An Open-Label Study of Two Different Doses of 3,3'-Dioxo-2,2'-Bisindolylidene-5,5'-Disulfonate Disodium 0.8% Solution When Used as an Aid for Ureteral Patency
Phase: PHASE3
Conditions: Ureter Injury
Sponsor: Prove pharm

Clinical Study Protocol 
An Open-Label, Randomized, Multicenter Study to Evaluate the 
Safety, Efficacy, Pharmacokinetics and Physician Satisfaction of two 
different doses of Indigo Carmine Injection 0.8% solution when used 
as an aid in the determination of ureteral patency. 
PVP-19IC01 
Development Phase: 
3 
Investigational Product: 
Indigo Carmine 0.8% Injection, USP 
Indication: 
Determination of ureteral patency 
Sponsor: 
Provepharm Inc.  
100 Springhouse Drive, Suite 105 
Collegeville PA 19426 
Telephone: 1-610-601-8600 
IND Number 
137856 
Protocol Date: 
1 July 2019 
Conduct: In accordance with the ethical principles that originate from the Declaration of 
Helsinki and that are consistent with The International Conference on Harmonization of 
Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) 
Guidelines on Good Clinical Practice (ICH E6 GCP) and regulatory requirements as 
applicable. 
CONFIDENTIAL INFORMATION 
This document is the sole property of Provepharm Inc. This document and any and all 
information contained herein has to be considered and treated as strictly confidential. This 
document shall be used only for the purpose of the disclosure herein provided. No disclosure 
or publication shall be made without the prior written consent of Provepharm Inc. 
NCT#: 04228445

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ABBREVIATIONS 
λz 
The Apparent Plasma Terminal Phase Rate Constant 
AAGL 
American Association of Gynecological Laparoscopists 
AE 
Adverse Event 
ALP 
Alkaline Phosphatase 
ALT  
Alanine Aminotransferase 
AST 
Aspartate Aminotransferase 
AUC 
Area Under the Curve 
BP 
Blood Pressure 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
Clast 
Concentration at Last Quantifiable Time Point 
Cmax 
Maximum Plasma Concentration 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Clinical Report Form 
CS 
Clinically Significant 
CSR 
Clinical Study Report 
CV 
Curriculum Vitae 
e.g. 
For Example 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
FDA 
Food and Drug Administration 
FPFV 
First Patient First Visit 
GCP 
Good Clinical Practices 

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GEE 
Generalized Estimating Equation 
GFR 
Glomerular Filtration Rate 
GLP 
Good Laboratory Practices 
GMP  
Good Manufacturing Practices 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
IC 
Indigo Carmine 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonization 
IM 
Intramuscular 
IMP 
Investigational Medicinal Product 
IRB 
Institutional Review Board 
ITT 
Intent to Treat 
IV 
Intravenous 
IVRS/IWRS 
Interactive Voice Response System/Interactive Web Response System 
LH 
Laparoscopic Hysterectomy 
m2 
Meters Squared 
MDRD 
Modification of Diet in Renal Disease 
MEDRA 
Medical Dictionary for Regulatory Activities 
mg 
Milligram 
mL 
Milliliter 
N 
Number 
N/A 
Not Applicable 
NCS 
Not Clinically Significant 

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NIMP 
Non-Investigation Medicinal Product 
PH 
Proportional Hazard Assumption 
PK 
Pharmacokinetics 
PSAS 
Physician Satisfaction Agreement Scale 
PV 
Pharmacovigilance 
QS 
Quantity Sufficient  
RBC 
Red Blood Cell 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SmPC 
Summary of Product Characteristics 
SoA 
Schedule of Assessments 
t½z 
The Terminal Half-Life 
TAH 
Total Abdominal Hysterectomy 
Tlast 
Time to Reach Last Quantifiable Time Point 
Tmax 
Time to Reach Maximum Plasma Concentration 
TTV 
Time from Drug Administration to Visualization 
TVH 
Total Vaginal Hysterectomy 
US 
United States 
USP 
United States Pharmacopoeia 
UOVS 
Ureteral Orifice Visualization Scale 
VS 
Vital Signs 
WBC 
White Blood Cell 
WOCBP 
Woman of Childbearing Potential 

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Table of Contents 
Sponsor Signature Page .................................................................................................................2 
ABBREVIATIONS ........................................................................................................................3 
1 
PROTOCOL SUMMARY .............................................................................................9 
1.1. 
Protocol Synopsis .............................................................................................................9 
1.2. 
Study Schema .................................................................................................................16 
1.3. 
Schedule of Assessments (SoA) .....................................................................................17 
2. 
INTRODUCTION ........................................................................................................18 
2.1. 
Study Rationale ...............................................................................................................18 
2.2. 
Background .....................................................................................................................19 
3. 
OBJECTIVES AND ENDPOINTS .............................................................................20 
3.1. 
Objectives .......................................................................................................................20 
3.1.1. 
Primary Objective ...........................................................................................................20 
3.1.2. 
Secondary Objectives ......................................................................................................20 
3.2. 
Endpoints ........................................................................................................................21 
3.2.1. 
Efficacy Endpoints ..........................................................................................................21 
3.2.2. 
Safety Endpoints .............................................................................................................21 
4. 
STUDY DESIGN ..........................................................................................................22 
4.1. 
Overall Design ................................................................................................................22 
4.2. 
Scientific Rationale for Study Design ............................................................................22 
4.3. 
Justification for Dose ......................................................................................................23 
4.4. 
End of Study Definition ..................................................................................................23 
5. 
STUDY POPULATION ...............................................................................................24 
5.1. 
Inclusion Criteria ............................................................................................................24 
5.2. 
Exclusion Criteria ...........................................................................................................24 
5.3. 
Screen Failures ................................................................................................................25 
6. 
STUDY DRUG ..............................................................................................................26 
6.1. 
Study Drug(s) Administered ...........................................................................................26 
6.2. 
Preparation/Handling/Storage/Accountability ................................................................27 
6.3. 
Measures to Minimize Bias: Randomization and Blinding ............................................27 
6.4. 
Study Drug Compliance..................................................................................................28 
6.5. 
Concomitant Therapy .....................................................................................................28 
7. 
DISCONTINUATION OF STUDY DRUG AND SUBJECT 
DISCONTINUATION/WITHDRAWAL ...................................................................29 
7.1. 
Subject Discontinuation/Withdrawal from the Study .....................................................29 
7.2. 
Lost to Follow up ............................................................................................................29 
8. 
STUDY ASSESSMENTS AND PROCEDURES .......................................................31 
8.1. 
Efficacy Assessments .....................................................................................................32 
8.1.1. 
Ureter Assessment ..........................................................................................................32 
8.1.2. 
Physician Satisfaction Agreement Scale .........................................................................33 
8.1.3. 
Videography specifics .....................................................................................................34 
8.2. 
Safety Assessments .........................................................................................................34 

