Conditions: Coronary Artery Disease
Keywords: Lumason Dobutamine stress echo stress echo coronary artery disease
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 175
Healthy volunteers: no

Brief summary:
The purpose of this study was to assess the safety and efficacy of Lumason-enhanced dobutamine stress echo (CE-DSE) in subjects having a suboptimal left ventricular endocardial border delineation (LV EBD) at rest and who were scheduled for coronary angiography.

Detailed description:
The study was designed to assess the safety and efficacy of Lumason at improving the visualization of the LV EBD during pharmacologic stress echocardiography examinations and for detection or exclusion of the coronary artery disease (CAD). The study population consisted of adult subjects referred for pharmacological stress echocardiography and with suboptimal image quality during unenhanced ultrasound imaging at rest who had known or suspected CAD. Subjects enrolled in the study represented subjects who could benefit most from contrast-enhanced ultrasound (CEUS) stress echocardiography.

Arms:
arm 1: Lumason (sulfur hexafluoride lipid-type A microspheres) 2 mL IV injection

Interventions: intervention 1: Lumason
Intervention descriptions: intervention 1: Lumason (sulfur hexafluoride-type A microspheres) an ultrasound contrast agent was administered as 2 single 2-mL IV injections during rest and stress echocardiography

Protocol document:
Prot_SAP_000.pdf: 
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
Confidential
Page 1
SonoVue
Protocol GM&RA
CONFIDENTIAL
A Prospective Multicenter Phase III Clinical Evaluation of the Safety and 
Efficacy of Lumason™/SonoVue® in Subjects Undergoing Pharmacologic Stress 
Echocardiography with Dobutamine for the Diagnosis of Coronary Artery 
Disease
Lumason™
Protocol No.:
IND No.:
EudraCT No.:
BR1-141
46,958
2015-001713-28
Final Version
30 April 2015
SPONSOR MEDICAL EXPERT: 
Melda S. Dolan, MD, FECC, FESE
 
Bracco Imaging S.p.A.
Via Folli 50
20134 Milan Italy 
With offices at:
Via Caduti di Marcinelle 13
20134 Milan, Italy
Bracco Diagnostics Inc.
The information contained in this document is the property of Bracco Group.  It contains strictly confidential 
information and no part of this document will be published or disclosed without Bracco Group prior written 
permission except that this document may be disclosed to appropriate Institutional Review Board/Ethics 
Committee/Regulatory Authorities/Competent Authorities as long as they are required to keep it confidential.


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
Confidential
Page 2
Table of Contents
List of Tables .................................................................................................................................. 5
List of Figures................................................................................................................................. 6
List of Appendices.......................................................................................................................... 7
Abbreviations and Definition of Terms.......................................................................................... 8
1
Title of Study....................................................................................................................... 11
2
Protocol Number ................................................................................................................. 11
3
Introduction ......................................................................................................................... 11
4
Study Objectives.................................................................................................................. 12
5
Investigational Plan ............................................................................................................. 13
5.1
Overall Study Design Description................................................................................ 13
5.2
Discussion of Study Design ......................................................................................... 14
5.2.1
Risk-Benefit Considerations ................................................................................. 17
5.3
Study Duration ............................................................................................................. 18
5.4
Study Population .......................................................................................................... 18
5.4.1
Inclusion Criteria................................................................................................... 19
5.4.2
Exclusion Criteria.................................................................................................. 19
5.4.3
Discontinuation Criteria........................................................................................ 20
6
Investigational Product/Investigational Medicinal Product ................................................ 20
6.1
Description and Labeling ............................................................................................. 21
6.2
Storage.......................................................................................................................... 21
6.3
Blinding and Randomization........................................................................................ 21
6.4
Handling and Preparation............................................................................................. 21
6.5
Administration.............................................................................................................. 22
6.6
Accountability .............................................................................................................. 22
7
Non-Investigational Medicinal Products............................................................................. 22
7.1
Dobutamine .................................................................................................................. 22
7.2
Atropine........................................................................................................................ 23
8
Methodology........................................................................................................................ 23
8.1
Study Schedule............................................................................................................. 23
8.2
Written Informed Consent............................................................................................ 23
8.3
Subject Numbering....................................................................................................... 23
8.4
Subject Evaluations...................................................................................................... 23
8.4.1
Demographics........................................................................................................ 23
8.4.2
Medical History..................................................................................................... 24
8.4.3
Pregnancy Test ...................................................................................................... 24
8.4.4
Concomitant Medications ..................................................................................... 24
8.4.5
Safety Assessments ............................................................................................... 24
8.4.5.1
Adverse Events............................................................................................... 24
8.4.5.2
Physical Examination..................................................................................... 25
8.4.5.3
Vital Signs...................................................................................................... 25
8.4.5.4
Laboratory Evaluations .................................................................................. 25


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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8.4.5.5
Electrocardiograms ........................................................................................ 26
8.4.5.6
Continuous Electrocardiograms..................................................................... 27
8.4.5.7
Pulse Oximetry............................................................................................... 27
8.5
Imaging Procedures...................................................................................................... 27
8.5.1
Rest Echocardiography ......................................................................................... 27
8.5.2
Pharmacologic Stress with Dobutamine ............................................................... 28
8.5.3
Stress Echocardiography....................................................................................... 28
8.6
Efficacy Evaluations..................................................................................................... 29
8.6.1
On-Site .................................................................................................................. 29
8.6.2
Off-Site.................................................................................................................. 29
8.6.3
Efficacy Measurements......................................................................................... 30
8.6.3.1
Endocardial Border Delineation..................................................................... 30
8.6.3.2
LV Wall Motion............................................................................................. 30
8.6.3.3
Overall Diagnostic Conclusion ...................................................................... 31
8.6.4
Truth Standard....................................................................................................... 31
8.6.4.1
Coronary Angiography................................................................................... 31
8.6.4.2
6 Month Follow Up to Collect Cardiac Events.............................................. 32
8.6.5
Off-Site Assessment Methodology ....................................................................... 32
9
Reporting Safety Information.............................................................................................. 32
9.1
Adverse Events............................................................................................................. 33
9.1.1
Definitions............................................................................................................. 33
9.1.2
Expedited Reporting a Serious Adverse Event to Sponsor................................... 34
9.1.3
Breaking the Study Blind...................................................................................... 35
9.1.4
Data Collection...................................................................................................... 35
9.1.5
Subject Follow-Up ................................................................................................ 38
9.2
Special Situations ......................................................................................................... 38
9.3
Laboratory Evaluations ................................................................................................ 39
9.3.1
Reporting and Evaluation of Central Laboratory Test Results ............................. 39
9.3.2
Repeat Testing....................................................................................................... 39
9.3.3
Emergency Laboratory Analysis........................................................................... 40
9.4
Electrocardiograms....................................................................................................... 40
9.5
Continuous ECGs......................................................................................................... 40
9.6
Physical Examinations ................................................................................................. 40
9.7
Vital Signs.................................................................................................................... 40
9.8
Pulse Oximetry............................................................................................................. 41
10
Statistical Methods .............................................................................................................. 41
10.1
Subject Disposition and Demographic and Baseline Characteristics........................... 41
10.2
Analysis Population...................................................................................................... 41
10.3
Concomitant Medications............................................................................................. 41
10.4
Extent of Exposure....................................................................................................... 42
10.5
Safety Analysis............................................................................................................. 42
10.6
Efficacy Analysis ......................................................................................................... 42
10.6.1
Primary Efficacy Endpoint – Sensitivity and Specificity for Detection and 
Exclusion of CAD................................................................................................. 42


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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10.6.1.1
Truth Standard Diagnosis............................................................................... 42
10.6.1.2
Evaluation of CAD......................................................................................... 43
10.6.2
Co-Primary Efficacy Endpoint – Critical Shift of Subjects with Suboptimal 
Images to Adequate Images .................................................................................. 43
10.6.3
Secondary Efficacy Endpoints .............................................................................. 44
10.6.4
Success Criteria..................................................................................................... 44
10.7
Sample Size.................................................................................................................. 44
10.8
Data Handling............................................................................................................... 45
10.9
Interim Analyses........................................................................................................... 46
11
Ethics and Good Clinical Practice....................................................................................... 46
11.1
Ethical and Regulatory Compliance............................................................................. 46
11.2
Informed Consent and Subject Information................................................................. 46
11.3
Institutional Review Board/Ethics Committee/Research Ethics Board Approval....... 46
11.4
Financial Disclosure..................................................................................................... 47
11.5
Safety Monitoring Boards ............................................................................................ 47
12
Administrative Considerations............................................................................................ 47
12.1
Regulatory Requirements–Sponsor/Investigator Obligations...................................... 47
12.2
Sponsor Regulatory Obligations for SUSARs Reporting ............................................ 47
12.3
Protocol Amendment.................................................................................................... 47
12.4
Curriculum Vitae.......................................................................................................... 47
12.5
Administrative Structure .............................................................................................. 48
12.6
Protocol Deviations, Violations and Exceptions.......................................................... 48
12.7
Monitoring Procedures................................................................................................. 48
12.7.1
Study Monitoring .................................................................................................. 48
12.7.2
Case Report Form.................................................................................................. 49
12.7.3
Inspection and Auditing ........................................................................................ 49
12.8
Archiving of Records ................................................................................................... 49
12.9
Study Results................................................................................................................ 49
12.10 Use and Publication of Study Results .......................................................................... 50
12.11 Financing...................................................................................................................... 50
12.12 Change in Investigator.................................................................................................. 50
12.13 Definition of the End of the Study ............................................................................... 50
12.14 Premature Termination of the Study ............................................................................ 50
12.15 Information Material .................................................................................................... 51
12.16 Insurance Policy ........................................................................................................... 51
13
Confidentiality..................................................................................................................... 51
14
Protocol Acceptance............................................................................................................ 52
15
References ........................................................................................................................... 53


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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Page 5
List of Tables
Table A: Description of Investigational Product/Investigational Medicinal Product................... 21
Table B: Laboratory Analytes....................................................................................................... 26
Table C: Overall Diagnostic Conclusion Based on LV Wall Motion .......................................... 31
Table D: Serious Adverse Event Reporting – Sponsor Contact Personnel .................................. 35
Table E: Cross Tabulation of Diagnosis: Truth Standard vs. Stress Echocardiography .............. 43


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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List of Figures
Figure 1: Imaging Procedures....................................................................................................... 29
Figure 2: 17-Segment Model ........................................................................................................ 30


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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Page 7
List of Appendices
Appendix A: Investigational Product/Investigational Medicinal Product Labeling
Appendix B: LUMASON/SONOVUE Reconstitution
Appendix C: Study Schedule
Appendix D: Serious Adverse Event Report
Appendix E: Investigator Statement
Appendix F: Administrative Structure


Protocol BR1-141
Final Version
30 April 2015
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Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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Abbreviations and Definition of Terms
ACC
American College of Cardiology
AE
Adverse Event
AHA
American Heart Association
ALT
Alanine aminotransferase
ASE
American Society of Echocardiography
AST
Aspartate aminotransferase
BMI
Body Mass Index
ºC
Degrees Centigrade
CA
Competent Authority
CAD
Coronary Artery Disease
CD
Compact Disc
CE
Contrast Enhanced
CE-DSE
Contrast Enhanced-Dobutamine Stress Echocardiography
CEUS
Contrast-Enhanced Ultrasound
CFR
Code of Federal Regulations
CI
Confidence Interval
CK-MB
Creatine Kinase-Myocardial B
CMRI
Cardiac Magnetic Resonance Imaging
CRF
Case Report Form
CRM
Clinical Research Manager
CT
Computed Tomography
CTR
Clinical Trial Report
DSE
Dobutamine Stress Echocardiography
DVD
Digital Versatile Disc
EAE
European Association of Echocardiography
EB
Endocardial Border
EBD
Endocardial Border Delineation
EC
Ethics Committee
ECG
Electrocardiogram
EudraCT
Database of European Clinical Trials
EU
European Union
ºF
Degrees Fahrenheit
FN
False Negative
FP
False Positive
FDA
Food and Drug Administration
GGT
Gamma-Glutamyl Transpeptidase
h
Hour
IB
Investigator’s Brochure
ICH
International Conference on Harmonization


Protocol BR1-141
Final Version
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Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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IND
Investigational New Drug
Investigational product
Compound under investigation
IP/IMP
Investigational Product/Investigational Medicinal Product
IRB
Institutional Review Board
ITD
Intention to Diagnose
kg
Killogram
LAD
Left Anterior Descending
LCx
Left Circumflex
LM
Left Main
LV
Left Ventricle
LVEF
Left Ventricular Ejection Fraction
LVO
Left Ventricle Opacification
MedDRA
Medical Dictionary for Regulatory Activities
mg
Milligram
MI
Mechanical Index
min
Minute
mL
Milliliter
mmHg
Millimeter of Mercury
MOD
Magnetic Optical Disc
MR
Magnetic Resonance
MRI
Magnetic Resonance Imaging
mSv
Millisievert
NIMP
Non-Investigational Medicinal Product
PI
Package Insert
RBC
Red Blood cell Count (total erythrocyte count)
RCA
Right Coronary Artery
REB
Research Ethics Board
ROW
Rest of the World – countries outside of Americas, the European Union, and Japan.
S
Second
SAE
Serious Adverse Event
SAER
Serious Adverse Event Report
SAP
Statistical Analysis Plan
SF6
Sulfur Hexafluoride
SPC/SmPC
Summary of Product Characteristics
SPECT
Single Photon Emission Computed Tomography
SUSAR
Suspected Unexpected Serious Adverse Reaction
TN
True Negative
TP
True Positive
UCA
Ultrasound Contrast Agent
UE
Unenhanced
UE-DSE
Unenhanced-Dobutamine Stress Echocardiography 
UEUS
Unenhanced Ultrasound


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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US
Ultrasound
USA
United States of America
USP
United States Pharmacopeia
WBC
White Blood Cell Count (total leukocyte count)
WHO
World Health Organization
βHCG
Beta Human Chorionic Gonadotropin
2D
2 Dimensional


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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1
Title of Study
A Prospective Multicenter Phase III Clinical Evaluation of the Safety and 
Efficacy of Lumason™/SonoVue® in Subjects Undergoing Pharmacologic Stress 
Echocardiography with Dobutamine for the Diagnosis of Coronary Artery 
Disease
2
Protocol Number
This study is being conducted under protocol number: BR1-141.  
IND number: 46,958
EudraCT number: 2015-001713-28.
3
Introduction
Stress echocardiography is an established imaging modality for the detection of coronary artery 
disease (CAD). 1,2,3,4,5,6,7 The primary indication for conventional stress echocardiography is to 
indirectly detect angiographically significant (≥50% diameter) stenoses by inducing ischemia or 
to assess the patient’s risk for a cardiac event.5,8,9,10 Increasingly stress echo techniques have been 
shown to offer valuable prognostic information in the risk evaluation of stable chronic 
CAD.5,10,11 Significant studies (> 100 patients) of dobutamine stress echocardiography (DSE) 
show a range for sensitivity of between 61–95%, while that for specificity ranged from 51–
95%11 for the diagnosis of CAD.1 The detection of CAD with stress echocardiography is based 
on the observation of contractile dysfunction in any myocardial segment at rest or with stress; 
therefore, complete visualization of all left ventricular (LV) endocardial borders (EB) is 
necessary to document or exclude abnormalities of regional myocardial wall thickening 
confidently.12,13 Interpretation of wall thickening is qualitative, highly dependent on the skill and 
experience of the reviewing physician, and is affected considerably by quality of endocardial 
border delineation (EBD).12,13 Numerous patient factors may produce suboptimal images with 
poor EBD. Given, in addition, the challenges imposed by excessive cardiac motion due to 
hyperventilation and tachycardia at peak stress, non-diagnostic or poor-quality images may occur 
in up to 30% of patients.12,13 The advent of digital side-by-side analysis, standardized reporting 
criteria, and generalized use of tissue harmonic imaging has reduced, but not overcome, this 
problem.12
Use of ultrasound contrast agents (UCAs) has shown to improve EBD during stress 
echocardiography and is recommended for use in patients with poor unenhanced image quality 
by both the American Society of Echocardiography (ASE) and the European Association of 
Echocardiography (EAE).12,13  However, while Lumason™ (sulfur hexafluoride lipid-type A
microspheres) with an approved trade name in the USA/SonoVue® (sulfur hexafluoride 
microbubbles) with an approved trade name for everywhere else in the world  referred to 
hereafter as LUMASON, is approved for use in echocardiography both at rest and stress in the 


