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of a medical (investigational) product, whether or not related to the 
medicinal (investigational) product (ICH Guidance E2A 1995). 
 
Serious Adverse Event:  An SAE is an event that: 
 
 Results in Death 
 Is immediately life threatening 
 Requires inpatient hospitalization or prolongs existing hospitalization 
 Results in Persistent or significant disability and/or incapacity, or 
 Is a congenital anomaly/birth defect 
 Is an important medical event; Note:  Important medical events that may not result in 
death, be life threatening, or require hospitalization may be considered an SAE when, 
based upon appropriate medical judgment, they may jeopardize the subject and may 
require medical or surgical intervention to prevent 1 of the outcomes listed in this 
definition. 
 
Hospitalizations, which are the result of elective or previously scheduled surgery for pre-
existing conditions, which have not worsened after initiation of treatment, should not be 
classified as SAEs.  However, complication(s) resulting from a hospitalization for an elective or 
previously scheduled surgery that meets serious criteria must be reported as SAE(s).   
 
Assessment of Severity 
 


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The severity of AEs/SAEs must be recorded during the course of the event including the start 
and stop dates for each change in severity.  An event that changes in severity should be 
captured as a new event.  Worsening of pre-treatment events, after initiation of investigational 
product, must be recorded as new AE(s). 
 
The medical assessment of severity is determined by using the following definitions: 
 
 Mild:  A type of AE that is usually transient and may require only minimal treatment or 
therapeutic intervention.   
 
 Moderate:  A type of AE that is usually alleviated with specific therapeutic intervention.  
The event interferes with usual activities of daily living, causing discomfort but poses no 
significant or permanent risk of harm to the research subject. 
 
 Severe:  A type of AE that significantly affects clinical status, or may require intensive 
therapeutic intervention. 
 
Assessment of Causality 
 
 Definite: Events that, after careful medical evaluation, are considered definitely related 
to the drug treatment and other conditions (concurrent illness, progression/expression of 
disease state, or concurrent medication reaction) do not appear to explain the event.  
 
 Probable: Events that, after careful medical evaluation, are considered with a high 
degree of certainty to be related to the study drug. The following characteristics will 
apply: 
  
• 
A reasonable temporal relationship exists between the event and exposure to the 
study drug, and 
• 
The event is a known reaction to the study drug that can be explained by an 
alternative cause commonly occurring in the population or individual, or 
• 
The event is not a known reaction to the study drug but cannot be reasonably 
explained by an alternative cause.  
 
 Possible: Events that, after careful medical evaluation, do not meet the criteria for a 
definite or probable relationship to the study drug, but for which a connection cannot be 
ruled out with certainty. The following characteristics will apply: 
 
• 
The event occurs after exposure to the study drug  
• 
There is a reasonable temporal relationship to the study drug, but the event is not a 
known reaction to the study drug and could be explained by a commonly occurring 
alternative cause, or  
• 
In the absence of a reasonable temporal relationship, the event cannot be explained 
by an alternative cause.  
 
 Unlikely: Events that, after careful medical evaluation, do not meet the criteria for a 
possible or probable relationship to the study drug and for which a connection is 
unlikely. The following characteristics will apply:  


20 
Protocol Version 3.0 
 
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• 
The event does not follow a reasonable temporal sequence from study drug dosing, 
or 
• 
The event may have been produced by environmental factors, and  
• 
There is no apparent pattern of response to the study drug.  
 
 Not related: Events in this category will have either of the following characteristics: 
 
• 
The event occurs before exposure to the study drug, or 
• 
The event does not have a reasonable temporal relationship to study drug and can 
be explained by a commonly occurring alternative cause. 
 