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8.2.1. 
Physical Examinations ....................................................................................................34 
8.2.2. 
Vital Signs .......................................................................................................................34 
8.2.3. 
Electrocardiograms .........................................................................................................35 
8.2.4. 
Clinical Safety Laboratory Assessments ........................................................................35 
8.3. 
Adverse Events and Serious Adverse Events .................................................................36 
8.3.1. 
Time Period and Frequency for Collecting AE and SAE 
Information .....................................................................................................................36 
8.3.2. 
Method of Detecting AEs and SAEs ..............................................................................37 
8.3.3. 
Follow-up of AEs and SAEs ...........................................................................................37 
8.3.4. 
Regulatory Reporting Requirements for SAEs ...............................................................37 
8.3.5. 
Pregnancy ........................................................................................................................37 
8.4. 
Pharmacokinetics ............................................................................................................38 
9. 
STATISTICAL CONSIDERATIONS ........................................................................40 
9.1. 
Statistical Hypotheses .....................................................................................................40 
9.2. 
Sample Size Determination ............................................................................................40 
9.2.1. 
Table 1  Empirical Distribution of Proportion of Subjects in Each 
UOVS Categories and Effect Size between the IC Dose Group vs 
Saline...............................................................................................................................40 
9.2.2. 
Table 2  Number of Subjects Per Group Using Two-group Chi-
square Test Comparing Proportions in 3 Categories [1, 2] .............................................40 
9.3. 
Populations for Analyses ................................................................................................41 
9.4. 
Statistical Analyses .........................................................................................................42 
9.4.1. 
Type 1 Error Control for the Primary Efficacy Endpoint ...............................................42 
9.4.2. 
Treatment Effect Based on the Operating Surgeon’s UOVS 
Scores ..............................................................................................................................42 
9.4.3. 
Treatment Effect on UOVS Based on Central Rater’s 
Assessment ......................................................................................................................43 
9.4.4. 
Treatment Effect on Time to Visualization ....................................................................43 
9.4.5. 
Assessment of PSAS with IC Treatment ........................................................................43 
9.4.6. 
Assessment of Covariates ...............................................................................................44 
9.4.7. 
Safety Analysis ...............................................................................................................44 
9.4.8. 
PK Analysis ....................................................................................................................44 
10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS ....................................................................................................45 
10.1. 
Regulatory, Ethical, and Study Oversight Considerations .............................................45 
10.1.1. Regulatory and Ethical Considerations ...........................................................................45 
10.1.2. Financial Disclosure ........................................................................................................45 
10.1.3. Informed Consent Process ..............................................................................................46 
10.1.4. Institutional Review Board/Independent Ethics Committee (IRB) ................................46 
10.1.5. Data Protection................................................................................................................47 
10.1.6. Dissemination of Clinical Study Data .............................................................................47 
10.1.7. Data Quality Assurance ..................................................................................................48 
10.1.8. Source Documents ..........................................................................................................48 
10.1.9. Study and Site Start and Closure ....................................................................................49 
10.1.10. Publication Policy ...........................................................................................................49 

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10.2. 
Adverse Events: Definitions and Procedures for Recording, 
Evaluating, Follow-up, and Reporting ............................................................................50 
10.2.1. Definition of AE .............................................................................................................50 
10.2.2. Definition of SAE ...........................................................................................................51 
10.2.3. Recording and Follow-Up of AE and/or SAE ................................................................52 
10.2.4. Reporting of SAEs ..........................................................................................................54 
11. 
REFERENCES .............................................................................................................56 
 

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1 
PROTOCOL SUMMARY 
1.1. 
Protocol Synopsis 
 
