Conditions: Laparoscopic/Minimally Invasive Colorectal Surgery
Keywords: ASP5354 ureter visualization imaging agent near infrared fluorescence
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 13
Healthy volunteers: no

Brief summary:
The primary purpose of this study was to determine the optimal dose of ASP5354 for ureter visualization in participants undergoing laparoscopic/minimally invasive colorectal surgery This study also investigated the safety, tolerability and the pharmacokinetics of ASP5354 in participants undergoing laparoscopic/minimally invasive colorectal surgery.

Detailed description:
Participants were randomly assigned at each dose level (dose A, B, C). During a standard minimally invasive surgery, visualization of the surgical field was assessed following the placement of the near infrared fluorescence (NIR F) imaging system proximal to the ureter of interest and then ASP5354 was administered.

Based on Visualization Review Committee (VRC) review of the initial 3 dose levels, if none of the doses selected had visualization, then additional two dose levels (dose D and E) was planned to be added; if 1 dose selected has visualization, then the dose level D was planned to be added. The dose level F was planned to be added if only the dose E level has visualization.

Arms:
arm 1: Participants received single dose of pudexacianinium chloride at dose level A by intravenous (IV) bolus infusion on day 1 once the surgical area of interest is in view. arm 2: Participants received single dose of pudexacianinium chloride at dose level B by IV bolus infusion on day 1 once the surgical area of interest is in view. arm 3: Participants received single dose of pudexacianinium chloride at dose level C by IV bolus infusion on day 1 once the surgical area of interest is in view. arm 4: Participants who were enrolled in the dose expansion group received single dose of pudexacianinium chloride at dose level B by IV bolus infusion on day 1 once the surgical area of interest is in view.

Interventions: intervention 1: Pudexacianinium chloride
Intervention descriptions: intervention 1: Intravenous

Protocol document:
Prot_000.pdf: 
Sponsor: API 
ISN/Protocol 5354-CL-0201 
- CONFIDENTIAL - 
21 Jul 2021 
Astellas 
Page 1 of 82 
Version 3.0 Incorporating Substantial Amendment 2 
A Phase 2 Randomized Open-label, Dose-ranging Study for 
Ureter Visualization, Using ASP5354 in Subjects Undergoing 
Laparoscopic/Minimally Invasive Colorectal Surgery 
 
Protocol for Phase 2 Study of ASP5354 
 
ISN/Protocol 5354-CL-0201 
Version 3.0 
Incorporating Substantial Amendment 2 [See Section 13] 
21 Jul 2021 
 
IND 140758 
 
Sponsor: 
Astellas Pharma Inc. 
2-5-1, Nihonbashi-Honcho, Chuo-ku, 
Tokyo 103-8411, Japan 
 
 
 
Protocol History: 
Version 1.0 [08 May 2019] 
Version 1.1 Incorporating Nonsubstantial Amendment 1 [27 Aug 2020] 
Version 2.0 Incorporating Substantial Amendment 2 [08 Feb 2021] 
 
 
The information contained in this document is supplied as a background for clinical investigations.  This document contains 
confidential information, which is the intellectual property of Astellas.  By accepting or reviewing this document, you agree 
to hold this information in confidence and not copy or disclose it to others or use it for unauthorized purposes except (1) as 
otherwise agreed to in writing; (2) where required by applicable law; (3) where disclosure is directly related to the care and 
safety of the research subject; and (4) where disclosure of such information is made to a member of the investigator’s team 
who agrees to hold this information in confidence.   


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Version 3.0 Incorporating Substantial Amendment 2 
Table of Contents 
SIGNATURES ································
································
································
 8 
CONTACT DETAILS OF SPONSOR’S KEY PERSONNEL ································
·····  10 
1 
PROTOCOL SUMMARY ································
································
·······  12 
1.1 
Synopsis ································
································
························
 12 
1.2 
Study Schema ································
································
··················
 18 
1.3 
Schedules of Assessments ································
································
····  20 
1.3.1 
Sample Collection and Timed Procedure Schedule ································
· 22 
2 
INTRODUCTION ································
································
·················
 23 
2.1 
Background ································
································
····················
 23 
2.1.1 
Nonclinical and Clinical Data ································
··························
 24 
2.1.1.1 
Summary of Nonclinical Data ································
····················
 24 
2.1.1.2 
Summary of Clinical Data ································
························
 26 
2.1.2 
Summary of Key Safety Information for Investigational Product(s) ··············
 26 
2.2 
Study Rationale ································
································
················
 27 
2.3 
Risk Benefit Assessment ································
································
·····  28 
3 
STUDY OBJECTIVE(S) AND ENDPOINT(S) ································
··············
 29 
4 
STUDY DESIGN AND DOSE RATIONALE ································
················
 29 
4.1 
Study Design ································
································
···················
 29 
4.2 
Dose Rationale ································
································
················
 32 
4.3 
End of Study Definition ································
································
······  32 
5 
STUDY POPULATION ································
································
··········
 32 
5.1 
Inclusion Criteria ································
································
··············
 32 
5.2 
Exclusion Criteria ································
································
·············
 33 
5.3 
Restrictions During the Study ································
·······························
 34 
5.4 
Screen Failures ································
································
················
 34 
5.4.1 
Rescreening ································
································
··············
 34 
6 
INVESTIGATIONAL PRODUCT(S) ································
·························
 34 
6.1 
Investigational Product Administered ································
······················
 34 
6.2 
Preparation/Handling/Storage/Accountability ································
·············
 35 
6.2.1 
Packaging and Labeling ································
································
 35 
6.2.2 
Handling, Storage and Accountability ································
················
 36 


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6.3 
Randomization and Blinding ································
································
 36 
6.4 
Investigational Product Compliance ································
························
 36 
6.5 
Previous and Concomitant Treatment (Medication and Nonmedication Therapy) ····  36 
6.5.1 
Previous Medication ································
································
····  36 
6.5.2 
Concomitant Medication ································
·······························
 36 
6.5.3 
Prohibited Medication ································
································
··  37 
6.5.4 
Alternative Therapy for Visualization ································
················
 37 
6.6 
Dose Modification ································
································
············
 37 
6.7 
Criteria for Continuation of Treatment ································
·····················
 37 
7 
STUDY PROCEDURES AND ASSESSMENTS ································
············
 37 
7.1 
Efficacy Assessments ································
································
·········  37 
7.1.1 
Primary Efficacy Assessments ································
·························
 37 
7.1.1.1 
Anatomical Visualization of the Index Ureter(s) Using ASP5354···········
 37 
7.1.2 
Exploratory Assessments ································
·······························
 38 
7.1.2.1 
Intensity of Fluorescence ································
·························
 38 
7.1.2.2 
Duration of Ureter Visualization ································
·················
 38 
7.1.2.3 
Signal Background Ratio ································
·························
 38 
7.1.2.4 
Binary Questions (Yes/No) for the Benefit of Visualization by 
ASP5354 during Surgery ································
·························
 39 
7.1.2.5 
Ability to Visualize the Contralateral Ureter When Amenable to 
Visualization ································
································
·······  39 
7.2 
Safety Assessments ································
································
···········
 39 
7.2.1 
Adverse Events ································
································
··········
 39 
7.2.2 
Laboratory Assessments ································
································
 40 
7.2.3 
Vital Signs, Height and Weight ································
························
 40 
7.2.4 
Physical Examination ································
································
···  40 
7.2.5 
Electrocardiogram ································
································
·······  40 
7.2.6 
Order of Assessments ································
································
···  41 
7.3 
Adverse Events and Other Safety Aspects ································
·················
 41 
7.3.1 
Time Period for Collecting Adverse Event and Serious Adverse Event 
Information ································
································
···············
 41 
7.3.2 
Method of Detecting Adverse Events and Serious Adverse Events ···············
 41 
7.3.3 
Follow-up of Adverse Events ································
··························
 42 
7.3.4 
Reporting of Serious Adverse Events ································
·················
 42 


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7.3.5 
Disease-related Events and/or Disease-related Outcomes Not Qualifying as 
Adverse Events or Serious Adverse Events ································
··········
 42 
7.3.6 
Special Situations ································
································
········  42 
7.3.7 
Supply of New Information Affecting the Conduct of the Study ··················
 43 
7.3.8 
Urgent Safety Measures ································
································
 43 
7.3.9 
Reporting Urgent Safety Measures ································
····················
 43 
7.4 
Pharmacokinetics ································
································
··············
 44 
7.4.1 
Analysis of ASP5354 in Plasma ································
·······················
 44 
7.4.2 
Analysis of ASP5354 in Urine ································
·························
 44 
7.5 
Pharmacodynamics ································
································
···········
 45 
7.6 
Electronic Clinical Outcome Assessment ································
··················
 45 
7.7 
Other Assessments ································
································
············
 45 
7.7.1 
Green Discoloration of Urine ································
··························
 45 
7.8 
Total Amount of Blood ································
································
·······  45 
8 
DISCONTINUATION ································
································
············
 45 
8.1 
Discontinuation of Individual Subject(s) From Study Treatment ·······················
 45 
8.2 
Discontinuation of Individual Subject(s) From Study ································
····  45 
8.2.1 
Lost to Follow-up································
································
········  46 
8.3 
Discontinuation of the Study Site ································
···························
 46 
8.4 
Discontinuation of the Study································
································
· 46 
9 
STATISTICAL METHODOLOGY ································
···························
 46 
9.1 
Sample Size ································
································
····················
 46 
9.2 
Analysis Sets ································
································
···················
 47 
9.2.1 
Full Analysis Set ································
································
········  47 
9.2.2 
Safety Analysis Set ································
································
······  47 
9.2.3 
Pharmacokinetic Analysis Set ································
·························
 47 
9.3 
Demographics and Baseline Characteristics ································
···············
 47 
9.3.1 
Subject Disposition ································
································
······  47 
9.3.2 
Previous and Concomitant Treatment (Medication and Nonmedication 
Therapy) ································
································
··················
 47 
9.3.3 
Medical History ································
································
··········
 47 
9.3.4 
Investigational Product Exposure ································
·····················
 47 
9.4 
Analysis of Efficacy ································
································
··········
 48 
9.4.1 
Analysis of Primary Endpoint ································
·························
 48 


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9.4.1.1 
Primary Analysis ································
································
···  48 
9.4.1.2 
Secondary Analysis ································
································
 48 
9.4.1.3 
Subgroup Analysis ································
································
· 48 
9.4.2 
Analysis of Secondary Endpoints ································
·····················
 48 
9.4.3 
Analysis of Exploratory Endpoints ································
····················
 48 
9.5 
Analysis of Safety ································
································
·············
 48 
9.5.1 
Adverse Events ································
································
··········
 48 
9.5.2 
Laboratory Assessments ································
································
 49 
9.5.3 
Vital Signs ································
································
················
 49 
9.5.4 
Physical Examination ································
································
···  49 
9.5.5 
Electrocardiogram ································
································
·······  49 
9.5.5.1 
Routine 12-lead Electrocardiogram ································
·············
 49 
9.6 
Analysis of Pharmacokinetics ································
·······························
 49 
9.6.1 
Estimation of Pharmacokinetic Parameters ································
··········
 49 
9.7 
Analysis of Pharmacodynamics ································
·····························
 50 
9.8 
Other Analyses ································
································
················
 50 
9.8.1 
Green Discoloration of Urine ································
··························
 50 
9.9 
Major Protocol Deviations ································
································
···  50 
9.10 
Interim Analysis (and Early Discontinuation of the Study) ······························
 50 
9.11 
Additional Conventions ································
································
······  50 
10 
OPERATIONAL CONSIDERATIONS ································
·······················
 51 
10.1 
Data Collection ································
································
················
 51 
10.2 
Demographics and Baseline Characteristics ································
···············
 51 
10.2.1 
Demographics ································
································
············
 51 
10.2.2 
Medical History ································
································
··········
 51 
10.2.3 
Surgical Procedure and Indication ································
·····················
 51 
10.3 
Major Protocol Deviations ································
································
···  51 
10.4 
Study Organization ································
································
············
 52 
10.4.1 
Visualization Review Committee ································
·····················
 52 
11 
REFERENCES ································
································
····················
 53 
12 
APPENDICES ································
································
·····················
 54 
12.1 
Ethical, Regulatory and Study Oversight Considerations ································
 54 
12.1.1 
Ethical Conduct of the Study ································
···························
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12.1.2 
Institutional Review Board/Independent Ethics Committee/Competent 
Authorities ································
································
················
 54 
12.1.3 
Protocol Amendment and/or Revision ································
················
 54 
12.1.4 
Financial Disclosure ································
································
····  55 
12.1.5 
Informed Consent of Subjects ································
·························
 55 
12.1.5.1 
Subject Information and Consent ································
················
 55 
12.1.5.2 
Supply of New and Important Information Influencing the Subject’s 
Consent and Revision of the Written Information ·····························
 55 
12.1.6 
Source Documents ································
································
······  56 
12.1.7 
Record Retention ································
································
········  56 
12.1.8 
Subject Confidentiality and Privacy ································
···················
 56 
12.1.9 
Arrangement for Use of Information and Publication of the Study ···············
 57 
12.1.10 
Signatory Investigator for Clinical Study Report ································
····  57 
12.2 
Procedure for Study Quality Control ································
·······················
 58 
12.2.1 
Study Monitoring ································
································
········  58 
12.2.2 
Direct Access to Source Data/Documents ································
············
 58 
12.2.3 
Data Management ································
································
·······  58 
12.2.4 
Quality Assurance ································
································
·······  58 
12.3 
Contraception Requirements ································
································
· 60 
12.4 
Adverse Events: Definitions and Procedures for Recording, Evaluating, Follow-
up and Reporting ································
································
··············
 62 
12.4.1 
Definition of Adverse Events ································
··························
 62 
12.4.1.1 
Abnormal Laboratory Findings ································
··················
 62 
12.4.1.2 
Potential Cases of Drug-induced Liver Injury ································
··  62 
12.4.2 
Definition of Serious Adverse Events································
·················
 62 
12.4.3 
Criteria for Causal Relationship to Investigational Product ························
 63 
12.4.4 
Criteria for Defining the Severity of an Adverse Event ····························
 64 
12.4.5 
Reporting Procedures for Serious Adverse Events ································
··  65 
12.4.6 
Reporting Procedures for Special Situations ································
·········  66 
12.4.6.1 
Pregnancy ································
································
···········
 66 
12.4.6.2 
Medication Error, Overdose and “Off-label Use” ·····························
 67 
12.4.6.3 
Misuse/Abuse ································
································
······  67 
12.4.6.4 
Occupational Exposure ································
···························
 67 
12.4.6.5 
(Suspicion of) Transmission of Infectious Agent ······························
 67 
12.4.6.6 
Suspected Drug-drug Interaction ································
················
 68 
12.5 
Liver Safety Monitoring and Assessment ································
··················
 69 


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12.6 
List of Excluded Concomitant Medications································
················
 71 
12.7 
Laboratory Assessments ································
································
······  72 
12.8 
List of Abbreviations and Definition of Key Study Terms ·······························
 73 
13 
ATTACHMENT 1:   SUBSTANTIAL AMENDMENT 2 ································
· 76 
14 
SPONSOR SIGNATURE ································
································
········  82 
 


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SIGNATURES 
1.  SPONSOR’S SIGNATURES 
Required signatures (e.g., protocol authors and contributors, etc.) are located in [Section 14 
Sponsor’s Signatures]. 
 


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2.  INVESTIGATOR’S SIGNATURE 
A Phase 2 Randomized Open-label, Dose-ranging Study for Ureter Visualization, Using 
ASP5354 in Subjects Undergoing Laparoscopic/Minimally Invasive Colorectal Surgery 
ISN/Protocol 5354-CL-0201 
Version 3.0, Incorporating Substantial Amendment 2 
21 Jul 2021 
I have read all pages of this protocol for which Astellas is the sponsor.  I agree to conduct the 
study as outlined in the protocol and to comply with all the terms and conditions set out 
therein.  I confirm that I will conduct the study in accordance with International Council for 
Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good 
Clinical Practice (GCP) guidelines and applicable local regulations.  I will also ensure that 
subinvestigator(s) and other relevant members of my personnel have access to copies of this 
protocol and the ICH GCP guidelines to enable them to work in accordance with the 
provisions of these documents. 
Principal Investigator: 
Signature: 
 
 
 
 
Date (DD Mmm YYYY) 
Printed Name: 
 
  
<Insert name and qualification of the investigator> 
Address: 
 
 
 
 
 
 
 
 
 


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CONTACT DETAILS OF SPONSOR’S KEY PERSONNEL 
24-hour Contact for 
Serious Adverse Events 
 
See [Section 12.4.5 
Reporting Procedures for 
Serious Adverse Events] 
Please fax or email the serious adverse events/special 
situations worksheet to: 
Astellas Pharma Global Development Inc. 
North America fax number: +1-888-396-3750 
North America alternate fax number: +1-847-317-1241 
International fax number: +44-800-471-5263 
Email: safety-us@astellas.com 
Medical Monitor 
 
 
ICON Clinical Research LLC 
Office: Av. Fondo de la Legua 1171 Martinez, Buenos Aires, 
Argentina 
 
 
Clinical Research Contacts 
 
 
, Development 
Astellas Pharma Inc. 
2-5-1, Nihonbashi-Honcho, Chuo-ku,  
Tokyo, 103-8411, Japan  
 
 
 
 
 
 Development 
Astellas Pharma Inc. 
2-5-1, Nihonbashi-Honcho, Chuo-ku,  
Tokyo, 103-8411, Japan  
 
 
 
 
 
 Development 
Astellas Pharma Inc. 
2-5-1, Nihonbashi-Honcho, Chuo-ku,  
Tokyo, 103-8411, Japan  
 
 
 
PPD
PPD
PPD
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Sponsor: API 
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ICON Clinical Research LLC 
79 T.W. Alexander Drive 
4401 Research Commons Bldg 
Suite 330, PO Box 14353 
Durham, North Carolina 
United States, 22709 
 
 
PPD
PPD


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1 
PROTOCOL SUMMARY 
1.1 
Synopsis 
Date and Version of Protocol Synopsis: 
21 Jul 2021, Version 3.0 
Sponsor: 
Astellas Pharma Inc. (API) 
Protocol Number: 
5354-CL-0201 
Compound Name: 
ASP5354 
Phase of Development: 
Phase 2 
Title of Study: 
A Phase 2 Randomized Open-label, Dose-ranging Study for Ureter Visualization, Using ASP5354 in 
Subjects Undergoing Laparoscopic/Minimally Invasive Colorectal Surgery 
Planned Study Period: 
From approximately 1Q2020 to 4Q2021. 
Study Objectives and Endpoints:  
Objectives 
Endpoints 
Primary 
● 
To determine the optimal dose of ASP5354 
for ureter visualization in subjects 
undergoing laparoscopic/minimally invasive 
colorectal surgery. 
● 
Anatomical visualization of the index ureter(s) 
30 minutes after dosing of ASP5354 and the end of 
surgery (Yes/No). 
Secondary 
 
● 
To investigate the safety and tolerability of 
ASP5354 in subjects undergoing 
laparoscopic/minimally invasive colorectal 
surgery. 
 
 
 
 
 
 
● 
To investigate the pharmacokinetics of 
ASP5354 in subjects undergoing 
laparoscopic/minimally invasive colorectal 
surgery. 
Safety and Tolerability 
● 
Vital signs (blood pressure, pulse and respiratory 
rate) 
● 
Routine 12-lead electrocardiograms (ECGs) 
● 
Clinical laboratory tests (hematology [complete 
blood count], serum chemistry and urinalysis) 
● 
Nature, frequency and severity of 
treatment-emergent adverse events (TEAEs) and 
serious adverse events 
Pharmacokinetics 
● 
Plasma and urine concentrations of ASP5354 
● 
Amount of ASP5354 excreted in urine (Ae) during 
surgery 
● 
Percentage of drug dose excreted into urine (Ae%) 
during surgery 
Table continued on next page 
 
 


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Objectives 
Endpoints 
Exploratory 
● 
To investigate the relationship between 
pharmacokinetics and pharmacodynamics of 
ASP5354 in subjects undergoing 
laparoscopic/minimally invasive colorectal 
surgery. 
● 
To explore fluorescence intensity and 
visualization duration of ASP5354 in subjects 
undergoing laparoscopic/minimally invasive 
colorectal surgery. 
● 
To explore the benefit of visualization during 
surgery of ASP5354 in subjects undergoing 
laparoscopic/minimally invasive colorectal 
surgery. 
● 
The Likert Scale for qualitative response about the 
intensity of fluorescence at the predefined time 
points during surgery 
● 
Duration of anatomical visualization of the index 
ureter(s) from the first time of positive 
visualization to the last time point of positive 
visualization 
● 
Signal background ratio (SBR) 
● 
Binary questions (Yes/No) for the benefit of 
visualization by ASP5354 during surgery 
● 
Ability to visualize the contralateral ureter when 
amenable to visualization 
Ae: amount of ASP5354 excreted in urine; Ae%: percentage of drug dose excreted into urine; BMI: body 
mass index; ECGs: electrocardiograms; SAEs: serious adverse events; SBR: signal background ratio; 
TEAEs: treatment-emergent adverse events 
Planned Total Number of Study Sites and Location(s): 
Approximately 2 study sites in the US 
Study Population: 
Male or female subjects ≥ 18 years of age scheduled to undergo laparoscopic/minimally invasive 
colorectal surgery. 
Number of Subjects to be Enrolled/Randomized:  
Dose Arm 
ASP5354 Dose Level 
Number of Subjects 
1 
 
Up to 15 
2 
 
Up to 15 
3 
 
Up to 15 
Total 
- 
Up to 45 
 
Additional Dose Arm 
ASP5354 Dose Level 
Number of Subjects 
4 
 
Up to 15 
5 
 
Up to 15 
6 
 
Up to 15 
* Based on VRC review of the initial 3 dose levels, if none of the doses selected have visualization, then 
 
and 
 dose levels will be added; if 1 dose selected has visualization, then the 
 dose level will be added.  
The 
 dose level will only be added if only the 
 dose level has visualization. 
Study Design Overview: 
The study is a randomized open-label, dose-ranging study in adult subjects undergoing 
laparoscopic/minimally invasive colorectal surgery in which the need for anatomical visualization of 
the ureter is anticipated.  
Subjects will be randomly assigned to receive single doses of ASP5354 (
,  or 
) which will be 
administered as an iv bolus to evaluate the anatomical visualization of the index ureter(s) (and 
contralateral ureter when feasible).  Safety, tolerability and pharmacokinetics in the study population 
will also be assessed. 
Up to 15 subjects will be randomly assigned at each dose level.  During a standard minimally invasive 
surgery, visualization of the surgical field will be assessed following the placement of the 
near-infrared fluorescence (NIR-F) imaging system (with FDA 510[k] cleared optical device system) 
proximal to the ureter of interest and then ASP5354 will be administered.   
● 
Intraoperative ureter fluorescence visualization will be assessed at approximately 10, 20, 30, 45 
and 60 minutes post administration of ASP5354, and then every 30 minutes thereafter and/or at 
the last time point before removal of visualization instruments (end of surgery). 
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● 
The contralateral ureter will also be visualized when feasible, per the investigator’s judgment, at 
similar time points.  Fluorescence images will be captured on a bright field, NIR-F and overlay 
view (if available) and recorded during the entire surgery and archived. 
● 
The anatomical visualization of the index ureter(s) will be assessed by the investigator 
intraoperatively using a binary “Yes or No” question on the ability to visualize the ureter at each 
time point. 
● 
The fluorescence intensity of the ureter will be qualitatively assessed using a Likert Scale 
(0 = None, 1 = Mild, 2 = Moderate and 3 = Strong). 
● 
The duration of anatomical visualization of the index ureter(s) will be from the first time of 
positive visualization to the last time point of positive visualization.  
● 
Recorded images will be used to determine the SBR. 
The visualization data will be assessed by a Visualization Review Committee (VRC), consisting of 
the investigator(s) and the sponsor’s medical representative.  The VRC will be held at the following 
time points, but ad-hoc meetings may occur until the optimal dose is determined:  
1) after the initial 3 subjects have completed the surgical procedures at each of the dose levels; 
and  
2) then in increments of 3 subjects (up to a total of 15 subjects per dose level) for those who 
have completed surgical procedures at the expanded dose levels. 
The visualization data for the subjects, at each interim time point, who completed the surgical 
procedure will be assessed to decide whether to:  
1) expand a dose under evaluation;   
2) stop a dose under evaluation in which case additional subjects would not be added to a dose 
level; or 
3) define a dose as optimal. 
Once the optimal dose is determined, the optimal dose cohort may continue enrollment up to the 
maximum 15 subjects. 
The VRC assessment to stop, to continue or to define an optimal dose level will be based on the 
totality of the collected data, including but not limited to the following: 
1) anatomical visualization of the ureter at both 30 minutes after ASP5354 administration and at 
the end of surgery, or 
2) fluorescence intensity based on the Likert Scale (0 to 3).  
In the case where 2 doses perform equally, the lower dose may be selected. 
Based on VRC review of the initial 3 dose levels, if none of the doses selected have visualization, 
then 
 and 
 dose levels will be added; if 1 dose selected has visualization, then the 
 
dose level will be added.  The 
 dose level will only be added if only the 
 dose level has 
visualization.  For further details of the VRC, refer to the VRC charter. 
If the investigator judges that anatomical visualization of ASP5354 is not sufficient for the subject at 
30 minutes after administration, the investigator can proceed with surgery using other modalities for 
visualization. 
Safety and tolerability will be assessed by recording TEAEs and adverse reactions associated with the 
use of an investigational product (IP), clinical laboratory evaluations (hematology, serum chemistry 
and urinalysis), 12-lead ECGs, vital signs measurements and physical examination.  Blood and urine 
samples for pharmacokinetic assessment will be collected pre- and post-administration of ASP5354 at 
defined time points, up to the end of surgery.  In cases where surgery is completed in less than 
105 minutes, blood samples will also be collected at 180 minutes after administration. 
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Inclusion/Exclusion Criteria:  
Inclusion Criteria: 
1. Institutional Review Board (IRB)/Independent Ethics Committee (IEC) approved written 
informed consent and privacy language as per national regulations (e.g., Health Insurance 
Portability and Accountability Act authorization for US study sites) must be obtained from the 
subject prior to any study-related procedures (including withdrawal of prohibited medication, if 
applicable). 
2. Subject is scheduled to undergo laparoscopic/minimally invasive colorectal surgery. 
3. Subject will need visualization of the ureter(s). 
4. Subject is considered an adult (≥ 18 years of age) according to local regulation at the time of 
signing the informed consent form. 
5. Female subject is not pregnant (see [Appendix 12.3 Contraception Requirements]) and at least 1 
of the following conditions apply: 
a. Not a woman of childbearing potential (WOCBP; see [Appendix 12.3 Contraception 
Requirements]) 
b. WOCBP who agrees to follow the contraceptive guidance (see [Appendix 12.3 
Contraception Requirements]) from the time of informed consent through at least 30 days 
after final study treatment administration. 
6. Female subject must agree not to breastfeed starting at screening and throughout the study period. 
7. Female subject must not donate ova starting at first dose of IP and throughout the study period 
and for 30 days after final study treatment administration. 
8. Male subject with female partner(s) of childbearing potential (including breastfeeding partner) 
must agree to use contraception (see [Appendix 12.3 Contraception Requirements]) throughout 
the treatment period and for 30 days after final study treatment administration. 
9. Male subject must not donate sperm during the treatment period and for 30 days after final study 
treatment administration. 
10. Male subject with pregnant partner(s) must agree to remain abstinent or use a condom for the 
duration of the pregnancy throughout the study period and for 30 days after final study treatment 
administration. 
11. Subject agrees not to participate in another interventional study while participating in the present 
study. 
Subjects enrolled after optimal dose determination: 
12. Subject has any of the following values: 
• 
Body mass index > 25 
• 
Estimated glomerular filtration rate (eGFR) ≥ 15 mL/min/1.73 m2 and < 60. Subjects with an 
eGFR ≥ 60 mL/min/1.73 m2 may be considered after discussion with the medical monitor. 
Waivers to the inclusion criteria will NOT be allowed. 
 


