Clinical trial NCT03549507
Conditions: Necrotizing Enterocolitis of Newborn Bowel Ischemic
Keywords: Ultrasound Contrast-enhanced ultrasound
Sponsor: Children's Hospital of Philadelphia
Start date: 2020-11-04; Completion date: 2024-05-07
Enrollment: 1 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
There is no bedside imaging technique that can quantify dynamic bowel perfusion with high soft tissue contrast and sensitivity in necrotizing enterocolitis (NEC). Our goal is to assess the feasibility of utilizing contrast-enhanced ultrasound (CEUS) in bedside monitoring of bowel perfusion in NEC. Patients with suspected or diagnosed NEC will be recruited for the study. Following parental consent, the subject will undergo CEUS, performed separately from any clinically indicated conventional US, in the ICU. Subjects will be scanned with CEUS at two different time-points (at the time NEC is first suspected or diagnosed and at time of MRI scan). The CEUS scans will be interpreted by the sponsor-investigator.

The study will be conducted at one site, The Children's Hospital of Philadelphia. It is expected that up to 100 subjects will be enrolled per year, for up to two years, for a total enrollment of up to 200 subjects.

Detailed description:
Early detection of ischemia and necrotic bowel which leads to perforation is vital in improving morbidity and mortality associated with NEC. Abdominal radiography, the standard imaging algorithm for monitoring of NEC, has a low sensitivity of 40% in the diagnosis of severe NEC with necrotic bowel. There is a dire need to introduce better imaging tools such as CEUS to the clinical setting that can detect NEC at an early stage and prompt therapeutic implementation. CEUS enables safe, serial monitoring of dynamic quantification of bowel perfusion at the bedside.

Sulfur hexafluoride lipid-type A microspheres (LumasonTM, Bracco Inc) is an FDA-approved ultrasound contrast agent. The study duration per subject will be approximately 15 minutes including the time to prepare LumasonTM contrast agent and perform the CEUS, as well as the 60 minute monitoring period after the first and second injection of LumasonTM. CEUS will be performed at the time of suspected or diagnosed NEC, and at the time of surgery (for subjects undergoing surgery as part of clinical care) or at short-term follow-up of clinical condition (approximately 1 week after the first scan) for a total of two CEUS exams of 1 hour and 15-minute duration each.

Injection of LumasonTM contrast agent will be performed via the existing peripheral intravenous line or central line using the FDA-recommended dose of up to 0.03 mg/kg. Contrast-agent injection will be performed twice per CEUS scan to ensure image quality and test reproducibility. In the case of more stable patients without an IV line, a peripheral IV line will be started to conduct the investigational CEUS. Two bolus injections will performed to evaluate for dynamic bowel perfusion and several 2-minute cine clips as well as static images will be acquired during the exam.

Qualitative analysis with visual assessment and quantitative analysis of the acquired CEUS scans will be performed and interpreted by the PI. The scans will be assessed for diagnostic quality of images, artifacts encountered, and the presence of additional contributory diagnostic information.

Arms: arm 1: Intravenous administration of contrast agent Sulfur hexafluoride lipid-type A microspheres before performing contrast-enhanced ultrasound (CEUS). In pediatric patients, after reconstitution 0.03 mL per kg is administered intravenously. The weight-based dose of 0.03 mL per kg will be repeated one time during a single examination. Following each injection, an intravenous flush of 0.9% Sodium Chloride is injected. The study duration per subject will be approximately 15 minutes including the time to prepare the contrast agent and perform the CEUS, as well as the 60 minute monitoring period after the first and second injection (if there are two injections of contrast) of the contrast agent.
Interventions: intervention 1: Sulfur hexafluoride lipid-type A microspheres
Intervention descriptions: intervention 1: Injection of Sulfur hexafluoride lipid-type A microspheres (Lumason) contrast agent will be performed via the existing peripheral intravenous line using the FDA-recommended dose of 0.03 mg/kg. Two bolus injections will be performed to evaluate for dynamic bowel perfusion and several 2-minute cine clips as well as static images will be acquired during the exam.

Protocol documents:
Prot_SAP_000.pdf: 
Title: 
Evaluation of Bowel Perfusion with Contrast-Enhanced 
Ultrasound in Necrotizing Enterocolitis 
Short Title 
Contrast-Enhanced Ultrasound Evaluation of Bowel 
Perfusion in Necrotizing Enterocolitis 
 
 
 
 
Drug Name(s): 
Sulfur hexafluoride lipid-type A microspheres (LumasonTM) 
FDA IND 
137874 
Regulatory Sponsor: 
Misun Hwang, MD 
eIRB Number 
IRB 18-014910  
Protocol Date: 
January 10, 2018 
Amendment 1 Date: 27 April 2018 
Amendment 3 Date: 11 March 2019 
Amendment 2 Date: 27 November 2018 
Amendment 4 Date: 10 October 2019 
Amendment 5 Date: 15 April 2020              
Amendment 7 Date: 7 February 2022 
 
Amendment 9 Date: 31 October 2023 
Amendment 6 Date: 11 January 2021 
Amendment 8 Date: 11 April 2022 
 
 
 
 
 
Sponsor-Investigator 
Misun Hwang, MD 
The Children’s Hospital of Philadelphia 
Department of Radiology 
Philadelphia, PA, 19104 
Phone 267.425.7110 
email: Hwangm@chop.edu  
 


2 
 
 
 
 
 
 
TABLE OF CONTENTS 
 
Table of Contents .......................................................................................................... 2 
Abbreviations and Definitions of Terms ......................... Error! Bookmark not defined. 
Abstract .......................................................................................................................... 6 
ABBREVIATIONS AND DEFINITIONS OF TERMS ....................................................... 5 
Protocol Synopsis ......................................................................................................... 7 
1 
Background Information and Rationale ............................................................. 12 
1.1 
Introduction .................................................................................................................... 12 
1.2 
Name and Description of Investigational Product or Intervention ................................ 13 
1.3 
Compliance Statement.................................................................................................... 13 
2 
STUDY OBJECTIVES ........................................................................................... 14 
2.1 
Primary Objective (or Aim) ........................................................................................... 14 
2.2 
Secondary Objective (or Aim) ....................................................................................... 14 
3 
INVESTIGATIONAL PLAN .................................................................................... 14 
3.1 
General Schema of Study Design................................................................................... 14 
3.1.1 
Screening Phase ...................................................................................................... 14 
3.1.2 
Study Treatment Phase ........................................................................................... 15 
3.2 
Allocation to Treatment Groups and Blinding ............................................................... 15 
3.3 
Duration of Study Participation...................................................................................... 15 
3.4 
Total Number of Study Sites/Total Number of Subjects Projected ............................... 15 
3.5 
Study Population ............................................................................................................ 15 
3.5.1 
Inclusion Criteria .................................................................................................... 15 
3.5.2 
Exclusion Criteria ................................................................................................... 16 
4 
STUDY PROCEDURES ......................................................................................... 16 
4.1 
Screening ........................................................................................................................ 16 
4.2 
Study Treatment Phase ................................................................................................... 16 
4.2.1 
CEUS #1 ................................................................................................................. 18 
4.2.2 
CEUS #2 ................................................................................................................. 18 


3 
 
4.3 
Concomitant Medication ................................................................................................ 18 
4.4 
Rescue Medication Administration ................................................................................ 19 
4.5 
Subject Completion/Withdrawal .................................................................................... 19 
4.5.1 
Early Termination Study Visit ................................................................................ 19 
4.5.2 
Review of medical records from EPIC and/or other sources .................................. 19 
4.5.3 
Review of diagnostic images from the PACS (iSite Radiology or iSite Enterprise)
 