Title 
An Open-Label, Randomized, Multicenter Study to Evaluate the Safety, 
Efficacy, Pharmacokinetics and Physician Satisfaction of two different 
doses of Indigo Carmine Injection 0.8% solution when used as an aid in the 
determination of ureteral patency. 
Phase  
3 
Objectives 
Primary Objective 
To determine whether the use of Indigo Carmine 0.8% Injection, USP 
solution for injection (IC) provides a visualization advantage compared to 
saline when used as an aid in the determination of ureteral patency. 
Visualization will be measured by a 3-point Ureteral Orifice Visualization 
Scale (UOVS):  
1 = non-visualization 
2 = inadequate/equivocal visualization 
3 = adequate/unequivocal visualization of the urine jet stream 
after administration of test agent 
Secondary Objectives 
1. To evaluate the safety profile of IC when used as an aid in the 
determination of ureteral patency.  
2. To describe the time to visualization (TTV) of ureteral jets during 
urological and gynecological surgical procedures  
3. To determine physicians’ overall satisfaction with the IC treatment 
by assessing the proportion of surgeons who agree using the 5-point 
Physician Satisfaction Agreement Scale (PSAS) with the statement:  
“Compared to the saline treatment, my ability to assess ureter patency 
was improved after the addition of IC.”  
1 = Strongly Agree 
2 = Agree 
3 = Neither Agree nor Disagree 
4 = Disagree  
5 = Strongly Disagree   
A surgeon’s evaluation is considered satisfactory if the rating is either a 
1 (strongly agree) or 2 (agree).    
4. To determine the IC pharmacokinetic profile in a subset of subjects 
from 2 investigational sites. 

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5. An exploratory comparison will be done on the difference between 
the IC high dose vs IC low dose.  Surgeons will be blinded to the 
dose of IC. 
Methodology 
This is an open-label, randomized, multicenter study to evaluate the 
efficacy, safety, and pharmacokinetics of two doses (2.5 mL and 5.0 mL) of 
Indigo Carmine (IC) 0.8% Injection, USP solution for injection when used 
as an aid in the determination of ureteral patency. Study will enroll up to 
116 subjects from approximately 10 study centers in the United States. 
Subjects scheduled for urological or gynecological surgical procedures, age 
18 to 85 years inclusive, will be screened for participation.  Screening will 
occur within 30 days before study drug administration (Day of Surgery).  
After signing the informed consent, medical history, physical examination, 
baseline laboratory testing, pregnancy testing, 12-lead ECG, and vital sign 
measurements will be completed during the screening visit. 
On the day of surgery (Day 1) subjects will be evaluated for eligibility for 
randomization.  Eligible subjects will be randomized to receive a dose of 
either IC high dose (5 mL) or IC low dose (2.5 mL).    All randomized 
subjects will serve as his/her own control by receiving a dose of normal 
saline prior to receiving the randomized IC dose.  The surgeon will be 
blinded to the IC dose a subject receives.  Time of injection of saline and IC 
will be captured.   
Assessment will be recorded by videography. To evaluate the efficacy 
outcomes, each subject will first be injected intravenously with 5 mL 0.9% 
saline. The ureteral orifices/flow will be observed for up to 10 mins or until 
adequate/unequivocal visualization has occurred (whichever occurs first).  
The time period that will be captured on video is from injection up to 10 
minutes or until adequate/unequivocal visualization of both ureter orifices. 
If both ureters cannot be visualized simultaneously, then alternating 15-30 
second images of each ureter or ureteral orifice will be obtained. The 
surgeon will rate his/her ability to visualize the ureteral jet stream indicating 
ureteral patency for each ureter according to the scale above.  The process 
will be repeated in the same patient for the IC dose.  Hence, each subject 
will have 4 assessments for the ureter patency measurement. 
After the procedure, the surgeon performing the procedure will provide 
his/her overall satisfactory assessment by rating his/her agreement with the 
statement “Compared to the saline treatment, my ability to assess ureteral 
patency was improved after the addition of IC” using the PSAS: 1=Strongly 
Agree, 2=Agree, 3=Neither Agree nor Disagree, 4=Disagree, 5=Strongly 
Disagree. 
The videos will be sent to a central imaging group who will pool and blind 
the videos. Videos will then be assessed by a blinded central reviewer for 
assessment of ureteral patency using the same 3-point UOVS. 

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In a subset of subjects from 2 sites (about 16 subjects), subjects will be 
consented to participate in the pharmacokinetic (PK) portion of the study.  
Once consented, 13 blood samples will be taken from each subject at the 
scheduled timepoints post IC treatment for PK analysis.  Urine and stool 
samples will also be collected for analysis in this PK group at specified time 
points. 
All treated subjects will have a follow-up visit 7 to 30 days (± 2 days) after 
the procedure.  A final telephone follow-up call will occur on Day 30 (± 2 
days) in subjects who have the follow-up visit before Day 28. 
Safety assessments will include monitoring of AEs during and post the 
procedure, clinical laboratory tests, 12-Lead ECG, and vital sign 
measurements.   
Study 
Medication 
Indigo Carmine 0.8% Injection, USP (Provepharm supplied) 
5 mL Pre-filled 0.9% Saline Syringes (Provepharm supplied)  
Number of 
Subjects 
Up to 116 subjects will be enrolled from approximately 10 sites, including 
16 subjects to participate in PK/metabolite analysis at 2 sites 
Length of 
participation 
Each subject participation is expected to be up to 60 days, to include: 
• Up to 30-day screening 
• Day of procedure 
• 30-day follow-up 
Dose, Dosage 
Form, and 
Route of 
Administration 
Each ampule of IC single-dose ampule contains 40 mg indigo carmine in 
5 mL water for injection QS   
Each subject will receive: 
- 5 mL 0.9% normal saline injected over 1 minute via IV administration and  
- 2.5 mL or 5 mL IC injected over 1 minute via IV administration (dose will 
be blinded except to unblinded administrator) 
Sample Size 
A total of 96 subjects will be enrolled in the study; 48 subjects randomly 
assigned to 2.5 mL IC and 48 subjects assigned to 5 mL IC. This sample 
size calculation was determined based on two-group Chi-square test 
comparing proportions in 3 categories at 0.05 significance level. 
The sample size does not account for dropouts, protocol deviations, 
withdrawal of consent, etc. Up to an additional 20% (20) subjects may be 
enrolled to account for protocol deviations, withdrawal of consent, etc.  
A subgroup of subjects at 2 sites (total N=16, 8 from each IC dose level) 
will be evaluated for pharmacokinetics/metabolites. 