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Exclusion Criteria: 
1. Subject is anticipated to require ureteral stenting during surgery. 
2. Subject has a history of known retroperitoneal fibrosis. 
3. Subject has an active urinary tract infection. 
4. Subject has received investigational therapy within 28 days or 5 half-lives, whichever is longer, 
prior to screening. 
5. Subject has any condition that, in the investigator’s opinion, makes the subject unsuitable for 
study participation. 
6. Subject has a known or suspected hypersensitivity to ASP5354, indocyanine green (ICG) or any 
components of the formulation used. 
7. Subject has had previous exposure to ASP5354. 
8. Subject has moderate to severe cardiac disease that limits daily functioning (New York Heart 
Association Class III-IV) or other medical conditions that the investigator feels would impact 
safety or study compliance. 
9. Subject has a mean resting heart rate ≤ 45 bpm or ≥ 115 bpm, mean systolic blood pressure 
≥ 160 mmHg or mean diastolic blood pressure ≥ 100 mmHg on day -1.  If the mean blood 
pressure exceeds the limits above, repeat readings can be taken.  Subject who has adequately 
controlled blood pressure is eligible. 
10. Subject has a mean corrected QT interval (Triplicate ECG) using Fridericia’s formula (QTcF) 
> 430 msec (for male subjects) and > 450 msec (for female subjects) on day -1.  If the mean 
QTcF exceeds the limits above, the mean of 1 additional triplicate ECG can be taken. 
11. Subject has any of the following screening laboratory values: 
● Hemoglobin ≤ 9 g/dL 
● Absolute neutrophil count ≤ 1500/µL 
● Platelet count ≤ 100000/µL 
● eGFR < 60 mL/min/1.73 m2 (Not applicable to subjects enrolled after optimal dose 
determination.) 
● Serum bilirubin ≥ 2 × upper limit of normal (ULN) 
● Aspartate aminotransferase or serum glutamic oxaloacetic transaminase ≥ 2.5 × ULN 
● Alanine aminotransferase or serum glutamic pyruvic transaminase ≥ 2.5 × ULN 
12. Subject has taken ICG or other NIR-F imaging agents within 48 hours prior to study treatment 
administration. 
13. Subject has taken diuretics or inhibitors of renal transporters defined by FDA (see [Appendix 12.6 
List of Excluded Concomitant Medications]) within 48 hours prior to study treatment 
administration. 
14. Subject has used any illicit drugs, unless legally prescribed and is not being abused 
(amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine and opiates) within 30 days 
prior to day -1.  
15. Subject has a history of alcohol abuse.  Subject should not have consumed any alcohol within 
48 hours of surgery. 
Waivers to the exclusion criteria will NOT be allowed. 
Investigational Product:  
Name: 
ASP5354 
Use: 
ASP5354 solution for administration will be supplied by the sponsor or designee as a 
 
aqueous solution for iv administration.  The NIR-F intensity of ASP5354 will be measured at 
wavelength 820 nm when irradiated with near-infrared excitation light at wavelength 780 nm. 
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Dose(s): 
ASP5354, 
,  or 
 will be administered as a single dose once the surgical area of interest is in 
view. 
Mode of Administration: 
ASP5354 will be administered as a single iv bolus administration under fasting conditions. 
Dose Modifications: 
Since this is a single dose study, dose modification is not applicable. 
Concomitant Treatment (Medication and Nonmedication Therapy) Restrictions or 
Requirements: 
The following concomitant medications will not be allowed from 48 hours prior to the IP 
administration until the completion of the surgical procedure: 
● 
ICG unless used for anastomotic evaluation 
● 
Other NIR-F imaging agent 
● 
Diuretics 
● 
Inhibitors for renal transporters defined by FDA (see [Appendix 12.6 List of Excluded 
Concomitant Medications]) 
Duration of Treatment: 
Each subject will receive a single dose of ASP5354. 
Treatment Discontinuation Criteria: 
Since this is a single dose study, treatment discontinuation is not applicable. 
Statistical Methods: 
Sample Size Justification: 
The sample size is expected to provide adequate information to determine the optimal dose of 
ASP5354 for ureter visualization.  This sample size is not based on statistical power calculation. 
Efficacy: 
The success rate of anatomical visualization of the index ureter(s) 30 minutes after dosing of 
ASP5354 and at the end of surgery will be estimated with exact 95% confidence interval for the full 
analysis set (FAS).  The FAS consists of all randomized subjects who receive ASP5354 and have at 
least 1 assessment of ureter visualization during surgery.  The FAS will be used for the summary of 
efficacy. 
Safety and Tolerability: 
The safety and tolerability endpoints will be summarized by descriptive statistics for the safety 
analysis set (SAF).  The SAF consists of all randomized subjects who receive ASP5354. 
Pharmacokinetics: 
A listing and summary statistics of the individual plasma and urine concentrations and sampling times 
by treatment and assessment time interval will be prepared.  A total of Ae, Ae% and urine volume 
during surgery will be listed and summarized by treatment. 
Pharmacokinetic parameters will be evaluated by a population pharmacokinetic approach.  Details of 
population analyses will be described in a separate analysis plan and a separate report. 
Pharmacodynamics: 
The relationship between ureter visualization (Yes/No, Likert Scale, visualization duration, etc) and 
pharmacokinetics will be evaluated by a population pharmacokinetic/pharmacodynamic approach.  
Details of the population analyses will be described in a separate analysis plan and a separate report. 
Interim Analyses: 
The determination of dose(s) to stop, continue or define a dose as optimal will occur when every 3 
subjects randomly assigned to each dose level have the data for anatomical visualization during 
surgery reviewed via the VRC. The VRC will be held to review subject data until an optimal dose is 
determined. 
 
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1.3 
Schedules of Assessments 
Table 1 
Schedule of Assessments 
Assessments 
Screening 
Period 
Treatment Period 
Follow-up Period 
Day 
-28 to -1 
1 (Operation) 
7 (Outpatient) 13 
Preoperative 
Intraoperative 
Postoperative 
Window  
- 
- 
- 
- 
+3 
Visit Number 
1 
2 
3 
Informed Consent 
X 
 
 
 
 
Inclusion/Exclusion Criteria 
X 
 
 
 
 
Medical History 
X 
 
 
 
 
Demographics 
X 
 
 
 
 
Drug and Alcohol Screen 1 
X† 
 
 
 
 
Randomization 
 
X 
 
 
 
Physical Examination 2 
X 
X 
 
X 
X 
Vital Signs 3 
X 
X 
 
X 12 
X 
Clinical Laboratory Tests 4 
X† 
X 
 
X 12 
X 
Height and Body Weight 3 
X 
 
 
 
X 
Serum Pregnancy Test 5 
X† 
 
 
 
X 
Routine 12-lead ECG 6 
X 
X 
 
X 
X 
Dosing ASP5354 
 
 
X 
 
 
Blood Sampling for ASP5354 Pharmacokinetics and Metabolite Profiling 7 
 
X 
X 
X 
 
Urine Point Sampling for ASP5354 Pharmacokinetics and Metabolite 
Profiling 7 
 
X 
X 
 
 
Total Urine Sampling in Catheter Bag and Amount of ASP5354 Excreted in 
Urine During Surgery 8 
 
 
X 
 
 
Confirmation of urine discoloration 9 
 
 
→ 
→ 
→ 
Investigator Evaluation for Ureteral Visualization 10 
 
 
X 
 
 
Record Surgery 
 
 
X 11 
 
 
Previous/Concomitant Medications 
X 
X 
X 
X 
X 14 
AE Assessment 
X 
X 
X 
X 
X 14 
AE: adverse event, ECG: electrocardiogram 
†These assessments will be performed by local laboratory and the results of these assessments have to be obtained prior to subject randomization. 
Footnotes continued on next page 


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1. 
Drug screen (e.g., amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine and opiates) and alcohol screen will be performed by the study site’s preferred method. 
2. 
A full physical examination will be performed at screening (visit 1).  Body systems to be evaluated include general appearance, skin, lymphatic, head and neck, ears, nose and 
throat, chest and lungs, cardiovascular, abdomen, extremities, musculoskeletal and neuromuscular.  At visit 2 and follow-up (visit 3), a symptom-directed physical examination 
will be performed. 
3. 
Vital signs include blood pressure, pulse and respiratory rate.  All vital signs will be measured with the subject in the sitting or supine position.  Height and weight will be 
measured using standard institution practice and equipment. 
4. 
Clinical laboratory tests include blood collection for hematology (complete blood count) and serum chemistry and urine samples for urinalysis. 
5. 
This assessment will be required only for subjects who are women of childbearing potential (see [Appendix 12.3 Contraception Requirements]). 
6. 
Routine 12-lead ECGs will be taken in triplicate. 
7. 
Blood and urine samples for pharmacokinetics of investigational product and possible metabolite(s) (if applicable) will be collected from every subject.  A single pharmacokinetic 
blood sample will be collected at each of the following time points: prior to dose, approximately 10 minutes postdose, either approximately 30 or 60 minutes postdose and at the 
end of surgery.  In cases where surgery is completed in less than 105 minutes, blood samples will also be collected at approximately 180 minutes after administration.  Urine point 
sampling will be performed prior to dose and at the end of surgery (the end of surgery is an optional time point if a sample is available).  For a detailed procedure of 
pharmacokinetic sampling, refer to the Sample Handling and Processing Manual. 
8. 
Total urine volume of urine in the catheter bag will be calculated by dividing the weight of urine in the catheter bag by predefined density.  Urine volume will be recorded in 
millimeters, and urine sample for pharmacokinetics of investigational product and possible metabolite(s) (if applicable) will be collected from the urine in the catheter bag to 
calculate Ae and Ae%.  For a detailed procedure of total urine volume calculation, refer to the Sample Handling and Processing Manual. 
9. 
The incidence of green discoloration of the urine will be recorded any time after dose administration.  If it occurs, the color of urine will be assessed during surgery and every 
120 minutes (± 30 minute window) after the end of surgery until it resolves or the subject is discharged, whichever is earlier.  The start and stop date and time will also be 
recorded. 
10. Surgeon evaluation for ureteral visualization includes incidence of anatomical visualization of the ureter(s), questions for the surgical benefit of ASP5354 and the Likert Scale for 
qualitative response about the intensity of fluorescence at the predefined time points during surgery. 
11. Fluorescence images will be captured and recorded during the entire surgery and archived. 
12. Postoperative vital signs and clinical laboratory tests will be collected within 2 hours after the surgery. 
13. If a subject discontinues early from the study, day 7 outpatient visit procedures will be performed upon discontinuation. 
14. If a subject experiences an AE or change in concomitant medications during days 2 to 6, he or she should call the study site to inform any of these changes.  Should subjects 
develop a hypersensitivity reaction, an additional blood sample for determination of histamine and tryptase concentrations should be taken as soon as possible after the onset of 
the hypersensitivity reaction. 
 


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1.3.1 
 
Sample Collection and Timed Procedure Schedule 
Table 2 
Sample Collection and Timed Procedure Schedule 
Day 
Operation 
Time Point 
Sample Collection 
Efficacy Assessment 
Collection Window 
Blood Sampling for 
Pharmacokinetics 
Urine Point Sampling 
for Pharmacokinetics 
Surgeon Evaluation for 
Ureteral Visualization 
Day 1 
Preoperative 
Predose 
X 
X 
 d 
Within 1 h before dosing 
Intraoperative 
10 min a 
X 
 
X 
5 min ≤ and <15 min 
20 min a 
 
 
X 
15 min ≤ and < 25 min 
30 min a 
(X) e 
 
X 
25 min ≤ and < 38 min 
45 min a 
 
 
X 
38 min ≤ and < 53 min 
60 min a 
(X) e 
 
X 
53 min ≤ and < 75 min 
Τ min a, b 
 
 
X 
Τ-15 min ≤ and < Τ+15 min 
End of surgery 
- 
X 
X f 
X 
- 
Postoperative 
180 min c  
X 
 
 
105 min ≤ and < 240 min 
a. 
If end of surgery is earlier than this time point, this time point(s) would not need to be collected. 
b. 
This time point(s) would need to be evaluated every 30 minutes from 60 minutes after administration. 
c. 
In cases where the surgery is completed in less than 105 minutes, blood samples would be collected at approximately 180 minutes after administration. 
d. 
At the predose time point, fluorescence images will be captured and recorded on a bright field, near infrared fluorescence and overlay view (if available) for signal background 
ratio analysis, as well as the other time points. 
e. 
Either 30 or 60 minutes after administration need to be collected. 
f. 
Urine collection at end of surgery time point is optional (if a sample is available).  The urine sample for the amount of ASP5354 excreted in urine during surgery will be collected 
from the urine in the catheter bag after the end of surgery. 
 


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2 
INTRODUCTION 
2.1 
Background 
Iatrogenic ureteral injury (IUI) is a rare but serious complication of abdominal or pelvic 
surgery.  More than half of IUIs occur during gynecological procedures, followed by 
colorectal and vascular surgeries and urologic procedures [Burks & Santucci, 2014].  
In patients undergoing hysterectomy for benign disease, minimally invasive hysterectomy 
was associated with increased risk in IUI relative to open procedures [Packlam et al, 2016].  
In a US national series of 101021 patients, rates of ureteric repair were 0.5% for laparoscopic 
hysterectomy compared with 0.3% for open hysterectomy and 0.1% for vaginal hysterectomy 
[Wallis et al, 2016].  Laparoscopic procedure (versus vaginal), uterine size and endometriosis 
were associated with an increased risk for urologic intervention posthysterectomy. 
In a 10-year retrospective analysis of ureteral injury in colorectal surgery, 6027 (0.28%) cases 
were identified in approximately 2 million US procedures [Halabi et al, 2014].  Though rare, 
IUIs were independently associated with higher mortality, morbidity, longer length of 
hospital stay and increased hospital charges.  The highest risk factors for ureteral injuries 
were rectal cancer, adhesions and metastatic cancer.  Postoperative complications associated 
with IUI included acute renal failure, urinary tract infection, wound complications and 
anastomotic leak. 
Intraoperative diagnoses of IUIs are important to both patients and surgeons to minimize 
postoperative complications and improve surgical outcome; however, most IUIs are not 
detected intraoperatively [Burks and Santucci, 2014].  The identification of ureters can often 
be challenging in patients with advanced endometriosis, previous surgery or radiotherapy.  
Laparoscopic procedures may make it more difficult to detect the ureter using visual 
inspection and palpation, potentially increasing the risk for IUI [Slooter et al, 2019]. 
Preoperative ureteral stenting has long been proposed as a technique to identify the ureters 
and avoid IUI.  However, this is an invasive procedure with associated complications, and 
reduction in the injury rate is not significant.  Other preoperative techniques to reduce the risk 
of ureteral injury require radiation or complex nuclear medical equipment [Yeung et al, 2016]. 
Image guided surgery using near-infrared fluorescence (NIR-F) imaging is a promising 
technique that offers real-time visual information for surgeons and potentially makes 
operations safer for patients.  Two dyes, methylene blue (MB) and indocyanine green (ICG), 
are commercially available but each has limitations with respect to intraoperative 
identification of the ureter [Slooter et al, 2019]. 
MB can be used for the identification of ureters during surgery [Verbeek et al, 2013]; 
however, the penetration depth of MB imaging is not sufficient for the identification of 
ureters under some circumstances due to its 700 nm excitation wavelength.  Improved 
contrast agents with 800 nm fluorescence, a higher extinction coefficient and a higher 
quantum yield would be needed [Verbeek et al, 2013]. 


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ICG is the only approved iv administered NIR-F imaging agent for intraoperative 
fluorescence imaging such as angiography.  It has an NIR-F wavelength, which permits 
photons to penetrate relatively deeply through tissue and blood without altering the surgical 
field.  However, ICG is minimally excreted into urine due to its lipophilicity; therefore it is 
not suitable for ureteral imaging. 
ASP5354 is an ICG-derivative which shows hydrophilic properties by conjugating with 
cyclodextrins while maintaining the near-infrared fluorescent property similar to ICG.  
ASP5354 has an absorption of 780 nm and an emission of 820 nm, similar to that of ICG.  
ASP5354 is almost entirely excreted into the urine through the kidneys after iv administration 
due to its hydrophilic nature.  Therefore, the detection of excretory flow of ASP5354 from 
the kidneys to the bladder using NIR-F imaging technology has the potential to facilitate 
accurate identification of the ureter during surgery. 
2.1.1 
Nonclinical and Clinical Data 
Nonclinical and clinical data for ASP5354 available as of the writing of this protocol are 
summarized below.  Please refer to the current version of the ASP5354 Investigator’s 
Brochure (IB) for the most recent data. 
2.1.1.1 Summary of Nonclinical Data 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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). 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Please refer to the current ASP5354 IB for the most recent nonclinical data.  
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2.1.1.2 Summary of Clinical Data 
2.1.1.2.1 
Phase 1 Results (Study 5354-CL-0001) 
As of the writing of this protocol, 1 clinical study of ASP5354 has completed dosing and 
study report preparation is ongoing.  Study 5354-CL-0001 is a phase 1, double-blind, 
randomized, placebo controlled, single ascending dose study assessing the safety and 
pharmacokinetics of ASP5354 in healthy adult volunteers.  IP was administered as a single iv 
bolus injection under fasting conditions. 
2.1.1.2.1.1 
Pharmacokinetics and Product Metabolism in Humans 
Following a single iv administration, ASP5354 was rapidly eliminated.  The observed 
pharmacokinetic parameters suggested a dose proportional increase in the investigated dose 
range from 
 to 
.  Almost all fraction of ASP5354 administered was excreted 
unchanged in urine.  Renal clearance was nearly equivalent to the total body clearance.   
For details please refer to the current version of the ASP5354 IB. 
2.1.1.2.1.2 
Safety 
Single doses of up to 
 were considered safe and tolerable, with no patterns noted for 
safety concerns in healthy subjects.  Twenty subjects received ASP5354 at doses of 
, 
, 
  and 
 (each in 4 subjects).  Ten subjects received placebo for ASP5354 (2 subjects 
per cohort). 
Of the 30 subjects treated with ASP5354 or placebo, 5 subjects experienced at least 
1 treatment-emergent adverse event (TEAE; oral herpes, pyelonephritis, urinary tract 
infection, headache, presyncope, dysuria, nausea, vomiting and incontinence [each in 
1 subject]).  Two (20.0%) subjects who received placebo experienced TEAEs of nausea, 
vomiting and incontinence (each in 1 subject); 1 (25.0%) subject who received 
 
ASP5354 experienced TEAEs of oral herpes and presyncope; 1 (25.0%) subject who received 
 ASP5354 experienced a TEAE of urinary tract infection; and 1 (25.0%) subject who 
received 
 ASP5354 experienced TEAEs of dysuria, headache and pyelonephritis.  
None of the subjects experienced TEAEs considered to be at least possibly related to 
ASP5354.  One subject who received ASP5354, 
 experienced a ≥ grade 3 TEAE; the 
grade 3 event of pyelonephritis was also an SAE that was considered by the investigator to be 
not related to ASP5354; rather, it was assessed as related to the use of a urinary catheter.  
TEAEs leading to withdrawal of IP were not applicable, as it was a single dose study.  No 
deaths were reported in the study. 
For details please refer to the current version of the ASP5354 IB. 
2.1.2 
Summary of Key Safety Information for Investigational Product(s)  
The safety profile of ASP5354 is based on the results of the nonclinical studies and the 
clinical phase 1 study (Study 5354-CL-0001).  Please refer to the current version of the 
ASP5354 IB for the most recent information. 
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In nonclinical studies, ASP5354 caused very slight macrophage accumulation in the alveolus 
in the lungs of rats and increased plasma histamine levels in dogs.  Abnormal urine and skin 
color (pale green, greenish yellow or green) and green discoloration of the kidneys, 
epididymides and/or lymph nodes in gross pathology were also observed in rats and 
monkeys.  These findings disappeared with discontinuation of drug administration.  These 
findings were considered to be caused due to the green color of ASP5354 and were not 
considered toxicologically significant. 
Though very slight macrophage accumulation in the alveolus in the lung was observed in the 
4-week repeated iv dose toxicity study in rats (Study 5354-TX-0004), the possibility that 
ASP5354 induces this effect in the clinic is considered to be low.   
Increased plasma histamine levels and clinical signs which are associated with increased 
plasma histamine concentration levels were induced by the single administration in dogs; 
however, the possibility of ASP5354 inducing this effect in the clinic is considered to be low. 
ASP5354 has shown no discernible genotoxicity, phototoxicity or embryofetal toxicity 
potential in the nonclinical species tested. 
As of the writing of this protocol, 1 phase 1 first-in-human study of ASP5354 has completed 
dosing, and study report preparation is ongoing (Study 5354 CL 0001).  ASP5354 was well 
tolerated, with no IP-related TEAEs, or deaths reported in the study up to 
.  ASP5354 
had no clinically significant effect on vital signs, electrocardiogram (ECG), blood chemistry, 
hematology or urinalysis. 
In conclusion, the nonclinical and phase 1 clinical safety data support the initiation of a 
phase 2 dose-finding study in patients undergoing abdominal surgery. 
2.2 
Study Rationale 
IUI is a devastating rare but serious complication of abdominal or pelvic surgery.  More than 
half of IUIs occur during gynecological procedures, followed by colorectal surgery and 
vascular surgeries and urologic procedures [Burks and Santucci, 2014].  In retrospective 
analysis of ureteral injury in colorectal surgery, IUIs were independently associated with 
higher mortality, morbidity, longer length of hospital stay and increased hospital charges 
[Halabi et al, 2014].  The highest risk factors for ureteral injuries were rectal cancer, adhesion 
and metastatic cancer.  Postoperative complications associated with IUI included acute renal 
failure, urinary tract infection, wound complications and anastomotic leak.  
Intraoperative diagnoses of IUIs are important to both patients and surgeons to minimize 
postoperative complications and improve surgical outcomes; however, most IUIs are not 
detected intraoperatively [Burks and Santucci, 2014].  Preoperative ureteral stenting has long 
been proposed as a technique to identify the ureters and avoid IUI.  However, this is an 
invasive procedure with associated complications and reduction in the injury rate is not 
significant; in addition, there are increased costs related to the procedure as well as increased 
operative times [Yeung et al, 2016].  Image guided surgery using NIR-F imaging is a 
promising technique that offers real-time visual information for surgeons and potentially 
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make operations safer for patients.  There are 2 commercially available dyes, MB and ICG, 
but each has limitations with respect to intraoperative identification of the ureter [Slooter et al, 
2019].  Neither are FDA approved for ureter visualization. 
ASP5354 has hydrophilic properties, unlike ICG, with an absorption and emission similar to 
that of ICG.  After IV administration, ASP5354 is mostly excreted through the kidney and 
into the urine.  Therefore, the detection of excretory flow of ASP5354 from the kidneys to the 
bladder using NIR-F imaging technology can enable the surgeon, through a noninvasive 
means, to accurately and conveniently identify the ureters during surgery. 
Based on this preclinical data confirming ureter visualization and the safety data in healthy 
volunteers, there is a strong rationale to define the dose in subjects undergoing minimally 
invasive surgery.  As no noninvasive imaging agents are FDA approved for ureter imaging, 
ASP5354 provides a simplified and safe way to visualize ureters without adding operating 
time and expense or risk to subjects. 
2.3 
Risk Benefit Assessment 
As no FDA approved imaging agents for ureter identification are available, current options 
include stents or in some cases, off-label use of NIR-F agents.  Stents have the issue of 
increased time in the operating room and medical costs, as well as potential for ureteral injury.  
ASP5354 provides a noninvasive way to visualize the ureters intraoperatively.  ASP5354 has 
been safely administered up to 
.  ASP5354 was well tolerated, with no IP-related 
TEAEs reported.  As such, there are no safety concerns for its use in subjects undergoing 
minimally invasive surgery.  The benefits of avoiding ureters intraoperatively will minimize 
ureteral injuries and associated complications, potentially decrease operating times, avoid 
repeat surgeries and not increase medical costs like stents. 
One important potential risk, hypersensitivity reactions including anaphylactic reactions with 
fatal outcome, are reported to have occurred after treatment with iodine based ICG.  The 
reported incidence for ICG-related anaphylactic reactions is 1:42000 administrations or lower.  
Hypersensitivity reactions are well known to occur, in varying frequencies, with all kinds of 
contrast agents (iodinated contrast agents for computed tomography, gadolinium based 
contrast agents for magnetic resonance imaging and contrast agents for image enhancement 
during ultrasound).  No such reactions have been observed for ASP5354 during the phase 1 
study (Study 5354-CL-0001).  The risk with ASP5354 is estimated to be lower, as ASP5354 
is not iodinated, whereas ICG is. 
The benefit of ASP5354 outweighs the anticipated risk, as no toxicities have been seen in 
doses up to 
.  Safety will be monitored during the study for all enrolled subjects. 
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3 
STUDY OBJECTIVE(S) AND ENDPOINT(S) 
Table 3 
Study Objectives and Endpoints 
Objectives 
Endpoints 
Primary 
● 
To determine the optimal dose of ASP5354 for 
ureter visualization in subjects undergoing 
laparoscopic/minimally invasive colorectal 
surgery. 
● 
Anatomical visualization of the index ureter(s) 
30 minutes after dosing of ASP5354 and the end 
of surgery (Yes/No). 
Secondary 
 
● 
To investigate the safety and tolerability of 
ASP5354 in subjects undergoing 
laparoscopic/minimally invasive colorectal 
surgery. 
 