19 
5 
STUDY EVALUATIONS AND MEASUREMENTS ................................................ 19 
6 
STATISTICAL CONSIDERATIONS ...................................................................... 20 
6.1 
Primary Endpoint ........................................................................................................... 20 
6.2 
Secondary Endpoints ...................................................................................................... 21 
6.3 
Statistical Methods ......................................................................................................... 21 
6.3.1 
Baseline Data .......................................................................................................... 21 
6.3.2 
Safety Analysis ....................................................................................................... 21 
6.4 
Sample Size and Power .................................................................................................. 21 
7 
STUDY DRUG ....................................................................................................... 22 
7.1 
Description ..................................................................................................................... 22 
7.2 
Dosing ............................................................................................................................ 22 
7.3 
Investigational Drug Handling and Accountability ....................................................... 23 
8 
SAFETY MANAGEMENT ...................................................................................... 23 
8.1 
Clinical Adverse Events ................................................................................................. 23 
8.2 
Adverse Event Reporting ............................................................................................... 24 
8.3 
Definition of an Adverse Event ...................................................................................... 24 
8.4 
Definition of a Serious Adverse Event (SAE) ............................................................... 24 
8.4.1 
Relationship of SAE to study drug or other intervention ....................................... 25 
8.5 
IRB/IEC Notification of SAEs and Other Unanticipated Problems .............................. 25 
8.5.1 
Follow-up report ..................................................................................................... 26 
8.6 
Notifications of SAEs/IND Safety Reports to the FDA ................................................. 26 
8.7 
Medical Emergencies ..................................................................................................... 26 
8.8 
Study Stopping Rules ..................................................................................................... 26 
9 
STUDY ADMINISTRATION ................................................................................... 27 
9.1 
Data Collection and Management .................................................................................. 27 
9.2 
Confidentiality ................................................................................................................ 27 
9.3 
Data and Safety Monitoring Plan ................................................................................... 27 
9.4 
Regulatory and Ethical Considerations .......................................................................... 28 


4 
 
9.4.1 
Risk Assessment ..................................................................................................... 28 
9.4.2 
Potential Benefits of Trial Participation ................................................................. 29 
9.4.3 
Risk-Benefit Assessment ........................................................................................ 30 
9.5 
Recruitment Strategy ...................................................................................................... 30 
9.6 
Informed Consent and HIPAA Authorization ................................................................ 30 
9.7 
Payment to Subjects/Families ........................................................................................ 30 
10 PUBLICATION ....................................................................................................... 30 
11 REFERENCES....................................................................................................... 30 
 
 
 
 


5 
 
 
ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
 
 
AE 
Category B 
Drug 
 
CEUS 
CFR 
CHOP 
CT 
DSMP 
FDA 
HIPAA 
ICU 
IDS 
IND 
IV 
IRB 
LumasonTM 
 
MRI 
NEC 
ORC 
PI 
Pneumatosis 
intestinalis 
PHI 
SAE 
US 
XR 
PACS 
 
Adverse Event 
Animal reproduction studies have failed to demonstrate a risk to the 
fetus and there are no adequate and well-controlled studies in 
pregnant women 
Contrast-Enhanced Ultrasound 
Code of Federal Regulations 
Children’s Hospital of Philadelphia 
Computed Tomography 
Data Safety Monitoring Plan 
Food and Drug Administration 
Health Insurance Portability and Accountability Act 
Intensive Care Unit 
CHOP Investigational Drug Service 
Investigational New Drug 
Intravenous 
Institutional Review Board 
FDA-approved for contrast-enhanced ultrasound; the investigational 
drug 
Magnetic Resonance Imaging 
Necrotizing Enterocolitis 
The CHOP Office of Research Compliance 
Principal Investigator 
Gas cysts in the intestinal wall, sometimes indicative of necrotizing 
enterocolitis 
Protected Health Information 
Serious Adverse Event 
Conventional Ultrasound Scan 
Abdominal Radiography 
Picture Archiving and Communication System 
 
 


6 
 
ABSTRACT 
 
Context: 
 
There is no bedside imaging technique that can quantify dynamic bowel perfusion with 
high soft tissue contrast and sensitivity. Our goal is to assess the feasibility of utilizing 
contrast-enhanced ultrasound (CEUS) in bedside monitoring of bowel perfusion in 
neonatal necrotizing enterocolitis. 
 
Objectives: 
 
• Primary: Assess bowel perfusion characteristics in necrotizing enterocolitis using 
CEUS. 
• Secondary: Optimize the CEUS scanning technique and quantification methods 
for clinical translation.  
 
Study Design: 
 
Single site, open-label clinical study. 
 
Setting/Participants: 
 
Subjects in the neonatal or pediatric intensive care unit aged 1.5 years or younger with 
suspected or diagnosed necrotizing enterocolitis. 
 
The study will be performed at Children’s Hospital of Philadelphia (CHOP). CHOP will 
be the only site of the study.  
 
Study Interventions and Measures:  
 
Contrast-enhanced ultrasound scan with a duration of approximately 20 minutes. 
Qualitative analysis with visual assessment and quantitative analysis of the acquired 
scans will be performed by the sponsor-investigator. The scans will be assessed for 
diagnostic quality of images, artifacts encountered, and the presence of additional 
contributory diagnostic information.  
 
 


7 
 
PROTOCOL SYNOPSIS 
 
Study Title 
Evaluation of Bowel Perfusion with Contrast-Enhanced 
Ultrasound in Necrotizing Enterocolitis 
Funder 
Children’s Hospital of Philadelphia 
Clinical Phase 
Phase II 
Study Rationale 
Necrotizing enterocolitis (NEC) is a devastating disease of the 
newborn affecting 1-5% of all neonatal Intensive care unit 
(NICU) admissions and up to 10% of neonates under 1500 g 
with mortality rates of 50% or higher depending on severity [1] 
Bowel wall perforation occurs in 12%-31% of patients with 
NEC, increasing the mortality rate from 30% to 64% [2]. 
Therefore, early detection of ischemia and necrotic bowel 
which leads to perforation is vital in improving morbidity and 
mortality associated with NEC. Abdominal radiography, the 
standard imaging algorithm for monitoring of NEC, has a low 
sensitivity of 40% in the diagnosis of severe NEC with 
necrotic bowel [1]. 
 
Currently, there is no bedside tool with high soft tissue 
contrast to accurately monitor and quantify bowel perfusion in 
pediatric patients. Bowel perfusion is altered in developing 
ischemia which is important for early diagnosis and 
intervention in neonatal necrotizing enterocolitis, a disease of 
high mortality and morbidity if diagnosed late. Note that the 
current standard algorithm for diagnosis of necrotizing 
enterocolitis is abdominal radiography, which misses up to 
60% of advanced necrotizing enterocolitis. If the disease is 
missed at an early stage, highly morbid surgeries consist of 
extensive surgical resection of bowel which are not without 
serious potential complications. Abdominal ultrasound can be 
obtained to assess for gross complications of advanced 
necrotizing enterocolitis such as pneumatosis intestinalis or 
complex ascites but its diagnostic sensitivity for early 
necrotizing enterocolitis is limited. As compared to CEUS, 
color Doppler quantification of bowel perfusion offers 
suboptimal soft tissue contrast and inaccuracies related to 
artifact. As compared to CEUS, MRI is not easily obtainable in 
neonates and infants due to cost, support staff, transport 
and/or sedation requirements. CEUS on the other hand 
permits serial monitoring of bowel perfusion at the bedside 
with high soft tissue contrast and at lower cost than MRI. 
Further validation of this technique and standardization of 


8 
 
quantification methods will prove to be of significant clinical 
value 
 
There is a dire need to introduce better imaging tools such as 
contrast-enhanced ultrasound to the clinical setting that can 
detect NEC at an early stage and prompt therapeutic 
implementation. In this regard, contrast-enhanced ultrasound 
(CEUS) enables safe, serial monitoring of dynamic 
quantification of bowel perfusion at the bedside. 
 
Safety of intravenous use of Sulfur hexafluoride lipid-type A 
microspheres was based on evaluation of published literature 
involving use of LumasonTM in over 900 pediatric patients, as 
noted on the 2016 FDA product label. Non-fatal anaphylaxis 
was reported in one pediatric patient, but none in a neonate. 
Animal data of daily intravenous administration of Sulfur 
hexafluoride lipid-type A microspheres to rats (administered 
up to 10 times the recommended maximum human dose) and 
rabbits (administered up to 20 times the recommended 
maximum human dose) for 30 consecutive days and 14 
consecutive days, respectively, resulted in no toxicity to the 
fetus in animal studies, as noted on the 2016 FDA product 
label. Specifically pertaining to the use of Sulfur hexafluoride 
lipid-type A microspheres in bowel imaging in pediatric 
patients, we expect a similar risk of adverse events. For the 
proposed study, the same pediatric dosage, route of 
administration, safety monitoring guidelines, and low 
mechanical index used for contrast ultrasound settings will be 
used. 
 