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Inclusion 
Criteria 
I01.    Subjects between ≥ 18 and ≤ 85 years old 
I02.    Subjects who signed written, IRB approved, informed consent form 
I03.    Subjects scheduled for urological or gynecological surgical 
procedures in which the patency of the ureter must be assessed by 
the surgeon during the procedure 
Exclusion 
Criteria 
E01.   Subjects with stage 4 or 5 Chronic Kidney Failure as evidenced by a 
GFR <30 mL/min/1.73m2 (using the MDRD) or need for dialysis in 
the near future, or having only 1 kidney 
E02. Subjects with known severe hypersensitivity reactions to IC or other 
dyes including contrast agents 
E03. Known history of drug or alcohol abuse within 6 months prior to the 
time of screening visit 
E04. Subjects, as assessed by the Investigator, with 
conditions/concomitant diseases precluding their safe participation 
in this study (e.g. major systemic diseases); 
E05. Unable to meet specific protocol requirements (e.g., scheduled 
visits) or subject is uncooperative or has a condition that could lead 
to non-compliance with the study procedures 
E06. Subject is the Investigator or any Sub-Investigator, research 
assistant, pharmacist, study coordinator, other staff or relative 
thereof directly involved in the conduct of the protocol; 
E07. Subjects with life expectancy < 6 months; 
E08. Requirement for concomitant treatment that could bias primary 
evaluation. 
E09.    Subjects who are pregnant or breast feeding. 
Efficacy 
Endpoints     
Primary Efficacy Endpoint 
Surgeon’s assessment of each ureter’s patency by using the UOVS :   
1 = Not visualized – I cannot see the ureteral jet flow  
2 = Inadequately visualized or equivocal – I am less than 
completely confident that the ureter is patent  
3 = Adequately visualized or unequivocal – I am completely 
confident that the ureter is patent  
Secondary Efficacy Endpoints 
1. Time (minutes) to visualization (TTV) of the ureteral jets after each 
study drug administration 
2. Physician’s overall satisfaction with the IC treatment as an aid for 
the assessment of ureter patency 

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Other Endpoints 
1. UOVS scores assessed by the blinded central reviewer 
2. Concordance of the surgeons’ UOVS scores and the blinded central 
reviewer’s UOVS score. 
Safety 
Endpoints 
 Safety Endpoints included 
1. Incidence of treatment emergent adverse events 
2. Changes in safety laboratory tests and vital signs 
3. Changes clinically significant abnormal 12-ECG 
PK Samples 
The following PK samples will be collected from approximately 16 subjects 
from 2 study centers: 
1. Plasma samples will be collected prior to and post IC injection at 2, 
5, 7, 10, 15, 20, 30, and 40 mins, then at 1, 2, 3, and 4 hours after IC 
administration.  
2. Urine collection will occur by a voided sample within 1 hour prior 
to the surgery and post IC injection for the following time periods 0-
2 hours (including any urine drained during surgery), 2-6 hours, and 
6-12 hours. 
3. The first post-op stool will be collected and sent for analysis for IC 
and/or its breakdown products.  
PK 
Parameters  
The pharmacokinetic (PK) analysis will include at least the following 
parameters:  
1. AUC0-t, AUC0-t(90%subj), AUC0-∞  
2. AUCextr, AUC%extr,  
3. Cmax, Clast, Tmax, and Tlast  
4. λz: the apparent plasma terminal phase rate constant. 
5. t½z: the terminal half-life, where possible, calculated as 0.693/ λz. 
6. Total excretion in urine and in stool   
PK Analysis 
PK samples will be prepared to analyze for plasma concentration of IC and 
any major metabolites.  Non-compartmental analysis will be used to 
calculate PK parameters using the observed concentrations and actual 
sampling times.  All bioanalytical analyses will be completed to GLP 
standards using validated assays.  The Bioanalytical Report and PK Report 
will be provided as appendices of the final study report. 
Videography 
Subjects will have video recordings of the ureteral orifices beginning with 
the injection of 5 mL saline and ending up to 10 minutes after the injection 
of IC or once both ureters have been adequately visualized.  If both ureters 
cannot be visualized and recorded simultaneously then the camera will be 

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switched between ureters every 15-30 seconds until adequate/unequivocal 
visualization or 10 minutes have passed.  Once adequate/unequivocal 
visualization of a ureter has been obtained, then the camera may remain 
focused on the other ureter. 
Procedure 
Summary 
1. Subjects meeting all the inclusion and none of the exclusion criteria will 
be randomized to receive either 2.5 or 5 mL of IC injection. 
2. The videography will be completed during the ureteral patency check. 
3. The subject will be injected with 5 mL of 0.9% saline for injection 
intravenously over 1 minute, noting the time of injection, and the 
surgeon will assess the patency of the ureters by identifying the efflux 
of urine from the ureteral orifices for up to 10 minutes for each 
injection.   
a. If both ureteral jets cannot be assessed on camera 
simultaneously, then the camera will be switched between 
ureteral orifices every 15-30 seconds for the 10-minute 
observation period or until adequate/unequivocal visualization is 
observed.  Then camera may remain focused on the other 
ureteral orifice until adequate/unequivocal visualization is seen 
or the completion of the 10-minute observation period has 
lapsed. 
b. If visualization of the ureter is adequate/unequivocal and the 
time is less than 10 minutes, then the time of visualization is to 
be noted for each ureter and the video may be stopped. 
4. The surgeon will document the UOVS score assessing the patency of 
the ureters following the saline injection. 
5. After the normal saline observation period of up to 10 minutes, a 
blinded dose of IC (2.5 or 5 mL, based on randomization) will be 
administered over 1 minute intravenously by the non-surgeon unblinded 
administrator, noting the exact time of injection.   
6. Again, the surgeon will assess the patency of the ureters by identifying 
the efflux of blue urine from each ureteral orifice for up to 10 minutes 
using the same procedure as described above. 
7. Once identified, the surgeon will note the time of identification of efflux 
of the IV IC injection. 
8. The surgeon will document the UOVS score assessing the patency of 
the ureters following the IC injection. 
9. Subjects participating in the PK arm, at pre-determined time points will 
have blood drawn. All urine will be collected in separate containers for 
designated periods. Stool will be collected for all bowel movements 
within the first 24-hours post-surgery in this same subgroup. 