Event reporting 
 
All AEs, and SAEs should be documented on the appropriate adverse event logs/forms, 
entered into Redcap, and reported as outlined below. 
 All SAEs should be reported by the clinical site to the project manager (J Asselin) or the 
project PI (Cindy McEvoy, MD) within 1 business day. 
o The medical monitor (Dr. Sue Ann Smith, a neonatologist uninvolved in care of 
recruited patients) will then be notified and will review the event.  After the 
medical monitor has reviewed the event, additional notifications will be made to 
the DSMB, IRB, and FDA as necessary.  
o Sites should report events to their IRB according to IRB guidelines and 
regulations.   
o When deemed necessary by the medical monitor, the DSMB will be notified of 
any significant events immediately and a meeting will take place to determine 
appropriate actions. 
 All other AEs should be entered into Redcap within 7 days. 
The DSMB will meet after each dosing group of 8 patients and will review all other AEs/SAEs 
at that time. The DSMB will be composed of Dr. Patricia Spitale (neonatologist), Dr. Thuy 
Nguyen (pediatric pharmacist), and Zunqiu Chen (biostatistician). Dr. Sue Ann Smith will be 
the medical monitor for the study. 
Safety Monitoring 
 
Monitoring for SAEs and AEs will be performed throughout the study period.  The site will 
submit completed SAE CRFs to the data coordinator and/or the project PI, Dr. McEvoy, within 
1 business day.  Any additional background information will be collected about the event and 
the medical monitor will be notified.  The medical monitor will review the event information and 
determine additional action as necessary.  The medical monitor may stop the study or put the 
study on hold pending further review by the DSMB as the medical monitor deems necessary. 
 
The DSMB will meet before subject recruitment begins and will subsequently meet after each 
group of 8 patients reaches 28 days of age to review study progress and AE/SAE reports.  
Additionally, they may meet as needed to review SAEs when the medical monitor deems it 
necessary. 
 


21 
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Table 3. Study Procedure Table 
 
Study Day 
Pre-
Study 
First 
dose 
15 
min 1h 4h 12h 24h 48h 
Add’l 
doses 
2-5 
Through 
28 days 
36 wks 
PMAf 
40 
wks 
PMAf  
24 
months 
of age 
Informed 
consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Dose with 
Budesonide 
in 
calfactanta 
 
X 
 
 
 
 
 
 
X 
 
 
 
 
Tracheal 
Aspirateb 
 
X 
 
 
 
X 
X 
X 
X 
 
 
 
 
Dried blood 
spots from 
central linec 
 
 
X 
X 
X 
 
 
 
Xd 
 
 
 
 
Clinical 
data 
collection 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
PFTe 
 
X 
 
 
 
 
X 
 
 
 
X 
 
 
AE and 
SAE 
collection 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Respiratory 
status 
 
 
 
 
 
 
 
 
 
 
X 
 
X 
 
Medical 
record 
review 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
a) If remains intubated and mechanically ventilated may get up to 5 total doses 24-48 hours 
apart 
b) For the first dose, TA is obtained at 0 h, 12 h, and prior to a second dose.  When multiple 
doses occur back to back, TA is collected before each dose and at 24 and 48 h after the final 
dose. 
c)  If no central line, samples will be done at 1h and 4h  


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d)  If further daily doses given a single daily sample will be done before dosing 
e)  PFTs will be done at OHSU only.  Tests will be performed prior to the first dose of study 
medication, 24 hours after the first dose, prior to extubation and then at discharge at 34-36 
weeks of postmenstrual age 
f) PMA is postmenstrual age  
 
6. Data and Specimens 
 
a. Handling of Data and Specimens 
Tracheal Aspirate (TA) Samples:  Each TA sample is centrifuged within 20 minutes after 
collection to provide a cell pellet and a supernatant fraction.   Both the supernatant and cell 
pellet are frozen until shipment to UCSF laboratory via Fed Ex utilizing dry ice to keep samples 
frozen.  After shipment to UCSF, the supernatant fraction is assayed for selected cytokines 
using ELISA .  The TA cell pellet is used for isolation of DNA to quantitate cell content and to 
develop a repository for genetic studies of gene variants.   
 
