Clinical trial NCT04560179
Conditions: Bronchopulmonary Dysplasia
Sponsor: Erik Allen Jensen
Start date: 2022-03-22; Completion date: 2024-05-17
Enrollment: 27 participants; Allocation: NON_RANDOMIZED; Intervention model: SEQUENTIAL

Brief summary:
This study is an open-label, phase 1, sequential dose escalation trial seeking to establish preliminary tolerability, efficacy, and pharmacokinetic data for up to 4 different doses of inhaled tobramycin administered to very preterm infants with BPD who are receiving invasive mechanical ventilation and have a pathogenic Gram-negative organism detected by tracheal aspirate culture.

Detailed description:

Arms: arm 1: The phase 1 trial will begin with a dose of 78mg. All treatment arms will administer study drug every 12 hours for up to 14 days (28 doses). Up to 6 infants in this arm will receive the 78mg dose of tobramycin solution for inhalation administered via vibrating mesh nebulizer. During the trial, infants in each treatment arm will undergo blood and tracheal aspirate sampling and respiratory mechanics measurements at pre-specified time points to assess dose safety and potential efficacy. Continuous pulse oximetry monitoring for the duration of the trial will also occur. Clinical data will also be recorded daily throughout the trial in all participants. arm 2: If tolerability is demonstrated in the 78mg treatment arm following the 3+3 trial design, up to 6 infants will then be enrolled and receive 150mg of tobramycin solution for inhalation every 12 hours. The same monitoring procedures will be performed in this treatment arm as in the prior. arm 3: If tolerability is demonstrated in the 150mg treatment arm following the 3+3 trial design, up to 6 infants will then be enrolled and receive 150mg of tobramycin solution for inhalation every 12 hours. The same monitoring procedures will be performed in this treatment arm as in the prior. arm 4: If tolerability is demonstrated in the 216mg treatment arm following the 3+3 trial design, up to 6 infants will then be enrolled and receive 150mg of tobramycin solution for inhalation every 12 hours. The same monitoring procedures will be performed in this treatment arm as in the prior. arm 5: Enrolled infants who are eligible to participate in the phase-1 trial may be enrolled in an untreated observational cohort at parental discretion. This cohort will undergo collection of clinical and respiratory mechanics data for 14 days after enrollment but will not receive the study drug.
Interventions: intervention 1: Tobramycin solution for inhalation 78mg dose intervention 2: Tobramycin solution for inhalation 150mg dose intervention 3: Tobramycin solution for inhalation 216mg dose intervention 4: Tobramycin solution for inhalation 300mg dose
Intervention descriptions: intervention 1: Inhaled tobramycin - 78mg administered every 12 hours for 14 days. intervention 2: Inhaled tobramycin - 150mg administered every 12 hours for 14 days. intervention 3: Inhaled tobramycin - 216mg administered every 12 hours for 14 days. intervention 4: Inhaled tobramycin - 300mg administered every 12 hours for 14 days.

Protocol documents:
Prot_SAP_000.pdf: 
 
 
Title: Phase 1 Feasibility Trial of Inhaled Tobramycin in Preterm Infants with Bronchopulmonary 
Dysplasia 
 
ClinicalTrials.gov ID: NCT04560179  
 
Sponsor: Erik Allen Jensen, MD 
 
Document Date:  June 20, 2023 
 
 
 


 
 
 
 
 
 
 
 
ii
 
Title: 
Phase 1 Feasibility Trial of Inhaled Tobramycin in Preterm 
Infants with Bronchopulmonary Dysplasia  
Short Title 
Phase 1 Trial of Inhaled Tobramycin in BPD 
Drug or Device 
Name(s): 
Tobramycin solution (300mg/5mL) for inhalation USP 
FDA IND 
145836 
Funding agency 
National Heart Lung and Blood Institute (R34HL155690) 
Regulatory Sponsor: 
Erik Jensen, MD, MSCE 
eIRB Number 
IRB 19-016800 
Protocol Date: 
July 22, 2019 
Amendment 1 Date:  October 18, 2019 
Amendment 5 Date: November 12, 2021 
Amendment 2 Date:  December 23, 2019 
Amendment 6 Date: January 25, 2022 
Amendment 3 Date: April 3, 2020 
Amendment 7 Date: March 2, 2022 
Amendment 4 Date: June 2, 2020 
Amendment 8 Date: April 6, 2022 
Amendment 9 Date: June 20, 2023 
 
Study Principal Investigator  
Erik Jensen, MD MSCE 
Children’s Hospital of Philadelphia 
Roberts Center for Pediatric Research 
734 Schuylkill Ave – Suite 19364  
 Philadelphia, PA, 19130 
Phone 267-648-2720 
email: jensene@chop.edu 
 
 


 
 
 
 
 
 
 
 
iii
TABLE OF CONTENTS 
Table of Contents .................................................................................................................. iii 
Abbreviations and Definitions of Terms............................................................................. vi 
Abstract ................................................................................................................................ vii 
Protocol Synopsis ................................................................................................................... x 
Table 1: Schedule of Study Procedures ............................................................................. xv 
Figure 1: Study enrollment and phases flow diagram…………………………………..xii 
Figure 2: 3+3 dose escalation design schematic .............................................................. xvii 
1 
BACKGROUND INFORMATION AND RATIONALE ....................................................................... 1 
1.1 
INTRODUCTION ..................................................................................................................................... 1 
1.2 
BURDEN OF BRONCHOPULMONARY DYSPLASIA (BPD) ......................................................................... 1 
1.3 
PATHOGENIC BACTERIAL COLONIZATION OF THE AIRWAY AND BPD ................................................... 1 
1.4 
DIFFICULTY TREATING AIRWAY INFECTIONS WITH SYSTEMICALLY ADMINISTERED ANTIBIOTICS ......... 1 
1.5 
NAME AND DESCRIPTION OF INVESTIGATIONAL PRODUCT ................................................................... 2 
1.6 
FINDINGS FROM CLINICAL STUDIES ..................................................................................................... 2 
1.6.1 
Human pharmacokinetics ................................................................................................................ 2 
1.6.2 
Potential toxicity with inhaled tobramycin ...................................................................................... 6 
1.7 
RATIONALE FOR DRUG DOSAGES AND DURATION UNDER INVESTIGATION ............................................ 8 
1.8 
COMPLIANCE STATEMENT .................................................................................................................... 9 
2 
STUDY OBJECTIVES .............................................................................................................................. 9 
2.1 
PRIMARY OBJECTIVE ............................................................................................................................ 9 
2.2 
SECONDARY OBJECTIVES ..................................................................................................................... 9 
3 
INVESTIGATIONAL PLAN .................................................................................................................. 10 
3.1 
GENERAL SCHEMA OF STUDY DESIGN ............................................................................................... 10 
3.1.1 
Screening Phase ............................................................................................................................ 10 
3.1.2 
Phase 1 Trial ................................................................................................................................. 10 
3.1.3 
Observational Cohort .................................................................................................................... 11 
3.1.4 
Follow-up Phase ........................................................................................................................... 11 
3.2 
ALLOCATION TO TREATMENT AND MASKING .................................................................................... 12 
3.3 
STUDY DURATION, ENROLLMENT, AND NUMBER OF SITES ................................................................ 12 
3.3.1 
Duration of Study Participation .................................................................................................... 12 
3.3.2 
Total Number of Study Sites/Total Number of Subjects Projected ................................................ 12 
3.4 
STUDY POPULATION ........................................................................................................................... 12 
3.4.1 
Inclusion Criteria .......................................................................................................................... 12 
3.4.2 
Exclusion Criteria ......................................................................................................................... 13 
3.4.3 
Definition of Pathogenic GNR Airway Bacteria ........................................................................... 13 
4 
STUDY PROCEDURES .......................................................................................................................... 13 
4.1 
SCREENING PHASE ............................................................................................................................. 14 
4.2 
OBSERVATIONAL COHORT .................................................................................................................. 14 
4.3 
PHASE 1 TRIAL OF TOBRAMYCIN SOLUTION FOR INHALATION ........................................................... 14 
4.3.1 
Blood draws .................................................................................................................................. 15 
4.3.2 
Tracheal aspirate collection .......................................................................................................... 16 
4.3.3 
Respiratory mechanics measurements .......................................................................................... 16 
4.4 
FOLLOW-UP PHASE ............................................................................................................................. 17 
4.5 
CONCOMITANT MEDICATIONS............................................................................................................ 18 
4.6 
RESCUE MEDICATION ADMINISTRATION ............................................................................................ 18 
4.7 
SUBJECT COMPLETION/WITHDRAWAL ............................................................................................... 18 
4.7.1 
Early Termination Procedures ...................................................................................................... 18 
4.7.2 
Addition of Trial Participants after Early Termination ................................................................ 18 


  
 
 
 
 
 
 
 
iv
5 
STUDY EVALUATIONS AND MEASUREMENTS ............................................................................ 19 
5.1 
SCREENING AND MONITORING EVALUATIONS AND MEASUREMENTS ................................................ 19 
5.1.1 
Medical Record Review ................................................................................................................. 19 
5.1.2 
Clinical Data Warehouse………………………………………………………………………………….19 
5.1.3 
Clinical Laboratory Evaluations ................................................................................................... 20 
5.1.3 
Research Laboratory Evaluations ................................................................................................. 21 
5.1.4 
Respiratory mechanics measurements .......................................................................................... 22 
5.2 
SAFETY EVALUATIONS ....................................................................................................................... 22 
5.2.1 
Primary composite safety endpoint ............................................................................................... 22 
5.2.2 
Secondary safety endpoints ........................................................................................................... 23 
5.2.3 
Limitations of Audiology Testing in Hospitalized Infants ............................................................. 23 
5.2.4 
Individual Subject Stopping Rules ................................................................................................. 25 
5.2.5 
Study Stopping Rules ..................................................................................................................... 25 
5.3 
EFFICACY EVALUATIONS ................................................................................................................... 26 
5.4 
PHARMACOKINETIC EVALUATION ...................................................................................................... 28 
6 
STATISTICAL CONSIDERATIONS .................................................................................................... 28 
6.1 
PRIMARY ENDPOINT ........................................................................................................................... 28 
6.2 
SECONDARY ENDPOINTS .................................................................................................................... 28 
6.3 
STATISTICAL METHODS...................................................................................................................... 29 
6.3.1 
Baseline Data ................................................................................................................................ 29 
6.3.2 
Safety Analysis .............................................................................................................................. 29 
6.3.3 
Efficacy Analysis ........................................................................................................................... 29 
6.3.4 
Pharmacokinetic analysis ............................................................................................................. 31 
6.4 
SAMPLE SIZE AND POWER .................................................................................................................. 32 
6.5 
INTERIM ANALYSIS ............................................................................................................................ 32 
7 
STUDY MEDICATION (TOBRAMYCIN INHALATIONAL SOLUTION, USP) ........................... 32 
7.1 
DESCRIPTION ...................................................................................................................................... 32 
7.1.1 
Packaging ...................................................................................................................................... 33 
7.1.2 
Labeling ........................................................................................................................................ 33 
7.1.3 
Dosing ........................................................................................................................................... 33 
7.1.4 
Drug delivery via vibrating mesh nebulizer .................................................................................. 33 
7.1.5 
Treatment Compliance and Adherence ......................................................................................... 34 
7.1.6 
Drug Accountability ...................................................................................................................... 34 
8 
SAFETY MANAGEMENT ..................................................................................................................... 34 
8.1 
CLINICAL ADVERSE EVENTS .............................................................................................................. 34 
8.2 
ADVERSE EVENT REPORTING ............................................................................................................. 35 
8.3 
DEFINITION OF AN ADVERSE EVENT .................................................................................................. 35 
8.4 
DEFINITION OF A SERIOUS ADVERSE EVENT (SAE) ........................................................................... 36 
8.4.1 
Relationship/Severity of SAE to Study Drug or Other Intervention .............................................. 36 
8.5 
IRB/IEC NOTIFICATION OF SAES AND OTHER UNANTICIPATED PROBLEMS ...................................... 36 
8.5.1 
Follow-up report ........................................................................................................................... 36 
8.6 
INVESTIGATOR REPORTING OF A SERIOUS ADVERSE EVENT TO SPONSOR .......................................... 37 
8.7 
SPONSOR REPORTING OF SERIOUS ADVERSE EVENTS TO FDA .......................................................... 37 
8.8 
MEDICAL EMERGENCIES .................................................................................................................... 37 
9 
STUDY ADMINISTRATION ................................................................................................................. 37 
9.1 
TREATMENT ASSIGNMENT METHODS................................................................................................. 37 
9.2 
DATA COLLECTION AND MANAGEMENT ............................................................................................ 37 
9.3 
CONFIDENTIALITY .............................................................................................................................. 37 
9.4 
REGULATORY AND ETHICAL CONSIDERATIONS.................................................................................. 38 
9.4.1 
Data and Safety Monitoring Plan ................................................................................................. 38 
9.4.2 
Risk Assessment ............................................................................................................................. 41 


  
 
 
 
 
 
 
 
v
9.4.3 
Potential Benefits of Trial Participation ....................................................................................... 42 
9.4.4 
Risk-Benefit Assessment ................................................................................................................ 42 
9.5 
RECRUITMENT STRATEGY .................................................................................................................. 42 
9.6 
INFORMED CONSENT/ASSENT AND HIPAA AUTHORIZATION ............................................................ 43 
9.6.1 
Waiver of Assent ............................................................................................................................ 44 
9.7 
PAYMENT TO SUBJECTS/FAMILIES ..................................................................................................... 44 
10 
PUBLICATION ............................................................................................................................... 44 
11 
REFERENCES ................................................................................................................................ 45 
Appendix ............................................................................................................................... 52 
 


  
 
 
 
 
 
 
 
vi
ABBREVIATIONS AND DEFINITIONS OF TERMS 
a-TEOAE 
 
Automated transient evoked otoacoustic emissions 
AAP 
 
American Academy of Pediatrics 
ABR 
 
Auditory brainstem response 
AE 
 
Adverse event 
AKI 
 
Acute kidney injury 
BAEP 
 
Brainstem auditory evoked potential 
BAL 
 
Bronchoalveolar lavage 
BPD 
 
Bronchopulmonary dysplasia 
CCP 
 
Center for Clinical Pharmacology 
CF 
 
Cystic fibrosis 
CFU 
 
Colony forming units 
CHOP 
 
Children’s Hospital of Philadelphia 
CI 
 
Confidence interval 
DSMB 
 
Data safety monitoring board 
ET 
 
Endotracheal tube 
FDA 
 
Federal Drug Administration 
FiO2 
 
Fraction of inspired oxygen 
GNR  
 
Gram-negative rod 
HPLC-MS/MS 
 
High-performance liquid chromatography tandem mass spectrometry 
IDS 
 
Investigational drug services 
IND 
 
Investigational new drug 
IV 
 
Intravenous 
JCIH 
 
Joint Committee on Infant Hearing 
KDIGO 
 
Kidney disease improving global outcomes 
MAP 
 
Mean airway pressure 
MIC 
 
Minimum inhibitory concentration 
MTD 
 
Maximum tolerated dose 
NeoCLD 
 
Newborn and Infant chronic lung disease (program at CHOP) 
N/IICU 
 
Newborn and infant intensive care unit 
OAE 
 
Otoacoustic emissions 
OI 
 
Oxygenation index 
PI 
 
Primary investigator 
PK 
 
Pharmacokinetics 
PMA 
 
Postmenstrual age 
RSS 
 
Respiratory severity score 
SAE 
 
Serious adverse event 
SGOT/AST 
 
Aspartate aminotransferase 
SGPT/ALT 
 
Alanine aminotransferase 
SpO2 
 
Pulse oxygen saturation 
TSI 
 
Tobramycin solution for inhalation 
USP 
 
United States pharmacopeia 
VROA 
 
Visually reinforced orientation audiometry 
 
 
 


 
 
 
 
 
 
 
 
 
 
vii 
ABSTRACT 
Context:  
Bronchopulmonary dysplasia (BPD) is among the most common and consequential chronic 
complications associated with preterm birth. Among infants with BPD who continue to 
require invasive mechanical ventilation near term corrected gestation, airway colonization 
with a pathogenic Gram-negative rod (GNR) bacteria is an independent risk factor for 
prolonged need for supplemental oxygen and home oxygen therapy. Inhaled tobramycin is 
an effective therapy for treating airway infections secondary to GNR bacteria and has 
shown clinical benefit when used in several pediatric and adult chronic respiratory 
conditions. The safety and efficacy of inhaled tobramycin in preterm infants with BPD and 
GNR bacteria present in the airways is unknown.  
 
Objectives: 
The objective of this open-label, phase 1, inter-patient dose escalation feasibility trial is to 
identify a safe, well tolerated dose of inhaled tobramycin for use in very preterm infants with 
BPD receiving invasive mechanical ventilation who have evidence of a pathogenic GNR 
detected by tracheal aspirate culture.  
 
Study Design:  
Prospective open-label, phase 1, inter-patient 3+3 dose escalation feasibility trial. An 
untreated, observational cohort of eligible infants will also be enrolled. 
 
Setting/Participants: 
This trial will take place in the newborn and infant intensive care unit (N/IICU) at the 
Children’s Hospital of Philadelphia. This will be the only site. Up to 103 infants will be 
enrolled in this study to produce up to 24 infants with evaluable trial data and up to 28 
participants in the observational cohort. The trial will use the following inclusion/exclusion 
criteria: 
 
Inclusion Criteria: 
1. Male or female infants born <32 weeks’ gestation 
2. Diagnosed with BPD (use of supplemental oxygen or respiratory support at 36 
weeks postmenstrual age1) 
3. Postmenstrual age ≥36 weeks at study enrollment 
4. Treatment with invasive mechanical ventilation at enrollment without planned 
tracheal extubation within 7 days after enrollment 
5. Tracheal aspirate culture positive for a pathogenic GNR bacteria (see list of eligible 
organisms in section 3.4.3) within 7 days of beginning the study phase – this testing 
will be performed during the screening phase and must be met to participate in the 
phase 1 trial or observational cohort  
6. Parental/guardian permission (informed consent). 
Exclusion Criteria: 
1. Serum creatinine >0.4mg/dL within 14 days prior to enrollment 
2. Congenital or acquired disease of the kidney or renal collecting system that 
adversely affects renal function 
3. Congenital or acquired hepatobiliary disease that adversely affects liver function 
4. Treatment with a systemic antibiotic within 7 days prior to enrollment 
 
1 Postmenstrual age (PMA) is calculated as the gestational age at birth plus the chronological age  


 
 
 
 
 
 
 
 
 
 
viii 
5. Treatment with a nephrotoxic medication (see list in appendix A), excluding 
diuretics, within 48 hours prior to enrollment 
6. Treatment with a continuous infusion of a neuromuscular blocker (see list in 
Appendix B) within 48 hours prior to enrollment 
7. Known intolerance to aminoglycoside antibiotics 
8. Current treatment with high frequency or other oscillating mechanical ventilation 
9. Presence of a cancer diagnosis 
10. Maternal family history of early onset hearing loss defined as the need for an 
assistive hearing device prescribed before 30 years of age  
11. Persistent endotracheal tube leak >20%.  
12. Any prior use of an investigational drug as part of an FDA approved IND protocol.  
13. A subject who, in the judgement of the Investigator, is not an appropriate candidate 
for this research study. 
Study Interventions and Measures:  
The study will investigate tobramycin solution for inhalation (300mg/5mL). Up to 4 different 
dosages will be investigated: 78mg, 150mg, 216mg, and 300mg, all administered every 12 
hours for up to 14 days.  
 
The primary study outcome is a composite safety endpoint defined by the presence of any 
of the following events occurring at any time point during the 14-day trial: 
1) Trough serum tobramycin level (measured 11 hours after the administered dose) 
≥1mcg/mL 
2) Increase in serum creatinine level by ≥0.3mg/dL above pre-trial baseline 
3) Increase in serum creatinine level >1.5-fold above pre-trial baseline 
4) Urine output <0.5mL/kg/hr for 12 consecutive hours 
5) Any serious adverse event possibly attributable to the study drug 
 
Occurrence of the primary outcome in ≥2 infants at a single dosage level will define the 
“maximum tolerated dose” and result in cessation of the trial.  
 