Study Objective(s) 
Primary  
• 
Assess bowel perfusion characteristics in necrotizing 
enterocolitis using CEUS. 
 
Secondary 
• 
Optimize the CEUS scanning technique and 
quantification methods for clinical translation. 
 
Test Article(s) 
 
Sulfur hexafluoride lipid-type A microspheres (LumasonTM, 
Bracco Inc) is an FDA-approved ultrasound contrast agent 
which consists of active ingredients including Sulfur 
hexafluoride (strength 60.7 mg in 1 mg), 
Distearoylphosphatidylcholine, DL- (strength 0.19 mg in 1 
mg), 1,2-Dipalmitoyl-Sn-Glycero-3-Phospho-(1’-Rac-


9 
 
Glycerol), Sodium Salt (0.19 mg in 1 mg). Inactive ingredients 
include Polyethylene Glycol 4000 (strength 24.56 mg in 1 mg) 
and Palmitic Acid (0.04 mg in 1 mg). The Sulfur hexafluoride 
lipid microspheres are composed of SF6 (molecular weight 
145.9) gas in the core surrounded by an outer shell 
monolayer of phospholipids consisting of 1,2-Distearoyl-sn-
glycero-3-phosphocholine (DSPC) and 1,2-Dipalmitoyl-sn-
glycero-3-phosphoglycerol, sodium salt (DPPG-Na) with 
palmitic acid as stabilizer. Sulfur hexafluoride lipid-type A 
microspheres fall under Category B, that is, animal 
reproduction studies have failed to demonstrate a risk to the 
fetus and there are no adequate and well-controlled studies in 
pregnant women.1,2-Distearoyl-sn-glycero-3-phosphocholine 
(DSPC), with empirical formula C44H88NO8P, has a 
molecular weight of 790.6. 1,2-Dipalmitoyl-sn-glycero-3-
phospho-rac-glycerol sodium (DPPG-Na), with empirical 
formula C38H74 NaO10P, has a molecular weight of 745. 
 
Study Design 
 
Single site, open-label clinical study. 
Subject Population 
key criteria for 
Inclusion and 
Exclusion: 
Inclusion Criteria 
1. Males and females aged 1.5 years or younger 
2. Post menstrual age of 29 weeks or older  
3. Patients with suspected or diagnosed necrotizing 
enterocolitis 
4. Patient in the CHOP NICU or PICU 
5. Parental permission 
Exclusion Criteria 
1. Medical history of Lumason hypersensitivity 
2. Hemodynamic instability as defined by rapid escalation 
of cardiopulmonary support in the past 12-24 hours, as 
defined by the clinical care team including >1 intensive 
care physician not part of the study team 


10 
 
3. Pulmonary insufficiency as defined by FiO2 
requirements of >40% and/or subjects with pulmonary 
hypertension requiring nitric oxide 
 
Number Of Subjects  
 
200; CHOP will be the only site of the study. 
Study Duration 
The study duration per subject will be approximately 20 
minutes including the time to prepare LumasonTM contrast 
agent and perform the pre-contrast imaging and the CEUS, 
as well as the 60 minute monitoring period after the first and 
second injection of LumasonTM. A second CEUS may be 
performed at the time of suspected or diagnosed NEC, and at 
the time of surgery (for subjects undergoing surgery as part of 
clinical care) or at short-term follow-up of clinical condition 
(approximately within 1-2 weeks from the first scan) for a total 
of two CEUS exams of 1 hour and 15-minute duration each.  
 
Study participation will be complete when the 60-minute 
monitoring period after the last CEUS performed is complete 
(after the first CEUS in patients who undergo one exam, or 
after the second CEUS is complete in patients who undergo 
two exams). 
 
Study Treatment 
CEUS has a total duration of 1 hour and 20 minutes: CEUS 
has a duration of approximately 20 minutes for the CEUS and 
60 minutes for post-examination monitoring of potential 
adverse events. 
Efficacy Evaluations 
There are no efficacy evaluations for this diagnostic study. 
The endpoints of this study are: 
 
Primary Endpoint  
• 
The primary objective of this study is to evaluate 
whether CEUS technique can be utilized to detect early 
alterations in bowel perfusion in NEC. The differences in 
bowel perfusion between normal subjects (those with 
suspected or at risk of necrotizing enterocolitis but turn out to 
be normal on imaging and clinical evaluation) versus NEC 
patients will be assessed qualitatively and quantitatively using 
the CEUS technique, as detailed above. 
 


11 
 
Secondary Endpoint 
• 
Diagnostic quality of the CEUS exams will be 
evaluated with the scoring system of non-diagnostic (1), 
moderate artifacts degrading diagnostic quality (2), mild 
artifacts without degradation of diagnostic quality (3), and 
diagnostic (4). 
Pharmacokinetic 
Evaluations 
There are no pharmacokinetic evaluations. 
Safety Evaluations 
All subjects entered into the study and receiving at least one 
injection of investigational drug will be included in the safety 
analysis. The frequencies of AEs by type, severity, and 
temporal relationship to the CEUS scan will be summarized. 
SAEs (if any) will be described in detail. 
Statistical And 
Analytic Plan 
Baseline and demographic characteristics will be summarized 
by standard descriptive summaries. All subjects entered into 
the study and receiving at least one injection of investigational 
drug will be included in the safety analysis. The frequencies of 
AEs by type, severity, and temporal relationship to the CEUS 
scan will be summarized. SAEs (if any) will be described in 
detail. Details of sample size and power calculations for this 
study are described in Section 6 of the protocol. 
DATA AND SAFETY 
MONITORING PLAN 
The safety monitoring for this study is the primary 
responsibility of the sponsor-investigator. Monitoring the 
safety outcomes following IV administration of the 
investigational drug will be conducted primarily by the 
Principal Investigator and/or specifically designated study 
personnel. An independent safety monitor will be designated 
to oversee the safety reports, and help adjudicate attribution 
of serious adverse events, should they occur. Regular 
meetings to discuss the outcomes of the study, and of the 
safety events, will be conducted by the study team. The 
occurrence of adverse events, serious adverse events and 
unanticipated events will be reported by the study team in 
accordance with federal and institutional guidelines, as 
outlined in Section 8 of this clinical study. 
 
Prior to study initiation, the Office of Research Compliance 
(ORC) will conduct a pre-trial monitoring visit, to assess trial 
readiness of the study staff. Once the pre-trial monitoring visit 
has been successfully completed, the ORC will also monitor 
the IND study on at least an annual basis.    
 


12 
 
1 
Background Information and Rationale 
 
1.1 Introduction 
Necrotizing enterocolitis (NEC) is a devastating disease of the newborn affecting 1-5% 
of all neonatal Intensive care unit (NICU) admissions [1] and up to 10% of neonates 
under 1500 g. Bowel wall perforation occurs in 12%-31% of patients with NEC, 
increasing mortality rate from 30% to 64% [2]. Therefore, early detection of ischemia 
and necrotic bowel which leads to perforation is vital in improving morbidity and 
mortality associated with NEC. Despite extensive research and improvements in the 
field of neonatal care, the morbidity and mortality associated with NEC have remained 
unchanged over the last 3 decades [3]. Strategies and sensitive/specific tests for 
predicting and preventing NEC are lacking.  
 
Contrast enhanced ultrasound (CEUS) is a novel technique in pediatrics which has 
proven itself as a safe and effective modality with benefits over conventional ultrasound 
(US) by its improved real-time evaluation of the micro- and macro-vascularity of 
normally and abnormally perfused tissue [4]. CEUS does not depend on ionizing 
radiation, can be performed bedside and the ultrasound contrast agent used is not 
associated with renal toxicity. There have been no studies on the use of CEUS to 
evaluate NEC.  Despite the favorable safety profile and diagnostic benefits, the paucity 
of CEUS studies for NEC in the U.S. resulted from the off-label usage of this novel 
contrast agent. 
 
