Clinical trial NCT04185246
Conditions: Cardiac Disease
Keywords: Echo Echocardiogram Cardiac ultrasound suboptimal contrast agent enhancing agent LVO LVEBD
Sponsor: Trust Bio-sonics, Inc.
Start date: 2019-11-04; Completion date: 2020-07-01
Enrollment: 36 participants; Allocation: NA; Intervention model: SEQUENTIAL

Brief summary:
NH002 (Perflutren Lipid Microspheres) Injectable Suspension is an ultrasound contrast agent for use in patients with suboptimal echocardiograms to opacify the left ventricular (LV) chamber and to improve the delineation of the LV endocardial border. The primary objective of this study is to evaluate the safety and tolerability of 3 ascending doses of NH002.

Detailed description:

Arms: arm 1: Subjects will be enrolled with sequential allocation to 1 of 3 cohorts with the following intravenous (IV) doses of NH002: 2.5 µl/kg, 5.0 µl/kg, or 10.0 µl/kg.

Each patient will undergo an unenhanced ultrasound examination and a NH002 contrast-enhanced examination on the same day
Interventions: intervention 1: NH002 (Perflutren Lipid Microspheres) Injectable Suspension intervention 2: Echocardiogram
Intervention descriptions: intervention 1: NH002 is formulated as a microbubble injectable suspension for intravenous administration. NH002 requires an activation process prior to use. intervention 2: Each patient will undergo an unenhanced ultrasound examination and a NH002 contrast-enhanced examination on the same day.

Protocol documents:
Prot_000.pdf: 
 
 
CLINICAL STUDY PROTOCOL 
A Multicenter, Open-Label, Dose Ascending Study to Evaluate the Safety of 
NH002 as a Contrast Agent in Cardiac Echocardiography 
 
 
Protocol Number: 
NH002-LV 
EudraCT Number: 
Not Applicable 
Investigational Product: 
NH002 (Perflutren Lipid Microspheres) Injectable 
Suspension 
Phase: 
1 
Sponsor: 
Trust Bio-sonics, Inc. 
Room D105, No. 2, Sec. 2, Shengyi Rd. 
Zhubei City, Hsinchu County 30261 
Taiwan (R.O.C.) 
Protocol Date: 
13 Mar 2019 
Protocol Version: 
1.0 
 
 
 
 
 
 
CONFIDENTIAL 
This protocol may not be reproduced or communicated to a third party without the 
written permission of Trust Bio-sonics, Inc. 
 


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1 
PROTOCOL APPROVAL SIGNATURES 
Protocol Title: 
A Multicenter, Open-Label, Dose Ascending Study to Evaluate the 
Safety of NH002 as a Contrast Agent in Cardiac Echocardiography 
Protocol Number: 
NH002-LV 
This study will be conducted in compliance with the clinical study protocol (and 
amendments), International Council for Harmonisation (ICH) guidelines for current Good 
Clinical Practice (cGCP) and applicable regulatory requirements. 
Sponsor Signatory 
Ting-Yu Huang 
Project Director 
Room D105, No. 2, Sec. 2, Shengyi Rd. 
Zhubei City, Hsinchu County 30261 
Taiwan (R.O.C.) 
Signature 
Date 
  


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2 
SYNOPSIS 
Protocol Number: 
NH002-LV 
Title: 
A Multicenter, Open-Label, Dose Ascending Study to Evaluate the Safety of NH002 as a Contrast Agent in Cardiac 
Echocardiography 
Investigational Product: 
NH002 (Perflutren Lipid Microspheres) Injectable Suspension 
Phase: 
Phase 1 
Objectives: 
Primary Objective:  
To evaluate the safety and tolerability of 3 different ascending doses of NH002. 
Secondary Objective: 
To evaluate the preliminary diagnostic efficacy of NH002 as a contrast agent in echocardiography.  
Study Design: 
This is a phase 1, multicenter, open-label clinical study to evaluate the safety and tolerability of 3 ascending doses of 
NH002. Up to 36 eligible subjects will be enrolled with sequential allocation to 1 of 3 cohorts with the following 
intravenous (IV) doses of NH002: 2.5 µl/kg, 5.0 µl/kg, or 10.0 µl/kg. Each patient will undergo an unenhanced 
ultrasound examination and a NH002 contrast-enhanced examination on the same day at Day 1. Subjects will remain 
at the clinical site for 240 minutes of observation after NH002 administration.   
All AEs will be evaluated during the successive groups of subjects, per dose level. In general, enrollment will be 
halted and safety data reviewed for termination or continuation with the occurrence of any serious adverse event 
(SAE) considered at least possibly related to study agent administration (in any 1 subject) or the occurrence of 
clinically significant toxicity listed in Section 7.5.1.2 in 30% of subjects (within any 1 of the 3 dose levels). See 
Section 7.5.1.2 for details on stopping criteria for dosing for this trial. 
There will be a screening period (28 days before NH002 administration), a dosing period (during Day 1) which will 
include a single dose administration of NH002, and a 7-day follow-up period for each cohort. Continuous 
echocardiographic imaging will be performed at specified time points before, during, and after dose administration. 
The contrast agent will be prepared and administered by designated pharmacy technician(s), study nurse(s), 
sonographer, or radiologist. Subjects will be observed at a clinical site for at least 240 minutes (ie, 4 hours) and 
evaluated for AEs. Subjects will be contacted by phone after the end of treatment at 72 hours (ie, Day 4 [+3]) and at 
Day 7 (+3), for a follow-up safety assessment. Any drug-related AE or SAE will be followed until resolution.   
High ultrasound mechanical index (MI) values may cause microsphere cavitation or rupture and lead to ventricular 
arrhythmias. Additionally, end-systolic triggering with high MIs has been reported to cause ventricular arrhythmias. 
The safety of NH002 at MIs greater than 0.8 will not be evaluated. 
Number of Subjects:  
Up to 36 subjects will be enrolled. If a subject is withdrawn from the study, except for those subjects who will be 
terminated early due to AEs, the subject may be replaced as necessary with another subject assigned to the same 
dose. Further subjects may be enrolled at a given dose level if additional data are necessary to establish safety and 
tolerability prior to dose escalation. 


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Treatment: 
The contrast agent will be administered by IV bolus injection within 30 to 60 seconds, followed by a 10 mL saline 
flush over 10 seconds. The following dose regimens will be tested: 2.5 µl/kg, 5.0 µl/kg, 10.0 µl/kg. 
Subjects will be enrolled with sequential allocation to the NH002 doses. Each patient will undergo an unenhanced 
ultrasound examination and an NH002 contrast-enhanced examination on the same day at Day 1. Each subject will 
receive 1 of the following 3 NH002 doses: 2.5 µl/kg, 5.0 µl/kg, 10.0 µl/kg IV bolus. Sequential allocation is as 
follows, if all 3 dose levels exhibit acceptable toxicity: the first 12 subjects will receive 2.5 µl/kg, the next 12 will 
receive 5.0 µl/kg, and the final 12 will receive 10.0 µl/kg. (See Section 7.5.1.2 for further details, including the 
definition of unacceptable toxicity for this trial.) The 12 subjects in each ascending cohort will be enrolled only if no 
serious adverse reactions or toxicity reactions have been observed within 7 days from completion of the safety 
observation period for the last subject in the preceding cohort.  
A dose escalation committee (DEC) will evaluate clinical, laboratory, and electrocardiographic safety data before 
advancing to the next higher dose level. If stopping criteria for dosing as defined in Section 7.5.1.2 is observed in a 
specific cohort, the previous dose level will be considered to be the maximum tolerated dose and no further 
increases in dose will be included in the study. 
Duration of Treatment: 
The duration of treatment is expected to last between 4 to 5 hours during 1 day, barring any unexpected adverse 
reactions. 
Study Population: 
To be eligible for study entry, subjects must satisfy all of the following inclusion criteria: 
1. Male or female subjects 20 years of age or older 
2. Ability to understand and the willingness to provide written informed consent 
3. Suspected of having cardiac disease or undergoing evaluation of cardiac anatomy for congenital heart 
disease 
4. Negative pregnancy test within 24 hours of the procedure if subject is a woman of childbearing potential 
Subjects will be excluded from the study if one or more of the following exclusion criteria is applicable: 
1. Known right-to-left, bidirectional, or transient right-to-left cardiac shunt(s) 
2. Chronic obstructive pulmonary disease  
3. Ejection fraction at screening or baseline < 40% 
4. Uncontrolled serious ventricular arrhythmias or at high risk for arrhythmias due to prolongation of the QT 
interval (QT corrected by Fridericia’s formula > 450 msec in males and > 470 msec in females) 
5. Clinically significant abnormality in renal and liver function tests (alanine aminotransferase/aspartate 
aminotransferase > 2 × upper limit of normal [ULN]; serum creatinine > 1.5 × ULN) 
6. Known or suspected hypersensitivity to one or more of the ingredients of NH002, perflutren, or other 
ultrasound contrast agents 
7. Received an investigational compound within 30 days before enrolling in the study 
8. Received any contrast agent either intravascularly or orally within 48 hours before NH002 administration 
9. Pregnant or lactating female (conception during the study should be avoided) 
10. Serious medical or psychiatric illness/condition likely, in the judgment of the investigator, to interfere with 
compliance to protocol treatment/research 
11. Clinically unstable cardiopulmonary conditions considered not suitable for participation in the trial, in the 
judgment of the investigator 


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12. Uncontrolled arterial hypertension (defined as systolic blood pressure ≥ 200 mmHg or diastolic blood 
pressure ≥ 110 mmHg) or arterial hypotension (defined as systolic blood pressure ≤ 90 mmHg) 
Primary Endpoint(s): 
• 
Adverse event reporting and changes in physical examination findings, vital signs, clinical laboratory 
evaluations, oxygen saturation, and electrocardiograms (ECGs). 
Secondary Endpoint(s):  
• 
The percentage of subjects with moderate or complete left ventricular opacification (LVO), defined by an 
LVO grade of 2 (moderate) or 3 (complete), as assessed by the blinded central reader(s); 
• 
The percentage of subjects with complete LVO, defined by an LVO grade of 3 (complete), as assessed by 
the blinded central reader(s); 
• 
The change from baseline on the left ventricular endocardial border delineation (LVEBD) score, defined 
using a standard 12-segment model, as assessed by the blinded central reader(s). The left ventricular (LV) 
endocardium of the 4- or 2- chamber apical views are divided into 6 segments, with 2 basal, mid- and 
apical segments in each view. For each segment, LVEBD is graded as follows: 0= inadequate border 
(border not visible); 1= sufficient (border barely visible); 2= good (border clearly visible). A total 
delineation score (0-24) is obtained by adding the scores from the 6 individual segments in each of the 2 
views. 
• 
The changes from baseline on LVEBD score of subjects with suboptimal LVEBD at baseline, as defined by 
2 or more contiguous segments of 6 segments that cannot be visualized reliably in either the apical 
4- and/or the 2-chamber view, as assessed by the blinded central reader(s);  
• 
The duration of clinically useful contrast calculated by measuring the time between the disappearance of 
shadowing effect (useful effect starts) and the time when moderate or full LV enhancement and contrast 
enhancement are no longer adequate (useful effect ends), as assessed by the blinded central reader(s).  
Statistical Analysis: 
Sample Size Determination 
It is anticipated that 36 subjects will be enrolled (with up to 12 subjects per NH002 dose). Each subject will receive 
1 dose of NH002. If a subject is withdrawn from the study, the subject may be replaced with another subject 
assigned to the same dose, except for those who are discontinued due to AEs, as necessary. Further subjects may be 
enrolled at a given dose level if additional data are necessary to establish safety and tolerability prior to dose 
escalation. As this is a dose-finding study, a formal sample size calculation was not required.    
A Dose Escalation Committee 
To enhance the safety and integrity of the study data, a DEC will evaluate clinical, laboratory, and 
electrocardiographic safety data, routinely before advancing to the next higher dose level/cohort. The DEC will 
consist of sponsor personnel, the independent medical monitor, and the investigator(s) at the study site. A sentinel 
review approach will be followed whereby the first review will be conducted at the end of the safety follow-up 
observation period for the first subject treated in Cohort 1 to determine if subsequent subjects may be dosed in the 
cohort. To review all cumulative safety data for Cohort 1, the next review will be conducted at the end of the safety 
follow-up observation period for the last subject treated in Cohort 1. This sentinel review approach will continue to 
be followed for all successive cohorts (ie, Cohort 2 and Cohort 3), to review safety data and to provide a 
recommendation on study continuation, recommended dose, or early termination in case there is a concern regarding 
safety. 
Statistical Analysis: 
The safety assessments, including AEs, ECGs, and clinical laboratory evaluations, where indicated, will be 
presented using descriptive statistics for each dose of NH002. Data will be summarized for each dose group and 
overall. 


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Safety Evaluations 
Each subject must be carefully monitored for the development of any AEs throughout the study from the signing of 
the informed consent form to 7 days after receipt of the dose of study agent (ie, Day 7 Follow-up call). Safety 
evaluations will be based on 12-lead ECGs, changes in physical examinations performed, including vital signs and 
changes in laboratory parameters. These safety evaluations will also be assessed and recorded at specified time 
points: prior to injection, during injection and imaging, and up to 7 days post-injection.  
 


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3 
TABLE OF CONTENTS 
1 
Protocol Approval Signatures ....................................................................................... 2 
2 
Synopsis ........................................................................................................................... 3 
3 
Table of Contents ............................................................................................................ 7 
4 
List of Abbreviations .................................................................................................... 11 
5 
Introduction................................................................................................................... 13 
5.1 
Background ................................................................................................................... 13 
5.2 
NH002 ............................................................................................................................ 14 
5.2.1 
Nonclinical studies ......................................................................................... 16 
5.2.2 
Clinical studies ............................................................................................... 21 
5.3 
Risk/Benefit Assessment ............................................................................................... 22 
6 
Study Objectives & Endpoints .................................................................................... 24 
6.1 
Primary Objective ........................................................................................................ 24 
6.2 
Primary Endpoint ......................................................................................................... 24 
6.3 
Secondary Objective ..................................................................................................... 24 
6.4 
Secondary Endpoints .................................................................................................... 24 
7 
Investigational Plan ...................................................................................................... 25 
7.1 
Overall Study Design and Plan: Description .............................................................. 25 
7.1.1 
Imaging Guidelines ........................................................................................ 26 
7.1.2 
Ventricular Arrhythmia Related to High Mechanical Index ......................... 28 
7.2 
Schedule of Assessments............................................................................................... 29 
7.3 
Discussion of Study Design .......................................................................................... 31 
7.3.1 
Study Design .................................................................................................. 31 
7.3.2 
Quality Management and Risk Evaluation .................................................... 32 
7.4 
Selection of Study Population ...................................................................................... 32 
7.4.1 
Number of Planned Subjects ......................................................................... 32 
7.4.2 
Inclusion Criteria ........................................................................................... 32 
7.4.3 
Exclusion Criteria .......................................................................................... 33 
7.4.4 
Removal of Subjects From Therapy or Assessments .................................... 33 
7.4.5 
Enrollment Guidelines ................................................................................... 34 
7.5 
Investigational Products ............................................................................................... 35 
7.5.1 
Investigational Products Administered .......................................................... 35 


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7.5.1.1 
NH002 ............................................................................................................ 35 
7.5.1.2 
Stopping Criteria for Dosing ......................................................................... 35 
7.5.1.3 
Microspheres and Contrast Agent Activation ................................................ 36 
7.5.1.4 
Injection Volume(s) ....................................................................................... 37 
7.5.1.5 
Injection Route and Rate ............................................................................... 37 
7.5.1.6 
Emergency Event Management ..................................................................... 37 
7.5.2 
Identity of Investigational Products ............................................................... 37 
7.5.2.1 
NH002 (perflutren lipid microspheres) Injectable Suspension ..................... 37 
7.5.3 
Packaging and Labeling ................................................................................. 39 
7.5.3.1 
NH002 ............................................................................................................ 39 
7.5.4 
Selection of Doses in the Study ..................................................................... 39 
7.5.5 
Selection and Timing of Dose for Each Subject ............................................ 39 
7.5.6 
Prior and Concomitant Therapy ..................................................................... 39 
7.5.6.1 
Prohibited Medication/Therapy ..................................................................... 40 
7.5.6.2 
Rescue Medication ......................................................................................... 40 
7.5.7 
Dosing Compliance ....................................................................................... 40 
8 
Timing of Study Procedures ........................................................................................ 40 
8.1 
Pre-dosing ...................................................................................................................... 40 
8.1.1 
Visit 1: Screening .......................................................................................... 40 
8.2 
Dosing Period ................................................................................................................ 41 
8.2.1 
Visit 2: Day 1, Dosing ................................................................................... 41 
8.2.1.1 
Before Dosing ................................................................................................ 41 
8.2.1.2 
5 Minutes Before Dosing ............................................................................... 42 
8.2.1.3 
Dosing ............................................................................................................ 42 
8.2.1.4 
10 Minutes After Dosing ............................................................................... 42 
8.2.1.5 
30 Minutes After Dosing ............................................................................... 43 
8.2.1.6 
60 Minutes After Dosing ............................................................................... 43 
8.2.1.7 
120 Minutes After Dosing ............................................................................. 44 
8.2.1.8 
240 Minutes After Dosing ............................................................................. 44 
8.3 
Safety Follow-up Period ............................................................................................... 44 
8.3.1 
Day 4 (+3) ...................................................................................................... 44 
8.3.2 
Day 7 (+3) ...................................................................................................... 44 
8.4 
Duration of Treatment ................................................................................................. 45 
9 
Efficacy and Safety Variables ...................................................................................... 45 
9.1 
Efficacy and Safety Measurements Assessed ............................................................. 45 
9.1.1 
Efficacy Variables ......................................................................................... 45 
9.1.1.1 
Echocardiographic Imaging ........................................................................... 45 
9.1.2 
Safety Assessments ........................................................................................ 45 
9.1.2.1 
Overall Study Stopping Rules ....................................................................... 46 
9.1.2.2 
Adverse Events .............................................................................................. 47 