Additional exploratory secondary safety endpoints are: 
1) New onset or worsened coughing associated with a change in respiratory status 
(SpO2 <80% for >10 seconds; need for increase in FiO2 by >20%) 
2) Obstruction of the endotracheal tube requiring tube replacement 
3) Unplanned tracheal extubation 
4) Desaturation (SpO2 <80% for >10 seconds) during administration of inhaled 
tobramycin 
5) Rates of pre-discharge failed audiology examinations 
6) Rates of new intra-patient microbial resistance to tobramycin 
 
The exploratory secondary efficacy endpoints are: 
 
1) Tracheal aspirate tobramycin level 
2) Change in tracheal aspirate pathogenic bacterial colony forming unit (CFU) counts 
measured by high resolution, quantitative culture 
3) Change in the following measures of administered respiratory support: fraction of 
inspired oxygen (FiO2), ventilator mean airway pressure (MAP), and respiratory 
severity score (MAP x FiO2) 


  
 
 
 
 
 
 
 
ix
4) Change in the following measures of hypoxemia (SpO2<80%): Intermittent 
hypoxemia (events lasting 10s-3min), prolong hypoxemia (events lasting >1min), 
and daily proportion of time in hypoxemia 
5) Change in tracheal aspirate cytokine levels, neutrophil to total WBC ratio, and 
patterns in the airway microbiome 
6) Change in dynamic lung compliance, airway resistance, peak expiratory flow, and 
carbon dioxide (CO2) elimination 
 
 


  
 
 
 
 
 
 
 
x
PROTOCOL SYNOPSIS 
Study Title 
Phase 1 Feasibility Trial of Inhaled Tobramycin in Infants with 
Bronchopulmonary Dysplasia 
Funder 
Children’s Hospital of Philadelphia  
Clinical Phase 
Phase I 
Study Rationale 
Bronchopulmonary dysplasia (BPD) is among the most common 
and consequential chronic complications of preterm birth. 
Colonization or infection of the airway by a pathogenic Gram-
negative rod (GNR) bacteria in preterm infants with BPD receiving 
invasive mechanical ventilation is an independent risk factor for 
prolonged use of supplemental oxygen and home oxygen therapy. 
Respiratory infection with Pseudomonas aeruginosa (the most 
common GNR detected in airways of infants with BPD) is a known 
risk factor for poor pulmonary function and adverse clinical 
outcomes in several chronic respiratory conditions.  
 
Tobramycin is an aminoglycoside antibiotic that has been shown to 
eradicate bacterial airway colonization with P. aeruginosa and other 
pathogenic GNR bacteria in older children and adults when 
delivered to the lungs via nebulization. The safety and efficacy of 
inhaled tobramycin in preterm infants with BPD is unknown and 
there are no robust data that inform the optimal dosing strategy for 
use in this population. 
Study Objective(s) 
Primary  
• To determine the maximum, safely tolerated dose of tobramycin 
solution for inhalation (selected between 78mg, 150mg, 216mg, 
and 300mg administered every 12 hours) in very preterm infants 
with BPD who are actively receiving invasive mechanical 
ventilation and have evidence of a pathogenic GNR bacteria 
(listed in section 3.4.3) detected by tracheal aspirate culture.   
Secondary 
• To evaluate the association between the dose of inhaled 
tobramycin, plasma tobramycin pharmacokinetics, and peak 
tracheal aspirate tobramycin levels. 
• To compare the change (pre vs. post study) in tracheal aspirate 
pathogenic bacterial colony forming unit (CFU) counts measured 
by high resolution culture, (1) between participants in the phase 
1 trial and untreated infants in the observational cohort, and (2) 
between trial participants stratified by the administered dose of 
inhaled tobramycin. 
• To compare the change in measures of administered respiratory 
support (FiO2, ventilator mean airway pressure, and respiratory 
severity score) during the 14-day study period, (1) between 
participants in the observational cohort and phase 1 trial, and (2) 
between trial participants stratified by the administered dose of 
inhaled tobramycin. 


  
 
 
 
 
 
 
 
xi
• To evaluate changes (pre vs. post treatment) in airway cytokine 
levels, neutrophil/WBC ratio, and patterns in the airway 
microbiome. 
• To compare changes in the following measures of hypoxemia 
(SpO2<80%): Intermittent hypoxemia (events lasting 10s-3min), 
prolong hypoxemia (events lasting >1min), and daily proportion 
of time in hypoxemia during the 14-day study period, (1) 
between participants in the observational cohort and phase 1 
trial, and (2) between trial participants stratified by the 
administered dose of inhaled tobramycin. 
 
• To evaluate longitudinal change in dynamic lung compliance, 
airway resistance, peak expiratory flow, and CO2 elimination 
during the 14-day study period, (1) between participants in the 
observational cohort and phase 1 trial, and (2) between trial 
participants stratified by the administered dose of inhaled 
tobramycin. 
Study Drug 
Tobramycin solution (300mg/5mL) for inhalation USP 
Study Design 
Open-label, inter-patient 3+3 dose escalation trial. An untreated 
observational cohort of eligible infants will also be enrolled and 
outcomes compared to trial participants. 
Subject Population 
key criteria for Inclusion 
and Exclusion: 
Inclusion Criteria 
1. 
Male or female infants born <32 weeks’ gestation 
2. 
Diagnosed with BPD (use of supplemental oxygen or 
respiratory support at 36 weeks postmenstrual age) 
3. 
Postmenstrual age ≥36 weeks at study enrollment 
4. 
Treatment with invasive mechanical ventilation at enrollment 
without planned tracheal extubation within 7 days after 
enrollment 
5. 
Tracheal aspirate culture positive for a pathogenic GNR 
bacteria (eligible organisms listed in section 3.4.3) within 7 
days of beginning the study phase – this testing will be 
performed during the screening phase and must be met to 
participate in the phase 1 trial or observational cohort 
6. 
Parental/guardian permission (informed consent). 
Exclusion Criteria 
1. 
Serum creatinine >0.4mg/dL within 14 days prior to enrollment 
2. 
Congenital or acquired disease of the kidney or renal 
collecting system that adversely affects renal function 
3. 
Congenital or acquired hepatobiliary disease that adversely 
affects liver function 
4. 
Treatment with a systemic antibiotic within 7 days prior to 
enrollment 
5. 
Treatment with a potentially nephrotoxic medication (see list in 
Appendix A), excluding diuretics, within 48 hours prior to 
enrollment 


 
 
 
 
 
 
 
 
 
 
xii 
6. 
Treatment with a continuous infusion of a neuromuscular 
blocker (see list in Appendix B) within 48 hours prior to 
enrollment 
7. 
Known intolerance to aminoglycoside antibiotics 
8. 
Current treatment with high frequency or other oscillating 
mechanical ventilation 
9. 
Presence of a cancer diagnosis 
10. Maternal family history of early onset hearing loss defined as 
the need for an assistive hearing device prescribed before 30 
years of age  
11. Persistent endotracheal tube leak >20%.  
12. Any investigational drug use (as part of an FDA approved IND 
protocol) prior to enrollment.  
13. A subject who, in the judgement of the Investigator, is not an 
appropriate candidate for this research study. 
Number of Subjects  
 
Up to 103 infants will be enrolled in this study to produce up to 24 
infants with evaluable trial data and up to 28 participants in the 
observational cohort. The Children’s Hospital of Philadelphia will be 
the only study site. 
Study Duration 
Each subject’s participation will last from enrollment until hospital 
discharge. The phase 1 trial will last up to 14 days. 
The entire study is expected to last up to 3 years.  
Study Phases 
Screening Phase 
 
 
 
 
 
Study Phase 
Phase 1 Trial 
 
 
 
 
 
 
 
 
 
 
Observational (non-
treated) Cohort 
 
Potential subjects will be screened using daily census logs and the 
protocol inclusion and exclusion criteria. Infants who meet study 
eligibility criteria will be enrolled if a parent or guardian consents to 
once-weekly collections of a screening tracheal aspirate culture. If a 
screening culture is positive for a pathogenic GNR, the infant will be 
eligible to participate in the phase 1 trial or observational (non-
treated) cohort, but can be enrolled in only one. 
 
The phase 1 trial will last up to 14 days, with each infant receiving a 
specified dose of tobramycin solution for inhalation every 12 hours, 
administered via vibrating mesh nebulizer. The drug dose (78mg, 
150mg, 216mg, or 300mg) will be determined as per the inter-
patient dose escalation 3+3 design protocol. If the 300mg dose 
level is completed without evidence of safety concerns (i.e., 
stopping rules are not met), up to 3 additional infants will be 
enrolled to receive 300mg to provide a more robust assessment of 
drug dose tolerability and PK. During the trial, each infant will 
undergo continuous pulse oximetry, and blood and tracheal 
aspirate sampling and respiratory mechanics measurements at pre-
specified time points to assess dose safety and potential efficacy. 
Clinical data will also be recorded daily throughout the trial.  
 
Infants enrolled in the untreated observational cohort will undergo 
continuous pulse oximetry for 14 days after enrollment. Respiratory 


 
 
 
 
 
 
 
 
 
 
xiii 
 
 
 
Follow-Up Phase  
mechanics measurements will be performed at set timepoints. 
Clinical data will be collected daily from the medical record.  
 
All infants enrolled in the phase 1 trial and observational cohorts 
will be followed until hospital discharge. Pre-defined clinical data 
will be abstracted periodically from the medical record. For infants 
enrolled in the phase 1 trial, tracheal aspirate samples for high 
resolution culture, 16s sequencing, and cytokine level 
measurement will be collected ≤48 hours and 14 days after 
receiving the final dose of inhaled tobramycin. Infants enrolled in 
the observational cohort will undergo tracheal aspirate collection for 
culture ≤48 hours after completing the 14-day study phase. Infants 
in both cohorts will undergo respiratory mechanics measurements 
≤48 hours after completing the study phase. 
Safety Evaluations 
The primary study endpoint of the phase 1 trial is a safety outcome 
defined as the occurrence of any of the following: 
• Trough serum tobramycin level (measured 11 hours after the 
last administered dose) ≥1µg/mL 
• Increase in serum creatinine level by ≥0.3mg/dL above the pre-
trial level 
• Increase in serum creatinine >1.5-fold above the pre-trial level 
• Urine output <0.5mL/kg/hr for 12 hours 
• Any serious adverse event possibly attributable to the study drug 
 
Additional exploratory secondary safety endpoints are: 
• New onset or worsened coughing associated with a change in 
respiratory status (SpO2 <80% for >10 seconds; need for 
increase in FiO2 by >20%) 
• Obstruction of the endotracheal tube requiring tube replacement 
• Unplanned tracheal extubation 
• Desaturation (SpO2 <80% for >10 seconds) during 
administration of inhaled tobramycin 
• Rates of pre-discharge failed audiology examinations 
• Rates of new intra-patient microbial resistance to tobramycin 
Efficacy Evaluations 
The exploratory secondary efficacy endpoints are: 
• Tracheal aspirate tobramycin levels 
• Change in tracheal aspirate pathogenic bacterial colony forming 
unit (CFU) counts measured by high resolution, quantitative 
culture 
• Change in the following measures of administered respiratory 
support: fraction of inspired oxygen (FiO2), ventilator mean 
airway pressure (MAP), respiratory severity score (FiO2 x MAP) 
• Change in the following measures of hypoxemia (SpO2<80%): 
Intermittent hypoxemia (events lasting 10s-3min), prolonged 
hypoxemia (events lasting >1min), and daily proportion of time in 
hypoxemia 
• Change in tracheal aspirate cytokine levels, neutrophil/WBC 
ratio, and patterns in the airway microbiome 
• Change in dynamic lung compliance and airway resistance 


 
 
 
 
 
 
 
 
 
 
xiv
Pharmacokinetic 
Evaluations 
Blood and tracheal aspirate samples for pharmacokinetic (PK) 
analyses will be collected. Patient specific PK parameters will be 
calculated using log-linear regression based on plasma peak and 
trough tobramycin concentrations.  
 
Population PK analyses will be conducted using nonlinear mixed 
effects modeling. Mean population PK parameters (clearance, 
volume of distribution) will be estimated using the first-order 
conditional estimation with interaction method. A 2-compartment 
model with first order absorption will be utilized based on prior 
knowledge of inhaled tobramycin pharmacokinetics.  
Statistical and Analytic 
Plan 
All subjects entered into the phase 1 trial will be included in the 
primary safety analysis. The primary study outcome, listed above 
under “Safety Evaluations,” will be evaluated as a dichotomous 
outcome based on the presence of any of the listed outcomes. A 
3+3 design will be followed to determine enrollment and trial 
progression based on the results of each infant’s safety analysis. 
DATA AND SAFETY 
MONITORING PLAN 
A 4-member independent data safety monitoring board (DSMB) 
consisting of 2 neonatologists, 1 statistician, and 1 audiologist will 
monitor trial data, safety, progress throughout the trial. 


 
xv  
 
 
 
 
TABLE 1: SCHEDULE OF STUDY PROCEDURES  
Study Phase 
Screening 
Phase 1 Trial 
Follow-up 
Study Days 
 
0a 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15b 
>15 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Review inclusion & exclusion criteria 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pre-trial medical record review 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prior & concomitant medications 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Dispense study drug 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Review current medications 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Review all laboratory, imaging data 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Continuous pulse oximetry 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Pulmonary mechanics 
measurements 
 
X 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
Tracheal aspirate 
X 
X 
 
 
 
X 
 
 
5X 
 
 
X 
 
 
 
X 
2X 
2Xc 
Blood draw for laboratory testing 
- Serum tobramycin trough 
- Comprehensive metabolic paneld 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 
X 
 
 
Blood peak/trough for PK 
 
 
 
 
 
 
 
 
2X 
 
 
 
 
 
 
 
 
 
Collect blood sample during clinical 
draws for PK analysese 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Assessment of respiratory support 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Adverse event assessment (daily)  
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Adverse event assessment (weekly) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Review clinical audiology testing 
results 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Discharge medical record review 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Observational (non-treated) cohort 
 
 
Tracheal aspirate 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Continuous pulse oximetry 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Assessment of respiratory support 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Pulmonary mechanics 
measurements 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
Review clinical audiology testing 
results 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Discharge medical record review 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
a “Day 0” procedures to be completed ≤7 days prior to trial Day 1 
b “Day 15” procedures to be completed ≤48 hours after the final administered dose of inhaled tobramycin (phase 1 trial) or study day 14 (observational cohort) 
c Tracheal aspirate sample collected 14 days after the final administered dose of inhaled tobramycin or on the day of planned tracheal extubation, if earlier 
d Serum magnesium and phosphorus levels will be checked with the comprehensive metabolic panel on trial days 7 and 14. 
e Up to 6 blood samples (200µL per sample) for pharmacokinetic analyses (PK) will be collected per infant concurrent with clinically ordered blood draws. 


 
page xvi 
  
 
 
 
 
 
 
 
 
 
Figure 1. Study enrollment and phases flow diagram. 
 
* Details of both the phase 1 trial and observational (non-treated) cohort will be discussed with eligible 
families during the consent conversations. Eligible infants may participate in either cohort but can be 
enrolled in only one cohort.  
 
 


 
page xvii 
  
 
 
 
 
 
 
 
 
 
Figure 2. 3+3 dose escalation design schematic. MTD: maximum tolerated dose 
 
Up to 4 different dosages of tobramycin solution for inhalation will be investigated using the 
above 3+3 trial design (78mg, 150mg, 216mg, and 300mg; all administered every 12 hours for 
up to 14 days). If the 300mg dose level is completed in 3 participants and none show evidence 
of a toxic response, up to 3 additional infants will be enrolled to receive to receive 300mg. The 
trial will be stopped if 2 of the additional infants receiving 300mg develop a toxic response and 
the 216mg dose will be used as the maximum tolerated dose in subsequent trials.    
 
A toxic response is defined as the occurrence of any of the following safety endpoints during the 
14-day phase 1 trial (see individual subject and full study stopping rules in section 5.2.4 and 
5.2.5 for more details): 
• 
Trough serum tobramycin level (measured 11 hours after the last administered dose) 
≥1µg/mL 
• 
Increase in serum creatinine level by ≥0.3mg/dL above the pre-trial level1-3 
• 
Increase in serum creatinine >1.5-fold above the pre-trial level1-3 
• 
Urine output <0.5mL/kg/hr for 12 hours1-3 
• 
Any serious adverse event possibly attributable to the study drugb 
 
 
b The likelihood an adverse event is attributable to a study intervention will be defined as outlined in section 8.3. 


 
  
 
 
 
1
1 
BACKGROUND INFORMATION AND RATIONALE 
1.1 
Introduction 
The objective of this phase 1, 3+3 dose escalation clinical trial is to identify a well-tolerated 
dose of tobramycin solution for inhalation for use in very preterm infants with BPD who are 
actively receiving invasive mechanical ventilation and have evidence of a pathogenic Gram-
negative rode (GNR) bacteria detected by tracheal aspirate culture.  
 
1.2 
Burden of bronchopulmonary dysplasia (BPD) 
BPD is the most common and consequential complication of prematurity. BPD affects 
approximately 50% of extremely preterm infants, is a strong risk factor for life-long 
cardiopulmonary and neurodevelopmental impairment, and is a leading pediatric cause of 
disability-adjusted life years lost.4-7 Disease acuity in BPD, defined by the respiratory 
support administered at 36 weeks postmenstrual age (PMA), predicts the degree of 
childhood impairment. Very preterm infants treated with invasive mechanical ventilation at 
36 weeks PMA (grade 3 BPD) are 5 times more likely to undergo tracheostomy than very 
preterm infants with less severe disease.8 Over 60% of very preterm infants with grade 3 
BPD demonstrate severe cognitive impairment at 2 years and 1 in 5 die before their second 
birthday.8 Finally, the risk of late death or severe neurodevelopmental impairment increases 
the longer an infant is exposed to invasive mechanical ventilation after 36 weeks PMA.9 
These data suggest that interventions that promote lung disease recovery and shorten the 
duration of invasive ventilation may produce long-term benefit among infants with the most 
severe forms of BPD.   
 
1.3 
Pathogenic bacterial colonization of the airway and BPD 
Colonization of the airways in very preterm infants with pathogenic bacterial organisms and 
imbalance in the microbial communities in the airway (dysbiosis) have been implicated as 
inflammatory triggers and important risk factors in the development and exacerbation of 
BPD.10-12 Airway colonization by a pathogenic GNR organism is associated with a 2-fold 
increase in the rates of supplemental oxygen use at 36 weeks PMA and discharge to 
home.11 In an analysis of 121 very preterm infants receiving mechanical ventilation at 36 
weeks PMA in the newborn and infant intensive care unit (N/IICU) at the Children’s Hospital 
of Philadelphia (CHOP), airway colonization with a GNR bacteria was associated with a 6-
fold increase in the odds of death or need for home supplemental oxygen therapy (adjusted 
odds ratio 6.2, 95% CI 1.8-21).10 Pseudomonas aeruginosa was the most commonly 
cultured GNR (30% of study infants) and was independently associated with increased risk 
for death or prolonged oxygen therapy (adjusted odds ratio 4.9, 95% CI 1.1-22).10 In 
contrast, airway colonization with a Gram-positive organism was not a significant risk factor 
for respiratory morbidity.10 
 
1.4 
Difficulty treating airway infections with systemically administered antibiotics 
Bacterial lower respiratory tract infections and aberrant colonization of the airways with 
pathogenic bacterial organisms present a significant therapeutic challenge. Systemically 
administered antimicrobials have limited penetration into the lung parenchyma and the 
airway lining epithelial fluid.13 The utility of systemic antibiotics to treat respiratory infections 
may be further impaired in BPD, owing to the frequent presence of pulmonary hypertension 
and hypoxemia.14 These physiologic perturbations can lead to pulmonary vasoconstriction 
and shunting of blood flow away from respiratory sites targeted for antimicrobial therapy.14  
 
In contrast to systemically administered agents, inhaled antibiotics may maximize intra-
pulmonary drug concentrations, optimize pharmacokinetic indices, and substantially reduce 


 
  
 
 
 
2
systemic exposure and toxicity. Inhaled tobramycin has been shown to effectively decrease 
pathogenic bacterial density in the airways, reduce pulmonary exacerbation frequency, and 
improve the quality of life and lung function in patients with cystic fibrosis (CF) and non-CF 
bronchiectasis whose respiratory tract is colonized with P. aeruginosa.15,16 Owing to the 
favorable risk:benefit ratio, inhaled antibiotics are also gaining popularity for other disease 
states such ventilator associated tracheobronchitis and pneumonia.17-20 
 
1.5 
Name and Description of Investigational Product 
This study will utilize tobramycin solution for inhalation USP (United States Pharmacopeia). 
Tobramycin solution for inhalation USP is a sterile, clear, slightly yellow, non-pyrogenic, 
aqueous solution with the pH and salinity adjusted specifically for administration by 
nebulization. The chemical formula for tobramycin, USP is C18H37N5O9 and the molecular 
weight is 467.52. Tobramycin, USP is O-3-amino-3-deoxy-α-D-glucopyranosyl-(1→4)-O-
[2,6-diamino-2,3,6-trideoxy-α-D-ribo-hexopyranosyl-(1→6)]-2-deoxy-L-streptamine. The 
structural formula for tobramycin, USP is shown in Figure 3. Each 5mL ampule contains 
300mg of tobramycin, USP and 11.25mg sodium chloride in sterile water. Sulfuric acid and 
sodium hydroxide are added to adjust the pH to 6. Nitrogen is used for sparging. All 
ingredients meet USP requirements. The formulation contains no preservatives.  
 
 
Figure 3. Structural formula for Tobramycin, USP 
 
Tobramycin is a member of the aminoglycoside class of antibiotics. This class exert their 
antimicrobial activity by binding to the 30s subunit of the bacterial ribosome and inhibiting 
protein synthesis, thus inhibiting growth and division of the bacteria. Aminoglycosides, 
including tobramycin are not absorbed enterally, therefore must be administered locally 
such as through nebulization or intravenous instillation.  
 