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10. The surgeon will document his/her overall satisfaction by completing 
overall Physician Satisfaction Assessment for each subject. 
11. Subjects will be followed for 30 days (± 2 days) for adverse events. 
12. The videos will be sent to a central imaging group who will pool and 
blind the videos. The videos will then be assessed by a blinded central 
reviewer for ureteral patency using the same UOVS. 
The concordance in the UOVS scores between the surgeon’s assessment 
and the central blinded reader will be evaluated.   
Statistical 
Analysis 
Methodology 
The statistical analysis for this study will be carried out using SAS® 9.3 or 
later and described in a detailed statistical analysis plan (SAP), which will 
be finalized prior to first patient first visit. Any deviations from the planned 
analysis as described in the SAP will be justified and recorded in the final 
clinical study report (CSR). 
References 
1. Barikmo K, Muffly TM, C. Faulkner IC et al.  Excretion of colored 
urine after intravenous injection of indigo carmine dye. Poster 23. 
Female Pelvic Medicine & Reconstructive Surgery 2010;16, Suppl, 25-
26 
2. Chi AM, Curran DS, Morgan DM et al. Universal cystoscopy after 
benign hysterectomy examining the effects of an institutional policy. 
Obstet Gynecol 2016;127:369 – 375 
3. Ibeanu O, Chesson RR, Echols KT et al. Urinary tract injury during 
hysterectomy based on universal cystoscopy. Obstet Gynecol 
2009;113:6–10 
4. Cited in NIH (National Institute of Health), National Institute of 
Diabetes and Digestive and Kidney Diseases.  Estimating glomerular 
filtration rate.  https://www.niddk.nih.gov/health-
information/communication-programs/nkdep/laboratory-
evaluation/glomerular-filtration-rate/estimating 
5. Vakili B, Chesson RR, Kyle BL et al. The incidence of urinary tract 
injury during hysterectomy: A prospective analysis based on universal 
cystoscopy Am J Obstet Gynecol 2005;192:1599 – 604 

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1.3. 
Schedule of Assessments (SoA) 
 
 
Screening 
period 
Day -30 to -
11 
Randomization/ 
Treatment 
Day 1 
Day 
2 
PK 
Follow-up 
Visit Day 7 
to Day 32 or 
Early 
Termination2 
Telephone 
follow-up 
when 
Follow-up 
visit 
occurred 
prior to 
Day 28 
Informed Consent 
X 
 
 
 
 
Inclusion / Exclusion Criteria 
X 
X 
 
 
 
Medical, surgical history, demography, 
medication review 
X 
 
 
 
 
Physical Examination 
X 
 
 
X 
 
Height 
X 
 
 
 
 
Weight 
X 
 
 
X 
 
Vital Signs 
X 
X3 
 
X 
 
12 Lead ECG 
X 
 
 
X 
 
IVRS/IWRS Contact 
X 
X 
 
X 
X 
Concomitant Medications 
X 
X 
 
X 
X 
AE/SAE 
 
X 
X 
X 
X 
Blood Safety Labs4 
X 
 
 
X5 
 
PK Blood6 
 
X 
X 
 
 
PK Urine7 
 
X 
X 
 
 
Bowel Prep8 
X (Day -1) 
 
 
 
 
Stool collection for assay9 (PK group) 
 
X 
X 
 
 
Surgery 
 
X 
 
 
 
0.9% saline injection 
 
X 
 
 
 
0.8% IC injection 
 
X 
 
 
 
Video filming of ureteral jet flow with 
saline and with IC  
 
X 
 
 
 
Urine Pregnancy Test10 
 
X 
 
 
 