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9.1.3 
Appropriateness of Measurements ................................................................ 52 
9.2 
Dose Escalation Committee (DEC) ............................................................................. 53 
10 
Statistical Methods ........................................................................................................ 53 
10.1 Statistical and Analytical Plans ................................................................................... 53 
10.2 Datasets or Populations Analysed ............................................................................... 53 
Full Analysis Set ............................................................................................................ 53 
Per Protocol Set ............................................................................................................. 54 
10.3 Demographic and Other Baseline Characteristics .................................................... 54 
10.4 Safety Assessments ........................................................................................................ 54 
10.4.1 
Safety Variables ............................................................................................. 54 
10.5 Efficacy Variables ......................................................................................................... 54 
10.5.1 
Definition of Primary Efficacy Endpoint ...................................................... 54 
10.5.2 
Definition of Secondary Efficacy Endpoints ................................................. 55 
10.6 Methods of Analysis ...................................................................................................... 55 
10.7 Handling of Missing Data ............................................................................................ 55 
10.8 Determination of Sample Size ...................................................................................... 56 
10.9 Protocol Deviations ....................................................................................................... 56 
11 
Quality Assurance and Quality Control ..................................................................... 56 
11.1 Audit and Inspection .................................................................................................... 56 
11.2 Monitoring ..................................................................................................................... 56 
11.3 Data Management and Coding .................................................................................... 56 
11.4 Quality Management and Risk Evaluation ................................................................ 57 
12 
Records and Supplies ................................................................................................... 58 
12.1 Drug Accountability ..................................................................................................... 58 
12.2 Financing and Insurance .............................................................................................. 58 
13 
Ethics .............................................................................................................................. 59 
13.1 Independent Ethics Committee or Institutional Review Board ............................... 59 
13.2 Regulatory Authorities ................................................................................................. 59 
13.3 Ethical Conduct of the Study ....................................................................................... 59 
13.4 Informed Consent ......................................................................................................... 59 
13.5 Subject Confidentiality ................................................................................................. 60 


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14 
Reporting and Publication, Including Archiving ...................................................... 61 
15 
References ...................................................................................................................... 62 
16 
Appendices..................................................................................................................... 64 
16.1 Image Evaluation Guidelines ....................................................................................... 65 
16.2 Contrast Administration Guidelines for NH002 ........................................................ 68 
16.3 Investigator Signature Page ......................................................................................... 69 
 
List of Tables 
Table 1: The Phospholipid Materials of NH002 ........................................................................... 15 
Table 2: Product Features of NH002 ............................................................................................ 16 
Table 3: Nonclinical Studies with NH002 Microbubble Injectable Suspension .......................... 19 
Table 4: NH002 Administration ................................................................................................... 35 
Table 5: NH002 Injection Volume(s) ........................................................................................... 37 
 
List of Figures 
Figure 1: Imaging Results with Saline in Dog Contrast Mode ..................................................... 17 
Figure 2: Imaging Results with NH002 in Dog (Contrast Mode) ................................................ 17 
Figure 3: Pre-injection Imaging Results in Dog (B-mode) ........................................................... 18 
Figure 4: Enhanced EBD by NH002 as Compared to DEFINITY ............................................... 21 
Figure 5: Imaging Guidelines ....................................................................................................... 27 
Figure 6: Structure of NH002 ....................................................................................................... 38 
Figure 7: NH002 Sample Packaging Label .................................................................................. 39 
 
 
 




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WoCBP 
woman/women of childbearing potential 


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5 
INTRODUCTION 
 
Background 
5.1
Echocardiography serves as one of the first-line imaging modalities in the evaluation of cardiac 
disease, mainly due to its ability to evaluate both the anatomy and function of the 
heart.1 Echocardiography remains a standard of care due to its portability, accuracy, and cost-
effectiveness in the rapid diagnosis and follow-up of most cardiac diseases. Even so, suboptimal 
left ventricular (LV) cavity visualization and endocardial border delineation (EBD) often 
compromise the clinical utility of echocardiography.2  
Improved delineation of endocardial border is associated with more accurate assessments of LV 
systolic function. The use of contrast enhancement improves echocardiographic assessment of 
wall motion at rest, and increases the accuracy of LV volume and ejection fraction 
measurements.3  
In echocardiography, ultrasound contrast agents (UCAs), when used with contrast-specific 
imaging techniques have an established role for diagnostic cardiovascular imaging.4 The clinical 
need for such agents has risen, as more than 30% of certain populations use echocardiograms 
that are considered technically difficult or uninterpretable.5 Ultrasound contrast agents are 
composed of acoustically-active gas-filled microspheres (microbubbles) which optimize their 
detection by increasing the signal strength of ultrasound waves.6  
In October 2007, the US Food and Drug Administration (FDA) imposed additional product label 
warnings that included serious cardiopulmonary reactions, several new disease-state 
contraindications, and a mandated 30-minute post-procedure monitoring period for UCAs.7 
These additional warnings were prompted by reports of cardiopulmonary reactions that were 
temporally related, but were not clearly attributable. Subsequent published reports over the 
following months established the safety and the improved efficacy of clinical ultrasound 
applications with UCAs. Ultimately, in October 2011, the FDA further downgraded the warnings 
after hearing the results of the post-marketing data, which revealed continued safety and 
improved efficacy.8 
Since then, UCAs have demonstrated their ability to reduce variability in echocardiography 
interpretation and also to reduce exposure to the ionizing radiation that is associated with other 
imaging modalities.9 There remains an important opportunity to improve the clinical safety and 
efficacy of UCAs in association with echocardiography. Providing investigators with the option 
of a more appropriate UCA will potentially improve diagnostic decisions and better serve 
patients with cardiac conditions. 


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NH002 
5.2
NH002 is a UCA developed for use in patients with suboptimal echocardiograms to opacify the 
LV chamber, and to improve the delineation of the left ventricular endocardial border (LVEBD). 
Relative to the approved UCAs on the market, NH002 is expected to have a longer half-life, an 
improved safety profile, and effectiveness at a lower dose. 
NH002 is formulated as a microbubble injectable suspension in single-use, 1-mL lipid solution in 
 in headspace for intravenous (IV) administration. The active 
pharmaceutical ingredients API in NH002 are the gas core: perflutren (C3F8), 
 
 
 
For more details regarding the manufacturing process, also refer to the current version of the 
Investigator’s Brochure for NH002. 
NH002 also requires an activation process for the perflutren (C3F8) and lipids to be mixed and 
assembled into microbubbles. The activation method required utilizes agitation with the 
TRANSMIXTM device. TRANSMIXTM is
 and a mechanical 
agitator for the 
 mixing of the lipids solution and C3F8 for gas encapsulation. 
Prior to activation, the initial C3F8 content of NH002 is 
. After activation, the C3F8 
content encapsulated in microbubble suspension of NH002 is 
  
The shell coat of other clinically available microspheres (eg, DEFINITY®) is composed of 
biodegradable materials (such as galactose, albumin, phospholipids, or polymers) which slow 
down gas diffusion and dissolution to improve the circulation time in vivo. Currently, 
phospholipids are the most commonly used shell material. Additionally, lipid coatings oscillate 
flexibly in response to ultrasound exposure, and thus exhibit excellent acoustic properties.7 
The formulation of NH002 differs in that its lipid shell is composed of 
 According to the Handbook of Pharmaceutical Excipients, these lipid materials 
exhibit relatively low toxicity and good biological tolerance (at doses of g/kg range).10 
Moreover, NH002 contains 
, and is thus believed to have minimal 
additional safety concerns (Table 1). 




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Table 2 provides product features of NH002. 
 
Table 2: Product Features of NH002 
 
NH002 
API content (Headspace) 
C3F8 
 
Volume 
1-mL solution in a 2-mL vial 
Appearance 
before activation 
clear colorless liquid 
after activation 
homogenous, opaque, milky white injectable 
microsphere suspension 
Activation method 
Agitation (using TRANSMIXTM) 
 
5.2.1 Nonclinical studies 
Nonclinical studies have been conducted to evaluate the pharmacology, toxicology, and 
genotoxicity of NH002 (Table 3). NH002 appears to have an acceptable safety profile to support 
the first-in-human study. Preliminary imaging results of the dog studies showed significant 
enhancement with low-dose NH002 as compared to saline. See the following figures (Figure 1 
and Figure 2) for comparison. A pre-injection image in B-mode is provided for additional 
consideration (see Figure 3).  


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Figure 1: Imaging Results with Saline in Dog Contrast Mode 
 
Figure 2: Imaging Results with NH002 in Dog (Contrast Mode) 
 


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Figure 3: Pre-injection Imaging Results in Dog (B-mode) 
 
For more details regarding the study protocols for the nonclinical studies, refer to the current 
version of the Investigator’s Brochure for NH002.  


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Table 3: Nonclinical Studies with NH002 Microbubble Injectable Suspension 
Study No. 
Study Type 
Strain/Species 
Route/Doses 
Study Outcome 
Pharmacology 
Pharmacokinetics 
 


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Toxicology 
Other Studies (Hemolysis) 




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Risk/Benefit Assessment 
5.3
Perflutren has been used as a UCA in the US since 1997 (Optison™, GE Healthcare, NDA 
020899). The safety of perflutren contrast agents for echocardiography has been established in 
the published literature. A retrospective study in 63,189 adults undergoing transthoracic 
echocardiograms (TTEs) over 5 years was conducted to examine mortality within 1 day of the 
procedure. Of the 2518 TTEs that a perflutren contrast agent was used, 10 deaths occurred, and 
the investigator concluded that the use of contrast enhancement was not associated with 
increased mortality.14  
Other studies have concluded both the safety of contrast agents for enhancing imaging during 
echocardiography, and the outweighing benefit of improved diagnostic capabilities over the 
relatively low risk of adverse reactions.14,15,16 In 2013, Platts et al. performed a multicenter 
retrospective analysis on consecutive patients in Australia who received DEFINITY and were 
monitored for serious adverse events (SAEs) over a 5-year period.15 A total of 5956 studies with 
UCAs were performed, most of which were outpatient stress tests. There were 16 SAEs related 
to UCAs (0.27%), all of which were mild and transient, with the most common reactions being 
back pain and rash. There were no cases of serious anaphylaxis or death within 30  minutes of the 
contrast administration.15,16 
Since the time of the most recent change to the package labels for both Optison™ and 
DEFINITY, there have also been several published articles and reviews detailing both the safety 
and efficacy of UCAs in a variety of patient populations. In 2012, Wever-Pinzon et al. published 
a retrospective series on 1513 consecutive inpatients with documented pulmonary hypertension 
who had received UCAs. These patients were followed for 24  hours post-UCA administration 
for SAEs, including respiratory decompensation, hypotension, arrhythmias, syncope, 
convulsions, anaphylactic reactions, or death. Out of the 1513 patients, only 3 patients had an 
SAE after the administration of the UCA, none of these events was directly attributed to the 
UCA itself.17 
Kitzman et al, demonstrated adequate-to-full LV opacification (LVO) in the apical 4-chamber 
view in 87% of their patients given 5 ml/kg of perflutren, 91% of patients given 10 ml/kg of 
perflutren, and 0% of patients given placebo (p, 0.01 for both perflutren dose groups vs placebo). 
Similar results were seen in the apical 2-chamber view. Overall, 91% of all patients who 
received perflutren also showed improved EBD after the injection compared with 12% of 
patients who received placebo (p, 0.001).2 
Fifty-six percent of the patients (30 of 53) who received perflutren and who had nondiagnostic 
echocardiographic images in the apical 4-chamber view were converted to diagnostic 
echocardiograms. In the overall group, salvage was demonstrated in 48% of patients with 
nondiagnostic echocardiograms. Improved confidence in image interpretation as determined by 
the site investigators also occurred in 86% of all patients receiving perflutren. The ability to 


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detect wall motion abnormalities was considered enhanced in 87% of all patients receiving 
perflutren. Site investigators judged that the [perflutren] injection provided new information that 
was likely to result in a change in patient management in 57% of all patients who received 
perflutren, and to eliminate the need for an additional test in 60% of all patients who received 
perflutren.2 
This study has been designed to evaluate the safety and tolerability of NH002 in patients, while 
minimizing any potential risks. Although the potential benefits in patients are unknown at this 
time, prior early clinical signs, and nonclinical data provide a sound basis supporting the 
hypothesis that perflutren microspheres are a complementary diagnostic strategy. Thus, the 
benefit/risk assessment for this phase 1 study is anticipated to be favorable based on the lack of 
effective alternative treatments, the limited life expectancy due to malignant disease, and the 
strength of the scientific hypothesis under evaluation. 


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6 
STUDY OBJECTIVES & ENDPOINTS 
 
Primary Objective 
6.1
To evaluate the safety and tolerability of 3 different ascending doses of NH002. 
 
Primary Endpoint 
6.2
Adverse event (AE) reporting and changes in physical examination findings, vital signs (VS), 
clinical laboratory evaluations, oxygen saturation, and electrocardiograms (ECGs). 
 
Secondary Objective 
6.3
To evaluate the preliminary diagnostic efficacy of NH002 as a contrast agent in 
echocardiography. 
 
Secondary Endpoints 
6.4
• 
The percentage of subjects with moderate or complete LVO, defined by an LVO grade of 
2 (moderate) or 3 (complete), as assessed by the blinded central reader(s); 
• 
The percentage of subjects with complete LVO, defined by an LVO grade of 3 
(complete), as assessed by the blinded central reader(s); 
• 
The change from baseline on the left ventricular EBD (LVEBD) score, defined using a 
standard 12-segment model, as assessed by the blinded central reader(s). The LV 
endocardium of the 4- or 2-chamber apical views are divided into 6 segments, with 2 
basal, mid- and apical segments in each view. For each segment, LVEBD is graded as 
follows: 0 = inadequate border (border not visible); 1 = sufficient (border barely visible); 
2 = good (border clearly visible). A total delineation score (0-24) is obtained by adding 
the scores from the 6 individual segments in each of the 2 views; 
• 
The changes from baseline on LVEBD score of subjects with suboptimal LVEBD at 
baseline, as defined by 2 or more contiguous segments of 6 segments that cannot be 
visualized reliably in either the apical 4- and/or the 2-chamber view, as assessed by the 
blinded central reader(s);  
• 
The duration of clinically useful contrast calculated by measuring the time between the 
disappearance of shadowing effect (useful effect starts) and the time when moderate or 
full LV enhancement and contrast enhancement are no longer adequate (useful effect 
ends), as assessed by the blinded central reader(s). 


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7 
INVESTIGATIONAL PLAN 
 
Overall Study Design and Plan: Description 
7.1
This is a phase 1, multicenter, open-label clinical study to evaluate the safety and tolerability of 
3 ascending doses of NH002. Up to 36 eligible subjects will be enrolled with sequential 
allocation to 1 of 3 cohorts with the following IV doses of NH002: 2.5 µl/kg, 5.0 µl/kg, or 
10.0 µl/kg. Each patient will undergo an unenhanced ultrasound examination and a NH002 
contrast-enhanced examination on the same day at Day 1. Subjects will remain at the clinical site 
for up to 240 minutes of observation after NH002 administration.   
All AEs will be evaluated during the successive cohorts of subjects, per dose level. In general, 
enrollment will be halted and safety data reviewed for termination or continuation with the 
occurrence of any SAE considered at least possibly related to study agent administration (in any 
1 subject) or clinically significant toxicity listed in Section 7.5.1.2 in 30% of subjects (within any 
1 of the 3 dose levels). Please see Section 7.5.1.2 for details on treatment stopping criteria for 
this trial. 
There will be a screening period (28 days before NH002 administration), a dosing period (during 
Day 1) that will include a single dose administration of NH002, and a 7-day follow-up period for 
each cohort. Continuous echocardiographic imaging will be performed at specified time points 
before, during, and after dose administration. The contrast agent will be prepared and 
administered by designated pharmacy technician(s), study nurse(s), sonographer, or radiologist. 
Subjects will be observed at a clinical site for at least 240 minutes (4 hours) and evaluated for 
AEs. Subjects will be contacted by phone after the end of treatment at 72 hours (ie, Day 4 [+3]) 
and at Day 7 (+3), for a follow-up safety assessment. Any drug-related AE or SAE will be 
followed until resolution.   
High ultrasound mechanical index (MI) values may cause microsphere cavitation or rupture and 
lead to ventricular arrhythmias. Additionally, end-systolic triggering with high MIs has been 
reported to cause ventricular arrhythmias. The safety of NH002 at MIs greater than 0.8 will not 
be evaluated. 
In accordance with protocol guidelines for imaging, each study center will follow standard 
practice for (transthoracic) echocardiographic imaging, pre- and post-dosing. Efficacy 
evaluations will be based on blinded central echocardiographic imaging assessments.   
To enhance the safety and integrity of the study data, a dose escalation committee (DEC) will 
evaluate clinical, laboratory, and electrocardiographic safety data, routinely before advancing to 
the next higher dose level/cohort. The DEC will consist of sponsor personnel, the independent 
medical monitor, and the investigator(s) at the study site. A sentinel review approach will be 
followed whereby the first review will be conducted at the end of the safety follow-up 
observation period for the first subject treated in Cohort 1 to determine if subsequent subjects 
may be dosed in the cohort. To review all cumulative safety data for Cohort 1, the next review 
will be conducted at the end of the safety follow-up observation period for the last subject treated 
in Cohort 1. This sentinel review approach will continue to be followed for all successive cohorts 
(ie, Cohort 2 and Cohort 3), to review safety data and to provide a recommendation on study 
continuation, recommended dose, or early termination in case there is a concern regarding safety. 
If treatment stopping criteria, as defined in Section 7.5.1.2, is observed in a specific dosing 




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focal depth, time gain compensation, and dynamic range will be optimized for each subject and 
kept constant during the imaging.  
For each dose, two-dimensional transthoracic echocardiography will be performed using both B-
mode ultrasonography and contrast-specific ultrasonography before agent administration and 
using only contrast-specific ultrasonography after agent administration. The pre-injection 
imaging will be performed once in both apical 4- and 2- chamber views, first using B-mode 
ultrasonography and then contrast-specific ultrasonography. Switch between both apical views or 
imaging modes will be performed in 30-second intervals. Upon NH002 administration, the post-
injection imaging will be subsequently performed in both apical 4- and 2- chamber views using 
only contrast-specific ultrasonography. During this period, alternation of both apical views will 
be performed in 30-seconds until 10 minutes after NH002 administration. In both imaging 
sessions, the 4-chamber view will be performed first.  
DICOM cineloop sequences will be stored. Each cineloop sequence will be acquired for a 
duration of 5 seconds whenever the imaging mode or the imaging view is changed. 
 