1.6 
Findings from Clinical Studies  
1.6.1 
Human pharmacokinetics 
Wang et al. studied the pharmacokinetics (PK) of tobramycin solution for inhalation in 
patients aged between 6 months and 44 years using data from 4 clinical trials investigating 
300mg of tobramycin administered via nebulized aerosolization.21 The final PK model used 
a 2-compartment, first-order absorption model.21 Serum tobramycin levels reached near 
undetectable trough levels 10-12 hours after administration in children <7 years of age 
(Figure 4A) and peak serum tobramycin levels were below 1-2µg/mL in all children <2years 
of age (Figure 4B).21 Relative bioavailability in patients between 6 months and 7 years of 
age increased with age (i.e. younger children demonstrated lower serum concentration 
levels).21 Importantly, the investigators noted that systemic exposure is not predictive of 
clinical efficacy due to direct dosing at the infection site (lung).21 They concluded that P. 
aeruginosa eradication rates and safety profiles in patients aged <7 years were similar to 
older patients and that no dose adjustment is needed in young children.21 


 
  
 
 
 
3
 
Figure 4. (A) Visual prediction check of the final PK model for children <7 years of age. Open circles are observed 
concentrations. Central solid line is the median of observed concentrations, dashed lines are the 2.5% and 97.5% 
quantile of observed concentrations. The central shaded area represents the 95% confidence interval (CI) of the 
median of the simulated profiles and the upper and lower 95% CI for the 2.5% and 97.5% quartiles, respectively. (B) 
Effect of age on steady-state exposure of tobramycin using simulations (semi-log view). PK profiles of 1000 subjects 
were simulated per clearance per age group. Central solid lines represent the median concentration; shaded areas 
frame the 50% (25th-75th percentile) and 90% (5th-95th percentile) of the subjects. Horizontal dashed lines represent 
the safe serum trough threshold utilized by the study authors. Top panel shows simulations using data form 3 trials22-
24 and the bottom simulation data from an additional trial25. 
 
In a separate study, Rosenfeld et al. found that peak serum concentrations occurred 
approximately 1 hour (range 0.5-2hr) after administration of 300mg of tobramycin solution 
for inhalation to children aged 0.6-5.8 years (Figure 5).26 The average peak serum 
concentration of 0.6µg/mL (range: <0.2 to 1.2µg/mL) was within the acceptable, safe trough 
range for intravenous tobramycin. Serum tobramycin levels reached negligible values in 
most study patients within 4 hours after administration (Figure 5).26  
Figure 5. Serum tobramycin concentration – time profiles in 19 children (age range 0.6-5.8yr) treated with 300mg of 
inhaled tobramycin. Time shown along the x-axis indicates minutes since administration of the inhaled dose. In 3 
patients, all levels were below the detectable limit of 0.2µg/mL (dotted line).26 


 
  
 
 
 
4
1.6.1.1 
Clinical Studies in Older Children (>6 years) and Adults 
Tobramycin solution for inhalation is approved by the US Food and Drug Administration for 
the treatment of respiratory tract infections by P. aeruginosa in patients with CF who are at 
least 6 years of age. The CF Foundation “strongly recommends” the use of inhaled 
tobramycin for the treatment of “initial or new growth of P. aeruginosa from an airway 
culture.”16 They rate the certainty of benefit with this therapy as “high,” and estimate the net 
benefit as “substantial.”16 An expert panel from the European CF Society also recommends 
the use of inhaled tobramycin for the treatment of newly acquired P. aeruginosa infection.27 
Multiple controlled trials (Table 2) conducted in older children and adults support the safety 
and efficacy of inhaled tobramycin in this population. Several of these trials, combined with 
data from younger children (Table 3, section 1.6.1.2) were included in a recent Cochrane 
review. This meta-analysis supports the use of inhaled tobramycin in children with CF who 
have culture proven respiratory tract infections with P. aeruginosa.28 Inhaled tobramycin 
has also demonstrated efficacy for the treatment of respiratory tract infections due to P. 
aeruginosa and other GNR bacteria in non-CF bronchiectasis (Table 2), chronic obstructive 
pulmonary disease (COPD), and ventilator-associated infections.18,29-31 
 
1.6.1.2 
Clinical Studies in Infants and Young Children (≤ 6 years) 
Several clinical trials have demonstrated the safe use of inhaled tobramycin in infants (<1 
year of age) and young children (Table 3). The Australian Cystic Fibrosis Bronchoalveolar 
Lavage (BAL) study enrolled 170 infants (mean age 3.6 ± 1.6 months at enrollment).40,41 In  
 
Table 2. Published randomized trials evaluating tobramycin solution for inhalation (TSI) in older children (≥6 years) or adult 
patients with P. aeruginosa present in the sputum.  
Author / 
Year 
Dose / 
Frequency 
Disease 
Participants’ 
ages 
Study duration 
Trial 
size 
Key findings 
Ramsey 
199932 
300mg BID 
CF 
≥ 6 years 
 
24 weeks 
(cycled 28 days 
on, 28 days off) 
N=520 
• Improved pulmonary 
function 
• Decreased 
hospitalizations 
Murphy 
200433 
300mg BID 
CF 
6-15 years 
56 weeks 
(cycled 28 days 
on, 28 days off) 
N=184 
• Decreased 
hospitalization rates 
MacLuskey 
198934 
80mg TID 
CF 
7-24 years 
32 months 
N=27 
• Stability in pulmonary 
function, controls 
showed decline 
Smith 
198935 
600mg TID 
CF 
Mean age 
17.3 ± 6.1 
years 
12 weeks 
N=22 
• Improved symptoms, 
decrease in bacterial 
density 
Ramsey 
199336 
600mg TID 
CF 
Mean age 
17.1 ± 1.3 
years 
12 weeks 
(cycled 28 days 
on, 28 days off) 
N=71 
• Improved pulmonary 
function 
Scheinberg 
200537 
300mg BID 
non-CF 
bronchiectasis 
≥ 18 years 
 
12 weeks 
(cycled 14 days 
on, 14 days off) 
N=41 
• Improvements in 
respiratory symptoms 
Drobnic 
200538 
300mg BID 
non-CF 
bronchiectasis 
38-75 years 
6 months 
N=30 
• Decrease in the 
number of hospital 
admissions 
• Shorter duration of 
hospitalizations 
Barker 
200039 
300mg BID 
non-CF 
bronchiectasis 
Mean age 67 
± 13 years 
28 days 
N=74 
• Decreased sputum 
bacterial load 
• Improvement in 
respiratory symptoms 
• No new bacterial 
resistance 


 
  
 
 
 
5
 
total, 46 of the study infants were treated with 2 weeks of systemic antibiotic therapy 
followed by inhaled tobramycin (300mg twice daily) for 2 months.40,41 No adverse effects 
secondary to inhaled tobramycin were reported in the trial.41 A recent retrospective cohort 
study reported the use of inhaled tobramycin in 32 infants (<1 year of age) with CF.47 The 
mean age at initiation was 4 months, with 72% aged <6 months at first treatment.47 The 
median dose of inhaled tobramycin was 80mg (IQR 80-120); 4/32 (22%) were treated for 14 
days and the remainder (78%) were treated for 28 days.47 Most (83%) were free of P. 
aeruginosa by the end of the treatment course and (53%) remained free 6 months after 
treatment.47 Overall, inhaled tobramycin was well tolerated.47 Only 1 patient required 
therapy discontinuation owing to cough.47  
 
The limited available data from infants without CF support the safe use of inhaled 
tobramycin when administered to infants who: (1) do not have pre-existing renal disease, 
Table 3. Published randomized trials of tobramycin solution for inhalation (TSI) in young children with P. aeruginosa 
present in the sputum.  
Author / 
Year 
Dose / 
Frequency 
Disease 
Eligible 
participants’ ages 
Study duration 
Trial 
size 
Key findings 
Ratjen 
201924 
300mg BID 
CF 
3 months to >7 
years 
28 days with 
optional cross-
over for 
additional 28 
days 
N=51 
• Effective eradication of 
early P. aeruginosa 
infection 
• Favorable safety profile 
Ratjen 
201025 
300mg BID 
CF 
≥6 months (42% of 
cohort aged <6 
years) 
28 days vs. 56 
days 
N=88 
• >90% of participants free 
of P. aeruginosa after 1 
month of treatment 
• Favorable safety profile 
Gibson 
200342 
300mg BID 
CF 
6 months to 6 
years (29% of the 
cohort aged <3 
years) 
28 days 
N=21 
• Reduced density of P. 
aeruginosa in the lower 
airway with TSI 
• Reassuring mean peak 
(1.0 ± 0.4 mcg/mL) and 
trough (0.4 ± 0.5 mcg/mL) 
serum tobramycin levels  
• No change in renal or 
auditory function with TSI 
Treggiari 
201143 
300mg BID 
CF 
1-12 years 
(30% of the cohort 
aged 1-3 years) 
28 days +/- oral 
ciprofloxacin. 
Treatment 
provided either 
quarterly or only 
with positive 
sputum culture 
over 18 months 
period 
N=304 
• No benefit with addition of 
oral ciprofloxacin 
• Minimal emergence of 
antibiotic resistant strains 
• No difference in renal, 
liver, or audiometric 
function in between trial 
arms 
Taccetti 
201244 
300mg BID 
CF 
>1 year  
(35% in the TSI 
arm aged ≤5 
years) 
28 days TSI + 
oral ciprofloxacin 
vs. inhaled 
colistin + oral 
ciprofloxacin 
N=223 
• TSI with similar efficacy 
as colistin 
• 65% free of P. aeruginosa 
6 months after TSI + 
ciprofloxacin therapy 
• No serious adverse 
events 
Wiesemann 
199845 
80mg BID 
CF 
>4 years 
12 months 
N=22 
• Shorter time to 
conversion to P. 
aeruginosa negative 
respiratory culture 
Proesmans 
201346 
300mg BID 
CF 
0-18 years (7% <2 
years of age) 
 
28 days 
N=58 
• TSI vs. inhaled Na-
colistimethate resulted in 
similar eradication of P. 
aeruginosa (80-90%) 


 
  
 
 
 
6
and (2) are not receiving concurrent nephrotoxic intravenous antibiotics. A single center 
retrospective cohort study from the University of Oklahoma identified 40 pediatric patients 
without CF who received inhaled tobramycin for pneumonia or tracheitis during invasive 
mechanical ventilation.48 The median age at treatment was approximately 1 year with an 
interquartile range extending as low as 6 months.48 Most of the described patients received 
300mg twice daily with a low interquartile range of 60mg twice daily.48 Although the authors 
do not report respiratory outcomes, they indicate inhaled tobramycin continues to be one 
therapy used for the treatment of respiratory infections in young children receiving invasive 
respiratory support at their institution. The authors report that two infants developed acute 
kidney injury while receiving inhaled tobramycin (one after 5 days of therapy, the other after 
17 days of therapy).48 Both received 300mg twice daily during the treatment course.48 One 
received intravenous gentamicin within 48 hours of initiating tobramycin (last gentamicin 
trough 1.3mcg/mL) and the second had recently undergone surgical palliation for complex 
congenital heart disease.48 Both infants had detectable serum tobramycin trough levels (1.9 
and 3.2 mcg/mL).48 Two additional case reports describe the use of inhaled tobramycin 
(300mg twice daily) in preterm infants without CF (32 weeks’ gestation, 2.5 months of age 
treatment; 34 weeks’ gestation, 1 month of age at treatment).49,50 Although both infants 
developed detectable tobramycin levels above the goal trough range, they also both 
received concurrent IV doses of nephrotoxic antibiotics (including IV tobramycin in one 
case) and were diagnosed with pre-existing renal disease.49,50 Finally, analysis of data from 
the Pediatric Health Information System (PHIS) database suggests that inhaled tobramycin 
in used some infants with severe BPD. A recent study found that 3% (93/3252) of very 
preterm infants with severe BPD (gestational age <32 weeks; <1 year of age at treatment) 
in the dataset were prescribed inhaled tobramycin between 2007-2016. Taken together, 
these data indicate that: (1) inhaled tobramycin is used in clinical practice to treat infants 
(some as young as 3 months of age) with and without CF – including those with severe 
BPD, and (2) administration of inhaled tobramycin at doses ≤300mg, coupled with close 
monitoring of serum levels, is likely to be safe in preterm infants diagnosed with severe 
BPD who are ≥36 weeks PMA at the time of treatment.  
 
1.6.2 
Potential toxicity with inhaled tobramycin 
1.6.2.1 
Nephrotoxicity 
Nephrotoxicity was not seen during clinical trials of tobramycin solution for inhalation in 
children or adults. However, nephrotoxicity has been associated with aminoglycosides as a 
class. Aminoglycosides in systemic circulation are eliminated via glomerular filtration and 
can induce toxicity to the proximal tubule. The acute tubular necrosis that can occur is most 
commonly mild and reversible (owing to the ability of the proximal tubules to regenerate).51 
The considerably reduced serum exposure to tobramycin with inhalational administration 
substantially lowers the risk of acute kidney injury (AKI).52 At present, evidence suggesting 
a possible link between inhaled tobramycin and AKI in infants and young children is limited 
to case reports.48,53 In nearly all of these cases, there was evidence of renal dysfunction 
prior to the initiation of inhaled tobramycin and/or concomitant treatment with additional 
nephrotoxic agents.48,53  
 
1.6.2.2 
Ototoxicity 
Ototoxicity, as measured by complaints of hearing loss or by audiometric evaluations, did 
not occur in any of the clinical trials evaluating tobramycin solution for inhalation in children 
or adults.54 Moreover, there is no convincing evidence that inhaled tobramycin administered 
without concurrent systemic aminoglycoside therapy results in ototoxicity in pediatric 


 
  
 
 
 
7
patients. A multicenter European trial comparing a 28-day and a 56-day course of inhaled 
tobramycin (300mg twice daily) in children as young as 6 months of age with CF did not find 
a single case of hearing loss during extended follow-up.25 Similarly, a 2018 Cochrane 
review evaluating long-term use of inhaled tobramycin in CF patients did not identify a 
single case of hearing loss among 258 study participants.15 In a multicenter Australian 
study evaluating audiometric function before and after treatment with inhaled tobramycin 
(treatment duration ranged 59-119 days) in 142 children <6 years of age, only 1 child 
receiving both inhaled tobramycin and intermittent intravenous tobramycin developed mild, 
unilateral sensorineural hearing loss.55 This child inadvertently received a 300mg 
(21.3mg/kg) IV tobramycin dose, instead of a nebulized dose, in addition to the concurrently 
prescribed IV tobramycin dose.55 
 
In addition to the lack of data linking inhaled tobramycin to ototoxicity in young children, the 
overwhelming abundance of data indicate that systemically administered aminoglycosides 
do not cause hearing loss in infants when used as directed. A single center study of 2347 
infants found no increased risk of an abnormal audiology evaluation among infants who 
received gentamicin compared to those who did not.56 Two studies, including one 
evaluating 84,808 hospitalized infants, found no relationship between the presence of 
sensorineural hearing loss and the cumulative aminoglycoside dose, the average daily 
dose, or the duration of administration.57,58  
 
One small observational study (n=81) reported an increased risk of sensorineural hearing 
loss in infants who received systemic aminoglycoside therapy during concurrent treatment 
with a neuromuscular blocking agent.59 Whether this finding represents a true toxicity or is 
confounded by unmeasured illness severity is unknown. Notably, this study found no 
association between the dosage or duration of systemic aminoglycoside therapy and the 
risk for sensorineural hearing when the antibiotic was used in infants who were not co-
treated with a paralytic agent.59 Out of an abundance of caution, this phase 1 trial will 
exclude infants treated with a neuromuscular blocker within 48 hours of enrollment. 
 
Genetic risk for aminoglycoside induced ototoxicity: Homoplasmic A1555G and 
C1494T mutations in the 12s mitochondrial rRNA gene may predispose to aminoglycoside-
induced hearing loss.60,61 These mutations are present in approximately 0.2% of infants.62 
Although clinical testing for these maternally-inherited mutations is not yet performed in an 
anticipatory manner, avoiding use of aminoglycosides in patients with a maternal family 
history of early onset sensorineural hearing loss may be beneficial. 
 
Vestibular toxicity following treatment with aminoglycoside antibiotics: Vestibular 
toxicity, leading to symptoms such as dizziness, imbalance, and nausea, is a rare 
complication (~2%) of systemically administered aminoglycosides reported in adults.63 To 
our knowledge, there are only 3 single-patient case reports, all describing medically 
complex adults, reporting vestibular dysfunction after inhaled tobramycin use.63-66 There are 
no reports of vestibular toxicity in children receiving inhaled tobramycin. There are no 
diagnostic tests capable of accurately quantifying vestibular function in hospitalized infants. 
As per standard of care for very preterm infants with BPD in the CHOP N/IICU, all infants 
enrolled in the phase 1 trial will be referred to a neonatal follow-up clinic and undergo 
standardized cognitive and motor function testing through the first 2 years of life.  
 


 
  
 
 
 
8
1.6.2.3 
Additional adverse reactions 
Data from randomized, placebo-controlled trials of inhaled tobramycin in children and adults 
with CF indicate inhaled tobramycin is generally well tolerated. In 2 studies inclusive of 258 
patients who received inhaled tobramycin (300mg twice daily with alternating periods of 28 
days on and off treatment for 24 weeks), voice alteration (13% vs. 7% in the placebo group) 
and tinnitus without loss of hearing (3% vs 0% in the placebo group) were the only adverse 
experiences reported by significantly more patients treated with inhaled tobramycin than 
placebo.54 Changes in voice/cry are not expected in this study of infants receiving invasive 
mechanical ventilation. Additional reported adverse events that may be related to inhaled 
tobramycin use include cough, increased sputum production, bronchospasm, and 
hemoptysis. Allergic reactions and anaphylaxis are possible adverse reactions of any drug. 
 
1.7 
Rationale for drug dosages and duration under investigation 
The recommended dose of inhaled tobramycin in children with CF who are ≥6 years of age 
is 300mg administered every 12 hours for at least 28 days. Randomized trials and 
observational studies have evaluated the safety and efficacy of this regimen in children as 
young as 6 months of age with CF and not found evidence of toxicity.25,26,55,67 At present, 
there are no universally established dosing recommendations for inhaled tobramycin in 
children without CF. Published reports in young children without CF describe safe 
administration with doses ranging from 80-300mg every 12 hours.48,68,69 A consensus 
summary from the Society of Infectious Disease Pharmacists recommends a dose of 80mg 
every 12 hours for acute exacerbations of non-CF bronchiectasis.17  
 
This trial will investigate up to 4 different doses of inhaled tobramycin: 78mg, 150mg, 
216mg, and 300mg – each administered every 12 hours. A dose of 80mg has been shown 
to be safe and effective in children <6 years of age.45-47 This trial will investigate an initial 
dose of 78mg rather than 80mg (a 2.5% decrease in dose) to enable accurate dilution from 
a 300mg/5mL drug vial using a 3mL syringe (see section 7.1.3 for further details on drug 
dosing and aliquoting).  
 
The FDA-approved dose of intravenous (IV) tobramycin for infants is 7.5mg/kg per day. 
This IV dose far exceeds the amount of drug expected to reach the systemic circulation with 
nebulized therapy (Table 4). During nebulization, less than 15% of the administered drug 
reaches the airways. This equates to ≤12mg of tobramycin for each 78mg dose. The 
available data indicate that <5% of inhaled tobramycin that reaches the airways is absorbed 
into systemic circulation, resulting in serum peak levels that are approximately 95% lower 
with inhaled versus intravenous administration. Based on these data, we anticipate that a 
starting dose of 78mg of inhaled tobramycin administered every 12 hours will not generate 
detectable serum tobramycin trough levels or result in toxicity. This study will investigate 
doses as high as 300mg twice daily if the 3+3 dose escalation design does not raise 
concern for potential toxicity at lower doses. This maximum possible dose was chosen as 
the available PK data indicate there is no need to decrease the dose of inhaled tobramycin 
below the recommended dose of 300mg when treating children <6 years of age (see 
section 1.6.1).21  
 
Although the current recommended duration of therapy for the eradication of a P. 
aeruginosa lung infection in young children with CF is a 28-day treatment course, studies 
have demonstrated clinical benefit after 14 days of therapy.32,36,47 These data suggest that 
longer treatment courses may not be necessary. Therefore, a 14-day course is expected to 


 
  
 
 
 
9
generate sufficient data to evaluate dose safety and tolerability and provide preliminary 
efficacy data to inform the design of possible future phase 2 and phase 3 trials.   
 
1.8 
Compliance Statement 
This study will be conducted in full accordance of all applicable CHOP Research Policies 
and Procedures and all applicable federal and state laws and regulations including 45 CFR 
46, 21 CFR Parts 50, 54, 56, 312, and the Good Clinical Practice (GCP): Consolidated 
Guideline approved by the International Conference on Harmonization (ICH). Adverse 
event reporting will be targeted, so that potential adverse events arising from the use of 
inhaled tobramycin (for example, inhalational side-effects as well as renal adverse events) 
will be reported, while other adverse effects attributed to the underlying disease or clinical 
care will not be reported. All episodes of noncompliance will be documented. 
 