Abdominal radiography (XR) and computed tomography (CT) have been the diagnostic 
gold standard for NEC but are far from flawless [5, 6]. Pneumoperitoneum is the only 
radiologic sign that has been universally agreed upon as an indication for surgical 
intervention (i.e. laparotomy or peritoneal drainage) for patients with NEC, however not 
all neonates with bowel necrosis and perforation have free air on XR [1, 7]. Bowel 
dilation is a nonspecific finding in NEC that remains best appreciated on XR and is 
sometimes the only sign present in NEC [8]. Additional findings suggestive of NEC on 
XR include intramural air and portal venous air. Abdominal XR remains a necessary, 
primary imaging modality for the evaluation of NEC. However, signs of NEC on XR may 
be subtle, can easily be missed, especially in the early stages of NEC and consequently 
provide limited information that could lead to clinical interventions that may prevent or 
predict the onset of NEC [6]. 
 
Conventional abdominal ultrasound (US) has been implemented as a useful adjunct to 
XR for the early detection of intramural air, bowel wall edema, decreased peristalsis, 
and decreased or absent perfusion that suggests bowel wall ischemia/necrosis. 
Furthermore, hyperechogenic air bubbles may be seen within the diseased bowel wall. 
US may show pathology before XR becomes abnormal. Near-infrared spectroscopy 
(NIRS) is another non-invasive technique that has shown promise in improving early 
detection of NEC but suffers from limited depth penetration and poor soft tissue contrast 
[6]. CT and magnetic resonance imaging (MRI) have a very limited role in the diagnostic 
work up of NEC because the critically sick children have to be transported to either the 
CT or MRI suite. Furthermore, CT uses ionizing radiation which should be avoided 


13 
 
when possible in these very young, radiation sensitive neonates and MRI studies suffer 
from the longer acquisition times and image degradation due to the susceptibility 
artifacts from the air inclusions and motion. The use of contrast enhanced ultrasound is 
an exciting new modality that shows promise in early and reliable prediction of NEC. 
These imaging modalities all leave something to be desired when it comes to the 
evaluation, prediction and prevention of NEC. 
 
In summary, the current imaging algorithm is not sensitive enough to detect early NEC 
and prevent high mortality and morbidity associated with the disease. There is therefore 
a dire need to introduce better imaging tools to the clinical setting that can detect NEC 
at early stage and prompt therapeutic implementation. 
 
1.2 Name and Description of Investigational Product or Intervention 
Sulfur hexafluoride lipid-type A microspheres (LumasonTM, Bracco Inc) is FDA-approved 
ultrasound contrast agent which consist of active ingredients including Sulfur 
hexafluoride (strength 60.7 mg in 1 mg), Distearoylphosphatidylcholine, DL- (strength 
0.19 mg in 1 mg), 1,2-Dipalmitoyl-Sn-Glycero-3-Phospho-(1’-Rac-Glycerol), Sodium 
Salt (0.19 mg in 1 mg). Inactive ingredients include Polyethylene Glycol 4000 (strength 
24.56 mg in 1 mg) and Palmitic Acid (0.04 mg in 1 mg). The sulfur hexafluoride lipid 
microspheres are composed of SF6 (molecular weight 145.9) gas in the core 
surrounded by an outer shell monolayer of phospholipids consisting of 1,2-Distearoyl-
sn-glycero-3-phosphocholine (DSPC) and 1,2-Dipalmitoyl-sn-glycero-3-
phosphoglycerol, sodium salt (DPPG-Na) with palmitic acid as stabilizer. Sulfur 
hexafluoride lipid-type A microspheres fall under Category B, that is, animal 
reproduction studies have failed to demonstrate a risk to the fetus and there are no 
adequate and well-controlled studies in pregnant women.1,2-Distearoyl-sn-glycero-3-
phosphocholine (DSPC), with empirical formula C44H88NO8P, has a molecular weight 
of 790.6. 1,2-Dipalmitoyl-sn-glycero-3-phospho-rac-glycerol sodium (DPPG-Na), with 
empirical formula C38H74 NaO10P, has a molecular weight of 745. In pediatric 
patients, after reconstitution 0.03 mL per kg is administered intravenously. The weight-
based dose of 0.03 mL per kg will be repeated twice during a single examination. 
Following each injection, an intravenous flush of 0.9% Sodium Chloride is injected.  
 
1.3  Compliance Statement 
This study will be conducted in full accordance of all applicable Children’s Hospital of 
Philadelphia Research Policies and Procedures and all applicable federal and state 
laws and regulations including 45 CFR 46. All episodes of noncompliance will be 
documented. Research will also be conducted in full accordance with Food and Drug 
Administration (FDA) regulations 21 CFR 50 (Protection of Human Subjects), 21 CFR 
56 (Institutional Review Boards) and 21 CFR 312 (Investigational New Drug). 
 
The investigators will perform the study in accordance with this protocol, will obtain 
consent, and will report unanticipated problems involving risks to subjects or others in 
accordance with The Children’s Hospital of Philadelphia IRB Policies and Procedures 
and all federal requirements. Collection, recording, and reporting of data will be 


14 
 
accurate and will ensure the privacy, health, and welfare of research subjects during 
and after the study. 
2 
STUDY OBJECTIVES 
 
The purpose of the study is to evaluate bowel perfusion in neonatal necrotizing 
enterocolitis using the CEUS technique and optimize the technique for clinical use.  
 
2.1 Primary Objective (or Aim) 
The primary objective of this study is to evaluate the altered bowel perfusion in neonatal 
necrotizing enterocolitis using the CEUS technique. 
 
2.2 Secondary Objective (or Aim) 
The secondary objective is to optimize and standardize the CEUS scanning technique 
and quantification method.  
3 
INVESTIGATIONAL PLAN 
 
3.1 General Schema of Study Design 
Patients with suspected or diagnosed necrotizing enterocolitis will be recruited for the 
study. Following parental consent, the subject will undergo an investigational CEUS 
exam, which will be performed separately from any clinically indicated conventional 
ultrasound. CEUS duration of approximately 20 minutes, followed by 60 minutes of 
monitoring. A second CEUS may be performed at the time of surgery (for subjects 
undergoing surgery as part of clinical care) or at short-term follow-up of clinical condition 
(approximately within 1-2 weeks from the first scan) for a total of two CEUS exams of 1 
hour and 20-minute duration each. The CEUS exam includes a pre-contrast ultrasound 
evaluation with FDA-approved technologies (e.g. gray-scale ultrasound, Doppler 
ultrasound, microvascular imaging). 
 
3.1.1 Screening Phase 
Potential subjects will be identified by: 1) Identification by neonatologists of patients 
requiring abdominal ultrasound during the neonatal or pediatric intensive care unit stay 
for suspected, at risk of or diagnosed necrotizing enterocolitis. 
 
Before discussing participation in the study, we will confirm eligibility by reviewing the 
subjects’ medical records. Participation will be discussed between the PI and the 
referring neonatologist. Participation will be discussed with the parents/guardian by the 
neonatologist and/or radiologist after identification and confirmation of eligibility. 
Consent of the parents/guardian will be obtained prior to the exam by the PI, co-
investigator, or neonatologist co-investigator in a private setting. Questions will be 
answered by the PI, co-investigator, or referring neonatologist co-investigator.  
 


15 
 
3.1.2 Study Treatment Phase 
Investigational CEUS scan will be performed separately from any clinically indicated 
conventional US, in the ICU. A second CEUS may be performed at the time of 
suspected or diagnosed NEC, and at the time of surgery (for subjects undergoing 
surgery as part of clinical care) or at short-term follow-up of clinical condition 
(approximately within 1-2 weeks from the first scan) for a total of two CEUS exams of 1 
hour and 20-minute duration each. Injection of LumasonTM contrast agent will be 
performed via the existing peripheral intravenous line or central line using the FDA-
recommended dose of up to 0.03 mg/kg. Contrast-agent injection will be performed 
twice per CEUS scan to ensure image quality and test reproducibility. In the case of 
more stable patients without an IV line, a peripheral IV line will be started to conduct the 
investigational CEUS. Two bolus injections will performed to evaluate for dynamic bowel 
perfusion and several 2-minute cine clips as well as static images will be acquired 
during the exam. 
 
3.2 Allocation to Treatment Groups and Blinding 
Not applicable. The CEUS scan will be interpreted by the sponsor-investigator only.  
 