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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European Union (EU) and Canada, no UCA is currently approved in the United States of 
America (USA) for use in stress echocardiography to improve LV EBD in patients with 
suboptimal unenhanced imaging.
Although generalized use of tissue harmonic imaging has improved image quality, interpretation 
of stress echocardiograms may still be limited in patients with poor acoustic windows.12,14
Recent studies indicate that contrast-enhanced 2 dimensional (2D) echocardiography with 
harmonic imaging had excellent correlation with radionuclide, magnetic resonance (MR) and 
computed tomographic (CT) measurements of LV volumes and left ventricular ejection fraction 
(LVEF) with improved inter-observer agreement and physician interpretation confidence 
compared to unenhanced harmonic imaging in adult population.15,16 Increasing accuracy of 
LVEF measurements have been associated with contrast-enhanced harmonic imaging over 
unenhanced harmonic imaging and fundamental imaging, respectively.17,18
Non-diagnostic or poor-quality images may occur in up to 30% of patients during stress because 
of challenges imposed by excessive cardiac motion due to hyperventilation and tachycardia at 
peak stress.  There are documented benefits through published data regarding the value of the use 
of contrast agents in stress echocardiography with respect to improvement in image quality and 
EBD, which further translates into enhanced interpretation of regional and global function with 
decreased inter- and intra-observer variability.4,12  Previous investigations have demonstrated 
that improving the quality of stress echocardiography images, using contrast agents enables 
superior visualization of EBD at rest and peak stress across a range of unenhanced baseline 
image quality (i.e., greatest improvement is seen in patients with the poorest baseline 
images)with completeness of wall-segment visualization, at both rest and peak stress.6,19The 
ASE1 and American College of Cardiology (ACC)20 recommendations for use of contrast 
enhancement in stress testing have been supported by peer reviewed publications where the 
importance of adequate visualization of segments for accurate diagnosis have been emphasized.
1,4,6,19 ,20
Due to the increased routine use of DSE as a non-invasive cardiac imaging tool in adults with 
suspected or known CAD for the detection and/or evaluation of CAD and an increasing number 
of suboptimal, non-diagnostic quality images mostly with respect to the challenges imposed by 
excessive cardiac motion due to hyperventilation and tachycardia at peak stress, we aim to study 
the safety and efficacy of LUMASON-enhanced stress echocardiography with dobutamine in 
subjects with suboptimal LV EBD at unenhanced echocardiography.  This study is being 
conducted as part of the Phase III program in support of a supplemental New Drug Application 
(sNDA) to be filed with the Food and Drug Administration (FDA).
4
Study Objectives
Primary Objectives:
To assess the efficacy of LUMASON-enhanced dobutamine stress echocardiography (DSE)
in subjects with suspected or known CAD having suboptimal left ventricular (LV) 
endocardial border delineation (EBD) at unenhanced echocardiography in terms of:


Protocol BR1-141
Final Version
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Echocardiography
Lumason™
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Sensitivity and specificity for the detection or exclusion of CAD in unenhanced versus 
LUMASON-enhanced DSE using coronary angiography or clinical follow-up as the 
truth standard;
Critical shift from suboptimal (≥2 adjacent segments inadequate on any apical view) at 
unenhanced dobutamine stress echocardiography (UE-DSE) to adequate images 
(reduction of suboptimal adjacent segments) for LV EBD at contrast-enhanced 
dobutamine stress echocardiography (CE-DSE).
Secondary Objectives:
Change from peak stress non-contrast ultrasound (UE-DSE) versus peak stress contrast-
enhanced ultrasound (CE-DSE) in total LV EBD score.
To obtain safety data in subjects undergoing DSE with LUMASON.
5
Investigational Plan
5.1
Overall Study Design Description
This is a Phase III, prospective, multicenter, within-patient comparison of UE-DSE and CE-DSE 
that will be conducted at 10-15 sites in subjects with a suboptimal LV EBD on non-contrast 2D 
transthoracic echocardiography with harmonic imaging at rest and that are scheduled for 
coronary angiography. Suboptimal LV EBD is defined as published in guidelines, having ≥2 
adjacent segments on any apical view that are suboptimal1,12 during the rest non-contrast 
ultrasound examination.
Imaging conditions will be representative of those used in routine clinical practice and will 
include LV EBD, and LV wall motion using harmonic imaging modality with commercially 
available equipment.
Subjects who are enrolled in the study will undergo a rest non-contrast echocardiogram (UEUS) 
with the standard apical 4-, 2-, and 3-chamber views obtained with harmonic imaging. A 2 mL 
dose of LUMASON will be administered as an intravenous bolus injection during rest contrast 
enhanced echocardiography (CEUS).  A recording of 2D transthoracic harmonic 
echocardiography images will be performed for both UEUS and CEUS rest imaging. At least 30 
minutes after the completion of the rest unenhanced echocardiography (UEUS) and contrast 
enhanced-echocardiography (CEUS) the dobutamine infusion will begin.  Once peak stress is 
achieved (with or without the addition of atropine), subjects will undergo unenhanced 
dobutamine stress echocardiography (UE-DSE).  This will be followed by the contrast-enhanced 
dobutamine stress echocardiography (CE-DSE) with the administration of 2 mL of LUMASON.  
A recording of 2D transthoracic images will be performed for both UE-DSE and CE-DSE 
imaging. The on-site Investigators will provide an overall diagnosis of CAD for each subject 
from the unenhanced (UEUS and UE-DSE) and contrast-enhanced (CEUS and CE-DSE)
echocardiographic images.  


Protocol BR1-141
Final Version
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Echocardiography
Lumason™
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The truth standard in this study will be coronary angiography.  If coronary angiography is not 
performed or is indeterminate subjects will be followed for up to 6 months to collect cardiac 
events.
An off-site assessment of the echocardiographic images will be performed by 3 blinded readers 
unaffiliated with any of the investigational sites, blinded to the subject’s clinical information. 
The efficacy analysis will be based on the blinded reader evaluations of LV EBD, wall motion 
abnormalities and diagnosis of CAD.
A fourth blinded reader will assess the coronary angiography images and provide a diagnosis of 
CAD. The fourth reader will be blinded to the specifics of the protocol including the patient 
population and the results of the ultrasound examination, but will receive clinical information 
such as the medical history on the subject.  
Safety will be evaluated in this study by the collection of concomitant medications and the 
monitoring of adverse events, vital signs, electrocardiograms (discrete & continuous), laboratory 
tests and continuous pulse oximetry.
This study is planned to be conducted in the United States, Canada and Europe.
5.2
Discussion of Study Design
The use of UCAs has shown to improve EBD during stress echocardiography and is 
recommended for use in patients with poor unenhanced image quality by both the ASE and the 
EAE.12,13,21 However, while LUMASON (under the trade name SonoVue) is approved for use in 
echocardiography both at rest and stress in the EU, no UCA is currently approved in the USA for 
use in stress echocardiography to improve LV EBD in patients with suboptimal unenhanced 
imaging. 
The mechanism of action of LUMASON during stress echocardiography would be exactly the 
same as during an exam at rest. LUMASON would be used in subjects with suboptimal images 
to enhance the LV cavity and improve the detection of the EB. This Phase III study will, 
therefore, be conducted with a consistent design (dose, study population and endpoints of both 
LV EBD score and Sensitivity/Specificity for detection of CAD) based on the Phase III studies 
previously conducted to obtain approval for the use of LUMASON in echocardiography at 
rest22,23,24 and a previous large, multicenter, dose-ranging study of LUMASON in stress 
echocardiography25.
The study is designed to assess the safety and efficacy of LUMASON at improving the 
visualization of the LV EBD during pharmacologic stress echocardiography examinations and 
for detection or exclusion of CAD.  
The study population will consist of adult subjects referred for pharmacologic stress 
echocardiography and with suboptimal image quality during unenhanced ultrasound imaging at 
rest who have known or suspected CAD. Subjects to be enrolled in the current study are 
representative of subjects who could benefit most from CEUS stress echocardiography.
Imaging conditions will be representative of those used in routine clinical practice and will 
include LV EBD using harmonic imaging modality. Each subject will undergo an unenhanced


Protocol BR1-141
Final Version
30 April 2015
Lumason™/SonoVue® in Stress 
Echocardiography
Lumason™
Document ID: BDI-PTR-AC2940.15-BR141PT-B054714-1.0
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echocardiogram (UEUS) followed by a LUMASON-enhanced echocardiogram (CEUS) both at 
rest. The same applies to the stress echocardiogram when echocardiographic images will be 
obtained immediately after dobutamine administration (UE-DSE) and then LUMASON will be 
administered and LUMASON-enhanced stress images (CE-DSE) will be captured. Thus, each 
subject will serve as its own control and is designed as an intra-patient controlled study.
The current guidelines of American Heart Association (AHA)26 and ASE12 while they suggest 
ultrasound contrast use in difficult to image patients with suboptimal image quality at their 
presenting rest echocardiography exams, their definition of a suboptimal study is when 2 
contiguous segments are not visualized in any of the standard 3 apical views. The same criteria 
will be applied in adults during peak stress imaging, representative of those used in routine 
clinical practice and will include assessment of LV EBD using harmonic imaging modality.
Taking into consideration the clinical importance, both the diagnostic performance and the LV 
EBD visualization are defined as co-primary efficacy endpoints for the study.  

First co-primary efficacy endpoint is a comparison of diagnostic performance (sensitivity 
and specificity) for the detection or exclusion of CAD in unenhanced (UE-DSE) versus 
Lumason-enhanced DSE (CE-DSE) using coronary angiography or clinical events based 
on 6 month follow-up as the truth standard.  Subjects will be considered to have positive 
CAD if they have at least one vessel with stenosis ≥ 50% based on the assessment of off-
site reader for coronary angiography or have one of the clinical events (cardiac death, 
non-fatal myocardial infarction, revascularization and need for revascularization) within 
the 6 month follow-up.

Second co-primary efficacy endpoint is critical shift from suboptimal (≥2 adjacent 
segments inadequate on any apical view) at unenhanced dobutamine stress 
echocardiography (UE-DSE) to adequate images (reduction of suboptimal adjacent 
segments) for LV EBD at contrast-enhanced dobutamine stress echocardiography (CE-
DSE).
The secondary efficacy endpoint of LUMASON studies (change from baseline in total of LV 
EBD score) is the same endpoint for adult studies for LV EBD indication at rest echo 
examinations.
The muscle and cavity of the LV will be divided into 17 segments (17-segment model).  The 
17-segment model will be utilized as recommended by the ASE guidelines. 1,12,27  The standard 
views of a 2D echocardiogram as defined by the ASE are all used as part of a 2D 
echocardiogram at rest and stress.26  The clinical utility of improvement in LV EBD resulting 
from opacification of the LV as an aid in determination of more clinically relevant parameters is 
recognized by the 2008 ASE consensus statement as leading to improvement in feasibility, 
accuracy, and reproducibility of echocardiography for the qualitative and quantitative assessment 
of LV structure and function.12
Unevaluable image are defined as images with inadequate visualization of segments comprising 
all coronary artery territories due to 1 or more of the following artifacts: motion artifacts, lateral 
wall drop out, swirling or attenuation artifacts.  From the published literature6 and previous 
Bracco-sponsored study25 8% of the UE-DSE images are expected to be unevaluable.


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Stress can be induced by either physical exercise or pharmacologic agents. Physical stress was 
the first reported and used form of stress echocardiography, but it has several disadvantages 
since during exercise it is not possible to perform close echocardiographic monitoring and 
recordings are not reliable because of hyperventilation. Pharmacologic stress overcomes these 
disadvantages.
The pharmacologic stress agent used in this study will be dobutamine.  Dobutamine is a direct-
acting inotropic agent whose primary activity results from stimulation of the beta-
adrenoreceptors of the heart while producing comparatively mild chronotropic, hypertensive, 
arrhythmogenic, and vasodilative effects. Dobutamine acts primarily on beta-1 adrenergic 
receptors, with little effect on beta-2 or alpha receptors. In the USA, dobutamine is indicated 
when parenteral therapy is necessary for inotropic support in the short-term treatment of patients 
with cardiac decompensation due to depressed contractility resulting either from organic heart 
disease or from cardiac surgical procedures. In the EU and other countries, dobutamine is also 
indicated for use in stress echocardiography as an alternative to exercise in patients for whom 
routine exercise cannot be satisfactorily performed. Even if not approved for use in stress 
echocardiography in the USA, dobutamine is recommended as the pharmacologic stressor of 
choice for stress echocardiography by the ACC Foundation Appropriateness20 Criteria Task 
Force, the ASE, and the AHA. 1,26 Dobutamine became the preferred stressor in USA as the 
AHA/ACC guidelines stated that “dobutamine stress echo has higher sensitivity than vasodilator 
stress echo for detection of CAD.”28,29 The recent 2007 recommendations on stress 
echocardiography of the ASE conclude that “although vasodilators may have advantages for 
assessment of myocardial perfusion, dobutamine is preferred when the test is based on 
assessment of regional wall motion.” 1
Based on the Phase III studies conducted to obtain approval for use of LUMASON in 
echocardiography at rest22,23,24 and a previous large, multicenter, dose-ranging study of 
LUMASON in stress echocardiography25 the proposed dose of LUMASON is 2 mL bolus 
injection during the rest examination, and then again as a single 2 mL bolus injection after 
administration of the stress agent, dobutamine, and the UEUS stress examination is completed. 
There will be at least a 30 minute period between rest and stress LUMASON injections.
The off-site assessments of echocardiographic images will be performed by experienced 
cardiologists, unaffiliated with the enrolment centers and blinded to the subject’s clinical 
information. Blinded off-site assessments are used to evaluate diagnostic agents in order to 
eliminate the potential bias associated with on-site assessments due to the investigator’s 
knowledge of the subject’s medical history, clinical findings from other modalities, or other 
clinical information. Thus, the off-site assessments allow for a blinded evaluation of efficacy 
which is not possible for the on-site assessments.
All subjects will have a truth standard of either coronary angiography or, if coronary 
angiography is indeterminate or not performed, they will be followed for up to 6 months to 
collect cardiac events. Although we will study the sensitivity and specificity in detecting 
anatomic stenoses ≥50% in severity, not all the subjects will undergo coronary angiography. 
Therefore, subjects who do not undergo coronary angiography in the study will be followed for 
cardiac events (cardiac death, non-fatal myocardial infarction, revascularization and need for 