The investigators will perform the study in accordance with this protocol, will obtain 
informed consent, and will report unanticipated problems involving risks to subjects or 
others in accordance with the CHOP IRB Policies and Procedures and all federal 
requirements. Collection, recording, and reporting of data will be accurate and will ensure 
the privacy, health, and welfare of research subjects during and after the study.  
 
2 
STUDY OBJECTIVES 
The objective of this trial is to evaluate the safety and tolerability of tobramycin solution for 
inhalation in very preterm infants with BPD who have evidence of a pathogenic GNR 
detected by tracheal aspirate culture.  
 
2.1 
Primary Objective 
The primary objective of this study is to determine the maximum well-tolerated dose of 
tobramycin solution for inhalation (selected between 78mg, 150mg, 216mg, and 300mg 
administered twice daily) administered to very preterm infants with BPD who are actively 
receiving invasive mechanical ventilation and have evidence of a pathogenic GNR detected 
by tracheal aspirate culture.   
 
2.2 
Secondary Objectives 
The secondary, exploratory objectives are to: 
• 
Evaluate the association between the dose of inhaled tobramycin, plasma 
tobramycin pharmacokinetics, and peak tracheal aspirate tobramycin levels. 
Table 4. Anticipated maximum peak serum tobramycin concentrations and daily IV dose equivalence for the 
drug dosage under investigation in this phase 1 trial  
Inhaled tobramycin 
dose 
Anticipated maximum peak serum 
concentration (per dose)a 
Approximate IV dose equivalents based on 
anticipated systemic exposureb  
78mg 
0.31 µg/mL 
0.24 mg/kg/day 
150mg 
0.6 µg/mL 
0.46 mg/kg/day 
216mg 
0.86 µg/mL 
0.66 mg/kg/day 
300mg 
1.2 µg/mL 
0.92 mg/kg/day 
a Calculated using data from Rosenfeld et al. indicating maximum peak serum tobramycin levels were 
≤1.2µg/mL following treatment with 300mg of inhaled tobramycin in young children.26 
b Intravenous (IV) dose equivalents calculated based on the anticipated maximum peak serum 
concentrations shown in the table and observed peak serum concentrations of 26.0 ± 2.1µg/mL after 
administration of a 10mg/kg intravenous dose of tobramycin in pediatric patients.70    


 
  
 
 
 
10 
• 
Compare the change (pre vs. post study) in tracheal aspirate pathogenic bacterial 
colony forming unit (CFU) counts measured by high resolution culture, (1) between 
participants in the phase 1 trial and untreated infants in the observational cohort, 
and (2) between trial participants stratified by the administered dose of inhaled 
tobramycin. 
• 
Compare the longitudinal change in measures of administered respiratory support 
(FiO2, ventilator mean airway pressure, and respiratory severity score) during the 
14-day study period, (1) between participants in the phase 1 trial and observational 
cohort and (2) between trial participants stratified by the administered dose of 
inhaled tobramycin. 
• 
Compare changes (pre vs. post treatment) in tracheal aspirate cytokine levels, 
neutrophil/WBC ratio, and patterns in the airway microbiome, stratified by the 
administered dose of inhaled tobramycin. 
• 
Change in the following measures of hypoxemia (SpO2<80%): Intermittent 
hypoxemia (events lasting 10s-3min), prolonged hypoxemia (events lasting >1min), 
and daily proportion of time in hypoxemia 
 
• 
Compare the longitudinal change in dynamic lung compliance, airway resistance, 
peak expiratory flow, and CO2 elimination during the 14-day study period, (1) 
between participants in the observational cohort and phase 1 trial and (2) between 
trial participants stratified by the administered dose of inhaled tobramycin. 
3 
INVESTIGATIONAL PLAN 
3.1 
General Schema of Study Design 
This study is an open-label, phase 1, inter-patient 3+3 dose escalation feasibility trial. A 
prospective, observational (non-treated) cohort of eligible infants will also be enrolled and 
outcomes compared to the trial participants. The study phases are depicted in Figure 6 and 
described in the sections that follow. 
 
3.1.1 
Screening Phase 
Potential subjects will be screened using daily census logs and the protocol inclusion and 
exclusion criteria. Male and female infants who meet the criteria will be eligible to undergo a 
once-weekly screening tracheal aspirate culture while receiving invasive mechanical 
ventilation. Infants with a screening culture that is positive for a pathogenic GNR bacteria 
will become eligible to enroll in the phase 1 trial of inhaled tobramycin or the untreated, 
observational cohort. Infants must begin the phase 1 trial or observational (non-treated) 
cohort 14-day study period within 7 days of collection of the screening tracheal aspirate 
sample that is positive for a pathogenic GNR (Figure 6). Infants whose screening culture(s) 
is/are negative for a pathogenic GNR bacteria will continue to be screened weekly for as 
long as they meet study eligibility criteria. This plan for serial screening is consistent with 
the regular surveillance used to monitor for Pseudomonas infection in children with CF and 
reflects the increasing risk of developing a GNR respiratory infection with longer duration of 
invasive mechanical ventilation.10,71,72 
 
3.1.2 
Phase 1 Trial 
The phase 1 trial will last up to 14 days, with each infant receiving an inhalational dose of 
tobramycin solution administered every 12 hours via vibrating mesh nebulizer. The dose 
(78mg, 150mg, 216mg, or 300mg) will be determined as per the 3+3 design protocol 
(Figure 2). During the investigational drug trial, each infant will undergo blood and tracheal 


 
  
 
 
 
11 
aspirate sampling and respiratory mechanics measurements at pre-specified time points to 
assess dosage safety and potential efficacy. Continuous pulse oximetry will be performed 
beginning 24-48hr prior to initiating the study drug and continued until completion of the 14-
day trial. Review of clinical data and assessment for adverse events will be conducted daily 
throughout the 14-day trial.  
  
3.1.3 
Observational (non-treated) Cohort 
Infants enrolled in the observational (non-treated) cohort will undergo 14 days of clinical 
data collection. During the study phase, respiratory mechanics measurements will be 
performed on study days 1 and 7. Respiratory mechanics measurements will also be 
performed ≤48 hours after the final study day as indicated in section 3.1.4. Pulse oximetry 
will be performed throughout the 14-day study period.  
 
 
Figure 6. Diagram of study phases. GNR, Gram-negative rod. * Details of both the phase 1 trial and observational (non-
treated) cohort will be discussed with eligible families during the consent conversations. Eligible infants may participate in 
either cohort but can be enrolled in only one cohort. Infants will enter into one of the two study cohorts within 7 days from the 
collection of the screening tracheal aspirate.  
 
3.1.4 
Follow-up Phase  
All enrolled study infants will be followed until hospital discharge with pre-defined clinical 
data abstracted periodically from the medical record. For infants enrolled in the phase 1 
trial, tracheal aspirate samples for microbiology testing and cytokine level measurements 
will be collected ≤48 hours and 14 days (or on the day of planned tracheal extubation, if 
earlier) after receiving the final dose of inhaled tobramycin. For infants enrolled in the 
observational cohort, a tracheal aspirate for high resolution culture will be collected ≤48 
hours after study day 14. Infants enrolled in both cohorts will undergo the third and final 
respiratory mechanics measurements ≤48 hours after the final study day.  
 


 
  
 
 
 
12 
3.2 
Allocation to Treatment and Masking 
Infants who satisfy the inclusion and exclusion criteria and a parent or guardian consents 
for the infant to participate in the phase 1 trial will be allocated to receive up to 14 days of 
tobramycin solution for inhalation at the dosage determined by the 3+3 trial design. Drug 
dosage will not be masked (blinded). 
 
3.3 
Study Duration, Enrollment, and Number of Sites 
3.3.1 
Duration of Study Participation 
The study duration for each subject will last from enrollment until hospital discharge. For 
infants who participate in the phase 1 trial of inhaled tobramycin, the drug trial will last up to 
14 days. The last tracheal aspirates obtained for research purposes will be collected up to 
14 days after completing the final administered dose of inhaled tobramycin. For infants who 
participate in the observational (non-treated) cohort, clinical data will be collected daily for 
14 days beginning after enrollment. Respiratory mechanics data will be recorded at study 
entry (day 0 for the trial participants, day 1 for the observational cohort), study day 7, and 
≤48 hours after the final study day.    
  
3.3.2 
Total Number of Study Sites/Total Number of Subjects Projected 
Study site: Neonatal/infant intensive care unit at the Children’s Hospital of Philadelphia.  
 
Up to 103 infants will be enrolled in the study to produce up to 52 evaluable subjects (phase 
1 trial + observational cohort). These numbers are estimated according to the enrollment 
flow diagram shown in Figure 7. Up to 26 infants are expected to receive the study drug to 
produce up to 24 infants with evaluable trial data. These numbers for the phase 1 trial 
account for the 24 infants needed to enroll up to 6 infants per each of the 4 evaluated 
doses (assuming 1 infant with a toxic response per dose level) plus withdrawal of up to 2 
infants (~10%) prior to receiving 14 drug doses for reasons other than a toxic response. We 
will target a minimum of 12 infants in the observational cohort. Enrollment in the 
observational (non-treated) cohort will be capped at 28 infants.  
 
 
Figure 7. Flow diagram of estimated maximum enrollment numbers 
 
3.4 
Study Population 
3.4.1 
Inclusion Criteria 
1. 
Male or female infants born <32 weeks’ gestation 


 
  
 
 
 
13 
2. 
Diagnosed with BPD (use of supplemental oxygen or respiratory support at 36 
weeks postmenstrual age) 
3. 
Postmenstrual age ≥36 weeks at study enrollment 
4. 
Treatment with invasive mechanical ventilation at enrollment without planned 
tracheal extubation within 7 days after enrollment 
5. 
Tracheal aspirate culture positive for a pathogenic GNR bacteria (see list of eligible 
organisms in section 3.4.3) within 7 days of beginning the study phase – this testing 
will be performed during the screening phase and must be met to participate in the 
phase 1 trial or observational cohort 
6. 
Parental/guardian permission (informed consent). 
 
3.4.2 
Exclusion Criteria 
1.    Serum creatinine >0.4mg/dL within 14 days of enrollment 
2. 
Congenital or acquired disease of the kidney or renal collecting system that 
adversely affects renal function 
3. 
Congenital or acquired hepatobiliary disease that adversely affects liver function 
4. 
Treatment with a systemic antibiotic within 7 days prior to enrollment 
5. 
Treatment with a potentially nephrotoxic medication (see list in Appendix A), 
excluding diuretics, within 48 hours prior to enrollment 
6. 
Treatment with a continuous infusion of a neuromuscular blocker (see list in 
Appendix B) within 48 hours prior to enrollment 
7. 
Known intolerance to aminoglycoside antibiotics 
8. 
Current treatment with high frequency or other oscillating mechanical ventilation 
9. 
Presence of a cancer diagnosis 
10. Maternal family history of early onset hearing loss defined as the need for an 
assistive hearing device prescribed before 30 years of age  
11. Endotracheal tube leak >20%.  
12. Any investigational drug use (as part of an FDA approved IND protocol) prior to 
enrollment.  
13. A subject who, in the judgement of the Investigator, is not an appropriate candidate 
for this research study. 
 
Subjects that do not meet all of the above criteria may not be enrolled. Any violations of 
these criteria must be reported in accordance with IRB Policies and Procedures.  
3.4.3 
Definition of Pathogenic GNR Airway Bacteria  
One or more of the following pathogenic GNR organisms must be cultured from a tracheal 
aspirate sample within 7 days prior to enrollment to meet trial eligibility: 
• 
Pseudomonas aeruginosa 
• 
Klebsiella species 
• 
Enterobacter species 
• 
Stenotrophomonas maltophilia 
• 
Escherichia coli 
• 
Acinetobacter baumannii 
• 
Serratia marcescens 
4 
STUDY PROCEDURES 
Below is a summary of the study procedures that will occur in each study phase. A list of 
main study procedures can also be found in Table 1 (page xiv). 
 


 
  
 
 
 
14 
4.1 
Screening Phase 
Census documents in the CHOP N/IICU will be reviewed bi-weekly during the study period 
to identify potentially eligible subjects. For subjects likely to be eligible, medical records will 
be reviewed using the inclusion and exclusion criteria. Study participation and enrollment 
criteria will first be discussed with the subject’s attending physician. After obtaining approval 
of the attending physician, the parent(s) or guardian(s) will be approached, and the details 
of the study will be discussed. First, consent will be obtained to collect a screening tracheal 
aspirate culture up to once weekly for as long as eligibility is maintained and a pathogenic 
Gram-negative rod organism is not identified. The parent or guardian will be informed that 
the results of the tracheal aspirate culture(s) will be used to determine eligibility for the 
phase 1 trial of inhaled tobramycin and the observational cohort. Details of the two study 
cohorts will be discussed during the initial consent conversations. However, a second 
consent discussion for participation in the inhaled tobramycin trial or observational (non-
treated) cohort will take place for those in whom the tracheal aspirate culture is positive for 
a pathogenic GNR. After learning of the study goals, procedures, risks and benefits, the 
parent(s) of eligible infants will be invited to participate in the phase 1 trial or observational 
cohort and if interested, informed consent will be obtained. Informed consent will be 
obtained prior to any study related procedures being performed. All screening culture 
results will be disclosed to families of enrolled infants. See section 9.6 for a description of 
the informed consent procedure.  
 
4.2 
Observational (non-treated) cohort 
The medical record of each infant enrolled in the observational cohort will be reviewed 
regularly until hospital discharge to collect pre-specified clinical data. Each infant in the 
observational cohort will also undergo respiratory mechanics measurements on study days 
1, 7 and ≤48 hours after completing the 14-day study period (see section 4.3.3). A tracheal 
aspirate for culture will also be collected ≤48 hours after completing the 14-day study period 
(see section 4.3.2). Oxygen saturation data (SpO2) will be recorded using continuous pulse 
oximetry. To collect this data, a research pulse oximeter will be placed in addition to the 
pulse oximeter used for clinical monitoring. This second device will enable reliable data 
collection, storage, and analysis. Nursing and medical staff will be instructed to use the 
clinical oximeter for all clinical monitoring.   
 
4.3 
Phase 1 Trial of Tobramycin Solution for Inhalation 
The phase 1 trial of inhaled tobramycin will utilize a 3+3 inter-patient dose escalation design 
(Figure 8). The safety and tolerability of up to 4 different doses of tobramycin will be 
evaluated: 78mg, 150mg, 216mg, and 300mg. Each dose will be administered every 12 
hours for up to 14 days via vibrating mesh 
nebulizer. If the 300mg dose level is 
completed in 3 participants and none show 
evidence of a toxic response, up to 3 
additional infants will be enrolled to receive 
to receive 300mg. The trial will be stopped 
if 2 of the additional infants receiving 
300mg develop a toxic response and the 
216mg dose will be used as the maximum 
tolerated dose in subsequent trials. The 
study procedures utilized to monitor drug 
safety and efficacy are listed in Table 5. 
 
 
 
Figure 8. 3+3 dose escalation design schematic. MTD: maximum 
tolerated dose 


 
  
 
 
 
15 
 
 
4.3.1 
Blood draws 
All blood samples required as part of this research protocol will be collected by heel stick, 
venipuncture, or from an indwelling catheter (e.g., arterial line) by nursing staff or CHOP 
phlebotomy, in adherence with standardized, written hospital guidelines. Samples will then 
be transferred to the CHOP clinical or research laboratories for analysis. Required blood 
sample volumes per laboratory test are: serum tobramycin level (0.5mL), comprehensive 
metabolic panel (1mL), PK blood samples (200µL). In addition to the pre-specified study 
blood draws, 200µL of blood will be collected for PK analyses on up to 6 different occasions 
during the phase 1 trial at the times of clinically ordered blood draws. In total, the amount of 
blood drawn for this study will not exceed 9.1mL over the 14-day study period. This amount 
of blood extraction is not expected to incur risk to the study patients. As an additional step 
to protect the safety of enrolled subjects, study team members, in consultation with nursing 
staff and the medical team, will ensure blood volume extraction (for clinical + research 
purposes) does not exceed the maximum allowable weight-based limits specified by local 
and NIH guidelines (no more than 5 mL/kg drawn in a single day). 
 
Table 5. Study procedures utilized to monitor drug safety, tolerability, and efficacy for participants in the phase 1 trial.  
 
 
Study Day 
Follow-up 
Procedure 
0a 
1 
4 
7 
10 
14 
All 
(1-14) 
15b 
28C 
Tracheal aspirate 
X 
 
X – after 
7th dose 
X – after 
14th dose 
 
 
 
X 
X 
Test: 
 
 
 
 
 
 
 
 
 
Quantitative culture 
X 
 
 
 
 
 
 
X 
X 
Cell count differential 
X 
 
 
 
 
 
 
X 
X 
Cytokine levels 
X 
 
 
X 
 
 
 
X 
X 
16s sequencing 
X 
 
 
X 
 
 
 
X 
X 
Tobramycin leveld 
 
 
X 
5X 
X 
X 
 
 
 
Blood draw 
 
X – after 
2nd dose 
X – after 
7th dose 
X – after 
14th dose 
X – after 
20th dose 
X – after 
28th dose 
 
 
 
Test: 
 
 
 
 
 
 
 
 
 
Tobramycin troughe 
 
X 
X 
X 
X 
X 
 
 
 
Comprehensive 
metabolic panelf 
 
X 
X 
X 
X 
X 
 
 
 
Tobramycin peakg 
 
 
 
X 
 
 
 
 
 
Random tobramycin 
levelsh 
 
 
 
 
 
 
X 
 
 
Continuous pulse 
oximetry 
X 
 
 
 
 
 
X 
 
 
Pulmonary mechanics 
measurements 
X 
 
 
X 
 
 
 
X 
 
Daily medical record 
review 
 
 
 
 
 
 
X 
 
 
Weekly medical 
record reviewi 
 
 
 
 
 
 
 
X 
X 
a “Day 0” procedures to be completed ≤7 days prior to administration of the first study drug dose. The quantitative tracheal aspirate culture 
and cell count differential obtained during the screening phase will be used for the laboratory values. Prior to initiating study drug therapy, 
one additional tracheal aspirate will be collected for cytokine level measurements and 16s sequencing. 
b “Day 15” procedures to be completed ≤48 hours after administration of the last study drug dose 
c Follow-up tracheal aspirate for quantitative culture will be collected 14 days after the last administered dose of inhaled tobramycin or on the 
day of planned tracheal extubation if earlier   
d Tracheal aspirate for tobramycin level measurements obtained on trial day 7 will be collected approximately 1hr, 3hr, 6hr, 9hr, and 11hr 
after drug administration. Samples collected on trial days 4, 10, and 14 will be collected approximately 11hr after administration of doses 
number 8, 20 and 28, respectively.  
e Serum and blood tobramycin trough levels will be drawn concurrently, 11 hours after administration of the previous dose 
f Blood draws for comprehensive metabolic panels should be bundled with draws for tobramycin trough levels or other clinically ordered 
blood draws. Comprehensive panel to include a magnesium and phosphorus level on day 7 and 14.  
g Serum and blood tobramycin peak levels will be drawn concurrently, 1 hour after completion of the previous dose 
h Up to 6 blood tobramycin levels per infant will be drawn concurrent with clinically ordered blood draws.   
i Weekly medical record review to assess for adverse events will take place until hospital discharge 


 
  
 
 
 
16 
4.3.2 
Tracheal aspirate collection 
Following bedside review of the collection procedures, nursing or respiratory staff will 
collect all tracheal aspirates in a standardized manner under the direct (in-person) guidance 
of a study team member. Our research team has extensive experience coordinating 
endotracheal sample collection for microbiome and cytokine analyses. In most cases, the 
timing of tracheal aspirate collection for research can be planned by the investigative and 
nursing teams to coincide with routine, clinical suctioning of the endotracheal (ET) tube. 
However, if the collection of a tracheal aspirate sample must occur at a set time (e.g., timed 
after an inhaled tobramycin dose) and routine clinical suctioning will not occur at that time, a 
research only collection will be performed. Clinical and research suctioning of the 
endotracheal tube will be performed as described in the paragraph below. In the CHOP 
N/IICU, infants with BPD who are receiving invasive mechanical ventilation undergo routine 
endotracheal suctioning several times per day. Most undergo clinical suctioning at least 
every 3 hours, and often more frequently (as much as every 20-30 minutes) owing to the 
high secretion burden in infants with GNR bacteria in the airways. 
Tracheal aspirate samples will be collected by advancing a suction catheter just proximal to 
the end of the ET tube (length of ET tube is indicated at the bedside for all intubated 
infants). Tracheal secretions will be aspirated into a sterile Lukens trap. After tracheal 
secretion collection and removal of the suction catheter from the ET tube, the suction 
catheter will be cleared by aspiration of 1mL of sterile 0.9% saline (note: saline should not 
be instilled directly into the ET tube). This is the standard clinical procedure used to collect 
airway samples in the CHOP N/IICU. A minimum of 0.5mL of tracheal fluid will be collected 
– resulting in a total of approximately 1.5mL of fluid in the Lukens trap after flushing the 
suction catheter with 1mL of saline. In our experience, this procedure provides sufficient 
sample recovery for the specified analyses (see sections 5.1.2 and 5.1.3) and is consistent 
with tracheal aspirate volumes collected in prior studies.73-75 If less than 0.5mL of tracheal 
aspirate is recovered after a single suctioning, tracheal suctioning will be repeated at least 1 
hour later, at the time of the next clinical suctioning. Of note, saline lavage is not performed 
prior to ET tube suctioning as this does not result in greater recovery of mucous material in 
infants and may adversely affect clinical stability.76 A sterile (new) suction catheter will be 
used for all microbiological sampling as per unit protocol. Tracheal aspirate samples for 
tobramycin assays may be collected with a new suction catheter or the existing in-line 
suction catheter.  
4.3.3 
Respiratory mechanics measurements 
Respiratory mechanics data will be recorded at 3 timepoints: study entry (day 0 for 
participants in the phase 1 trial, day 1 for participants in the observational cohort), on study 
day 7, and ≤48 hours after completion of the 14-day study period. Respiratory mechanics 
will be measured using a Philips Respironics NM3 respiratory profile monitor placed in-line 
with the ventilator circuitry. Each recording will last for up to 4 hours. The Respironics 
sensor will be placed between the Neoflow sensor, if present, or directly to the endotracheal 
tube if not, and the “y” connector of the ventilator tubing. This device is FDA approved for 
use in infants (510 (k) K091459). The Respironics NM3 continuously monitors several 
mechanical and physiological respiratory parameters including minute ventilation, dynamic 
lung compliance, airway resistance, peak expiratory flow, and carbon dioxide elimination. 
These data will be downloaded to a dedicated, password protected study computer 
following each recording session. Respiratory mechanics measurements will be timed to 
correspond with quiet sleep periods.  
 