3.3 Duration of Study Participation 
The study duration per subject will be approximately 20 minutes including the time to 
prepare LumasonTM contrast agent, perform the pre-contrast imaging, and perform the 
CEUS, as well as the 60 minute monitoring period after the first and second injection of 
LumasonTM. CEUS will be performed at the time of suspected or diagnosed NEC, a 
second CEUS may be performed, and at the time of surgery (for subjects undergoing 
surgery as part of clinical care) or at short-term follow-up of clinical condition 
(approximately within 1-2 weeks from the first scan) for a total of two CEUS exams of 1 
hour and 20-minute duration each. 
 
Study participation will be complete when the 60 minute monitoring period after the last 
CEUS performed is complete (after the first CEUS in patients who undergo one exam, 
or after the second CEUS is complete in patients who undergo two exams). 
 
3.4 Total Number of Study Sites/Total Number of Subjects Projected 
The study will be conducted at one site, The Children’s Hospital of Philadelphia. It is 
expected 200 subjects will be enrolled to produce 100 evaluable subjects.  
 
3.5 Study Population 
3.5.1 Inclusion Criteria 
1. Males and females aged 1.5 years or younger 
2. Post menstrual age of 29 weeks or older  


16 
 
3. Patients with suspected or diagnosed necrotizing enterocolitis 
4. Patient in the CHOP NICU or PICU 
5. Parental permission 
3.5.2 Exclusion Criteria 
1. Medical history of Lumason hypersensitivity 
2. Hemodynamic instability as defined by rapid escalation of 
cardiopulmonary support in the past 12-24 hours, as defined by the clinical 
care team including >1 intensive care physician not part of the study team 
3. Pulmonary insufficiency as defined by FiO2 requirements of >40% and/or 
subjects with pulmonary hypertension requiring nitric oxide 
In this regard, there is a published report of infusion of the investigational drug in twelve 
neonates, ranging from 26.9 to 41 weeks gestational age, including four premature 
infants from 26.9 to 29.5 weeks gestational age [9] as well as 3 additional premature 
subjects from unpublished data of the sponsor. The infusion of the investigational drug 
in these neonates and premature infants was safe, and imaging results of good quality. 
 
Subjects who do not meet all of the enrollment criteria may not be enrolled. Subjects will 
be excluded from the study, if in the judgement of the primary clinical team, they are too 
unstable to tolerate the procedure.  
 
Any violations of these criteria must be reported in accordance with IRB Policies and 
Procedures.  
4 
STUDY PROCEDURES 
 
4.1 Screening 
• 
Identify patients scheduled for abdominal ultrasound for suspected or 
diagnosed necrotizing enterocolitis  
• 
Review of medical records (for inclusion and exclusion criteria) 
• 
Discussion of the case with neonatologists and study team to determine 
eligibility 
• 
If subject is considered eligible, written or electronic consent can be obtained 
at this stage by PI, co-investigator, or neonatologist co-investigator  
• 
Coordinate abdominal ultrasound schedule 
 
4.2 Study Treatment Phase 


17 
 
Subjects enrolled to this clinical trial are anticipated to receive the study drug and have 
CEUS imaging conducted in the ICU setting. In this case, the study drug and US 
imaging unit will be brought to the ICU for study drug administration and imaging. Any 
clinically indicated imaging must be performed prior to contrast-enhanced abdominal 
ultrasound so as not to delay clinical care. 
 
Pre-injection documentation 
Prior to injection, vital signs and baseline assessment will be recorded and documented 
from the medical record and clinically in-place monitoring. Baseline assessment will 
consist on neurological status as described in the clinical evaluations recorded in the 
medical chart prior to intervention.  
 
Pre-injection evaluation 
The exam will include a pre-contrast injection evaluation with FDA-approved 
technologies (e.g. gray-scale ultrasound, Doppler ultrasound, microvascular imaging) to 
assess anatomical structures and guide contrast evaluation. This will last approximately 
5 minutes.   
 
CEUS scan 
CEUS duration is of approximately 20 minutes. The CEUS examination will be 
terminated if there is a deterioration in the subject’s clinical status during imaging.  
 
60-minute monitoring period 
The study team, who are composed of personnel trained in recognizing signs of infusion 
reaction, will conduct the 60-minute monitoring period and record any untoward reaction 
that may be related to the infusion of the contrast drug. The 60 minute monitoring post-
CEUS will be conducted by study team members. 
 
Vital signs will be recorded and documented from the medical record and clinically in-
place monitoring at 1) 30 minutes post-scanning, and 2) 60 minutes post-scanning. 
During monitoring, subjects will be assessed for rash, allergic reactions, anaphylaxis, 
and abrupt deviations from the subject's baseline hemodynamic parameters trend not 
related to medical intervention. 
 
Adverse event assessment and documentation 
Adverse events will be recorded at 1) 60 minutes post-scanning when the monitoring 
period is completed, and 2) through 48 hours post-scanning, with documentation at 48 
hours post-scanning if no adverse event presents until this point. During the 48-hour AE 
assessment period, the following AEs of special interest will also be assessed: 
a. 
worsening cardiopulmonary status,  
b. 
worsening pulmonary hypertension, which may be suggested by new 
requirement of nitric oxide use, elevated pulmonary artery pressures on 
echocardiography, or differential limb pulse oximetry measurements,  
c. 
worsening neurological status, and 
d. 
serious gastrointestinal complications 
 


18 
 
4.2.1 CEUS #1 
• 
Obtain consent prior to scheduled CEUS exam (if not previously obtained by, 
PI, co-investigator, or neonatologist co-investigator) 
• 
Record vital signs and baseline assessment prior to injection of contrast 
• 
Perform pre-contrast injection evaluation. 
 
• 
CEUS scan performed, at the time of suspected or diagnosed necrotizing 
enterocolitis 
• 
Monitor patients for 60 minutes following the CEUS scan for documentation 
and treatment of potential adverse events 
• 
Documentation of any adverse events through 48 hours post-scanning 
 
Subjects may undergo surgery as part of clinical care. A second CEUS may be 
performed at the time of surgery for subjects undergoing surgery as part of clinical care, 
or at short-term follow-up of clinical condition (approximately within 1-2 weeks after the 
first scan) for a total of two CEUS exams of 1 hour and 20-minute duration each. 
 
4.2.2 CEUS #2 
• 
Record vital signs and baseline assessment prior to injection of contrast 
• 
Perform pre-contrast injection evaluation. 
 
• 
CEUS scan performed at the time of surgery for subjects undergoing surgery 
as part of clinical care, or at short-term follow-up of clinical condition 
(approximately within 1-2 weeks from  the first scan)  
• 
Monitor patients for 60 minutes following the scan for documentation and 
treatment of potential adverse events 
• 
Documentation of any adverse events through 48 hours post-scanning 
 
Study participation will be complete when the 60-minute monitoring period of the last 
CEUS performed is complete (after the first CEUS in patients who undergo one exam or 
after the second CEUS is complete in patients who undergo two exams). 
 
The study team will not use sedation or general anesthesia to conduct the research 
CEUS scans. 
 
The results of the CEUS imaging will be collected for research purposes only. The 
results of the CEUS will not be used to direct clinical care decisions, without 
confirmation of diagnosis by another medically established diagnostic product or 
procedure. 
 
4.3 Concomitant Medication 
No concomitant medications will be recorded, with the exception of rescue medications, 
as noted below. 
 


19 
 
4.4 Rescue Medication Administration 
All the rapid response equipment and resuscitation staff are readily available 24/7 in the 
intensive care unit setting at Children’s Hospital of Philadelphia. In the rare event that a 
significant allergic reaction occurs, or anaphylaxis results following injection of 
LumasonTM, standard clinical medication to treat the subject will be administered. For 
presumed allergic reactions, medications may include intravenous diphenhydramine 
and bolus corticosteroids (prednisolone), based on clinical care. For anaphylaxis, 
medications may include epinephrine as well as fluid and oxygen administration for 
emergency treatment, based on clinical care decision making and on the severity of 
symptoms. Any severe allergic or anaphylactic reaction will be reported to both the IRB 
and the FDA. 
 
4.5 Subject Completion/Withdrawal 
Subjects’ families may withdraw their child from the study at any time without prejudice 
to their child’s care. A study investigator may withdraw a subject to protect the subject 
for reasons of safety or for administrative reasons. 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, 
the adverse events will be recorded and reported. 
 