All tracheal aspirate samples will be labeled by study ID number and date/time, without other 
identifiers.  The study samples will be stored in a locked freezer and access to the Ballard 
laboratory requires a coded identification card that is available only to approved laboratory 
personnel.  Analyses of data from study samples as related to clinical parameters will be 
performed with assistance from the project manager. Should any portion of samples remain, 
we will request that it be stored, labeled only by study number, in the laboratory of  Dr. P. 
Ballard for further testing of substances identified as related to airway inflammation and the 
development of  BPD (this is addressed specifically in the consent form and reflected in the 
CRF). 
 
Dried Blood Spot (DBS) Samples:   DBS samples will be labelled with the subject study ID and 
the date and time the sample was collected.   DBS samples will be placed in DBS envelope 
and kept in ambient temperature in a secure location until samples can be batched and mailed 
to the University of Utah’s Center  for Human Toxicology Laboratory. Promptly upon receipt of 
the samples, they will be catalogued and stored until they are analyzed via LC/MS/MS. After 
the budesonide concentrations have been established via LC/MS/MS, the data will be used to 
construct non-compartmental and population pharmacokinetic models. These models will be 
constructed according to FDA guidelines and industry-standard best practices and will be used 
to quantify the extent of budesonide exposure.46 
 
Once all testing has been completed, any leftover samples will be shipped to Dr. Cindy 
McEvoy at OHSU. 
 
b. Sharing of Results with Subjects 
TA and blood spot results will not be shared with subjects or site investigators.   Each tracheal 
aspirate sample and blood spot will be coded with subject study ID only.  Patient name, initials, 
birth date and other potential identifiers are known only to investigators at each study site and 
this information will not be available to Dr. P. Ballard or other laboratory personnel.  
 
 


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7. Data Analysis 
a. Primary outcome/objective 
Efficacy will be measured and summarized within each dosing level.  Dose 
escalation will be defined as effective at the dose level in which 5 of 8 infants 
achieve: 
 At least 1 of the following: 
1) Extubation within 72 hours of first dose or after < 3 doses-without re-
intubation before 28 days of age  
2) RSS on nasal continuous positive airway pressure (NCPAP) < 1.5 or 
on nasal cannula Fi02 <25% at < 2L/min at 28 days of age, persisting 
for at least 72 hours. 
3)  Cumulative supplemental oxygen < 4.2 from time of enrollment to 28 
days of  age4 (see calculation below) 
4) No respiratory support at 28 days, including no supplemental oxygen by 
nasal cannula  
 AND a > 50% suppression of the tracheal aspirate interleukin-8 (IL-8) or CCL2 
(chemokine ligand 2) at 24-72 hours (or prior to extubation if occurs at <24 hours 
after dosing) after the initial dose in 5 of 8 of the infants (this may be a different 
combination of 5 patients than those who met the above criteria).   
Note: If a patient is re-intubated before 28 days of age for non-respiratory reasons (e.g. 
required for surgery), these additional days of respiratory support will not be included  when 
analyzing the primary outcome. 
As the design of this study is for dose escalation, analyses will likely be under-powered to 
detect significant differences, yet these will provide valuable data for future studies.   
 
Cytokine values will be compared between infants in this study and a matched comparison 
group from the TOLSURF study.3  If 32 patients are enrolled and 32 matched patients are 
used, this study will have 80% power to detect a significant (α<0.05) change of 71 pg/ml/mg 
urea (estimated SD 100) in IL-8 (as compared to zero) from before the first dose to 12 hours 
post-dose. This is based on a previous finding of a significant difference (p=0.03) of 145 
pg/ml/mg urea IL-8 between treated and untreated infants at 12 hours after treatment with 
budesonide.  Likewise, this study will have 80% power to detect a significant (α<0.05) change 
of 0.14 in the ratio CCL2 secretion relative to pre-dosing at 24 hours after the first dose. This is 
based on a significant finding of a 90% reduction in CCL2 as compared to controls after 
budesonide treatment of lung explants. 
 