                                                 
1 Bloodwork/ECG completed as part of the  pre-operatively work-up (within 30 days of surgery) will be acceptable for study use and will not 
have to be repeated for study participation. 
2 Subjects who have onsite visit between Day 7 and 27 may have safety follow-up completed with an additional telephone call between Day 28-
32. 
3 Continuous monitoring, including heart rate and rhythm, will be required during the procedure and in the immediate post-operative period, vital 
signs will be collected for the study at the following specific time points: immediately prior to each injection and 5 minutes after each injection 
and thereafter approximately every 15 minutes through 1 hour following the procedure 
4 Blood Safety Labs: hematology = White blood cell count (WBC), Red blood cell count (RBC), Hemoglobin, Hematocrit, platelets, differential 
blood count (Neutrophils, lymphocytes, monocytes, eosinophils, basophils). Serum chemistry = total bilirubin, AST, ALT, alkaline phosphatase 
(ALP), creatinine, blood urea nitrogen, sodium, chloride, potassium, bicarbonate, phosphorus, calcium, glucose, albumin. 
5 Must be performed at the same laboratory as the initial bloodwork. 
6 For a total of 16 Subjects at 2 planned sites blood plasma collection will occur prior to and post IC injection at 2, 5, 7, 10, 15, 20, 30, and 40 
mins, then at 1, 2, 3, 4 hours after IC administration. 
7 For  a total of 16 Subjects at 2 planned sites urine collection will occur by a voided sample within 1 hour prior to the surgery and post IC 
injection for the following time periods 0-2 hours (including any urine drained during surgery), 2-6 hours, and 6-12 hours. 
8 Bowel prep will be required preoperatively (Day -1) for Subjects in the PK/Breakdown product collection patients. 
9 The first post-op stool will be collected and sent for analysis for IC and/or its breakdown products. 
10 Urine Pregnancy Tests will be done on day of surgery on all women of childbearing potential. 

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2. 
INTRODUCTION 
 
Indigo carmine (IC) was introduced into clinical practice in 1903 by Voelcker and Joseph and 
was originally used as a test of renal function (Lacy 1955).  In the 100+ years since then, indigo 
carmine has been used as a vital dye during surgery to identify vessels, tissues, and fistulae, and 
to help visualize ureteral urine ejection jets after urological or gynecological surgery.  All these 
indications rely on IC’s deep blue color to identify tissues and structures. Indigo carmine is a 
contrast stain that produces a very vivid coloration and is not absorbed by cells (Jung 1999).  
 
It is currently marketed as an unapproved drug product in the United States (US) but is approved 
in the United Kingdom, France, Germany, Belgium, Luxemburg, and the Netherlands.  The 
intention of this development program is to obtain an approval for indigo carmine when used as 
an aid in the determination of ureteral patency. 
 
2.1. 
Study Rationale 
Gynecologic and urologic surgery may lead to injury of the ureters and bladder because of the 
close anatomic locations of these structures to other genitourinary structures. Although 
gynecologic and urologic surgeons are highly trained in surgical techniques to reduce the 
occurrence of complications, urinary tract injuries still occur in many surgical procedures. The 
current estimates of urinary tract injuries with all types of gynecologic surgery range from 0.2 to 
15 per 1000 cases. Two of the largest reported series of hysterectomies suggest that urinary tract 
injuries are more common with laparoscopic hysterectomy (LH) than with abdominal (total 
abdominal hysterectomy (TAH)) or total vaginal hysterectomy (TVH) (AAGL 2012). Clinical 
experience and published literature of case studies suggest that intraoperative detection and 
repair of urinary tract injuries significantly reduces morbidity and improves outcomes after such 
complications of gynecologic surgery.  
 
Most studies of laparoscopic surgery with known risk for lower urinary tract injury have 
included an evaluation for the recognition of urinary tract injuries using intraoperative 
cystoscopy. These studies have demonstrated that many urinary tract injuries are not recognized 
at the time of hysterectomy when specific measures are not taken to confirm ureteral patency and 
absence of bladder injury (AAGL 2012). In a study by Gilmour et al (Gilmour 2006) less than 
50% of cases of ureteral injuries were detected intraoperatively when intraoperative cystoscopy 
was not performed. For bladder injuries, less than 25% of cases were detected intraoperatively 
when intraoperative cystoscopy was not performed. When intraoperative cystoscopy was 
performed at the time of laparoscopic hysterectomy (LH), 90-100% of ureteral injures and 80% 
of bladder injuries were detected intraoperatively. The current literature suggests that routine 
intraoperative cystoscopy at the time of LH is cost-effective, beneficial, and is of low risk to the 
patient.  
 

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The “gold standard” for detecting ureteral patency during gynecological and urological surgery is 
use of a blue dye, typically indigo carmine. However, due to drug shortages in the supply of 
indigo carmine to the US, some hospitals have utilized inferior alternative agents such as 
methylene blue, preoperative oral phenazo-pyridine and sterile water or a 10% dextrose solution 
(Barbieri 2014). The advantages of using indigo carmine compared to these other products is its 
excellent  safety profile and the fact that it is not absorbed by cells and is readily excreted giving 
surgeons a rapid indication of ureteral patency. 
 
2.2. 
Background 
Indigo carmine (IC) has a long history of use in intraoperative cystoscopy as the standard of care 
following surgeries with high risk of injury to the urinary tract such as in hysterectomies, pelvic 
organ prolapses, and anti-incontinence operations. In the US, approximately 600,000 
hysterectomies, 226,000  prolapse repairs, and 135,000 stress incontinence operations are 
performed annually (Gilmour 2006). There have been very few adverse events reported in the 
literature with the use of IC use across applications.  
 
Cystoscopy is a procedure that allows for visual examination of the lower urinary tract in both 
males and females. Under direct visualization, the  cystoscope  is inserted into the urethra and 
advanced up to the bladder. A rigid or flexible cystoscope allows complete visual inspection of the 
urethra, bladder and ureteral orifices. Intraoperative injuries to the bladder and urethra can be seen, 
which then enables immediate repair. Ligation of the ureter manifests as failure of urine to be 
propelled into the bladder . Failure to identify a “jet” of urine emanating from the ureteral orifice 
intraoperatively suggests a ureteral injury. 
 