4.5.1 Early Termination Study Visit 
A subject may be withdrawn by a parent prior to or during a CEUS scan, provided the 
60 minute monitoring is completed after injection of the investigational drug.  The CEUS 
examination will be terminated if there is a deterioration in the subject’s clinical status 
during imaging. 
 
4.5.2 Review of medical records from EPIC and/or other sources 
• 
Date of birth 
• 
Weight 
• 
Clinical diagnosis 
• 
Treatment history (medications, chemotherapy, antibiotics, steroids)  
• 
Surgical history  
• 
Pathology report 
 
4.5.3 Review of diagnostic images from the PACS (iSite Radiology or iSite 
Enterprise) 
• 
Review of abdominal ultrasound and CEUS images (iSite) 
5 
STUDY EVALUATIONS AND MEASUREMENTS 
 
The results of the pre-contrast injection and CEUS imaging will be collected for research 
purposes only. The results of the CEUS will not be used to direct clinical care decisions, 


20 
 
without confirmation of diagnosis by another medically established diagnostic product or 
procedure. 
 
Qualitative analysis 
Visual rating by 2 teams consisting of primary investigator and second radiologist (co-
investigator). Each scan will be rated for diagnostic quality and qualitative rating of 
bowel perfusion. The visual rating scale used will be: 0 (absent flow), 1 (decreased 
flow), 2 (normal flow), 3 (increased flow).  
 
Quantitative analysis 
Qontrast (Bracco Diag Inc., 510K regulatory status granted in 2004) contrast 
quantification software or similar software will be used to analyze the obtained CEUS 
scans. For each scan, wash-in and wash-out curves will be generated to quantify the 
rate of wash-in, time to peak intensity, peak intensity, and area under the curve. 
 
The CEUS scans will be interpreted by the sponsor-investigator, and a second 
interpretation by a “second reader” in Radiology, who is part of a group of radiologists 
with sufficient training and expertise to read CEUS scans. 
 
Monitoring After Investigational Drug Administration: 
 
The monitoring post-administration will encompass 60 minutes, during which time the 
subject will be observed for the occurrence of infusion reactions. Any concern by the 
investigative team or attending staff for severe allergic reaction or anaphylaxis will be 
managed as all similar reactions are managed as part of clinical care, with close 
observation and treatment as clinically indicated, which may include diphenhydramine, 
corticosteroids, fluids, oxygen and epinephrine. Post-infusion reactions will be recorded 
and reported following established IRB and FDA reporting guidelines. 
6 
STATISTICAL CONSIDERATIONS 
 
Up to 100 evaluable patients should be adequate for assessment of clinical feasibility 
and optimization of protocol. Qualitative analysis will be used as detailed above. 
Quantitative analysis will be performed by drawing a region of interest in bowel 
segments and compared to control subjects (those with suspected or at risk of 
necrotizing enterocolitis but turns out to be normal on imaging and clinical evaluation) 
using non-parametric Wilcoxon testing. 
 
6.1 Primary Endpoint 
The primary objective of this study is to evaluate whether CEUS technique can be 
utilized to detect early alterations in bowel perfusion in NEC. The differences in bowel 
perfusion between normal subjects (those with suspected or at risk of necrotizing 
enterocolitis but turn out to be normal on imaging and clinical evaluation) versus NEC 
patients will be assessed qualitatively and quantitatively using the CEUS technique, as 
detailed above.  
 


21 
 
6.2 Secondary Endpoints 
Diagnostic quality of the CEUS exams will be evaluated with the scoring system of non-
diagnostic (1), moderate artifacts degrading diagnostic quality (2), mild artifacts without 
degradation of diagnostic quality (3), and diagnostic (4). As a gold standard for NEC 
diagnosis, surgical findings (in those subjects undergoing surgery as part of clinical 
care) or Bell’s staging criteria for NEC in combination with radiographic and clinical 
signs (for subjects not undergoing surgery) will be used. 
 
6.3 Statistical Methods 
6.3.1 Baseline Data  
Baseline and demographic characteristics will be summarized by standard descriptive 
summaries. 
 
6.3.2 Safety Analysis 
All subjects entered into the study, who receive at least one dose of investigational drug 
per CEUS scan will be included in the safety analysis. The frequencies of AEs by type, 
severity, and temporal relationship to the CEUS scan will be summarized. SAEs (if any) 
will be described in detail. 
 
Adverse events will be recorded at 1) 60 minutes post-scanning when the monitoring 
period is completed, and 2) through 48 hours post-scanning, with documentation at 48 
hours post-scanning if no adverse event presents until this point. 
 
6.4 Sample Size and Power 
As noted in the main text, no previous study has used contrast ultrasound to examine 
bowel wall perfusion. For power analyses, estimates of group differences were obtained 
from a study that provided mean perfusion values for a control group using Doppler 
ultrasound. Although contrast ultrasound and Doppler ultrasound rely on different 
methods to quantify perfusion (contrast quantifies by absolute intensity and Doppler 
quantifies by counting the color signal) the methods are similar enough that Doppler 
findings can inform the present study (and of note, given that contrast ultrasound is 
more sensitive, power analysis based on the Doppler findings are considered to be 
conservative). Given the reported bowel perfusion mean and standard deviation value 
for controls of 3.78 (SD = 1.1) [1], assuming a moderate effect (Cohen’s D = 0.71), a 
sample size of 40 per group would provide power = 0.88 (alpha = 0.05). Since each 
subject has a clinical care US (control group) followed by a research CEUS 
(experimental group), each subject will serve as its own control. Since the CEUS 
method has not been applied to NEC imaging before, an interim statistical analysis may 
be considered to review image quality and comparisons between the results of each 
subject’s CEUS scans and any available clinical US exams, to better inform the number 
of subjects required for statistical significance.   


22 
 
7 
STUDY DRUG 
 
7.1 Description 
LumasonTM is currently FDA approved for use in the pediatric population for 
echocardiography and evaluation of focal hepatic lesions, and very recently (Jan 2017) 
approved for use in children for the evaluation of the urinary tract in pediatric patients 
with known or suspected vesicoureteral reflux. Previously, the presence of cardiac 
shunts was a contraindication for its use, but this was recently cancelled by the FDA as 
of December 2016. However, for this clinical study, the FDA has recommended that 
subjects with hemodynamically significant PDA and right-to-left cardiac shunts be 
excluded from study participation. Ultrasound contrast agents have been approved for 
use in Europe for almost two decades. In the case of SonoVue (now called LumasonTM), 
a second-generation lipid/sulfur hexafluoride US contrast agent (Bracco, Milan, Italy), 
the European Union approved its intravenous use in adults in 2001. The FDA in the 
United States just recently approved the use of LumasonTM for evaluation of focal 
hepatic lesions in the pediatric population in 2016. Through decades of clinical 
utilization of ultrasound contrast agents, there are established recommended doses for  
the intravenous route of administration. Recommended intravenous dose for 
LumasonTM is weight-based, 0.03mL/kg as an intravenous injection. As an example, for 
the intended study population it may be roughly estimated that the maximum weight of 
an infant 1 year of age may be up to approximately 10 kg, for a maximum dose of 0.30 
mL. Two injections per exam will be performed. 
 
7.2 Dosing 
Through decades of clinical utilization of ultrasound contrast agents, there are 
established recommended doses for the intravenous route of administration. 
Recommended intravenous dose for LumasonTM is weight-based, 0.03mL/kg as an 
intravenous injection, up to a maximum of 2.4mL per injection. As an example, for the 
intended study population it may be roughly estimated that the maximum weight of an 
infant 1 year of age may be up to approximately 10 kg, for a maximum dose of 0.30 mL. 
Two injections per exam will be performed. Since investigational findings are subject to 
pre-analytic variability, performing two contrast-agent injections facilitates validation of 
findings if reproducibility is found. No increased risk was found at initial experience at 
Johns Hopkins Hospital, where 5 of 10 patients required double injection of contrast-
agent. Clinical cases at Johns Hopkins Hospital and The Children’s Hospital of 
Philadelphia have required double injection due to variable factors such as microbubble 
trapping within intravenous tubing. Adverse effects are not dose-dependent, thus risk is 
not increased by modifying the timing of interventions or increasing the number of 
contrast-agent injections. 
 