 
  
 
 
 
17 
The Respironics NM3 monitor sensor adds a negligible amount of dead space to the 
ventilator circuitry (0.8mL, <3% of inspired tidal volume for eligible study participants) and 
has been successfully used without adverse effects in infant studies,77,78 including those 
conducted in the CHOP N/IICU (IRB 12-008686). The same procedure for collecting 
respiratory mechanics data will be used for infants enrolled in the phase 1 trial and those 
enrolled in the observational cohort. 
 
4.3.4 
Continuous pulse oximetry 
Continuous peripheral oxygen saturation (SpO2) measurements will be obtained beginning 
24-48hr prior to initiating the study drug and continued throughout the 14-day study period 
using a Masimo Rad-G pulse oximeter. A 2-second averaging time and 2-second sample 
rate will be used for all oximeter recordings. These parameters provide a more reliable 
measurement of the number and duration of hypoxemic events compared to the oximeters 
used for clinical care, which employ longer averaging times and less frequent sampling. 
The study pulse oximetry probe may be placed on any extremity in accordance with unit 
policy. The study pulse oximetry probe and connected pulse oximeter will be used in 
addition to the probe and oximeter used for routine clinical care. The study pulse oximeter 
will be used for research purposes only. The study pulse oximeter will not have 
programmed alarms, however saturation data will be displayed to ensure the oximetry 
probe and oximeter are functioning properly. Raw data will be extracted from the pulse 
oximeter every 72 hours and stored on a single password protected CHOP workstation for 
cleaning and analysis. The oximeter data will not be provided to the clinical team. 
 
4.4 
Follow-up Phase  
The medical records of each enrolled subject will be periodically reviewed until hospital 
discharge for the following information: 
 
• 
Occurrence of adverse events that may be related to the investigational drug 
• 
Weekly abstraction of respiratory support and microbiological data (including all 
culture results and use of any antimicrobials) 
• 
Weekly abstraction of respiratory medication administration (drug name and class) 
• 
Discharge review of respiratory support data, audiology screening results, 
medications prescribed for home use, and discharge disposition 
 
For the infants enrolled in the phase 1 trial, tracheal aspirate samples will be collected at 2 
separate time points during the follow-up phase (as shown in Table 5). The first time point 
will be within 48 hours of completing the last dose of inhaled tobramycin. The second time 
point will be 14 days after the last administered dose of inhaled tobramycin or on the day of 
tracheal extubation, if earlier. Two tracheal aspirate samples will be collected at each 
timepoint to obtain enough fluid from the respiratory tract to complete the described 
laboratory testing (one sample will be analyzed in the clinical laboratory– see section 
5.1.3.3; and the second is used for research laboratory analyses – see section 5.1.3.1). For 
infants enrolled in the observational (non-treated) cohort, a single tracheal aspirate for high 
resolution culture will be obtained within 48 hours after study day 14, or at the time of 
extubation, if earlier. The procedures described in section 4.2.2 will be used to obtain these 
samples. The final respiratory mechanics measurements in both study cohorts be obtained 
≤48 hours after completing the 14-day study period.  
 


 
  
 
 
 
18 
4.5 
Concomitant Medications 
All medications administered within 7 days prior to enrollment through the end of the phase 
1 trial will be recorded for infants who participate in the drug trial. All antibiotics 
administered to infants enrolled in the observational cohort will be recorded. The dates of 
administration and drug dosages will be recorded. 
  
4.6 
Rescue Medication Administration 
Subjects may receive rescue medications for adverse reactions to the study medication at 
the discretion of the treating medical team. Data on the administered medications and 
dosages will be recorded.  
 
4.7 
Subject Completion/Withdrawal 
Subjects will be withdrawn from the phase 1 trial for any of the following reasons: 
 
• 
Occurrence of any of the primary safety endpoints listed in section 5.2.1 
• 
Initiation of an intravenous antibiotic, continuous infusion of a neuromuscular 
blocking agent, or a new cancer diagnosis 
• 
Tracheal extubation 
o Infants who have not yet completed the phase 1 trial and are re-intubated 
≤48 hours after extubation may restart the trial procedures at the same stage 
as when the extubation occurred. 
o Infants will not resume the investigational drug trial if 2 instances (combined) 
of endotracheal tube obstruction or unplanned extubation occur during the 
14-day phase 1 trial. 
 
Infants enrolled in the observational (non-treated) cohort will discontinue respiratory 
mechanics measurements if invasive mechanical ventilation is halted during the 14-day 
study period and not resumed within 48 hours. Clinical data collection will continue as 
appropriate.  
 
Withdrawal from the study is also permitted at any time by parent or physician request or at 
the discretion of the investigator to protect the subject for reasons of safety (i.e., presence 
of adverse events) or for administrative purposes. Early withdrawal will not result in any 
prejudice to the subject’s care. It will be documented whether or not each subject completes 
the clinical study. If the Investigator becomes aware of any serious, related adverse events 
after the subject completes or withdraws from the study, they will be recorded in the source 
documents and on the case report forms and reported to the IRB and DSMB as described 
in section 8. 
 
4.7.1 
Early Termination Procedures 
Subjects who are removed from the phase 1 trial early will undergo measurement of at least 
1 serum tobramycin trough, a comprehensive metabolic panel, and collection of 2 tracheal 
aspirate samples after administration of the final tobramycin dose – unless these 
procedures are refused by the parent or physician. Laboratory testing may be repeated at 
the discretion of the research and medical teams for safety purposes. Review of the 
medical record will also continue as appropriate unless refused by the parent or guardian. 
 
4.7.2 
Addition of Trial Participants after Early Termination 
Additional research subjects will be added to the phase 1 trial as per the 3+3 design 
(Figure 8). In addition, subjects who are removed early from the trial for reasons other than 


 
  
 
 
 
19 
a toxic response or safety concern related to the investigational drug will be replaced 1:1 if 
fewer than 14 doses of inhaled tobramycin were administered. Up to 2 subjects total and no 
more than 1 per dose level, who receive ≥14 doses of inhaled tobramycin, are removed 
early from the trial for reasons other than a toxic response or safety concern related to the 
investigational drug, and do not meet criteria for any of the primary tolerability endpoints will 
inform progression through the 3+3 trial design without requiring participant replacement. 
This is supported by the available data which show that elevated serum tobramycin trough 
levels in infants and young children are typically observed within the first 7 days of 
inhalational administration during every 12-hour dosing.48-50 PK analyses in young children 
demonstrate similar serum tobramycin levels are obtained after 28 days and after 56 days 
of inhaled tobramycin use suggesting negligible serum drug accumulation or impaired renal 
function when initial levels are reassuring.21  
 
At least 3 infants must receive the full 14-day treatment course (28 doses) of the dose that 
is ultimately defined as the maximum tolerated dose at the end of this trial. Participants will 
be added as needed to fulfill this requirement. If the 300mg dose level is completed in 3 
participants and none show evidence of a toxic response, up to 3 additional infants will be 
enrolled to receive to receive 300mg. This will provide a more robust assessment of drug 
tolerability and PK. The trial will be stopped if 2 of the additional infants receiving 300mg (2 
out of 5 or 2 out of 6) develop a toxic response. In that event, the 216mg dose will be 
considered the maximum tolerated identified in this study. 
 
5 
STUDY EVALUATIONS AND MEASUREMENTS 
5.1 
Screening and Monitoring Evaluations and Measurements 
5.1.1 
Medical Record Review 
5.1.1.1 
Screening Phase 
The following data will be abstracted from the medical record during the screening phase: 
• 
Date of birth 
• 
Gestational age 
• 
Birth weight 
• 
Sex 
• 
Race/ethnicity 
• 
Prenatal exposure to corticosteroids 
• 
Postmenstrual age at study enrollment 
• 
Weight, length, and head circumference at enrollment 
• 
Respiratory support (including settings and FiO2) at enrollment 
• 
Currently prescribed medications and those administered with 7 days prior to 
enrollment (name, dose, route, and interval).  
• 
Prior microbiological data from birth through enrollment, including culture results and 
antibiotic exposures (drug, duration, dates of exposure) 
• 
Results of any prior audiological examination or testing 
• 
Diagnoses present in the active problem list 
 
5.1.1.2 
Phase 1 Trial 
The medical record will be reviewed as follows throughout the phase 1 trial: 
• 
Daily review of the medical record and discussion with the medical team, nursing, 
and respiratory staff to assess for adverse events 


 
  
 
 
 
20 
• 
Daily review of weight, vital signs (heart rate, blood pressure, oxygen saturation), 
and fluid balance (including urine output) recorded by nursing staff in the electronic 
medical record (EPIC) flow sheet 
• 
Daily respiratory support data (mode of ventilation, settings, FiO2, and calculated 
respiratory severity score: mean airway pressure x FiO2) recorded in EPIC  
• 
Daily review of administered medications (name, dose, route, and interval)  
• 
Daily review of any clinically ordered laboratory tests and radiographic imaging. 
 
5.1.1.3 
Observational (non-treated) cohort 
The medical records of infants who are enrolled in the observational cohort will be reviewed 
daily for 14 days beginning after enrollment. Respiratory support data (mode of ventilation, 
settings, FiO2, and calculated respiratory severity score: mean airway pressure x FiO2) will 
be abstracted from EPIC. 
 
5.1.1.4 
Follow-up Phase 
The medical record will be reviewed from the end of the 14-day phase 1 trial/observational 
period until hospital discharge as follows: 
• 
Weekly review of the medial record to assess for serious adverse events (only 
infants enrolled in the phase 1 trial) 
• 
Results of all audiology evaluations conducted prior to hospital discharge 
• 
Date, age, and postmenstrual age at hospital discharge 
• 
Medications and respiratory support prescribed for home use 
 
5.1.2 
Continuous pulse oximetry 
A research pulse oximeter (see section 4.3.4) will be used throughout the 14-day study 
period to measures reliable SpO2 levels. Oximeter monitoring will begin 24-48 hours prior 
beginning the phase 1 drug trial to establish baseline values.  
 
5.1.3 
Clinical Data Warehouse 
Routine physiologic monitoring data (heart rate, oxygen saturation, blood pressure) will be 
obtained from the clinically prescribed bedside multiparameter monitor on phase 1 trial and 
observational cohort study participants. This data will be downloaded from CHOP data 
warehouse and analyzed by the study team. These data will be stored on the CHOP 
research server. 
 
5.1.4 
Clinical Laboratory Evaluations 
5.1.4.1 
Screening phase 
Tracheal aspirate sampling will be performed for the following laboratory evaluations during 
the screening phase to establish eligibility:  
• 
Tracheal aspirate cell count with differential 
• 
Tracheal aspirate quantitative culture with antimicrobial sensitivities 
 
Of note, this sampling will be performed even in infants with a recent clinically obtained 
tracheal aspirate culture as quantitative cultures are not part of routine clinical practice. 
 


 
  
 
 
 
21 
5.1.4.2 
Phase 1 Trial 
Blood sampling will be performed for the following laboratory evaluations: 
• 
Serum tobramycin level monitoring (note: this sampling differs from blood 
tobramycin levels used for the PK analyses described in section 5.1.4.2) 
• 
Serum electrolytes 
• 
Liver function assessment 
• 
Renal function assessment 
Tracheal aspirate sampling will be performed for the following laboratory evaluations: 
• 
Tracheal aspirate cell count with differential 
• 
Tracheal aspirate quantitative culture with antimicrobial sensitivities 
 
The laboratory tests listed in sections 5.1.3.1 and 5.1.3.2 (and shown in Table 6) will be 
performed in the clinical laboratory at CHOP. All laboratory tests performed in the CHOP 
clinical lab meet the regulatory requirements of the State Health Department, CLIA 88, 
FDA, AABB, CAP and JCAHO. The timing of collection of these tests is outlined in Table 5 
and in sections 4.2 and 4.3 
 
Table 6: Clinical Laboratory Tests 
 
5.1.4.3 
Follow-up Phase 
For the phase 1 trial participants, tracheal aspirate sampling will be performed for the 
following laboratory evaluations at two timepoints during the follow-up phase (≤48 hours 
after receiving the final dose of inhaled tobramycin and 14 days after receiving the final 
dose of inhaled tobramycin or on the day of planned tracheal extubation, if earlier): 
• 
Tracheal aspirate cell count with differential 
• 
Tracheal aspirate quantitative culture with antimicrobial sensitivities 
 
For observational cohort participants, tracheal aspirate sampling for the above laboratory 
tests will be performed once during the follow-up phase, ≤48 hours after day 14 of the 
observation period. 
5.1.5 
Research Laboratory Evaluations 
5.1.5.1 
Tracheal Aspirates 
The following laboratory analyses will be performed using tracheal aspirate samples 
collected during the study treatment phase: 
• 
Tobramycin level 
• 
Cytokine level analysis (including: IL 1β, 6, 8, 17A/F, 18, 22, 23, TNF-α, MIF, IFN-γ) 
• 
16s microbial sequencing 
 
Additional tracheal aspirate samples will be collected during the follow-up phase for the 
following laboratory analyses: 
• 
Cytokine level analysis (including: IL 1β, 6, 8, 17A/F, 18, 22, 23, TNF-α, MIF, IFN-γ) 
Category 
Tests 
Drug monitoring  
Metabolic panel 
 
Liver function tests 
Renal function tests 
Microbiology 
Body fluid cell count 
Serum tobramycin 
Serum sodium, potassium, chloride, bicarbonate, glucose, calcium, total 
protein, albumin. Magnesium and phosphorus will be collected on day 7, 14 
SGOT/AST, SGPT/ALT, total Bilirubin, alkaline phosphatase 
BUN, creatinine 
Quantitative culture of tracheal aspirate fluid with antimicrobial sensitivities 
Cell count and differential of tracheal aspirate fluid 


 
  
 
 
 
22 
• 
16s microbial sequencing 
 
Cytokine level analyses will be performed in the CHOP Translational Core Laboratory. 
Owing to inevitable variation in dilution during the collection process, cytokine level will be 
normalized to total protein levels in the tracheal aspirate fluid and compared to typical 
ranges found in published studies to ensure validity.73,79 Tracheal aspirate tobramycin level 
analyses will be performed in the laboratories of University of Florida College of Pharmacy. 
Whether tracheal aspirate drug levels require correction for dilution is uncertain due to a 
lack of available evidence. We will explore differences in the study results with and without 
urea correction for dilution and compare the results to laboratory controls. The 16s 
microbial sequencing will be performed in the laboratories of the CHOP/Penn Microbiome 
Program. All laboratory procedures will be conducted using validated assays. The results of 
these laboratory tests will be reported as part of the data collection process for the research 
study, but will not appear in the medical record nor be used to make clinical decisions.  
 
5.1.5.2 
Blood Sampling for Pharmacokinetic Analyses 
Blood (0.2mL) will be collected for pharmacokinetic analyses at 2 set time points on day 7 
plus up to 6 random collections coincident with clinically obtained draws. The lower limit of 
quantification for serum tobramycin concentrations in the CHOP clinical laboratory is 0.6 
µg/mL. Based on this quantification limit, we anticipate that the majority of subjects will 
have clinically undetectable trough and/or peak levels, limiting the ability to characterize 
serum pharmacokinetics. Measurement of blood plasma tobramycin levels will be 
performed at the University of Florida College of Pharmacy using high-performance liquid 
chromatography tandem mass spectrometry (HPLC-MS/MS). This will enable quantification 
of drug levels down to a level of 50-100 ng/mL and facilitate estimation of patient-specific 
pharmacokinetics. Individually identifiable will be removed from samples and the samples 
will be labeled with unique identifiers. The CHOP study team will be able to link the unique 
identifiers to individually identifiable information, but the individually identifiable information 
will not be available to outside researchers. 
 
5.1.6 
Respiratory mechanics measurements 
A Philips Respironics NM3 respiratory profile monitor will be used to collect respiratory 
mechanics data. Measurements will be performed at 3 time points: at study entry (study day 
0 for infants enrolled in the phase 1 trial, study day 1 for infants enrolled in the 
observational cohort), study day 7, and ≤48 hours after completing the 14-day study period. 
Each recording period will last 4 hours and coincide with a period of quiet sleep. The NM3 
monitor is FDA approved for use in infants (510 (k) K091459). The Respironics NM3 
monitor will be used to collect data on the following endpoints: dynamic compliance (mL/cm 
H2O), peak expiratory flow (L/min), airway resistance (cm H2O/L/sec), and carbon dioxide 
elimination (mL/min). These data will be downloaded to a dedicated, password protected 
study computer following each recording session. For this analysis, only results obtained 
from mandatory, ventilator driven non-pressure support breaths will be used.  
 
5.2 
Safety Evaluations 
5.2.1 
Primary composite safety endpoint 
The primary objective of this trial is to assess the safety and tolerability of up to 4 different 
dose levels of tobramycin solution for inhalation in preterm infants receiving invasive 
mechanical ventilation with a tracheal aspirate culture positive for a pathogenic GNR. 
Following the 3+3 trial design (Figure 8), tolerability of each dose will be defined as no 


 
  
 
 
 
23 
more than 1 in 6 infants per dose level developing any of the following safety endpoints 
during the 14-day trial: 
 
• Serum tobramycin trough (measured 11 hours after the prior dose) ≥1µg/mL 
Serum tobramycin trough levels will be performed as per the schedule outlined in section 
4.2. Quantification of serum tobramycin levels will be performed in the CHOP Clinical 
Laboratory as described in section 5.1.3. Current standards (including pharmacy 
guidelines at CHOP for conventional dosing strategies) and manufacturer labelling for 
intravenous tobramycin recommend maintaining serum tobramycin trough levels 
<2µg/mL.80 Out of an abundance of caution, any measured trough value ≥1µg/mL will 
define a toxic response. 
• Increase in serum creatinine level by ≥0.3mg/dL above pre-trial level 
• Increase in serum creatinine >1.5-fold above the pre-trial level 
• Urine output <0.5mL/kg/hr for 12 hours 
 
These 3 measures of reduced kidney function indicate stage 1 (the least severe stage) 
of acute kidney injury (AKI) as defined by the Kidney Disease Improving Global 
Outcomes (KDIGO) practice guidelines.1-3 Serum creatinine levels will be measured as 
per the schedule outlined in section 4.2. Quantification of serum creatinine levels will be 
performed by the CHOP Clinical Laboratory as described in section 5.1.2. 
 
Nursing staff monitor urine output of all infants in the CHOP N/IICU throughout each day 
by weighing every soiled diaper. Typically, this is performed every 3 hours, coinciding 
with nursing care times. As per routine clinical care, “combo” diapers containing both 
urine and stool will be utilized when calculating total urine output. Data on urine output is 
recorded in the electronic medical record (EPIC) and will be reviewed by the study and 
clinical teams prior to the administration of each investigational drug dose.      
 
• Any serious adverse event possibly attributable to the study drug.  
 
The occurrence of adverse events identified by clinical monitoring or laboratory testing 
(in addition to those described above) will be monitored throughout the trial and reported 
as described in section 8. A serious adverse event will constitute a toxic response. 
 
5.2.2 
Secondary exploratory safety endpoints 
Additional exploratory safety endpoints that will be recorded are: 
 
• New onset or worsened coughing associated with a concomitant drop in SpO2 to <80% 
for >10 seconds, need for an increase in FiO2 by an absolute change >20%, or need 
clinical recovery using positive pressure support delivered via manual bag ventilation 
• Obstruction of the endotracheal tube requiring tube replacement 
• Unplanned tracheal extubation 
• Desaturation (SpO2 <80% for >10 seconds) during administration of inhaled tobramycin 
• Rates of pre-discharge failed audiology examinations 
• New intra-patient antimicrobial resistance 
 
5.2.3 
Limitations of Audiology Testing in Hospitalized Infants 
Inhaled tobramycin therapy in infants with BPD is not expected to result in ototoxicity. 
Ototoxicity, as measured by complaints of hearing loss or by audiometric evaluations, did 


 
  
 
 
 
24 
not occur with tobramycin inhalation solution therapy during clinical studies.54 Moreover, 
intravenous aminoglycoside use has not been shown to result in hearing loss in preterm 
infants when used as directed (see section 1.6.2.2 for a discussion of these studies). 
 