 
 See Figure 5 for details.   
Figure 5: Imaging Guidelines 
 
 
Each subject should have 24 DICOM file(s) for the NH002 dosing and the unenhanced imaging. 
These files will be exported to the USB flash drive or CD from the ultrasound machine. 
The video for NH002 dosing will be recorded by an external video recorder in mp4 format. The 
sponsor will provide each site with a recording instrument confirmed to be compatible with 
. 
All clinical sites will use the same model of the recording instruments. The recording duration 
will be at least 15 minutes for each dosing period (starting from approximately 5 minutes before 


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and continuing until 10 minutes after dosing). Each subject should have at least 1 video file 
stored in a USB flash drive or CD (for the NH002 dosing and the unenhanced ultrasound 
imaging). The sponsor will also provide USB flash drives or CDs to sites.  
Central image evaluations will occur offsite to ensure that the blinded reader(s) perform their 
image evaluations independently. The blinded central reader(s) will not have any knowledge of 
subject identifiers nor dose level received to obtain an image. DICOM image groups obtained in 
each subject will be merged and presented to the blinded reader(s) in a random sequence. Each 
blinded evaluation will be locked in the dataset shortly after it is obtained and before additional 
types of image evaluations are performed, if indicated and applicable.  
If indicated and applicable, consensus image evaluations (or consensus reads) can be performed 
after the individual readings (if applicable) are completed and locked. (These consensus reads 
will NOT serve as the primary image evaluation used to demonstrate any preliminary efficacy of 
NH002.) If used, the consensus reads will also be locked once obtained and before additional 
types of blinded readings are performed. 
The blinded central reviewer(s) with appropriate training, education, and qualifications will 
assess the echocardiographic images for this study. The blinded central reader(s) will be 
provided with redacted USB flash drive(s) or CD(s) in order to access and score randomized 
video image groups without knowledge/information of dosing or subject identifiers. Refer to 
Image Evaluation Guidelines for more information. 
7.1.2 Ventricular Arrhythmia Related to High Mechanical Index 
High ultrasound MI values may cause microsphere cavitation or rupture and lead to ventricular 
arrhythmias. Additionally, end-systolic triggering with high MIs has been reported to cause 
ventricular arrhythmias.  
The MI (transmit power) control is important to adjust for penetration needs while limiting 
bubble destruction. Mechanical index settings vary by patient. The MI should be set at 0.8 or 
below. 
Therefore, NH002 is not recommended for use at MIs greater than 0.8.  
 




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for the conditionally triggered standard 12-lead ECG examination. All the identified ECG changes from the modified 12-lead ECG monitoring will be recorded 
in a timely manner. The results of the modified 12-lead ECG will also be included into the subject’s study file as reference. See Section 9.1.2.2.7. Further, safety 
assessments will be based on the results of the standard 12-lead ECG. If such ECG changes occur during both the modified 12-lead ECG monitoring and the 
subsequent confirmative standard 12-lead ECG examination in 2 or more subjects during the study, the protocol will be revised to require a standard 12-lead 
ECG examination be performed for all subsequent subjects at an earlier post-injection time point when these early ECG changes may potentially occur. 
4 Complete PE at screening to include assessments of general appearance, skin (turgor, pallor, cyanosis, erythema, lesions), head (eyes, ear, nose, and throat), 
neck, lungs and heart (observation, palpation, percussion, auscultation), abdomen, back, lymph nodes, and extremities and a neurological exam. 
5 Focused PE (all PEs after screening) to include cardiopulmonary (observation, palpation, percussion, auscultation) and skin (turgor, pallor, cyanosis, erythema, 
lesions) assessments. 
6 B-mode echocardiography and contrast-specific ultrasonography will be employed for all pre-injection and unenhanced images, while post-injection images 
will be acquired through contrast-specific ultrasonography only. See Section 7.1.1 for details. 
7 Urine or serum (β-HCG) pregnancy test is required for WoCBP at screening; it is not required for postmenopausal or surgically-sterilized women. For WoCBP, 
urine and serum pregnancy tests are both required before dosing on Day 1. 
8 CBC to include HCT, Hgb, WBC with differential, ANC, and PLT. 
9 Metabolic panel to include sodium, potassium, chloride, calcium, magnesium, phosphorus, glucose, BUN, creatinine, total protein, albumin, ALP, ALT, AST, 
total bilirubin, and bicarbonate. 
10 Cardiac troponin I levels should be measured before dosing and once again at 4 hours after dosing in all subjects.


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Discussion of Study Design 
7.3
7.3.1 Study Design 
The objectives of this study are to evaluate the safety and tolerability of 3 ascending doses of 
NH002. This will be done through this phase 1, multicenter, open-label, dose escalation study.  
The selected endpoints for demonstrating safety of NH002 are in accordance with current 
standards.7,14-17 Prespecified safety follow-up evaluations will monitor subjects for any AEs and 
potential adverse allergic reactions during and after dosing. 
The evaluable population of eligible subjects with potential cardiac conditions are well suited for 
exploration. Subjects with clinically unstable cardiopulmonary conditions, considered unsuitable 
as determined by the investigator(s), will be excluded from trial participation. These include 
conditions such as, marked ventricular ectopy, atrial fibrillation with variable ventricular 
response, myocardial infarction or unstable angina pectoris (within 6 months prior), acute 
decompensated heart failure, acute renal failure and respiratory failure of any cause, moderate to 
severe pulmonary hypertension, and acute pulmonary embolus or pulmonary infarction. A dose-
finding strategy will be employed, which concentrates patient dosing assignments at and around 
the dose which has the best efficacy/tolerability profile based on a utility function (ie, LVO).18  
The rationale for the design of this phase 1 trial is to appropriately evaluate and explore the 
selected doses of NH002. NH002 is expected to provide improved ultrasound image quality in 
subjects with sub-optimal image(s) at baseline, and better product potency, in addition to 
decreasing the risk of gas embolism in pulmonary circulation.13 Stopping guidelines will also be 
used to adapt to any perceived changes in safety and tolerability. See Section 7.5.1.2 and 
Section 9.1.2.1. 
The clinical relevance for selecting the endpoints of LVO and duration of clinically useful 
contrast is founded upon multiple studies that LV cavity visualization and EBD are often 
compromised.2,3,5,19 A recent report indicates that up to 20% of routine TTE in the clinic setting 
may have poor EBD and could be regarded as nondiagnostic. 19 Multiple studies also reported by 
Chong et al, have demonstrated that microbubble contrast combined with harmonic imaging for 
LVO improves the diagnostic accuracy, confidence, and interobserver agreement in assessment 
of regional systolic function or myocardial thickening in technically difficult-to-image cases. 
19 Intravenous administration of microbubble contrast agents facilitates delineation of the EBD. 
This procedure has been shown to consistently increase the number of myocardial segments that 
can be interpreted, to improve accuracy of assessing regional and global left ventricular function, 
to decrease inter-interpreter variability, to increase interpreter confidence, and to be a cost-
effective strategy.20 
Patient factors further contribute to the significant challenges in poor image quality. It is 
recognized that the clinical utility of echocardiography, LVO, and EBD need to be improved to 
enhance overall endocardial visualization. Accurate assessments of LV volumes also require 
adequate EBD. Kitzman et al demonstrated that contrast-enhanced images resulted in the 
conversion of 48% of non-diagnostic examinations (defined as ≥ 4 of 6 nonevaluable segments 
in a single apical view) into “salvaged” studies (where ≤ 1 poorly visualized segment remained 


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on the same comparative view) following LVO.2 By improving the quality of images acquired, it 
is expected to restore adequate diagnostic utility. 
Refer also to Section 5 of the protocol. 
7.3.2 Quality Management and Risk Evaluation 
This protocol was evaluated to identify processes and data that were critical to assure human 
subject protection and reliability of study results. 
Predefined quality tolerance limits have been established, taking into consideration the medical 
and statistical characteristics of the variables as well as the statistical design of the study, to 
identify systematic issues that could impact subject safety or data integrity. Detection of 
deviations from the predefined quality tolerance limits will trigger an evaluation to determine if 
action is needed. Any important deviations from the predefined quality tolerance limits and 
remedial actions taken will be described in the clinical study report. 
Risk control measures will be periodically reviewed to ascertain whether the implemented 
quality management activities remain effective and relevant, taking into account emerging 
knowledge and experience. 
 
Selection of Study Population 
7.4
7.4.1 Number of Planned Subjects  
It is estimated that 36 subjects will be enrolled in the study.  
If a subject is withdrawn from the study, except for those subjects who will be terminated early 
due to AEs, the subject may be replaced as necessary with another subject assigned to the same 
dose, except for those who are discontinued due to AEs, as necessary. Further subjects may be 
enrolled at a given dose level if additional data are necessary to establish safety and tolerability 
prior to dose escalation. 
Refer to the statistical considerations on which the numbers are based in Section 10.8.  
7.4.2 Inclusion Criteria 
To be eligible for study entry subjects must satisfy all of the following inclusion criteria: 
1. Male or female subjects 20 years of age or older 
2. Ability to understand and the willingness to provide written informed consent 
3. Suspected of having cardiac disease or undergoing evaluation of cardiac anatomy for 
congenital heart disease 
4. Negative pregnancy test within 24 hours of the procedure if subject is a woman of 
childbearing potential (WoCBP) 


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7.4.3 Exclusion Criteria 
Subjects will be excluded from the study if one or more of the following exclusion criteria is 
applicable: 
1. Known right-to-left, bidirectional, or transient right-to-left cardiac shunt(s) 
2. Chronic obstructive pulmonary disease  
3. Ejection fraction at screening or baseline < 40% 
4. Uncontrolled serious ventricular arrhythmias or at high risk for arrhythmias due to 
prolongation of the QT interval (QT corrected by Fridericia’s formula > 450 msec in males 
and > 470 msec in females) 
5. Clinically significant abnormality in renal or liver function tests (alanine transferase 
[ALT]/aspartate aminotransferase [AST] > 2 × upper limit of normal [ULN]; serum 
creatinine > 1.5 × ULN) 
6. Known or suspected hypersensitivity to one or more of the ingredients of NH002, perflutren, 
or other ultrasound contrast agents 
7. Received an investigational compound within 30 days before enrolling in the study 
8. Received any contrast agent either intravascularly or orally within 48 hours before NH002 
administration 
9. Pregnant or lactating female (conception during the study should be avoided) 
10. Serious medical or psychiatric illness/condition likely, in the judgment of the investigator, to 
interfere with compliance to protocol treatment/research 
11. Clinically unstable cardiopulmonary conditions considered not suitable for participation in 
the trial, in the judgment of the investigator 
12. Uncontrolled arterial hypertension (defined as systolic blood pressure ≥ 200 mmHg or 
diastolic blood pressure ≥ 110 mmHg) or arterial hypotension (defined as systolic blood 
pressure ≤ 90 mmHg) 
7.4.4 Removal of Subjects From Therapy or Assessments 
Subjects may stop study agent and withdraw from the study for any of the following reasons: 
• 
Subject request 
• 
Use of nonpermitted concurrent therapy 
• 
Non-compliance with the study agent or study schedule 
• 
Lost to follow-up 
• 
Occurrence of AEs not compatible with the continuation of subject participation in the 
study, in the investigator’s opinion, or unacceptable to the subject to continue 
• 
Investigator request 
• 
Intercurrent illness 
• 
Sponsor request 
• 
Pregnancy 


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Subjects who do not comply with the protocol or who withdraw consent will be replaced. 
Subjects who stop study agent for any other reason (ie, AE) will not be replaced.  
Subjects are free to withdraw from the study at any time without providing reason(s) for 
withdrawal and without prejudice to further research treatment. The reason(s) for withdrawal 
will be documented in the case report form (CRF). 
Subjects withdrawing from the study will be encouraged to complete the same final evaluations 
as subjects completing the study according to this protocol, particularly safety evaluations. The 
aim is to record data in the same way as for subjects who completed the study. 
Reasonable efforts will be made to contact subjects who are lost to follow-up. These efforts must 
be documented in the subject’s file.  
The sponsor has the right to terminate the study at any time in case of SAEs or if special 
circumstances concerning the study agent or the company itself occur, making further research 
treatment of subjects impossible. In this event, the investigator(s) will be informed of the reason 
for study termination. 
Pregnancy 
Only female subjects with negative pregnancy tests will be allowed to enroll in the study. 
Subjects will be instructed that known or suspected pregnancy occurring during the study in 
female subjects should be confirmed and reported to the investigator, who will then withdraw the 
subject from the study without delay. Upon discontinuation from the study, only those 
procedures that would not expose the subject to undue risk will be performed.  
The investigator should also be notified of pregnancy occurring during the study but confirmed 
after completion of the study.  
In the event that a subject is subsequently found to be pregnant after inclusion in the study, any 
pregnancy will be followed to term, and the status of mother and child will be reported to the 
sponsor after delivery. 
Full details will be recorded on the withdrawal page of the CRF, or an SAE report will be 
completed if the subject has completed the study. Procedures to be followed if a pregnancy 
occurs are provided in Section 9.1.2.2.1. 
7.4.5 Enrollment Guidelines 
All eligibility requirements must be reviewed before the subject may enter the study. The 
following information must also be provided to the sponsor and/or their representative, as 
requested: 
1. Completed and signed protocol-specific eligibility checklist. 
2. All pages of the original signed informed consent forms (ICFs), including Health Insurance 
Portability and Accountability Act (HIPAA)21 Form for US sites. 


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3. Relevant source documents such as: subject medical history and physical exam, admission or 
discharge notes, diagnostic reports, confirmation of diagnosis, and relevant subject-specific 
written communication. 
 
Investigational Products 
7.5
7.5.1 Investigational Products Administered 
7.5.1.1 NH002 
Each subject will receive 1 of 3 NH002 doses (2.5 µl/kg, 5.0 µl/kg, or 10.0 µl/kg) by IV bolus 
injection. The subjects will be allocated sequentially to each single dose as follows: initial 12 
subjects receive 2.5 µl/kg, the next 12 receive 5.0 µl/kg, and final 12 receive 10.0 µl/kg. See 
Table 4. 
Table 4: NH002 Administration 
Agent  
Subjects 
Dose (µl/kg) IV bolus 
Schedule 
NH002 
First set of 12 
2.5   
Day 1  
Next set of 12 
5.0   
Final set of 12 
10.0   
 
7.5.1.2 Stopping Criteria for Dosing 
All AEs will be evaluated during the successive cohorts of subjects, per dose level. Intrasubject 
dose escalations are not permitted. 
Dose escalation will proceed successively with each cohort as described in previous sections 
(Section 7.5.1.1), unless any of the following criteria is/are exhibited:  
• The occurrence of any SAE considered at least possibly related to study agent 
administration in any 1 subject who received study agent. The occurrence of such an SAE 
will supersede all other stopping rules. 
• The occurrence of the following in 30% of subjects (within any one of the 3 dose levels): 
o moderate or severe clinically significant nonhematologic lab toxicity, including 
cardiac disorder(s) 
o moderate or severe clinically significant hematologic lab toxicity 
o mild, moderate, or severe clinically significant anaphylactic reaction  
Supportive care is permissible at the discretion and clinical judgment of the treating investigator, 
until symptoms are resolved to baseline.  
If any of the stopping criteria for dosing are observed, the study will be halted, and the risk to 
other subjects evaluated, prior to a decision as to whether to terminate the study (See also 
Section 9.1.2.1 for overall study stopping rules). 




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elastomeric septum. It is essential to follow directions for activation carefully and to adhere to 
strict aseptic procedures during preparation. 
7.5.1.4 Injection Volume(s) 
The injection volume (µl) for each subject will be calculated based on their body weight (kg). 
See Table 5.  
Table 5: NH002 Injection Volume(s) 
NH002 Dose (µl/kg) 
NH002 volume (µl) based on subject weight (X kg) 
10.0 
 = X × 10.0 
5.0 
= X × 5.0 
2.5   
= X × 2.5  
 
7.5.1.5 Injection Route and Rate 
For this trial, NH002 must be injected by an IV bolus method. Do not administer by intra-
arterial injection. 
Slow injection is important for safety (according to prior experience in animal studies). Sites 
should always have cardiopulmonary resuscitation personnel and equipment readily 
available before administration and monitor all subjects for acute reactions. 
NH002 will be injected as an IV bolus, within 30 to 60 seconds, followed by a 10 mL saline 
flush over 10 seconds.  
7.5.1.6 Emergency Event Management 
Subjects will be closely monitored by a medical team during the injection and for at least 4 hours 
(240 minutes) following dosing for the presence of symptoms of allergic or hypersensitivity 
reaction(s). Subjects who receive 4-hour monitoring after the dose on Day 1 will be encouraged 
to quickly report any symptoms at the time during this period. The necessary rescue material, 
equipment, and appropriate medications will be available in the clinic to allow rapid intervention 
in case of emergency.   
7.5.2 Identity of Investigational Products 
7.5.2.1 NH002 (perflutren lipid microspheres) Injectable Suspension 
Formulation, Dosage and Strength 
The gas core in NH002 is perflutren. The structure is a gas core surrounded by a lipid shell 
(Figure 6). NH002 is formulated as a microbubble injectable suspension of perflutren in single-
use, 1-mL lipid solution in 2-mL vials with 
 C3F8 in headspace for IV administration. 
Chemical Name  


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NH002 (perflutren lipid microspheres) 
Chemical Structure 
Molecular formula: C3F8   
How Supplied 
NH002 is supplied as a single use, 1-mL lipid solution in 2-mL clear glass vial 
containing clear liquid in packages of 4 single-use vials.  
Storage and Handling 
Store NH002 between 2-8°C (36°-46°F). 




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7.5.6.1 Prohibited Medication/Therapy 
Subjects must not have received an investigational compound within 30 days before enrolling in 
the study. Subjects must also not have received any contrast agent either intravascularly or orally 
within 48 hours before NH002 administration. Concomitant medications and procedures will be 
recorded beginning at the time of screening, as well as during and after research treatment.   
Subjects with known right-to-left, bidirectional, or transient right-to-left cardiac shunt(s) are 
excluded from this study, but it is important to note the following: When administering NH002 
to subjects with a cardiac shunt, the microspheres can bypass filtering by the lung and enter the 
arterial circulation. Subjects with shunts should be assessed for embolic phenomena following 
NH002 administration.  
7.5.6.2 Rescue Medication 
Slow injection is important for safety (according to prior experience in animal studies). Sites 
should always have cardiopulmonary resuscitation personnel and equipment readily available 
before administration and monitor all subjects for acute reactions. 
7.5.7 Dosing Compliance 
On the day of scheduled dosing (ie, Day 1) dosing compliance will be assessed by the 
administering clinician(s). At a minimum, date, time, duration, and dose should be recorded in 
the treatment record(s) to confirm that each dose of NH002 was administered as per protocol.  
8 
TIMING OF STUDY PROCEDURES 
Before performing any study-specific procedure or assessment, written informed consent and 
authorization for use of protected health information (HIPAA) 21 in the US, must be obtained in 
accordance with all application policies, regulations, and laws. The planned study assessments 
are in Section 7.2. 
 