 
 
 
 
 
● 
To investigate the pharmacokinetics of ASP5354 
in subjects undergoing laparoscopic/minimally 
invasive colorectal surgery. 
Safety and Tolerability 
● 
Vital signs (blood pressure, pulse and respiratory 
rate) 
● 
Routine 12-lead ECGs 
● 
Clinical laboratory tests (hematology [complete 
blood count], serum chemistry and urinalysis) 
● 
Nature, frequency and severity of TEAEs and 
SAEs 
Pharmacokinetics 
● 
Plasma and urine concentrations of ASP5354 
● 
Ae during surgery 
● 
Ae% during surgery 
Exploratory 
● 
To investigate the relationship between 
pharmacokinetics and pharmacodynamics of 
ASP5354 in subjects undergoing 
laparoscopic/minimally invasive colorectal 
surgery. 
● 
To explore the fluorescence intensity and 
visualization duration of ASP5354 in subjects 
undergoing laparoscopic/minimally invasive 
colorectal surgery. 
● 
To explore the benefit of visualization during 
surgery of ASP5354 in subjects undergoing 
laparoscopic/minimally invasive colorectal 
surgery. 
● 
The Likert Scale for qualitative response about 
the intensity of fluorescence at the predefined 
time points during surgery 
● 
Duration of anatomical visualization of the index 
ureter(s) from the first time of positive 
visualization to the last time point of positive 
visualization 
● 
SBR 
● 
Binary questions (Yes/No) for the benefit of 
visualization by ASP5354 during surgery 
● 
Ability to visualize the contralateral ureter when 
amenable to visualization 
Ae: amount of ASP5354 excreted in urine; Ae%: percentage of drug dose excreted into urine; 
ECGs: electrocardiograms; SAEs: serious adverse events; SBR: signal background ratio; 
TEAEs: treatment-emergent adverse events 
4 
STUDY DESIGN AND DOSE RATIONALE 
4.1 
Study Design 
The study is a randomized open-label, dose-ranging study in adult subjects undergoing 
laparoscopic/minimally invasive colorectal surgery in which the need for anatomical 
visualization of the ureter is anticipated.  This study will be conducted at approximately 
2 study sites in the US.  


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Subjects will be randomly assigned to receive single doses of ASP5354 (
,  or 
) 
which will be administered as an iv bolus to evaluate the anatomical visualization of the 
index ureter(s) (and contralateral ureter when feasible).  Safety, tolerability and 
pharmacokinetics in the study population will also be assessed. 
Up to 15 subjects will be randomly assigned at each dose level.   
Dose Arm 
ASP5354 Dose Level 
Number of Subjects 
1 
 
Up to 15 
2 
 
Up to 15 
3 
 
Up to 15 
Total 
- 
Up to 45 
 
Additional Dose Arm 
ASP5354 Dose Level 
Number of Subjects 
4 
 
Up to 15 
5 
 
Up to 15 
6 
 
Up to 15 
* Based on VRC review of the initial 3 dose levels, if none of the doses selected have visualization, then 
 
and 
 dose levels will be added; if 1 dose selected has visualization, then the 
 dose level will be added. 
The 
 dose level will only be added if only the 
 dose level has visualization. 
During a standard minimally invasive surgery, visualization of the surgical field will be 
assessed following the placement of the NIR-F imaging system (with FDA 510[k] cleared 
optical device system) proximal to the ureter of interest and then ASP5354 will be 
administered.   
● 
Intraoperative ureter fluorescence visualization will be assessed at approximately 10, 20, 
30, 45 and 60 minutes postadministration of ASP5354, and then every 30 minutes 
thereafter and/or at the last time point before removal of visualization instruments (end 
of surgery). 
● 
The contralateral ureter will also be visualized when feasible, per the investigator’s 
judgment, at similar time points.  Fluorescence images will be captured on a bright field, 
NIR-F and overlay view (if available) and recorded during the entire surgery and 
archived. 
● 
The anatomical visualization of the index ureter(s) will be assessed by the investigator 
intraoperatively using a binary “Yes or No” question on the ability to visualize the ureter 
at each time point. 
● 
The fluorescence intensity of the ureter will be qualitatively assessed using a Likert Scale 
(0 = None, 1 = Mild, 2 = Moderate and 3 = Strong). 
● 
The duration of anatomical visualization of the index ureter(s) will be from the first time 
of positive visualization to the last time point of positive visualization. 
● 
Recorded images will be used to determine the signal background ratio (SBR). 
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The visualization data will be assessed by a Visualization Review Committee (VRC), 
consisting of the investigator(s) and the sponsor’s medical representative.  The VRC will be 
held at the following time points, but ad-hoc meetings may occur until the optimal dose is 
determined:  
1) after the initial 3 subjects have completed the surgical procedures at each of the 
dose levels; and 
2) then in increments of 3 subjects (up to a total of 15 subjects per dose level) for those 
who have completed surgical procedures at the expanded dose levels. 
The visualization data for the subjects, at each interim time point, who completed the surgical 
procedure will be assessed to decide whether to:  
1) expand a dose under evaluation;  
2) stop a dose under evaluation in which case additional subjects would not be added 
to a dose level; or 
3) define a dose as optimal.   
Once the optimal dose is determined, the optimal dose cohort may continue enrollment up to 
the maximum 15 subjects. 
The VRC assessment to stop, to continue or to define an optimal dose level will be based on 
the totality of the collected data, including but not limited to the following: 
1) anatomical visualization, defined as positive visualization of the ureter at both 
30 minutes after ASP5354 administration and at the end of surgery; or 
2) fluorescence intensity based on the Likert Scale (0 to 3).  
In the case where 2 doses perform equally, the lower dose may be selected. 
Based on VRC review of the initial 3 dose levels, if none of the doses selected have 
visualization, then 
 and 
 dose levels will be added; if 1 dose selected has 
visualization, then the 
 dose level will be added.  The 
 dose level will only be 
added if only the 
 dose level has visualization.  For further details of the VRC, refer to 
the VRC charter. 
If the investigator judges that anatomical visualization of ASP5354 is not sufficient for the 
subject at 30 minutes after administration, the investigator can proceed with surgery using 
other modalities for visualization. 
Safety and tolerability will be assessed by recording TEAEs and adverse reactions associated 
with the use of an IP, clinical laboratory evaluations (hematology, serum chemistry and 
urinalysis), 12-lead ECG, vital signs measurements and physical examination.  Blood and 
urine samples for pharmacokinetic assessment will be collected pre- and post-administration 
of ASP5354 at defined time points, up to the end of surgery.  In cases where surgery is 
completed in less than 105 minutes, blood samples will also be collected at 180 minutes after 
administration. 
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4.2 
Dose Rationale 
A dose of 
 was considered to provide sufficient ureter visualization for 3 hours after iv 
administration, based on ureter visualization data in nonclinical studies and pharmacokinetic 
data of the phase 1 healthy volunteer study (Study 5354-CL-0001).  ASP5354 is confirmed 
safe and well tolerated up to 
 in the phase 1 study.  Since a large safety margin is 
confirmed, 3 dose levels of 
,  and 
 were proposed as initial doses in this phase 2 
study by selecting 
 as the central dose and using a common ratio of “3” to differentiate 
between doses and to determine the optimal dose. 
4.3 
End of Study Definition  
The study start is defined as the date the first subject signs informed consent.  The end of the 
study is defined as the last visit or scheduled procedure shown in the Schedule of 
Assessments for the last subject in the study. 
5 
STUDY POPULATION 
All screening assessments must be completed and reviewed to confirm the potential subject 
meets all eligibility criteria.  Prospective approval of protocol deviations to eligibility criteria 
(also known as protocol waivers or exemptions) is not permitted. 
5.1 
Inclusion Criteria 
Subject is eligible for participation in the study if all of the following apply: 
1. Institutional Review Board (IRB)/Independent Ethics Committee (IEC) approved written 
informed consent and privacy language as per national regulations (e.g., Health 
Insurance Portability and Accountability Act authorization for US study sites) must be 
obtained from the subject prior to any study-related procedures (including withdrawal of 
prohibited medication, if applicable). 
2. Subject is scheduled to undergo laparoscopic/minimally invasive colorectal surgery. 
3. Subject will need visualization of the ureter(s). 
4. Subject is considered an adult (≥ 18 years of age) according to local regulation at the 
time of signing the informed consent form (ICF). 
5. Female subject is not pregnant (see [Appendix 12.3 Contraception Requirements]) and at 
least 1 of the following conditions apply: 
a. Not a woman of childbearing potential (WOCBP; see [Appendix 12.3 Contraception 
Requirements]) 
b. WOCBP who agrees to follow the contraceptive guidance (see [Appendix 12.3 
Contraception Requirements]) from the time of informed consent through at 
least 30 days after final study treatment administration. 
6. Female subject must agree not to breastfeed starting at screening and throughout the 
study period. 
7. Female subject must not donate ova starting at first dose of IP and throughout the study 
period and for 30 days after final study treatment administration. 
8. Male subject with female partner(s) of childbearing potential (including breastfeeding 
partner) must agree to use contraception (see [Appendix 12.3 Contraception 
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Requirements]) throughout the treatment period and for 30 days after final study 
treatment administration. 
9. Male subject must not donate sperm during the treatment period and for 30 days after 
final study treatment administration. 
10. Male subject with pregnant partner(s) must agree to remain abstinent or use a condom 
for the duration of the pregnancy throughout the study period and for 30 days after final 
study treatment administration. 
11. Subject agrees not to participate in another interventional study while participating in the 
present study. 
Subjects enrolled after optimal dose determination: 
12. Subject has any of the following values at screening: 
● 
Body mass index > 25 
● 
Estimated glomerular filtration rate (eGFR) ≥ 15 mL/min/1.73 m2 and < 60. 
Subjects with an eGFR ≥ 60 mL/min/1.73 m2 may be considered after discussion 
with the medical monitor. 
Waivers to the inclusion criteria will NOT be allowed. 
5.2 
Exclusion Criteria 
Subject will be excluded from participation in the study if any of the following apply: 
1. Subject is anticipated to require ureteral stenting during surgery. 
2. Subject has a history of known retroperitoneal fibrosis. 
3. Subject has an active urinary tract infection. 
4. Subject has received any investigational therapy within 28 days or 5 half-lives, 
whichever is longer, prior to screening. 
5. Subject has any condition that, in the investigator’s opinion, makes the subject unsuitable 
for study participation. 
6. Subject has a known or suspected hypersensitivity to ASP5354, ICG or any components 
of the formulation used. 
7. Subject has had previous exposure to ASP5354. 
8. Subject has moderate to severe cardiac disease that limits daily functioning (New York 
Heart Association Class III-IV) or other medical conditions that the investigator feels 
would impact safety or study compliance. 
9. Subject has a mean resting heart rate ≤ 45 bpm or ≥ 115 bpm, mean systolic blood 
pressure (SBP) ≥ 160 mmHg or mean diastolic blood pressure (DBP) ≥ 100 mmHg on 
day -1.  If the mean blood pressure exceeds the limits above, repeat readings can be 
taken.  Subject who has adequately controlled blood pressure is eligible. 
10. Subject has a mean corrected QT interval (Triplicate ECG) using Fridericia’s formula 
(QTcF) > 430 msec (for male subjects) and > 450 msec (for female subjects) on day -1.  
If the mean QTcF exceeds the limits above, the mean of 1 additional triplicate ECG may 
be taken. 
11. Subject has any of the following screening laboratory values: 
● Hemoglobin ≤ 9 g/dL 


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● Absolute neutrophil count ≤ 1500/µL 
● Platelet count ≤ 100000/µL 
● eGFR < 60 mL/min/1.73 m2 (Not applicable to subjects enrolled after optimal 
dose determination.) 
● Serum bilirubin ≥ 2 × upper limit of normal (ULN) 
● Aspartate aminotransferase (AST) or serum glutamic oxaloacetic transaminase 
≥ 2.5 × ULN 
● Alanine aminotransferase (ALT) or serum glutamic pyruvic transaminase 
≥ 2.5 × ULN 
12. Subject has taken ICG or other NIR-F imaging agents within 48 hours prior to study 
treatment administration. 
13. Subject has taken diuretics or inhibitors of renal transporters defined by FDA (see 
[Appendix 12.6 List of Excluded Concomitant Medications]) within 48 hours prior to 
study treatment administration. 
14. Subject has used any illicit drugs, unless legally prescribed and is not being abused 
(amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine and opiates) within 
1 month prior to day -1. 
15. Subject has a history of alcohol abuse.  Subject should not have consumed any alcohol 
within 48 hours of surgery. 
Waivers to the exclusion criteria will NOT be allowed. 
5.3 
Restrictions During the Study 
Not applicable. 
5.4 
Screen Failures 
A screen failure is defined as a potential subject who signed the ICF, but did not meet 1 or 
more criteria required for participation in the study and was not randomized. 
For screen failures, the demographic data, date of signing the ICF, inclusion and exclusion 
criteria, AEs up to the time of screen failure and reason for screen failure will be collected in 
the electronic case report form (eCRF). 
5.4.1 
Rescreening 
Rescreening is not allowed.  However, in case that the results of screening assessments that 
do not meet the parameters required by the eligibility criteria (e.g., clinical laboratory tests, 
vital signs, physical examination, ECGs, etc) may be repeated once within the screening 
period without the need to register the subject as a screen failure. 
6 
INVESTIGATIONAL PRODUCT(S) 
6.1 
Investigational Product Administered 
The assigned doses of ASP5354 will be administered as a single dose once the surgical area 
of interest is in view.  


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Table 4 
Investigational Product(s) 
Name 
ASP5354 
Use 
Test Product 
Dosage Formulation 
Solution for injection 
Physical Description 
Clear deep green solution 
Unit Dose Strength 
 
Packaging and Labeling 
ASP5354 solution will be provided in 10-mL amber glass vials and stored 
according to the instructions on the label 
Route of Administration 
Intravenous bolus infusion 
Administration Instruction 
Administered as a single iv bolus administration under fasting conditions, once 
the surgical area of interest is in view 
IP or Non-IP 
IP 
Sourcing 
Provided by sponsor 
IP: investigational product 
 
ASP5354 solution for injection should be stored between -25° and -15°C.  ASP5354 vials 
must be allowed to thaw for at least 1.5 hours at controlled room temperature or may be 
thawed overnight (not more than 12 hours) under refrigerated conditions (approximately 2°to 
8°C).  ASP5354 vials must be used for preparation of administration within 4 hours after they 
are removed from the freezer or refrigerator, respectively.  After thawing, the vials should be 
gently rotated several times to ensure a uniform concentration and to avoid foaming (the 
concentration in the vials is not uniform after thawing).  The thawed ASP5354 is filtered and 
further diluted with Dextrose 5% in water.  The diluted ASP5354 shall be stored at controlled 
room temperature (20° to 25°C) prior to administration and must be administered within (not 
more than) 4 hours after penetration of vials. 
Refer to the pharmacy manual for detailed information regarding preparation, handling and 
storage of the IP. 
6.2 
Preparation/Handling/Storage/Accountability 
6.2.1 
Packaging and Labeling 
All IP used in this study will be prepared, packaged and labeled under the responsibility of 
qualified personnel at Astellas Pharma Inc. (API), Astellas US Technologies (AUST) or 
sponsor’s designee in accordance with API, AUST or sponsor’s designee standard operating 
procedures (SOPs), Good Manufacturing Practice (GMP) guidelines, International Council 
for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) 
Good Clinical Practice (GCP) guidelines and applicable local laws/regulations. 
Each vial will bear a label conforming to regulatory guidelines, GMP and local laws and 
regulations that identifies the contents as an investigational drug. 
Refer to the pharmacy manual for detailed information regarding packaging and labeling of 
the IP. 
CCI


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6.2.2 
Handling, Storage and Accountability 
1. The investigator or designee must confirm appropriate temperature conditions have been 
maintained during transit for all IP received and any discrepancies are reported and 
resolved before use of the IP. 
2. Only subjects enrolled in the study may receive IP and only authorized study site 
personnel may supply or administer IP.  Only IP with appropriate expiry/retest dating 
may be dispensed. 
3. All IP must be stored in a secure, environmentally controlled and monitored (manual or 
automated) area in accordance with the labeled storage conditions and access must be 
limited to the investigator and authorized study site personnel. 
4. The investigator, institution or the head of the medical institution (where applicable) is 
responsible for accountability, reconciliation and record maintenance (i.e., receipt, 
reconciliation and final disposition records). 
5. Further guidance and instruction on final disposition of used and unused IP is provided in 
the pharmacy manual. 
Refer to the pharmacy manual for detailed information regarding handling, storage and 
accountability of the IP. 
6.3 
Randomization and Blinding 
This is an open-label study; however, enrollment, randomization and dispensation of IP will 
be performed via the interactive response technology (IRT) system.  Prior to initiation of 
treatment, study site personnel will obtain the randomization number and treatment 
assignment from the IRT system.  Specific IRT procedures will be described in the respective 
study manual. 
6.4 
Investigational Product Compliance 
IP will be administered intravenously at the study site by study site personnel.  The exact date 
and time (in minutes) of IP administration will be documented. 
6.5 
Previous and Concomitant Treatment (Medication and 
Nonmedication Therapy) 
6.5.1 
Previous Medication 
The investigator must record the use of all previous (from day -28 or informed consent date, 
whichever is later, prior to visit 2) treatment in the eCRF, both medication and 
nonmedication treatments. 
6.5.2 
Concomitant Medication 
The investigator must record the use of all concomitant (any medication between visit 2 and 
follow-up [visit 3]) treatment in the eCRF, both medication and nonmedication treatments. 


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6.5.3 
Prohibited Medication 
The following concomitant medications will not be allowed from 48 hours prior to the IP 
administration until the completion of the surgical procedure: 
● 
ICG unless used for anastomotic evaluation 
● 
Other NIR-F imaging agents 
● 
Diuretics 
● 
Inhibitors for renal transporters defined by FDA (see [Appendix 12.6 List of Excluded 
Concomitant Medications]) 
A list of excluded concomitant medications is provided in [Appendix 12.6 List of Excluded 
Concomitant Medications]. 
6.5.4 
Alternative Therapy for Visualization 
If the investigator judges that anatomical visualization of ASP5354 is not sufficient for the 
subject at 30 minutes after administration, the investigator can proceed with surgery using 
other modalities for visualization. 
6.6 
Dose Modification 
Since this is a single dose study, dose modification is not applicable. 
6.7 
Criteria for Continuation of Treatment  
Not applicable, as ASP5354 is a single dose anatomical imaging agent and has no therapeutic 
indications. 
7 
STUDY PROCEDURES AND ASSESSMENTS 
7.1 
Efficacy Assessments 
7.1.1 
Primary Efficacy Assessments 
7.1.1.1 Anatomical Visualization of the Index Ureter(s) Using ASP5354 
The investigator will select the index ureter (i.e., right or/and left) before surgery.  The 
incidence of anatomical visualization of ureter(s) will be assessed at approximately 10, 20, 
30, 45 and 60 minutes postadministration of ASP5354, and then every 30 minutes thereafter 
and/or at the last time point before removal of visualization instruments (end of surgery) by 
the investigator intraoperatively using a binary “Yes or No” question on the ability to 
visualize the ureter: “Can the ureter be adequately visualized with NIR-F”?   
● 
If the answer to the question 30 minutes after ASP5354 administration and at the end of 
surgery = Yes, then anatomical visualization of the index ureter(s) was a success. 
● 
All other cases will be handled as “Not a success”. 


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If the anatomical visualization of the index ureter(s) was not assessed, “Not Done” will be 
entered in the eCRF.  However, in the only case that the anatomical visualization of the index 
ureter(s) was not assessed at 30 minutes after ASP5354 administration, the answer will be 
imputed as follows: 
● 
If the answer at the nearest time points before and after 30 minutes both = Yes, then the 
answer to the question 30 minutes after ASP5354 administration will be imputed 
as = Yes. 
● 
If the answer at the nearest time points before and after 30 minutes both = No, then the 
answer to the question 30 minutes after ASP5354 administration will be imputed 
as = No. 
● 
All other cases will be handled as “Not Done” at 30 minutes after ASP5354 
administration, and imputing will not occur. 
7.1.2 
Exploratory Assessments 
7.1.2.1 Intensity of Fluorescence 
The intensity of fluorescence will be qualitatively assessed during surgery by the investigator, 
by direct examination of the ureter.  A Likert Scale (0 = None, 1 = Mild, 2 = Moderate and 
3 = Strong) will be used to determine the intensity of fluorescence at each time point.  The 
contralateral ureter will also be visualized when feasible, per the investigators judgment, at 
similar time points. 
7.1.2.2 Duration of Ureter Visualization 
Intraoperative ureter fluorescence visualization will be assessed at approximately 10, 20, 30, 
45 and 60 minutes postadministration of ASP5354, and then every 30 minutes thereafter 
and/or at the last time point before removal of visualization instruments (end of surgery).  
The duration of visualization of the index ureter(s) will be based on the result from the first 
positive visualized time point to the last positive visualized time point.  Images will be 
captured on a bright field, NIR-F and overlay view (if available).  The contralateral ureter 
will also be visualized when feasible, per the investigators judgment, at similar time points.  
7.1.2.3 Signal Background Ratio 
Fluorescence images will be captured and recorded during the entire surgery and archived.  
Images will be captured on a bright field, NIR-F and overlay view (if available).  SBR will be 
calculated using fluorescence images at predose, approximately 10, 20, 30, 45 and 60 minutes 
postadministration of ASP5354, and then every 30 minutes thereafter and/or at the last time 
point before removal of visualization instruments (end of surgery), as follows 
[Hoogstins et al, 2019]: 
SBR =  
mean region of interest 
mean signal background 
Additional detail will be provided in the Imaging Charter. 