Some studies evaluating inhaled tobramycin in older children and adults performed 
audiometric testing prior to and after completing a drug course and did not find significant 
evidence of persistent hearing loss.55,81,82 However, such an approach is not appropriate for 
hospitalized preterm infants. Physiologic test measures which include Auditory Brainstem 
Response (ABR) or Otoacoustic Emissions (OAE), must be used to screen newborns and 
infants for hearing loss. Newborns with tracheostomy or ventilator dependency may be 
screened later due to their complex medical conditions. Patient awake state, movement and 
electrical interference from equipment can impact testing. In addition, chronic fluid or 
abnormal middle ear pathology, common in this population, may cause hearing loss that 
can be transient or chronic in nature.   
 
Complex medical presentations common in this population and environmental influences 
may impact initial test results, supporting the need for continued audiologic follow-up. One 
study of 78 preterm infants with BPD found auditory brainstem response (ABR) 
abnormalities in 22% of participants.83 All subjects who failed the ABR demonstrated 
normal hearing by visual reinforcement orientation audiometry (VROA) at 8-12 months.83 
Notably, 10 of 14 infants (71%) who failed the gold standard VROA at follow-up had a 
normal ABR at hospital discharge.83 Another study of 216 hospitalized infants compared the 
diagnostic reliability of automated transient evoked otoacoustic emissions (a-TEOAE), 
automated auditory brainstem response (a-ABR), and conventional brainstem auditory 
evoked potential (BAEP/ABR).84 These tests produced modest specificity (71-91%) and 
poor positive predictive value (9-25%) when compared to repeat testing BAEP/ABR 
conducted 2-3 months later.84 Finally, BAEP/ABR can only detect hearing abnormalities 
occurring between 0.5-4hz.85 Most ototoxic medications affect frequencies >4hz.85  
 
The American Academy of Pediatrics (AAP) recommends that all infants at risk of hearing 
loss should undergo subsequent screening by 24 to 30 months of age. Importantly, 
recommendations for audiologic follow-up should be customized based on individual patient 
risk factors and likelihood of delayed-onset hearing loss. Accordingly, the Joint Committee 
on Infant Hearing (JCIH) recommend beginning in 2019 that that all infants with a risk 
indicator for hearing loss who pass the initial newborn hearing screen should be referred for 
an audiologic assessment by 9 months of age.86 
 
Owing to challenges of completing audiologic testing in preterm infants who are receiving 
intensive care (e.g., ongoing invasive mechanical ventilation) and obstacles that contribute 
to obtaining these tests in this population (infants with severe BPD in the CHOP N/IICU 
require a median of 3 attempts at audiology screening before a test can be successfully 
performed – regardless of the eventual result), all audiologic screening in study participants 
will be performed prior to hospital discharge as per standard of care in the CHOP N/IICU. 
All study infants will undergo pre-discharge ABR and OAE testing as well as tympanometry 
as indicated by the audiologic evaluation. The results of these evaluations will be recorded 
as part of the study data. The rate of failed pre-discharge hearing tests in a recent cohort of 
infants who received care in the CHOP N/IICU and were similar to those eligible for this 
study was 36%.87 Based on these data, a rate of pre-discharge hearing screening 
abnormalities ≥50% among the infants treated with inhaled tobramycin will trigger adverse 
event reporting to the IRB and FDA. In addition, the rates of failed pre-discharge 
audiometric evaluations will be compared between infants who participate in the 


 
  
 
 
 
25 
investigational drug trial and those who are enrolled in the study but do not participate in the 
drug trial (e.g., lack of GNR in the airway or parental refusal to participate in the phase 1 
trial). A statistically significant difference in the rates of failed evaluations between these 
two groups will also trigger adverse event reporting to the IRB and FDA.  
 
In the CHOP N/IICU, all infants with abnormal audiologic test results are referred for 
additional evaluations by Audiology and Otolaryngology. Consistent with the above 
recommendations from the JCIH86, infants who attend the CHOP newborn follow-up 
program and pass their newborn hearing screening are also referred for repeat audiologic 
testing by at least 9-12 months of age or as soon as possible thereafter if discharge occurs 
at a later age. In addition, all infants seen in the CHOP newborn follow-up program undergo 
detailed neurologic evaluations including standardized motor and cognitive testing. 
  
5.2.4 
Individual Subject Stopping Rules 
A study subject will be removed from the phase 1 trial and will not receive additional doses 
of the study drug if he/she develops a toxic response defined as any of the following: 
• 
Trough serum tobramycin level (measured 11 hours after the last administered 
dose) ≥1µg/mL 
• 
Increase in serum creatinine level by ≥0.3mg/dL above the pre-trial level1-3 
• 
Increase in serum creatinine >1.5-fold above the pre-trial level1-3 
• 
Urine output <0.5mL/kg/hr for 12 hours1-3 
• 
Any other serious adverse event possibly attributable to the investigational drug  
 
Subjects may also be withdrawn from the study for reasons that do not meet the above 
criteria as described in section 4.7. 
 
If an infant commences the trial and the primary medical team subsequently chooses to 
prescribe a systemic antibiotic (e.g., for the positive tracheal aspirate or another suspected 
infection that was unknown/not present at the time of enrollment), the infant will stop the 
phase 1 trial early. No additional doses of inhaled tobramycin will be administered but final 
laboratory testing will be performed. 
 
5.2.5 
Study Stopping Rules 
The study stopping rules will follow 
the 3+3 dose escalation design 
shown in Figure 9. The study will 
be stopped if more than 1 subject 
develops a drug-related toxic 
response (any of the safety 
endpoints listed under section 
5.2.1) at a single dosage level. 
Further enrollment will be stopped, 
but data analysis for those enrolled 
will continue until completed.   
 
                                                                                  Figure 9. 3+3 dose escalation design schematic. MTD: maximum 
tolerated dose 
 


 
  
 
 
 
26 
5.3 
Efficacy Evaluations 
Measures of potential drug efficacy will be assessed as secondary, exploratory endpoints. 
These will include the following outcomes: 
 
• 
Change (pre vs post treatment) in tracheal aspirate pathogenic bacterial colony forming 
unit (CFU) counts measured by high resolution culture 
Tracheal aspirates for high resolution culture will be obtained ≤7 days prior to initiating 
inhaled tobramycin (as part of the screening procedures), ≤48 hours after completing 
the final drug dose, and 14 days after completing the final drug dose (or on the day of 
planned tracheal extubation if earlier) (see schedule under section 4.3). For infants 
enrolled in the observational (non-treated) cohort, tracheal aspirate for culture will be 
obtained ≤7 days prior to initiating the 14-day observation period (as part of the 
screening procedures) and ≤48 hours after completing the observation period. These 
tests will be performed in the CHOP clinical laboratory as described in section 5.1.3.  
Reduction in number and eradication of pathogenic GNR bacteria from the airways is 
anticipated to result in clinical benefit. Evidence from preterm infants with BPD indicates 
the presence of these organisms in the airway is associated with worse clinical 
outcomes.10,11 Moreover, data from children with CF demonstrate that early treatment of 
P. aeruginosa infection of the respiratory tract leads to improved clinical 
outcomes.16,88,89 As such, change in quantitative tracheal aspirate culture results 
following treatment with inhaled tobramycin is anticipated to be a suitable surrogate 
marker to assess the likelihood of clinical benefit. For this phase 1 feasibility trial, we will 
evaluate the relative intra-patient change in bacterial CFU counts and assess the 
association between the longitudinal change in CFU count, airway cytokine levels, and 
the RSS.  
• 
Tracheal aspirate tobramycin levels 
Tracheal aspirate samples used for the measurement of tobramycin levels will be 
collected at the following times: (1) a single tracheal aspirate will be collected on days 4, 
10, and 14, 11-hours after completion of the 8th, 20th, and 28th doses respectively; and 
(2) 5 samples will be collected on trial day 7, approximately 1-hour, 3-hours, 6-hours, 9-
hours, and 11-hours after completion of the 14th tobramycin dose (see section 4.3) 
Tracheal aspirate drug levels will be measured in the laboratory of the University of 
Florida College of Pharmacology (see section 5.1.3). Tracheal aspirate drug levels will 
be compared to published concentrations needed to overcome the minimum inhibitory 
concentration necessary to kill 90% of detected bacterial isolates (MIC90).90,91 
Longitudinal change in tracheal aspirate levels will be modeled as part of the PK 
analyses. Correction for serum urea levels will be performed.  
• 
Longitudinal change in the following respiratory support measures during the 14-day 
study period: 
o 
Fraction of inspired oxygen (FiO2) 
o 
Ventilator mean airway pressure (MAP) 
o 
Respiratory severity score (MAP x FiO2) 
 
FiO2 and mean airway pressure (MAP) are established clinical measures of respiratory 
disease severity. They characterize the amount of respiratory support (supplemental 
oxygen and positive airway pressure) required to maintain cardiorespiratory stability in 
infants receiving invasive respiratory support. The respiratory severity score (RSS) is a 


 
  
 
 
 
27 
simplified severity score calculated as the MAP multiplied by the FiO2. The RSS is used 
in place of the oxygenation index (OI) owing to the infrequent availability of data on the 
partial pressure of arterial oxygen (PaO2) in hospitalized infants. The RSS correlates 
well with OI in infants receiving mechanical ventilation and is a useful predictor of poor 
respiratory outcomes in preterm infants.92-94 FiO2 and MAP values (measured by the 
mechanical ventilator and reported on the ventilator graphical interface), are recorded at 
least hourly in the respiratory flow sheet in the electronic medical record (EPIC). These 
data will be used to calculate daily, time-weighted values for FiO2, MAP, and RSS. 
Longitudinal change in these respiratory support measures during the 14-day study 
period will be compared (1) between infants who participate in the phase 1 trial and 
those enrolled in the observational (non-treated) cohort, and (2) among participants in 
the phase 1 trial, stratified by the administered dose of inhaled tobramycin.  
 
Owing to the expected heterogeneity in lung disease and respiratory support 
requirements of the enrolled infants, a protocolized ventilator management algorithm 
will not be utilized during this study. However, routine practice in our unit is to wean 
FiO2 in infants with BPD to maintain SpO2 ≥90%. Most infants with BPD who require 
invasive respiratory in our unit are supported using a time-cycled, pressure-limited, 
volume-guarantee mode of ventilation that delivers 8-10mL/kg of tidal volume at 
mandated rates of 15-25 breaths per minute. The Draeger V500 ventilator is used in all 
infants receiving acute care in the CHOP N/IICU who are prescribed intermittent 
mechanical ventilation. On this mode of support, the ventilator automatically adjusts the 
positive inspiratory pressure (PIP) – within set limits – to deliver the goal tidal volume. 
Change in the PIP along with any clinical adjustments in the positive end expiratory 
pressure (PEEP) will be the primary determinants of any observed changes in the 
delivered MAP. Only infants with an endotracheal tube leak ≤20% will be enrolled in this 
trial to avoid ventilator leak compensation and artificial inflation of the PIP levels.95 Of 
note, the Draeger V500 ventilator calculates the endotracheal leak proportion during all 
modes of intermittent ventilation employed in our unit.  
 
• 
Change (pre vs post) in tracheal aspirate cytokine levels, neutrophil/WBC ratio, and 
patterns in the airway microbiome: 
Tracheal aspirates collected ≤7 days prior to initiating inhaled tobramycin, ≤48 hours 
after completing the final drug dose, and 14 days after completing the final drug dose 
will be analyzed. Patterns in the airway microbiome will be assessed using 16s 
sequencing performed in the laboratories of the CHOP/Penn Microbiome Program. The 
following cytokine levels will be measured in tracheal aspirate samples: IL 1β, 6, 8, 
17A/F, 18, 22, 23, TNF-α, MIF, IFN-γ. These analyses will be performed in CHOP 
Translational Core Laboratory. Tracheal aspirate cell counts will be obtained by the 
CHOP clinical laboratory. Changes in the observed patterns in the airway microbiome, 
neutrophil/WBC ratio, and tracheal aspirate cytokine levels (compared to the pre-
treatment baseline) will be assessed, stratified by the treatment dosage level (see 
section 6.3 for further details of the analytical plan). For this phase 1 feasibility trial, we 
will evaluate the relative, intra-patient change in these measures as the association 
between these measures and clinical benefit in the study population is uncertain. 
• 
Longitudinal change in the following measures of respiratory mechanics during the 14-
day study period: 
o 
Dynamic compliance (mL/cm H2O) 
o 
Peak expiratory flow (L/min),  


 
  
 
 
 
28 
o 
Airway resistance (cm H2O/L/sec) 
o 
Carbon dioxide elimination (mL/min). 
 
The Respironics NM3 respiratory parameter monitor will be used to record these data 
for 4 hours periods at study entry, on study day 7, and ≤48 hours after completing the 
14-day study period. These data will provide important information on the potential 
effects of inhaled tobramycin on respiratory function. 
 
• 
Longitudinal change in the following measures of hypoxemia (SpO2<80%): 
o Intermittent hypoxemia (SpO2<80% lasting for 10s-3min) 
o Prolonged hypoxemia (SpO2<80% lasting for >1min) 
o Daily proportion of time with hypoxemia 
 
Daily frequency of intermittent and prolonged hypoxemic events are established, real-
time indicators of cardiorespiratory stability in BPD.96 Cumulative burden of hypoxemia 
predicts the severity of childhood neurodevelopmental delay.97 These data will be 
recorded continuously throughout the study using a research pulse oximeter that will be 
placed in addition to the clinical multiparameter monitor. Nursing and medical staff will 
be instructed to use the clinically placed monitor for all care decisions.  
 
5.4 
Pharmacokinetic Evaluation 
Each infant participating in the phase 1 trial will undergo blood sampling on day 7 to 
measure serum tobramycin peak and trough levels. Up to 6 random blood samples (200µL 
per sample) will also be collected concurrent with clinical blood draws. Based on prior 
studies, we anticipate that most trial participants, particularly those receiving the lowest 
dosages of inhaled tobramycin, will have serum trough levels below the lower limit of 
quantification in the clinical lab (0.6µg/mL).21,48,90 Therefore, we will collect 200µL of blood 
for high resolution measurement of peak, trough and random plasma tobramycin levels as 
per section 6.3.4. Blood samples collected for PK analyses will be stored at -80c in vials 
labelled with a study ID number only. Sample batches will be shipped to and subsequently 
processed at the University of Florida College of Pharmacy. This laboratory has extensive 
experience with high-resolution drug level testing and has established assays for 
tobramycin. 
 
6 
STATISTICAL CONSIDERATIONS 
6.1 
Primary Endpoint 
The primary study endpoint is a composite safety endpoint. The presence of any of the 
following events occurring at any timepoint during the 14-day phase 1 trial will define a toxic 
drug response: 
• 
Trough serum tobramycin level (measured 11 hours after the last administered 
dose) ≥1µg/mL 
• 
Increase in serum creatinine level by ≥0.3mg/dL above the pre-trial level 
• 
Increase in serum creatinine >1.5-fold above the pre-trial level 
• 
Urine output <0.5mL/kg/hr for 12 hours 
• 
Any serious adverse event possibly attributable to the investigational drug 
 
6.2 
Secondary Endpoints 
Additional exploratory secondary safety endpoints are: 
• 
New onset or worsened coughing associated with a change in respiratory status 
(SpO2 <80% for >10 seconds; need for increase in FiO2 by >20%) 


 
  
 
 
 
29 
• 
Obstruction of the endotracheal tube requiring tube replacement 
• 
Unplanned tracheal extubation 
• 
Desaturation (SpO2 <80% for >10 seconds) during administration of inhaled 
tobramycin 
• 
Rates of pre-discharge failed audiology examinations 
• 
Rates of new intra-patient microbial resistance to tobramycin 
o Bacterial antimicrobial sensitivities reported for the pre-treatment tracheal 
aspirate culture will be compared to the tracheal aspirate cultures collected 
during and post treatment. The presence of new-onset microbial resistance to 
tobramycin within a patient – a change from susceptible to resistant by the same 
organism – will be recorded 
 
The exploratory secondary efficacy endpoints are: 
• 
Tracheal aspirate tobramycin levels 
• 
Change in tracheal aspirate pathogenic bacterial colony forming unit (CFU) counts 
measured by high resolution, quantitative culture 
• 
Longitudinal change in the following measures of administered respiratory support: 
FiO2, ventilator mean airway pressure (MAP), respiratory severity score (MAP x 
FiO2) 
• 
Change in the following measures of hypoxemia (SpO2<80%): intermittent 
hypoxemia (events lasting 10s-3min), prolonged hypoxemia (events lasting >1min), 
and daily proportion of time with hypoxemia 
• 
Change in tracheal aspirate cytokine levels, neutrophil/WBC ratio, and patterns in 
the airway microbiome 
• 
Longitudinal change in dynamic compliance, peak expiratory flow, airway 
resistance, and carbon dioxide elimination 
 
6.3 
Statistical Methods 
6.3.1 
Baseline Data  
Baseline and demographic characteristics will be summarized using standard descriptive 
statistics (e.g., means and standard deviations for continuous variables such as age and 
percentages for categorical variables such as gender). 
 
6.3.2 
Safety Analysis 
All subjects entered into the phase 1 investigational drug trial of inhaled tobramycin will be 
included in the safety analysis. The primary study outcome is a safety endpoint and will be 
evaluated as a dichotomous outcome based on the presence of any of the events listed in 
section 6.1. The 3+3 design flow diagram shown in Figure 8 will be followed to determine 
enrollment and trial progression based on the results of each infant’s safety analysis. 
In addition, the frequencies of AEs by type, body system, severity, and relationship to the 
study drug will be summarized. SAEs (if any) will be described in detail. 
 
6.3.3 
Exploratory Secondary Safety and Efficacy Analyses 
The exploratory secondary safety and efficacy endpoints listed in section 6.2 will be 
analyzed as described in Table 7 (next page). In brief, all study data will first be 
summarized using standard descriptive statistics as appropriate based on data type and 
distribution. Assessments of change over time for all longitudinal secondary outcome data 
will first be assessed graphically, with and without stratification by the administered 
tobramycin dose. Next, paired t-tests or Wilcoxon sign rank tests will be used to compare 


 
  
 
 
 
30 
pre-trial baseline values to those recorded at later time points. Non-paired tests will be used 
to compare data baseline and subsequent data points between the infants in the phase 1 
trial and observational (non-treated) cohorts.  
Table 7. Planned statistical techniques and data availability for the phase 1 trial outcomes 
 
Values available for:  
 
Study outcome 
Phase 1 
trial cohort 
Observational 
cohort 
Statistical approach 
Secondary safety 
endpoints 
Yes 
Yes, for some 
endpoints 
• Rates of the secondary tolerability endpoints will be summarized 
for each dose level. Descriptive, narrative details of these 
endpoints will also be provided as appropriate.  
• Data on the rates of endotracheal tube obstruction, unplanned 
tracheal extubation, and audiology testing results will be compared 
between the trial and observational cohorts. 
• Planned comparisons of audiology testing results are described in 
the Protection of Human Subjects document. 
Secondary efficacy 
endpoints 
 
 
 
Negative TA culture 
for the target GNR 
Yes 
No 
• Primary exploratory efficacy endpoint. 
• Rates will be summarized for the trial cohort as a whole and 
stratified by drug dose level.  
Change in TA GNR 
pathogen CFU 
Yes 
No 
• Absolute (pre vs post) and relative change ([post trial – pre trial] / 
pre trial) in the target organism CFU/mL on the log10 scale will be 
calculated for each phase 1 trial participant. Paired t-tests or 
Wilcoxon sign rank tests as appropriate will be used to compare 
pre- vs. post-trial absolute CFU/mL values for the whole cohort and 
stratified by drug dose level.  
Tracheal aspirate 
tobramycin levels 
Yes 
No 
• Values, stratified by drug dose, will be compared to established 
concentrations necessary to overcome the minimum inhibitory 
concentration needed to kill 90% of detected bacterial isolates 
(MIC90).90,91 Values will also be used in our PK analyses.  
Tracheal aspirate 
cytokine levels 
Yes 
No 
• Individual cytokine levels measured pre-trial, ≤48 hours after the 
final dose, and 14 days after the final drug dose will be 
summarized. Transformation (e.g. logarithmic) or categorization by 
centiles will be considered based on the data distribution. 
Correction for total protein levels in the tracheal aspirate will be 
employed to account for differences in dilution during sample 
collection.  
• The relative intra-patient change for the individual cytokine levels 
from pre-trial baseline to the 2 post-trial timepoints will be 
calculated and compared between dose levels. 
• The association between change in cytokine levels and tobramycin 
dose will be explored using bivariate and multivariate correlation 
analyses with Bonferroni correction for multiple testing.100  
• We will consider exploratory cluster and linear discriminate 
analyses to identify airway immunological profiles associated with 
differences in the response to inhaled tobramycin (e.g. change in 
CFU/mL).101,102  
Respiratory support 
measures 
 
Hypoxemia (SpO2 
<80%) 
Yes 
Yes 
• Continuously recorded FiO2, MAP, and RSS values will be 
converted to time-weighted daily averages for each study day.  
• Continuously recorded SpO2 data will be used to calculate daily 
rates of intermittent and prolonged hypoxemia and daily total 
hypoxemia exposure. 
• Assessment of longitudinal change using linear mixed effects 
models will be considered.  
Pulmonary 
mechanics 
Yes 
Yes 
• Infants in both study cohorts will undergo pulmonary mechanics 
measurements pre-trial, after 7d, and after 14d. 
• Intra-patient change, relative to pre-trial baseline, will be calculated 
for each participant and compared (1) between the two cohorts and 
(2) stratified by drug dose.  
• Assessment of longitudinal change using linear mixed effects 
models will be considered. 
CFU, colony forming units; GNR, Gram-negative rod; TA, tracheal aspirate  


 
  
 
 
 
31 
We will consider constructing exploratory linear mixed effects models to evaluate the 
longitudinal change in repeated measures outcome data. Owing to the modest total study 
sample size and low numbers of infants treated with each tobramycin dose, these analyses 
will only be performed on an exploratory basis if graphical evaluations of the study data 
suggest strong evidence of a linear change over the course of the study period. These 
models will be considered: (1) for the phase 1 trial participants as a whole, (2) with 
stratification by the administered dose of inhaled tobramycin, and (3) between infants who 
participate in the phase 1 trial and those enrolled in the observational (non-treated) cohort 
(for the study outcome data collected in both cohorts). Treatment dose group and treatment 
week will be included in the models as independent variables (the latter is added to account 
for natural fluctuation in disease acuity over time). Random effects for intercept +/- slope 
will be considered based on their effect on model fit assessed using the Akaike information 
criterion. Backwards, stepwise elimination techniques to determine inclusion of possible 
confounding, independent covariates will be employed to account for measured differences 
between comparison groups. Adherence to accepted numbers of independent covariates 
based on the study sample size will be used to avoid model over-fitting.98 If longitudinal 
models are performed, 95% confidence intervals for all estimates of effect will be reported 
and caution will be urged when interpreting these hypothesis generating results.   
 