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revascularization) at 6 months.  The proposed cardiac events as hard end points in the study are 
widely used, well recognized, robust, measurements of a non-invasive test to predict cardiac 
events.5,7,10
The coronary angiography images will be reviewed by a single, off-site reader who is an
experienced cardiologist to provide a centralized reading for the confirmation of the diagnosis of 
CAD. He/she will be blinded to the results of the stress echo examination. Medical historical 
information will be provided to the reader. This will allow the gold standard to achieve the 
maximum accuracy and reliability. It will also serve to reduce the variability of readings 
performed by many different unblinded readers on-site across all study sites.
5.2.1
Risk-Benefit Considerations
Despite substantial technical improvements in imaging modalities that have an impact on image 
quality on the equipment side, approximately 30% of patients continue to undergo stress 
echocardiographic examinations that are non-diagnostic or inconclusive due to poor image 
quality, with subsequent poor evaluation of cardiac function, leading to the need for more 
invasive techniques. 1,12 ,30  These patients would benefit from a contrast agent that can opacify 
the LV and improve the delineation of EBs.
Given the fact that the subjects to be enrolled in the present clinical trial will be subjects already 
having suboptimal echocardiographic images and have no evidence of intracardiac shunts, 
inclusion in the study is unlikely to add any risk for the subjects.  Due to the fact that subjects 
who are contraindicated for dobutamine will already be excluded from this study, dobutamine 
used as the stressor in this study will not add any additional risk to the subjects.
Even though the subjects have been scheduled for coronary angiography most likely have 
already undergone other diagnostic imaging modalities such as DSE or SPECT, the contrast 
enhanced DSE will still add incremental value with better EBD which will translate into better 
recognition of wall motion abnormalities. Furthermore, CEUS stress echocardiography will
prevent the risk of those subjects being exposed to radiation through other imaging modalities in 
the down streaming of appropriate assessment of cardiac function and structures.
LUMASON efficacy and safety is based on data from 75 clinical trials in 6307 subjects and post-
marketing surveillance over 12 years of market use in an estimated 2.7 million patients. 
Hypersensitivity to any of the components of LUMASON, as for other contrast agents, cannot be 
excluded, and there is potential for unpredictable, serious hypersensitivity reactions, including 
anaphylactoid reactions. Further, such reactions may lead to life-threatening conditions if they 
occur in critically ill patients. However, serious hypersensitivity reactions after LUMASON 
administration are rare (estimated occurrence of approx. 1 in 10,000 exposures) and suboptimal 
EBD and inaccurate assessment of LV function (global and regional wall motion assessment) 
and/or cardiac anatomy are frequent in a patient population with suspected CAD.12,13,31
Echocardiography is an imaging modality that represents a fundamental investigation used in the 
initial and ongoing management of patients with suspected or known CAD because it is 
noninvasive, provides easy access, anytime and any day, can be performed at the bedside of the 
patient, and can be repeated as often as needed for monitoring. The balance of the benefit of 
LUMASON in comparison with its risks is also positively affected by the availability of acute


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care settings and physicians that can promptly and effectively recognize and manage serious 
adverse events.
Although optional noninvasive modalities (SPECT, CMRI and CT angiography) may provide 
diagnostic accuracy, valuable and detailed data, all of them pose greater risk than transthoracic 
echocardiograms.32 33 Probably the most critical drawback is the time delay in obtaining the data 
needed for urgent risk assessment and treatment, but safety concerns including exposure to 
radioactive material and risky patient transportation to the nuclear/MRI department are also 
present.21 Furthermore, if an imaging agent is used, these imaging modalities carry the same risk 
of rare, serious hypersensitivity reactions and the same risk of serious, life-threatening 
complications.
Of note, at the patient level, the effective dose of a single nuclear cardiology stress imaging scan 
ranges from 10 to 27 mSv.  The corresponding equivalent dose exposure is 500 chest X-rays 
[Cardiolite® (Tc-99m Sestamibi)], 1200 chest X-rays (Thallium) and 1300 chest X-rays (with 
dual imaging protocol).  According to the latest estimates the risk of cancer for a middle aged 
patient ranges from 1 in 1000 (for Cardiolite) to 1 in 400 (for a dual isotope scan).21 Therefore, 
contrast-enhanced stress echocardiography should be preferred due to wider availability and for 
its radiation free nature.  Overall, the effectiveness and suitability of LUMASON as a contrast 
agent for use with echocardiography to obtain improve LV EBD has been demonstrated. 
Clinical studies have shown LUMASON to be safe and well tolerated with minimal risk to 
subjects. In conclusion, the benefit-to-risk ratio for LUMASON is high, indicating an advantage 
to subjects undergoing stress echocardiography.
With the marked increase in costs associated with cardiovascular care and interventions, it is 
vital that non-invasive imaging procedures be highly accurate in detecting whether a patient is at 
risk for coronary events, not just whether they have a significant coronary stenosis. It is also vital 
that these stress imaging procedures be equally accurate in identifying which patients are at risk 
for future cardiac events. 
5.3
Study Duration
The procedure associated with the LUMASON administration will be completed within 
approximately 1 hour.  Safety monitoring will begin at the time of signing Informed Consent and 
continue for 72 hours after the last LUMASON administration or until the subject undergoes 
cardiac intervention, whichever comes first.  Subjects will be followed up to 6 months to collect 
truth standard data (coronary angiography or cardiac events) as described in Section 8.6.4.
5.4
Study Population
The study will be conducted in subjects who have suboptimal image quality on UEUS 
echocardiography at rest and are scheduled for coronary angiography. It is expected that 10-15 
study centers will participate in the study. Approximately 175 subjects will be enrolled in order 
to obtain 45 subjects with positive CAD and 45 with negative CAD based on the truth standard.  
The prevalence of positive CAD subjects will be tracked based on the onsite diagnosis of CAD 
from the truth standard.  If the overall yield of positive CAD subjects is lower than the assumed 


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30%,34 then additional subjects will need to be enrolled in order to obtain a sufficient number of 
positive CAD subjects.
5.4.1
Inclusion Criteria
Enroll a subject in this study if the subject meets the following inclusion criteria:
Provides written Informed Consent and is willing to comply with protocol requirements;
Is at least 18 years of age;
Has suspected or known CAD and is scheduled to undergo coronary angiography within 6 
months after the LUMASON DSE.
Has undergone a previous echocardiography prior to enrollment; resulting in suboptimal 
unenhanced images at rest, defined as ≥ 2 suboptimal adjacent segments in any apical 
view.
5.4.2
Exclusion Criteria
Exclude a subject from this study if the subject does not fulfill the inclusion criteria, or if any of 
the following conditions are observed:
Is a pregnant or lactating female. Exclude the possibility of pregnancy:
by testing on site at the institution (serum or urine βHCG) within 24 hours prior to the 
start of LUMASON administration(s),
by surgical history (e.g., tubal ligation or hysterectomy),
post menopausal with a minimum 1 year without menses;
Has any known hypersensitivity to 1 or more ingredients of LUMASON (sulfur 
hexafluoride or to any components of LUMASON);
Has any known hypersensitivity to dobutamine;
Has an ongoing or recent (within the last 30 days) acute myocardial infarction;
Has known right-to-left, bidirectional or transient cardiac shunt (ruled out with agitated 
saline study performed before administration of LUMASON);
Has electrolyte (especially potassium and magnesium) abnormalities;
Has unstable pulmonary and/or systemic hemodynamic conditions e.g.:
decompensated or inadequately controlled congestive heart failure (NYHA Class IV); 
hypovolemia; 
uncontrolled hypertension, i.e. resting systolic blood pressure >200 mmHg or diastolic 
blood pressure >110 mmHg; 
unstable angina; 
acute coronary syndrome;
aortic dissection; 
acute pericarditis, 
myocarditis, or endocarditis;
stenosis of the main left coronary artery; 


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hemodynamically significant outflow obstruction of the left ventricle, including 
hypertrophic obstructive cardiomyopathy; 
hemodynamically significant cardiac valvular defect; 
acute pulmonary embolism;
Has uncontrolled cardiac arrhythmias;
Has significant disturbance in conduction; 
Has hypertrophic subaortic stenosis;
Has an acute illness (e.g., infections, hyperthyroidism, or severe anemia);
Was previously entered into this study or received an investigational compound within 30 
days before admission into this study;
Has been treated with any other contrast agent either intravascularly or orally within 48 
hours of the first LUMASON administration;
Has any medical condition or other circumstances which would significantly decrease the 
chances of obtaining reliable data, achieving study objectives, or completing the study 
and/or postdose follow-up examinations;
In addition, due to the use of Atropine in subjects who have not reached targeted heart rate with 
peak dobutamine infusion, subjects with the following will be excluded:
Glaucoma;
Pyloric stenosis; 
Prostatic hypertrophy.
5.4.3
Discontinuation Criteria
Clearly document the reason for the subject’s discontinuation on the Case Report Form (CRF). 
Discontinued subjects are not replaced.  Discontinue a subject from the study if the subject:
Withdraws consent;
No longer meets the Inclusion Criteria;
Experiences any of the Exclusion Criteria;
Has an adverse event that, in the opinion of the Investigator, requires the subject’s 
discontinuation. Perform subject follow-up in accordance with Section 9.1.5.
6
Investigational Product/Investigational Medicinal Product
The investigational product (IP)/investigational medicinal product (IMP) is 
LUMASON/SONOVUE. 
In the USA and Canada, LUMASON will be supplied as a single 3-part kit with the following 
components:

1 clear glass vial labeled as LUMASON (sulfur hexafluoride lipid-type A microspheres) 
for injectable suspension,


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
1 prefilled syringe containing 5 mL Sodium Chloride 0.9% Injection, USP (Diluent) and 
its plunger rod

1 Mini-spike
In Europe, SONOVUE will be supplied in a kit which includes 2 individual plastic containers 
each with the following components:

1 vial containing 25 mg of lyophilized powder labeled as SONOVUE

1 pre-filled syringe containing 5 mL sodium chloride NaCl 0.9%w/v and its plunger rod

1 MiniSpike transfer system
6.1
Description and Labeling
LUMASON is formulated as a 25-mg sterile, non-pyrogenic lyophilized powder in a septum-
sealed vial. The ingredients in each vial of LUMASON, after reconstitution, are listed in Table A
Table A: Description of Investigational Product/Investigational Medicinal Product
Ingredient
Concentration / Amount per Unit
Polyethylene glycol (PEG) 4000
4.91 mg/ml
Phospholipids (DSPC/DPPG 1:1 w/w)
0.075 mg/ml 
Palmitic Acid
0.008 mg/ml
Sulfur Hexafluoride (SF6)
8 µL/ml*
*Contents in the microspheres/microbubbles
Each vial of LUMASON will bear a two-part label with the information detailed in Appendix A.
Affix the second part of the two-part label from the vial to the vial label page.
6.2
Storage
Store the LUMASON kits at controlled room temperature at 25°C (77°F) excursions are 
permitted to 15-30 °C (59-86°F).  No special storage conditions are required for SONOVUE
containers.  Do not freeze.  All kits/containers must be stored in a secure area with limited 
access.
6.3
Blinding and Randomization
This is an open-label, nonrandomized study.
6.4
Handling and Preparation
Prior to administration, the lyophilized powder should be reconstituted under aseptic technique
conditions by injecting the prefilled 5 mL sodium chloride syringe into the 
LUMASON/SONOVUE vial. After adding the sodium chloride solution, the vial should be 
shaken vigorously for 20 seconds, after which a homogeneous white milky liquid is obtained. 


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Detailed instructions for the reconstitution of both LUMASON and SONOVUE are presented in 
Appendix B.  
Store the reconstituted IP/IMP at room temperature in the vial supplied. Although the suspension 
is useable for up to 3 hours in the USA and up to 6 hours in Canada/ Europe the more 
conservative time period of 3 hours will be used in this study. Upon standing for more than 15 
minutes, buoyancy causes some of the larger microspheres/microbubbles to rise to the surface. 
Therefore, gently agitate the reconstituted vial in a top-to-bottom manner to resuspend the sulfur 
hexafluoride microspheres/microbubbles before administration.
A separate kit/container should be used for the rest (CEUS) and stress (CE-DSE) LUMASON 
administrations. In the case of incomplete use of vial contents, the product cannot be reused.
6.5
Administration
Once reconstituted, LUMASON will be administered as a single 2 mL bolus injection during the 
rest examination, and then again as a single 2 mL bolus injection after peak stress is achieved 
with the administration of the stress agent, dobutamine (with or without the addition of atropine), 
and the UE-DSE stress examination is completed.  Each LUMASON injection will be performed 
intravenously through a peripheral line using a 20 gauge catheter followed immediately with 5 
mL of saline in order to flush the intravenous line of any remaining contrast agent. There will be 
at least a 30 minute period between the rest and the stress LUMASON injections.
6.6
Accountability
In accordance with International Conference of Harmonization (ICH) and United States (US)
FDA requirements, the Investigator and/or Drug Dispenser must at all times be able to account 
for all IP/IMP furnished to the institution.  The appropriate site personnel must sign, date and 
immediately forward to the Sponsor the IP/IMP receipt form included with each shipment.
No IP/IMP is to be used outside of this study.  Record the use of the IP/IMP on the appropriate 
Drug Accountability Record.  All containers of IP/IMP must be accounted for, whether used or 
unused, during the course of and at the conclusion of the study.  At the conclusion of the study, 
all shipments of IP/IMP to the Investigator must be returned to the Sponsor accompanied by a 
completed, signed copy of the appropriate IP/IMP inventory reconciliation form.
7
Non-Investigational Medicinal Products
7.1
Dobutamine
Dobutamine is a non-investigational medicinal product (NIMP) in this study due to its use as the 
pharmacologic stress agent during stress echocardiographic imaging. Refer to the dobutamine 
Package Insert (PI)/Summary of Product Characteristics (SPC) for details on the storage, 
handling, preparation and administration.


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7.2
Atropine
Atropine is a non-investigational medicinal product (NIMP) in this study due to its use as an 
adjunct to dobutamine in achieving peak stress during DSE. Refer to the atropine PI/SPC for 
details on the storage, handling, preparation and administration.
8
Methodology
8.1
Study Schedule
See Appendix C for the timing of required observations and evaluations.
8.2
Written Informed Consent
Obtain written Informed Consent from the subject prior to the implementation of study 
procedures required by the protocol.  The subject must sign the Informed Consent approved by 
the Institutional Review Board (IRB)/Ethics Committee (EC)/Research Ethics Board (REB)
(including specific local requirements e.g., data protection where applicable).  Give the subject a 
copy of the signed and dated written informed consent form including any other written 
information regarding the study. Document the Informed Consent process in detail in the source 
record at the site.
8.3
Subject Numbering
Assign a four digit subject number for each subject who qualifies for the study and signs the 
written Informed Consent.  The first two digits of the subject number will be the investigational 
site number assigned by Sponsor.  For site numbers less than ten use a leading zero. The third 
and fourth digits will be a number starting with 01 for the first subject enrolled and incrementing 
by “1” for each sequential subject.
For example, at site 01, the investigative site will assign the first subject 0101; the second subject 
0102, etc.
Never reassign subject numbers.  In the event that a subject withdraws from the study, the 
number assigned to that subject is retired and the next subject receives the next sequential 
number.
8.4
Subject Evaluations
8.4.1
Demographics
Obtain demographic information on each subject (including; height, weight, age, sex and race) 
and record on the CRF.