Change in CCL2 and IL8 within patients will also be assessed via statistical analysis. If 32 
patients are enrolled, this study will have 80% power to detect a significant (α<0.05) change of 
73 pg/ml/mg urea (estimated SD 150) in IL-8 (as compared to zero) from before the first dose 
to 12 hours post-dose. This is based on a previous finding of a significant difference (p=0.03) 
of 145 pg/ml/mg urea IL-8 between treated and untreated infants at 12 hours after treatment 
with budesonide.  Likewise, this study will have 80% power to detect a significant (α<0.05) 
change of 0.1 in the ratio CCL2 secretion relative to pre-dosing at 24 hours after the first dose. 
This is based on a significant finding of a 90% reduction in CCL2 as compared to controls after 
budesonide treatment of lung explants. 
 


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Comparisons of major co-morbidities of prematurity (PDA/NEC/IVH/sepsis/ ROP) between 
treated infants in this study and the matched comparison group will be descriptive and include 
comparisons of the co-morbidities and overall outcomes between the different dosing levels 
and compared using Fisher’s exact test. 
 
b. Secondary outcomes 
The secondary objectives will quantify safety criteria including clinical parameters that may be 
associated with potentially elevated systemic glucocorticoid concentrations.  Serial budesonide 
levels will also be collected to perform pharmacokinetic modeling.   
 
c. Clinical parameters including blood pressure, glucose, sodium and potassium 
levels 
Clinical parameters potentially consistent with elevated glucocorticoid concentrations will be 
collected and analyzed descriptively within each infant, dosing group, and across the total 
study population.  Comparisons of clinical parameters (continuous and normal/abnormal) will 
be made between dosing levels. These clinical parameters will be collected after the first dose 
of the study drug and up to 7 days after the final dose. 
 
d. Budesonide pharmacokinetic modeling 
Blood concentrations of budesonide will be examined over time (15 minutes, 1 hour, and 4 
hours after each dose) to evaluate the pharmacokinetics of the budesonide at each dosing 
level.  Based on preliminary data in lambs, we could expect a Cmin of 1.9 ng/mL at 9 hours 
post-dosing.  Non-compartmental and population pharmacokinetic analyses will be performed 
to establish the extent of budesonide exposure.  Commonly reported pharmacokinetic 
parameters will be calculated including:  clearance, volume of distribution, area under the 
curve (AUC), half-life, peak concentration (Cmax), and trough concentration (Cmin). 
 
e. Clinical respiratory status at 28 days of age 
Using descriptive statistics and basic inferential statistics RSS at 28 days of age and BPD 
status at 36 and 40 weeks will be compared between dosing groups and to case matched 
patients from the TOLSURF study.3 For normally distributed data an ANOVA will be 
conducted, and for non-parametric data a Kruskal-Wallis ANOVA will be conducted.  Any 
pairwise comparisons will be controlled for using a multiple testing correction to protect against 
Type I error.  As this is a secondary objective, analyses will likely be underpowered to detect 
significant differences, yet these analyses will provide valuable data for future studies.  The 
difference in change in RSS between the first day of treatment and day 28 will also be 
evaluated between groups using multiple paired t-tests and a correction for multiple testing to 
control Type I error. In addition, the decrease in RSS over the period of medication dosing will 
be compared/associated with the measured plasma concentrations of budesonide. The 
incidence of BPD at 36 and 40 weeks will be compared to historical case matched infants from 
the TOLSURF study.3 
 
Individual patient’s decrease in RSS at 28 days of age will be correlated with their measured 
budesonide levels at each level after the first dose.  If all 32 patients are enrolled, the study will 
have 80% power to detect a significant (α<0.05) correlation between RSS and budesonide 
of ρ ≥ ± 0.48.  If 16 patients are enrolled, the study will have 80% power to detect a significant 
correlation between RSS and budesonide of ρ ≥ ± 0.65. 
 