Even though the initial dosing of investigational drug for this study is 0.03 mL/kg, it is 
possible that the optimal dose for CEUS imaging of hypoxic ischemic injury may be less 
than 0.03 mL/kg. Since there would be no apparent safety concern regarding the 
administration of a lower dose of the investigational drug, the study team would proceed 
with a lower dose administration, if initial imaging studies suggest that a dose less than 


23 
 
0.03 mL/kg may provide more optimal imaging results. Therefore, the study team 
proposes that a dose range of 0.01-0.03 mL/kg be considered for dose optimization of 
the initial subjects, as indicated. Based on the published report of safe infusion of the 
investigational drug in four premature infants from 26.9 to 29.5 weeks gestational age 
[15] as well as 3 additional premature subjects from unpublished data of the sponsor, 
this dose range seems appropriate. For each subject, the dose per injection, as well as 
the total dose delivered, will be recorded in the study file for each administration of 
study drug. The expectation is that the intravenous injection of this ultrasound contrast 
agent will permit noninvasive, non-ionizing delineation of physiology and 
pathophysiology with higher resolution and accuracy than conventional ultrasound 
techniques. Since preliminary studies at Hopkins showed that half of neonates required 
two injections of contrast agent to achieve evaluable CEUS images, two injections will 
be performed per CEUS scan in all subjects to ensure adequate image quality and 
reproducibility. 
 
7.3 Investigational Drug Handling and Accountability 
Bulk supplies of drug will be directly shipped to IDS, by drug supplier. On an as-needed 
basis, only 1 box of either 5 or 20 vials, will be ordered from IDS by the principal 
investigator. The 1 box supply will be located in a storage unit, inside the Ultrasound 
Suite, with access limited to study personnel and dedicated Ultrasound personnel. 
 
The investigational imaging drug, LumasonTM, will be maintained as a separate supply 
from the Radiology Departments and central supply’s LumasonTM that is used for clinical 
care purposes. Specifically, the investigational drug LumasonTM for this IND will be 
physically segregated from the clinical use LumasonTM, and stored in a location that 
identifies the study drug as “Investigational Drug LumasonTM, For IND Research Only” 
so that all Radiology personnel know to limit the use of the investigational drug 
LumasonTM for study in this particular IND research exclusively.   
 
The investigational drug LumasonTM will be labeled according to FDA regulations, and 
identified as for IND research use only. The study team will use the investigational drug 
exclusively for the purposes of this IND study, and will not distribute or administer the 
investigational drug to persons not participating in the clinical study.  
 
The study team will ensure that each LumasonTM vial used in the IND clinical trial is 
entered into an investigational drug log that contains at least the following details of 
each vial: Lot #number and expiration date. For each subject, the LumasonTM lot 
#number, expiration date, # of vials used, disposition of unused LumasonTM, and 
discard procedure, will be recorded as part of the study record. Expired lots of 
LumasonTM will not be used in the clinical trial, and will be discarded. 
8 
SAFETY MANAGEMENT 
 
8.1 Clinical Adverse Events 


24 
 
Clinical adverse events (AEs) will be monitored throughout the study. Subjects enrolled 
to this clinical trial are anticipated to receive the study drug and have CEUS imaging 
conducted in the ICU setting. In this case, the study drug and US imaging unit will be 
brought to the ICU for study drug administration and imaging. CEUS has a duration of 
approximately 20 minutes. The 60 minute monitoring post-CEUS will be conducted by 
the study team members. The study team, who are composed of personnel trained in 
recognizing signs of infusion reaction (rash, allergic reactions, anaphylaxis) will conduct 
the 60 minute monitoring period and record any untoward reaction that may be related 
to the infusion of the contrast drug. Adverse events will be recorded at 1) 60 minutes 
post-scanning when the monitoring period is completed, and 2) through 48 hours post-
scanning, with documentation at 48 hours post-scanning if no adverse event presents 
until this point. All adverse events suspected of being related to study drug infusion will 
be reported to the regulatory authorities. 
 
In addition to assessing each patient after dosing, after 10 subjects, a complete safety 
analysis to determine the safety of continuing the study will be performed. 
 
 
8.2 Adverse Event Reporting 
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.  
 
AEs will be recorded and graded per the International Neonatal Consortium (INC) 
Neonatal AE Terminology. 
 
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), 
symptom, or disease temporally associated with the use of a medicinal product, whether 
or not 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. Adverse events will be recorded at 1) 60 minutes post-scanning 
when the monitoring period is completed, and 2) through 48 hours post-scanning, with 
documentation at 48 hours post-scanning if no adverse event presents until this point. 
 
8.4 Definition of a Serious Adverse Event (SAE) 


25 
 
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), requires inpatient hospitalization or prolongation of existing hospitalization, a 
persistent or significant disability/incapacity, or a congenital anomaly/birth defect in the 
offspring of a subject.   
 
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. 
A distinction should be drawn 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 of SAE to study drug or other intervention 
The relationship of each SAE to the study intervention should be characterized using 
one of the following terms in accordance with CHOP IRB Guidelines: definitely, 
probably, possibly, unlikely or unrelated.  
 
8.5 IRB/IEC Notification of SAEs and Other Unanticipated Problems 
The Investigator will promptly notify the IRB 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 below. 
External SAEs that are both unexpected and related to the study intervention will be 
reported promptly after the investigator receives the report. 
 
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 
 


26 
 
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 new or reassessed information (e.g., concomitant medication, medical history) 
should be submitted to the IRB. The investigator is responsible for ensuring that all SAE 
are followed until either resolved or stable.  
8.6 Notifications of SAEs/IND Safety Reports to the FDA 
Unexpected fatal or life-threatening adverse events that are related to the study drug, 
will be reported to FDA as soon as possible but no later than 7 calendar days following 
the sponsor’s initial receipt of the information. 
 
Unexpected serious adverse events that are related to the study drug but not fatal or 
life-threatening, will be reported to FDA as soon as possible but no later than within 15 
calendar days following the sponsor’s initial receipt of the information. 
 
Follow-up reporting: Any relevant additional information obtained by the sponsor that 
pertains to a previously submitted IND safety report will be submitted as a Follow-up 
IND Safety Report. Such report will be submitted as soon as the information is 
available, but no later than 15 calendar days after the sponsor receives the information. 
 
All other adverse events, will be reported to the FDA at or by the time of the Annual 
Report. 
 
8.7 Medical Emergencies 
Any medical emergencies that develop following injection of the investigational drug will 
be managed according to clinical care. See protocol Section 4.4 for a more complete 
description of clinical care management for serious adverse events following infusion of 
the investigational drug.   
 
8.8 Study Stopping Rules 
The study will be stopped for image futility, if non-diagnostic imaging is obtained in the 
first 3 subjects. 
 
There are some circumstances which potentially may arise, requiring temporary study 
stop. The FDA requires that the study be stopped for all patients (to allow for review of 
the protocol and procedures based on study related events) after one episode of 
anaphylaxis or death or other serious adverse event, regardless of relation to study 
drug. Therefore, anaphylaxis or death or another SAE will prompt a temporary stop to 
formally discuss the event with the FDA. As such an event (if deemed unrelated) may 
not meet the prompt reporting criteria for the IRB, the IRB will be notified as applicable 
(in accordance with CHOP IRB SOP 408). The study will proceed only with documented 
concurrence of the FDA (and the IRB, as applicable).  
 


27 
 
The occurrence of two non-fatal SAEs directly related to use of the study drug or one 
death directly attributed to use of the study drug will stop the study. 
9 
STUDY ADMINISTRATION 
 
9.1 Data Collection and Management 
Confidentiality: All subjects will be assigned a number unrelated to their medical record 
number and this will be kept in a master list. All of the data collected will be recorded 
using the respective “research number” to maintain anonymity. Only the master list will 
contain patient identifiers and a link to the research-specific code, and the data 
collection sheet will not contain patient identifiers.  
 
Security:  All files (master list and data collection sheet) will be password protected and 
will be stored on a password protected computer at CHOP on the secure storage 
network on the secure Hospital server. A password protected excel spreadsheets will be 
used for data collection. We will also store data in REDCap, a secure online tool for 
data collection and storage. The paper scoresheets will be identifiable only by the coded 
# of each subject and will be stored in a binder in a locked cabinet located in the study 
coordinator's office. The only way we will use to transfer information between co-
investigators will be [send secure] emails using the study members @email.chop.edu 
account. These e-mails will be only accessed from CHOP network computers and will 
be erased after the download of the password-protected excel sheet. All computers will 
meet CHOP IT Policy A-3-6: Acceptable Use of Technology Resources. 
 