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8.4.2
Medical History
Obtain a general medical history with relevant findings along with a special focus on cardiac 
history after the subject has signed the Informed Consent and within 24 hours prior to the first 
LUMASON administration.  Record the subject’s medical history/cardiac history on the 
appropriate Medical History or Cardiac Medical History sections of the CRF.
8.4.3
Pregnancy Test
If the subject is female, and of child bearing potential, exclude the possibility of pregnancy:
by testing (serum or urine βHCG) within 24 hours prior to the start of IP/IMP
administration;
by surgical history (e.g., tubal ligation or hysterectomy);
post-menopausal with a minimum of 1 year without menses.
8.4.4
Concomitant Medications
Record all medications (prescription and over-the-counter) taken within 24 hours prior to the 
first LUMASON administration in the Concomitant Medication section of the CRF.  
Additionally, record newly prescribed pharmacological treatments in this section up through 24 
hours after the last LUMASON administration. Any medication taken for treatment of an adverse 
event that occurred after the subject signed the informed consent form should be recorded.
Record administration time and dosage information for medications administered as part of the 
pharmacologic stress procedure in a separate section on the CRF. Do not record on the 
concomitant medication CRF page.
8.4.5
Safety Assessments
8.4.5.1
Adverse Events
Monitor subjects for any untoward medical occurrences from the time of signing of Informed 
Consent through 72 hours after the last LUMASON administration or until the subject undergoes 
cardiac intervention, whichever comes first. Adverse events will be collected at the 24 hour 
postdose visit with a follow up phone call/contact at 72 hours.  Any untoward medical 
occurrences as a result of the administration of the NIMP(s) (dobutamine or atropine) will also 
be considered adverse events.  However, the known response pattern resulting from the 
administration of NIMP(s) for the purpose used in this study will not be recorded as adverse 
events. 
Record all untoward medical events in the Adverse Event section of the CRF as specified in 
Section 9.  Only postdose untoward medical occurrences will be tabulated as adverse events.
All serious adverse events that occur during the study monitoring period are required to be 
collected regardless of the relationship to LUMASON or the NIMPs on the Serious Adverse 
Event Report (SAER) Form.


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In addition, the investigator should report any serious adverse events that occur after the 
monitoring period that he/she believes may be related to the LUMASON or the NIMPs on the 
SAER Form.
8.4.5.2
Physical Examination
Perform a physical examination within 24 hours prior to the first LUMASON administration and 
record any significant changes at 24 hours after the last LUMASON administration on the 
Adverse Event CRF.  The subject’s baseline physical examination will be recorded on the 
Medical/Physical exam section of the CRF.
8.4.5.3
Vital Signs
Collect blood pressure and heart rate at the time points listed below.  Obtain all the vital signs in 
a position that is consistent for all time points for each subject.
Rest echocardiography:
within 1 hour prior to the Rest LUMASON (CEUS) administration
30 minutes after the Rest LUMASON (CEUS) administration
Stress echocardiography:
immediately prior to the Stress LUMASON (CE-DSE)
administration
30 minutes after the Stress LUMASON (CE-DSE) administration
1 hour after the Stress LUMASON (CE-DSE) administration*
24 hours after the Stress LUMASON (CE-DSE) administration*
*If stress echocardiography (CE-DSE) is not performed, obtain vitals at 1 and 24 hours after the 
rest LUMASON administration.
8.4.5.4
Laboratory Evaluations
Collect blood samples within 24 hours prior to the first LUMASON administration and 24 hours 
after the last LUMASON administration.  A central laboratory  will perform evaluations for the 
analytes listed in Table B:


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Table B: Laboratory Analytes
Hematology
Clinical Chemistry
Cardiac Biomarker
Hematocrit
Sodium
AST/SGOT
Cardiac Troponin
Hemoglobin
Potassium
ALT/SGPT
RBC count
Chloride
Alkaline 
Phosphatase
WBC count
Glucose
GGT
differential WBC count
Urea Nitrogen
Uric Acid
Platelets
Creatinine
Total Protein
Total Bilirubin
Albumin
LDH
Calcium
Magnesium
Prepare blood samples and ship via overnight courier per the Instruction Manual provided by the 
central laboratory.
8.4.5.5
Electrocardiograms
A 12-Lead electrocardiogram (ECG) will be performed in all subjects at the following time 
points:
Rest echocardiography:
within 1 hour prior to the Rest LUMASON (CEUS) administration
30 minutes after the Rest LUMASON (CEUS) administration
Stress echocardiography:
immediately prior to the Stress LUMASON (CE-DSE)
administration
30 minutes after the Stress LUMASON (CE-DSE) administration
1 hour after the Stress LUMASON (CE-DSE) administration*
24 hours after the Stress LUMASON (CE-DSE) administration*
*If stress echocardiography (CE-DSE) is not performed, obtain ECGs at 1 and 24 hours after 
the rest LUMASON (CEUS) administration.
ECG examinations will be evaluated by a central ECG laboratory  that will provide the ECG 
equipment to the site and will employ a cardiologist to perform the centralized ECG evaluation 
containing the following information:

RR interval

PR interval

QRS interval

QT and QTc interval (Bazett’s and Fridericia’s)

Overall diagnostic assessment

Comparison to Baseline


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Attach a photocopy of the ECG reports to the appropriate page of the CRF. A dual snap electrode 
will be supplied to the site by the central ECG laboratory in order to allow the acquisition of both 
discrete (static) 12-lead ECGs and continuous ECG monitoring described in section 8.4.5.6.
8.4.5.6
Continuous Electrocardiograms
Continuous ECG monitoring will be performed starting 10 minutes prior to the first 
administration of LUMASON (CEUS) and will continue through 30 minutes after the last 
administration of LUMASON.
Continuous ECG examinations will be evaluated on site by the Investigator.
8.4.5.7
Pulse Oximetry
Pulse oximetry will be monitored starting at 10 minutes prior to the first administration of 
LUMASON (CEUS) and continuing through 30 minutes after the last LUMASON 
administration (CE-DSE).  
Pulse oximetry will be evaluated by the on-site Investigator. 
8.5
Imaging Procedures
At each site, the same commercially available echocardiographic equipment and transducers will 
be used for all subjects. Subjects will be examined in either the supine or lateral decubitus 
position. The transmit power (≤ 0.8 MI) and the gain setting will be adjusted at the beginning of 
each procedure to obtain optimal endocardial visualization. Both settings must be kept constant 
throughout each subject’s evaluation. Focus will be set at the far field below the mitral valve. 
Every effort must be made to keep the optimal transducer position for standard views. At each 
site, image acquisition should be performed by the same sonographer throughout each subject’s
study. See Figure 1 for the order of imaging procedures.
Both the rest (UEUS and CEUS) and the stress (UE-DSE and CE-DSE) examinations must be 
identified by subject number and stored as DICOM clips for transfer to the central imaging lab 
according to the instructions in the provided Imaging Manual. The investigational sites are also 
required to maintain a copy of the subject’s examination as a record of the study.
Prior to rest administration of LUMASON (CEUS), further evaluation for intracardiac shunt will 
be demonstrated with agitated saline study to assess the interatrial septum, unless an intracardiac 
shunt has been previously ruled out. If no evidence of intracardiac shunt is confirmed by the 
cardiologist, the subject will further undergo a CEUS echocardiography examination.
8.5.1
Rest Echocardiography
At rest, UEUS will be acquired using harmonic imaging with the standard apical 4-, 2-, and 3-
chamber views. Following the rest UEUS echocardiogram, a rest CEUS echocardiogram will be 
performed with administration of a single 2 mL bolus injection of LUMASON. A recording of 
2D transthoracic harmonic echocardiography imaging will be performed from 30 seconds prior 
to injection of LUMASON and continue until all images are acquired. For the rest CEUS images, 


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the apical 4-chamber view will be acquired first followed by the apical 2- and 3-chamber views.
8.5.2
Pharmacologic Stress with Dobutamine
For stress, dobutamine will be administered intravenously by infusion pump at least 30 minutes 
after the rest LUMASON administration starting at a rate of 5 µg/kg/min. At 3 minute intervals, 
the infusion rate can be increased to 10, 20, 30, and 40 µg/kg/min to reach one of the following 
peak stress endpoints:
Target heart rate:  0.85 x (220 - age);
Development of new or worsening regional wall motion abnormalities;
Peak dose;
Significant arrhythmias;
Severe hypertension (systolic blood pressure >200 mmHg or diastolic blood pressure >110 
mmHg);
Hemodynamically significant hypotension;
Intolerable symptoms.
If a peak stress endpoint is reached, the test can be stopped before reaching the maximal dose of 
dobutamine. If no peak stress endpoint is reached after the maximum dose of dobutamine, 0.25 
mg to 0.5 mg of atropine can be administered intravenously at peak infusion to increase heart 
rate. If peak stress is still not reached, atropine, in divided doses of 0.25 to 0.5 mg up to a total of 
2.0 mg, should be used as needed to achieve target heart rate.
8.5.3
Stress Echocardiography
Immediately after the administration of dobutamine, with or without the addition of atropine, a 
stress UE-DSE echocardiogram will be acquired at peak stress using harmonic imaging with the 
standard apical 4-, 2-, and 3-chamber views. Following the stress UE-DSE echocardiogram, a 
stress CE-DSE echocardiogram will be performed with administration of a single 2 mL bolus 
injection of LUMASON. A recording of 2D transthoracic harmonic echocardiography imaging 
will be performed from 30 seconds prior to injection of LUMASON and continue until all 
images are acquired. For both US-DSE and CE-DSE images, the apical 4-chamber view will be 
acquired first followed by the apical 2- and 3-chamber views.


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Figure 1: Imaging Procedures
8.6
Efficacy Evaluations 
8.6.1
On-Site
The on-site Investigators will provide an overall diagnosis of CAD (Section 8.6.3.3) for each 
subject based on wall motion abnormalities from the results of the unenhanced (UEUS & UE-
DSE) and contrast-enhanced (CEUS and CE-DSE) echocardiographic images.
8.6.2
Off-Site
The off-site efficacy evaluations of the echocardiographic images will be performed by 3 
independent, experienced readers, unaffiliated with the investigational sites and blinded to any 
clinical information for subjects enrolled in this study.  These off-site blinded readers will score 
EBD (Section 8.6.3.1), LV wall motion (Section 8.6.3.2) and overall diagnostic conclusion 
(Section 8.6.3.3).


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8.6.3
Efficacy Measurements
If the subject does not complete the echocardiography examinations (rest, stress or any views are 
missing) the subject will not be included in the efficacy evaluation for on- or off-site.
8.6.3.1
Endocardial Border Delineation
The assessment of EBD will be performed by dividing the heart into 17 segments using the 
standard apical 4-, 2-, and 3-chamber views (see Figure 2) according to the ASE1,12,27 guidelines:
Figure 2: 17-Segment Model
LV EBD will be graded for each segment of the 17-segment model shown in Figure 2 by each 
off-site reader using the following 3-point scale:
0 = Inadequate (endocardial border not visible)
1 = Sufficient (endocardial border barely visible)
2 = Good (endocardial border clearly visible)
For LV EBD, separate assessments will be collected for the unenhanced (UE-DSE) and contrast-
enhanced (CE-DSE) images at peak stress.  
8.6.3.2
LV Wall Motion
LV wall motion will be graded by each off-site reader using the following 3-point scale at rest 
and peak stress for unenhanced (UEUS and UE-DSE) and contrast-enhanced (CEUS and CE-
DSE) images:
1 = Normal
2 = Abnormal (Mild-Moderate, Severe Hypokinesis, Akinetic and/or Dyskinetic)
3 = Unevaluable


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8.6.3.3
Overall Diagnostic Conclusion
The overall diagnostic conclusion will be determined by the on-site Investigator and the off-site 
reader using the scoring system in Table C: 
1 = Normal
2 = Abnormal (Mild-Moderate, Severe Hypokinesis, Akinetic and/or Dyskinetic)
3 = Unevaluable (Inadequate visualization of segments comprising all coronary 
artery territories due to motion artifacts, lateral wall drop out, swirling or 
attenuation artifacts) 
Table C: Overall Diagnostic Conclusion Based on LV Wall Motion
Rest
Stress
Overall Diagnostic 
Conclusion 
1 Normal
1 Normal
Normal
1 Normal
2 Abnormal 
Abnormal
2 Abnormal 
2 Abnormal (no change or 
worsening)
Abnormal
3 Unevaluable
3 Unevaluable
Unevaluable*
*A score of 3 – unevaluable at either rest or stress will result in an overall diagnostic conclusion
of unevaluable.
Separate assessments will be collected for unenhanced (UEUS and UE-DSE) and contrast-
enhanced (CEUS and CE-DSE).
8.6.4
Truth Standard
8.6.4.1
Coronary Angiography
The results of coronary angiography will be collected on-site for any subject who has the 
procedure within 6 months following the LUMASON administration(s).  All coronary 
angiography images should be archived and stored on dedicated digital media according to the 
standard practice at the institution.  Investigational sites are required to maintain the original
images as a record of the study. A copy of the images shall be archived on portable digital media 
(e.g., digital versatile disc [DVD], magnetic optical disc [MOD], compact disc [CD], etc.) that 
will be forwarded to the central imaging laboratory according to the instructions in the Imaging 
Manual. If a subject does not undergo coronary angiography or coronary angiography is 
indeterminate, 6 month follow-up data will be collected to determine if the subject has CAD 
based on clinical information (see Section 8.6.4.2).
A fourth blinded reader will assess the coronary angiography images and provide a diagnosis of 
CAD. The off-site reader will be blinded to the specifics of the protocol including the patient 
population and the results of the echocardiographic examination, but will receive clinical 
information such as the medical history.


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For subjects with coronary angiography as the truth standard, a subject will be diagnosed by the 
blinded off-site coronary angiography reader as Positive if percentage diameter stenosis of any of 
the vessels (RCA, LAD, LCx and LM) is ≥50%; otherwise, a subject will be considered as 
Negative.
8.6.4.2
6 Month Follow Up to Collect Cardiac Events
A 6 month follow up will be required for those subjects who have not had a coronary 
angiography or had coronary angiography with indeterminate results within the 6 months 
following the LUMASON administration(s).
Subjects requiring 6 month follow up will be monitored by a review of their hospital and clinic 
records as well as a phone call follow-up at 6 months to collect cardiac events, as defined by the 
incidence of: a) cardiac death; b) non-fatal myocardial infarction as defined by a greater than two 
fold serial elevation in serum creatine kinase-myocardial B (CK-MB) fraction levels; c) coronary 
revascularization; and d) documentation of the need for coronary revascularization. If a coronary 
angiography is performed during the 6 month follow-up, this will be collected and accepted as 
the truth standard for the subject. Each site will have study personnel who will be responsible 
for collecting phone/clinical record or coronary angiography images during the follow up period.
8.6.5
Off-Site Assessment Methodology
Digital recordings of the echocardiographic images will be blinded, randomized and assessed 
off-site at a core laboratory.  The off-site assessment of the digital recordings will be performed 
by 3 independent, experienced readers, unaffiliated with the investigational sites and blinded to 
any clinical information for subjects enrolled in this study.
In addition, the coronary angiography images will be evaluated by a fourth blinded reader who 
will provide a diagnosis of CAD. This off-site reader will be blinded to the specifics of the 
protocol including the patient population and the results of the ultrasound examination, but will 
receive clinical information such as the medical history.
A separate off-site methodology document will be prepared and will contain the details regarding 
how the images will be anonymized, randomized and presented to the readers. 
Changes to the off-site image assessment will either be reviewed and approved via a protocol 
amendment or described in the separate off-site image assessment document.
9
Reporting Safety Information
The investigator is responsible for the detection, documentation and reporting of adverse events.
AE collection begins when a subject signs the Informed Consent and continues through the 
follow-up period defined in the section 8.4.5.1 of the protocol. In addition, an investigator should 
report any serious AEs that occur after this time period that he/she believes may be related to the 
IP/IMP and/or NIMP(s).