Pre-dosing 
8.1
8.1.1 Visit 1: Screening  
The following assessment(s) must be collected/performed within 28 days prior (unless specified) 
to Day 1. Clinical and laboratory evaluations performed as part of routine standard of care do not 
need to be repeated if performed within the appropriate window. 
• 
Eligibility: Assess against the inclusion and exclusion criteria 
• 
Medical history: Relevant history including description of all prior therapies, and signs or 
symptoms 
• 
Vital signs: Measurements include body temperature, blood pressure (BP), heart rate (HR), 
and respiratory rate (RR)  
• 
Demography 
• 
Height (cm) 
• 
Weight (kg) 


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• 
Standard 12-lead ECG 
• 
Physical exam (PE): A complete PE will include assessments of general appearance, skin 
(turgor, pallor, cyanosis, erythema, lesions), head (eyes, ear, nose, and throat), neck, lungs 
and heart (observation, palpation, percussion, auscultation), abdomen, back, lymph nodes, 
extremities, and neurological exam 
• 
A urine or serum (beta-human chorionic gonadotropin [β-HCG]) pregnancy test is required 
for WoCBP; it is not required for postmenopausal or surgically sterilized women.  
• 
Concomitant medications and procedures: Concomitant medications and procedures will 
be recorded beginning at the time of screening. 
 
Dosing Period 
8.2
Collection of concomitant medications and AEs as defined in Section 9.1.2 should occur 
throughout the study. 
For imaging guidelines, refer to Section 7.1.1. 
8.2.1 Visit 2: Day 1, Dosing  
8.2.1.1 Before Dosing 
The following procedures will be performed before administration of the NH002 contrast 
injection on Day 1: 
• 
Eligibility: Assess against the inclusion and exclusion criteria 
• 
Medical history, including baseline signs and symptoms should be documented and 
continually reassessed throughout the study 
• 
VS: Measurements include BP, body temperature, HR, and RR  
• 
Weight (kg) 
• 
Standard 12-lead ECG 
• 
Focused PE: A focused PE will include cardiopulmonary (observation, palpation, 
percussion, auscultation) and skin (turgor, pallor, cyanosis, erythema, lesions) assessments. 
• 
Both a urine and a serum (β-HCG) pregnancy test are required for WoCBP before dosing 
on Day 1. (Note that at screening, only a urine or serum test is necessary; before dosing on 
Day 1, both tests are necessary.) 
• 
Complete blood count (CBC) to include hematocrit (HCT), hemoglobin (Hgb), white blood 
cell (WBC) count with differential, absolute neutrophil count (ANC), and platelet count 
(PLT) 
• 
Coagulation studies: Thrombin time (TT) and activated partial thromboplastin time (aPTT) 
• 
Metabolic panel: Metabolic panel evaluations to include sodium, potassium, chloride, 
calcium, magnesium, phosphorus, glucose, blood urea nitrogen (BUN), creatinine, total 
protein, albumin, alkaline phosphatase (ALP), ALT, AST, total bilirubin, and bicarbonate  
• 
Cardiac troponin I 
• 
Pulse oximetry: SpO2 
• 
Pretreatment AEs: Baseline events, findings, and conditions before NH002 initiation 
should be documented and continually reassessed throughout the study. 


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• 
Concomitant medications and procedures: Concomitant medications and procedures will 
continue to be recorded. 
8.2.1.2 5 Minutes Before Dosing 
The following procedures will be performed approximately 5 minutes before dosing: 
• 
Imaging should proceed as specified in Section 7.1.1. 
• 
Modified 12-lead ECG monitoring should be conducted, from 5 minutes before dosing to 
10 minutes after dosing. See Section 9.1.2.2.7. 
• 
Pulse oximetry: SpO2 should be measured and recorded, from 5 minutes before dosing to 
10 minutes after dosing.  
• 
VS: BP, body temperature, HR, and RR should be measured and recorded, from 5 minutes 
before dosing to 10 minutes after dosing. 
• 
Record any AEs that may occur and any changes in concomitant medication. 
 
8.2.1.3 Dosing 
After all procedures have been performed, NH002 will be administered. Each subject will 
receive an assignment to 1 of the 3 NH002 dose levels (2.5 µl/kg, 5.0 µl/kg, or 10.0 µl/kg IV 
bolus). See Section 7.5.1 for details. The following procedures will be performed after the 
contrast injection:  
• 
Imaging should proceed as specified in Section 7.1.1. 
• 
Modified 12-lead ECG monitoring to continue until completion of imaging (ie, to 10-
minutes after dosing). Any clinically significant ECG changes indicative of arrhythmia or 
S-T segment change identified after dosing from the modified 12-lead ECG monitoring 
will be recorded in a timely manner, and a standard 12-lead ECG will be conducted 
subsequently (when the first recorded ECG change occurs). The imaging will be 
temporarily stopped for the conditionally triggered standard 12-lead ECG examination. 
The results of the modified 12-lead ECG will also be included into the subject’s study file 
as reference. See Section 9.1.2.2.7. 
• 
Pulse oximetry: SpO2 should also be measured and recorded until completion of imaging 
(ie, 10 minutes after dosing).  
• 
VS: BP, body temperature, HR, and RR should be measured and recorded until completion 
of imaging (ie, 10 minutes after dosing). 
• 
Record any AEs that may occur and any changes in concomitant medication. 
8.2.1.4 10 Minutes After Dosing 
The following procedures will be performed approximately 10 minutes after the contrast 
injection: 
• 
VS: Measurements include BP, body temperature, HR, and RR  
• 
Modified 12-lead ECG monitoring to continue until completion of imaging (ie, to 10-
minutes post dosing); See Section 9.1.2.2.7. 


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• 
Standard 12-lead ECG. Site(s) should make every effort to target the timing to be as close 
to the end of the modified 12-lead ECG monitoring (10 minutes after dosing) as possible. 
• 
Pulse oximetry: SpO2 
• 
Imaging should proceed as specified in Section 7.1.1.    
• 
Record any AEs that have occurred since the previous visit and any changes in 
concomitant medication. 
8.2.1.5 30 Minutes After Dosing 
The following procedures will be performed approximately 30 minutes after the contrast 
injection (+5 minutes): 
• 
VS: Measurements include BP, body temperature, HR, and RR  
• 
Standard 12-lead ECG 
• 
Focused PE: A focused PE will include cardiopulmonary (observation, palpation, 
percussion, auscultation) and skin (turgor, pallor, cyanosis, erythema, lesions) assessments. 
• 
Pulse oximetry: SpO2 
• 
Record any AEs that have occurred since the previous visit and any changes in 
concomitant medication. 
Site(s) should make every effort to target the timing as possible. However, given that these 
procedures may be performed in different examination rooms in the study sites, a time window 
of +5 minutes is permitted. 
8.2.1.6 60 Minutes After Dosing 
The following procedures will be performed approximately 60 minutes after the contrast 
injection (+5 minutes): 
• 
VS: Measurements include BP, body temperature, HR, and RR  
• 
Standard 12-lead ECG 
• 
Focused PE: A focused PE will include cardiopulmonary (observation, palpation, 
percussion, auscultation) and skin (turgor, pallor, cyanosis, erythema, lesions) assessments. 
• 
CBC to include HCT, Hgb, WBC count with differential, ANC, and PLT 
• 
Coagulation studies: TT and aPTT 
• 
Metabolic panel: Metabolic evaluations include sodium, potassium, chloride, calcium, 
magnesium, phosphorus, glucose, BUN, creatinine, total protein, albumin, ALP, ALT, 
AST, total bilirubin, and bicarbonate 
• 
Pulse oximetry: SpO2 
• 
Record any AEs that have occurred since the previous visit and any changes in 
concomitant medication. 
Site(s) should make every effort to target the timing as possible. However, given that these 
procedures may be performed in different examination rooms in the study sites, a time window 
of +5 minutes is permitted. 


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8.2.1.7 120 Minutes After Dosing 
The following procedures will be performed approximately 120 minutes after the contrast 
injection (+5 minutes): 
• 
VS: Measurements include body temperature, BP, HR, and RR  
• 
Focused PE: A focused PE will include cardiopulmonary (observation, palpation, 
percussion, auscultation) and skin (turgor, pallor, cyanosis, erythema, lesions) assessments. 
• 
CBC to include HCT, Hgb, WBC count with differential, ANC, and PLT 
• 
Coagulation studies: TT and aPTT 
• 
Metabolic panel: Metabolic panel evaluations include sodium, potassium, chloride, 
calcium, magnesium, phosphorus, glucose, BUN, creatinine, total protein, albumin, ALP, 
ALT, AST, total bilirubin, and bicarbonate  
• 
Pulse oximetry: SpO2 
• 
Record any AEs that have occurred since the previous visit and any changes in 
concomitant medication. 
8.2.1.8 240 Minutes After Dosing 
The following procedures will be performed approximately 240 minutes after the contrast 
injection (+5 minutes): 
• 
CBC to include HCT, Hgb, WBC count with differential, ANC, and PLT 
• 
Coagulation studies: TT and aPTT 
• 
Cardiac Troponin I 
• 
Record any AEs that have occurred since the previous visit and any changes in 
concomitant medication. 
 
Safety Follow-up Period 
8.3
8.3.1 Day 4 (+3)  
Subjects will be contacted by telephone at least 72 hours after NH002 dosing (ie, Day 4 [+3]) for 
a follow-up assessment of safety. An additional 3-day window is permitted for this Safety 
Follow-up, if site(s) have difficulty reaching the subject on Day 4. The following procedures will 
also be performed during the Safety Follow-up call: 
• Record any AEs that have occurred since the previous visit and any changes in concomitant 
medication. Any AE or SAE should be followed up until resolved.   
8.3.2 Day 7 (+3) 
Subjects will be contacted by telephone again 7 days after the end of their treatment for another 
follow-up assessment of safety. An additional 3-day window is permitted for this second Safety 
Follow-up, if site(s) have difficulty reaching the subject on Day 7. The following procedures will 
be also performed during the Safety Follow-up call: 


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• Record any AEs that have occurred since the previous visit and any changes in concomitant 
medication. Any AE or SAE should be followed up until resolved.   
 
Duration of Treatment 
8.4
The duration of treatment is expected to last between 4 to 5 hours during 1 day, barring any 
unexpected adverse reactions.  
9 
EFFICACY AND SAFETY VARIABLES  
The planned schedule of assessments is in Section 7.2. 
 
Efficacy and Safety Measurements Assessed  
9.1
9.1.1 Efficacy Variables   
9.1.1.1 Echocardiographic Imaging   
Efficacy evaluations will be based on blinded central echocardiographic imaging assessments of 
LVO, duration of useful contrast enhancement, LVEBD, and diagnostic confidence: 
• 
Left ventricular opacification (LVO): The degree of LVO following each injection was 
graded according to the following four-point rating scale: 0= none (no visible contrast 
within the LV cavity); 1= faint (weak or trace effect of contrast within the LV cavity); 2= 
moderate (some areas of the LV cavity fully opacified but without a time when the whole 
cavity was filled with contrast to the same high density); or 3= complete (homogeneous 
and high intensity effect).  
• 
Duration of clinically useful contrast enhancement: The time between the disappearance 
of shadowing effect and the time when moderate or full LV enhancement and contrast 
enhancement are no longer adequate (useful effect ends). 
• 
Left ventricular endothelial border delineation (LVEBD): To determine the impact of 
NH002 on EBD, a standard 12-segment model will be used, dividing the LV 
endocardium of the 4- or 2-chamber apical views into 6 segments, with 2 basal, mid- and 
apical segments in each view.    
9.1.2 Safety Assessments 
Each subject must be carefully monitored for the development of any AEs throughout the study 
from the signing of the ICF to at least 7 days after receipt of the study agent (ie, Day 7 Follow-up 
call). Safety evaluations will also be assessed and recorded at specified time points: before 
injection, during injection and imaging (beginning approximately 5 minutes prior to and 
continuing until 10 minutes after injection), and up to 7 days post-injection.  
Safety parameters to be evaluated will include: 
• 
Standard 12-lead ECGs assessed pre-injection, and at 10, 30, and 60 minutes post-
injection; during imaging, a standard 12-lead ECG will be conditionally performed at the 


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earliest time point when any clinically significant ECG changes indicative of arrhythmia 
or S-T segment change are identified within 10 minutes after injection from the modified 
12-lead ECG monitoring; each standard 12-lead ECG assessment will be scored as either 
normal or abnormal;  
• 
Changes in VS assessed pre-injection, during injection and imaging (beginning 
approximately 5 minutes prior to and continuing until 10 minutes after), and at 30, 60, 
and 120 minutes post-injection; VS measurements will include body temperature, HR, 
RR, and BP; 
• 
The following parameters will be included in the complete PE (at screening): General 
appearance, skin (turgor, pallor, cyanosis, erythema, lesions), head (including ear, eyes, 
nose, and throat), neck, lungs and heart (observation, palpation, percussion, auscultation), 
abdomen, back lymph nodes, extremities, and a neurological exam. A focused PE (after 
screening) will include cardiopulmonary (observation, palpation, percussion, 
auscultation) and skin (turgor, pallor, cyanosis, erythema, lesions) assessments; each PE 
assessment will be scored as either normal or abnormal; 
• 
Changes in hematology and metabolic laboratory parameters taken pre-injection and 60, 
120, and 240 minutes post-injection; 
• 
TT and aPTT pre-injection and 60, 120, and 240 minutes post-injection; 
• 
Cardiac troponin I pre-injection and 240 minutes (4 hours) post-injection; 
• 
SpO2 assessed pre-injection, during injection and imaging (beginning approximately 5 
minutes prior to and continuing until 10 minutes after), and at 30, 60, and 120 minutes 
post-injection; 
• 
The assessment of any potential adverse allergic reactions during and post-injection. 
9.1.2.1 Overall Study Stopping Rules 
The estimated duration of the study is contingent on safety and the number of dose levels 
evaluated in the dose escalation. See Section 7.5.1.2 for stopping criteria for dosing. The study 
will be stopped in the event of any new findings that indicate a relevant deterioration of the risk-
benefit relationship that would render continuation of the study unjustifiable. Study 
administration with NH002 will be halted and the DEC will convene to determine if the study 
should be terminated for any safety concerns. See Section 9.2 for DEC details. 
The sponsor may also end the study for administrative reasons.  
Should the study be terminated prematurely, the sponsor will provide written notification to all 
investigators and regulatory authorities specifying the reason(s) for early termination. The 
investigator must inform the institutional review board (IRB)/independent ethics committees 
(IEC) promptly and provide the reason(s) for the termination. Previously dosed subjects will be 
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9.1.2.2 Adverse Events 
Adverse Event Definition 
An AE is defined as any untoward medical occurrence in a clinical study subject administered a 
medicinal product which does not necessarily have a causal relationship with this treatment. An 
AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of a medicinal (investigational) 
product, whether or not it is related to the medicinal (investigational) product. This includes an 
exacerbation of pre-existing conditions or events, intercurrent illnesses, drug interaction or the 
significant worsening of the indication under investigation that is not recorded elsewhere in the 
CRF under specific efficacy assessments. Anticipated fluctuations of pre-existing conditions, 
including the disease under study, that do not represent a clinically significant exacerbation or 
worsening need not be considered AEs. 
It is the responsibility of the investigator to document all AEs that occur during the study. Any 
AEs will be elicited by asking the subject a nonleading question, for example, “Have you 
experienced any new or changed symptoms since we last asked/since your last visit?”. All AEs 
should be reported on the appropriate page of the CRF. 
Assessment of Severity 
Each AE will be assigned a category by the investigator as follows: 
Mild: 
An AE that is easily tolerated by the subject, causes minimal discomfort, 
and does not interfere with everyday activities. 
Moderate: 
An AE that is sufficiently discomforting to interfere with normal 
everyday activities; intervention may be needed. 
Severe: 
An AE that prevents normal everyday activities; treatment or other 
intervention usually needed. 
If there is a change in severity of an AE, it must be recorded as a separate event. 
Assessment of Causality 
Every effort will be made by the investigator to assess the relationship of the AE, if any, to the 
study agent. Causality should be assessed using the categories presented in the following: 
Unrelated: 
Clinical event with an incompatible time relationship to study agent 
administration, and that could be explained by underlying disease or 
other drugs or chemicals or is incontrovertibly not related to the study 
agent. 
Unlikely: 
Clinical event whose time relationship to study agent administration 
makes a causal connection improbable, but that could plausibly be 
explained by underlying disease or other drugs or chemicals. 


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Possible: 
Clinical event with a reasonable time relationship to study agent 
administration, but that could also be explained by concurrent disease 
or other drugs or chemicals. 
Probable: 
Clinical event with a reasonable time relationship to study agent 
administration, and is unlikely to be attributed to concurrent disease 
or other drugs or chemicals. 
Very 
Likely/Certain: 
Clinical event with plausible time relationship to study agent 
administration, and that cannot be explained by concurrent disease or 
other drugs or chemicals. 
Action Taken 
The investigator will describe the action taken (with regard to the study agent and/or the subject) 
in the appropriate section of the CRF, as follows: 
• 
None 
• 
Study agent stopped 
• 
Study agent temporarily interrupted 
• 
Concomitant medication 
• 
Other, specify. 
Follow-up of Adverse Events 
All investigators should follow up subjects with AEs until the event is resolved or until, in the 
opinion of the investigator, the event is stabilized or determined to be chronic. Details of AE 
resolution must be documented in the CRF. 
Subjects should also be followed up 3 days (+3) and 7 days (+3) after receiving NH002 (ie, 
Day 4 and Day 7), and any AEs that occur during this time should be reported according to the 
procedures outlined in this section. 
Documentation and Reporting of Adverse Events 
All AEs should be reported and documented in accordance with the procedures outlined in this 
section. All AEs occurring during the study must be documented on the relevant CRF pages. The 
following data should be documented for each AE: 
• 
Description of the symptom event 
• 
Classification of ‘serious’ or ‘not serious’ 
• 
Severity 
• 
Date of first occurrence and date of resolution (if applicable) 
• 
Action taken 
• 
Causal relationship 
• 
Outcome of event (unknown, recovered, not yet recovered, recovered with sequelae, death 
[with date and cause reported]) 


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9.1.2.2.1 Serious Adverse Events 
Serious Adverse Event Definition 
An SAE is any untoward medical occurrence or effect that, at any dose, 
• 
Results in death. 
• 
Is life-threatening (an AE is life-threatening if the subject was at immediate risk of death 
from the event as it occurred; ie, it does not include a reaction that might have caused death 
if it had occurred in a more serious form). 
• 
Requires or prolongs inpatient hospitalization. (Complications occurring during 
hospitalization are AEs and are SAEs if they cause prolongation of the current 
hospitalization. Hospitalization for elective treatment of a pre-existing nonworsening 
condition is not, however, considered an AE. The details of such hospitalizations must be 
recorded on the medical history or physical examination page of the CRF). 
• 
Results in persistent or significant disability/incapacity. (An AE is incapacitating or 
disabling if it results in a substantial and/or permanent disruption of the subject’s ability to 
carry out normal life functions). 
• 
Results in a congenital anomaly/birth defect. 
In addition, medical and scientific judgement is required to decide if prompt notification is 
required in situations other than those defined for SAEs above. This may include any event that 
the investigator regards as serious that did not strictly meet the criteria above but may have 
jeopardized the subject or required intervention to prevent one of the outcomes listed above, or 
that would suggest any significant hazard, contraindication, side effect, or precaution that may be 
associated with the use of the investigational product. 
Reporting of Serious Adverse Events 
Any SAE must be reported by the investigator if it occurs during the clinical study or within 4-7 
days of receiving the study agent, whether or not the SAE is considered to be related to the 
investigational product. An SAE report consists of the SAE form, the AE form, and the 
concomitant medication form. A copy of these forms must be emailed within 24 hours for the 
attention of the product safety scientist at: 
Name: Ting-Yu Huang 
Company: Trust Bio-sonics, Inc. 
E-mail: 
  