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f.  
Safety Analyses  
A review of all adverse events will be conducted at a minimum after each set of 8 patients by 
the DSMB. 
 
g. 
Exploratory Outcomes 
 
Serial PFT measurements within each patient will be analyzed using ANOVA for repeated 
measures.  The analyses of samples forwarded to Dr. Ambalavanan’s laboratory will 
primarily be descriptive and provide new findings on the scope and differential levels of 
cytokine suppression in infants treated with budesonide suspended in surfactant as well 
as potential modulation of the mucosal microbiome. 
 
 
8. Privacy, Confidentiality, and Data Security 
Upon enrollment, subjects will be assigned a code that will be used instead of their name, 
medical record number or other personally identifying information.  Electronic files for data 
analysis will contain only the subject code.  Codes will not contain any part of the 18 HIPAA 
identifiers (initials, DOB, MRN).  The key associating the codes and the subjects personally 
identifying information will be restricted to the PI and study staff. The key will be kept secure as 
outlined below. Data will be entered into the REDCap system by site study personnel. 
 
Standard practices will be followed to maintain the confidentiality and security of data collected 
in this study.  Study staff will be trained with regard to these procedures.  Paper files will be 
stored in locked filing cabinets in restricted access locations.  Electronic data will be stored in a 
web-accessible encrypted REDCap database housed on UCSF secure server.  Access to 
data/specimens is restricted to study personnel.      
 
9. Provisions to Monitor the Data to Ensure the Safety of Subjects 
We will monitor the accuracy of data entry by the sites, internally, by regular review of the CRF 
completion in REDCAP by Jeanette Asselin the project manager (PM). Ms. Asselin will 
communicate regularly with each study site (coordinator and respiratory staff) and will resolve 
any data concerns or issues on those calls.  She will perform a site visit to each study site as 
well as external monitoring. All data will be monitored for meeting entry criteria, adherence to 
protocol, primary and secondary outcomes, and adverse event reporting 
 
10. Risks and Benefits 
a. Risks to Subjects 
Surfactant 
The most common side effects reported when premature infants are given surfactant are:  
decreases in their oxygen saturation, plugging of their breathing tube, decreases in heart rate, 
reflux of surfactant into the breathing tube, and the need for extra mechanical breaths to be 
given from the breathing machine. There is also a chance that during this procedure that the 
infant may require reintubation. These events are generally brief and not associated with 
serious complications or death. The surfactant is given by skilled care givers in the NICU who 
can respond to all of these potential side effects.  
 
Budesonide 


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When the steroid is administered in high doses and is found in the infant’s blood stream 
(systemic circulation), potential adverse effects include:  decrease in growth, hyperglycemia, 
hypertension, a negative effect on brain development or ability to respond to stress.  Infants 
who were given high doses either intravenously or by mouth, and given this medication over 
42 days of treatment with a slow decrease each day were more likely to have cerebral palsy at 
follow-up.  In this study, we will start at 0.025 mg/kg of budesonide given onto the surface of 
the lung with surfactant and do not expect much to be absorbed into the bloodstream.  This is 
1/10 of the dose used in previous studies of about 400 total premature babies. We will closely 
monitor the amount of steroid being absorbed into the infants’ blood. Previous studies in 
babies giving 10 x the dose of steroid in surfactant did not show any adverse developmental 
outcomes at follow-up at 2-3 years of age.   
 
 
 
 
 
 
Tracheal Aspirate 
This will be collected at the time of routine suctioning as much as possible.  The discomfort 
from this procedure is minimal, although can result in transient decrease in oxygen saturation 
and occasionally dislodgement of the endotracheal tube, requiring reintubation.    . 
 
Plasma Samples 
Blood draws are common from central lines that premature babies often have in place during 
the first 1-2 weeks of life. Collecting study samples from the central line will not cause any 
additional pain. If a central line is not available, blood samples will be collected via heel prick. 
We will time study blood draws with testing done for clinical care as much as possible.  The 
maximum total blood collected for pharmacokinetic analysis is 7 total samples of 0.2 ml or a 
total of 1.4  ml.  This would be 2.8 ml/kg for a 500 gm newborn over a five day period which 
does not represent an excessive blood loss.   
 