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Any untoward medical occurrence that occurs from the time of signed Informed Consent to the 
time immediately prior to IP/IMP administration will be tabulated in the Clinical Trial Report as 
a “predose event.”  
9.1
Adverse Events
9.1.1
Definitions
An adverse event (AE) is any untoward medical occurrence in a subject or a clinical trial subject 
administered a medicinal product and which does not necessarily have to have a causal 
relationship with the use of the product. 
Any AE that occurs after the follow-up period defined in the protocol is not required to be 
collected in the AE section of the CRF.
An existing condition, which is detected by the diagnostic procedure conducted to test the 
efficacy of an investigational contrast agent, is not considered an AE.
Symptoms or medically significant laboratory or instrumental (e.g., electrocardiographic) 
abnormalities of a pre-existing condition, such as cancer or other disease, should not be 
considered an AE. However, new symptoms and laboratory or instrumental abnormalities, as 
well as worsening of pre-existing ones are considered AEs.
A serious adverse event (SAE) is any untoward medical occurrence that at any dose: 
results in death, 
is life-threatening (i.e. the subject was at risk of death at the time of the event/reaction; it 
does not refer to an event/reaction which hypothetically might have caused death if it were 
more severe),
requires in subject hospitalization or prolongation of existing hospitalization, 
results in persistent or significant disability/incapacity (where disability is defined as a 
permanent or substantial disruption of ability to carry out normal life functions, either 
reported or defined as per clinical judgment),
is a congenital anomaly/birth defect, 
is an important medical event that may not result in death, be life-threatening, or require 
hospitalization but may be considered serious when, based upon appropriate medical 
judgment, it may jeopardize the subject and may require medical or surgical intervention to 
prevent any of the outcomes listed in the definition above.  Examples of such events 
include allergic bronchospasm requiring intensive treatment in an emergency room or at 
home, and blood dyscrasias or convulsions that do not result in hospitalization, or 
development of drug dependency or drug abuse. 
Any suspected transmission via a medicinal product of an infectious agent is also considered an
SAE.
A non-serious AE is any AE that does not meet the criteria listed above for a SAE.
The reference safety document will be the same for the whole clinical trial.


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An unexpected AE is one where the nature, severity, specificity or outcome is not consistent with 
the applicable product reference document.
In particular, the sponsor will determine the expectedness of the AEs reported during this clinical 
trial according to the following reference document(s):
LUMASON: latest version of the Investigator’s Brochure (IB),
Dobutamine: latest version of PI/SPC approved in the country where the site is located which
administered the NIMP to the study subject,
Atropine: latest version of PI/SPC approved in the country where the site is located which 
administered the NIMP to the study subject.
If an updated version of the reference safety document becomes available during the conduction 
of the study and before the last subject out, the Sponsor will provide the new version to all 
investigators. For Europe, the new version will be provided to all investigators, ECs and CAs.
The new version will become the reference safety document for assessing the expectedness of 
AEs.
An AE with fatal outcome should be considered unexpected unless the reference safety 
document specifically states that the event might be associated with a fatal outcome. 
When it is uncertain whether an AE is expected, the AE should be treated as unexpected.
9.1.2
Expedited Reporting a Serious Adverse Event to Sponsor
The Investigator must report all serious adverse events within 24 hours, by telephone or by fax, 
to both the Drug Safety group and the Clinical Manager of the Sponsor as listed in Table D. 
A Serious Adverse Event Report (SAER, see Appendix D) must be completed by the 
Investigator and faxed to the Sponsor (both the Drug Safety group and the Clinical Manager) 
within 3 calendar days after the Investigator first became aware of the serious event.  The top 
two copies of the completed SAER should also be sent to the Sponsor by traceable mail.  The 
bottom copy remains on-site with the CRF. 
In case of death, a comprehensive narrative report of the case should be prepared by the 
Investigator and sent to both the Drug Safety group and the Clinical Manager of the Sponsor by 
traceable mail together with the SAER, with a copy retained on-site with the CRF.  If an autopsy 
is performed, a copy of the autopsy report should be actively sought by the Investigator and sent 
to the Sponsor as soon as available with a copy retained on-site with the CRF.
A follow-up SAER should be filled in by the Investigator if important follow-up information 
(e.g., diagnosis, outcome, causality assessment, results of specific investigations) becomes
available after submission of the initial SAER.  The follow-up SAER should be sent to the 
Sponsor in the same manner and following the same timeline as described for the initial.
If the Investigator becomes aware of any serious adverse events within the follow-up window 
established in the protocol following IP/IMP administration, they will be reported to both the
Drug Safety group and the Clinical Manager of the Sponsor as described above.


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If outside the follow-up window established in the protocol, any serious adverse events are 
reported to the Investigator, which he/she believes are related to the administration of the IP/IMP
and/or NIMP(s), it is the Investigator’s responsibility to report this serious adverse event to both 
the Drug Safety group and the Clinical Manager of the Sponsor.  Such serious adverse events 
will be reported using a Serious Adverse Event Report or any other way chosen by the 
Investigator.  Do not use the Case Report Form.
The names, telephone, fax numbers, and email address of the contact persons are given below in 
Table D.
Table D: Serious Adverse Event Reporting – Sponsor Contact Personnel
Name/Title
Office Telephone 
Number
Alternate Telephone 
Number
Fax Number / E-mail
Europe
 
 
 
 
 
* To be used outside the normal business hours.
For additional SAE questions call the Medical Expert. The contact details are on the cover page 
of the protocol.
9.1.3
Breaking the Study Blind
This is an open-label, nonrandomized study.
9.1.4
Data Collection
The Investigator will collect adverse events through non-leading questions and examination of 
the subjects/subjects.
For each event, record the following information in the Adverse Event section of the Case Report 
Form:
Classification of the Event: Classify the event as either serious or non-serious (see 
definitions in Section 9.1.1).
Description of Signs or Symptoms: Whenever possible, record a specific diagnosis for 
the event.  If a diagnosis cannot be made, then record each sign or symptom separately. If 
multiple episodes of an event occur, separated by an appropriate time interval to justify 


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considering the subsequent episodes as a repeat occurrence, record each episode separately 
on the Case Report Form.  Indicate if the event is local (i.e., occurring at the site of 
administration). For serious AEs only: provide a detailed chronological description of the 
clinical course of the event(s) and of all relevant signs and symptoms.
Onset Date and Time: Record the date and time the event started.  If a change from 
predose in a laboratory test is reported as an adverse event, record the start date as the date 
of collection of the first lab sample that shows the change.
Stop Date and Time: Record the date and time the event resolved.  If a change from 
predose in a laboratory test is reported as an adverse event, record the stop date as the date 
of collection of the first postdose sample that shows a return to the predose level.
Intensity: 
1. Mild: Event not resulting in disability/incapacity, which resolves without treatment.
2. Moderate: Event not resulting in disability/incapacity, which requires treatment.
3. Severe: Event resulting in temporary and/or mild disability/incapacity, which 
requires treatment.
Relationship to the IP/IMP/NIMP:  Make every effort to determine the cause of each 
adverse event.  Classify the correlation between the IP/IMP/NIMP and the adverse event as 
follows:


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1. Reasonable
Possibility
The event falls into one of the two following categories:
1) a) The event follows a reasonable temporal sequence 
from administration of the IP/IMP and/or NIMP; 
AND
   b) The event follows a known response pattern to the 
IP/IMP and/or NIMP but could have been produced by any 
of the following features: 
the subject’s clinical state, or
other therapy administered, or
the diagnostic/interventional procedure;
OR
   c) The event cannot be reasonably explained by any of the 
following features:
the subject’s clinical state, or
other therapy administered, or
the diagnostic/interventional procedure;
OR
       d) There is evidence of partial or complete disappearance    
of the event after withdrawal of the product (positive 
dechallenge).
2)    The report of the event contains:
       a) conflicting data 
AND/OR
       b) dubious or insufficient/poor evidence
2. No reasonable
Possibility
The event is either a predose event or is definitely due to 
causes separate from the administration of the IP/IMP, i.e.,
documented pre-existing condition
technical and manual procedural problems
concomitant medication and/or NIMP
the subject’s clinical state
the event is judged as not related and does not fall 
under either of the categories for “reasonable 
possibility”
erroneous administration of treatment
Action Taken: 
0. None
1. Change in the IP/IMP administration (including brief interruption of administration 
of total dose and early termination of administration, i.e., dose reduction)


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2. Drug treatment required (a medication was prescribed or changed; record in the 
Concomitant Medication section of the Case Report Form)
3. Non-drug treatment required (a non-drug treatment was prescribed or changed, record 
under “Comments” in the Adverse Event section of the Case Report Form)
4. Hospitalization or prolonged hospitalization
5. Diagnostic or clinical test(s) conducted (attach a copy of the results to the Case 
Report Form) 
6. Subject discontinued from the study
Subject Outcome:  
1. Recovered without sequelae
2. Recovered with sequelae (describe the sequelae under “Comments” in the Adverse 
Event section of the Case Report Form)
3. Not Recovered, event on-going (follow the subject until a definite outcome can be 
determined.  When follow-up data are collected, report follow-up information under 
“Comments” in the Adverse Event section of the Case Report Form; if the event is 
serious, fill in a follow-up Serious Adverse Event Report)
4. Died (list primary cause of death under “Event Description” in the Adverse Event 
section of the Case Report Form; if available, obtain a copy of the autopsy report to 
the Case Report Form and send a copy to Sponsor)
Comments:
Provide other pertinent clinical information and observations, and the rationale for the 
provided causality under “Comments” in the Adverse Event section of the Case Report 
Form.  For example, record predisposing or contributing conditions, such as previous 
history, concomitant diseases or medications, and/or procedural risks and explain the 
reasoning for attributing the event(s) to the cause chosen.
If the investigator states that there is no reasonable possibility that the event is related, 
he/she should provide details of an alternative explanation for the event. 
9.1.5
Subject Follow-Up
Make every attempt to follow the subject until the adverse event is resolved, stabilized, returned
to baseline or deemed irreversible.
9.2
Special Situations
The Investigator must report all special situations within 24 hours by telephone or by fax using 
the Special Situation form supplied by the Sponsor, to both the Drug Safety group and the 
Clinical Manager of the Sponsor as listed in Table D. 
Special situations include but are not limited to the following: accidental overdose of IP/IMP
and/or NIMP, medication error with IP/IMP and/or NIMP (such as administration of the wrong 
drug or wrong dose, wrong administration rate or wrong technique in drug usage process, 
administration via the wrong route, radiation under-dose, labeled drug-drug or drug-disease or 
drug-food interaction). In addition, any notification of lactation or pregnancy of a subject or 
partner must be reported by the Investigator to the Sponsor within 24 hours using the appropriate 


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(i.e., Lactation or Pregnancy Report) form.  Any associated SAEs must be reported concurrently 
using the SAER.  
In case urgent safety measures are taken during the conduct of the study, these will be 
communicated in a timely fashion by the Sponsor to the applicable regulatory authorities, 
IRBs/ECs/REBs and also to concerned investigators and subjects.
9.3
Laboratory Evaluations
9.3.1
Reporting and Evaluation of Central Laboratory Test Results
The central laboratory will send a report of the laboratory results to the Investigator within 48 
hours after the sample is picked up from the investigational site.  Attach a copy of the laboratory 
report to the Case Report Form. 
The Investigator or sub-investigator must review laboratory values within 24 hours of receipt of 
the laboratory report. After the review is completed, the Investigator must sign and date each 
laboratory report.
The central laboratory will provide normal reference ranges for the laboratory tests on the 
laboratory results report.  A value is normal when it falls on or within the upper and lower limits 
of the reference range.  A value is abnormal when it falls outside the upper or lower limit of the 
reference range.  The central laboratory will flag all abnormal values on the laboratory report and 
will verify that the result is not due to pre-analytical problems (e.g., sample taken improperly, 
sample stored incorrectly, sample labeled incorrectly) or to analytical problems (e.g., machine 
not accurately calibrated, technical problems with equipment or reagents, deterioration of 
analyte).  
The central laboratory has established ‘alert criteria’ for some laboratory tests and will make an 
immediate telephone call to the Investigator in the event that a laboratory result meets these 
criteria.
The Investigator must evaluate any change from predose to postdose in a laboratory test which 
represents a worsening of the subject’s clinical state as to whether it meets the definition of an 
adverse event.  Record all changes determined to meet the definition of an adverse event in 
the Adverse Event section of the Case Report Form.  
9.3.2
Repeat Testing
Collect additional samples to repeat the lab test that represents a worsening of the subject’s 
clinical state and meets the definition of an adverse event, until the value returns to the predose 
level or clinically stabilizes, or until the Investigator or physician of record determines that 
further follow-up is unnecessary.


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9.3.3
Emergency Laboratory Analysis
If a laboratory result needs to be obtained immediately, split the sample and send one-half to the 
local laboratory for immediate analysis and the other half to the central laboratory.  Attach 
results from both laboratories to the Case Report Form.
9.4
Electrocardiograms
If a worsening from predose to postdose is observed on an ECG, repeat the ECG at the discretion 
of the Investigator, in addition to obtaining an ECG at the postdose time points required by the 
protocol.  The Investigator will assess any worsening from predose to postdose in the ECG for 
clinical relevance (whether it meets the definition of an adverse event).  Record all changes 
determined to meet the definition of an adverse event in the Adverse Event section of the Case 
Report Form.
9.5
Continuous ECGs
If a worsening is observed during the continuous ECG monitoring, the Investigator will assess 
the worsening for clinical relevance (whether it meets the definition of an adverse event).  
Record all changes determined to meet the definition of an adverse event in the Adverse Event 
section of the CRF.
9.6
Physical Examinations
If a worsening from predose to postdose is observed in a physical examination, repeat the 
physical examination at the discretion of the Investigator, in addition to obtaining a physical 
examination at the postdose time points required by the protocol.  Document any change from 
predose to postdose in the Physical Examination section of the Case Report Form.  The 
Investigator will evaluate any worsening at the postdose physical examination for their clinical 
relevance and to determine whether they meet the definition of an adverse event.  Record all 
changes determined to meet the definition of an adverse event in the Adverse Event section of 
the Case Report Form.  Record specific signs, symptoms, and/or laboratory information 
supporting these changes.
9.7
Vital Signs
If a worsening from predose to postdose is observed in vital signs, repeat the vital sign 
measurement at the discretion of the Investigator, in addition to obtaining vital sign 
measurements at the postdose time points required by the protocol.  The Investigator will 
evaluate any worsening in vital signs for its clinical relevance as to whether it meets the 
definition of an adverse event.  Record all changes determined to meet the definition of an 
adverse event in the Adverse Event section of the Case Report Form.