The investigator should not wait to receive additional information to document fully the event 
before notification of a SAE, though additional information may be requested. Where applicable, 
information from relevant laboratory results, hospital case records, and autopsy reports should be 
obtained. 
Instances of death, congenital abnormality, or an event that is of such clinical concern as to 
influence the overall assessment of safety, if brought to the attention of the investigator at any 


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time after cessation of study agent administration and linked by the investigator to this study, 
should be reported to the study monitor. 
The sponsor and/or the appointed representative(s) will promptly notify all relevant investigators 
and the regulatory authorities of findings that could adversely affect the safety of subjects, 
impact on the conduct of the study, or alter the IEC/ IRB approval/favorable opinion of the 
study. In addition, the sponsor and/or the appointed representative(s), will expedite the reporting 
to all concerned investigators, to the IEC(s)/IRB(s), where required, and to the regulatory 
authorities of all adverse reactions that are both serious and unexpected. 
Details of the procedures to be followed if a pregnancy occurs are also provided in Section 7.4.4. 
9.1.2.2.2 Unexpected Adverse Reactions 
Unexpected Adverse Reaction Definition 
An unexpected adverse reaction is any untoward and unintended response that is related to the 
administration of the study agent, at any dose that is not consistent with the applicable product 
information (eg, current version of the Investigator's Brochure for an unauthorized 
investigational medicinal product or summary of product characteristics for an authorized 
product). 
All suspected unexpected serious adverse reactions (SUSARs) will be the subject of expedited 
reporting. The sponsor and/or the appointed representative(s) shall ensure that all relevant 
information about a SUSAR that is fatal or life-threatening is reported to the relevant competent 
authorities and IEC/IRB within 7 days after knowledge by the sponsor of such a case and that 
relevant follow-up information is communicated within an additional 8 days. All other SUSARs 
will be reported to the relevant competent authorities and IEC/IRB within 15 days after 
knowledge by the sponsor of such a case. All investigators should follow up SUSARs until the 
event is resolved or until, in the opinion of the investigator, the event is stabilized or determined 
to be chronic. Poststudy SUSARs that occur after the subject has completed the clinical study 
must be reported by the investigator to the sponsor. 
Warnings and Precautions  
Hypersensitivity Reactions: Do not administer NH002 to subjects with known or suspected 
hypersensitivity to perflutren.  
Serious Cardiopulmonary Reactions: Serious cardiopulmonary reactions including fatalities have 
occurred uncommonly during or shortly following perflutren-containing microsphere 
administration, typically within 30 minutes of administration. The risk for these reactions may be 
increased among subjects with unstable cardiopulmonary conditions (acute myocardial 
infarction, acute coronary artery syndromes, worsening or unstable congestive heart failure, or 
serious ventricular arrhythmias). Always have cardiopulmonary resuscitation personnel and 
equipment readily available before NH002 administration and monitor all subjects for acute 
reactions. 


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Systemic Embolization: Subjects with known right-to-left, bidirectional, or transient right-to-left 
cardiac shunt(s) are excluded from this study, but it is important to note the following. When 
administering study agent to subjects with a cardiac shunt, the microspheres can bypass filtering 
by the lung and enter the arterial circulation. Subjects with shunts should be assessed for embolic 
phenomena following study agent administration. The study agent is only for IV administration; 
do not administer by intra-arterial injection(s). 
Ventricular Arrhythmia Related to High Mechanical Index: High ultrasound MI values may 
cause microsphere cavitation or rupture and lead to ventricular arrhythmias. Additionally, end-
systolic triggering with high MIs has been reported to cause ventricular arrhythmias. Study agent 
is not recommended for use at MIs greater than 0.8. 
9.1.2.2.3 Clinical Laboratory Evaluation 
The hematology and clinical chemistry laboratory analyses will be performed at local 
laboratories. Reference ranges will be supplied by the local laboratories and used by the 
investigator to assess the laboratory data for clinical significance and pathological changes.  
Methods (including whether samples are taken fed or fasting) and timing for assessing, recording 
and analyzing each laboratory variable should follow local guidelines, within the confines of the 
protocol timeframes. The following laboratory safety tests will be performed at screening and 
treatment visits: 
Hematology 
Hgb, HCT, WBC count (total and differential), red blood cell count, PLT, mean cell volume, 
mean cell hemoglobin (MCH), and MCH concentration 
Clinical Chemistry 
Creatinine, BUN, AST, ALT, ALP, total bilirubin, albumin, total protein, sodium, potassium, 
chloride, glucose, bicarbonate, calcium, magnesium, and phosphorus 
9.1.2.2.4 Other Laboratory Variables 
Screening for pregnancy will be performed (serum or urine β-HCG at screening and serum and 
urine β-HCG before dosing on Day 1, for WoCBP only). 
Other laboratory variables collected are:  
• 
Coagulation studies of TT and aPTT 
• 
Cardiac troponin I 
9.1.2.2.5 Vital Signs 
Vital signs (BP, body temperature, RR, and HR) will be recorded at screening, before injection, 
during injection and imaging (beginning approximately 5 minutes prior to and continuing until 
10 minutes after injection), and 30, 60, and 120 minutes post-injection, in a standardized manner. 


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9.1.2.2.6 Pulse oximetry 
SpO2 will be assessed pre-injection, during injection and imaging (beginning approximately 5 
minutes prior to and continuing until 10 minutes after), and at 30, 60, and 120 minutes post-
injection. 
9.1.2.2.7 Physical Examination 
At screening, a complete physical examination will be performed. A focused physical 
examination will be performed before dosing and during dosing at the following time points for 
each contrast injection: 30, 60, and 120 minutes post-injection. Any changes will be recorded 
and assessed. Each PE assessment will be scored as either normal or abnormal. 
9.1.2.2.8 Electrocardiogram 
Standard 12-lead ECGs will be performed at screening, before dosing on Day 1, and at 10, 30, 
and 60 minutes post-injection. Safety assessments will be based on the results of the standard 12-
lead ECGs. The following parameters will be evaluated: First degree heart block (HB), second 
degree HB, complete HB, atrial arrhythmia, junctional rhythms, ventricular arrhythmia, LVH 
(left ventricular hypertrophy), other chamber enlargement, Q-wave infarct, ST-T wave changes; 
ischemia suspected, nonspecific ST-T wave changes, QTc interval prolongation. Each 12-lead 
ECG assessment will be scored as either normal or abnormal. 
In addition, modified 12-lead ECG monitoring will be performed during imaging to prevent the 
ECG electrodes from affecting the accessibility of the optimal imaging windows of 
echocardiography. Imaging can be performed with a modified 12-lead ECG, by repositioning the 
V5-V6 leads upward. If any clinically significant ECG changes indicative of arrhythmia or S-T 
segment change are identified within 10 minutes after dosing from the modified 12-lead ECG 
monitoring, an additional standard 12-lead ECG examination will be performed subsequently 
when the earliest ECG change occurs. 
To further enhance the quality of the modified 12-lead ECGs: 
1. 
The investigator should make best efforts to minimize the displacement of the electrodes 
for the modified 12-lead ECG compared with the standard 12-lead ECG. 
2. 
The repositioning of the ECG leads for echocardiography should only involve the V5-V6 
leads, which have been shown to have minimal effect on ECG waveform.22 
3. 
To describe the shifted leads, study personnel should make appropriate annotations in the 
study records/subject file. 
Refer to Image Evaluation Guidelines for information on central review procedures. 
9.1.3 Appropriateness of Measurements 
The efficacy and safety assessments planned for this study are generally recognized as reliable, 
accurate, and relevant to the diagnostic modality and underlying disease/condition. 


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Dose Escalation Committee (DEC) 
9.2
To enhance the safety and integrity of the study data, a DEC will evaluate clinical, laboratory, 
and electrocardiographic safety before advancing to the next dose level. It will consist of sponsor 
personnel, the independent medical monitor, and the investigator(s) at the study site.  
A sentinel review approach will be followed whereby the first review will be conducted at the 
end of the safety follow-up observation period for the first subject treated in Cohort 1 to 
determine if subsequent subjects may be dosed in the cohort. In order to review all cumulative 
safety data for Cohort 1, the next review will be conducted at the end of the safety follow-up 
observation period for the last subject treated in Cohort 1. This sentinel review approach will 
continue to be followed for all successive cohorts (ie, Cohort 2 and Cohort 3), in order to review 
safety data and to provide a recommendation on study continuation, recommended dose, or early 
termination in case there is a concern regarding safety. This will be repeated until the highest 
dose level has been evaluated.  
Further details regarding timelines and specific responsibilities of the DEC will be provided in a 
separate Charter. 
10 STATISTICAL METHODS 
The statistical planning and analysis of the trial will be performed by the biostatistics department 
of the sponsor or designated contract research organization. 
 Statistical and Analytical Plans 
10.1
A statistical analysis plan will be prepared and finalized prior to database lock of the study. The 
statistical analysis plan will include full details of all planned statistical analyses and the dose 
selection strategy. 
 Datasets or Populations Analysed 
10.2
Safety Set 
The Safety Set (SS) will consist of all subjects receiving at least 1 injection of NH002. 
Full Analysis Set 
The Full Analysis Set (FAS) will consist of all enrolled subjects who have completed all study 
evaluation periods, and received 1 dose injection of NH002. 


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Per Protocol Set 
The Per Protocol Set will be a subset of the Full Analysis Set and will include all subjects who 
completed all study evaluation periods, received 1 dose injection of NH002 and had no major 
protocol deviations. Further definition of the per protocol set will be specified in the statistical 
analysis plan. 
 Demographic and Other Baseline Characteristics 
10.3
Demographic and baseline characteristic data will be summarized for each cohort and overall. 
Descriptive statistics (N, mean, standard deviation, median, minimum, and maximum) will be 
presented for continuous variables and the number and percentage of subjects in each category 
will be presented for categorical variables. No formal testing of demographic or baseline 
characteristics will be performed. The impact of any imbalances in demographics or baseline 
characteristic variables on treatment effect may be investigated in exploratory analyses on select 
efficacy or safety variables; such analyses will be documented in the statistical analysis plan, if 
applicable. 
 Safety Assessments 
10.4
All safety assessments, including AEs, PEs, VS, ECGs, and clinical laboratory evaluations, 
where indicated, will be presented using descriptive statistics for each dose of NH002. Data will 
be summarized for each dose group and overall. 
10.4.1 Safety Variables 
• 
Adverse events recorded before injection, during injection and imaging, and post-
injection; 
• 
Any changes in PE findings 
• 
Standard 12-lead ECGs assessed before injection and at 10, 30, and 60 minutes post-
injection; an additional standard 12-lead ECG will also be conditionally performed at the 
earliest time point when any clinically significant ECG changes indicative of arrhythmia 
or S-T segment change are identified within 10 minutes after injection from the modified 
12-lead ECG monitoring; 
• 
Changes in VS assessed before injection, during injection and imaging (beginning 
approximately 5 minutes prior to and continuing until 10 minutes after), and at 30, 60, 
and 120 minutes post-injection; 
• 
Changes in hematology and metabolic laboratory parameters taken before injection and 
60, 120, and 240 minutes post-injection; 
• 
SpO2 assessed before injection, during injection and imaging (beginning approximately 5 
minutes prior to and continuing until 10 minutes after), and at 30, 60, and 120 minutes 
post-injection. 
 Efficacy Variables 
10.5
10.5.1 Definition of Primary Efficacy Endpoint 
There are no primary efficacy endpoints for this study. 


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10.5.2 Definition of Secondary Efficacy Endpoints 
• 
The percentage of subjects with moderate or complete LVO, defined by an LVO grade of 
2 (moderate) or 3 (complete), as assessed by the blinded central reader(s); 
• 
The percentage of subjects with complete LVO, defined by an LVO grade of 3 
(complete), as assessed by the blinded central reader(s); 
• 
The change from baseline on the LVEBD score, defined using a standard 12-segment 
model, as assessed by the blinded central reader(s). The LV endocardium of the 4- or 2- 
chamber apical views are divided into 6 segments, with 2 basal, mid-, and apical 
segments in each view. For each segment, LVEBD is graded as follows: 0 = inadequate 
border (border not visible); 1 = sufficient (border barely visible); 2 = good (border clearly 
visible). A total delineation score (0-24) is obtained by adding the scores from the 6 
individual segments in each of the 2 views. 
• 
The changes from baseline on LVEBD score of subjects with suboptimal LVEBD at 
baseline, as defined by 2 or more contiguous segments of 6 segments that cannot be 
visualized reliably in either the apical 4- and/or the 2-chamber view, as assessed by the 
blinded central reader(s);  
• 
The duration of clinically useful contrast calculated by measuring the time between the 
disappearance of shadowing effect (useful effect starts) and the time when moderate or 
full LV enhancement and contrast enhancement are no longer adequate (useful effect 
ends), as assessed by the blinded central reader(s).  
 
 Methods of Analysis 
10.6
The safety and efficacy variables of each dose of NH002 will be presented using descriptive 
statistics. A series of selection rules based on efficacy and safety will be applied to determine the 
maximum tolerated dose. If no single NH002 dose shows a clear benefit in safety or efficacy, the 
lowest dose of NH002 will be selected. 
Secondary endpoints will be summarized by descriptive statistics. Graphical presentations of the 
data will be provided, where appropriate. 
 Handling of Missing Data 
10.7
As all treatments are scheduled in a single study visit, the level of missing data for the primary 
endpoint is anticipated to be small. Every attempt will be made to avoid missing data. All 
subjects will be used in the safety analysis, using nonmissing data available. No imputation 
process will be used to estimate/substitute missing data. 


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 Determination of Sample Size 
10.8
Each of the expected 36 completed subjects will receive 1 dose of NH002 (with 12 subjects per 
NH002 cohort). As this is a dose finding study, a formal sample size calculation was not 
required. The number of subjects dosed within each cohort is based on safety, with the objective 
of exposing the lowest possible number of subjects to the investigational agent, while still being 
able to assess safety.  
 Protocol Deviations 
10.9
Major protocol deviations will be defined in the statistical analysis plan and agreed upon during 
a Data Review Meeting prior to database lock. 
11 QUALITY ASSURANCE AND QUALITY CONTROL 
 Audit and Inspection 
11.1
Study centers and study documentation may be subject to Quality Assurance audit during the 
course of the study by the sponsor or its nominated representative. In addition, inspections may 
be conducted by regulatory authorities at their discretion. 
 Monitoring 
11.2
Data for each subject will be recorded on a CRF. Data collection must be completed for each 
subject who signs an ICF and is administered study agent. 
In accordance with current good clinical practice (cGCP) and International Council for 
Harmonisation (ICH) guidelines, the study monitor will carry out source document verification 
at regular intervals to ensure that the data collected in the CRF are accurate and reliable.  
The investigator must permit the monitor, the IEC/IRB, the sponsor’s internal auditors, and 
representatives from regulatory authorities direct access to all study-related documents and 
pertinent hospital or medical records for confirmation of data contained within the CRFs.   
 Data Management and Coding 
11.3
The sponsor and/or the appointed representative(s) will be responsible for activities associated 
with the data management of this study. This will include setting up a relevant database and data 
transfer mechanisms, along with appropriate validation of data and resolution of queries. Data 
generated within this clinical study will be handled according to the relevant standard operating 
procedures of the data management and biostatistics departments of the sponsor and/or the 
appointed representative(s). 
Study centers will complete the CRF. Data entered into the CRF must be verifiable against 
source documents at the study center. Data to be recorded directly on the CRF will be identified 
and the CRF will be considered the source document. Any changes to the data entered into the 
data capture system will be recorded in the audit trail (and if electronic, will be FDA CFR 21 
Part 11 compliant). 


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Medical coding will use Medical Dictionary for Regulatory Activities for concomitant diseases 
and AEs and WHODrug for medications. 
Missing or inconsistent data will be queried in writing to the investigator for clarification. 
Subsequent modifications to the database will be documented. 
 Quality Management and Risk Evaluation 
11.4
Details are provided in Section 7.3.2. 


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12 RECORDS AND SUPPLIES 
 Drug Accountability 
12.1
On receipt of the study agent (including rescue medication, if relevant), the investigator (or 
designee) will conduct an inventory of the supplies and verify that study agent supplies are 
received intact and in the correct amounts before completing a supplies receipt. The investigator 
will retain a copy of this receipt at the study center and return the original receipt to the study 
monitor. The monitor may check the study supplies at each study center at any time during the 
study. 
It is the responsibility of the study monitor to ensure that the investigator (or designee) has 
correctly documented the amount of the study agent received, dispensed, and returned on the 
dispensing log that will be provided. A full drug accountability log will be maintained at the 
study center at all times. The study monitor will arrange collection of unused study agent 
returned by the subject. The study monitor will also perform an inventory of study agent at the 
close-out visit to the study center. All discrepancies must be accounted for and documented. 
 Financing and Insurance 
12.2
Financing and insurance of this study will be outlined in a separate agreement between the 
contract research organization and the sponsor. 


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13 ETHICS 
 Independent Ethics Committee or Institutional Review Board 
13.1
Before initiation of the study at each study center, the protocol, the ICF, other written material 
given to the subjects, and any other relevant study documentation will be submitted to the 
appropriate IEC/IRB. Written approval of the study and all relevant study information must be 
obtained before the study center can be initiated or the study agent is released to the investigator. 
Any necessary extensions or renewals of IEC/IRB approval must be obtained for changes to the 
study such as amendments to the protocol, the ICF or other study documentation. The written 
approval of the IEC/IRB together with the approved ICF must be filed in the study files. 
The investigator will report promptly to the IEC/IRB any new information that may adversely 
affect the safety of the subjects or the conduct of the study. The investigator will submit written 
summaries of the study status to the IEC/IRB as required. On completion of the study, the 
IEC/IRB will be notified that the study has ended. 
 Regulatory Authorities 
13.2
Relevant study documentation will be submitted to the regulatory authorities of the participating 
countries, according to local/national requirements, for review and approval before the beginning 
of the study. On completion of the study, the regulatory authorities will be notified that the study 
has ended. 
 Ethical Conduct of the Study 
13.3
The investigator(s) and all parties involved in this study should conduct the study in adherence to 
the ethical principles based on the Declaration of Helsinki, cGCP, ICH guidelines, and the 
applicable national and local laws and regulatory requirements. 
 Informed Consent 
13.4
The process of obtaining informed consent must be in accordance with applicable regulatory 
requirement(s) and must adhere to cGCP. 
The investigator is responsible for ensuring that no subject undergoes any study related 
examination or activity before that subject has given written informed consent to participate in 
the study. 
The investigator or designated personnel will inform the subject of the objectives, methods, 
anticipated benefits and potential risks and inconveniences of the study. The subject should be 
given every opportunity to ask for clarification of any points s/he does not understand and, if 
necessary, ask for more information. At the end of the interview, the subject will be given ample 
time to consider the study. Subjects will be required to sign and date the ICF. After signatures 
are obtained, the ICF will be kept and archived by the investigator in the investigator’s study file. 
A signed and dated copy of the subject ICF will be provided to the subject or their authorized 
representative. 