Identification of ureteral patency is done by visualizing ureteral ejection of blue dye after the 
intravenous injection of 5 mL of indigo carmine. Many clinicians also administer 5 mg of 
furosemide intravenously to hasten the excretion of the indigo carmine. If the ureter is patent, 
ejection of indigo carmine usually occurs 5 to 10 minutes after the intravenous infusion. Failure 
to see the dye within 20 to 30 minutes mandates further investigation by the surgeon. An 
intraoperative intravenous pyelogram or retrograde ureteropyelogram, and/or ureteral catheter 
placement is usually performed to verify ureteral integrity in these cases. Visualization of urinary 
jets may be seen without prior indigo carmine injection and indicates ureteral integrity. In many 
cases, the visualization of urinary jets is equivocal when indigo carmine or other agents to 
enhance detection of urinary jets is not administered  
 
Since indigo carmine is not metabolized, following IV administration it is quickly excreted by 
the kidneys . This allows for the urine to be sufficiently colored blue within approximately 10 
minutes (AAGL 2012).  
 

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3. 
OBJECTIVES AND ENDPOINTS 
3.1. 
Objectives 
3.1.1. 
Primary Objective 
To determine whether the use of Indigo Carmine 0.8% Injection, USP solution for injection (IC) 
provides a visualization advantage compared to saline when used as an aid in the determination 
of ureteral patency. Visualization will be measured by a 3-point Ureteral Orifice Visualization 
Scale (UOVS):  
1 = non-visualization 
2 = inadequate/equivocal visualization  
3 = adequate/unequivocal visualization of the urine jet stream after administration 
of test agent 
3.1.2. 
Secondary Objectives 
1. To evaluate the safety profile of IC when used as an aid in the determination of ureteral 
patency.  
2. To describe the time to visualization (TTV) of ureteral jets during urological and 
gynecological surgical procedures. 
3. To determine physician’s overall satisfaction with the IC treatment by assessing the 
proportion of surgeons who agree with the statement “Compared to the saline treatment, 
my ability to assess ureteral patency was improved after the addition of IC” using the 5-
point Physician Satisfaction Agreement Scale (PSAS):  
1 = Strongly Agree 
2 = Agree 
3 = Neither Agree nor Disagree 
4 = Disagree 
5 = Strongly Disagree 
A physician evaluation is considered satisfactory if the rating is either a 1 (strongly agree) 
or 2 (agree).    
4. To determine the IC pharmacokinetic profile in a subset of subjects from 2 
investigational sites. 
5. An exploratory comparison will be done on the difference between the IC high dose vs 
IC low dose. Surgeons will be blinded to the dose of IC. 
 

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3.2. Endpoints 
3.2.1. Efficacy Endpoints 
The primary efficacy endpoint is the surgeon’s assessment of ureteral patency by UOVS:   
1 = Not visualized – I cannot see the ureteral jet flow  
2 = Inadequately visualized or equivocal – I am less than completely confident 
that the ureter is patent  
3 = Adequately visualized or unequivocal – I am completely confident that the 
ureter is patent  
The secondary efficacy endpoints include: 
1. Time (minutes) to visualization (TTV) of the ureteral jets after each study drug 
administration 
2. Proportion of physicians who agree that compared to saline,  IC treatment improves 
visualization as an aid for the assessment of ureteral patency  
Other efficacy endpoints will include: 
1. UOVS scores assessed by the blinded central reviewer 
2. Concordance of the UOVS scores between the surgeons’ assessments and the blinded 
central reviewer assessments. 
3.2.2. Safety Endpoints 
The safety endpoints will include: 
1. Treatment emergent adverse events 
2. Proportion of subjects with clinically important changes in clinical safety laboratory tests 
after treatment 
3. Proportion of subjects with clinically important changes in vital signs after treatment 
4. Proportion of subjects with clinically important changes in ECG after treatment 
 

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4. 
STUDY DESIGN 
 
4.1. 
Overall Design 
This is a phase 3, prospective, multicenter, parallel-group study to evaluate the safety, efficacy, 
pharmacokinetics and physician satisfaction of Indigo Carmine injection 0.8% solution when 
used as an aid in the determination of ureteral patency.   
The study is unblinded for the primary comparison of saline vs IC due to the inherent properties 
of IC; each subject serves as his/her own control.  However, the surgeon is blinded to the IC dose 
received for assessing any effect on the determination of the ureteral patency. The subject is 
blinded to the randomized dose of IC treatment.  The central rater is blinded to the treatment and 
the IC dose when reviewing and rating the video. The preparer and administrator of IC will be 
unblinded. 
The study will be comprised of 3 periods: 
• Screening Period (up-to 30 days prior to surgery) 
o To coincide with the pre-operative testing period 
• Randomization / Dosing (Day 1) 
o Single doses of saline and IC will be administered on the day of dosing (i.e., 
the day of procedure) 
• 30-day Safety Follow-up Period 
o Safety follow-up procedures to coincide with postoperative follow-up 
 
4.2. 
Scientific Rationale for Study Design 
A recent review of the literature found that there were no prospective studies that directly 
demonstrated that IC produces an additive advantage as a visualization aid in determining 
ureteral patency in urological and gynecologic surgeries.  This clinical study will demonstrate 
that the addition of IC is additive in the identification of ureteral patency. A comparison of the 
use of the dye to an injection of 0.9% saline would allow physicians to directly compare the 
procedure without dye to the same procedure with the use of dye.   
Additionally, timing of the visualization of the dye is also broadly described in the literature.  
This study is designed to determine the mean time needed for the dye to be identified by the 
physician during a procedure.  This is important as excretion of the dye is thought to be 
concentration dependent, making the timing of the visualization of the dye in the bladder related 
to the dose administered (Oravisto, 1957). 
 