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9.8
Pulse Oximetry
If a worsening is observed during the continuous pulse oximetry monitoring, the Investigator will 
assess the worsening for clinical relevance (whether it meets the definition of an adverse event).  
Record all changes determined to meet the definition of an adverse event in the Adverse Event 
section of the CRF.
10
Statistical Methods
Summary statistics (mean, median, standard deviation, minimum, and maximum) will be 
provided for continuous variables, and the number and percentage of each category will be 
provided for categorical data. Unless otherwise specified, the statistical tests will be 2-sided at 
0.05 level of significance. 
Any changes in the original statistical methodology will be documented in the statistical analysis 
plan (SAP). 
All statistical analyses will be performed using SAS® software.
10.1
Subject Disposition and Demographic and Baseline Characteristics
Summary tables will be provided for the number of subjects who have been enrolled, dosed and 
completed according to the protocol. The number of subjects who prematurely discontinued the 
study and the reasons for their discontinuation will be summarized. Summary statistics will be 
presented for demographic and baseline characteristics, including age, sex, race, height, weight, 
BMI, and other relevant study entry criteria.
10.2
Analysis Population
Safety Analysis Population – all subjects who receive LUMASON will be included in the safety 
analysis population.
Efficacy Analysis Population – all subjects who receive LUMASON, and have data available at 
peak stress for both UE-DSE and CE-DSE and a definitive diagnosis (Positive, Negative) for 
CAD from truth standard (coronary angiography or 6 months follow-up data) will be included in 
the modified Intent-to-Diagnose (ITD) population.
10.3
Concomitant Medications
Concomitant medications will be coded according to therapeutic area using the WHO drug 
reference list. Concomitant medications will be presented in data listings and summarized by 
frequency counts according to anatomical and therapeutic area for all subjects dosed.


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10.4
Extent of Exposure
Descriptive statistics will be presented to summarize the volume (mL) of IP/IMP administered. 
Dose administration for IP/IMP will be listed for each injection by subject. The peak dose of 
dobutamine and total dose of atropine will be summarized and listed for each subject.
10.5
Safety Analysis
The safety data will be summarized for all subjects dosed. Summary tables, including clinically 
significant changes wherever applicable, will be presented for the following safety endpoints:

Adverse Events

Clinical Laboratory Evaluations

Electrocardiograms

Vital Signs

Special Situations
For vital signs and ECGs, the baseline for time points at rest will be the last measurement prior 
to the IP/IMP administration at rest; while the baseline for time points at stress will be the 
measurement which is collected after administration of the pharmacologic stress agent, 
dobutamine, and prior to LUMASON administration at stress (CE-DSE). All adverse events will 
be coded by MedDRA and summarized by system organ class and preferred term, by intensity 
and by causal relationship to the IP/IMP.  Adverse event will be summarized separately for the 
NIMP(s). Only those events which occur from the start of IP/IMP administration through the 
follow-up period defined in the protocol will be tabulated in the Clinical Trial Report as “adverse 
events” see Section 9.1.1. 
10.6
Efficacy Analysis 
The echocardiographic images will be assessed by 3 off-site blinded readers independent of the 
investigational sites; accordingly, the efficacy analysis will be performed separately for each off-
site reader. An additional, fourth, blinded reader will assess the coronary angiography images 
and provide a diagnosis of CAD.
10.6.1
Primary Efficacy Endpoint – Sensitivity and Specificity for Detection and 
Exclusion of CAD
10.6.1.1
Truth Standard Diagnosis
For subjects with coronary angiography as the truth standard, a subject will be diagnosed by the 
blinded off-site coronary angiography reader as Positive if percentage diameter stenosis of any of 
the vessels (RCA, LAD, LCx and LM) is ≥50%; otherwise, a subject will be considered as 
Negative. 
For the remaining subjects who didn’t have coronary angiography as the truth standard, a subject 
will be Positive if the subject had cardiac death, documented myocardial infarction, documented 


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revascularization or need for revascularization during follow up; otherwise, the subject will be 
considered as Negative.
10.6.1.2
Evaluation of CAD
The overall diagnostic echocardiographic conclusion will be classified by the off-site readers as 
Normal, Abnormal or Unevaluable for unenhanced (UEUS and UE-DSE) and LUMASON-
enhanced (CEUS and CE-DSE) echocardiography respectively. 
The diagnostic performance of echocardiography (UE-DSE and CE-DSE) will be derived based 
on the diagnosis from stress echocardiography and diagnosis from truth standard (see Table E). 
Table E: Cross Tabulation of Diagnosis: Truth Standard vs. Stress Echocardiography
Truth Standard 
(TS)
Stress Echocardiography
Normal
Unevaluable*
Abnormal
Negative
TN
FP
FP
Positive
FN
FN
TP
TN = true negative;  TP = true positive;  FN = false negative;  FP = false positive
*Unevaluable is defined as: inadequate visualization of segments comprising all coronary artery territories due to 
motion artifacts, lateral wall drop out, swirling or attenuation artifacts.
The null hypothesis is that there is no difference in sensitivity/specificity between CE-DSE and 
UE-DSE at peak stress; the alternative hypothesis is that CE-DSE is superior to UE-DSE in 
terms of higher sensitivity/specificity: 
H0:  DiagCE - DiagUE 0,
Ha:  DiagCE - DiagUE > 0; 
where DiagCE and DiagUE denote sensitivity/specificity for CE-DSE and UE-DSE at peak stress
respectively.
The sensitivity and the specificity along with their 95% confidence intervals (CIs) will be 
calculated, and the difference in sensitivity/specificity will be tested using McNemar's Chi-
square 2-sided test.
In addition, inter-reader agreement for the detection/exclusion of CAD for CE-DSE and UE-DSE 
will be evaluated by kappa statistic.
10.6.2
Co-Primary Efficacy Endpoint – Critical Shift of Subjects with Suboptimal 
Images to Adequate Images
The co-primary endpoint:  The proportion of subjects with suboptimal images (≥2 adjacent 
segments with inadequate LV EBD in any of the 3 apical views) at UE-DSE converted to 
adequate images (reduction of suboptimal adjacent segments to obtain adequate images) at CE-
DSE will be estimated along with the 95% CIs.


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Additionally, the following supportive analyses will be performed:

proportion of subjects with optimal images at UE-DSE to suboptimal (≥2 adjacent 
segments with inadequate LV EBD in any of the 3 apical views) at CE-DSE in terms of 
LV EBD visualization will be estimated along with the 95% CIs.

The percentage of patients with at least one segment with inadequate border delineation 
will also be summarized for each of the views for both UE-DSE and CE-DSE.

Inter-reader agreement among 3 off-site readers on the critical shift of subjects with 
suboptimal images at UE-DSE to adequate images at CE-DSE will be evaluated by kappa 
statistic.
10.6.3
Secondary Efficacy Endpoints
For the secondary efficacy endpoint of change in total LV EBD scores at peak stress from UE-
DSE to CE-DSE, the total LV EBD score is calculated as the sum of the individual scores (0, 1, 
or 2) assigned to each of the 17 ventricle segments with a range from 0 to 34. 
For each blinded reader assessment, paired t-test will be used to compare total LV EBD score 
between the CE-DSE and UE-DSE. Summary statistics will be provided for the total LV EBD 
score from UE-DSE and CE-DSE. The change in total LV EBD score from UE-DSE to CE-DSE
will be summarized along with the 95% CIs and tested by paired t-test. The cross tabulated (for 
UE-DSE vs. CE-DSE) distribution of the individual scores (0, 1, or 2) assigned to each of the 17 
ventricle segments will be presented.
Inter-reader agreement among 3 off-site readers on the EBD score for each segment from UE-
DSE and CE-DSE will be evaluated by kappa statistic.
10.6.4
Success Criteria
The study will be considered a success if:

for the primary efficacy endpoint of sensitivity/specificity, CE-DSE is superior to UE-
DSE for at least 2 out of the 3 blinded readers and for the same 2 readers a lower limit of 
2-sided 95% CI for sensitivity/specificity is ≥50% (a rate considered greater than 
chance);

for co-primary efficacy endpoint of proportion of subjects with critical shift of 
suboptimal images at UE-DSE to adequate images at CE-DSE, for at least 2 out of the 3 
blinded readers lower limit of 2-sided 95% CI is above 35%.
10.7
Sample Size
Software nQuery Advisor 7.0 is used for sample size determination in this study.
As suggested by FDA during the type C meeting on 13Aug2014, and as also reinforced and 
documented in the official meeting minutes issued by FDA on 24Sep2014, this study needs to 
demonstrate 1) a significant improvement in diagnostic performance endpoints, i.e., 
sensitivity/specificity from unenhanced stress imaging (UE-DSE) to contrast-enhanced stress 


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imaging (CE-DSE) in term of detection and exclusion of CAD, and 2) lower the CI of CE-DSE 
sensitivity and specificity greater than 50% (a rate greater than chance).
For significant improvement in diagnostic performance endpoints, the null hypothesis is that 
there is no difference in sensitivity/specificity between CE-DSE and UE-DSE at peak stress; the 
alternative hypothesis is that CE-DSE is superior to UE-DSE in terms of higher 
sensitivity/specificity: 
H0:  DiagCE - DiagUE 0,
Ha:  DiagCE - DiagUE > 0; 
where DiagCE and DiagUE denote sensitivity/specificity for CE-DSE and UE-DSE at peak stress
respectively.
With 2-sided significance level (alpha) of 5%, and the probability of a Type II error () of 0.20, 
assuming expected difference between CE-DSE and UE-DSE is 25%,1 and the proportion of 
discordant pairs is 40%, 45 subjects will be needed for sensitivity/specificity respectively. 
For lower CI of CE-DSE sensitivity/specificity greater than 50%, assuming sensitivity/specificity 
of CE-DSE = 65%, about 45 CAD positive subjects are needed to have 95% confidence that the 
lower limit of sensitivity >50%, and 45 CAD negative subjects are needed to have 95% 
confidence that lower limit of specificity>50%.  
Assuming approximately 30% of subjects enrolled will be CAD positive,13 in order to obtain 45 
CAD positive subjects, 150 subjects in total need to be enrolled. With a total of 150 subjects 
enrolled, this will also provide more than enough CAD negative subjects according to the above 
calculations.  The prevalence of positive subjects will be tracked based on on-site CAD diagnosis 
from the truth standard and if less than 30% of subjects are CAD positive, additional subjects 
will need to be enrolled to obtain sufficient CAD positive subjects.
For the co-primary endpoint of critical shift from suboptimal at UE-DSE images to adequate 
images at CE-DSE, sample size assumptions are based on the results from the previous pivotal 
phase III Bracco-sponsored study 
).22 This study had 44.8%-46.0% of the subjects 
converted from suboptimal at UEUS to adequate at LUMASON-enhanced echocardiography
(CEUS) at rest. When the sample size is 150, a 2-sided 95% CI for a single proportion using the 
large sample normal approximation will extend 8% from the observed proportion for an expected
proportion of 45% subjects with critical shift from suboptimal at UE-DSE images to adequate 
images at CE-DSE. This can be translated as lower limit of 95% CI >35% for the observed 
proportion of subjects with critical shift.
Considering approximately 15% of subjects enrolled are anticipated to dropout, approximately 
175 subjects will need to be enrolled.
10.8
Data Handling
All data collected will be entered into the database and displayed in the data listings and/or
tables. Details of the data handling procedures will be specified in the SAP.


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10.9
Interim Analyses
No interim analysis is planned.
11
Ethics and Good Clinical Practice
11.1
Ethical and Regulatory Compliance 
The study will be conducted in accordance with the protocol, ICH, Good Clinical Practice, FDA 
regulations, ethical principles that have their origin on the Declaration of Helsinki and all 
applicable local regulations, whichever offers greatest protection for the subject.
11.2
Informed Consent and Subject Information
All subjects must sign and personally date an approved Informed Consent Form after receiving 
detailed written and verbal information about the reason, the nature and the possible risks 
associated with the administration of the IP/IMP and NIMP(s).  This must be done according to 
the guidelines provided in the Declaration of Helsinki, ICH E6 Guideline for Good Clinical 
Practice, and as applicable, the requirements of Title 21 CFR 50.20 through 50.27 and the EU 
Directive 2001/20/EC, 2005/28/EC, and related guidance, and applicable laws and regulations 
of Health Canada.
The subject must be made aware and agree that personal information may be scrutinized during 
inspection or audit by competent authorities and properly authorized persons.  However, personal 
information will be treated as strictly confidential and will not be publicly available in 
accordance to the Guidelines for Data Processing within the Framework of Clinical Drug Trials 
24 July 2008.
11.3
Institutional Review Board/Ethics Committee/Research Ethics Board
Approval
The protocol, Informed Consent Form, Subject Information Sheet, if applicable, and any 
advertisement for the recruitment of subjects must be reviewed and approved by an appropriately 
constituted IRB/EC/REB, as required in chapter 3 of the ICH E6 Guideline, and as applicable, 
Title 21 CFR 56.107 through 56.115 and EU Directive 2001/20/EC, 2005/28/EC, and related 
guidance, and applicable laws and regulations of Health Canada. Written IRB/EC/REB approval 
must be obtained by Sponsor prior to shipment of IP/IMP or subject enrollment.  
The Investigator is committed in accordance with local requirements to inform the IRB/EC/REB
of any emergent problem, serious adverse events, and/or protocol amendments.


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11.4
Financial Disclosure
Financial support to Investigators/Sub-investigators other than the cost of conducting the clinical 
study or other clinical studies will be disclosed where applicable in accordance with Title 21 
CFR 54.2 to 54.6 and the EU Directive 2001/20/EC, 2005/28/EC and related guidance.
11.5
Safety Monitoring Boards
There will be no safety monitoring board for this study.
12
Administrative Considerations
12.1
Regulatory Requirements–Sponsor/Investigator Obligations
This study will be conducted in accordance with the Declaration of Helsinki, ICH E6 Guideline, 
and as applicable, Title 21 CFR 312.50 through 312.70, and EU Directive 2001/20/EC and 
2005/28/EC related guidance, and applicable laws and regulations of Health Canada. To ensure 
compliance the Investigator agrees, by written consent to this protocol, to fully cooperate with 
compliance checks by allowing access to all documentation by authorized individuals, and 
competent and regulatory authorities.
12.2
Sponsor Regulatory Obligations for SUSARs Reporting
The Sponsor shall ensure that all relevant information about suspected unexpected serious 
adverse reaction (SUSAR) which occurs during the course of the clinical trial, whether or not 
this event is fatal or life-threatening, will be reported to the applicable Regulatory 
Authority/Competent Authorities including but not limited to FDA, Health Canada or any 
European Economic Area State in which the trial is being conducted and to the relevant ECs in 
accordance with EU Directive 2011/C 172/01 about Communication from the Commission —
Detailed guidance on the collection, verification and presentation of adverse event/reaction 
reports arising from clinical trials on medicinal products for human use (‘CT-3’).
12.3
Protocol Amendment
No change to the protocol may be made without the joint agreement of both the Investigator and 
Sponsor.  Any amendment to the original protocol will be signed by both parties and submitted 
to the IRB/EC/Competent Authority (CA)/REB for approval or notification prior to 
implementation except in circumstances when it is necessary to implement urgent safety 
measures to remove an immediate hazard to the study subject(s).
12.4
Curriculum Vitae
The Investigator and any sub-investigator(s) must provide Sponsor with current copies of their 
own signed and dated curriculum vitae.