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It should be emphasized that the subject may refuse to enter the study or to withdraw from the 
study at any time, without consequences for their further care or penalty or loss of benefits to 
which the subject is otherwise entitled. Subjects who refuse to give or who withdraw written 
informed consent should not be included or continue in the study. 
If new information becomes available that may be relevant to the subject’s willingness to 
continue participation in the study, a new ICF will be approved by the IEC(s)/IRB(s) (and 
regulatory authorities, if required). The study subjects will be informed about this new 
information and reconsent will be obtained. 
 Subject Confidentiality 
13.5
Monitors, auditors, and other authorized agents of the sponsor and/or its designee, the 
IEC(s)/IRB(s) approving this research, and the United States (US) FDA, as well as that of any 
other applicable agency(ies), will be granted direct access to the study subjects’ original medical 
records for verification of clinical study procedures and/or data, without violating the 
confidentiality of the subjects to the extent permitted by the law and regulations. In any 
presentations of the results of this study or in publications, the subjects’ identity will remain 
confidential. 
All personal data collected and processed for the purposes of this study should be managed by 
the investigator and his/her staff with adequate precautions to ensure confidentiality of those 
data, and in accordance with the HIPAA, 21 applicable to national and/or local laws and 
regulations on personal data protection. 


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14 REPORTING AND PUBLICATION, INCLUDING ARCHIVING 
Essential documents are those documents that individually and collectively permit evaluation of 
the study and quality of the data produced. After completion of the study (end of study defined as 
the date of the last visit of the last subject), all documents and data relating to the study will be 
kept in an orderly manner by the investigator in a secure study file. This file will be available for 
inspection by the sponsor or its representatives. Essential documents should be retained for 
2 years after the final marketing approval in an ICH region or for at least 2 years since the 
discontinuation of clinical development of the investigational product. It is the responsibility of 
the sponsor to inform the study center when these documents no longer need to be retained. The 
investigator must contact the sponsor before destroying any study related documentation. In 
addition, all subject medical records and other source documentation will be kept for the 
maximum time permitted by the hospital, institution, or medical practice. 
The sponsor must review and approve any results of the study or abstracts for professional 
meetings prepared by the investigator(s). Published data must not compromise the objectives of 
the study. Data from individual study centers in multicenter studies must not be published 
separately. 


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15 REFERENCES 
1. Malik SB, Chen N, Parker RA 3rd, Hsu JY. Transthoracic echocardiography: pitfalls and 
limitations as delineated at cardiac CT and MR imaging. Radiographics. 2017;37(2):383-406.   
2. Kitzman DW, Goldman ME, Gillam LD, Cohen JL, Aurigemma GP, Gottdiener JS. Efficacy 
and safety of the novel ultrasound contrast agent perflutren (definity) in patients with 
suboptimal baseline left ventricular echocardiographic images. Am J 
Cardiol. 2000;86(6):669-74. 
3. Nanda NC, Wistran DC, Karlsberg RP, et al. Multicenter evaluation of SonoVue for 
improved endocardial border delineation. Echocardiography. 2002;19(1):27-36. 
4. Mulvagh SL, Rakowski H, Vannan MA, et al. American Society of Echocardiography 
consensus statement on the clinical applications of ultrasonic contrast agents in 
echocardiography. J Am Soc Echocardiogr. 2008;21(11):1179-201; quiz 1281. 
5. Reilly JP, Tunick PA, Timmermans RJ, Stein B, Rosenzweig BP, Kronzon I. Contrast 
echocardiography clarifies uninterpretable wall motion in intensive care unit patients. J Am 
Coll Cardiol. 2000;35(2):485-90. 
6. European Society of Cardiology. The physics of contrast imaging: an introduction to 
contrast. Available at: https://www.escardio.org/Education/Practice-Tools/EACVI-
toolboxes/Contrast-Echo/The-physics-of-Contrast-Imaging. Accessed on November 1, 2017. 
7. Definity package insert. Lantheus Medical Imaging. January 2017. Available 
at: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=8ab9c79c-1b5c-4e86-899c-
cc74686f070a. Accessed October 13, 2017.  
8. Appis AW, Tracy MJ, Feinstein SB. Update on the safety and efficacy of commercial 
ultrasound contrast agents in cardiac applications. Echo Res Pract. 2015;2(2):R55–R62. 
Published online April 22, 2015.  
9. Ferrara K, Pollard R, Borden M. Ultrasound microbubble contrast agents: fundamentals and 
application to gene and drug delivery. Annu Rev Biomed Eng. 2007;9:415-47. 
10. Rowe RC, Sheskey, PJ, Quinn, ME, ed. Handbook of pharmaceutical excipients, 6th ed. 
Washington, DC: American Pharmacists Association; 2009. 
11. 
  
12. Khismatullin DB. Resonance frequency of microbubbles: effect of viscosity. J Acoust Soc 
Am. 2004;116(3):1463-73. 


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13. 
 
  
 
14. Goldberg YH, Ginelli P, Siegel R, Ostfeld RJ, Schaefer M, Spevack DM. Administration of 
perflutren contrast agents during transthoracic echocardiography is not associated with a 
significant increase in acute mortality risk. Cardiology. 2012;122(2):119-25.  
15. Platts DG, Luis SA, Roper D, et al. The safety profile of perflutren microsphere contrast 
echocardiography during rest and stress imaging: results from an Australian multicentre 
cohort. Heart Lung Circ. 2013 Dec;22(12):996-1002.  
16. Hauben M, Hung EY, Hanretta KC, Bangalore S, Snow V. Safety of perflutren ultrasound 
contrast agents: a disproportionality analysis of the US FAERS database. Drug Saf. 
2015;38(11):1127-39.  
17. Wever-Pinzon O, Suma V, Ahuja A, et al. Safety of echocardiographic contrast in 
hospitalized patients with pulmonary hypertension: a multi-center study. Eur Heart J 
Cardiovasc Imaging. 2012 Oct;13(10):857-62. doi: 10.1093/ehjci/jes057. Epub 2012 Mar 16. 
18. Gao P, Liu L, Mehta C. Adaptive designs for noninferiority trials. Biom J. 2013;55(3):310-
21. doi: 10.1002/bimj.201200034.  
19. Chong A, Haluska B, Wahi S. Clinical application and laboratory protocols for performing 
contrast echocardiography. Indian Heart J. 2013;65(3):337-346. 
doi:10.1016/j.ihj.2013.04.002. 
20. Lindner JR and Lewis C. Contrast echocardiography: clinical utility for the evaluation of left 
ventricular systolic function. Am Heart Hosp J. 2004;2(1):16-20. 
21. US Department of Health and Human Services. The Health Insurance Portability and 
Accountability Act (HIPAA) of 1996 (P.L.104-191) [HIPAA]. 
http://aspe.hhs.gov/admnsimp/pl104191.htm. Effective August 21, 1996. 
22. Kania M, Rix H, Fereniec M, et al. The effect of precordial lead displacement on ECG 
morphology. Med Biol Eng Comput. 2014;52(2):109-119. doi: 10.1007/s11517-013-1115-9. 


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16 APPENDICES 
 
 


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 Image Evaluation Guidelines  
16.1
Sponsor-recommended* equipment/platforms:   
Ultrasound System 
 with contrast LVO mode 
Video Recording Instrument 
 
Image Evaluation Software 
Windows Media Player/DICOM viewer 
*Equipment other than the listed can be used after discussion and approval by the sponsor. 
 
Equipment technical settings to be used at each site: Site should follow standard institutional 
guideline(s) for equipment technical settings and subject preparation, positioning, and overall 
comfort measures.  
 
Site qualification process: Local site imaging technicians must be trained and qualified in 
equipment operation, including the ability to identify faulty or unacceptable images and 
understand the importance of repeating imaging when appropriate. (In the event of faulty or 
unacceptable images, an unscheduled visit can be scheduled to repeat all necessary procedures.) 
 
Acquisition quality control monitoring process: 
A checklist will be used for quality control. Checklist for each treatment: 
□ Subject ID and treatment number were input correctly on ultrasound imaging system and 
external video recorder 
□ Investigator confirmed settings of mechanical index, depth, focal depth, dynamic range, 
gain, and time gain compensation were optimized for each subject  
□ 4-chamber view of pre-injection B mode acquired 
□ 2-chamber view of pre-injection B mode acquired 
□ 4-chamber view of pre-injection contrast mode acquired 
□ 2-chamber view of pre-injection contrast mode acquired 
□ Treatment administered 
□ 4-chamber view of post-injection contrast mode acquired 
□ 2-chamber view of post-injection contrast mode acquired 
□ 19 transitions between 2-chamber and 4-chamber view post-injection. Two or less 
transitions may be skipped when a standard 12-lead ECG is conditionally triggered 
during the imaging session. 
□ A video containing consecutive pre-injection and 10-minute post-injection sessions 
acquired 
□ 4 pre-injection DICOM cineloop sequences acquired 
□ 20 post-injection DICOM cineloop sequences acquired (including 10 DICOM cineloop 
sequences of 4-chamber view and 10 DICOM cineloop sequences of 2-chamber view). 
Two or less sequences may be skipped when a standard 12-lead ECG is conditionally 
triggered during the imaging session.) 


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Data storage and transfer:  
USB flash disks or CD(s) labeled with subject ID will be used to store and transfer video files 
and DICOM files acquired in the study. Each subject’s image data will be stored in one USB 
flash disk or CD(s) and shipped to the sponsor or designated contract research organization for 
randomized sequencing (for central blinded reviews).  
 
Standards for Image Evaluation: 
(a) LVO:  
0 = none (no visible contrast within the LV cavity); 
1 = faint (weak or trace effect of contrast within the LV cavity);  
2 = moderate (some areas of the LV cavity fully opacified but without a time when the 
whole cavity was filled with contrast to the same high density); 
3 = complete (homogeneous and high intensity effect). 
(b) LVEBD: The apical 4-chamber and 2-chamber views of the left ventricle were divided into 
segments by using cardiac maps. A total delineation score (0-24) was obtained by adding the 
scores (0 = inadequate, ie, border not visible; +1 = sufficient, ie, border barely visible; +2 = 
good, ie, border clearly visible) from the six individual segments in each of the two views.
 
 
 
 
0 = inadequate, ie, border not visible; 
1 = sufficient, ie, border barely visible; 
2 = good, ie, border clearly visible. 
 
Image display sequence and randomization.   
The sponsor or designated contract research organization will merge the grouped images 
obtained in the study from each subject and present the merged set to the blinded reader in a 
random sequence. The whole recorded image data will be used for scoring by the blinded 
reader(s). The blinded reader(s) should document the time points of the best images used for 
LVEBD scoring as well as the start and end time points of useful contrast imaging.  
 
Central blinded reader training and qualification:  
Central blinded reader(s) have been selected to assess the echocardiographic images for this 
study because of their background training and qualifications. These selected blinded central 
reader(s) will also be trained in scoring procedures for LVO and LVEBD using sample images, 
prior to the central image evaluations. 
 


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Timing of image reads and the read process:  
Merged/randomized image scoring of blinded central reader(s) will be done after all the images 
in the study are obtained. 
 
Imaging data lock process: 
Each blinded evaluation will be locked in the dataset shortly after it is obtained and before 
additional types of image evaluations are performed. 
 
If indicated and applicable, consensus image evaluations (or consensus reads) can be performed 
after the individual readings (if applicable) are completed and locked. (These consensus reads 
will NOT serve as the primary image evaluation used to demonstrate any preliminary efficacy of 
NH002.) If used, the consensus reads will also be locked once obtained and before additional 
types of blinded readings are performed. 
 
Quality control of the image display and blinded interpretation process: 
Central image evaluations will occur offsite to ensure that the blinded reader(s) perform their 
image evaluations independently of other image evaluations. The blinded central reader(s) will 
not have any knowledge of subject identifiers nor dose level received to obtain an image. The 
blinded central reader(s) will also evaluate image groups in a randomized sequence. 
 
Imaging data transfer process to the sponsor: 
All USB flash disks or CDs with subject data will be returned to the sponsor or designated 
contract research organization after the completion of study (from both the local sites and the 
blinded central reader[s]). 
 
Archiving of images and blind image interpretations: 
Images and blind image interpretations will be archived in the sponsor or designated contract 
research organization’s database. 
 
 


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 Contrast Administration Guidelines for NH002  
16.2
NH002 Administration: for further details, see also the current version of the NH002 
Investigator’s Brochure. 
 
1. Obtain IV access or evaluate for an existing IV line. (May be given through a peripherally 
inserted central catheter [PICC] or central line if necessary.) 
2. NH002 is to
using the TRANSMIXTM vial 
 
3. NH002 is to be activated for
 TRANSMIX
 activation cycle, and will appear as 
a milky-white suspension. Do not use this drug unless it has completed a full
 
activation cycle in the TRANSMIXTM. Do not reactivate the vial if TRANSMIXTM did not 
complete a full 
; the product should not be used. Do not reactivate a 
successfully activated NH002 vial.  
4. Invert the vial and withdraw the activated milky white suspension using an 18- to 20-gauge 
syringe needle. Withdraw the material from the middle of the liquid in the inverted vial. Do 
not inject air into the vial. Use the product immediately after its withdrawal from the vial; do 
not allow the product to stand in the syringe. 
5. Administer activated NH002 into the IV port closest to the IV site.  
6. Injections are to be administered slowly, over 30 to 60 seconds, followed by a 10 mL saline 
flush over 10 seconds.  
 
NOTE: Contact the sponsor or clinical research associate for further instructions. Do not discard 
the vial(s). 
 
 


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 Investigator Signature Page 
16.3
Protocol Title: 
A Multicenter, Open-Label, Dose Ascending Study to Evaluate the 
Safety of NH002 as a Contrast Agent in Cardiac Echocardiography 
Protocol Number: 
NH002-LV 
Version: 
1.0 
Confidentiality and cGCP Compliance Statement 
I, the undersigned, have reviewed this protocol (and amendments), including appendices, and I 
will conduct the study as described in compliance with this protocol (and amendments), cGCP, 
and relevant ICH guidelines. 
Once the protocol has been approved by the IEC/IRB, I will not modify this protocol without 
obtaining prior approval of Trust Bio-sonics, Inc. and of the IEC/IRB. I will submit the protocol 
amendments and/or any ICF modifications to Trust Bio-sonics, Inc. and IEC/IRB, and approval 
will be obtained before any amendments are implemented. 
I understand that all information obtained during the conduct of the study with regard to the 
subjects’ state of health will be regarded as confidential. No subjects’ names will be disclosed. 
All subjects will be identified by assigned numbers on all CRFs, laboratory samples, or source 
documents forwarded to the sponsor. Clinical information may be reviewed by the sponsor or its 
agents or regulatory agencies. Agreement must be obtained from the subject before disclosure of 
subject information to a third party. 
Information developed in this clinical study may be disclosed by Trust Bio-sonics, Inc., to other 
clinical investigators, regulatory agencies, or other health authority or government agencies as 
required. 
Investigator Signature 
Date 
Printed Name 
Institution 
 

SAP_001.pdf: 
Sponsor: Trust Bio-sonics, Inc. 
Protocol No.:NH002-LV 
SAP Version 2.0 2020-12-07 
CONFIDENTIAL 
Page 1 of 28 
STATISTICAL ANALYSIS PLAN 
Version 2.0, 2020-12-07 
Protocol Title: A Multicenter, Open-Label, Dose Ascending Study to Evaluate the Safety of 
NH002 as a Contrast Agent in Cardiac Echocardiography 
Protocol No.: NH002-LV 
Protocol Version: 1.0 
Trust Bio-sonics, Inc. 
Room D105, No. 2, Sec. 2, Shengyi Rd. 
Zhubei City, Hsinchu County 30261 
Taiwan (R.O.C.) 
--------------------------------------------------------------------------------------------------------------------- 
This confidential document is the property of the sponsor.  No unpublished information 
contained in this document may be disclosed without prior written approval of the sponsor. 


Sponsor: Trust Bio-sonics, Inc. 
Protocol No.:NH002-LV 
SAP Version 2.0 2020-12-07 
CONFIDENTIAL 
Page 2 of 28 
Table of Contents 
Version history ...............................................................................................................................3 
1.
Introduction .....................................................................................................................4 
1.1. 
Flow Chart and Visit Schedule .........................................................................................5 
1.2. 
Study Design .....................................................................................................................7 
1.3. 
Randomization ..................................................................................................................7 
1.4. 
Objectives and Endpoints .................................................................................................8 
2.
Statistical Hypotheses ...................................................................................................10 
3.
Sample Size Determination ..........................................................................................11 
4.
Populations for Analysis ...............................................................................................12 
4.1. 
Full Analysis Set (FAS) ..................................................................................................12 
4.2. 
Per Protocol Set (PP) ......................................................................................................12 
4.2.1. 
Major Protocol Deviations ..............................................................................................12 
4.3. 
Safety Set (SS) ................................................................................................................12 
5.
Statistical Analyses .......................................................................................................13 
5.1. 
General Considerations ...................................................................................................13 
5.1.1. 
Baseline Definition .........................................................................................................13 
5.2. 
Participant Dispositions and Demographics ...................................................................13 
5.2.1. 
Disposition of Subjects ...................................................................................................13 
5.2.2. 
Demographic and Other Baseline Characteristics ..........................................................14 
5.2.3. 
Concomitant Medication .................................................................................................14 
5.2.4. 
Exposure .........................................................................................................................14 
5.3. 
Primary Endpoints Analysis ...........................................................................................15 
5.3.1. 
Adverse Events ...............................................................................................................15 
5.3.2. 
Laboratory Evaluation ....................................................................................................16 
5.3.3. 
Vital Signs .......................................................................................................................18 
5.3.4. 
Pulse oximetry ................................................................................................................19 
5.3.5. 
Physical Examination......................................................................................................19 
5.3.6. 
Electrocardiograms (ECGs) ............................................................................................19 
5.4. 
Secondary Endpoints Analysis .......................................................................................20 
5.4.1. 
Secondary Efficacy Endpoints ........................................................................................20 
5.5. 
Analysis of Other Variables ............................................................................................23 
5.5.1. 
Telephone Call ................................................................................................................23 
5.6. 
Dose Escalation Committee (DEC) ................................................................................23 
5.7. 
Handling of Missing Data ...............................................................................................24 
6.
References ......................................................................................................................25 
7.
Appendices .....................................................................................................................26 
7.1. 
Appendix 1: List of Abbreviations .................................................................................26 
7.2. 
Appendix 2: Adds to Protocol-Planned Analyses...........................................................27 
7.2.1. 
Re-screened Subjects ......................................................................................................27 
7.2.2. 
External Data Transfer ....................................................................................................27 
7.3. 
Appendix 3: Signatures ...................................................................................................28 


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Version history 
This Statistical Analysis Plan (SAP) for study NH002-LV is based on the protocol 
version 1.0.  
Table 1 SAP Version History Summary 
SAP 
Version 
Approval Date 
Change 
Rationale 
1 
2020-08-31 
Not Applicable 
Original version 
2 
1. Remove Section 5.5.1
LVEBD Score for
Subjects with
Suboptimal LVEBD
(LVEBD Score < 14 and
at Least 4 Segments
with a Delineation
Rating of +1 or +2) at
Baseline.
2. Add Section 7.2.2
External Data Transfer.
1. According to the protocol,
the Suboptimal LVEBD
at Baseline is as defined
by 2 or more contiguous
segments of 6 segments
that cannot be visualized
reliably in either the
apical 4- and/or the 2-
chamber view, as
assessed by the blinded
central reader(s).
2. The external data is
transferred by
Inc. and Trust Bio-sonics,
Inc.