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7.1.2.4 Binary Questions (Yes/No) for the Benefit of Visualization by ASP5354 during 
Surgery 
The investigator will be asked the following questions: 
● 
Was the location of the index ureter(s) as expected (Yes or No)? 
● 
Did visualization of the index ureter(s) occur with white light at 30 minutes after 
ASP5354 administration (Yes or No)? 
● 
Did visualization of the index ureter(s) occur with white light at the end of surgery (Yes 
or No)? 
● 
Was NIR-F superior to white light in terms of visualization of the index ureter(s) (Yes or 
No)? 
● 
Did the location of the ureter visualized by NIR-F alter the operative plan (Yes or No)? 
● 
Were any ureter anomalies or abnormalities observed (Yes or No)? 
7.1.2.5 Ability to Visualize the Contralateral Ureter When Amenable to Visualization 
The contralateral ureter will be assessed using the same binary questions for anatomical 
visualization and Likert Scale, when feasible, per the investigator’s judgment, at similar time 
points. 
7.2 
Safety Assessments 
Study procedures and their timing are summarized in the Schedule of Assessments [Table 1].  
Protocol waivers or exemptions are not allowed. 
Procedures conducted as part of a subject’s routine clinical management (i.e., standard of 
care) obtained before signing the ICF may be utilized for screening or baseline purposes, 
provided the procedures met the protocol-specified criteria and were performed within the 
time frame, as defined in the Schedule of Assessments [Table 1]. 
7.2.1 
Adverse Events 
Should subjects develop a visible adverse event (AE) of the skin, such as rash or 
discoloration, photographs of the skin should be obtained, if possible, starting as soon as 
possible after first presentation of the AE.  A calibrated color card and ruler for scale will be 
included in the photograph as a reference when taking the picture so that the skin color can be 
assessed. 
Should subjects develop a hypersensitivity reaction, an additional blood sample for 
determination of histamine and tryptase concentrations should be taken as soon as possible 
after the onset of the hypersensitivity reaction. 
See [Section 7.3 Adverse Events and Other Safety Aspects] for information regarding AE 
collection and data handling. 


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7.2.2 
Laboratory Assessments 
Laboratory assessments will be performed at a local laboratory.  Blood samples will be 
collected via a peripherally or centrally placed iv cannula, an intra-arterial cannula or by 
direct venipuncture in a suitable vein. 
Blood samples for hematology (complete blood count) and serum chemistry and urine 
samples for urinalysis will be collected as indicated in the Schedule of Assessments [Table 1].  
The clinical laboratory tests to be performed in the study are listed in [Appendix 12.7 
Laboratory Assessments]. 
Drugs of abuse (e.g., amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine 
and opiates) and alcohol tests will be performed on samples collected as indicated in the 
Schedule of Assessments [Table 1].  Drugs of abuse and alcohol tests will be performed 
according to the study site’s preferred method. 
Pregnancy tests will be performed on samples collected as indicated in the Schedule of 
Assessments [Table 1].  Pregnancy tests (WOCBP only) will be performed on serum samples. 
The investigator or subinvestigator must review the laboratory report and document this 
review. 
Clinical significance of out-of-range laboratory findings is to be determined and documented 
by the investigator or subinvestigator who is a qualified physician. 
7.2.3 
Vital Signs, Height and Weight 
Blood pressure (SBP and DBP), pulse and respiratory rate measurements will be taken as 
indicated in the Schedule of Assessments [Table 1].  All vital signs will be measured with the 
subject in the sitting or supine position. 
Height and weight will be measured using standard institution practice and equipment and 
recorded as indicated in the Schedules of Assessments [Table 1]. 
7.2.4 
Physical Examination  
Physical examinations will be performed as indicated in the Schedule of Assessments 
[Table 1] and whenever there is a medical indication. 
A full physical examination will include the following body systems: general appearance, 
skin, lymphatic, head and neck, ears, nose and throat, chest and lungs, cardiovascular, 
abdomen, extremities, musculoskeletal and neuromuscular.   
On day 1 (pre- and postoperatively) and at the follow-up visit, a symptom-directed physical 
examination will be performed. 
7.2.5 
Electrocardiogram  
Routine (12-lead) ECGs will be taken as indicated in the Schedule of Assessments [Table 1].  
Routine 12-lead ECGs will be taken after the subject has been resting in the supine position.  
Routine 12-lead ECGs will be taken in triplicate. 


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The investigator will review, sign and date the ECG after recording to ensure subject safety.  
The time of the ECG, the interval measurements, as well as an overall conclusion, will be 
documented.  This overall conclusion will be recorded as normal, abnormal not clinically 
significant or abnormal clinically significant in the eCRF.  Any clinically significant ECG 
abnormalities should be recorded as an AE. 
The anonymized ECG recordings will be printed, signed and saved in the source.  
Alternatively, per time point, the ECGs can be stored electronically and reviewed in a timely 
manner by the investigator. 
7.2.6 
Order of Assessments  
The following order should be followed when more than 1 assessment is required at a time 
point: 
1. 12-lead ECG 
2. Vital signs 
3. Blood or urine collection for pharmacokinetics 
4. Blood sampling for clinical laboratory tests 
7.3 
Adverse Events and Other Safety Aspects 
The definitions of an AE or SAE can be found in [Appendix 12.4 Adverse Events: 
Definitions and Procedures for Recording, Evaluating, Follow-up or Reporting]. 
The investigator and medically qualified designee(s) are responsible for detecting, 
documenting and recording events that met the definition of an AE or SAE. 
7.3.1 
Time Period for Collecting Adverse Event and Serious Adverse Event 
Information 
In order to identify any events that may be associated with study procedures and could lead to 
a change in the conduct of the study, Astellas collects AEs even if the subject has not 
received IP.  AE collection begins after the signing of the ICF and will be collected through 
to the last clinical study protocol-defined assessment or when the subject is determined to be 
a screen failure. 
7.3.2 
Method of Detecting Adverse Events and Serious Adverse Events 
The methods of recording, evaluating and assessing seriousness, causality and severity of 
AEs and SAEs are described in [Appendix 12.4 Adverse Events: Definitions and Procedures 
for Recording, Evaluating, Follow-up or Reporting].  Care will be taken not to introduce bias 
when detecting AEs and/or SAEs.  Open-ended and nonleading verbal questioning of the 
subject is the preferred method to inquire about AE occurrences. 
An AE with a change in severity is recorded as a new AE. 


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7.3.3 
Follow-up of Adverse Events 
All AEs occurring during or after the subject has discontinued the study are to be followed up 
until resolved or judged to be no longer clinically significant, or until they become chronic to 
the extent that they can be fully characterized by the investigator. 
If after the protocol-defined AE collection period (see [Section 7.3.1 Time Period for 
Collecting Adverse Event and Serious Adverse Event Information]), an AE progresses to an 
SAE, or the investigator learns of any (S)AE (serious adverse event or adverse event) 
including death, where he/she considers there is reasonable possibility it is related to the IP or 
study participation, the investigator must promptly notify the sponsor. 
7.3.4 
Reporting of Serious Adverse Events 
Prompt notification by the investigator to the sponsor of an SAE is essential, so that legal 
obligations and ethical responsibilities towards the safety of subjects and the safety of a study 
intervention under clinical investigation are met. 
In the case of an SAE, the investigator must contact the sponsor by fax or email immediately 
(within 24 hours of awareness). 
Procedures for reporting SAEs to the sponsor are described in [Section 12.4.5 Reporting 
Procedures for Serious Adverse Events]. 
7.3.5 
Disease-related Events and/or Disease-related Outcomes Not Qualifying as 
Adverse Events or Serious Adverse Events 
Green discoloration of the urine will not be considered an AE as this is an expected, known 
effect of short duration without any expected untoward clinical symptoms; however, this still 
needs to be recorded. 
Under this protocol, the following event will not be considered as an (S)AE: 
● 
Preplanned and elective hospital/clinical procedures/interventions or procedures for 
diagnostic, therapeutic or surgical procedures for a preexisting condition that did not 
worsen during the course of the study.  These procedures are collected in the eCRF. 
7.3.6 
Special Situations  
Certain special situations observed in association with the IP, such as incorrect administration 
(e.g., wrong dose of IP or background therapy) are collected in the eCRF, as protocol 
deviations per [Section 10.3 Major Protocol Deviations] or may require special reporting, as 
described below.  These special situations are not considered AEs, but do require to be 
communicated to Astellas as per the timelines defined below. 
If a special situation is associated with, or results in, an AE, the AE is to be assessed 
separately from the special situation and captured as an AE in the eCRF.  If the AE meets the 
definition of an SAE, the SAE is to be reported as described in [Section 12.4.5 Reporting 
Procedures for Serious Adverse Events] and the details of the associated special situation are 
to be included in the clinical description on the SAE worksheet. 


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The special situations are: 
● 
Pregnancy  
● 
Medication error, overdose and use outside protocol 
● 
Misuse/abuse 
● 
Occupational exposure 
● 
(Suspicion of) Transmission of infectious agent 
● 
Suspected drug-drug interaction 
Instructions and procedures for reporting special situations are provided in [Section 12.4.6 
Reporting Procedures for Special Situations]. 
7.3.7 
Supply of New Information Affecting the Conduct of the Study 
When new information becomes available that is necessary for conducting the study properly, 
the sponsor will inform all investigators involved in the study as well as the appropriate 
regulatory authorities.  Investigators should inform the IRB/IEC of such information when 
needed. 
The investigator will also inform the subjects, who will be required to sign an updated ICF in 
order to continue in the study. 
7.3.8 
Urgent Safety Measures 
An urgent safety measure (USM) is an intervention that is not defined by the protocol and 
can be put in place with immediate effect without needing to gain prior approval by the 
sponsor, relevant competent authorities (CA) or IRB/IEC, where applicable, in order to 
protect subjects from any immediate hazard to their health and/or safety.  Either the 
investigator or the sponsor can initiate a USM.  The cause of a USM can be safety-, product- 
or procedure-related. 
7.3.9 
Reporting Urgent Safety Measures 
In the event of a potential USM, the investigator must contact the study physician (within 
24 hours of awareness).  Full details of the potential USM are to be recorded in the subject’s 
medical records.  The sponsor may request additional information related to the event to 
support their evaluation. 
If the event is confirmed to be a USM, the sponsor will take appropriate action to ensure the 
safety and welfare of the subjects.  These actions may include but are not limited to a change 
in study procedures or study treatment, halting further enrollment in the study, or stopping 
the study in its entirety.  The sponsor or sponsor's designee will notify the relevant CA and 
concerned ethics committee within the timelines required per current local regulations, and 
will inform the investigators, as required.  When required, investigators must notify their 
IRB/IEC within timelines set by regional regulations. 


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7.4 
Pharmacokinetics 
7.4.1 
Analysis of ASP5354 in Plasma 
Blood samples for the analysis of ASP5354 in plasma will be collected as indicated in the 
Schedule of Assessments [Table 1] and Sample Collection and Timed Procedure Schedule 
[Table 2]. 
Blood samples will be collected via a peripherally or centrally placed iv cannula, an 
intra-arterial cannula or by direct venipuncture in a suitable vein.  Pharmacokinetic sample 
collection should not be taken from the same cannula used for the administration of the IP.  
Blood sampling, processing, storage and shipment instructions are provided in a Sample 
Handling and Processing Manual. 
When deemed appropriate at a later date, plasma samples remaining after the 
pharmacokinetic analysis may be used for exploratory metabolic profiling or exploratory 
biomarker analysis after the study.  These tests will be described in a separate report and will 
not be incorporated in the integrated clinical study report. 
7.4.2 
Analysis of ASP5354 in Urine 
Urine point samples for the analysis of ASP5354 in urine will be collected from the urine that 
collects in the catheter tubing at the time points indicated in the Schedule of Assessments 
[Table 1] and Sample Collection and Timed Procedure Schedule [Table 2].  The urine sample 
for the amount of ASP5354 excreted in urine during surgery will be collected from the urine 
in the catheter bag after the end of surgery. 
A urinary catheter will be inserted depending on the surgery procedure.  While the catheter is 
in place, urine will be collected in a catheter bag.  At the time of point urine collection, the 
catheter bag will be disconnected and the tubing will be allowed to fill with fresh urine.  The 
point of urine collection will be obtained from the urine that collects in the catheter tubing.  
Aliquots of at least 0.2 mL will be collected in polypropylene vials for the urine point 
samples.  The timing of urine point collections during the period of catheterization is detailed 
in Table 2.  Total urine volume of urine in the catheter bag will be calculated by dividing the 
weight of urine in the catheter bag by predefined density.  Urine sample for pharmacokinetics 
of the IP and possible metabolite(s) (if applicable) will be collected from the urine in the 
catheter bag. 
Urine sampling, calculation, processing, storage and shipment instructions are provided in a 
Sample Handling and Processing Manual. 
When deemed appropriate at a later date, urine samples remaining after the pharmacokinetic 
analysis may be used for exploratory metabolic profiling or exploratory biomarker analysis 
after the study.  These tests will be described in a separate report and will not be incorporated 
in the integrated clinical study report. 


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7.5 
Pharmacodynamics 
Pharmacodynamic variables will include the primary endpoint (anatomical visualization of 
the index ureter) and exploratory efficacy endpoints (Likert Scale, visualization duration and 
SBR [Section 7.1 Efficacy Assessments]). 
7.6 
Electronic Clinical Outcome Assessment 
Not applicable. 
7.7 
Other Assessments 
7.7.1 
Green Discoloration of Urine 
The incidence of green discoloration of the urine will be recorded any time after dose 
administration.  If it occurs, the color of urine will be assessed during surgery and every 
120 minutes (± 30 minute window) after the end of surgery until it resolves or the subject is 
discharged, whichever is earlier.  The start and stop date and time will also be recorded.  
7.8 
Total Amount of Blood 
The total amount of blood drawn for each subject will vary depending on the length of the 
surgery.  At any time during the study, if any laboratory abnormalities are found for a subject, 
additional blood may be drawn for safety monitoring.  The average expected blood draw is 
shown in Table 5. 
Table 5 
Blood Sampling 
Sample Type 
Number of Samples 
Sample Volume (mL) 
Total Volume (mL) 
Serum chemistry  
4 
8.5 
34.0 
Hematology 
4 
4.0 
16.0 
ASP5354 pharmacokinetic profiling 
4a 
4.0 
16.0 
Total 
66.0 
a 
The number of samples for pharmacokinetic profiling will depend on the duration of the surgery.  E.g., if 
surgery lasts for 120 minutes, samples will be taken pre-dose, 10 minutes postdose, either 30 or 60 minutes 
postdose and at the end of surgery. 
8 
DISCONTINUATION 
8.1 
Discontinuation of Individual Subject(s) From Study Treatment  
Since this is a single dose study, treatment discontinuation is not applicable. 
8.2 
Discontinuation of Individual Subject(s) From Study 
A discontinuation from the study is defined as a subject who enrolled in the study and for 
whom the study is permanently discontinued for any reason.   
The subject is free to withdraw from the study for any reason and at any time without giving 
reason for doing so and without penalty or prejudice.  The investigator is also free to 
terminate a subject’s involvement in the study at any time if the subject’s clinical condition 
warrants it. 


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The reason for discontinuation from the study must be documented in the subject’s medical 
records. 
A subject must discontinue the study for any of the following reasons: 
● 
Subject requests to stop the study.  
● 
Any clinical AE, laboratory abnormality or intercurrent illness that, in the opinion of the 
investigator, indicates to continue in the study is not in the best interest of the subject. 
● 
Subject signs informed consent, but surgery is performed without administration of 
ASP5354. 
8.2.1 
Lost to Follow-up 
Every reasonable effort is to be made to contact any subject lost to follow-up during the 
course of the study to complete study-related assessments and record outstanding data. 
8.3 
Discontinuation of the Study Site 
If an investigator intends to discontinue participation in the study, the investigator must 
immediately inform the sponsor. 
8.4 
Discontinuation of the Study 
The sponsor may terminate this study prematurely, either in its entirety or at any study site, 
for reasonable cause, including optimal dose determination, provided that written notice is 
submitted in advance of the intended termination.  Advance notice is not required if the study 
is stopped due to safety concerns.  If the sponsor terminates the study for safety reasons, the 
sponsor will immediately notify the investigator and subsequently provide written 
instructions for study termination. 
9 
STATISTICAL METHODOLOGY 
A statistical analysis plan (SAP) will be written to provide details of the analysis, along with 
specifications for tables, listings and figures to be produced.  The SAP will be finalized 
before first subject screened.  Changes from the planned analyses in the final SAP that impact 
the statistical analyses will be justified in the clinical study report. 
In general, continuous data will be summarized with descriptive statistics (number of 
subjects, mean, SD, minimum, median and maximum), frequency and percentage for 
categorical data. For analysis by dose group, unless specified otherwise, subjects are grouped 
by the dose received for those randomized before determination of optimal dose, and a 
separate group for additional subjects enrolled after determination of optimal dose.  
9.1 
Sample Size 
This sample size is not based on statistical power calculation.  The sample size is expected to 
provide adequate information to determine the optimal dose of ASP5354 for ureter 
visualization. 


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9.2 
Analysis Sets 
The allocation of subjects to analysis sets, except the pharmacokinetic analysis set (PKAS), 
will be determined prior to database hard-lock. 
9.2.1 
Full Analysis Set 
The full analysis set (FAS) will consist of all subjects who receive ASP5354 and have at least 
1 assessment of ureter visualization during surgery.  This will be the primary analysis set for 
efficacy analyses. 
9.2.2 
Safety Analysis Set 
The SAF consists of all subjects who receive ASP5354.  The SAF will be used for all 
summaries of the safety data, unless otherwise specified. 
9.2.3 
Pharmacokinetic Analysis Set 
The PKAS consists of all subjects who receive at least 1 dose of IP for which at least 
1 plasma or urine concentration data are available with the time of dosing and sampling.  
Inclusion of subjects in the PKAS with major protocol deviations will be considered by the 
pharmacokineticist on a case-by-case basis. 
The PKAS will be used for all summaries of the pharmacokinetic data. 
9.3 
Demographics and Baseline Characteristics  
Demographics and other baseline characteristics will be summarized by descriptive statistics 
by dose group and overall for the FAS and SAF. 
9.3.1 
Subject Disposition  
The number and percentage of subjects who completed and discontinued screening period 
and reasons for discontinuation will be presented for all subjects who signed the ICF.  A 
similar table for study disposition will also be presented by dose group and overall for the 
FAS and SAF.  All disposition details and dates of first and last evaluations for each subject 
will be listed. 
9.3.2 
Previous and Concomitant Treatment (Medication and Nonmedication 
Therapy)  
All previous and concomitant treatment will be presented in a listing. 
9.3.3 
Medical History  
Medical history will be coded using MedDRA and will be summarized by SOC and preferred 
term (PT) by dose group and overall for the SAF. 
Medical history for each subject will be listed. 
9.3.4 
Investigational Product Exposure 
Since this is a single dose study, the analysis of duration of exposure is not applicable. Study 
drug dosing information will be listed. 


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9.4 
Analysis of Efficacy  
Efficacy analysis will be conducted on the FAS.  No hypothesis testing will be performed. 
9.4.1 
Analysis of Primary Endpoint 
9.4.1.1 Primary Analysis 
The primary efficacy endpoint, the success rate of anatomical visualization of the index 
ureter(s) at 30 minutes after dosing and end of surgery, will be estimated with exact 95% 
confidence interval (CI) by dose group. 
9.4.1.2 Secondary Analysis 
The frequency and percentage of anatomical visualization of the index ureter(s) will be 
summarized by dose group and time point including 30 minutes after dosing and end of 
surgery. 
9.4.1.3 Subgroup Analysis 
No specific subgroup analysis is planned. 
9.4.2 
Analysis of Secondary Endpoints 
Since no efficacy endpoint is specified as secondary endpoint, it is not applicable. 
9.4.3 
Analysis of Exploratory Endpoints 
The Likert Scale of the intensity of the fluorescence will be summarized by frequency and 
percentage by dose group and time point for index ureter(s), and if applicable, for the 
contralateral ureter. 
The duration of anatomical visualization of the index ureter(s) will be summarized using 
descriptive statistics by dose group. 
The SBR for index ureter(s) will be summarized using descriptive statistics by dose group 
and time point. 
The binary questions for the benefit of visualization during surgery will be summarized by 
frequency and percentage by dose group. 
The success rate of anatomical visualization of the contralateral ureter at 30 minutes after 
dosing and end of surgery will be estimated with exact 95% CI by dose group, if applicable.  
In addition, the frequency and percentage of anatomical visualization of the contralateral 
ureter will be summarized by dose group and time point, if applicable. 
9.5 
Analysis of Safety 
Safety analyses will be conducted using the SAF, unless otherwise specified.  No hypothesis 
testing will be performed. 
9.5.1 
Adverse Events  
AEs will be coded using MedDRA and graded using the National Cancer Institute-common 
terminology criteria for AE (NCI-CTCAE, version 5.0). 


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A TEAE is defined as an AE observed after administration of the IP and up to the follow-up 
period.  An IP-related TEAE is defined as any TEAE with a causal relationship assessed as 
“yes” by the investigator. 
The number and percentage of subjects with TEAEs, IP-related TEAEs, serious TEAEs, 
IP-related serious TEAEs will be summarized by SOC, PT and dose group.  The number and 
percentage of TEAEs by toxicity grade will also be summarized.  The worst toxicity grade 
will be summarized if the same AE is recorded more than once for a subject. 
AE data will be listed. 
9.5.2 
Laboratory Assessments  
For quantitative clinical laboratory measurements, descriptive statistics will be used to 
summarize results and change from baseline by dose group and time point. 
Shift from baseline to the postbaseline worst grade based on NCI-CTCAE (version 5.0) until 
the follow-up period in laboratory tests will be tabulated. 
Laboratory data will be listed. 
9.5.3 
Vital Signs  
Descriptive statistics will be used to summarize vital sign results and changes from baseline 
by dose group and time point. 
Vital signs data will be listed. 
9.5.4 
Physical Examination  
Not applicable. 
9.5.5 
Electrocardiogram 
9.5.5.1 Routine 12-lead Electrocardiogram 
The routine 12-lead ECG results will be summarized by dose group and time point. 
Routine 12-lead ECG data and interpretations will be listed. 
9.6 
Analysis of Pharmacokinetics 
Descriptive statistics will be used to summarize plasma and urine concentrations of ASP5354 
and pharmacokinetic parameters by dose group and time point.  All concentrations and 
pharmacokinetic parameters will be listed. 
9.6.1 
Estimation of Pharmacokinetic Parameters  
Since the volume of urine point sample is very small, the volume of urine in the catheter bag 
will be used as the total urine volume.  The following pharmacokinetic parameters will be 
calculated using the urine collection data during surgery: 
● 
Amount of ASP5354 excreted in urine (Ae) during surgery 
● 
Percent of drug dose excreted into urine (Ae%) during surgery 


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Other pharmacokinetic parameters will be estimated by a population pharmacokinetics 
approach using concentrations of ASP5354.  All details of population analyses will be 
described in a separate analysis plan and a separate report will be written. 
9.7 
Analysis of Pharmacodynamics 
Pharmacodynamic analyses will be conducted on the FAS as efficacy analyses [Section 9.4 
Analysis of Efficacy]. 
The relationship between pharmacokinetics and anatomical visualization or other exploratory 
endpoints will be explored by a population pharmacokinetics/pharmacodynamics approach.  
All details of population analyses will be described in a separate analysis plan and a separate 
report will be written.  
9.8 
Other Analyses 
9.8.1 
Green Discoloration of Urine 
The frequency and percentage of patients who experience green discoloration of urine will be 
summarized by dose group.  The duration of green discoloration will be summarized using 
descriptive statistics by dose group. 
9.9 
Major Protocol Deviations 
Major protocol deviations as defined in [Section 10.3 Major Protocol Deviations] will be 
summarized for all randomized subjects by dose group, by study site and overall. 
Major protocol deviation data will be listed by subject. 
The major protocol deviation criteria will be uniquely identified in the summary table and 
listing. 
9.10 
Interim Analysis (and Early Discontinuation of the Study) 
The determination of dose(s) to stop, continue or define a dose as optimal will occur when 
every 3 subjects randomly assigned to each dose level have the data for anatomical 
visualization during surgery reviewed via the VRC.  The VRC will be held to review subject 
data until an optimal dose is determined. 
9.11 
Additional Conventions 
As a general principle, no imputation of missing data will be done.  Exceptions are the start 
and stop dates of AEs and concomitant medications if they are missing. 
For the primary efficacy endpoint of anatomical visualization at 30 minutes after dosing and 
end of surgery, imputation will be performed when the assessment is not done at 30 minutes 
after dosing. 
See the SAP for details of the definitions for analysis windows to be used for analyses by 
time point. 