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12.5
Administrative Structure
The administrative structure of the study (e.g., monitoring and vendor personnel, statistician, 
laboratory facilities, and clinical trial supply management) is presented in Appendix F.  A 
complete and controlled list of the Investigators, study sites, and IRBs/ECs/REBs involved in 
this study can be found in the Trial Master File maintained by the Sponsor.
12.6
Protocol Deviations, Violations and Exceptions
As a matter of policy, Bracco will not grant exceptions to protocol-specific entry criteria to allow 
subjects to enter a study.  If investigative center personnel learn that a subject who did not meet 
protocol eligibility criteria was entered in a study they must immediately inform Bracco.
If a violation is serious, and meet the definition of a serious breach the Clinical Research 
Manager (CRM) takes appropriate action according to the requirements and timelines stated the 
Code of Federal Regulations (CFR), European Guidance and local regulations as applicable.
12.7
Monitoring Procedures
12.7.1
Study Monitoring
An appropriate representative of Sponsor (Study Monitor) will maintain contact with the 
Investigator and will visit the study site for the purpose of discussing and/or retrieving data.
An initiation visit will be made by the Study Monitor to discuss the protocol and the obligations 
of both the Sponsor and the Investigator.  The Investigator must allow the Study Monitor to 
perform periodic, interim monitoring visits.  The purposes of these visits are to:
verify that written Informed Consent was obtained prior to each subject’s participation in 
the study;
assess the progress of the study;
review the compliance with the study protocol;
determine whether all adverse events were appropriately reported;
determine whether the Investigator is maintaining the essential documents;
discuss any emergent problem;
check the Case Report Forms for legibility, accuracy and completeness;
validate the contents of the Case Report Forms against source documents;
assess the status of IP/IMP storage, dispensing and retrieval.
All data required by the protocol must be reported accurately on the Case Report Form and must 
be consistent with the source documents.  Source documents are original documents, data and 
records (e.g., hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ 
diaries or evaluation checklists, pharmacy dispensing records, recorded data from automated 
instruments, copies, magnetic media, X-rays or other diagnostic images, subject files, laboratory 
records).  The Investigator will make available the source documents for inspection.  This 
information will be considered confidential.


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The Study Monitor will perform a close-out visit at the conclusion of the Investigator’s 
involvement in the study.
12.7.2
Case Report Form
The Sponsor will provide a Case Report Form for each subject.  The Investigator must record all 
data on the Case Report Form and archive a copy of the completed Case Report Form at the 
investigational site.  Case Report Forms must be completed for all subjects who sign Informed 
Consent even if the subject fails to complete the study.
If requested, copies of the Case Report Forms are to be made available to the appropriate 
Competent and Regulatory Authorities.
12.7.3
Inspection and Auditing
The Investigator/Institution will make available for direct access all trial related records
including source documentation for inspection by Competent and Regulatory Authorities, 
IRB/EC/REB and for auditing by the Sponsor.  This information will be considered confidential.
12.8
Archiving of Records
Essential documents (copies of the protocol, subject identification codes, CRF, source data, Informed 
Consent Form and other documents) pertaining to the study conduction must be kept for the 
maximum period of time as required by the study center and by the applicable local regulations. For 
non-European sites, this time period must be at least 2 years after the last approval of the 
marketing application of the IP/IMP in an ICH region and until there is no pending or 
contemplated marketing application in an ICH region or at least two years have elapsed since the 
formal discontinuation of clinical development of the IP/IMP. For EU sites, according to EU 
Directive 2005/28/EU this time period must be at least 5 years after study completion. This period 
may be extended if required by local law.
No study document should be destroyed without prior written agreement between Sponsor and the 
Investigator.
Originals of all documentation and copies of outgoing correspondence concerning the study will be 
stored and retained in a safe area in the Trial Master File of Sponsor for the lifetime of the product. In 
particular, the final report must be retained by Sponsor, or the subsequent owner, for five years 
beyond the lifetime of the IP/IMP.
12.9
Study Results
A final report of the study results will be written by Sponsor or its designee. 
The Clinical Trial Report (CTR) will be written within one year of the notification of the end of 
study and sent to the involved ECs and CAs. It will be reviewed and approved by the 
Investigator when required by local authorities.


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12.10 Use and Publication of Study Results
All unpublished documentation (including the Protocol, Case Report Form and Investigator’s 
Brochure) given to the Investigator is strictly confidential.  All recipients must agree not to 
disclose the information herein contained to any person without the prior written authorization of 
Sponsor.  The submission of these documents to the IRB/EC/CA/REB is expressly permitted.  
The Investigator agrees that Sponsor maintains the right to use the results of this study in their 
original form and/or in a global report for submission to governmental, competent, and 
regulatory authorities of any country.
The results of the study may be presented during scientific symposia or published in a scientific 
journal only after review by Sponsor in accordance with the guidelines set forth in the applicable 
publication or financial agreement. 
12.11 Financing
A financial agreement (separate from the protocol) will be made with the Investigator or 
designee.  Such agreement will be archived in the relevant file.
12.12 Change in Investigator
In the event that the Investigator is unable to continue the study, another suitable person at the 
site will become the designated Investigator, and documentation testifying to this will be 
submitted to Sponsor.  The new Investigator must be acceptable to both Sponsor and the 
IRB/EC/REB before the study can be continued.
12.13 Definition of the End of the Study
The end of the study is defined asthe last subject’s image review conducted by the off-site 
blinded assessor(s).
12.14 Premature Termination of the Study
If Sponsor, the Investigator, or IRB/EC/CA/REB should discover conditions arising during the 
study that indicate the study should be prematurely terminated, an appropriate schedule for 
termination will be instituted.  If the Investigator prematurely terminates the study, an 
explanatory letter must be provided to Sponsor.
Sponsor also reserves the right to discontinue this study for administrative reasons at any time.  
The Investigator will be reimbursed for reasonable expenses incurred, if it is necessary to 
prematurely terminate the study or an individual subject’s participation.  Sponsor will not 
reimburse the Investigator for the evaluation of subjects if the evaluations are not conducted in 
compliance with the present protocol.


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12.15 Information Material
Before the beginning of the study the Investigator will be given the current version of reference 
safety document, Investigator’s Brochure.  If the Investigator’s Brochure is revised during the 
study, the Investigator will receive a copy of the revised version.  The Investigator’s Brochure
and the protocol are confidential communications of Sponsor.  Acceptance constitutes the 
agreement by the recipient that no unpublished information herein contained will be published or 
disclosed without Sponsor’s prior written approval except that this document may be disclosed to 
appropriate IRB/EC/CA/REB as long as they are required to keep it confidential.
12.16 Insurance Policy
Whenever applicable, Sponsor will provide verification of insurance coverage for damages 
emerging from the study and involving test subjects treated with the IP/IMP.  The Investigator 
will be supplied with all data concerning the insurance company and policy number for a 
maximum sum insurable as stated in the Subject Information Sheet and Informed Consent Form.
13
Confidentiality
All information provided to the Investigator dealing with the IP/IMP will be regarded as 
confidential.  The members of the research team agree not to discuss such information in any 
way without prior written permission from Sponsor. 


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14
Protocol Acceptance
I agree to conduct this clinical study according to the above protocol and to make no additions or 
changes without prior consent of Sponsor and in accordance with good clinical practice and local 
requirements/regulations.
____________________________________
___________________
Investigator (Signature)
Date
____________________________________
Investigator (Printed Name)
____________________________________
___________________
For Bracco (Signature)
Date


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15
References
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Echocardiography Recommendations for Performance, Interpretation, and Application of 
Stress Echocardiography. J Am Soc Echocardiogr. 2007 Sep;20(9):1021-41.
2 Anantharam,B, Chahal N, Chelliah R, Ramzy I, Gani F, Senior R:Safety of Contrast in Stress 
Echocardiography in Stable Patients and in Patients With Suspected Acute Coronary 
Syndrome but Negative 12-Hour Troponin Am J Cardiol. 2009;104:14 –18.
3 Gaibazzi N, Rigo F, Reverberi C. Detection of Coronary Artery Disease by Combined 
Assessment of Wall Motion, Myocardial Perfusion and Coronary Flow Reserve: A 
Multiparametric Contrast Stress-Echocardiography Study. J Am Soc Echocardiogr 
2010;23:1242-50.
4 Dolan MS, Riad K, El-Shafei A, Puri S, Tamirisa K, Bierig M, et al. Effect of intravenous 
contrast for left ventricular opacification and border definition on sensitivity and specificity of 
dobutamine stress echocardiography compared with coronary angiography in technically 
difficult patients. Am Heart J. 2001;142:908-15.
5 Dolan MS, Gala SS, Dodla S, Abdelmoneim SS, Xie F, Cloutier D, et al. Safety and Efficacy of 
Commercially Available Ultrasound Contrast Agents for Rest and Stress Echocardiography. J 
AM Coll Cardiol. 2009;53(1):32-38.
6 Plana JC, Mikati IA, Dokainish H, Lakkis N, Abukhalil J, Davis R, et al. A randomized cross-
over study for evaluation of the effect of image optimization with contrast on the diagnostic 
accuracy of Dobutamine Echocardiography in coronary artery disease. J Am Coll Cardioll 
Img 2008;1:145-52.
7 Douglas PS, Hoffmann U, Patel MR, Mark DB, Al-Khalidi HR, Cavanaugh B, et al. Outcomes 
of Anatomical versus Functional Testing for Coronary Artery Disease. N Eng J Med. 
2015;372: 1291-1300.
8 Gibbons RJ. Noninvasive Diagnosis and Prognosis Assessment in Chronic Coronary Artery 
Disease: Stress Testing with and without Imaging Perspective. Circ Cardiovasc Imaging 
2008;1:257-69.
9 Kim C, Kwok YS, Heagerty P, Redberg R. Pharmacologic Stress testing for Coronary Disease 
diagnosis: A Meta-analysis. Am Heart J 2001;142:934-944.
10 Krivokapich J, Child JS, Walter DO, Garfinkel A. Prognostic Value of Dobutamine Stress 
Echocardiography in Prediciting Events in Pateints with Known or Suspected Coronary 
Artery Disease. J Am Coll Cardiol 1999;33:708-16.
11 Marwick, TH. Education in Heart Stress Echocardiography. Heart 2003;89:113-118.


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12 Mulvagh SL, Rakowski H, Vannan MA, Abdelmoneim SS, Becher H, Bierig M, et al. 
American Society of Echocardiography Consensus Statement on the Clinical Applications of 
Ultrasonic Contrast Agents in Echocardiography. J Am Soc Echocardiogr. 2008 
Nov;21(11):1179-201.
13 Senior R, Becher H, Monaghan M, Agati L, Zamorano J,Vanoverschelde JL, et al. EAE 
recommendations Contrast echocardiography: Evidence-based recommendations by European 
Association of Echocardiography. European Journal of Echocardiography 2009;10:194–212.
14 Brown AS, Calachinas M, Evdoridis C, Hancock J, Wild S, Prasan A, et al: SonoVue 
improves endocardial border detection and variability in assessing wall motion score and 
ejection fraction during stress echocardiography. Irish Journal of Medical Sciences. 
2004;173(1):13-7.
15 Thomson HL, Basmadjian AJ, Rainbird AJ, Razavi M, Avierinos JF, Pellikka PA, et al. 
Contrast echocardiography improves the accuracy and reproducibility of left ventricular 
remodeling measurements: a prospective randomly assigned, blinded study. J Am Coll 
Cardiol. 2001;38:867-75.
16 Hundley WG, Kizilbash AM, Afridi I, Franco F, Peshock RM, Grayburn PA. Effect of 
contrast enhancement on transthoracic echocardiographic assessment of left ventricular 
regional wall motion. Am J Cardiol. 1999;84:1365-8.
17 Yu EH, Sloggett CE, Iwanochko RM, Rakowski H, Siu SC. Feasibility and accuracy of left 
ventricular volumes and ejection fraction determination by fundamental, tissue harmonic and 
intravenous contrast imaging in difficult to image patients J Am Soc Echocardiogr. 
2000:13:216-24.
18 Dias BF, Yu EHC, Sloggett CE, Iwanochko RM, Rakowski H, Siu SC. Contrast enhanced 
quantitation of left ventricular ejection fraction: what is the best method? J Am Soc 
Echocardiogr. 2001;14:1183-90.
19 Rainbird AJ, Mulvagh SL, Oh JK, McCully RB, Klarich KW, Shub C, et al. Contrast 
dobutamine stress echocardiography: Clinical practice assessment in 300 consecutive patients. 
J Am Soc Echocardiog 2001;14:378-85.
20 Douglas PS, Khandheria B, Stainback RF, Weissman NJ. 
ACCF/ASE/ACEP/AHA/ASNC/SCAI/SCCT/SCMR 2008 appropriateness criteria for stress 
echocardiography. J Am Coll Cardiol 2008;51:1127– 47.
21 Sicari R, Nihoyannopoulos P, Evangelista A, Kasprzak J, Lancellotti P, Polderman D, et al. 
EAE GUIDELINES. Stress echocardiography expert consensus statement. European 
Association of Echocardiography (EAE) (a registered branch of the ESC). Eur J 
Echocardiogr. 2008;9:415–37.
22 Bracco Final Clinical Trail Report 
23 Bracco Final Clinical Trail Report 


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24 Bracco Final Clinical Trail Report 
25 Bracco Final Clinical Trial Report 
26 Douglas PS, Garcia MJ, Haines DE, Lai WW, Manning WJ, Patel AR, et al. 
ACCF/ASE/AHA/ASNC/HFSA/HRS/SCAI/SCCM /SCCT/ SCMR 2011 Appropriate Use 
Criteria for Echocardiography. A Report of the American College of Cardiology Foundation 
Appropriate Use Criteria Task Force, American Society of Echocardiography, American 
Heart Association, American Society of Nuclear Cardiology, Heart Failure Society of 
America, Heart Rhythm Society, Society for Cardiovascular Angiography and Interventions, 
Society of Critical Care Medicine, Society of Cardiovascular Computed Tomography, Society 
for Cardiovascular Magnetic Resonance. J am Coll Cardiol 2011;57(9):1126-66.
27 Lang R, Bierig M, Devereux R, Flachskampf F, Foster E, Pellikka P, et al. Recommendations 
for chamber quantification: a report from the American Society of Echocardiography’s 
guidelines and standards committee and the chamber quantification writing group, developed 
in conjunction with the European Association of Echocardiography, a branch of the European 
Society of Cardiology. J Am Soc Echocardiogr 2005;18:1440-63.
28 Cheitlin MD, Armstrong WF, Aurigemma GP, Beller GA, Bierman FZ, Davis JL, et al.
ACC/AHA/ASE 2003 guideline update for the clinical application of echocardiography: 
Summary article. A report of the American College of Cardiology/American Heart 
Association Task Force on Practice Guidelines (ACC/AHA/ASE Committee to Update the 
1997 Guidelines on the Clinical Application of Echocardiography) J Am Coll Cardiol. 
2003;42:954–970.
29 Mieres JH, Shaw LJ, Arai A, Budoff MJ, Flamm SD, Hundley WG, et al. Cardiac Imaging 
Committee, Council on Clinical Cardiology, and the Cardiovascular Imaging and Intervention 
Committee, Council on Cardiovascular Radiology and Intervention, American Heart 
Association Role of noninvasive testing in the clinical evaluation of women with suspected 
coronary artery disease: Consensus statement from the Cardiac Imaging Committee, Council 
on Clinical Cardiology, and the Cardiovascular Imaging and Intervention Committee, Council 
on Cardiovascular Radiology and Intervention, American Heart Association. Circulation. 
2005;111:682–96.
30 Senior R, Dwivedi G, Hayat S, Lim TK. Clinical benefits of contrast-enhanced 
echocardiography during rest and stress examinations. Eur J Echocardiography. 2005;6(Suppl
2):S6-S13.
31 Cosyns B, Roosens B, Hernot S, El Haddad P, Lignian H, Pierard L, et al. Use of contrast 
echocardiography in intensive care and at the emergency room. Curr Cardiol Rev. 
2011(7);157-62.
32 Paterson I, Mielniczuk LM, O'Meara E, So A, White JA. Imaging heart failure: current and 
future applications. Can J Cardiol. 2013 Mar;29(3):317-28.