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1. 
Introduction 
This Statistical Analysis Plan (SAP) contains a more technical and detailed elaboration of the 
principal features of the analysis described in the protocol, and includes detailed procedures for 
executing the statistical analysis of the primary and secondary variables and other data. The SAP 
is finalized and signed prior to hard lock of the database. 




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lead ECG examination in 2 or more subjects during the study, the protocol will be revised to require a standard 12-lead ECG examination be performed for all subsequent subjects at an earlier post-
injection time point when these early ECG changes may potentially occur.  
4 Complete PE at screening to include assessments of general appearance, skin (turgor, pallor, cyanosis, erythema, lesions), head (eyes, ear, nose, and throat), neck, lungs and heart (observation, 
palpation, percussion, auscultation), abdomen, back, lymph nodes, and extremities and a neurological exam.  
5 Focused PE (all PEs after screening) to include cardiopulmonary (observation, palpation, percussion, auscultation) and skin (turgor, pallor, cyanosis, erythema, lesions) assessments.  
6 B-mode echocardiography and contrast-specific ultrasonography will be employed for all pre-injection and unenhanced images, while post-injection images will be acquired through contrast-specific 
ultrasonography only.  
7 Urine or serum (β-HCG) pregnancy test is required for WoCBP at screening; it is not required for postmenopausal or surgically-sterilized women. For WoCBP, urine and serum pregnancy tests are 
both required before dosing on Day 1.  
8 CBC to include HCT, Hgb, WBC with differential, ANC, and PLT.  
9 Metabolic panel to include sodium, potassium, chloride, calcium, magnesium, phosphorus, glucose, BUN, creatinine, total protein, albumin, ALP, ALT, AST, total bilirubin, and bicarbonate.  
10 Cardiac troponin I levels should be measured before dosing and once again at 4 hours after dosing in all subjects. 


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1.2. 
Study Design 
This is a phase 1, multicenter, open-label clinical study to evaluate the safety and tolerability of 3 
ascending doses of NH002. Up to 36 eligible subjects will be enrolled with sequential allocation 
to 1 of 3 cohorts with the following intravenous (IV) doses of NH002: 2.5 μl/kg, 5.0 μl/kg, or 
10.0 μl/kg. Each patient will undergo an unenhanced ultrasound examination and a NH002 
contrast-enhanced examination on the same day at Day 1. Subjects will remain at the clinical site 
for 240 minutes of observation after NH002 administration.  
All AEs will be evaluated during the successive groups of subjects, per dose level. In general, 
enrollment will be halted and safety data reviewed for termination or continuation with the 
occurrence of any serious adverse event (SAE) considered at least possibly related to study agent 
administration (in any 1 subject) or the occurrence of clinically significant toxicity listed in the 
study protocol in 30% of subjects (within any 1 of the 3 dose levels). See Section 7.5.1.2 in the 
study protocol for details on stopping criteria for dosing for this trial.  
There will be a screening period (28 days before NH002 administration), a dosing period (during 
Day 1) which will include a single dose administration of NH002, and a 7-day follow-up period 
for each cohort. Continuous echocardiographic imaging will be performed at specified time 
points before, during, and after dose administration. The contrast agent will be prepared and 
administered by designated pharmacy technician(s), study nurse(s), sonographer, or radiologist. 
Subjects will be observed at a clinical site for at least 240 minutes (i.e., 4 hours) and evaluated 
for AEs. Subjects will be contacted by phone after the end of treatment at 72 hours (i.e., Day 4 
[+3]) and at Day 7 (+3), for a follow-up safety assessment. Any drug-related AE or SAE will be 
followed until resolution.  
High ultrasound mechanical index (MI) values may cause microsphere cavitation or rupture and 
lead to ventricular arrhythmias. Additionally, end-systolic triggering with high MIs has been 
reported to cause ventricular arrhythmias. The safety of NH002 at MIs greater than 0.8 will not 
be evaluated. 
 
1.3. 
Randomization 
This is a phase 1 dose ascending study without the use of an active control as well as a placebo 
control.  No dose or treatment randomization is applicable. 




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 The duration of clinically useful contrast 
calculated by measuring the time between the 
disappearance of shadowing effect (useful effect 
starts) and the time when moderate or full LV 
enhancement and contrast enhancement are no 
longer adequate (useful effect ends), as assessed 
by the blinded central reader(s). 
 


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2. 
Statistical Hypotheses 
Not applicable. 


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3. 
Sample Size Determination 
It is anticipated that 36 subjects will be enrolled (with up to 12 subjects per NH002 dose). Each 
subject will receive 1 dose of NH002. If a subject is withdrawn from the study, the subject may 
be replaced with another subject assigned to the same dose, except for those who are 
discontinued due to AEs, as necessary. Further subjects may be enrolled at a given dose level if 
additional data are necessary to establish safety and tolerability prior to dose escalation. As this 
is a dose-finding study, a formal sample size calculation was not required 


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4. 
Populations for Analysis 
In accordance with ICH recommendations in guidelines E3 and E9, the following analysis sets 
will be used for the analyses.  
The Full Analysis Set and Per Protocol Set will be used for summaries of demographic and 
baseline characteristics and efficacy variables. The Safety Set will be used for summaries of 
demographic and baseline characteristics and all safety variables. 
4.1. 
Full Analysis Set (FAS) 
The Full Analysis Set (FAS) will consist of all enrolled subjects who have completed all study 
evaluation periods, and received 1 dose injection of NH002. 
4.2. 
Per Protocol Set (PP)  
The Per Protocol Set will be a subset of the Full Analysis Set and will include all subjects who 
completed all study evaluation periods, received 1 dose injection of NH002 and had no major 
protocol deviations. 
4.2.1. 
Major Protocol Deviations 
The protocol deviations defined in Protocol Deviation Handling Plan1 (PDHP) Version 1.0 will 
be recorded. All protocol deviations will be discussed between CRO (Medical Monitor, Data 
Manager, Biostatistician) and the Sponsor and classified as “minor” or “major”. Major protocol 
deviations leading to the exclusion of a patient from the PP Set will be discussed between CRO 
(Medical Monitor, Data Manager, Biostatistician) and the Sponsor. Protocol Deviations will be 
assessed prior to database lock.  
4.3. 
Safety Set (SS) 
The Safety Set (SS) will consist of all subjects receiving at least 1 injection of NH002. 


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5. 
Statistical Analyses 
5.1. 
General Considerations 
The safety and efficacy variables of each dose of NH002 will be presented using descriptive 
statistics. For continuous variables, the descriptive statistics (N, mean, standard deviation, 
median, minimum, and maximum) will be presented for safety variables, and the descriptive 
statistics (N, mean, standard deviation, median, minimum, maximum, and 95% CI) will be 
presented for efficacy variables. For categorical variables, the number and percentage of subjects 
in each category will be presented. Data will be summarized for each dose group and overall. 
All data processing, summarization, and analyses will be performed using SAS Version 9.4. 
5.1.1. 
Baseline Definition 
For each analysis, the baseline value will be a non-missing value obtained from each assessment 
before dosing on Day 1. If baseline data is missing no derivation will be performed and will be set 
to missing. 
5.2. 
Participant Dispositions and Demographics 
5.2.1. 
Disposition of Subjects 
The following subject data will be summarized and presented: 
– 
Number of subjects screened for overall 
– 
Number of subjects re-screened for overall 
– 
Number of subjects received injection for each dose group and overall 
– 
Number and percentage of subjects who received injection completed the study phase 
– 
Number and percentage of subjects who received injection did not complete the study phase 
– 
Number and percentage of subjects in each analysis set. 
– 
Number and percentage of the reason for subjects excluded from PP analysis set. 
– 
Number and percentage of Major Protocol Deviations leading or not leading to exclusion 
from PP. 
 
Table 01.1: Summary of Subject’s Disposition, All Screened Subjects 
 
Table 01.2: Incidence of Major Protocol Deviations, All Screened Subjects 
 
Listing 01.1: Subject’s Disposition, All Screened Subjects  
 
Listing 01.2: Discontinued Patients, All Screened Subjects  
 
Listing 01.3: Inclusion/Exclusion Criteria Not Met, All Screened Subjects 
 
Listing 02: Protocol Deviations, All Screened Subjects 


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 
Listing 03: Exclusion from Analysis Population, Safety Set 
5.2.2. 
Demographic and Other Baseline Characteristics 
Demographic and baseline characteristics include age, sex, height, weight, BMI and subjects 
with suboptimal LVEBD. Age, sex, and height will be collected once at screening visit, and 
weight will be measured twice at screening visit and before dosing on Day 1. The baseline 
weight will be the measurement on Day 1. The Body Mass Index (BMI) will be derived by 
weight at baseline and height. These variables data will be summarized by each cohort and 
overall for the SS, the PP and the FAS population.  
Age=(Date of ICF signed-Date of Birth)/365.25, rounded down to the integer. 
BMI (kg/m2)=Body Weight (kg)/(Height (cm)/100)^2, rounded to the first decimal place. 
Descriptive statistics (n, mean, standard deviation, median, minimum, and maximum) will be 
presented for continuous variables and the number and percentage of subjects in each category 
will be presented for categorical variables. No formal testing of demographic or baseline 
characteristics will be performed.  
The number and percentage of subjects with at least one medical history will be tabulated by 
each dose group and overall. The medical history will be summarized by system organ class and 
preferred terms in Medical Dictionary for Regulatory Activities (MedDRA) Version 22.0. Also, 
medical history data will be listed with start date and ongoing at the start of study drug. 
 
Table 02.1.1: Summary of Demographics and Baseline Characteristics, Safety Set 
 
Table 02.1.2: Summary of Demographics and Baseline Characteristics, Per Protocol 
Set 
 
Table 02.1.3: Summary of Demographics and Baseline Characteristics, Full Analysis 
Set 
 
Table 02.2: Summary of Medical History, Safety Set 
 
Listing 04.1: Demographics and Baseline Characteristics, All Screened Subjects 
 
Listing 04.2: Medical History, All Screened Subjects 
5.2.3. 
Concomitant Medication 
Concomitant medication will be listed by subject identity, drug name, indication, dose, unit, 
frequency, route, start date and end date. 
 
Listing 05.1: Concomitant Medications, All Screened Subjects 
5.2.4. 
Exposure 
The study drug administration will be listed by subject identity, dosing date, drug activation 
time, start dosing time, end dosing time and study drug dose. 
 
Listing 05.2: Study Drug Administration, Safety Set 


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5.3. 
Primary Endpoints Analysis 
This is a Phase 1 study to evaluate the safety and tolerability of 3 ascending doses of NH002. 
Therefore, the primary endpoints are all safety assessment. 
All safety assessments, including Treatment Emergent Adverse event (TEAEs), physical 
examination findings, vital signs (VS), clinical laboratory evaluations, other laboratory 
evaluations, oxygen saturation and electrocardiograms (ECGs) where indicated, will be 
presented using descriptive statistics for each dose of NH002. Data will be summarized for each 
dose group and overall. 
5.3.1. 
Adverse Events 
Adverse events recorded before injection, during injection and imaging, and post-injection. 
Treatment emergent adverse events (TEAEs) are those with start date on or after dosing or those 
worsen after dosing.  
If the minutes of onset time is missing and the onset date and hour of onset time are on or after 
that of dosing date, the adverse event will be considered as a TEAE. 
If the onset time is missing and the onset date is on or after that of dosing date, the adverse event 
will be considered as a TEAE. 
If the onset date is a partial date (month/year) and month/year occurs on or after that of dosing 
date, the following cases will be considered:  
 
If month/year of the onset date is greater than the month/year of dosing date, the adverse 
event will be considered as a TEAE;  
 
If month/year of the onset date is equal to the month/year of dosing date, and the stopped 
date is on or after dosing date, the adverse event will be considered as a TEAE; 
 
If month/year of the onset date is equal to the month/year of dosing date, and the stopped 
date is a partial date, the adverse event will be considered as a TEAE. 
For safety analyses, the number and percentage of subjects with at least one TEAE will be 
tabulated by each dose group and overall. The TEAEs will be summarized by system organ class 
and preferred terms in Medical Dictionary for Regulatory Activities (MedDRA) Version 22.0. 
Similar summaries will also be provided for related TEAEs, serious TEAEs, treatment related 
serious TEAEs, TEAEs that lead to death and institution of concomitant medication. 
For the summary of TEAEs by severity, if a subject experienced more than one episode of an 
adverse event, the subject is counted only once within a preferred term by using the most severe 
episode. If a subject experienced more than one adverse event within a system organ class, the 
subject is counted once for each preferred term and once for the system organ class.  
The related TEAEs which are defined as the causal relationship in “Very Likely/Certain”, 
“Probably”, and “Possible” will also be summarized by each dose group and overall.  


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 
Table 03.1.1: Overview of Treatment Emergent Adverse Events, Safety Set 
 
Table 03.1.2: Summary of Treatment Emergent Adverse Events, Safety Set 
 
Table 03.1.3: Summary of Treatment Emergent Adverse Events with Severity, Safety 
Set 
 
Table 03.1.4: Summary of Related Treatment Emergent Adverse Events, Safety Set 
 
Table 03.1.5: Summary of Treatment Emergent Adverse Events Leading to Death, 
Safety Set 
 
Table 03.1.6: Summary of Treatment Emergent Serious Adverse Events, Safety Set 
 
Table 03.1.7: Summary of Related Treatment Emergent Serious Adverse Events, 
Safety Set 
 
Table 03.1.8: Summary of Treatment Emergent Adverse Events Institution of 
Concomitant Medication, Safety Set 
 
Table 03.2.1: Listing for Treatment Emergent Adverse Events Leading to Death, 
Safety Set 
 
Table 03.2.2: Listing for Treatment Emergent Serious Adverse Events Safety, Set 
 
Table 03.2.3: Listing for Treatment Emergent Adverse Events Institution of 
Concomitant Medication, Safety Set 
 
Listing 07: Adverse Events All Screened Subjects 
5.3.2. 
Laboratory Evaluation 
5.3.2.1. 
Clinical Laboratory Evaluation 
The clinical laboratory evaluations including hematology and clinical chemistry will be collected 
before dosing, 60 minutes, 120 minutes and 240 minutes after dosing at dosing period. 
 
Hematology  
Hematology include Hematocrit, Hemoglobin, RBC, MCV, MCH, MCH concentration, ANC, 
WBC, Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, and Platelet. ANC will be 
derived by the following formula:  
 
ANC=WBC (*10^3/mm3)* Neutrophils (%)*10 
 
Clinical Chemistry  
Clinical Chemistry include creatinine, BUN, AST, ALT, ALP, total bilirubin, albumin, total 
protein, sodium, potassium, chloride, glucose, bicarbonate, calcium, magnesium, and 
phosphorus. 
The results of clinical laboratory evaluations will be summarized using mean, standard deviation, 
median, minimum and maximum for each dose group and overall at each time point during 


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dosing period. Additionally, a within-subject change will be calculated as the post-baseline 
measurement minus the baseline measurement and summarized after dosing. Therefore, the 
change from baseline will also be summarized. 
The abnormal laboratory result will be noted as [L] and [H] for lower and for higher than the 
reference ranges, respectively, which supplied by the local laboratories. It will be displayed in 
listing. Shift tables will be produced by the non-missing results to compare the baseline with 
post-injection. 
Abnormal laboratory results will be listed in Table 03.3.1 and Table 03.3.2. 
 
Table 03.3.1: Listing for Abnormal Complete Blood Count Values, Safety Set 
 
Table 03.3.2: Listing for Abnormal Metabolic Panel Values, Safety Set 
 
Table 04.1: Summary of Complete Blood Count, Safety Set 
 
Table 04.2: Shift Table of Complete Blood Count, Safety Set 
 
Table 05.1: Summary of Metabolic Panel, Safety Set 
 
Table 05.2: Shift Table of Metabolic Panel, Safety Set 
 
Listing 08.1: Complete Blood Count Part 1, Safety Set 
 
Listing 08.2: Complete Blood Count Part 2, Safety Set 
 
Listing 08.3: Metabolic Panel Part 1, Safety Set 
 
Listing 08.4: Metabolic Panel Part 2, Safety Set 
 
Listing 08.5: Metabolic Panel Part 3, Safety Set 
5.3.2.2. 
Other Laboratory Variables 
Screening for pregnancy will be performed (serum or urine β-HCG at screening and serum and 
urine β-HCG before dosing on Day 1, for WoCBP only). A Detailed listing of all pregnancies 
will be provided. 
Other laboratory variables collected are: 
 
Coagulation studies of TT and aPTT 
Coagulation studies including Thrombin Time (TT) and Activated Partial Thromboplastin time 
(aPTT) will be measured before dosing, 60 minutes, 120 minutes and 240 minutes after dosing at 
dosing period. 
 
Cardiac troponin I/Cardiac troponin T 
Cardiac troponin I/Cardiac troponin T will be measured before dosing and 240 minutes after 
dosing at dosing period. 
The results of Coagulation studies and Cardiac troponin I/Cardiac troponin T will be summarized 
by the descriptive statistics (n, mean, standard deviation, median, minimum and maximum) for 


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each dose group and overall at each time point during dosing period. Also, the change from 
baseline will be summarized. 
The abnormal laboratory result will be noted as [L] and [H] for lower and for higher than the 
reference ranges, respectively, which supplied by the local laboratories. It will be displayed in 
listing. Shift tables will be produced to compare the baseline results with post-injection. 
Abnormal laboratory results will be listed in Table 03.3.3, Table 03.3.4 and Table 03.3.5. The 
results of pregnancy tests will be shown in Listing. 
 