Anonymization, de-identification, or destruction: After the study is finalized (results 
published in a scientific journal), the master list containing the reference to PHI will be 
archived in accordance with FDA and CHOP requirements. De-identified scoresheets 
and data gathered from the study will be archived in a password-protected folder on the 
primary computer of the P.I. These data will be destroyed only after a period of time 
compliant with federal and institutional guidelines.  
  
9.2 Confidentiality 
All data and records generated during this study will be kept confidential in accordance 
with Institutional policies and HIPAA on subject privacy and that the Investigator and 
other site personnel will not use such data and records for any purpose other than 
conducting the study. Data will be safeguarded by data entry into REDCap, a secure 
online data entry tool. Exported datasets will be stored on secure CHOP network drives. 
Patient names will be removed from images for use in the educational setting. 
   
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 (PHI limited to dates and zip codes).  
 
9.3 Data and Safety Monitoring Plan 


28 
 
The safety monitoring for this study is the primary responsibility of the sponsor-
investigator. Monitoring the safety outcomes following IV administration of the 
investigational drug will be conducted primarily by the Principal Investigator. An 
independent safety monitor has been designated to oversee the safety reports, and help 
adjudicate attribution of adverse events, should they occur. Regular meetings to discuss 
the outcomes of the study, and of the safety events, will be conducted by the study 
team. The occurrence of adverse events, serious adverse events and unanticipated 
events will be reported by the study team in accordance with federal and institutional 
guidelines, as outlined in Section 8 of this clinical study. 
 
Prior to study initiation, the Office of Research Compliance (ORC) will conduct a pre-
trial monitoring visit, to assess trial readiness of the study staff. Once the pre-trial 
monitoring visit has been successfully completed, the ORC will also, at minimum, 
monitor the IND study on an annual basis.    
 
9.4 Regulatory and Ethical Considerations   
9.4.1 Risk Assessment 
The pre-contrast scan is non-invasive and poses risk no greater than minimal. The 
potential risks associated with contrast-enhanced ultrasonography have been 
extensively studied, and the risk associated with the technique is less than that of CT or 
MRI contrast agents. The risk of adverse events is the lowest of all contrast agents 
available, with CT contrast being the highest (0.6%), followed by MR contrast (0.0088%) 
and ultrasound contrast (0.0086%) [10].  
 
Several studies detail the safety profile of ultrasound contrast agents in children and 
have shown minor adverse events including nausea, tinnitus, lightheadedness, altered 
taste sensation [11-13]. Serious cardiopulmonary reactions, including fatalities, have 
occurred uncommonly in adults that had complex comorbidities. Risk for these reactions 
may be increased among patients with unstable cardiopulmonary conditions. One 
documented severe reaction in a child documented symptoms of generalized pruritus, 
nausea, hypotension with tachycardia initially then bradycardia [14]. Management in this 
instance consisted of oxygen, intravenous epinephrine, and fluids (0.9% normal saline) 
with resolution of symptoms in two hours. Treatment of both minor, mild, and severe 
adverse reactions post LumasonTM administration are the same as that of CT or MRI 
contrast agents.  
 
In comparison to CT or MRI contrast agents, however, ultrasound contrast agents have 
proven to be much safer in children with only one serious adverse event over decades 
of its use to date (contrasting to approximately 15-20 adverse events per 2000 children 
if CT contrast agent were to be used). No serious adverse event has been reported in a 
neonate since its clinical use in this population. Animal studies on its toxicity profile also 
validate no fetal toxicity and the ultrasound contrast agent belongs to category B.  
 
In the case of SonoVue (now called LumasonTM), a second-generation lipid/sulfur 
hexafluoride US contrast agent (Bracco, Milan, Italy), the European Union approved its 


29 
 
intravenous use in adults in 2001. The FDA in the United States just recently approved 
the use of LumasonTM for evaluation of focal hepatic lesions and vesicoureteral reflux in 
pediatric population in 2016. For the remainder of clinical applications, the ultrasound 
contrast agents are being used off-label in both Europe and the United States. 
 
Risks of the administration of the study drug are considered a minor increase above 
minimal risk, without the prospect of direct benefit. Adverse effects are not dose-
dependent, thus risk is not increased by modifying the timing of interventions or 
increasing the number of contrast-agent injections. 
 
Another risk of the study includes the insertion of a peripheral IV line. This is a no 
greater than minimal risk procedure, with the main risks of discomfort, bruising, and 
infection which are generally self-limited. There is a no greater than minimal risk of 
breach of confidentiality, which is minimized by having all of study personnel undergo 
HIPAA training.  
 
Interference of CEUS with MRI is not expected since CEUS contrast-agent 
Lumason clears within minutes after injection. Elimination of Lumason (Sulfur 
Hexafluoride Lipid-Type A Microspheres) occurs via the lungs in the first minutes 
following contrast-agent injection (please see Package Insert's section 12.3 
Pharmacokinetics for more detail, attached in Application's section 12.02 
(3.0)). Additionally, Misun Hwang, the sponsor-investigator, has also 
previously performed brain CEUS before MRI in neonatal patients without adverse 
events. 
 
Steps Taken to Minimize Risks 
 
Parents and/or legal guardians of participants will be asked about contraindications to 
contrast enhanced ultrasonography examinations, as listed in the exclusion criteria. In 
order to appropriately treat potential rare adverse events, patients will be monitored by 
the study team for 60 minutes following contrast administration. 
 
Vital signs will be recorded from the medical chart and in-place monitoring and 
documented at 1) 30 minutes post-scanning, and 2) 60 minutes post-scanning. During 
monitoring, subjects will be assessed for rash, allergic reactions, anaphylaxis, and 
abrupt deviations from the subject's baseline hemodynamic parameters trend not 
related to medical intervention. 
 
Adverse events will be recorded at 1) 60 minutes post-scanning when the monitoring 
period is completed, and 2) through 48 hours post-scanning, with documentation at 48 
hours post-scanning if no adverse event presents until this point. 
 
9.4.2 Potential Benefits of Trial Participation 
The patient will not receive a direct benefit as a result of participating in the study. 
Indirect benefits may include improvement in current diagnostic algorithm for detection 


30 
 
and monitoring of NEC and downstream reduction of high mortality and morbidity 
associated with NEC. 
  
9.4.3 Risk-Benefit Assessment 
The benefit to society outweighs the risks of this study.   
 
9.5 Recruitment Strategy 
Potential subjects will be identified by reviewing the subjects’ medical charts to confirm 
eligibility by: 1) Neonatologists or 2) the radiologists to identify subjects with suspected 
or diagnosed necrotizing enterocolitis. Potential participation will be discussed by the PI 
with the referring neonatologist (co-investigator). Parents/legal guardians may be 
approached over phone/e-mail by a member of the study team to determine if they are 
interested in receiving more information about the study. Participation will be discussed 
with the parents/guardian by the neonatologist and/or radiologist. Parental/guardian 
permission (informed consent) will be obtained.   
 
Additionally, the CHOP Research Discovery Finder, e-mails, and a tearpad flyer will be 
used in the recruitment strategy 
 
9.6 Informed Consent and HIPAA Authorization 
Approved members of the study team will obtain written or electronic (e-consent) 
parental/guardian informed consent prior to the proposed study in a private setting. The 
electronic consent form will be administered in REDCap. The investigators will assure 
that parents/guardian comprehend the nature of the study, the study procedures and 
the risks and benefits of participation, steps that will be taken to avoid coercion and 
documentation of consent. A combined HIPAA consent-authorization document will be 
used.  
 
9.7 Payment to Subjects/Families 
Families will be offered a gift card of 50 USD value for participation in the study.  
10 PUBLICATION 
 
The investigative team plans to publish the data collected in a scientific journal.  Data 
may also be presented as abstract, podium presentation, or poster presentations at 
scientific meetings and conventions.  No patient identifying information will be used in 
publications. 
11 REFERENCES 
 
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assessment of bowel viability with color doppler US, Radiology, 235 (2005) 587-594. 


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