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33 Bamberg F, Marcus RP, Schlett CL, Schoepf UJ, Johnson TR, Nance JW , et al. Imaging 
evaluation of acute chest pain: systematic review of evidence base and cost-effectiveness. J 
Thorac Imaging. 2012 Sep;27(5):289-95.
34 Douglas PS, Patel MR, Bailey SR, Dai D, Kaltenbach L, Brindis RG, et al. Hospital variability 
in the rate of finding obstructive coronary artery disease at elective, diagnostic coronary 
angiography. J Am Coll Cardiol. 2011; 58:801-809.


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Appendix A: Investigational Product/Investigational Medicinal Product
Labeling


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Investigational Product/Investigational Medicinal Product Labeling
North America
The following 2-part label will be applied to the kit:
LUMASON Kit Label
Lumason™
IND 46,958
Protocol No. BR1-141
Contents:
1 Lumason™ vial of 25 mg lyophilized powder
1 pre-filled syringe of 5 mL Sodium Chloride injection USP,
0.9% Sodium Chloride (DILUENT)
1 mini-spike
Store at 25 C (77 F) excursions permitted to 15-30C (59-
86F) 
CAUTION: New Drug - Limited by Federal Law to 
Investigational Use
Batch No.:
Exp. Date:
Bracco Diagnostics Inc., Monroe Twp, NJ 08831
Lumason™
IND 46,958
Protocol No. BR1-141
Contents:
1 Lumason™ vial of 25 mg lyophilized powder
1 pre-filled syringe of 5 mL Sodium Chloride injection USP,
0.9% Sodium Chloride (DILUENT)
1 mini-spike
Store at 25 C (77 F) excursions permitted to 15-30C (59-
86F) 
CAUTION: New Drug - Limited by Federal Law to 
Investigational Use
Batch No.:
Exp. Date:
Bracco Diagnostics Inc., Monroe Twp, NJ 08831
The following 2-part label will be positioned on each vial of LUMASON:
LUMASON 2-Part Vial Label 
Lumason™
25mg
Protocol:  BR1-141
Store at 25 C (77 F) excursions permitted to 15-30C (59-
86F)
For i.v. use after reconstitution with 5 mL of saline.
CAUTION: New Drug - Limited by Federal Law to 
Investigational Use.
Subject No.
Batch No.:
Exp. Date:
Bracco Diagnostics Inc., Monroe Twp, NJ 08831
Lumason™
25mg
Protocol:  BR1-141
Store at 25 C (77 F) excursions permitted to 15-30C (59-
86F)
For i.v. use after reconstitution with 5 mL of saline
CAUTION: New Drug - Limited by Federal Law to 
Investigational Use.
Subject No.
Batch No.:
Exp. Date:
Bracco Diagnostics Inc., Monroe Twp, NJ 08831
The above labels are “master labels.”  The actual labels used for each country are filed in the 
Trial Master File of the study.


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Europe
The following label will be applied to the kit which includes 2 clear plastic containers:
SonoVue Kit Label
SonoVue® (BR1) - 8 µl/mL    
Dispersion for intravenous injection after reconstitution              Code number. Lxxx/xx
Study No. BR1-141
EudraCT No.
Centre No.
Subject No. 
Content: 2 vials of 25 mg lyophilized powder (SonoVue®)
2 pre-filled syringes with 5 mL NaCl 0.9% w/v
Batch No.:xxxxx/xxxxx
Expiry date: mm/yyyy – mm/yyyy
Direction for use: Administer within 3 hours after reconstitution.
See paragraphs 6.4-6.5 and Appendix B of the study protocol.
No special storage conditions are required. Do not freeze.
FOR CLINICAL TRIAL USE ONLY
Return to IMP Manufacturer all the material, including empty packaging and unused or partially 
used product
Sponsor: Bracco Imaging S.p.A. (+39-02-2177.1) Via Folli, 50 - I -20134 Milano
IMP Manufacturer: Bracco Imaging S.p.A. (+39-0125-56170.1) Via Ribes, 5 - I -10010 Colleretto Giacosa 
The following 2-part label will be positioned on each vial of SonoVue:
SonoVue Vial Label
SonoVue®(BR1) - 8 µl/mL        Code No. Lxxx/xx
25 mg lyophilized powder to be reconstituted 
with 5 mL NaCl 0.9% - For intravenous injection 
after reconstitution
Study No. BR1-141
Centre No.
Subject No.
Batch No: 
Exp. Date: mm/yyyy
No special storage conditions are required. 
FOR CLINICAL TRIAL USE ONLY
Sponsor: Bracco Imaging S.p.A - I - 20134 Milano
SonoVue®(BR1) - 8 µl/mL        Code No. Lxxx/xx
25 mg lyophilized powder to be reconstituted 
with 5 mL NaCl 0.9% - For intravenous injection 
after reconstitution
Study No. BR1-141
Centre No.
Subject No.
Batch No: 
Exp. Date: mm/yyyy
No special storage conditions are required. 
FOR CLINICAL TRIAL USE ONLY
Sponsor: Bracco Imaging S.p.A - I - 20134 Milano
The above labels are “master labels.”  The actual labels used for each country are filed in the 
Trial Master File of the study.


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Appendix B: LUMASON/SONOVUE Reconstitution


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LUMASON Reconstitution
LUMASON is supplied within a kit containing the following: 

a clear glass vial labeled as LUMASON (sulfur hexafluoride lipid microsphere) for 
Injectable Suspension,

a prefilled syringe labeled as Sodium Chloride Injection, USP, 0.9% Sodium Chloride 
(DILUENT), and 

a Mini-Spike.  
Glass vial
Prefilled syringe and 
its plunger rod
Mini-Spike 
Reconstitution steps:

Prior to LUMASON reconstitution, inspect the kit and its components for signs of damage.  Do 
not use the kit if the protective caps on the vial and prefilled syringe are not intact or if the kit 
shows other signs of damage.  

Perform all LUMASON reconstitution steps under aseptic conditions.  The LUMASON vial 
and the prefilled syringe do not contain a bacteriostatic preservative.  

LUMASON is reconstituted by injecting the prefilled syringe contents (5 mL saline) into the 
LUMASON vial using the following illustrated steps below.  
1. Connect the plunger rod to the prefilled 
syringe barrel by screwing it clockwise into the 
syringe (see Figure 1).
Figure 1.


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LUMASON Reconstitution (continued)
2. Open the Mini-Spike blister and remove the 
syringe tip cap (see Figure 2).
Figure 2.
3. Open the Mini-Spike green cap and connect 
the syringe to the Mini-Spike by screwing it in 
clockwise (see Figure 3).
Figure 3.
4. Remove the flip cap plastic protective cap 
from the vial, remove the Mini-Spike spike 
protection and position the spike in the center of 
the rubber stopper of the vial. Press firmly 
inward until the spike is fully inserted in the 
stopper (see Figure 4).
Figure 4.
5. Empty the content of the syringe into the vial 
by pushing on the plunger rod (see Figure 5). 
Figure 5.


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LUMASON Reconstitution (continued)
6. Shake vigorously for 20 seconds, mixing all 
the contents in the vial (see Figure 6). A 
homogeneous white milky liquid indicates 
formation of sulfur hexafluoride lipid 
microspheres. 
Figure 6.
7. Invert the system and slowly withdraw the 
needed volume of suspension into the syringe 
(see Figure 7).
Figure 7.
8. Unscrew the syringe from the Mini-Spike 
(see Figure 8). Immediately connect the syringe 
to the dose administration line (20 G) and 
administer as directed under Section 6.4.
Figure 8.


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SONOVUE Reconstitution
1
v1.0-08/2000 ©BRG 2000
2
3
4
5
6
7
8


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1. Connect the plunger rod by screwing it clockwise into the syringe.
2. Open the MiniSpike transfer system blister and remove syringe tip cap.
3. Open the transfer system cap and connect the syringe to the transfer system by screwing 
it in clockwise.
4. Remove the protective disk from the vial.  Slide the vial into the transparent sleeve of the 
transfer system and press firmly to lock the vial in place.
5. Empty the contents of the syringe into the vial by pushing on the plunger rod.
6. Shake vigorously for 20 seconds to mix all the contents in the vial to obtain a white milky 
homogeneous liquid.
7. Invert the system and carefully withdraw SONOVUE into the syringe.
8. Unscrew the syringe from the transfer system.
Do not use if the liquid obtained is clear and/or if solid parts of the lyophilisate are seen in 
the suspension.


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Appendix C: Study Schedule 


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Event
Rest 
Stress
Follow-Up
UEUS 
IP/IMP
CEUS
STRESSOR
UE-DSE
IP/IMP
CE-DSE
Within -24 hr
-1 hr
-10 min
Immed prior
0 min
30 min
Immed prior
0 min
30 min
1 hr
24 hr
72 hr
6 mos
Written Informed Consent a

Pregnancy Test (if necessary)

Adverse Events Monitoring b













Concomitant Medications c












Medical History d

Physical Examination


Vital Signs e,f






Laboratory Evaluations g


Electrocardiogram - discretef






Electrocardiogram – continuous








Pulse oximetry 








IP/IMP Administration


Imaging Procedure 




Dobutamine Administration h

Truth Standard



Follow-up Contact for AE

a
Obtain prior to implementation of any study procedure.
b
Start monitoring from the time of signing Informed Consent up to 72 h after the last LUMASON administration or until cardiac intervention, whichever comes first. Collect adverse events for LUMASON and 
NIMPs (dobutamine, atropine).
c
Record all medications (prescription and over-the-counter) taken within 24 hours prior to first LUMASON administration up through 24 hours after the last LUMASON administration. 
d
Includes Demographics, Cardiac Medical History and General Medical History.
e
Includes systolic and diastolic blood pressure, heart rate.
f
If stress echocardiography (CE-DSE) is not performed, obtain at 1 and 24 hours after the rest LUMASON administration.
g
Collect blood samples according to the instruction manual from the central laboratory.
h
Atropine may be administered with dobutamine if needed to achieve peak stress.


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Appendix D: Serious Adverse Event Report


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Protocol BR1-141
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Protocol BR1-141
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Protocol BR1-141
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Protocol BR1-141
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Appendix E: Investigator Statement


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INVESTIGATOR STATEMENT 
Page 1 of 2
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Investigational 
Product
Lumason™/SonoVue®
Protocol No.
BR1-141
Protocol 
Title:
A Prospective Multicenter Phase III Clinical Evaluation of 
the Safety and Efficacy of Lumason/SonoVue in Subjects 
Undergoing Pharmacologic Stress Echocardiography with 
Dobutamine for the Diagnosis of Coronary Artery Disease
Investigator:
Study Site:
COMMITMENTS
By signing this document, I agree to conduct the study as outlined in the protocol and in 
accordance with: Title 21 CFR 56 - Institutional Review Boards, the European Directive 
2001/20/EC, 2005/28/EC and related guidance, and the Declaration of Helsinki, as well as, all 
applicable government regulations, Good Clinical Practice and also
I declare:
1) I am well qualified by scientific training and experience to conduct investigational studies in 
the clinical area of the proposed study and I am affiliated with a recognized medical school 
or with an independent institution recognized for its excellence.
2) I have received and understand the information about pharmacology, toxicology and possible 
risks and side effects of the investigational product (e.g., as described in the Reference Safety 
Information).
3) I shall provide information to all staff members involved in the study about their obligations 
as described in this document.
4) I shall submit the protocol, Informed Consent Form/Subject Information Sheet and other 
required documentation to the IRB/EC for review and approval.
5) I shall make no changes to the protocol without formal amendment (prepared in agreement 
with the Sponsor), except when necessary to protect the safety, the rights or welfare of 
subjects. In this last case I will inform the Sponsor of the change. I shall report serious 
breaches according to applicable regulations.
6) I shall require Informed Consent from each subject prior to enrollment into the study.  The 
Informed Consent shall be documented by use of a written consent form approved by the 
IRB/EC. I shall also require a Data Protection Form.


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7) I shall use the investigational product only in compliance with the study protocol and I shall 
be responsible for the security and accountability of clinical study supplies.
8) I shall notify the Sponsor immediately or no later than 24 hours by telephone and/or by fax of 
serious adverse events and submit written reports of serious adverse events, as outlined in the 
protocol, to Sponsor.
9) (for sites outside the European Economic Area, add the following) I shall submit a written 
report of adverse events to the IRB/EC, as required by applicable regulatory requirements.
10) I shall complete the Sponsor’s Case Report Form (CRF) in a timely and legible manner.
11) I shall maintain accurate source records (hospital or other institutional records), which will 
support the data entered into Case Report Forms and I shall maintain these as specified in the 
protocol.
12) I shall retain essential documents (Investigator’s files) including study codes for at least 5 
years (or longer if required by law or agreements with the Sponsor) after completion or 
discontinuation of the trial and, in any case, no documentation will be destroyed without 
prior written agreement with the Sponsor.
13) I shall allow monitoring visits by Sponsor’s representatives a predetermined frequency.
14) I shall allow the authorized Sponsor representative and any competent and regulatory 
authorities to inspect the facilities and pertinent records at reasonable times and in a manner 
which ensure subject confidentiality.
15) I shall maintain confidentiality about all information concerning the investigational product, 
such as patent applications, formulas, manufacturing process, basic scientific data and 
formulation information supplied by the Sponsor and not previously published and I shall not 
disclose this information to a third party without the written consent of the Sponsor.
16) I shall permit the information developed in the clinical study to be used by the Sponsor in 
connection with the development of the investigational product and may be disclosed to the 
IRB/EC and competent and regulatory authorities.
Following completion of the study, the data may be considered for reporting at a scientific 
meeting and/or for publication in a scientific journal.  A copy of the manuscript or abstract will 
be provided to the Sponsor for review before submission to a scientific journal for publication 
and/or a scientific meeting selection committee for oral or poster presentation.  Subgroup or 
individual Investigator publications must not interfere or compromise publication of the multi-
center results of this clinical study.
____________________________________
___________________
Investigator
Date
____________________________________
Investigator (Printed Name)


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Appendix F: Administrative Structure 
Administrative Structure
North America
Clinical Research Personnel
 
m
CROs
Study Monitoring
Central Laboratory
Central ECG Laboratory
Central Imaging Laboratory
TBD
TBD
Europe
Clinical Research Personnel
CROs
Study Monitoring
Central Laboratory
TBD
Central ECG Laboratory
Central Imaging Laboratory
TBD
TBD