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10 
OPERATIONAL CONSIDERATIONS 
10.1 
Data Collection 
The investigator or site designee will enter data collected using an electronic data capture 
system.  In the interest of collecting data in the most efficient manner, the investigator or 
designee should record data (including clinical laboratory values, if applicable) in the eCRF 
within 5 days after the subject’s visit. 
The investigator or site designee is responsible to ensure that all data in the eCRFs and 
queries are accurate and complete and that all entries are verifiable with the source.  These 
documents should be appropriately maintained by the study site. 
The monitor should verify the data in the eCRFs with the source and confirm that there are no 
inconsistencies among them. 
Clinical laboratory tests are performed at the local laboratory.   
10.2 
Demographics and Baseline Characteristics 
10.2.1 Demographics 
Demographic information will be collected for all subjects at screening and will include age, 
sex, race, ethnicity, weight, height and body mass index.  
10.2.2 Medical History 
Medical history will be collected at screening in order to assess inclusion/exclusion criteria. 
10.2.3 Surgical Procedure and Indication 
Details on surgical procedure and indication will be collected at screening in order to assess 
inclusion/exclusion criteria. 
10.3 
Major Protocol Deviations 
A protocol deviation is generally an unplanned excursion from the protocol that is not 
implemented or intended as a systematic change.  All deviations from the protocol are to be 
recorded.  A protocol waiver is a documented prospective approval of a request from an 
investigator to deviate from the protocol.  Protocol waivers are strictly prohibited. 
The investigator is responsible for ensuring the study is conducted in accordance with the 
procedures and evaluations described in this protocol and must protect the rights, safety and 
well-being of subjects.  The investigator should not implement any deviation from, or 
changes of, the protocol, unless it is necessary to eliminate an immediate hazard to subjects. 
A major protocol deviation is one that may potentially impact the completeness, accuracy or 
reliability of data contributing to the primary endpoint or affect the rights, safety or 
well-being of a subject.  Major protocol deviations will have additional reporting 
requirements. 
When a major deviation from the protocol is identified for an individual subject, the 
investigator or designee must ensure the sponsor is notified.  The sponsor will follow up with 


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the investigator, as applicable, to assess the deviation and the possible impact to the safety 
and/or efficacy and/or pharmacokinetic parameters of the subject to determine subject 
continuation in the study. 
The major protocol deviation criteria that will be summarized at the end of the study are as 
follows: 
Protocol Deviation 1 - Entered into the study even though the subject did not satisfy entry 
criteria  
Protocol Deviation 2 - Developed withdrawal criteria during the study and was not 
withdrawn 
Protocol Deviation 3 - Received wrong treatment or incorrect dose 
Protocol Deviation 4 - Received excluded concomitant treatment 
The investigator will also assure that deviations meeting IRB/IEC and appropriate regulatory 
authorities’ criteria are documented and communicated appropriately.  All documentation and 
communications to the IRB/IEC and appropriate regulatory authorities will be provided to the 
sponsor and maintained within the Trial Master File. 
10.4 
Study Organization 
10.4.1 Visualization Review Committee 
The visualization data will be assessed by the principal investigator/investigator and the 
sponsor’s medical representative, who form the VRC.  The VRC will be held at the following 
time points, but ad-hoc meetings may occur until the optimal dose is determined: 1) after the 
initial 3 subjects have completed the surgical procedures at each of the dose levels, and 
2) then in increments of 3 subjects (up to a total of 15 subjects per dose level) for those who 
have completed the surgical procedures at each of the expanded dose levels.  For further 
details on the VRC, refer to the VRC charter.  


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11 
REFERENCES  
Burks FN and Santucci RA. Management of iatrogenic ureteral injury. Ther Adv Urol. 2014;6:115-24. 
Halabi WJ, Jafari MD, Nguyen VQ, Carmichael JC, Mills S, Pigazzi A, et al. Ureteral injuries in 
colorectal surgery: an analysis of trends, outcomes, and risk factors over a 10-year period in the 
United States. Dis Colon Rectum. 2014;57:179-86. 
Hoogstins C, Burggraaf JJ, Koller M, Handgraaf H, Boogerd L, van Dam G, et al.  Setting standards for 
reporting and quantification in fluorescence-guided surgery.  Mol Imaging Biol. 2019;21:11-8. 
Packlam VT, Cohen AJ, Pariser JJ, Nottingham CU, Faris SF, Bales GT.  The impact of minimally 
invasive surgery on major iatrogenic ureteral injury and subsequent ureteral repair during 
hysterectomy: a national analysis of risk factors and outcomes.  Urology. 2016;98:183-8. 
Slooter MD, Janssen A, Bemelman WA, Tanis PJ, Hompes R. Currently available and experimental dyes 
for intraoperative near-infrared fluorescence imaging of the ureters: a systematic review. Tech 
Coloproctol. 2019;23(4):305-13. 
Verbeek FP, van der Vorst JR, Schaafsma BE, Swijnenburg RJ, Gaarenstroom KN, et al. Intraoperative 
near infrared fluorescence guided identification of the ureters using low dose methylene blue: a 
first in human experience. J Urol. 2013;190(2):574-9. 
Wallis CJ, Cheung DC, Garbens A, Kroft J, Carr L, Nathens AB, et al. Occurrence of and risk factors for 
urological intervention during benign hysterectomy: analysis of the national surgical quality 
improvement program database.  Urology. 2016;97:66-72. 
Yeung TM, Volpi D, Tullis ID, Nicholson GA, Buchs N, Cunningham C, et al. Identifying ureters in situ 
under fluorescence during laparoscopic and open colorectal surgery. Ann Surg. 2016;263(1):e1-2. 


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12 
APPENDICES 
12.1 
Ethical, Regulatory and Study Oversight Considerations 
12.1.1 Ethical Conduct of the Study 
The study will be conducted in accordance with the protocol, ICH guidelines, applicable 
regulations and guidelines governing study conduct and the ethical principles that have their 
origin in the Declaration of Helsinki. 
12.1.2 Institutional Review Board/Independent Ethics Committee/Competent 
Authorities 
GCP requires that the protocol, any protocol amendments, IB, ICF and all other forms of 
subject information related to the study (e.g., advertisements used to recruit subjects) and any 
other necessary documents be reviewed by an IRB/IEC.  The IRB/IEC will review the 
ethical, scientific and medical appropriateness of the study before it is conducted.  IRB/IEC 
approval of the protocol, ICF and subject information and/or advertising, as relevant, will be 
obtained prior to initiation of any study-specific procedures. 
Any substantial amendments to the protocol will require CA and IRB/IEC approval before 
implementation, except for changes necessary to eliminate an immediate hazard to subjects. 
The investigator will be responsible for the following: 
● 
Providing written summaries of the status of the study to the IRB/IEC annually or more 
frequently in accordance with the requirements, policies and procedures established by 
the IRB/IEC. 
● 
Notifying the IRB/IEC of SAEs or other significant safety findings as required by 
IRB/IEC procedures. 
● 
Providing oversight of the conduct of the study at the study site and adherence to 
requirements of 21 Code of Federal Regulations, ICH guidelines, the IRB/IEC, EU 
Regulation No. 536/2014 for studies (if applicable), and all other applicable local 
regulations. 
12.1.3 Protocol Amendment and/or Revision 
Any changes to the study that arise after approval of the protocol must be documented as 
protocol amendments: substantial amendments and/or nonsubstantial amendments.  
Depending on the nature of the amendment, either IRB/IEC or CA approval or notification 
may be required.  The changes will become effective only after the approval of the sponsor, 
investigator, IRB/IEC and appropriate regulatory authorities. 
Amendments to this protocol must be signed by the sponsor and investigator.  Written 
verification of IRB/IEC approval will be obtained before any amendment is implemented.  
Modifications to the protocol that are administrative in nature do not require IRB/IEC 
approval, but will be submitted to the IRB/IEC for their information, if required by local 
regulations. 


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If there are changes to the ICF, written verification of IRB/IEC approval must be forwarded 
to the sponsor.  An approved copy of the new ICF must also be forwarded to the sponsor. 
12.1.4 Financial Disclosure 
Investigators and subinvestigators will provide the sponsor with sufficient, accurate financial 
information as requested to allow the sponsor to submit complete and accurate financial 
certification or disclosure statements to the appropriate regulatory authorities.  Investigators 
are responsible for providing information on financial interests during the course of the study 
and for 1 year after completion of the study. 
12.1.5 Informed Consent of Subjects 
12.1.5.1 Subject Information and Consent 
The investigator or his/her representative will explain the nature of the study to the subject 
and answer all questions regarding this study.  Prior to any study-related screening 
procedures being performed on the subject, the ICF will be reviewed, signed and dated by the 
subject, the person who administered the ICF and any other signatories according to local 
requirements.  A copy of the signed ICF will be given to the subject and the original will be 
placed in the subject’s medical record.  An entry must also be made in the subject’s dated 
source documents to confirm that the ICF was signed prior to any study-related procedures 
and that the subject received a signed copy of the ICF. 
The signed ICFs will be retained by the investigator and made available (for review only) to 
the study monitor, auditor and appropriate regulatory authorities and other applicable 
individuals upon request. 
12.1.5.2 Supply of New and Important Information Influencing the Subject’s Consent 
and Revision of the Written Information 
1. The investigator or his/her representative will immediately inform the subject verbally 
whenever new information becomes available that may be relevant to the subject’s 
consent or may influence the subject’s willingness to continue participating in the study 
(e.g., report of serious adverse drug reaction).  The communication must be documented 
in the subject’s medical records and whether the subject is willing to remain in the study 
or not must be confirmed and documented. 
2. The investigator must update the subject’s ICF and submit it for approval to the 
IRB/IEC.  The investigator or his/her representative must obtain written informed 
consent from the subject on all updated ICFs throughout their participation in the study.  
The investigator or his/her designee must reconsent subjects with the updated ICF even if 
relevant information was provided verbally.  The investigator or his/her representative 
who obtained the written informed consent and the subject should sign and date the ICF.  
A copy of the signed ICF will be given to the subject and the original will be placed in 
the subject’s medical record.  An entry must be made in the subject’s records 
documenting the reconsent process. 


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12.1.6 Source Documents 
Source data must be available at the study site to document the existence of the subjects and 
to substantiate the integrity of study data collected.  Source data must include the original 
documents relating to the study, as well as the medical treatment and medical history of the 
subject. 
The investigator is responsible for ensuring the source data are attributable, legible, 
contemporaneous, original, accurate and complete whether the data are handwritten on paper 
or entered electronically.  If source data are created (first entered), modified, maintained, 
achieved, retrieved or transmitted electronically via computerized systems (and/or other kinds 
of electronic devices) as part of regulated study activities, such systems must be compliant 
with all applicable laws and regulations governing use of electronic records and/or electronic 
signatures.  Such systems may include, but are not limited to, electronic medical/health 
records, protocol-related assessments, AE tracking, electronic clinical outcome assessment 
(eCOA) and/or drug accountability. 
Paper records from electronic systems used in place of electronic format must be certified 
copies.  A certified copy must be an exact copy and must have all the same attributes and 
information as the original.  Certified copies must include signature and date of the individual 
completing the certification.  Certified copies must be a complete and chronological set of 
study records (including notes, attachments, and audit trail information, if applicable).  All 
printed records must be kept in the subject file and be available for archiving. 
12.1.7 Record Retention 
The investigator will archive all study data (e.g., subject identification code list, source data 
eCRFs and investigator's file) and relevant correspondence.  These documents are to be kept 
on file for the appropriate term determined by local regulation (for US study sites, 2 years 
after approval of the NDA or discontinuation of the IND).  The sponsor will notify the study 
site/investigator if the NDA/MAA/J-NDA is approved or if the IND/IMPD/CHIKEN 
TODOKE is discontinued.  The investigator agrees to obtain the sponsor's agreement prior to 
disposal, moving or transferring of any study-related records.  The sponsor will archive and 
retain all documents pertaining to the study according to local regulations. 
Data generated by the methods described in the protocol will be recorded in the subject’s 
medical records and/or study progress notes. 
The documents of the VRC (minutes and SOPs and others) and the judgment committee 
outside the study sites (minutes and SOPs and others) shall be retained by the sponsor. 
12.1.8 Subject Confidentiality and Privacy 
Individual subject medical information obtained as a result of this study is considered 
confidential and disclosure to third parties is prohibited unless the subject provides written 
consent or approval.  Additional medical information may be given only after approval of the 
subject to the investigator or to other appropriate medical personnel responsible for the 
subject's well-being.  


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The sponsor shall not disclose any confidential information on subjects obtained during the 
performance of their duties in the study without justifiable reasons. 
Even though any individuals involved in the study, including the study monitors and auditors, 
may get to know matters related to a subject’s privacy due to direct access to source 
documents, or from other sources, they may not disclose the content to third parties. 
The sponsor affirms the subject's right to protection against invasion of privacy.  Only a 
subject identification number will identify subject data retrieved by the sponsor.  However, 
the sponsor requires the investigator to permit the sponsor, sponsor's representative(s), the 
IRB/IEC and when necessary, representatives of the regulatory health authorities to review 
and/or to copy any medical records relevant to the study. 
The sponsor agrees to comply and process personal data in accordance with all applicable 
privacy laws and regulations, including, without limitation, the Personal Information 
Protection Law in Japan and privacy laws in the US.  If the services will involve the 
collection or processing of personal data (as defined by applicable data protection legislation) 
within the European Economic Area (EEA), then the sponsor shall serve as the controller of 
such data, as defined by the EU Data Protection Directive (DPD), and investigator and/or 
third party shall act only under the instructions of the sponsor in regard to personal data.  If 
the sponsor is not based in the EEA, the sponsor must appoint a third party to act as its local 
data protection representative or arrange for a co-controller established in the EU for data 
protection purposes in order to comply with the DPD. 
12.1.9 Arrangement for Use of Information and Publication of the Study 
Information concerning the test product, patent applications, processes, unpublished scientific 
data, the IB and other pertinent information is confidential and remains the property of the 
sponsor.  Details should be disclosed only to the persons involved in the approval or conduct 
of the study.  The investigator may use this information for the purpose of the study only.  It 
is understood by the investigator that the sponsor will use the information obtained during the 
study in connection with the development of the drug and therefore may disclose it as 
required to other clinical investigators or to regulatory agencies.  In order to allow for the use 
of the information derived from this study, the investigator understands that he/she has an 
obligation to provide the sponsor with all data obtained during the study. 
Publication of the study results is discussed in the study agreement. 
12.1.10 Signatory Investigator for Clinical Study Report 
ICH E3 guidelines recommend and EU Directive 2001/83/EC requires that a final clinical 
study report that forms part of a marketing authorization application, be signed by the 
representative for the coordinating investigator(s) or the principal investigator(s).  The 
representative for the coordinating investigator(s) or the principal investigator(s) will have 
the responsibility to review the final study results to confirm to the best of his/her knowledge 
it accurately describes the conduct and results of the study.  The representative for the 
coordinating investigator(s) or the principal investigator(s) will be selected from the 
participating investigators by the sponsor prior to database hard-lock.  


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12.2 
Procedure for Study Quality Control 
12.2.1 Study Monitoring 
The sponsor or delegated contract research organization (CRO) is responsible for monitoring 
the study to ensure that the rights, safety and well-being of subjects are protected, the study is 
properly conducted in adherence to the current protocol and GCP and the study data reported 
by the investigator/subinvestigator are accurate, complete and verifiable with the source.  The 
sponsor is responsible for assigning the study monitor(s) to this study for proper monitoring.  
They will monitor the study in accordance with planned monitoring procedures. 
12.2.2 Direct Access to Source Data/Documents 
The investigator and the study site must accept monitoring and auditing by the sponsor or 
delegated CRO, as well as inspections from the IRB/IEC and appropriate regulatory 
authorities.  In these instances, they must provide all study-related records including source 
documents when they are requested by the sponsor monitors and auditors, the CRO, the 
IRB/IEC or appropriate regulatory authorities.  The confidentiality of the subject’s identity 
shall be well protected consistent with local and national regulations when the source 
documents are subject to direct access. 
12.2.3 Data Management 
Data management will be coordinated by the Data Science department of the sponsor in 
accordance with the SOPs for data management.  All study-specific processes and definitions 
will be documented by data management. eCRF completion will be described in the eCRF 
instructions.  Coding of medical terms and medications will be performed using MedDRA 
and the WHO Drug Dictionary, respectively. 
12.2.4 Quality Assurance 
The sponsor is implementing and maintaining quality assurance (QA) and quality control 
(QC) systems with written SOPs to ensure that studies are conducted and data are generated, 
documented, recorded and reported in compliance with the protocol, GCP and applicable 
regulatory requirement(s).  Where applicable, the QA and QC systems and written SOPs of 
the CRO will be applied. 
The sponsor or sponsor’s designee may arrange to audit the study at any or all study sites and 
facilities.  The audit may include on-site review of regulatory documents, eCRFs and source 
documents.  Direct access to these documents will be required by the auditors. 
To support quality around subject safety and reliability of study results, quality tolerance 
limits (QTLs) are defined and monitored.  QTLs represent the acceptable variation of study 
data, taking into consideration the current state of medical and statistical knowledge about the 
variables to be analyzed as well as the statistical design of the study.  It is a level point, or 
value associated with a parameter that should trigger an evaluation if a deviation is detected 
to determine if there is a possible systematic issue (i.e., a trend has occurred).  The QTLs 
defined for this study are provided below.  


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Table 6 
Quality Tolerance Limit 
QTL #: Name and Parameter 
Definition 
Parameter Justification 
QTL1: Sufficient data for 
assessment of anatomical 
visualization of the index ureter(s): 
% missing data of anatomical 
visualization of the index ureter at 
30 minutes after dosing ASP5354 
and/or end of surgery. 
Number of subjects who missed 
assessment of anatomical 
visualization of the index ureter(s) 
at 30 minutes after dosing 
ASP5354 and/or end of surgery per 
total number of randomized 
subjects (%). 
Since anatomical visualization of 
the index ureter(s) at 30 minutes 
after dosing ASP5354 and end of 
surgery is the primary endpoint to 
determine the optimal dose of 
ASP5354, missing data points 
would hinder evaluation of 
efficacy. 
QTL2: Sufficient data for PK 
modeling simulation: % missing 
data of plasma and urine PK data. 
Number of time points which 
missed both collection of plasma 
and urine PK and/or missed either 
collection of plasma or urine PK 
per total number of PK time points 
(%). Please note that the urine PK 
time point at end of surgery will 
not be included in QTL2 since this 
time point is optional. 
Since PK data will be correlative 
with anatomical visualization, PK 
modeling simulation would be 
useful to consider optimal dose. 
PK: pharmacokinetics; QTL: quality tolerance limit 
QTL Management Activities: 
● 
For control of risks associated with QTL1: Sufficient data for assessment of anatomical 
visualization of the index ureter(s), refer to [Section 7.1.1 Anatomical Visualization of 
the Index Ureter(s) Using ASP5354]. 
● 
For control of risks associated with QTL2: Sufficient data for assessment of anatomical 
visualization of the index ureter, refer to [Section 7.4.2 Analysis of ASP5354 in Urine]. 


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12.3 
Contraception Requirements 
WOCBP who are eligible for participation in the study, including those who choose complete 
abstinence, must have pregnancy tests as specified in the Schedule of Assessments.  
Pregnancy test results must confirm that the subject is not pregnant. 
WOMEN OF CHILDBEARING POTENTIAL DEFINITIONS AND METHODS OF 
CONTRACEPTION DEFINITIONS 
A female is considered fertile (i.e., WOCBP) following menarche and until becoming 
postmenopausal unless permanently sterile. 
Females in the following categories are not considered WOCBP 
● 
Premenopausal with 1 of the following (i.e., permanently sterile): 
o Documented hysterectomy 
o Documented bilateral salpingectomy 
o Documented bilateral oophorectomy 
● 
Postmenopausal 
A postmenopausal state is defined as at least 12 months after last menstrual bleeding without 
an alternative medical cause. 
In case the last menstrual bleeding cannot be clearly determined, confirmation with more 
than 1 follicle-stimulating hormone (FSH) measurement of at least > 40 IU/L (or higher per 
local institutional guidelines) is required. 
Females on hormone replacement therapy (HRT) and whose menopausal status is in doubt 
will be required to use 1 of the nonestrogen hormonal highly effective contraception methods 
if they wish to continue their HRT during the study.  Otherwise, they must discontinue HRT 
to allow confirmation of postmenopausal status by repeated FSH measurements before study 
enrollment. 
Documentation of any of these categories can come from the study site personnel’s review of 
the female subject’s medical records, medical examination or medical history interview. 
CONTRACEPTION GUIDANCE FOR FEMALE SUBJECTS OF CHILDBEARING 
POTENTIAL 
Female subjects of childbearing potential are eligible for participation in the study if they 
agree to use 1 of the highly effective methods of contraception listed below from the time of 
signing the ICF and until the end of relevant systemic exposure, defined as 30 days after the 
final IP administration.a 
Highly effective methods of contraception (failure rate of < 1% per year when used 
consistently and correctly)b: 
● 
Combined (estrogen- and progestogen-containing) hormonal contraception associated 
with inhibition of ovulation 
o Oral 


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o Intravaginal 
o Transdermal 
● 
Progestogen-only hormonal contraception associated with inhibition of ovulation 
o Oral 
o Injectable 
o Implantable 
● 
Other combined (estrogen- and progesterone-containing) methods 
o Vaginal ring 
o Injectable 
o Implantable 
o Intrauterine hormone-releasing system or intrauterine device 
● 
Bilateral tubal occlusion 
● 
Vasectomized partner 
A vasectomized partner is a highly effective contraception method provided that the 
partner is the sole male sexual partner of the WOCBP and the absence of sperm has been 
confirmed.  If not, an additional highly effective method of contraception should be used. 
● 
Sexual abstinence 
Sexual abstinence is considered a highly effective method only if defined as refraining 
from heterosexual intercourse during the entire period of risk associated with the test 
product.  The reliability of sexual abstinence needs to be evaluated in relation to the 
duration of the study and the preferred and usual lifestyle of the subject.  It is not 
necessary to use any other method of contraception when complete abstinence is elected. 
a Local laws and regulations may require use of alternative and/or additional contraception methods. 
b Typical use failure rates may differ from those when used consistently and correctly.  Use should be 
consistent with local regulations regarding the use of contraceptive methods for subjects 
participating in clinical studies. 
CONTRACEPTION GUIDANCE FOR MALE SUBJECTS WITH PARTNER(S) OF 
CHILDBEARING POTENTIAL. 
Male subjects with female partners of childbearing potential are eligible for participation in 
the study if they agree to the following during treatment and until the end of relevant 
systemic exposure defined as 30 days after final IP administration.a 
● 
Inform any and all partner(s) of their participation in a clinical drug study and the need to 
comply with contraception instructions as directed by the investigator. 
● 
Use a condom. 
● 
Female partners of male subjects who have not undergone a vasectomy with the absence 
of sperm confirmed or a bilateral orchiectomy should consider use of effective methods 
of contraception. 
a Local laws and regulations may require use of alternative and/or additional contraception methods. 


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12.4 
Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up and Reporting 
12.4.1 Definition of Adverse Events 
An AE is any untoward medical occurrence in a subject administered an IP, and which does 
not necessarily have to 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 (new or exacerbated) temporally associated with the use of IP whether 
or not considered related to the IP.  
12.4.1.1 Abnormal Laboratory Findings 
Any abnormal laboratory test result (e.g., hematology, biochemistry or urinalysis) or other 
safety assessment (e.g., vital signs, physical examination, ECGs or radiographic scans), 
including those that worsen from baseline, that is considered to be clinically significant in the 
medical and scientific judgment of the investigator and not related to underlying disease, is to 
be reported as an (S)AE. 
Any clinically significant abnormal laboratory finding or other abnormal safety assessment, 
which is associated with the underlying disease, does not require reporting as an (S)AE, 
unless judged by the investigator to be more severe than expected for the subject’s condition. 
Repeating an abnormal laboratory test or other safety assessment, in the absence of any of the 
above criteria, does not constitute an AE.  Any abnormal test result that is determined to be 
an error does not require reporting as an AE. 
12.4.1.2 Potential Cases of Drug-induced Liver Injury 
Refer to [Appendix 12.5 Liver Safety Monitoring and Assessment] for detailed instructions 
on drug-induced liver injury (DILI).  Abnormal values in AST and/or ALT concurrent or 
with abnormal elevations in TBL that meet the criteria outlined in [Appendix 12.5 Liver 
Safety Monitoring and Assessment], in the absence of other causes of liver injury, are 
considered potential cases of DILI (potential Hy’s Law cases) and are always to be 
considered important medical events and reported per [Section 12.4.5 Reporting Procedures 
for Serious Adverse Events]. 
12.4.2 Definition of Serious Adverse Events 
An AE is considered “serious” if, in the view of either the investigator or sponsor, the event:  
● 
Results in death 
● 
Is life-threatening (An AE is considered “life-threatening” if, in the view of either the 
investigator or sponsor, its occurrence places the subject at immediate risk of death; it 
does not include an AE that, had it occurred in a more severe form, might have caused 
death.) 
● 
Results in persistent or significant disability/incapacity or substantial disruption of the 
ability to conduct normal life functions  
● 
Results in congenital anomaly, or birth defect 


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● 
Requires inpatient hospitalization (except for planned procedures as allowed per study) 
or leads to prolongation of hospitalization (except if prolongation of planned 
hospitalization is not caused by an AE)  
● 
Other medically important events (defined in paragraph below) 
Medical and scientific judgment should be exercised in deciding whether expedited reporting 
is appropriate in other situations, such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization, but may jeopardize the 
subject or may require intervention to prevent one of the other outcomes listed in the 
definition above.  These events, including those that may result in disability/incapacity, 
usually are considered serious.  Examples of such events are intensive treatment in an 
emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsions that 
do not result in hospitalization, or development of drug dependency or drug abuse. 
12.4.3 Criteria for Causal Relationship to Investigational Product 
A medically qualified investigator is obligated to assess the relationship between IP and each 
occurrence of each (S)AE.  This investigator will use medical judgment as well as the 
reference safety information [Section 2.1.2 Summary of Key Safety Information for 
Investigational Product(s)] to determine the relationship.  The causality assessment is 1 of the 
criteria used when determining regulatory reporting requirements. 
The investigator is requested to provide an explanation for the causality assessment for each 
(S)AE and must document in the medical notes that he/she has reviewed the (S)AE and has 
provided an assessment of causality. 
Following a review of the relevant data, the causal relationship between the IP and each 
(S)AE will be assessed by answering “yes” or “no” to the question “Do you consider that 
there is a reasonable possibility that the event may have been caused by the IP?” 
When making an assessment of causality, the following factors are to be considered when 
deciding if there is evidence and/or arguments to suggest there is a “reasonable possibility” 
that an (S)AE may have been caused by the IP (rather than a relationship cannot be ruled out) 
or if there is evidence to reasonably deny a causal relationship: 
● 
Has the subject been administered IP? 
● 
Plausibility (i.e., could the event been caused by the suspect drug?  Consider biologic 
and/or pharmacologic mechanism, half-life, literature evidence, drug class, preclinical 
and study data, etc.) 
● 
Dechallenge/dose reduction/rechallenge: 
o Dechallenge: Did the (S)AE resolve or improve after only stopping the dose of the 
suspect drug without any treatment? 
o Dose reduction: Did the (S)AE resolve or improve after reducing the dose of the 
suspect drug? 
o Rechallenge: Did the (S)AE reoccur if the suspected drug was reintroduced after 
having been stopped? 