Table 03.3.3: Listing for Abnormal Coagulation Studies Values, Safety Set 
 
Table 03.3.4: Listing for Abnormal Cardiac Troponin I Values, Safety Set 
 
Table 03.3.5: Listing for Abnormal Cardiac Troponin T Values, Safety Set 
 
Table 06.1: Summary of Coagulation Studies, Safety Set 
 
Table 06.2: Shift Table of Coagulation Studies, Safety Set 
 
Table 07.1.1: Summary of Cardiac Troponin I, Safety Set 
 
Table 07.1.2: Summary of Cardiac Troponin T, Safety Set 
 
Table 07.2.1: Shift Table of Cardiac Troponin I, Safety Set 
 
Table 07.2.2: Shift Table of Cardiac Troponin T, Safety Set 
 
Listing 08.6: Coagulation Studies, Safety Set 
 
Listing 08.7.1: Cardiac Troponin I, Safety Set 
 
Listing 08.7.2: Cardiac Troponin T, Safety Set 
 
Listing 08.8: Pregnancy Test, Safety Set 
5.3.3. 
Vital Signs 
Vital signs (BP, body temperature, RR, and HR) will be recorded at screening, before injection, 
during injection and imaging (beginning approximately 5 minutes prior to and continuing until 
10 minutes after injection), and 30, 60, and 120 minutes post-injection, in a standardized manner. 
Blood pressures (SBP and DBP) will be measured only at 5 time points for every 2 minutes 
within 10 minutes after injection. Body temperature will be only collected twice at 5 minutes and 
10 minutes within 10 minutes after injection. 
The results of vital signs will be summarized using mean, standard deviation, median, minimum 
and maximum for each dose group and overall at screening visit and each time point during 
dosing period. The baseline for the vital signs will be the last measurement before dosing. Also, 
the change from baseline of the vital signs will be calculated as the post-baseline measurement 
minus the baseline measurement and summarized after dosing. 
 
Table 08: Summary of Vital Signs, Safety Set 


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 
Listing 08.9: Vital Signs, Safety Set 
5.3.4. 
Pulse oximetry  
SpO2 will be assessed pre-injection, during injection and imaging (beginning approximately 5 
minutes prior to and continuing until 10 minutes after), and at 30, 60, and 120 minutes post-
injection. 
The result of SpO2 will be summarized using mean, standard deviation, median, minimum and 
maximum for each dose group and overall at each time point during dosing period. Also, the 
change from baseline of SpO2 will be summarized after dosing. 
 
Table 11: Summary of Pulse Oximetry, Safety Set 
 
Listing 08.12: Pulse Oximetry, Safety Set 
5.3.5. 
Physical Examination 
At screening, a complete physical examination will be performed. A focused physical 
examination including skin and cardiopulmonary assessments will be performed before dosing 
and during dosing at the following time points after contrast injection: 30, 60, and 120 minutes 
post-injection. Each focused PE assessment will be scored as either normal or abnormal. The 
baseline will be the last assessments before dosing. Any changes with respect to the baseline will 
be recorded and assessed.  
Number and percent of subjects with normal and abnormal skin and cardiopulmonary conditions 
in physical examination will be tabulated. Shift tables for all categories of non-missing physical 
examination results will be produced to compare baseline results with post-injection results. 
Physical examination with abnormal findings will be provided in a listing.  No statistical 
analyses will be performed. 
 
Table 09.1: Summary of Physical Examination, Safety Set 
 
Table 09.2: Shift Table of Physical Examination, Safety Set 
 
Listing 08.10.1: Physical Examination Part 1, Safety Set 
 
Listing 08.10.2: Physical Examination Part 2, Safety Set 
 
Listing 08.10.3: Physical Examination Abnormal Findings, Safety Set 
5.3.6. 
Electrocardiograms (ECGs) 
Standard 12-lead ECGs will be performed at screening, before dosing on Day 1, and at 10, 30, 
and 60 minutes post-injection. Safety assessments will be based on the results of the standard 12-
lead ECGs. The ECG parameters include QTc, QT, and HR and ECG interpretation will be 
classified as either normal, abnormal but not clinically significant (NCS), or abnormal clinically 
significant (CS). 


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In addition, modified 12-lead ECG monitoring will be performed during imaging to prevent the 
ECG electrodes from affecting the accessibility of the optimal imaging windows of 
echocardiography. Imaging can be performed with a modified 12-lead ECG, by repositioning the 
V5-V6 leads upward. If any clinically significant ECG changes indicative of arrhythmia or S-T 
segment change are identified within 10 minutes after dosing from the modified 12-lead ECG 
monitoring, an additional standard 12-lead ECG examination will be performed subsequently 
when the earliest ECG change occurs. 
The descriptive statistics (n, mean, standard deviation, median, minimum, and maximum) of the 
ECG parameters at screening visit and each time point during dosing period including changes 
from baseline will be summarized and presented by each dose group and overall. The baseline 
will be the last measurements before dosing.  
Number and percent of subjects with normal, abnormal, NCS and abnormal, CS results for the 
12-lead ECG interpretation will be tabulated by each dosing group and overall. A shift table 
showing the changes of non-missing results in normal, abnormal, NCS and abnormal, CS from 
baseline to each post-baseline measurement will be displayed.   
 
Table 10.1: Summary of 12-Lead ECG Parameters, Safety Set 
 
Table 10.2: Summary of 12-Lead ECG Interpretation, Safety Set 
 
Table 10.3: Shift Table of 12-Lead ECG Interpretation, Safety Set 
 
Listing 08.11: 12-Lead ECG Test, Safety Set 
5.4. 
Secondary Endpoints Analysis 
5.4.1. 
Secondary Efficacy Endpoints 
5.4.1.1. 
Definition of endpoints 
 
The percentage of subjects with moderate or complete LVO, defined by an LVO grade of 2 
(moderate) or 3 (complete), as assessed by the blinded central reader(s);  
 
The percentage of subjects with complete LVO, defined by an LVO grade of 3 (complete), 
as assessed by the blinded central reader(s);  
 
The change from baseline on the LVEBD score, defined using a standard 12-segment 
model, as assessed by the blinded central reader(s). The LV endocardium of the 4- or 2- 
chamber apical views are divided into 6 segments, with 2 basal, mid-, and apical segments 
in each view. For each segment, LVEBD is graded as follows: 0 = inadequate border 
(border not visible); 1 = sufficient (border barely visible); 2 = good (border clearly visible). 
A total delineation score (0-24) is obtained by adding the scores from the 6 individual 
segments in each of the 2 views.  
 
The changes from baseline on LVEBD score of subjects with suboptimal LVEBD at 
baseline, as defined by 2 or more contiguous segments of 6 segments that cannot be 


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visualized reliably in either the apical 4- and/or the 2-chamber view, as assessed by the 
blinded central reader(s);  
 
The duration of clinically useful contrast calculated by measuring the time between the 
disappearance of shadowing effect (useful effect starts) and the time when moderate or full 
LV enhancement and contrast enhancement are no longer adequate (useful effect ends), as 
assessed by the blinded central reader(s).  
5.4.1.2. 
Main analytical approach 
 
LVO Grade 
A pair of apical 4-chamber and 2-chamber view images in each of the pre-injection and post-
injection imaging sessions will be subjected to LVO grade assessment by central reading.   
The highest LVO Grade among the pair of apical 4-chamber and 2-chamber view images of pre-
injection and post-injection will be summarized and presented by each dose group and overall. 
When the LVO grades of both apical 4-chamber and 2-chamber view images are the same, the 
LVO grade of the apical 4-chamber view image will be used. 
The following descriptive statistics will be summarized and presented: 
– 
Number and percentage of subjects in each LVO Grade. 
– 
Number and percentage of subjects with moderate or complete LVO. 
In addition, the bar chart for Cumulative Percent of LVO Grade will be presented by pre-dose 
and post-dose groups in each cohort and overall.  
Two LVO grades obtained for each session along with the corresponding times of images used 
will be listed.   
 
Table 12.1.1: Summary of LVO Grade, Full Analysis Set 
 
Figure 12.1.2: Cumulative Percent of LVO Grade, Full Analysis Set 
 
Table 12.2.1: Summary of LVO Grade, Per Protocol Set 
 
Figure 12.2.2: Cumulative Percent of LVO Grade, Per Protocol Set 
 
Listing 06.1: Image Evaluation - LVO Grade, Safety Set 
 
LVEBD Score 
The descriptive statistics (n, mean, standard deviation, median, minimum, maximum, and 95% 
CI) of LVEBD score for pre-injection, post-injection and the change from baseline will be 
summarized and presented by each dose group and overall. LVEBD scores for subjects with 
suboptimal LVEBD at baseline (as defined by 2 or more contiguous segments of 6 segments that 
cannot be visualized reliably in either the apical 4- and/or the 2-chamber view) will also be 
summarized. 


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Moreover, the boxplots of the LVEBD score for pre-injection, post-injection and the change of 
LVEBD score will be presented by each cohort and overall. 
Likewise, for subjects with suboptimal LVEBD at Baseline (as defined by 2 or more contiguous 
segments of 6 segments that cannot be visualized reliably in either the apical 4- and/or the 2-
chamber view), the boxplots of the LVEBD Score for pre-injection, post-injection and the 
change of LVEBD score will be presented by each cohort and overall. 
 
Table 13.1.1.1: Summary of LVEBD Score, Full Analysis Set 
 
Figure 13.1.1.2: LVEBD Score, Full Analysis Set 
 
Figure 13.1.1.3: Change of LVEBD Score, Full Analysis Set 
 
Table 13.1.2.1: Summary of LVEBD Score, Per Protocol Set 
 
Figure 13.1.2.2: LVEBD Score, Per Protocol Set 
 
Figure 13.1.2.3: Change of LVEBD Score, Per Protocol Set 
 
Table 13.2.1.1: Summary of LVEBD Score for Subjects with Suboptimal LVEBD at 
Baseline (as defined by 2 or more contiguous segments of 6 segments that cannot be 
visualized reliably in either the apical 4- and/or the 2-chamber view), Full Analysis Set 
 
Figure 13.2.1.2: LVEBD Score for Subjects with Suboptimal LVEBD at Baseline (as 
defined by 2 or more contiguous segments of 6 segments that cannot be visualized 
reliably in either the apical 4- and/or the 2-chamber view), Full Analysis Set 
 
Figure 13.2.1.3: LVEBD Score for Subjects with Suboptimal LVEBD at Baseline (as 
defined by 2 or more contiguous segments of 6 segments that cannot be visualized 
reliably in either the apical 4- and/or the 2-chamber view), Full Analysis Set 
 
Table 13.2.2.1: Summary of LVEBD Score for Subjects with Suboptimal LVEBD at 
Baseline (as defined by 2 or more contiguous segments of 6 segments that cannot be 
visualized reliably in either the apical 4- and/or the 2-chamber view), Per Protocol Set 
 
Figure 13.2.2.2: LVEBD Score for Subjects with Suboptimal LVEBD at Baseline (as 
defined by 2 or more contiguous segments of 6 segments that cannot be visualized 
reliably in either the apical 4- and/or the 2-chamber view), Per Protocol Set 
 
Figure 13.2.2.3: LVEBD Score for Subjects with Suboptimal LVEBD at Baseline (as 
defined by 2 or more contiguous segments of 6 segments that cannot be visualized 
reliably in either the apical 4- and/or the 2-chamber view), Per Protocol Set 
 
Listing 06.2: Image Evaluation - LVEBD Score, Safety Set 
 
Duration of Clinically Useful Contrast Enhancement 
The duration of clinically useful contrast enhancement is the number of minutes between start 
time point and end time point of useful contrast imaging, rounded to one decimal place. 
The formula for duration calculation is as below. 


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Duration (Minutes)=(End Time (hh:mm:ss) of Useful Contrast Imaging - Start Time (hh:mm:ss) 
of Useful Contrast Imaging)/60, rounded to one decimal place. 
The descriptive statistics (n, mean, standard deviation, median, minimum, maximum, and 95% 
CI) of the duration of clinically useful contrast enhancement will be presented by each dose 
group and overall. The unit of duration is minute. 
Furthermore, the boxplots of duration of clinically useful contrast enhancement will be presented 
by each cohort and overall. 
 
Table 14.1.1: Summary of Duration of Clinically Useful Contrast Enhancement, Full 
Analysis Set 
 
Figure 14.1.2: Duration of Clinically Useful Contrast Enhancement, Full Analysis Set 
 
Table 14.2.1: Summary of Duration of Clinically Useful Contrast Enhancement, Per 
Protocol Set 
 
Figure 14.2.2: Duration of Clinically Useful Contrast Enhancement, Per Protocol Set 
 
Listing 06.3: Image Evaluation - Duration of Clinically Useful Contrast Enhancement, 
Safety Set 
5.5. 
Analysis of Other Variables 
5.5.1. 
Telephone Call 
Telephone Call will be listed by subject identity, visit, day, date of contact, successfully reaching 
the subject, any new or changed symptoms experienced since last visit, and any new or changed 
medications used since last visit. 
 
Listing 09: Telephone Call, Safety Set 
5.6. 
Dose Escalation Committee (DEC) 
To enhance the safety and integrity of the study data, a DEC will evaluate clinical, laboratory, 
and electrocardiographic safety before advancing to the next dose level. It will consist of sponsor 
personnel, the independent medical monitor, and the investigator(s) at the study site.  
A sentinel review approach will be followed whereby the first review will be conducted at the 
end of the safety follow-up observation period for the first subject treated in Cohort 1 to 
determine if subsequent subjects may be dosed in the cohort. In order to review all cumulative 
safety data for Cohort 1, the next review will be conducted at the end of the safety follow-up 
observation period for the last subject treated in Cohort 1. This sentinel review approach will 
continue to be followed for all successive cohorts (i.e., Cohort 2 and Cohort 3), in order to 
review safety data and to provide a recommendation on study continuation, recommended dose, 
or early termination in case there is a concern regarding safety. This will be repeated until the 
highest dose level has been evaluated.  


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The tables and listings for DEC will follow the shells that only present the data of the dose in 
each cohort. 
Further details regarding timelines and specific responsibilities of the DEC will be provided in a 
separate Charter2. 
5.7. 
Handling of Missing Data 
As all treatments are scheduled in a single study visit, the level of missing data for the primary 
endpoint is anticipated to be small. Every attempt will be made to avoid missing data. All 
subjects will be used in the safety analysis, using non-missing data available. No imputation 
process will be used to estimate/substitute missing data. 


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6.
References
1.
Protocol Deviation Handling Plan (PDHP) Version 1.0.
2.
The Dose Escalation Committee Charter Version 1.0.
3.
NH002-LV_Protocol_v1.0.
4.
Data Transfer Specification TRUS-10006193, ECHO Final 3.0, 16 Sep 2020.
5.
Specification of Suboptimal Echocardiogram Data Transfer_20201116_TRUST. 


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7. 
Appendices 
7.1. 
Appendix 1: List of Abbreviations 
 
List of Abbreviations 
Abbreviations 
Description of abbreviations 
AE 
Adverse Event 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Aminotransferase 
ANC 
Absolute Neutrophil Count 
aPTT 
Activated Partial Thromboplastin Time 
AST 
Aspartate Aminotransferase 
BMI 
Body Mass Index 
β-HCG 
Beta-Human Chorionic Gonadotropin 
CBC 
Complete Blood Count 
CI 
Confidence Interval 
CS 
Clinically Significant 
DBP 
Diastolic Blood Pressure 
DEC 
Dose Escalation Committee 
EBD 
Endocardial Border Delineation 
ECG 
Electrocardiogram 
EOT 
End of Treatment 
FAS 
Full Analysis Set 
HCT 
Hematocrit 
HR 
Heart Rate 
ICF 
Informed Consent Form 
ICH 
International Council for Harmonisation 
IV 
Intravenous 
LV 
Left Ventricle [Ventricular] 
LVEBD 
Left Ventricular Endocardial Border Delineation 
LVO 
Left Ventricular Opacification 
MCH 
Mean Cell Hemoglobin 
MedDRA 
Medical Dictionary for Regulatory Activities 
NCS 
Not Clinically Significant 
PD 
Protocol Deviation 
PDHP 
Protocol Deviation Handling Plan 
PE 
Physical Examination 
PLT 
Platelet Count 
PP 
Per Protocol 
RR 
Respiratory Rate 
SAE 
Serious Adverse Event 


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Abbreviations 
Description of abbreviations 
SBP 
Systolic Blood Pressure 
SpO2 
Peripheral Oxygen Saturation 
SS 
Safety Set 
TEAE 
Treatment-Emergent Adverse Events 
TT 
Thrombin Time 
VS 
vital signs 
WBC 
white blood cell 
7.2. 
Appendix 2: Adds to Protocol-Planned Analyses 
7.2.1. 
Re-screened Subjects 
For re-screened subjects, information is taken from the last re-screening visit instead of the 
screening visit. 
The number of subjects re-screened are summarized in Table 01.1: Summary of Subject’s 
Disposition. 
7.2.2. 
External Data Transfer 
The external data transfer process not described in the study protocol3 is illustrated as below. 
The efficacy data including LVO grade, LVEBD score, and the useful contrast imaging start 
time/ end time assessed by the blinded central readers (assigned by 
Inc.) are not from 
the record on CRF but be transferred by 
 Inc. Further details are provided in the Data 
Transfer Specification4. 
For those subjects are suboptimal LVEBD at baseline, as defined by 2 or more contiguous 
segments of 6 segments that cannot be visualized reliably in either the apical 4- and/or the 2-
chamber view, Trust Bio-Sonics, Inc. transfers the Suboptimal Echo Classification Data 
according to the Specification of Suboptimal Echocardiogram Data Transfer5. 
All external data is cleaned and reconciled to ensure the data correctness. 
 
 


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7.3. 
Appendix 3: Signatures 
 
 
 
 
 
 
 
 
 
Prepared by: 
 
Date: 
 
 
Amy Lin 
Data Analyst 
 Inc. 
 
(YYYY-MM-DD) 
 
 
 
 
 
 
 
 
Approved by: 
 
Date: 
 
 
Sky Chen 
Director of Technical Operation 
 Inc. 
 
(YYYY-MM-DD) 
 
 
 
 
 
 
 
 
Reviewed by: 
 
Date: 
 
 
Shih-Tsung Kang 
Tech Development Manager 
Trust Bio-sonics, Inc. 
 
(YYYY-MM-DD)