Patterns in the airway microbiome will be assessed by comparing (pre vs. post treatment) 
measures of alpha diversity (e.g. species counts, Shannon index) and beta-diversity 
(Weighted and unweighted UniFrac distances99). Principal coordinate analyses and 
evaluation of dominant taxa will also be used to compare the microbiome before versus 
after completing treatment and with stratification by the administered dose of inhaled 
tobramycin. Linear mixed effects models, appropriate for repeated measures data, will also 
be constructed to measure the association between the dose of inhaled tobramycin and 
measures of alpha diversity.  
 
Lastly, hypothesis generating analyses will assess changes in bacterial CFU/mL (on the 
log10 scale) as a function of: (1) the GNR organism cultured from the tracheal aspirate, (2) 
pre-trial airway cytokine levels, (3) pre-trial RSS, and (4) pre-trial pulmonary mechanics 
measurements. These data will help inform the selection of pre-specified subgroups group 
analyses that will be performed in future randomized trials.  
 
6.3.4 
Pharmacokinetic analysis 
Patient specific pharmacokinetic parameters will be calculated using log-linear regression 
as the equation: Kel = (ln Cpmax/Cpmin) / t, where Kel is the elimination rate constant from 
plasma, Cpmax and Cpmin and the peak and trough concentrations, respectively, and t is the 
time between samples. Based on this equation, the estimated concentration at the time of 
tracheal aspirate sampling can also be calculated. The correlation between tracheal and 
plasma tobramycin concentrations will then be determined. 
 
Population pharmacokinetic (PK) analysis will be conducted using nonlinear mixed effects 
modeling with the program NONMEM (Icon Inc.). Dosing information and plasma 
tobramycin concentrations will be used to facilitate model development. Mean population 
PK parameters (clearance, volume of distribution) will be estimated using the first-order 
conditional estimation with interaction method. A 2-compartment model with first order 
absorption will be utilized based on prior knowledge of inhaled tobramycin.21 Between 
subject variability and covariate effects (body weight, PMA, renal function, sex) will be 
 


 
  
 
 
 
32 
assessed. Owing to a lack of existing data on the likely plasma tobramycin levels after 
inhalational administration in the target population, it is uncertain whether the sampling 
strategy implemented in this trial will enable formal population PK analysis. Therefore, this 
analysis will be conducted strictly as an exploratory aim. 
 
6.4 
Sample Size and Power 
The traditional 3+3 design is the prevailing method for conducting phase 1 drug trials.103,104 
The sample size for this design is dictated by the number of different dosages investigated 
in the trial and whether or not additional patients are added to individual dosage levels 
based on the occurrence of a toxic drug response. Up to 24 infants are expected to provide 
analyzable trial data as described in section 3.3.2. The purpose of the 3+3 trial design is to 
continue dose escalation (within set boundaries) until at least two patients among a cohort 
of three to six patients experience dose-limiting toxicities (i.e., ≥33% of patients with a dose-
limiting toxicity at that dose level).103,104 If the 300mg dose level is completed in 3 
participants and none show evidence of a toxic response, up to 3 additional infants will be 
enrolled to receive to receive 300mg. The trial will be stopped if 2 of the additional infants 
receiving 300mg develop a toxic response and the 216mg dose will be used as the 
maximum tolerated dose in subsequent trials. 
 
Enrollment in the observational cohort will run concurrent with enrollment in the phase 1 
trial. A minimum of 12 infants and a maximum of 28 infants will be enrolled in the 
observational cohort. This variable enrollment number is selected to avoid delay in study 
closure if enrollment in the trial cohort out paces the observational cohort. Importantly, 
these numbers are anticipated to provide adequate statistical power to measure clinically 
meaningful differences in the frequency of intermittent hypoxemia, an important measure of 
cardiorespiratory stability.96,97 Premature infants with BPD average 75 (SD ± 9) intermittent 
hypoxemic events (SpO2 <80% for 10s-3min) per day during the first several months of 
life.105,106 A sample size of 12 infants in the observational cohort and 12 in the trial cohort 
will provide >80% power (α=0.05) to detect a 15% difference in the mean daily frequency of 
intermittent hypoxemic events between the two groups. A sample size of 24 infants per 
group will provide >80% to detect an 11% difference in in the mean daily frequency of 
intermittent hypoxemic events.  
 
6.5 
Interim Analysis 
The 3+3 study design requires analysis of each study participant’s data in “real time” to 
determine trial progression. After each infant completes the trial, an interim analysis will be 
performed to determine whether to continue the trial at the present dosage and enrollment 
number, expand the number of infants enrolled at the present dosage, advance to the next 
dosage, or end the trial after reaching a maximum tolerated dosage (MTD) or maximum 
planned investigational dosage. 
 
7 
STUDY MEDICATION (TOBRAMYCIN INHALATIONAL SOLUTION, USP) 
7.1 
Description 
Tobramycin inhalational solution, USP is a tobramycin solution for inhalation. It is a sterile, 
clear, slightly yellow, non-pyrogenic, aqueous solution with the pH and salinity adjusted 
specifically for administration by a compressed air driven reusable nebulizer. The structural 
formula is shown in protocol section 1.5. Each single-use 5mL ampule contains 300mg 
tobramycin, USP and 11.25mg sodium chloride in sterile water for injection. Sulfuric acid 
and sodium hydroxide are added to adjust the pH to 6.0. Nitrogen is used for sparging. All 
ingredients meet USP requirements. The formulation contains no preservatives.  


 
  
 
 
 
33 
7.1.1 
Packaging 
Tobramycin inhalation solution comes in a single dose, ready-to-use ampule containing 
300mg tobramycin. Each foil pouch contains 4 ampules. 
  
7.1.2 
Labeling 
The product label for tobramycin inhalational solution, USP (300mg/5mL) is included in 
Appendix C. As the use of tobramycin in this clinical trial is investigational, the FDA label 
requirements per regulations will be affixed to the investigational product. 
 
7.1.3 
Dosing 
This phase 1, dose escalation trial will investigate up to 4 different dosages of tobramycin 
solution for inhalation: 78mg, 150mg, 216mg, and 300mg. Each dose will be administered 
every 12 hours. For administered doses less than 300mg, the investigational pharmacy at 
CHOP will draw-up the appropriate volume from the 300mg/5mL tobramycin inhalational 
solution, USP ampule (i.e., 78mg = 1.3mL; 150mg = 2.5mL; 216mg = 3.6mL) using a single 
3mL syringe (which indicate 0.1mL increments) or 5mL syringe (which indicate 0.2mL 
increments) as appropriate. The measured drug dose will then be diluted with sterile normal 
saline to produce a solution for aerosolization totaling 5mL. This will ensure that the same 
volume of liquid is aerosolized at each dosage level. All reconstituted drug product will be 
stored for not more than 4 hours at room temperature or 24 hours under refrigeration prior 
to administration. 
 
7.1.4 
Drug delivery via vibrating mesh nebulizer 
Each dose of tobramycin solution for inhalation will be administered using the Aerogen ® 
Nebulizer System (Aerogen solo nebulizing chamber and Pro-X Controller/power source). 
This system utilizes a vibrating mesh nebulizer and is the standard equipment used for the 
delivery of aerosolized medications in the CHOP N/IICU. The Aerogen ® Solo nebulizing 
chamber and Pro-X Controller/power source are suitable for use in infants receiving 
invasive mechanical ventilation and FDA approved for use in children >29 days of age (see 
FDA approval information and device manual included in Appendix D, E). All study 
participants will be greater than 29 days of age at study initiation owing to enrollment of 
infants who are <32 weeks gestational age and ≥36 weeks PMA (gestational age + 
chronological age). The Aerogen ® Nebulizer System does not contribute additional flow to 
the ventilator circuit.  
 
Vibrating mesh nebulizers employ a central aperture plate that is perforated with precisely 
formed holes. A piezo ring vibrates the aperture plate which acts as a micropump drawing 
liquid through the holes to generate aerosolized particles with a mass median aerodynamic 
diameter (MMAD) of 1-5µm for a range of different medications.107-110 Achieving particles of 
this size is essential as smaller particles are more prone to removal during exhalation and 
particles with an MMAD >5-10µm are susceptible to “rainout” in the nebulizer/ventilator 
circuitry and/or the large, conducting airways.111-113 
 
Bench studies using a pediatric lung model in a mechanically ventilated patient with an 
endotracheal tube (5mm inner diameter) and a tracheostomy tube (4.4mm inner diameter) 
have assessed the proportion of aerosolized drug delivered distal to the breathing tube 
using the Aerogen ® Solo vibrating mesh nebulizer.114,115 The maximum proportion of 
inhaled drug was achieved using a tracheostomy: 19.8% ± 3.0%, likely owing to its shorter 
length despite modestly small inner diameter.115 Using an endotracheal tube, 13.6% ± 1.3% 


 
  
 
 
 
34 
of the drug was delivered when the bias flow was set to 2L/min and 10.6% ± 0.3% when the 
bias flow was set to 5L/min.114 The ventilators used in the CHOP N/IICU to support infants 
with BPD use a bias flow of either 2.5L/min when set to the pediatric mode (most common 
for the target study population) or 4L/min when set to the neonatal mode. Extrapolating the 
published bench study data114,115 to the mechanical ventilators employed in this phase 1 
trial, without correction for additional loss owing to the smaller endotracheal tubes used in 
the study infants (3.5-4mm), we conservatively estimate that 11-13% of the aerosolized 
drug will reach the respiratory tract of enrolled study subjects. Further loss of drug 
may occur prior to the alveolar/capillary interface owing to the narrow caliber of the airways 
in infants and tighter bronchial branching patterns in small children.116-119 Despite this drug 
loss, the available data indicate the range of tobramycin dosages under investigation in this 
study will yield a dose that results in sufficient antimicrobial levels to significantly reduce the 
respiratory load of the targeted pathogenic organisms. In a study evaluating tobramycin 
levels in the epithelial lining fluid obtained by bronchoalveolar lavage 30-45 minutes after 
treatment with 300mg of inhaled tobramycin administered by jet nebulization to 12 young 
children with CF (mean age 3.3 years, range 0.9 to 5 years), the tobramycin concentration 
was within the target range in 11 or 12 patients, with a mean value of 90 µg/mL.26 This level 
is above the minimum inhibitory concentration of 2-4µg/mL necessary to kill 90% of 
pseudomonas isolates (MIC90) in vitro and the necessary sputum levels of approximately 
50µg/mL needed to effectively eradicate P. aeruginosa in the airway.90,91 Modern vibrating 
mesh nebulizers produce a 2-3 fold higher drug output than do jet nebulizers, suggesting 
similar respiratory drug levels may be reached at the lower drug doses under investigation 
in this study.108,120,121 
 
7.1.5 
Treatment Compliance and Adherence 
The drug dosages will be administered, according to the research protocol, by the clinically 
assigned respiratory therapist and documented in the medical record by the bedside 
nursing staff. The study team will also document each dosage in separate study logs. Drug 
administration will be open label.   
 
7.1.6 
Drug Accountability 
Adequate records of study drug receipt and disposition will be maintained. The CHOP 
Investigational Drug Service (IDS) Records of receipts, investigational drug orders, 
dispensing records, and disposition forms will be examined during the course of the study. 
The purpose of these records is to ensure regulatory authorities that the investigational new 
drug will not be distributed to any person who is not a study subject under the terms and 
conditions set forth in this protocol. The study medication will be prescribed by the 
Investigator or designee and may not be used for any purpose other than that described in 
this protocol.  
 
The CHOP IDS will purchase supplies for the study and partially used and empty containers 
will be discarded at the time of dose preparation to agree with IDS operating procedures. 
 
8 
SAFETY MANAGEMENT 
8.1 
Clinical Adverse Events 
Clinical adverse events (AEs) will be monitored throughout the study. These will constitute 
both the defined tolerability drug endpoints and additional events that are observed 
clinically or by diagnostic testing (imaging, laboratory analyses) throughout the trial and 
pose a risk to the study participant.  


 
  
 
 
 
35 
8.2 
Adverse Event Reporting 
The Primary Investigator will hold responsibility for recording and reporting unanticipated 
problems related to research that occur during and after study treatment. Unanticipated 
problems related to the research involving risks to subjects or others that occur during the 
course of this study (including SAEs) will be reported to the IRB in accordance with CHOP 
IRB SOP 408: Unanticipated Problems Involving Risks to Subjects. AEs that are not serious 
but that are notable and could involve risks to subjects will be summarized in narrative or 
other format and submitted to the IRB at the time of continuing review. Adverse event 
reporting will be targeted, so that potential adverse events arising from the use of inhaled 
tobramycin (for example, inhalational side-effects as well as renal adverse events) will be 
reported, while other adverse effects attributed to the underlying disease or clinical care will 
not be reported. All episodes of noncompliance will be documented. All adverse events 
reported to the CHOP IRB will also be reported in a timely manner to the study DSMB. 
 
8.3 
Definition of an Adverse Event 
An adverse event is any untoward medical occurrence in a subject who has received an 
intervention (drug, biologic, or other intervention). The occurrence does not necessarily 
have to have a causal relationship with the treatment. An AE can therefore be any 
unfavorable or unintended sign (including an abnormal laboratory finding, for example) or 
disease temporally associated with the use of a medicinal product, whether or not it is 
considered related to the medicinal product. 
 
All AEs (including serious AEs) will be noted in the study records and on the case report 
form with a full description including the nature, date and time of onset, determination of 
non-serious versus serious, intensity (mild, moderate, severe), duration, causality, and 
outcome of the event. 
 
The likelihood that the adverse event was related to the study drug or a study procedure will 
be classified using the following scheme: 
 
• 
Unrelated: There is no apparent relationship between the adverse event and a 
study intervention or procedure 
• 
Unlikely: It is unlikely that there could be any causal relationship between the 
adverse event and a study intervention or procedure 
• 
Possibly: It is a possibility that the adverse event is related to a study intervention 
or procedure 
• 
Probably: The adverse event is probably related to a study intervention or 
procedure 
• 
Definitely: The adverse event is definitely related to a study intervention or 
procedure  
 
The severity of an adverse event will be classified using the following scheme: 
• 
Mild: Asymptomatic or mild symptoms; clinical or diagnostic observation only; no 
intervention indicated 
• 
Moderate: Minimal, local, or non-invasive intervention indicated 
• 
Severe: Severe or medically significant, but not immediately life threatening; 
prolongation of hospitalization indicated/required; disabling. 
• 
Life-threatening: Life-threatening consequences; urgent intervention indicated 
 


 
  
 
 
 
36 
8.4 
Definition of a Serious Adverse Event (SAE) 
An SAE is any adverse drug experience occurring at any dose that results in any of the 
following outcomes:  
 
• 
death, 
• 
a life-threatening event (at risk of death at the time of the event),  
• 
prolongation of existing hospitalization beyond what would be expected for a 
preterm infant who requires extended invasive mechanical ventilation near and 
beyond term corrected gestation, or 
• 
a persistent or significant disability/incapacity. 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug event when, based upon 
appropriate medical judgment, they may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed in this definition. 
 
Of note, there is a distinction between serious and severe AEs. A severe AE is a major 
event of its type. A severe AE does not necessarily need to be considered serious. For 
example, nausea which persists for several hours may be considered severe nausea, but 
would not be an SAE. On the other hand, a stroke that results in only a limited degree of 
disability may be considered a mild stroke, but would be an SAE. 
  
8.4.1 
Relationship/Severity of SAE to Study Drug or Other Intervention 
In accordance with CHOP IRB Guidelines, the relationship of each SAE to the study 
intervention and the severity of the SAE will be characterized as described in section 8.3.  
 
8.5 
IRB/IEC Notification of SAEs and Other Unanticipated Problems 
The Investigator will promptly notify the IRB and DSMB of all on-site unanticipated, serious 
Adverse Events that are related to the research activity. Other unanticipated problems 
related to the research involving risk to subjects or others will also be reported promptly.  
 
Written reports will be filed using the eIRB system and in accordance with the timeline 
summarized on page 36. External SAEs that are both unexpected and related to the study 
intervention will be reported promptly after the investigator receives the report. 
 
8.5.1 
Follow-up report 
If an SAE has not resolved at the time of the initial report and new information arises that 
changes the investigator’s assessment of the event, a follow-up report including all relevant 
Type of Unanticipated 
Problem 
Initial Notification  
(Phone, Email, Fax) 
Written Report 
Internal (on-site) SAEs 
Death or Life Threatening  
24 hours 
Within 2 calendar days 
Internal (on-site) SAEs 
All other SAEs 
7 days 
Within 7 business days 
Unanticipated Problems 
Related to Research 
7 days  
Within 7 business days 
All other AEs 
N/A 
Brief Summary of important 
AEs may be reported at time 
of continuing review 


 
  
 
 
 
37 
new or reassessed information (e.g., concomitant medication, medical history) will be 
submitted to the IRB. The investigator will be responsible for ensuring that all SAEs are 
followed until either resolved or stable.  
 
8.6 
Investigator Reporting of a Serious Adverse Event to Sponsor 
Reporting will be consistent with regulatory and protocol-specified requirements. 
 
8.7 
Sponsor Reporting of Serious Adverse Events to FDA 
In the event that a serious adverse event attributed to the investigational drug or its delivery 
occurs, the reporting to the FDA will be by the study Sponsor according to FDA guidance.  
 
8.8 
Medical Emergencies 
All infants enrolled in the study will be hospitalized in the CHOP N/IICU throughout the trial. 
All medical emergencies will be handled by the primary medical team responsible for the 
care of each study participant. The study team will be available for consultation if needed. 
 
9 
STUDY ADMINISTRATION 
9.1 
Treatment Assignment Methods 
Participants in this open label 3+3 phase 1 inter-patient dose escalation trial will be 
assigned to the dosage of inhaled tobramycin based on the order in which they enter the 
study. No randomization or blinding will be employed in this phase 1 trial. 
  
9.2 
Data Collection and Management 
For each subject, the hospital medical record will serve as the primary record (source 
documents). Case report forms will be generated for each subject and maintained in the 
research-focused electronic data capture system REDCap. On occasion, paper case report 
documents may be generated, but will be destroyed as soon as possible after that data has 
been entered into REDCap. See section 9.3 for a description of data confidentiality 
protection measures. All data stored to REDCap is routinely backed-up to ensure 
recoverability in the event of malfunction of the primary server. 
 