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● 
Laboratory or other test results: a specific laboratory investigation supports the 
assessment of the relationship between the (S)AE and the IP (e.g., based on values 
pretreatment, during and posttreatment). 
● 
Available alternative explanations independent of IP exposure; such as other 
concomitant drugs, past medical history, concurrent or underlying disease, risk factors 
including medical and family history, season, location, etc., and strength of the 
alternative explanation. 
● 
Finally, judging which are more likely based on all the above contents, factors of 
reasonable possibility or confounding factors, comprehensive judgment of plausible 
temporal relationship between exposure to the IP and (S)AE onset and/or resolution will 
be provided.  Did the (S)AE occur in a reasonable temporal relationship to the 
administration of the IP? 
There may be situations in which an SAE has occurred and the investigator has minimal 
information to include in the initial report to the sponsor.  However, it is very important that 
the investigator always assesses causality for every event before the initial transmission of the 
SAE data to the sponsor.  With limited or insufficient information about the event to make an 
informed medical judgment and in absence of any indication or evidence to establish a causal 
relationship, a causality assessment of “no” is to be considered.  In such instance, the 
investigator is expected to obtain additional information regarding the event as soon as 
possible and to re-evaluate the causality upon receipt of additional information.  The 
medically qualified investigator may revise his/her assessment of causality in light of new 
information regarding the SAE and shall send an SAE follow-up report and update the eCRF 
with the new information and updated causality assessment.   
12.4.4 Criteria for Defining the Severity of an Adverse Event 
AEs, including abnormal clinical laboratory values, will be graded using NCI-CTCAE 
guidelines (version 5.0).  The items that are not stipulated in the NCI-CTCAE version 5.0 
will be assessed according to the criteria below and entered into the eCRF: 
Table 7 
Grading Scale Defining the Severity of an Adverse Event 
Grade 
Assessment Standard 
1 - Mild 
Asymptomatic or mild symptoms, clinical or diagnostic observations only; 
intervention not indicated 
2 - Moderate 
Minimal local or noninvasive intervention indicated; limiting age-appropriate 
instrumental ADLa 
3 - Severe 
Medically significant but not immediately life-threatening, hospitalization or 
prolonged hospitalization indicated; disabling; limiting self-care ADLb 
4 - Life-threatening 
Life-threatening consequences, urgent intervention indicated 
5 - Death 
Death related to AE 
ADL: activities of daily living; AE: adverse event 
a Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, managing 
money, etc. 
b Self-care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking medications 
and not bedridden. 


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12.4.5 Reporting Procedures for Serious Adverse Events 
The investigator must complete and submit an SAE worksheet containing all information that 
is required by local and/or regional regulations to the sponsor by fax or email immediately 
(within 24 hours of awareness). 
The SAE worksheet must be signed by a medically qualified investigator (as identified on 
delegation of authority log).  Signature confirms accuracy and completeness of the SAE data 
as well as the investigator causality assessment including the explanation for the causality 
assessment. 
For contact details, see [Contact Details of Sponsor's Key Personnel].  Fax or email the 
SAE/special situations worksheet to: 
Astellas Pharma Global Development Inc. 
Pharmacovigilance  
North America fax number: +1-888-396-3750 
North America alternate fax number: +1-847-317-1241 
International Fax: +44-800-471-5263 
Email: safety-us@astellas.com 
If there are any questions, or if clarification is needed regarding the SAE, please contact the 
sponsor’s medical monitor/study physician or their designee [Contact Details of Sponsor’s 
Key Personnel]. 
Follow-up information for the event should be sent promptly (as soon as available but no 
longer than within 7 days of the initial notification). 
Full details of the SAE should be recorded on the medical records, SAE/special situation 
worksheet and on the eCRF. 
The following minimum information is required: 
● 
International study number/study number  
● 
Subject number, sex and age 
● 
Date of report 
● 
Description of the SAE (event and seriousness criteria)  
● 
Causal relationship to the IP (including reason) 
● 
Drug provided (if any) 
The sponsor or sponsor’s designee will medically evaluate the SAE and determine if the 
report meets the requirements for expedited reporting based on seriousness, causality, and 
expectedness of the events (e.g., suspected unexpected serious adverse reaction [SUSAR] 
reporting) according to current local/regional regulatory requirements.  The sponsor or 
sponsor's designee will submit expedited safety reports to CA and concerned ethics 
committee per current local regulations, and will inform the investigators of such regulatory 
reports as required.  Investigators must submit safety reports as required by their IRB/IEC 
within timelines set by regional regulations (e.g., EMA, FDA) where required.  


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Documentation of the submission to and receipt by the IRB/IEC of expedited safety reports 
should be retained by the study site.  In the US, FDA expedited IND reporting guidelines will 
be followed. 
The sponsor will notify all investigators responsible for ongoing clinical studies with the test 
product of all SUSARs, which require submission per local requirements to the IRB/IEC. 
The investigator should provide written documentation of IRB/IEC notification for each 
report to the sponsor. 
The investigator may contact the sponsor’s medical monitor/study physician for any other 
problem related to the rights, safety or well-being of the subject. 
12.4.6 Reporting Procedures for Special Situations 
12.4.6.1 Pregnancy 
If a female subject becomes pregnant during the study dosing period or within 30 days from 
the discontinuation of dosing, the investigator is to report the information to the sponsor 
according to the timelines in [Section 12.4.5 Reporting Procedures for Serious Adverse 
Events] using the SAE worksheet as a special situation and in the eCRF. 
The investigator will attempt to collect pregnancy information on any female partner of a 
male subject who becomes pregnant during the study dosing period or within 30 days from 
the discontinuation of dosing and report the information to the sponsor according to the 
timelines in [Section 12.4.5 Reporting Procedures for Serious Adverse Events] using the SAE 
worksheet as a special situation. 
The expected date of delivery or expected date of the end of the pregnancy, last menstruation, 
estimated conception date, pregnancy result and neonatal data, etc., should be included in this 
information. 
While pregnancy itself is not considered to be an AE or SAE, any pregnancy complication or 
termination (including elective termination) of a pregnancy is to be reported for a female 
subject as an AE in the eCRF or SAE per [Section 12.4.5 Reporting Procedures for Serious 
Adverse Events].  For (S)AEs experienced by a female partner of a male subject, (S)AEs are 
to be reported via the SAE worksheet. 
Additional information regarding the outcome of a pregnancy when also categorized as an 
SAE is mentioned below: 
● 
“Spontaneous abortion” includes miscarriage, abortion and missed abortion. 
● 
Death of a newborn or infant within 1 month after birth is to be reported as an SAE 
regardless of its relationship with the IP. 
● 
If an infant dies more than 1 month after the birth, it is to be reported if a relationship 
between the death and intrauterine exposure to the IP is judged as “possible” by the 
investigator. 
● 
Congenital anomaly (including anomaly in miscarried fetus). 


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Unless a congenital anomaly is identified prior to spontaneous abortion or miscarriage, the 
embryo or fetus should be assessed for congenital defects by visual examination or other 
means as appropriate. (S)AEs experienced by the newborn/infant should be reported via the 
pregnancy reporting form.  Generally, follow-up will be no longer than 6 to 8 weeks 
following the estimated delivery date. 
12.4.6.2 Medication Error, Overdose and “Off-label Use” 
If a medication error (defined as an unintended failure in the treatment process that leads to, 
or has the potential to lead to harm to the subject; this includes the prescribing, storing, 
dispensing, preparation for administration or administration of a medicinal product), 
overdose or “off-label use” (i.e., use outside of what is stated in the protocol) is suspected, 
refer to [Section 10.3 Major Protocol Deviations].  Any associated (S)AEs are to be reported 
in the eCRF.  If the AE meets the definition of an SAE, the SAE is also to be reported as 
described in [Section 12.4.5 Reporting Procedures for Serious Adverse Events] together with 
the details of the medication error, overdose and/or “off-label use.” 
In the event of suspected ASP5354 overdose, the subject should receive supportive care and 
monitoring.  The medical monitor/expert should be contacted as applicable. 
12.4.6.3 Misuse/Abuse 
Definition of misuse: Situations where the IP is/are intentionally and inappropriately used not 
in accordance with the intended use as defined in the protocol. 
Definition of abuse: Persistent or sporadic, intentional excessive use of medicinal products 
which is accompanied by harmful physical or psychological effects. 
If misuse or abuse of the IP is suspected, the investigator must forward the special situation 
worksheet to the sponsor by fax or email immediately (within 24 hours of awareness).  Any 
associated (S)AEs are to be reported in the eCRF.  If the AE meets the definition of an SAE, 
the SAE is also to be reported as described in [Section 12.4.5 Reporting Procedures for 
Serious Adverse Events] together with details of the misuse or abuse of the IP. 
12.4.6.4 Occupational Exposure 
If occupational exposure (e.g., inadvertent exposure to the IP of study site personnel while 
preparing it for administration to the subject) to the IP occurs, the investigator must forward 
the special situation worksheet to the sponsor by fax or email immediately (within 24 hours 
of awareness).  Any associated (S)AEs occurring to the individual associated with or 
resulting from the special situation are to be reported on the special situations worksheet. 
12.4.6.5 (Suspicion of) Transmission of Infectious Agent  
If transmission of an infectious agent associated with the IP is suspected, the investigator 
must forward the special situation worksheet to the sponsor by fax or email immediately 
(within 24 hours of awareness) and any associated (S)AEs are to be reported in the eCRF If 
the AE meets the definition of an SAE, the SAE is also to be reported as described in 


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[Section 12.4.5 Reporting Procedures for Serious Adverse Events] together with the details of 
the suspected transmission of infectious agent.  
12.4.6.6 Suspected Drug-drug Interaction 
If a drug-drug interaction associated with the IP is suspected, the investigator must forward 
the special situation worksheet to the sponsor by fax or email immediately (within 24 hours 
of awareness).  Any associated (S)AEs are to be reported in the eCRF.  If the AE meets the 
definition of an SAE, the SAE is also to be reported as described in [Section 12.4.5 Reporting 
Procedures for Serious Adverse Events] together with details of the suspected drug-drug 
interaction.  


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12.5 
Liver Safety Monitoring and Assessment 
The purpose of this appendix is to provide guidance for the monitoring of DILI during the 
course of the study.  It should be noted that this section does not specify the end of study 
analyses of liver enzymes.  The end of study liver enzymes analyses will be described in the 
SAP.  Any subject enrolled in a study with active drug therapy and reveals an increase of 
serum aminotransferases (AT) to > 3 × ULN or TBL > 2 × ULN should undergo detailed 
testing for liver enzymes (including at least ALP, ALT, AST and TBL).  Testing should be 
repeated within 72 hours of notification of the test results.  For studies for which a central 
laboratory is used, alerts will be generated by the central laboratory regarding moderate and 
severe liver abnormality to inform the investigator and study team.  Subjects should be asked 
if they have any symptoms suggestive of hepatobiliary dysfunction. 
Definition of Liver Abnormalities 
Confirmed abnormalities will be characterized as moderate and severe where ULN is as 
shown below. 
Table 8 
Moderate and Severe Liver Abnormalities 
 
ALT or AST 
 
TBL 
Moderate 
> 3 × ULN 
or 
> 2 × ULN 
Severe 
> 3 × ULN 
and† 
> 2 × ULN 
ALT: alanine aminotransferase; AST: aspartate aminotransferase; TBL: total bilirubin; ULN: upper limit of 
normal 
†Samples taken simultaneously or within maximum 24 hours. 
In addition, the subject should be considered to have severe hepatic abnormalities for any of 
the following: 
● 
ALT or AST > 8 × ULN 
● 
ALT or AST > 5 × ULN for more than 2 weeks 
● 
ALT or AST > 3 × ULN and† and* TBL > 2 × ULN or international normalized ratio 
(INR) > 1.5 (if INR testing is applicable/evaluated) 
● 
ALT or AST > 3 × ULN with the appearance of fatigue, nausea, vomiting, right upper 
quadrant pain or tenderness, fever, rash and/or eosinophilia (> 5%)  
† Samples taken simultaneously or within a maximum of 24 hours. 
The investigator may determine that abnormal liver function results, other than as described 
above, may qualify as moderate or severe abnormalities and require additional monitoring 
and follow-up. 
Follow-up Procedures 
Confirmed moderate and severe abnormalities in hepatic functions should be thoroughly 
characterized by obtaining appropriate expert consultations, detailed pertinent history, 
physical examination and clinical laboratory tests.  The study site personnel are to complete 
the liver abnormality case report form (LA-CRF).  Subjects with confirmed abnormal liver 
function testing should be followed as described below. 


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Confirmed moderately abnormal liver function tests should be repeated 2 to 3 times weekly, 
and then weekly or less if abnormalities stabilize or the IP has been discontinued and the 
subject is asymptomatic. 
Severe hepatic liver function abnormalities as defined above, in the absence of another 
etiology, may be considered an important medical event and may be reported as a SAE.  The 
sponsor should be contacted and informed of all subjects for whom severe hepatic liver 
function abnormalities possibly attributable to IP are observed. 
To further assess abnormal hepatic laboratory findings, the investigator is expected to:  
● 
Obtain a more detailed history of symptoms and prior or concurrent diseases.  Symptoms 
and new-onset diseases are to be recorded as “AEs” within the eCRF.  Illnesses and 
conditions such as hypotensive events, and decompensated cardiac disease that may lead 
to secondary liver abnormalities should be noted.  Nonalcoholic steatohepatitis is seen in 
obese hyperlipoproteinemic and/or diabetic subjects, and may be associated with 
fluctuating AT levels.  The investigator should ensure that the medical history form 
captures any illness that predates study enrollment that may be relevant in assessing 
hepatic function. 
● 
Obtain a history of concomitant drug use (including nonprescription medication, 
complementary and alternative medications), alcohol use, recreational drug use and 
special diets.  Medications are to be entered in the eCRF.  Information on alcohol, other 
substance use and diet should be entered on the LA-CRF or an appropriate document. 
● 
Obtain a history of exposure to environmental chemical agents. 
● 
Based on the subject’s history, other testing may be appropriate including: 
o Acute viral hepatitis (A, B, C, D, E or other infectious agents) 
o Ultrasound or other imaging to assess biliary tract disease 
o Other clinical laboratory tests, including INR and direct bilirubin 
● 
Consider gastroenterology or hepatology consultations. 
● 
Submit results for any additional testing and possible etiology on the LA-CRF or an 
appropriate document. 
Study Treatment Discontinuation 
Since this is a single dose study, treatment discontinuation is not applicable. 


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12.6 
List of Excluded Concomitant Medications 
Please note that this is not an exhaustive list. Investigators should verify the subject’s 
concomitant medications for all prohibited and restricted drug classes.  
Prohibited Medications 
From 48 hours prior to the IP administration until the completion of the surgical procedure. 
Class 
Drug 
ICG 
Indocyanine green 
Other NIR-F imaging agent 
Methylene blue 
Other agents which emit fluorescent when excited by 
NIR-F spectrum 
Diuretics 
Loop 
E.g., bumetanide, furosemide, torsemide 
Osmotic 
E.g., isosorbide (also a vasodilator), glycerin, mannitol 
Thiazides 
E.g., hydrochlorothiazide, indapamide, metolazone 
Potassium Sparing 
E.g., eplerenone, spironolactone, triamterene 
Carbonic Anhydrase Inhibitors 
E.g., acetazolamide, dichlorphenamide, methazolamide 
Xanthines 
E.g., aminophylline, theophylline 
Inhibitors for 
renal transporters 
defined by FDA 
P-gp inhibitors 
Amiodarone 
Carvedilol 
Clarithromycin 
Dronedarone 
Itraconazole 
Lapatinib 
Lopinavir and ritonavir 
Propafenone 
Quinidine 
Ranolazine, ritonavir 
Saquinavir and ritonavir 
Telaprevir 
Tipranavir and ritonavir 
Verapamil 
Simeprevir 
OAT1 and OAT3 inhibitors 
P-aminohippuric acid (PAH) 
Probenecid 
Teriflunomide 
MATE1 and MATE2-K 
inhibitors 
Cimetidine 
Dolutegravir 
Isavuconazole 
Ranolazine 
Trimethoprim 
Vandetanib 
MATE: multidrug and toxin extrusion; OAT: organic anion transporter; P-gp: P-glycoprotein 
 


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12.7 
Laboratory Assessments 
Laboratory tests will be performed according to the Schedule of Assessments and sent to the 
local laboratory for analysis.  
Table 9 
Clinical Laboratory Tests 
Panel/Assessments 
Parameters to be Analyzed 
Hematology 
Hemoglobin 
Hematocrit 
Erythrocytes 
Leukocytes 
Differential leukocytes 
Platelets 
Biochemistry 
Albumin  
Alanine aminotransferase 
Alkaline phosphatase 
Aspartate aminotransferase 
Blood urea nitrogen 
Calcium 
Chloride 
Creatinine 
Creatinine kinase 
Gamma-glutamyl transferase 
Glucose 
Inorganic phosphorus 
Lactate dehydrogenase 
Magnesium 
Potassium 
Sodium  
Total bilirubin 
Total cholesterol 
Total protein 
Triglycerides 
Uric acid 
Follicle-stimulating hormone (postmenopausal female subjects only) 
 
Histamine and tryptase (only in subjects with hypersensitivity reaction) 
Urinalysis 
Urine, dipstick as 
applicable (if protein, 
blood, leukocytes or 
nitrites are abnormal, 
microscopy will be 
performed) 
Protein 
Glucose 
pH 
Blood 
Leukocytes 
Urobilinogen 
Bilirubin 
Ketones 
Nitrite 
Color 
 
Microscopy (optional) 
Casts 
Crystals 
Epithelial cells 
Leucocytes 
Erythrocytes 
Bacteria 
Pregnancy - Serum 
Human chorionic gonadotropin (female subjects only) 


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12.8 
List of Abbreviations and Definition of Key Study Terms 
List of Abbreviations 
Abbreviations 
Description of abbreviations 
ADL 
activities of daily living 
AE 
adverse event 
Ae 
amount of ASP5354 excreted in urine 
Ae% 
percentage of drug dose excreted into urine 
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
API 
Astellas Pharma Inc. 
AST 
aspartate aminotransferase 
AT 
aminotransferases 
AUC0-24 
area under the concentration-time curve from time zero to 24 hours postdose 
AUCinf 
area under the concentration-time curve from time zero to infinity 
AUClast 
area under the plasma concentration-time curve from time zero to the time of the 
last quantifiable concentration 
AUST 
Astellas US Technologies 
CA 
competent authorities 
CI 
confidence interval 
C0 
plasma concentration at time 0 
CRO 
contract research organization 
CV 
coefficient of variation 
CYP 
cytochrome P450 
DILI 
drug-induced liver injury 
DBP 
diastolic blood pressure 
DPD 
Data Protection Directive 
ECG 
electrocardiogram 
eCOA 
electronic clinical outcome assessment 
eCRF 
electronic case report form 
EEA 
European Economic Area 
eGFR 
estimated glomerular filtration rate 
FAS 
full analysis set 
FSH 
follicle-stimulating hormone 
GCP 
Good Clinical Practice 
GMP 
Good Manufacturing Practices 
HPWCs 
human peripheral white blood cells 
HRT 
hormone replacement therapy 
IB 
Investigator’s Brochure 
ICF 
informed consent form 
ICG 
indocyanine green 
ICH  
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 


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Abbreviations 
Description of abbreviations 
IEC 
Independent Ethics Committee 
INR 
international normalized ratio 
IP 
investigational product 
IRB 
Institutional Review Board 
IRT 
interactive response technology 
ISN 
international study number 
IUI 
iatrogenic ureteral injury 
iv 
intravenous 
LA-CRF 
liver abnormality case report form 
LC-MS/MS 
liquid chromatography with tandem mass spectrometry 
MATE 
multidrug and toxin extrusion 
max 
maximum 
MB 
methylene blue 
min 
minimum 
n 
number of subjects 
NCI-CTCAE 
National Cancer Institute - common terminology criteria for adverse events 
NIR-F 
near-infrared fluorescence 
OAT 
organic anion transporter 
P-gp 
P-glycoprotein 
PT 
preferred term 
QA 
quality assurance 
QC 
quality control 
QTcF 
corrected QT interval using Fridericia’s formula 
QTL 
quality tolerance limits 
(S)AE 
serious adverse event or adverse event 
SAE 
serious adverse event 
SAF 
safety analysis set 
SAP 
statistical analysis plan 
SBP 
systolic blood pressure 
SBR 
signal background ratio 
SOP 
standard operating procedure 
SUSAR 
suspected unexpected serious adverse reactions 
t½ 
apparent terminal elimination half-life 
TBL 
total bilirubin 
TEAE 
treatment-emergent adverse event 
ULN 
upper limit of normal 
USM 
urgent safety measure 
VRC 
Visualization Review Committee 
Vss 
volume of distribution at steady state 
WOCBP 
women of childbearing potential 


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Definition of Key Study Terms 
Terms 
Definition of Terms 
Baseline 
Assessments of subjects as they enter a study before they receive any treatment.  
Endpoint  
Variable that pertains to the efficacy or safety evaluations of a study. 
Note: Not all endpoints are themselves assessments since certain endpoints might 
apply to populations or emerge from analysis of results.  That is, endpoints might 
be facts about assessments (e.g., prolongation of survival). 
Enroll 
To register or enter a subject into a study. 
Randomization 
The process of assigning subjects to dose groups using an element of chance to 
determine assignments in order to reduce bias. 
Screen failure 
Potential subject who signed the ICF, but did not meet 1 or more criteria required 
for participation in the study and was not enrolled. 
Screening 
A process of active consideration of potential subjects for enrollment in a study. 
Screening period 
Period of time before entering the investigational period, usually from the time 
when a subject signs the consent form until just before the test product or 
comparative drug (sometimes without randomization) is given to a subject.  
Study period 
Period of time from the first study site initiation date to the last study site 
completing the study. 
Variable 
Any quantity that varies; any attribute, phenomenon or event that can have 
different qualitative or quantitative values. 


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13 
ATTACHMENT 1:   SUBSTANTIAL AMENDMENT 2 
I.  The purpose of this amendment is: 
Substantial Changes 
1. Add enrollment of subjects with high body mass index (BMI) and/or renal 
impairment after optimal dose is determined 
DESCRIPTION OF CHANGE:  
The study design is updated to allow the enrollment of additional subjects into the 
 arm 
after it is determined to be an optimal dose. This revision effects the following sections: 
• Study Schema (Figures 1 and 2) 
• Study Design 
Text is added to clarify that the optimal dose cohort may continue enrollment with up to a 
maximum of 15 subjects. 
Text is added to Section 9, Statistical Methodology, to clarify that there will be a separate 
group for the analysis of subjects enrolled after determination of optimal dose.  
 
RATIONALE: 
To obtain additional data of ASP5354 in the broadest subject population at the selected 
optimal dose. 
 
2. Add inclusion criterion for BMI and/or renal impairment 
DESCRIPTION OF CHANGE:  
An inclusion criterion is added to enroll subjects with a BMI > 25 and/or renal impairment 
after the optimal dose has been determined. 
 
RATIONALE: 
The BMI criterion is added to collect further information on visualization in subjects with 
high BMI. The estimated glomerular filtration rate (eGFR) criterion is added to collect 
further information on visualization and pharmacokinetic data in subjects with eGFR < 60. 
 
Nonsubstantial Changes 
1. Revise planned study period 
DESCRIPTION OF CHANGE:  
The completion of the planned study period is changed from 3Q2021 to 4Q2021. 
 
RATIONALE: 
This revision is made to reflect the estimated study completion. 
 
2. Clarify that VRC will be held until the optimal dose is determined 
CCI


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DESCRIPTION OF CHANGE:  
Text is added to clarify that the VRC will be held to review subject data until the optimal 
dose is determined. 
 