Pulmonary Function Tests: 
These tests are non-invasive, well tolerated, and can be performed in intubated and extubated 
patients. 9-12;45 They are routinely be performed in the neonatal intensive care unit at OHSU. 
There are no known specific risks of the lung function test. If the infant is on a ventilator there 
is the risk the ETT will become dislodged during the testing but this risk will be minimized by 
continuous and careful monitoring by the bedside skilled NICU personnel during testing. 
During the testing there may be small fluctuations in the infant’s oxygen saturation or the infant 
may become restless during testing. This will be monitored closely and appropriate actions 
taken if needed.   
 
Confidentiality 
There is a risk to confidentiality. This risk will be minimized by storing the subjects personal 
and medical information collected as part of this study in a secure location as detailed in 
section 8 above. 
 


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b. Potential Benefits to Subjects 
The subject may or may not benefit by taking part in this study. There is no guarantee that the 
infant will receive direct benefit from his/her participation in this study. The benefits of being in 
this study may be a chance that the study medication may decrease the inflammation in the 
lungs and help decrease their risk of developing BPD.  Participation may provide information 
that may benefit other premature babies by helping to determine which dose of steroid is best 
to decrease markers of inflammation. Still, the infant may get no direct benefit from this study.  
 
 
11. Drugs or Devices  
As these 2 FDA approved medications will be given in combination and given intratracheally, 
the study medication is investigational (IND number 128102).  This study will comply with all 
FDA requirements, IRB requirements, and ICH-GCP guidance.  We will follow applicable 
Research Pharmacy policies and procedures and will provide each research pharmacy with a 
manual for the study drug. 
 
 
 
 
12. Multi-Site Coordination  
 
Prior to study initiation, study template documents including template consent forms and 
CRF forms will be provided to the outside participating sites. The PIs at each of the 
participating sites will submit the protocol, consent, and other pertinent items to their 
respective IRBs. When approval is obtained, each recruitment site PI will forward the 
IRB approval letter and approved consent to Dr. McEvoy at OHSU. These items will be 
submitted as a modification to the OHSU submission for this study. Once the OHSU 
IRB approves these modifications the respective sites will be notified that the research 
can be initiated at that site. 
 
Prior to beginning the study, all sites will have a site initiation visit by the Project 
Manager (PM).  At that visit, the study protocol and procedures will be reviewed, with 
site sponsor, study coordinator, study respiratory staff, research pharmacy and other 
medical staff.  A manual of operations will be provided and reviewed.  PM will meet 
separately with study coordinator to review all regulatory and study procedures.  
Appropriate recording of clinical data as specified by FDA Regulations for Good Clinical 
Practice will be reviewed with study personnel. 
 
During subject enrollment, the PM will communicate regularly with site coordinators to 
review site screening, enrollment, and subject status and data completion.   When a 
dosing group has been enrolled (8 infants) she will communicate when study enrollment 
is on hold and when enrollment can be restarted.   
 
The project manager for the study is J Asselin at UCSF Benioff Children’s Hospital Oakland.  
Sites will complete data on case report forms (CRFs) and then enter data into a secure 
REDCap database housed on the UCSF server. Data will be assessed for quality via 
automated and manual processes.  Queries (data discrepancies) will be generated to identify 
potential errors in the study data and to resolve them in a timely manner.   Copies of the 
original site CRFs will be faxed or pdf to DCC on a scheduled basis for comparison with 


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REDCap entries.  Original CRF documents are kept by the site in a secure location for their 
records. 
The study coordinator at each site (or designate) will review all CRFs for completeness, 
accuracy and consistency prior to entry into REDCap or faxing to the DCC. Should any 
questions or changes arise during this review, all queries will be referred back to the site 
investigator for resolution prior to faxing the forms to the DCC. 
 
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