9.3 
Confidentiality 
All data and records generated during this study will be kept confidential in accordance with 
Institutional policies and HIPAA on subject privacy. The Investigator and other site 
personnel will not use such data and records for any purpose other than conducting the 
study. Research related records will be stored using the research-focused electronic data 
capture system REDCap. Study data may also be shared via secure email. Only the 
Investigator and other approved study personnel will have access to the password-
protected study database or data shared via secure email. Some data may initially be 
recorded on paper. As soon as possible, that data will be transcribed to the REDCap 
database and the paper copies will be destroyed. If storage of paper documents for any 
period of time is needed, those documents will be kept in locked file cabinets within the 
locked offices of the department of neonatology. Data files will be coded whenever possible 
to ensure that exposure of patient identifiers is kept to the minimum degree necessary to 
carry out the research goals. REDCap includes an automated export mechanism to 
common statistical packages. The export mechanism offers robust de-identification and 
coding options including removal all identifiers. When we export data for analysis, we will 
not include any individually identifiable private information. All study data (including PHI) will 
be maintained for the longer of either (1) 6 years after publication of the study results or (2) 
2 years after the last marketing approval for the investigational agent (study drug) or, if no 


 
  
 
 
 
38 
application is filed or approved, 2 years after the FDA is notified that the IND application is 
formally discontinued. Thereafter, the database with identifiers will be deleted from 
REDCap and only a de-identified version will be retained. All paper copies of case reports, 
if not destroyed during the course of the study, will be destroyed at this time.  
 
No identifiable data will be used for future study without first obtaining IRB approval. The 
investigator will obtain a data use agreement between the provider (the PI) of the data and 
any recipient researchers (including others at CHOP) before sharing a limited dataset. 
 
9.4 
Regulatory and Ethical Considerations 
9.4.1 
Data and Safety Monitoring Plan 
An independent 4-member Data Safety Monitoring Board (DSMB) will be convened to 
monitor for any safety events associated with this study. At the recommendation of the 
NHLBI safety review committee, the DSMB will consist of 2 neonatologists (1 will serve as 
chair), 1 audiologist, and 1 statistician. The DSMB may appoint additional members as they 
require.   
 
Data collection and management with specific focus on ensuring subject confidentiality will 
be carried out as described in section 9. Adverse events will be monitored for and reported 
as described in section 8. 
 
9.4.1.1 Responsibilities of the DSMB 
The DSMB is responsible for safeguarding the interests of study participants, assessing the 
safety and efficacy of study procedures, and for monitoring the overall conduct of the study.  
The DSMB will regularly review study safety data. As this is an open-label phase 1 dose 
escalation trial, all study data will be provided to the DSMB without masking of administered 
drug dose levels. 
 
The DSMB is an independent group advisory to the investigators and affiliated institution, 
and is required to provide recommendations about starting, continuing, and stopping the 
study. The DSMB makes recommendations about: 
• 
Participant safety and risk/benefit ratio of study procedures and interventions, 
including whether new data from other sources affects the study 
• 
Initial approval of the protocol and subsequent amendments (with specific attention 
to study population, intervention, and study procedures) 
• 
Initial approval of consent forms and subsequent amendments 
• 
Study burden on participants 
• 
Recruitment, and retention of participants 
• 
Completeness, quality, and planned analysis of data  
• 
Performance of individual centers and core labs 
• 
Review of plans for incidental findings  
• 
Ancillary study burden on participants and main study 
 
9.4.1.2 Communication plan 
The diagram shown below illustrates the flow of information between the DSMB and other 
entities in this study.   
 


 
  
 
 
 
39 
 
 
Communication with DSMB members will be primarily through the study PI. It is expected 
that study investigators will not communicate about the study with DSMB members outside 
of DSMB meetings. The investigator may contact the DSMB when needed for urgent 
concerns or clarifications of recommendations. 
 
9.4.1.3 Scheduling, timing, and organization of DSMB meetings 
The purpose of the first meeting is to review and discuss this charter, provide an overview 
of study activities, review and make recommendations about the protocol(s), informed 
consent materials, determine the frequency of interim analyses, finalize the DSMB data 
reporting template, and decide whether data will be unmasked to randomized group. 
Enrollment in a study cannot begin until the DSMB’s and IRB’s recommendations for 
approval. 
 
• 
Meetings (in person or by teleconference) will be held 2 times a year, with 
additional meetings scheduled as needed.  
 
• 
Summaries of interim data analyses will be provided to the DSMB chair prior to 
each drug dose escalation. The DSMB chair may choose to convene a full DSMB 
meeting to discuss these findings and place the study on temporary hold as needed. 
  
• 
The DSMB will conclude its operations when all study procedures and planned 
primary data analyses are completed. 
 
The agenda for DSMB meetings and calls may be drafted by the PI and study 
biostatistician, and finalized after consultation with the DSMB Chair. The agenda and 
meeting materials will be distributed by 2 weeks before each meeting. The NHLBI Program 
Office may receive this material at the same time as DSMB members. When the agenda is 
distributed, DSMB members will be asked to report any new conflicts of interest since the 
last DSMB meeting. New conflicts will be reviewed by the Chair and study staff to 
determine if the conflict limits the ability of the DSMB member to participate in the 
discussion; according to the conflict of interest policy at the grantee institution.  
 
The DSMB will review the following data: adverse events and other safety data, quality and 
completeness of study data, quality report(s) of any core(s), and enrollment data at each 
meeting to ensure proper trial conduct. The DSMB may also review formal interim analyses 
of drug tolerability endpoints prior to each planned drug dose escalation.  
The expertise of the attending members should be appropriate for the agenda of the 
meeting. It’s expected that all DSMB members will attend every meeting. The Chair may 
designate replacements for a meeting. For the purposes of voting on recommendations, a 
quorum is 3 members of the Board.   
 


 
  
 
 
 
40 
All standing Monitoring Board members are voting members. The Board may also decide in 
advance whether ad hoc members can vote. 
 
Meetings are organized into open, closed, and executive sessions. 
 
• 
Open session - information is presented to the DSMB by the study investigators, 
with time for discussion.   
 
• 
Closed session - the DSMB, select study staff, and NHLBI program staff (at the 
Chair’s discretion) discuss confidential data (any study data grouped by treatment 
arm), including information on drug dose safety.  
 
The principal investigator and staff involved in subject enrollment and treatment may 
not be present or review grouped data.   
 
• 
Executive session (optional) -- DSMB members, and NHLBI program staff (at the 
Chair’s discretion), may convene to discuss study issues independently.  
 
• 
Recommendations (optional) – Meeting attendees may be reconvened to receive 
the DSMB’s recommendations. 
 
If the executive session occurs on a conference call, steps will be taken to ensure that only 
the appropriate participants are on the call, and to invite others to re-join the call only at the 
conclusion of the executive session. 
 
9.4.1.4 Reports to the DSMB 
For each meeting, the study biostatistician will prepare summary reports and tables to 
facilitate the oversight role of the DSMB. The DSMB will discuss at the first or subsequent 
meetings what data they wish to review and how it should be presented.  
 
9.4.1.5 Statistical monitoring guidelines 
At the first meeting, review of the protocol will include review of the statistical analysis plan. 
The DSMB will discuss the adequacy of that plan. The DSMB will discuss the statistical 
monitoring procedures they propose to follow to guide their recommendations about 
termination or continuation of the trial. These procedures could include guidelines for early 
termination for safety reasons.  
 
9.4.1.6 DSMB recommendations 
Voting on recommendations will follow Roberts’ Rules of Order (Robert's Rules of Order 
Newly Revised (11th Edition) RONR. 
 
Board Recommendations signed by the DSMB Chair, will be sent to the investigators within 
7 calendar days after the meeting. Recommendations should include a statement as to 
whether the study, is approved to continue as planned, should continue with specified 
changes, or should be stopped. Requests for additional data from the investigators or study 
statistician should include an expected due date. In addition to recommendations memos 
issued to investigators (for review and IRB distribution), recommendations related to data 
analysis issues may be issued separately for statisticians. 
 


 
  
 
 
 
41 
Recommendations are distributed by the principal investigator to the NHLBI Program Office 
and the IRB.  
 
9.4.1.7 Additional monitoring 
Before the initiation of the clinical investigation, the sponsor‐investigator will arrange a 
pretrial monitoring visit with the Office of Research Compliance (ORC) to confirm clinical 
trial readiness. After enrolling and starting administration of the investigational agent to the 
first subject, the sponsor‐investigator will contact ORC to arrange a monitoring visit. 
Thereafter, ORC will monitor the study at least annually. Monitoring activities will be guided 
by ICH E6 section 5.18. Interim monitoring activities will include 100% review of regulatory 
files as well as a percentage of enrolled subject records, source documents, applicable 
informed consent forms, and case report forms. The percentage of subject data review may 
be amended throughout the course of the study based on an ongoing risk assessment. A 
tapered approach to monitoring may be employed, if conduct and documentation of the 
study reaches a level of reliability that would permit valid conclusions based upon a 
sampling of data. 
 
9.4.2 
Risk Assessment 
The risks of participation in the phase 1 trial are greater than minimal. The primary medical 
risk to subjects relates to the administration of inhaled tobramycin. The anticipated potential 
risks associated with this medication are described in section 1.6.2. There may also be 
other unforeseen risks in this patient population. Robust monitoring of adverse events, 
including serial measurement of serum tobramycin trough levels, will be utilized to mitigate 
risk to the study participants. Any infant with a serum tobramycin trough level equal to or 
above the currently accepted standard of 2µg/mL, or any evidence of significant, new onset 
end-organ injury as measured by laboratory testing or clinical examination will be removed 
from the trial and no further doses will be administered. 
 
Blood draws for research purposes may also pose a risk to the study participants. Infants 
with BPD receiving care in the CHOP N/IICU typically undergo multiple blood draws per 
week. Study team members will make every effort to coordinate with the medical team such 
that study blood collections might occur concurrent with other clinically indicated blood 
draws. All blood draws will be performed by nursing or phlebotomy staff according to unit 
guidelines, either via heel stick, venipuncture, or indwelling catheter. Phlebotomy may 
cause some pain, bleeding, or bruising. Rarely, taking blood may cause infection. To 
ensure collection of the necessary volume of blood for this research (up to 1.7mL per draw) 
does not incur risk to the patient, study team members, in consultation with nursing staff, 
will ensure that volume of blood extraction (for clinical + research purposes) does not 
exceed the maximum allowable weight-based limits specified by local and NIH guidelines 
(no more than 5 mL/kg drawn in a single day). In the event that excess blood draw is 
possible, the research team will coordinate with the medical team to prioritize laboratory 
testing to ensure patient safety. 
 
Suctioning of the endotracheal tube is not anticipated to incur additional risk to the study 
participants as this is a routine clinical procedure for infants receiving invasive mechanical 
ventilation in the CHOP N/IICU. Infants receiving invasive mechanical ventilation undergo 
ET tube suctioning as part of routine clinical care several times throughout the day. 
Whenever possible, study staff will coordinate with nursing and respiratory therapy to 
collect ET aspirate samples at the time of planned clinical suctioning. ET suctioning 


 
  
 
 
 
42 
involves interruption of mechanical ventilation for ≤5 seconds, equivalent to the interval 
between 1-2 breaths for many infants. 
 
Placement of the in-line sensor for the Philips Respironics NM3 respiratory parameter 
monitor requires brief (≤5 seconds) interruption of mechanical ventilation. Mechanical 
ventilation is briefly interrupted in infants with BPD on a daily basis as part of routine clinical 
care for reasons such as suctioning of the endotracheal tube, repositioning of the infant, 
and replacement or maintenance of the ventilator equipment. These brief interruptions are 
well tolerated in most infants. 
 
Pulse oximetry is part of routine clinical care. For this study, an additional research monitor 
will be used. The adhesive used can on rare occasions result in minor skin irritation.  
 
The other risk to participation in this study is breach of subject confidentiality. The study will 
employ extensive data security measures (see sections 9.2 and 9.3) to protect participant 
privacy. 
 
9.4.3 
Potential Benefits of Trial Participation 
Colonization/infection of the lungs with pathogenic GNR organisms in preterm infants with 
BPD, and in multiple other pediatric and adult respiratory conditions, is associated with 
respiratory morbidity and increased mortality.10,11,16,18 Inhaled tobramycin has shown benefit 
from improving respiratory function and clinical outcomes in children CF and airway 
colonization with P. aeruginosa (the most common GNR detected in the airways of infants 
receiving invasive mechanical ventilation for BPD).10,16,28 Based on these data, we 
anticipate that inhaled tobramycin may provide direct benefit to preterm infants with BPD 
who have a tracheal aspirate culture positive for a pathogenic GNR. Society may also 
benefit from the knowledge gained from this trial. This study will provide the first trial data 
on the safety, tolerability, and potential efficacy of inhaled tobramycin administered to 
preterm infants with BPD who require prolonged invasive mechanical ventilation. These 
data will inform the design of future phase 2 and 3 trials exploring this medication.  
 
9.4.4 
Risk-Benefit Assessment 
Participants in the phase 1 drug trial will be exposed to greater than minimal risk but with 
the prospect of direct benefit through the expected treatment of GNR bacteria in the lungs. 
Given the many safeguards in place to mitigate the study risks, the potential benefits of 
treatment with inhaled tobramycin outweigh the risks associated with participation in this 
trial. Participation in the observational cohort is expected to be minimal risk. 
 
9.5 
Recruitment Strategy 
All subjects will be recruited from the CHOP N/IICU. Unit census documents will be 
reviewed weekly to identify possible subjects. For subjects likely to be eligible, medical 
records will be further reviewed to assess eligibility (only the number of subjects screened 
for eligibility and no specific subject level data will be recorded prior to informed consent). 
The Investigator or a designated study team member will confirm eligibility with the potential 
subject’s attending physician and request permission to approach the parent(s) or 
guardian(s). If appropriate, the parent(s) or guardian(s) of eligible subjects will be 
approached and details of the study will be discussed. If interested, the parent(s) or 
guardian(s) will be invited to participate. Study personnel will then review the information in 
the consent form, answer any questions, and obtain informed consent. A study team 
physician will obtain consent for participation in the phase 1 trial.  


 
  
 
 
 
43 
Only 2 participants will be allowed in the 14-day phase 1 trial at the same time. A new 
participant will not be enrolled into a higher tobramycin dose level until all participants 
enrolled at the lower dose have completed the 14-day trial or been withdrawn. 
 
Most preterm infants with BPD who require prolonged invasive mechanical ventilation are 
followed by the CHOP Newborn and Infant Chronic Lung Disease (NeoCLD) Program. The 
NeoCLD program is comprised of physicians, respiratory therapists, and nurses who are 
jointly engaged in clinical care and research. Every year 60-70 infants are admitted to 
CHOP and cared for by the NeoCLD program. Another 15-20 admitted to the CHOP N/IICU 
require prolonged mechanical ventilation but do not receive care from the NeoCLD 
program. Together, these admission rates indicate up to 75-90 infants per year will be 
eligible to undergo a screening tracheal aspirate culture. During the past 2 years, over 60% 
of eligible infants were enrolled and retained in clinical trials conducted in the CHOP 
N/IICU. Based on this rate, we estimate that 30-50 infants will undergo a screening tracheal 
aspirate culture as part of this study each year. Our prior data show that 54-60% of infants 
in the BPD population at CHOP have tracheal aspirate cultures positive for pathogenic 
GNR bacteria. Accordingly, we expect to enroll 15-30 infants per year into the phase 1 trial 
or observational cohort. Based on these numbers we anticipate this study will require up to 
3 years to complete. If all enrolled infants successfully complete the 14-day phase 1 trial, 
we anticipate the study will require up to 18 months to complete.  
 
9.6 
Informed Consent/Assent and HIPAA Authorization 
After determining eligibility and obtaining permission from the attending physician, a study 
team member will contact a potential participant’s parent(s) or guardian(s) to obtain 
informed consent and HIPAA authorization. This study will obtain informed consent in 2 
phases. First, consent will be obtained to collect a screening tracheal aspirate culture to 
determine eligibility to enroll in the phase 1 trial of inhaled tobramycin or the untreated 
observational cohort. Second, for infants whose tracheal aspirate culture is positive for a 
pathogenic GNR organism, consent will be obtained to enroll in either the phase 1 trial or 
observational cohort.  
 
During the initial consent conversation(s), the study team will provide a description of the 
goals and study procedures involved in the phase 1 drug trial and observational cohort and 
the need to obtain a screening tracheal aspirate culture to determine eligibility to participate 
in either study cohort. For families who express interest in potential participation, we will 
obtain consent to collect a research tracheal aspirate sample for culture to determine 
eligibility. Consent to participate in the phase 1 trial or observational cohort will not be 
obtained until after the results of the tracheal aspirate culture are available. Our initial 
consent discussions will be conducted in the N/IICU in a private setting or via the 
telephone. Before collection of a screening tracheal aspirate culture or medical/PHI, the 
screening consent form must be signed by a legally acceptable surrogate and the 
investigator-designated research professional obtaining the consent if consent is obtained 
in person. If consent for screening procedures is obtained via the telephone, the consent 
form will be signed by the investigator-designated research professional obtaining the 
consent and a study team member or clinician witnessing and verifying consent. Parents or 
guardians providing consent via telephone will be provided a copy of the consent form 
(either via email or left at the baby’s bedside as per their preference).  
For those infants whose screening tracheal aspirate culture is positive for a pathogenic 
GNR, a second consent discussion will take place to cover the goals, procedures, risks, 
and benefits of the phase 1 trial of inhaled tobramycin and the observational cohort. This 
conversation will take place in in a private setting in the N/IICU or via telephone. Enrollment 


 
  
 
 
 
44 
in either study cohort will be offered but an infant may only be enrolled in one of the study 
cohorts (phase 1 trial or observational cohort). Consent for the observational cohort will be 
obtained either in person or via telephone, through a waiver of documentation of consent.   
 
Before any study procedures take place, all necessary consent forms must be signed by a 
legally acceptable surrogate and the investigator-designated research professional 
obtaining the consent.  
 
Consent to participate in the phase 1 drug trial will be obtained by a physician member of 
the study team. Consent for the phase 1 drug trial will be obtained either through written 
consent or through electronic consent (also known as eConsent). Electronic consent will 
utilize the REDCap survey method. The 21 Code of Federal Regulations (CFR) Part 11 
compliant version of REDCap will be used to obtain eConsent, and all associated 
regulations will be followed. If using the REDCap survey, the research team will email the 
eConsent to the parents. We will follow up with a telephone call or video conferencing to 
review the details of the trial and allow for all questions to be answered. Parents will then 
sign the eConsent, which is returned electronically back to the study team. PHI needed for 
emailing and returning the signed eConsent is only available in the PDF and not in the body 
of the email or the email subject header, thus maintaining subject confidentiality. REDCap’s 
eConsent automatically returns a signed copy to the parent if he/she agrees to participate.  
 
Parents/guardians with limited English proficiency will consent using the short form process. 
Short forms in the preferred language will be obtained from the CHOP IRB website. The 
Study Summary document will be signed by the study team member obtaining consent and 
the witness/interpreter. The short form document will be signed by the parent/guardian and 
the witness/interpreter. Consent will only occur in person with the study team, 
parent/guardian, and interpreter all present.  
 
Whenever feasible, consent conversations will take place in person. At the conclusion of all 
consent discussions, parent(s) or guardian(s) will be provided the opportunity to ask 
questions about the study and to discuss the study with their family, friends, and/or other 
medical professionals. As there are no negative repercussions for lack of participation, we 
do not anticipate a significant risk of coercion or undue influence to participate. 
 
9.6.1 
Waiver of Assent 
A waiver of assent is requested as all study participants are infants and incapable of 
providing assent under any circumstances.  
 
9.7 
Payment to Subjects/Families 
Subjects and/or families will not receive financial compensation for participation in this 
study. 
 
10 PUBLICATION 
Following completion of this study, results and analyses will be presented at research 
meetings and published in in scholarly journals. 
 
 


 
  
 
 
 
45 
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52 
APPENDIX 
 
Appendix A: List of nephrotoxic medications122 
 
Acyclovir  
Ambisome  
Amikacin  
Captopril  
Carboplatin  
Cefotaxime  
Ceftazidime  
Cefuroxime  
Cidofovir  
Cisplatin  
Colistimethate  
Cyclosporine  
Dapsone  
Enalapril 
Foscarnet  
Gadopentetate 
dimeglumine  
Ganciclovir  
Gentamicin  
Ibuprofen  
Ifosfamide  
Iodixanol  
Iohexol  
Iopamidol   
Ioversol  
Ketorolac  
Lisinopril  
Lithium 
Mesalamine  
Methotrexate  
Nafcillin  
Piperacillin  
Sirolimus  
Sulfasalazine  
Tacrolimus  
Ticarcillin/clavulanic acid  
Tobramycin  
Topiramate  
Valacyclovir  
Valganciclovir  
Vancomycin  
Zonisamide 
 
 
 
 


 
  
 
 
 
2
Appendix B: List of neuromuscular blocking agents123 
 
Altracurium 
 
Cisatracurium 
 
Doxacurium 
 
Metrocurine 
 
Mivacurium 
 
Pancuronium 
 
Pipercuronium 
 
Rapacuronium 
 
Rocuronium 
 
Succinylcholine 
 
Tubocurarine 
 
Vecuronium 
 
 
 


 
  
 
 
 
3
Appendix C: Inhaled tobramycin label 
 
Appendix D: Aerogen ® Solo FDA approval letter 
 
Appendix E: Aerogen ® Solo System Instruction Manual