RATIONALE: 
This revision is made for clarification. 
 
3. Minor Administrative-type Changes 
DESCRIPTION OF CHANGE:  
Include minor administrative-type changes (e.g., typos, format, numbering and consistency 
throughout the protocol) and add eGFR to List of Abbreviations.   
 
RATIONALE: 
To provide clarifications, administrative changes and corrections to the protocol. 
 
II.  Amendment Summary of Changes: 
IIA. Substantial Changes 
1 Protocol Summary 
1.2 Study Schema (Figure 1 Study Schema) 
WAS: 
• Up to 15 subjects will be randomly assigned to receive single doses of ASP5354 at each 
dose level (
,  or 
).  
 
IS AMENDED TO: 
BMI: body mass index; 
• Up to 15 subjects will be randomly assigned to receive single doses of ASP5354 at each 
dose level (
,  or 
). Once the optimal dose is determined, the optimal dose 
cohort may continue enrollment up to the maximum 15 subjects. 
 
1 Protocol Summary 
1.2 Study Schema (Figure 2 Dose Cohort Schema) 
WAS: 
• The visualization data for the subjects, at each interim time point, who completed the 
surgical procedure will be assessed to decide whether to: 1) expand a dose under 
evaluation; 2) stop a dose under evaluation in which case additional subjects would not be 
added to a dose level; or 3) define a dose as optimal. Visualization should occur at 30 
minutes post ASP5354 administration and end of surgery and Likert Scoring (0 to 3) for 
each time point should be recorded.  
 
IS AMENDED TO: 
BMI: body mass index; 
• The visualization data for the subjects, at each interim time point, who completed the 
CCI
CCI
CCI
CCI
CCI
CCI


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surgical procedure will be assessed to decide whether to: 1) expand a dose under 
evaluation; 2) stop a dose under evaluation in which case additional subjects would not be 
added to a dose level; or 3) define a dose as optimal. Visualization should occur at 30 
minutes post ASP5354 administration and end of surgery and Likert Scoring (0 to 3) for 
each time point should be recorded. Once the optimal dose is determined, the optimal 
dose cohort may continue enrollment up to the maximum 15 subjects. 
 
1 Protocol Summary and 4 Study Design and Dose Rationale 
1.1 Synopsis, 4.1 Study Design 
WAS: 
The visualization data will be assessed by a Visualization Review Committee (VRC), consisting of 
the investigator(s) and the sponsor’s medical representative.  The VRC will be held at the following 
time points, but ad-hoc meetings may occur:  
1) after the initial 3 subjects have completed the surgical procedures at each of the dose levels; 
and  
2) then in increments of 3 subjects (up to a total of 15 subjects per dose level) for those who 
have completed surgical procedures at the expanded dose levels. 
The visualization data for the subjects, at each interim time point, who completed the surgical 
procedure will be assessed to decide whether to:  
1) expand a dose under evaluation;  
2) stop a dose under evaluation in which case additional subjects would not be added to 
a dose level; or 
3) define a dose as optimal.   
 
IS AMENDED TO: 
The visualization data will be assessed by a Visualization Review Committee (VRC), consisting of 
the investigator(s) and the sponsor’s medical representative.  The VRC will be held at the following 
time points, but ad-hoc meetings may occur until the optimal dose is determined:  
1) after the initial 3 subjects have completed the surgical procedures at each of the dose levels; 
and  
2) then in increments of 3 subjects (up to a total of 15 subjects per dose level) for those who 
have completed surgical procedures at the expanded dose levels. 
The visualization data for the subjects, at each interim time point, who completed the surgical 
procedure will be assessed to decide whether to:  
1) expand a dose under evaluation;  
2) stop a dose under evaluation in which case additional subjects would not be added 
to a dose level; or 
3) define a dose as optimal.   
Once the optimal dose is determined, the optimal dose cohort may continue enrollment 
up to the maximum 15 subjects. 
 


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1 Protocol Summary and 5 Study Population 
1.1 Synopsis, 5.1 Inclusion Criteria 
ADDED: 
Subjects enrolled after optimal dose determination: 
12. Subject has any of the following values at screening: 
● 
Body mass index > 25 
● 
Estimated glomerular filtration rate (eGFR) ≥ 15 mL/min/1.73 m2 and < 60. 
Subjects with an eGFR ≥ 60 mL/min/1.73 m2 may be considered after 
discussion with the medical monitor. 
 
1 Protocol Summary and 5 Study Population 
1.1 Synopsis, 5.2 Exclusion Criteria 
WAS: 
Subject has any of the following screening laboratory values: 
● Hemoglobin ≤ 9 g/dL 
● Absolute neutrophil count ≤ 1500/µL 
● Platelet count ≤ 100000/µL 
● Estimated glomerular filtration rate < 60 mL/min/1.73 m2 
● Serum bilirubin ≥ 2 × upper limit of normal (ULN) 
● Aspartate aminotransferase or serum glutamic oxaloacetic transaminase 
≥ 2.5 × ULN 
● Alanine aminotransferase or serum glutamic pyruvic transaminase ≥ 2.5 × ULN 
 
IS AMENDED TO: 
Subject has any of the following screening laboratory values: 
● Hemoglobin ≤ 9 g/dL 
● Absolute neutrophil count ≤ 1500/µL 
● Platelet count ≤ 100000/µL 
● eGFREstimated glomerular filtration rate < 60 mL/min/1.73 m2 (Not applicable 
to subjects enrolled after optimal dose determination.) 
● Serum bilirubin ≥ 2 × upper limit of normal (ULN) 
● Aspartate aminotransferase or serum glutamic oxaloacetic transaminase 
≥ 2.5 × ULN 
● Alanine aminotransferase or serum glutamic pyruvic transaminase ≥ 2.5 × ULN 
 
9 Statistical Methodology 
 
ADDED: 
For analysis by dose group, unless specified otherwise, subjects are grouped by the dose 
received for those randomized before determination of optimal dose, and a separate 
group for additional subjects enrolled after determination of optimal dose. 
 
IIB. Nonsubstantial Changes 


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1 Protocol Summary 
1.1 Synopsis, Objectives and Endpoints 
ADDED: 
Ae: amount of ASP5354 excreted in urine; Ae%: percentage of drug dose excreted into 
urine; ECGs: electrocardiograms; SAEs: serious adverse events; SBR: signal 
background ratio; TEAEs: treatment emergent adverse events 
 
9 Statistical Methodology 
9.2.1 Full Analysis Set 
WAS: 
The full analysis set (FAS) will consist of all subjects who are randomized and receive 
ASP5354 and have at least 1 assessment of ureter visualization during surgery.  This will be 
the primary analysis set for efficacy analyses. 
 
IS AMENDED TO: 
The full analysis set (FAS) will consist of all subjects who are randomized and receive 
ASP5354 and have at least 1 assessment of ureter visualization during surgery.  This will be 
the primary analysis set for efficacy analyses. 
 
9 Statistical Methodology 
9.2.2 Safety Analysis Set 
WAS: 
The SAF consists of all randomized subjects who receive ASP5354.  The SAF will be used 
for all summaries of the safety data, unless otherwise specified. 
 
IS AMENDED TO: 
The SAF consists of all randomized subjects who receive ASP5354.  The SAF will be used 
for all summaries of the safety data, unless otherwise specified. 
 
1 Protocol Summary and 9 Statistical Methodology 
1.1 Synopsis, 9.10 Interim Analysis (and Early Discontinuation of the Study) 
ADDED: 
The VRC will be held to review subject data until the optimal dose is determined. 
 
1 Protocol Summary and 10 Operational Considerations 
10.4.1 Visualization Review Committee 
WAS: 
The visualization data will be assessed by the principal investigator/investigator and the 
sponsor’s medical representative, who form the VRC.  The VRC will be held at the following 
time points, but ad-hoc meetings may occur: 1) after the initial 3 subjects have completed the 
surgical procedures at each of the dose levels, and 2) then in increments of 3 subjects (up to a 


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total of 15 subjects per dose level) for those who have completed the surgical procedures at 
each of the expanded dose levels.  In addition, the VRC may meet on an ad-hoc basis.  For 
further details on the VRC, refer to the VRC charter. 
 
IS AMENDED TO: 
The visualization data will be assessed by the principal investigator/investigator and the 
sponsor’s medical representative, who form the VRC.  The VRC will be held at the following 
time points, but ad-hoc meetings may occur until the optimal dose is determined: 1) after 
the initial 3 subjects have completed the surgical procedures at each of the dose levels, and 2) 
then in increments of 3 subjects (up to a total of 15 subjects per dose level) for those who 
have completed the surgical procedures at each of the expanded dose levels.  In addition, the 
VRC may meet on an ad hoc basis.  For further details on the VRC, refer to the VRC charter. 
 
12 Appendices 
12.8 List of Abbreviations and Definition of Key Study Terms 
ADDED: 
eGFR 
estimated glomerular filtration rate 
 


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14 
SPONSOR SIGNATURE 
Astellas Signatories 
(Electronic signatures are attached at the end of the document.) 
 
Medical Science Oncology 
 
Biostatistics, Data Science 
 
PPD
PPD

SAP_001.pdf: 
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STATISTICAL ANALYSIS PLAN 
Version 4.0 dated 02 September 2021 
A Phase 2 Randomized Open-label, Dose-ranging Study for Ureter Visualization, Using 
ASP5354 in Subjects Undergoing Laparoscopic/Minimally Invasive Colorectal Surgery 
ISN: 5354-CL-0201 
IND number: 140758 
Sponsor: 
Astellas Pharma Inc. (API) 
2-5-1, Nihonbashi-Honcho, Chuo-ku,
Tokyo 103-8411, Japan
This document contains confidential information which is the intellectual property of Astellas. By accepting or reviewing 
this document, you agree to hold this information in confidence and not copy or disclose it to others or use it for 
unauthorized purposes except (1) as otherwise agreed to in writing; (2) where required by applicable law; (3) where 
disclosure is directly related to the care and safety of the research participant; and (4) where disclosure of such information 
is made to a member of the investigator’s team who agrees to hold this information in confidence. 


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Table of Contents 
I. 
LIST OF ABBREVIATIONS AND KEY TERMS ································
···········
 4 
1 
INTRODUCTION ································
································
··················
 6 
2 
STUDY OBJECTIVE(S) AND DESIGN ································
·······················
 6 
2.1 
Study Objective(s) ································
································
·············
 6 
2.1.1 
Primary Objective ································
································
········  6 
2.1.2 
Secondary Objectives ································
································
····  6 
2.1.3 
Exploratory Objectives ································
································
··  6 
2.2 
Study Design ································
································
····················
 6 
2.3 
Randomization ································
································
··················
 8 
3 
SAMPLE SIZE································
································
······················
 8 
4 
ANALYSIS SETS ································
································
··················
 8 
4.1 
Full Analysis Set ································
································
···············
 8 
4.2 
Safety Analysis Set ································
································
············
 9 
4.3 
Pharmacokinetic Analysis Set ································
································
 9 
5 
ANALYSIS ENDPOINTS ································
································
········  9 
5.1 
Primary Efficacy Endpoint ································
································
····  9 
5.2 
Secondary Efficacy Endpoints ································
·······························
 10 
5.3 
Exploratory Efficacy Endpoints ································
·····························
 10 
5.4 
Safety Endpoints ································
································
··············
 10 
5.4.1 
Adverse Event ································
································
···········
 10 
5.4.2 
Clinical Laboratory Variables ································
··························
 11 
5.4.3 
Vital Signs, Height and Weight ································
························
 11 
5.4.4 
Routine 12-lead electrocardiograms································
···················
 11 
5.4.5 
Physical Examination ································
································
···  11 
5.5 
Other Endpoints ································
································
···············
 11 
5.5.1 
Pharmacokinetic Endpoints ································
····························
 11 
5.5.2 
Pharmacodynamic Endpoints ································
··························
 11 
5.5.3 
Green Discoloration of Urine ································
··························
 12 
6 
STATISTICAL METHODOLOGY ································
···························
 12 
6.1 
General Considerations ································
································
·······  12 
6.2 
Study Population ································
································
··············
 12 


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6.2.1 
Disposition of Subjects ································
································
· 12 
6.2.2 
Protocol Deviations ································
································
·····  13 
6.2.3 
Demographic and Other Baseline Characteristics ································
···  13 
6.2.4 
Previous and Concomitant Medications ································
··············
 14 
6.2.5 
Non-medication Therapies ································
·····························
 14 
6.3 
Study Drugs Exposure and Compliance ································
····················
 14 
6.4 
Analysis of Efficacy ································
································
··········
 14 
6.4.1 
Analysis of Primary Efficacy Endpoint ································
···············
 14 
6.4.2 
Analysis of Secondary Efficacy Endpoints ································
···········
 15 
6.4.3 
Analysis of Exploratory Efficacy Endpoints ································
·········  15 
6.5 
Analysis of Safety ································
································
·············
 16 
6.5.1 
Adverse Events ································
································
··········
 16 
6.5.2 
Clinical Laboratory Evaluation ································
························
 16 
6.5.3 
Vital Signs ································
································
················
 17 
6.5.4 
Electrocardiograms ································
································
······  18 
6.5.5 
Pregnancies ································
································
···············
 18 
6.6 
Analysis of Pharmacokinetics ································
·······························
 18 
6.7 
Analysis of Pharmacodynamics ································
·····························
 19 
6.7.1 
Concentration-response Relationship Analysis ································
······  19 
6.8 
Other Analyses ································
································
················
 19 
6.8.1 
Green Discoloration of Urine ································
··························
 19 
6.9 
Interim Analysis (and Early Discontinuation of the Clinical Study) ····················
 19 
6.10 
Additional Conventions ································
································
······  19 
6.10.1 
Analysis Windows ································
································
······  19 
6.10.2 
Imputation Rules for Incomplete Dates ································
···············
 20 
7 
REVISION AND RATIONALE ································
································
 21 
8 
REFERENCES ································
································
····················
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9 
APPENDICES ································
································
·····················
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9.1 
Appendix 1: Author and Approver Signatures ································
············
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I. 
LIST OF ABBREVIATIONS AND KEY TERMS 
List of Abbreviations 
Abbreviations 
Description of abbreviations 
AE 
adverse event 
Ae 
amount of ASP5354 excreted in urine 
Ae% 
percentage of ASP5354 excreted in urine 
ALP 
Alkaline Phosphatase 
ALT 
Alanine Transaminase 
AST 
Aspartate Transaminase 
ATC 
anatomical therapeutic chemical 
BMI 
body mass index 
CSR 
clinical study report 
%CV 
coefficient of variation 
DBP 
diastolic blood pressure 
ECG 
electrocardiograms 
FAS 
full analysis set 
Geo %CV 
geometric coefficient of variation 
GM 
geometric mean 
ICH 
international conference on harmonization 
IP 
investigational product 
IRT 
interactive response technology 
MedDRA 
Medical Dictionary for Regulatory Activities 
NCI-CTCAE 
National Cancer Institute - common terminology criteria for adverse events 
NIR-F 
near-infrared fluorescence 
PKAS 
pharmacokinetic analysis set 
PT 
preferred term 
SAE 
serious adverse event 
SAF 
safety analysis set 
SAP 
statistical analysis plan 
SBP 
systolic blood pressure 
SBR 
signal background ratio 
SD 
standard deviation 
SOC 
system organ class 
TEAE 
treatment-emergent adverse event 
TLF 
Tables Listings Figures 
ULN 
upper limit of normal 
VRC 
visualization review committee 
WHO 
world health organization 
 
 


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List of Key Terms 
Terms 
Definition of terms 
Baseline  
Assessments of subjects as they enter a trial before they receive any 
treatment.  
Endpoint  
Variable that pertains to the efficacy or safety evaluations of a trial. 
Enroll 
To register or enter a subject into a study.  
Intervention 
The drug, device, therapy or process under investigation in a clinical study 
that is believed to have an effect on outcomes of interest in a study (e.g., 
health-related quality of life, efficacy, safety, pharmacoeconomics).  
Investigational period 
Period of time where major interests of protocol objectives are observed, 
and where the test drug or comparative drug (sometimes without 
randomization) is usually given to a subject, and continues until the last 
assessment after completing administration of the test drug or comparative 
drug. 
Post investigational 
period 
Period of time after the last assessment of the protocol. Follow-up 
observations for sustained adverse events and/or survival are done in this 
period. 
Randomization 
The process of assigning trial subjects to treatment or control groups using 
an element of chance to determine assignments in order to reduce bias. 
Screening 
A process of active consideration of potential subjects for enrollment in a 
trial. 
Screen failure 
Potential subject who did not meet 1 or more criteria required for 
participation in a trial. 
Screening period 
Period of time before entering the investigational period, usually from the 
time when a subject signs the consent until just before the test drug or 
comparative drug (sometimes without randomization) is given to a 
subject.  
Study period 
Period of time from the first site initiation date to the last site completing 
the study. 
Study treatment  
ASP5354 
Variable 
Any entity that varies; any attribute, phenomenon or event that can have 
different qualitative or quantitative values. 


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1 
INTRODUCTION 
This Statistical Analysis Plan (SAP) contains technical and detailed elaboration of the 
principal features of the analysis described in the protocol, and includes procedures for 
executing the statistical analysis to fulfil the objectives of the study. 
The final SAP will be approved prior to first subject screened. 
Changes from the planned analyses in the final SAP that impact the statistical analyses will 
be documented in the Clinical Study Report (CSR).  
2 
STUDY OBJECTIVE(S) AND DESIGN 
2.1 
Study Objective(s) 
2.1.1 
Primary Objective 
The primary objective is to determine the optimal dose of ASP5354 for ureter visualization in 
subjects undergoing laparoscopic/minimally invasive colorectal surgery. 
2.1.2 
Secondary Objectives 
Secondary objectives are to investigate the safety and tolerability as well as pharmacokinetics 
of ASP5354 in subjects undergoing laparoscopic/minimally invasive colorectal surgery. 
2.1.3 
Exploratory Objectives 
Exploratory objectives are the following: 
● 
To investigate the relationship between pharmacokinetics and pharmacodynamics of 
ASP5354 in subjects undergoing laparoscopic/minimally invasive colorectal surgery. 
● 
To explore the fluorescence intensity and visualization duration of ASP5354 in subjects 
undergoing laparoscopic/minimally invasive colorectal surgery. 
● 
To explore the benefit of ASP5354 in subjects undergoing laparoscopic/minimally 
invasive colorectal surgery. 
2.2 
Study Design 
The study is a randomized open-label, dose-ranging study in adult subjects undergoing 
laparoscopic/minimally invasive colorectal surgery in which the need for anatomical 
visualization of the ureter is anticipated. 
Subjects will be randomly assigned to receive a single dose of ASP5354 (
,  or 
) 
which will be administered as an iv bolus to evaluate the anatomical visualization of the 
index ureter(s) (and contralateral ureter when feasible). The index ureter(s) is selected by the 
investigator before surgery. The opposite ureter is the contralateral ureter, if applicable. 
Safety, tolerability and pharmacokinetics in the study population will also be assessed. 
Up to 15 subjects will be randomly assigned at each dose level.  
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Table 1 
Dose Arms, ASP5354 dose levels and number of randomized subjects 
Dose Arm 
ASP5354 Dose Level 
Number of Subjects 
1 
 
Up to 15 
2 
 
Up to 15 
3 
 
Up to 15 
Total 
- 
Up to 45 
 
 
 
Additional Dose Arm 
ASP5354 Dose Level 
Number of Subjects 
4 
 
Up to 15 
5 
 
Up to 15 
6 
 
Up to 15 
* Based on Visualization Review Committee (VRC) review of the initial 3 dose levels, if 
none of the doses selected have visualization, then 
 and 
 dose levels will be added; 
if 1 dose selected has visualization, then 
 dose level will be added. The 
 dose level 
will only be added if only 
 dose level has visualization. 
During a standard minimally invasive surgery, visualization of the surgical field will be 
assessed following the placement of the NIR-F imaging system (with FDA 510[k] cleared 
optical device system) proximal to the ureter of interest and then ASP5354 will be 
administered. 
The visualization data will be assessed by a VRC, consisting of the investigator(s) and the 
sponsor’s medical representative. The VRC will be held at the following time points, but ad-
hoc meetings may occur until the optimal dose is determined: 
1. After the initial 3 subjects have completed the surgical procedures at each of the dose 
levels; and 
2. then in increments of 3 subjects (up to a total 15 subjects per dose level) for those who 
have completed surgical procedures at the expanded dose levels. 
The visualization data for the subjects, at each interim time point, who completed the surgical 
procedure will be assessed to decide whether to: 
1. expand a dose under evaluation; or 
2. stop a dose under evaluation in which case additional subjects would not be added to a 
dose level; or 
3. define a dose as optimal. 
Once the optimal dose is determined, the optimal dose cohort may continue enrollment up to 
the maximum 15 subjects.  
The VRC assessment to stop, to continue or to define an optimal dose level will be based on 
the totality of the collected data, including but not limited to the following: 
1. anatomical visualization of the ureter at both 30 minutes after ASP5354 administration 
and at the end of surgery; or 
2. fluorescence intensity based on the Likert Scale (0 to 3). 
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In the case where 2 doses perform equally, the lower dose may be selected. 
Based on VRC review of the initial 3 dose levels, if none of the doses selected have 
visualization, then 
 and 
 dose levels will be added; if 1 dose selected has 
visualization, then the 
 dose level will be added. The 
 dose level will only be 
added if only the 
 dose level has visualization. For further details of the VRC, refer to 
the VRC charter. 
2.3 
Randomization 
This is an open-label study; however, enrollment, randomization and dispensation of 
investigational product (IP) will be performed via the interactive response technology (IRT) 
system.  
The treatment is assigned in a 1:1:1 ratio to: 
● 
 ASP5354 
● 
 ASP5354 
● 
 ASP5354 
Doses of  
 and/or 
 will be added if needed as outlined in SAP Section 2.2. Prior to 
initiation of treatment, study site personnel will obtain the randomization number and 
treatment assignment from the IRT system. Specific IRT procedures will be described in the 
respective study manual. 
No stratification factors will be used. 
3 
SAMPLE SIZE 
This sample size is not based on statistical power calculation. The sample size is expected to 
provide adequate information to determine the optimal dose of ASP5354 for ureter 
visualization. 
Up to 15 subjects will be randomly assigned at each dose level. This results in a total 
expected sample size of a maximum of 45 subjects. In case, additional dose groups are added, 
sample size can increase to a maximum of 90 subjects (15 subjects in each of the six possible 
doses). 
4 
ANALYSIS SETS 
In accordance with International Conference on Harmonization (ICH) recommendations in 
guidelines E3 and E9, the following analysis sets will be used for the analyses. 
The determination of whether subjects are included or excluded from the analysis sets except 
pharmacokinetic analysis set (PKAS) will be made prior to database hard-lock. 
4.1 
Full Analysis Set  
The full analysis set (FAS) will consist of all subjects who receive ASP5354 and have at least 
1 assessment of ureter visualization during surgery. This will be the primary analysis set for 
efficacy analyses. 
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4.2 
Safety Analysis Set 
The safety analysis set (SAF) consists of all subjects who receive ASP5354. The SAF will be 
used for all summaries of the safety data, unless otherwise specified. 
4.3 
Pharmacokinetic Analysis Set 
The PKAS consists of all subjects who receive ASP5354 for which at least 1 plasma or urine 
concentration data are available with the time of dosing and sampling. Inclusion of subjects 
in the PKAS with important protocol deviations will be considered by the pharmacokineticist 
on a case-by-case basis. 
The PKAS will be used for all summaries of the pharmacokinetic data. 
5 
ANALYSIS ENDPOINTS  
5.1 
Primary Efficacy Endpoint 
The primary endpoint is the anatomical visualization of the index ureter(s) 30 minutes after 
dosing of ASP5354 and the end of surgery. 
The incidence of anatomical visualization of ureter(s) will be assessed at approximately 10, 
20, 30, 45 and 60 minutes postadministration of ASP5354, and then every 30 minutes 
thereafter and/or at the last time point before removal of visualization instruments (end of 
surgery) by the investigator intraoperatively using a binary “Yes” or “No” question on the 
ability to visualize the ureter: “Can the ureter be adequately visualized with near-infrared 
fluorescence (NIR-F)?” 
● 
If the answer to the question 30 minutes after ASP5354 administration and at the end of 
surgery is equal to “Yes”, then anatomical visualization of the index ureter(s) was a 
success. 
● 
All other cases will be handled as “Not a success”. 
If both ureters are index per investigator, both need to have successful visualization for a 
subject to have successful visualization. 
In the case that if the anatomical visualization of the index ureter(s) was not assessed at 
30 minutes after ASP5354 administration, the answer will be imputed as follows: 
● 
If the answer at the nearest time points before and after 30 minutes both = Yes, then the 
answer to the question 30 minutes after ASP5354 administration will be imputed as = 
Yes. 
● 
If the answer at the nearest time points before and after 30 minutes both = No, then the 
answer to the question 30 minutes after ASP5354 administration will be imputed as = 
No. 
● 
All other cases will be handled as “Not Done” at 30 minutes after ASP5354 
administration, and imputing will not occur. 


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5.2 
Secondary Efficacy Endpoints 
Since no efficacy endpoint is specified as secondary endpoint, it is not applicable. 
5.3 
Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are the following: 
● 
The Likert Scale for qualitative response about the intensity of fluorescence at the 
predefined time points during surgery 
● 
Duration of anatomical visualization of the index ureter(s) from the first time of positive 
visualization to the last time point of positive visualization 
● 
Signal background ratio (SBR) 
● 
Binary questions (Yes/No) for the benefit of ASP5354 
● 
Ability to visualize the contralateral ureter when amenable to visualization 
5.4 
Safety Endpoints 
Safety and tolerability endpoints include adverse events (AEs), laboratory test results, vital 
signs, electrocardiograms (ECGs) and findings in physical examination. 
5.4.1 
Adverse Event 
AE will be assessed by evaluation of the following variables: 
● 
Treatment-emergent adverse events (TEAEs; frequency, severity, seriousness, and 
relationship to study drug) 
○ TEAE is defined as an AE observed after ASP5354 administration and up to 
the follow-up period (up to Day 10). 
○ If the AE occurs on Day 1 and the onset check box is marked “Onset after first 
study drug taken” or the both onset check boxes “Onset before first study drug 
taken” and “Onset after first study drug taken” are marked or left blank, then 
the AE will be considered treatment emergent.  
○ If the AE occurs on Day 1 and the onset check box is marked “Onset before 
first study drug taken”, then the AE will not be considered treatment 
emergent.  
○ If a subject experiences an event both during the pre-investigational period 
and during the investigational period, the event will be considered as TEAE 
only if it is reported with a new start date (i.e., as a new AE).  
○ Any AEs with onset dates completely missing will be considered TEAEs in 
summaries unless the investigator has noted on the CRF the event began prior 
to treatment. AEs with partially missing onset dates will be assumed TEAEs 
unless the available portion of the date indicates that the onset was strictly 
before start of study medication. 
○ A drug-related TEAE is defined as any TEAE with possible relationship to 
study treatment as assessed by the investigator or with missing assessment of 
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● 
Serious adverse events (SAEs) include adverse events that are flagged as serious by the 
investigator on eCRF, or upgraded by the Sponsor based on review of the Sponsor’s list 
of Always Serious terms. 
5.4.2 
Clinical Laboratory Variables 
Refer to protocol sections 7.2.2 and protocol appendix 12.7 for a table of the laboratory tests 
that will be performed during the conduct of the study. Refer to the protocol schedule of 
assessments for the evaluations’ schedule.  
5.4.3 
Vital Signs, Height and Weight 
Vital signs will include blood pressure (systolic blood pressure (SBP) and diastolic blood 
pressure (DBP)), pulse and respiratory rate. Vital signs, height and weight will be measured 
as indicated by the protocol schedule of assessments. 
5.4.4 
Routine 12-lead electrocardiograms 
A routine 12-lead ECG will be performed at the time points outlined in the protocol schedule 
of assessments. ECGs will be taken in triplicate. 
To calculate QTcF (corrected QT interval using Fridericia’s formula) the following formula 
will be applied: 
𝑄𝑇𝐶𝐹= 𝑄𝑇
√𝑅𝑅
1 𝑠
3
 
 
5.4.5 
Physical Examination 
Physical examinations will be performed as indicated in the protocol schedule of assessments 
and whenever there is a medical indication. On day 1 (pre- and postoperatively) and at the 
follow-up visit, a symptom-directed physical examination will be performed. If clinically 
significant worsening of findings from baseline is noted, the changes will be documented as 
AEs on the AE eCRF. 
5.5 
Other Endpoints 
5.5.1 
Pharmacokinetic Endpoints 
Pharmacokinetic endpoints are: 
● 
Plasma and urine concentrations of ASP5354 
● 
Amount of ASP5354 excreted in urine (Ae) during surgery 
● 
Percentage of ASP5354 excreted into urine (Ae%) during surgery 
5.5.2 
Pharmacodynamic Endpoints 
Pharmacodynamic variables will include the primary endpoint (anatomical visualization of 
the index ureter) and exploratory efficacy endpoints (Likert Scale, visualization duration and 
SBR).  


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5.5.3 
Green Discoloration of Urine 
The incidence and the duration of green discoloration of urine will be analyzed. It will be 
recorded any time after dose administration. If it occurs, the color of urine will be assessed 
during surgery and every 120 minutes (± 30 minutes window) after the end of surgery until it 
resolves or the subject is discharged, whichever is earlier. The start and stop date and time 
will also be recorded. 
6 
STATISTICAL METHODOLOGY 
6.1 
General Considerations 
Continuous data will be summarized descriptively including the number of subjects, mean, 
standard deviation (SD), median, minimum and maximum. In addition, for continuous PK 
variables, and PK parameters, coefficient of variation (%CV), geometric mean (GM), and 
geometric coefficient of variation (Geo %CV) will also be calculated. GM and Geo %CV 
will not be calculated if at least one value is below the quantification limit (BQL). 
Categorical data will be summarized by frequencies and percentages. Percentages by 
categories will be based on the number of subjects with no missing data, i.e. the percentages 
for the non-missing categories will add up to 100%. All non-coded free-text variables will be 
displayed in data listings only. 
Summaries based on FAS (e.g., disposition, baseline characteristics and efficacy endpoints), 
safety summaries based on SAF and summaries based on PKAS will be presented by actual 
treatment dose received.  
All data summarization and analyses will be performed using SAS® Version 9.4 or higher on 
Red Hat Enterprise Linux. Specifications for table, figures, and data listing formats can be 
found in the TLF specifications document for this study. 
Unless otherwise specified, all summaries will be presented by dose group. Subjects who 
were randomized before determination of optimal dose are grouped by the dose received. 
Additional subjects who were enrolled after determination of optimal dose are in a separate 
group. Apart from the overall group, a separate overall group for the optimal determined dose 
will be presented when specified in the TLF specifications document. 
6.2 
Study Population 
In general, data such as patient disposition, demographics and baseline characteristics will be 
summarized for FAS and SAF by dose group and overall, unless specifically stated 
otherwise. In the event when FAS is identical to SAF (i.e., all randomized subjects have at 
least 1 assessment of ureter visualization during surgery), then these data summaries will not 
need to be repeated for SAF. 
6.2.1 
Disposition of Subjects 
Disposition of subjects will be summarized for the FAS and SAF by dose group and overall, 
unless otherwise specified. The following analyses will be performed: 


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● 
Number and percentage of subjects with informed consent, discontinued prior to 
randomization (screen failures), randomized for all subjects with informed consent.  
● 
Number and percentage of subjects who were randomized, took IP, did not take IP 
(including the reason why IP was not taken), in each analysis set by dose group and 
overall for all randomized subjects.  
● 
Number and percentage of subjects who completed/discontinued the screening period, 
and primary reason for discontinuation for all subjects with informed consent. 
● 
Number and percentage of subjects who completed and discontinued the study, and 
primary reason for discontinuation by dose group and overall for all randomized 
subjects, FAS and SAF.  
6.2.2 
Protocol Deviations 
The number and percentage of subjects with the following important protocol deviation 
criteria will be summarized for each criterion and overall, by dose group and total as well as 
by investigative site, for all randomized subjects. Subjects deviating from a criterion more 
than once will be counted once for the corresponding criterion. Any subjects who have more 
than one important protocol deviation will be counted once in the overall summary.  
The unique identifiers will be as follows: 
PD1 – Inclusion/Exclusion,  
PD2 – Withdrawal Ciriteria, 
PD3 – Study Intervention, 
PD4 – Excluded Concomitant Medication, 
PD5 – Informed Consent, 
PD6 – Safety Reporting, 
PD7 – Procedures/Tests. 
A data listing will be provided by investigative site and subject. 
6.2.3 
Demographic and Other Baseline Characteristics 
Demographic variables (sex, age, ethnicity, race), age categories (>=18 to <65, >=65), 
EudraCT age categories (>=18 to <65, <=65 to <85, >=85), height, weight and body mass 
index (BMI), BMI categories (< 25, >=25 to <30, >=30 to <40, >=40) will be summarized by 
dose group and overall for FAS and SAF.  
Medical history is coded in Medical Dictionary for Regulatory Activities (MedDRA), and 
will be summarized by System Organ Class (SOC) and Preferred Term (PT) by dose group 
and overall for the SAF. 
Drug and alcohol history and medical history for each subject will be listed. 


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6.2.4 
Previous and Concomitant Medications 
Previous medications are defined as medications that patients started and ended prior to IP 
administration. Concomitant medications are defined as any medications that patients took at 
or after the day of IP administration. Medications that started prior to IP administration and 
continued after IP administration will be counted in both previous and concomitant 
medications.  
Previous and concomitant medications will be summarized in separate tables by therapeutic 
subgroup (anatomical therapeutic chemical (ATC) 2nd level) and chemical subgroup (ATC 
4th level) and preferred world health organization (WHO) name by dose group and overall 
for the SAF. Subjects taking the same medication multiple times will be counted once per 
medication. A medication that can be classified into several chemical and/or therapeutic 
subgroups is presented in all chemical and therapeutic subgroups. 
All previous and concomitant medications will be presented in a listing. 
6.2.5 
Non-medication Therapies 
The frequency and percentage of subjects with previous and/or concomitant non-medication 
therapies will be summarized along with the reason for use for SAF by dose group and 
overall. 
All non-medication therapies will be presented in a listing. 
6.3 
Study Drugs Exposure and Compliance 
Since this is a single dose study, the analysis of duration of exposure is not applicable. 
Study drug dosing information which includes dosing date and time, total volume prepared 
and actual volume administered in ml, total test drug used in mg and the compliance in % 
will be listed only. 
6.4 
Analysis of Efficacy  
Efficacy analyses will be conducted on the FAS. No hypothesis testing will be performed. 
6.4.1 
Analysis of Primary Efficacy Endpoint 
The primary endpoint is the anatomical visualization of the index ureter(s) 30 minutes after 
dosing of ASP5354 and the end of surgery. 
6.4.1.1 Primary Analysis for Primary Efficacy Endpoint  
The success rate of anatomical visualization of the index ureter(s) at 30 minutes after dosing 
and end of surgery, will be estimated with exact 95% confidence interval (CI) by dose group. 
SAS code for constructing 95% confidence interval for the success rate by Clopper-Pearson 
method is given as follows: 
proc freq; 
    tables success / binomial (level=’1’); 


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    exact binomial; 
run; 
where the numerical variable success is either 1, in case of success, or 0 otherwise. 
6.4.1.2 Secondary Analysis for Primary Efficacy Endpoint 
The frequency and percentage of anatomical visualization of the index ureter(s) will be 
summarized by dose group and all assessed time points (including 30 minutes after dosing 
and end of surgery). 
6.4.2 
Analysis of Secondary Efficacy Endpoints  
Since no efficacy endpoint is specified as secondary endpoint, it is not applicable. 
6.4.3 
Analysis of Exploratory Efficacy Endpoints 
Exploratory efficacy endpoints are listed in Section 5.5. 
The Likert Scale of the intensity of the fluorescence will be summarized by frequency and 
percentage by dose group and time point for index ureter(s), and if applicable, for the 
contralateral ureter. 
The duration of anatomical visualization of the index ureter(s) will be summarized using 
descriptive statistics by dose group. The duration of anatomical visualization of the index 
ureter(s) in minutes will be from the first time of positive visualization to the last time of 
positive visualization: 
𝐷𝑢𝑟𝑎𝑡𝑖𝑜𝑛= 𝑙𝑎𝑠𝑡 𝑡𝑖𝑚𝑒 −𝑓𝑖𝑟𝑠𝑡 𝑡𝑖𝑚𝑒+ 1 
If there are missing assessments of the anatomical visualization, then the last observed time 
of positive visualization will be used. 
Based on fluorescence images recorded during the entire surgery (predose and approximately 
10, 20, 30, 45 and 60 minutes postadministration of ASP5354, and then every 30 minutes 
thereafter and/or at the last time point before removal of visualization instruments), the SBR 
will be derived as follows: 
𝑆𝐵𝑅= 𝑚𝑒𝑎𝑛 𝑟𝑒𝑔𝑖𝑜𝑛 𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡
𝑚𝑒𝑎𝑛 𝑠𝑖𝑔𝑛𝑎𝑙 𝑏𝑎𝑐𝑘𝑔𝑟𝑜𝑢𝑛𝑑 
based on Hoogstins et al, 2019. The SBR for index ureter(s) will be summarized using 
descriptive statistics by dose group and time point. 
The investigator will be asked the following questions: 
● 
Was the location of the index ureter(s) as expected (Yes or No)? 
● 
Did visualization of the index ureter(s) occur with white light at 30 minutes after 
ASP5354 administration (Yes or No)? 
● 
Did visualization of the index ureter(s) occur with white light at the end of surgery (Yes 
or No)? 


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● 
Was NIR-F superior to white light in terms of visualization of the index ureter(s) (Yes or 
No)? 
● 
Did the location of the ureter visualized by NIR-F alter the operative plan (Yes or No)? 
● 
Were any ureter anomalies or abnormalities observed (Yes or No)? 
These binary questions for the benefit will be summarized by frequency and percentage by 
dose group. 
The success rate of anatomical visualization of the contralateral ureter at 30 minutes after 
dosing and end of surgery will be estimated with exact 95% CI by dose group (for SAS code, 
see Section 6.4.1.1), if applicable. In addition, the frequency and percentage of anatomical 
visualization of the contralateral ureter will be summarized by dose group and time point, if 
applicable. 
6.5 
Analysis of Safety 
Safety analyses will be conducted on the SAF by dose group and overall, unless otherwise 
specified. No hypothesis testing will be performed.  
6.5.1 
Adverse Events 
AEs will be coded using MedDRA and graded using the National Cancer Institute-common 
terminology criteria for AE (NCI-CTCAE, version 5.0). 
A TEAE is defined as an AE observed after administration of the IP and up to the follow-up 
period. An drug-related TEAE is defined as any TEAE with a causal relationship assessed as 
“yes” by the investigator. 
An overview summary table will include number and percentages of subjects with TEAEs, 
drug-related TEAEs, serious TEAEs, drug-related serious TEAEs, TEAEs leading to death, 
drug-related TEAEs leading to death and of all deaths. Frequencies and percentages of 
TEAEs and drug-related TEAEs by worst NCI CTCAE will be presented in the overview, 
too. 
The number and percentage of subjects with TEAEs, drug-related TEAEs, serious TEAEs, 
drug-related serious TEAEs will be summarized by SOC and PT. The number and percentage 
of TEAEs by NCI CTCAE grade will also be summarized. The worst toxicity grade will be 
summarized if the same AE is recorded more than once for a subject.  
All AE data will be listed. 
6.5.2 
Clinical Laboratory Evaluation 
Baseline is the last measurement taken prior to IP administration. 
For quantitative clinical laboratory measurements (hematology, biochemistry, urinalysis), 
descriptive statistics will be used to summarize results and change from baseline by dose 
group and time point (no overall presentation). 
Quantitative laboratory test results by NCI-CTCAE Grade (version 5.0) will be summarized 
with frequencies and percentages by time point. Shift from baseline to the postbaseline worst 
grade based on NCI-CTCAE until the follow-up period in laboratory tests will be tabulated. 


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All laboratory data will be listed. 
6.5.2.1 Liver Safety Assessment  
The liver safety assessments will be summarized by the following categories below based on 
the measurements from Alkaline Phosphatase (ALP), Alanine Transaminase (ALT), total 
bilirubin, Aspartate Transaminase (AST) and their combination.  
The subject’s highest value post-baseline of each parameter will be used; (upper limit of 
normal (ULN).   
● 
ALT: > 3xULN, > 5xULN, > 10xULN, > 20xULN 
● 
AST: > 3xULN, > 5xULN, > 10xULN, > 20xULN  
● 
ALT or AST: > 3xULN, > 5xULN, > 10xULN, > 20xULN 
● 
ALP: > 1.5xULN 
● 
Total Bilirubin: > 2xULN 
● 
(ALT or AST > 3xULN) and Total Bilirubin > 2xULN 
● 
(ALT or AST > 3xULN) and Total Bilirubin > 2xULN and ALP < 2xULN 
The last 2 criteria where 2 or more parameters are evaluated will use the measurements on 
the same day or up to 1 day apart. The number and percentage of subjects meeting the criteria 
post-baseline will be summarized. 
6.5.3 
Vital Signs 
Descriptive statistics will be used to summarize vital sign results and changes from baseline 
by dose group and time point. Baseline is the last measurement taken prior to IP 
administration. 
Tables for potentially clinically significant vital signs will be generated using baseline value 
and worst value obtained post-baseline for each subject. If the criterium is checking for 
values greater (or equal) a specific value, the worst value is referring to the highest value 
obtained. If the criterium is checking for values less (or equal) a specific value, the worst 
value is referring to the lowest value obtained. If the criterium is a combination of two 
conditions, the conditions need to be fulfilled for values obtained on the same post-baseline 
time points to meet the criterium. 
The following potentially clinically significant criteria are defined for each parameter: 
Vital Sign Variable 
Criteria 
SBP 
≥180 mmHg AND ≥20 mmHg change from baseline  
SBP  
< 80 mmHg 
DBP 
≥105 mmHg AND ≥15 mmHg change from baseline 
Pulse Rate 
≥120 bpm AND ≥15 bpm change from baseline 
All vital signs data will be listed. 


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6.5.4 
Electrocardiograms  
ECG will be taken in triplicate. The mean values of available triplicate results will be used in 
the analyses. 
Descriptive statistics will be used to summarize routine 12-lead ECG results and changes 
from baseline by dose group and time point. Baseline is the last measurement taken prior to 
IP administration. 
Number and percentage of subjects with normal and abnormal results for the overall 
interpretation will be tabulated by time point. A shift analysis table showing shift in overall 
ECG interpretation from baseline to each time point will be provided. Percent of subjects on 
different kind of abnormality will also be reported.   
The QT interval corrected by Fridericia's Correction formula (QTcF interval) will be 
summarized using frequency tables for values of clinical importance using the range criteria 
below. 
QTcF Interval Criteria 
QTcF Interval Value (msec) 
Normal 
Borderline 
Prolonged 
Clinically significant 
≤ 450 
> 450 to <=480 
> 480 to <=500 
> 500 
QTcF interval: Fridericia-corrected QT interval 
Cumulative tabulation for >450, >480 and >500 msec will also be presented. 
The QTcF interval will also be summarized by the frequencies of subjects with a change 
from baseline of clinical importance using the criteria identified below.  
Variable 
Change from Baseline 
QTcF Interval (msec) 
<=0 
>0 to <=30 
>30 to <=60 
> 60 
QTcF interval: Fridericia-corrected QT interval 
Baseline value is latest pre-dose assessment. Cumulative tabulation for >0, >30 and >60 msec 
will also be presented. 
All routine 12-lead ECG data and interpretations will be listed. 
6.5.5 
Pregnancies  
A listing of all pregnancy tests will be provided. 
6.6 
Analysis of Pharmacokinetics  
Descriptive statistics will be used to summarize plasma and spot urine concentrations of 
ASP5354 by dose group and time point. In addition, for each dose, descriptive statistics will 
be used to summarize Ae and Ae%. 


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All sampling dates, times, urine volumes and plasma and urine concentrations will be listed. 
Pharmacokinetics will be evaluated by a population pharmacokinetics approach using 
concentrations of ASP5354. All details of population analyses will be described in a separate 
analysis plan and a separate report will be written. 
6.7 
Analysis of Pharmacodynamics 
6.7.1 
Concentration-response Relationship Analysis 
The relationship between pharmacokinetics and anatomical visualization or other exploratory 
endpoints will be explored by a population pharmacokinetics/pharmacodynamics approach. 
All details of population analyses will be described in a separate analysis plan and a separate 
report will be written. 
6.8 
Other Analyses 
6.8.1 
Green Discoloration of Urine 
The frequency and percentage of patients who experience green discoloration of urine will be 
summarized by dose group. The duration of green discoloration in hours will be summarized 
using descriptive statistics by dose group. The duration is calculated from first date/time 
point of green discoloration to last date/time point of green discoloration. If the green 
discoloration is still present at discharge, the duration is from first date/time point of green 
discoloration to the date/time of discharge. 
Additional to descriptive statistics, if applicable, the duration will be analyzed using Kaplan 
Meier estimation. For this purpose, subjects with green discoloration of urine at discharge are 
censored at the time point of discharge.  
6.9 
Interim Analysis (and Early Discontinuation of the Clinical Study) 
The determination of dose(s) to stop, continue or define a dose as optimal will occur when 
every 3 subjects randomly assigned to each dose level have the data for anatomical 
visualization during surgery reviewed via the VRC.  
6.10 
Additional Conventions 
As a general principle, no imputation of missing data will be done. Exceptions are the start 
and stop dates of AEs and concomitant medications if they are missing (see Section 6.10.2). 
For the primary efficacy endpoint of anatomical visualization at 30 minutes after dosing and 
end of surgery, imputation will be performed in special cases when the assessment is not 
done at 30 minutes after dosing (see Section 5.1). 
6.10.1 Analysis Windows 
For efficacy analysis of anatomical visualization, assessments are summarized per time point 
mapped according to the following table: 


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Table 2 
Mapping actual assessments times to time points 
Time Point (post ASP5354 administration) 
Collection Window 
10 min 
5 min ≤ and < 15 min 
20 min 
15 min ≤ and < 25 min 
30 min 
25 min ≤ and < 38 min 
45 min 
38 min ≤ and < 53 min 
60 min 
53 min ≤ and < 75 min 
T min (every 30 minutes from 60 minutes) 
T-15 min ≤ and < T+15 min 
End of surgery 
- 
If more than one record is mapped to a specific time point, the record which is closest to the 
scheduled time point will be used in the analysis. In case of ties, the earlier one will be used. 
For safety analyses,  no mapping to analysis windows will be performed. The visit as 
collected in CRF will be used as analysis visit. Baseline is the last non missing assessment 
before ASP5354 administration. 
In case of unscheduled assessments on Day 1 for vital signs, they will not be taken into 
account to derive Baseline, because unscheduled vital signs assessments do not provide 
information about the assessment time. 
In safety analyses of post-baseline results (i.e. potentially clinical significant vital signs), not 
by time point, both scheduled and unscheduled assessments will be included to define the 
worst case. See also Section 6.5.2, Section 6.5.3 and Section 6.5.4. 
Both scheduled and unscheduled assessments will be listed. 
6.10.2 Imputation Rules for Incomplete Dates 
In case of missing partial start and stop dates for concomitant medications, the following 
rules will be used: 
If the start date is missing or partial: 
● 
if the month is missing, use January 
● 
if the day is missing, use the first day of the month under consideration 
● 
if the year is missing, use year of the informed consent date 
● 
if the entire date is missing then: 
a) If the concomitant medication did not start more than 28 days prior to treatment 
administration (operation day), use the informed consent date.  
b) Otherwise, use the 29th day prior to treatment administration. 
If the stop date is missing or partial and the concomitant medication is not ongoing then: 
● 
if the month is missing, use December 
● 
if the day is missing, use the last day of the month under consideration 
● 
if the year or the entire date is missing, set the stop date to December 31st, 2099 
If the imputed start date is after the stop date, then the imputed start date will be 1 day prior 
to the stop date.  


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For AEs, a missing or incomplete onset date will be imputed according to the following 
conventions. 
If only the year is known for the AE onset date, the imputed onset date will be the latest of 
the following non-missing dates: 
● 
Date of first dose of study drug  
● 
January 1 of the year of AE onset date  
If only the month and year is known for the onset date, set the surrogate onset date to the first 
day of that month and then apply the following rules. 
● 
If the month and year of the onset date is prior to the month and year of the first dose of 
study drug, then the surrogate onset date will be the imputed onset date. 
● 
If the month and year of the onset date is on or after the month and year of the first dose 
of study drug, then the imputed onset date will be the latest of the following non-missing 
dates: 
○ Date of first dose of study drug 
○ Surrogate onset date 
If the imputed onset date is after the adverse event end date, the imputed onset date will be 
the same as the adverse event end date. 
7 
REVISION AND RATIONALE 
All updates in the revision of SAP v1.0 only have clarification purpose, e.g. detailed visit 
windows and updated imputation rules for missing AE and concomitant medication dates. 
SAP v2.0 does not include major revisions compared to SAP v1.0.  SAP v3.0 includes 
updates of the protocol substantial amendment 1, updates of unique identifiers for protocol 
deviations and removal of mapping to analysis window for safety analyses. SAP v4.0 
includes updates of the protocol substantial amendment 2. 
8 
REFERENCES 
Hoogstins C, Burggraaf JJ, Koller M, Handgraaf H, Boogerd L, van Dam G, et al. Setting 
Standards for Reporting and Quantification in Fluorescence-Guided Surgery. Mol 
Imaging Biol. 2019;21:11-18. 
ICH Harmonized Tripartite Guideline E 3. Structure and Content of Clinical Study Reports, 
November 1995. (www.ich.org; Guidelines; "Efficacy" Topics) 
ICH Harmonized Tripartite Guideline E 9. Statistical Principles for Clinical Trials, 
February 1998. (www.ich.org; Guidelines; "Efficacy" Topics) 
9 
APPENDICES 
9.1 
Appendix 1: Author and Approver Signatures 
(E-signatures are attached at the end of document.)