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4.3. 
Justification for Dose 
The 5 mL dose is the IV dose most frequently mentioned in the literature (AAGL, 2012; Gill, 
2001; Harris, 1997; Jabs, 2001; Jelovsek, 2007; Lee, 1996; O‘Brien, 1990; Pettit, 1994; Ribiero, 
1999; Speights, 2000)  and is the recommended dose in the label of the marketed unapproved  
US product and the approved non-US product (American Regent Package insert, 2017; Serb 
SmPC, 2015). 
 
 
 
 
 
 
 
4.4. 
End of Study Definition 
A subject is considered to have completed the study if he/she has completed all phases of the 
study including the follow-up safety procedures and 30-day safety check.  The end of study 
participation date for a subject is the date of the final safety follow-up visit.  
The end of the study date for the study is defined as the date of last scheduled procedure shown 
in the Schedule of Assessments from the last subject in the trial. 
 

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5. 
STUDY POPULATION 
 
Prospective approval of protocol deviations to recruitment and enrollment criteria, also known as 
protocol waivers or exemptions, is not permitted. 
 
5.1. 
Inclusion Criteria 
Subjects are eligible to be included in the study only if all the following criteria apply: 
I01.  Subjects between ≥ 18 and ≤ 85 years old 
I02.  Subjects who signed written, IRB approved, informed consent form 
I03.  Subjects scheduled for urological or gynecological surgical procedures in which the 
patency of the ureter must be assessed by the surgeon during the procedure 
 
5.2. 
Exclusion Criteria  
Subjects are excluded from the study if any of the following criteria apply: 
E01.   Subjects with stage 4 or 5 Chronic Kidney Failure as evidenced by a GFR <30 
mL/min/1.73m2 (using the MDRD11) or need for dialysis in the near future or having 
only 1 kidney 
E02. Subjects with known severe hypersensitivity reactions to IC or other dyes, including 
contrast dyes 
E03. Known history of drug or alcohol abuse within 6 months prior to the time of 
screening visit 
E04. Subjects, as assessed by the Investigator, with conditions/concomitant diseases 
precluding their safe participation in this study (e.g. major systemic diseases) 
E05. Unable to meet specific protocol requirements (e.g., scheduled visits) or subject is 
uncooperative or has a condition that could lead to non-compliance with the study 
procedures 
E06. Subject is the Investigator or any Sub-Investigator, research assistant, pharmacist, 
study coordinator, other staff or relative thereof directly involved in the conduct of 
the protocol 
E07. Subjects with life expectancy < 6 months 
E08. Requirement for concomitant treatment that could bias primary evaluation. 
                                                 
11 GFR (mL/min/1.73 m2) = 175 × (Scr)-1.154 × (Age)-0.203 × (0.742 if female) × (1.212 if African American) 

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E09. Subjects who are pregnant or breast feeding 
5.3. 
Screen Failures 
 
"Enrolled" means a subject's, or their legally acceptable representative’s, agreement to 
participate in a clinical study following completion of the informed consent process and the 
subject has been randomized to receive study drug. Potential subjects who are screened for the 
purpose of determining eligibility for the study, but fail to meet all inclusion/exclusion criteria  to 
participate in the study, or who are randomized but did not have a surgical procedure to assess 
ureteral patency are considered screen failures, unless otherwise specified by the protocol.  
Screen failures include subjects who consent to participate in the clinical study but are not 
subsequently dosed with IC. A minimal set of screen failure information is required to ensure 
transparent reporting of screen failure subjects to meet the Consolidated Standards of Reporting 
Trials (CONSORT) publishing requirements and to respond to queries from regulatory 
authorities. Minimal information includes demography, screen failure details, eligibility criteria, 
and any serious adverse event (SAE). Individuals who do not meet the criteria for participation in 
this study (screen failure) may not be rescreened.  Subjects may not be enrolled more than once 
nor receive study drug more than once. 
 

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6. 
STUDY DRUG 
 
Study drug is defined as any investigational drug(s), marketed product(s), placebo, or medical 
device(s) intended to be administered to a study subject according to the study protocol. 
For this study, it is the administration of 5 mL of 0.9% saline IV followed by the blinded dose of 
0.8% IC of either 2.5 mL or 5 mL. 
 
6.1. 
Study Drug(s) Administered 
 
ARM Name 
Control (open label) 
Low Dose (blinded) 
High Dose (blinded) 
Drug Name 
0.9% Saline 
Indigo Carmine 
Indigo Carmine 
Type  
Drug 
Drug 
Drug 
Dose 
Formulation 
Pre-filled Syringe 
Ampule 
Ampule 
Unit Dose 
Strength(s) 
0.9% saline for 
injection 
40 mg/ 5mL 
40 mg/5mL 
Dosage 
Level(s) 
5 mL 
2.5 mL 
5 mL 
Route of 
Administration 
IV injection over 1 
minute 
IV injection over 1 
minute 
IV injection over 1 
minute 
Use 
placebo-comparator 
experimental 
experimental 
IMP and 
NIMP 
NIMP 
IMP 
IMP 
Sourcing 
Provided centrally by 
the Sponsor 
Provided centrally by 
the Sponsor  
Provided centrally by 
the Sponsor  
Packaging and 
Labeling 
N/A 
Indigo Carmine 40 
mg/5 mL solution for 
injection is supplied in 
5 mL ampules 
Indigo Carmine 40 
mg/5 mL solution for 
injection is supplied in 
5 mL ampules.  
 
 
 

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6.2. 
Preparation/Handling/Storage/Accountability 
1. 
Both 0.9% Saline and IC are to be administered as IV injections.  The IC is not to be 
diluted.  The start and stop time for each injection is to be captured down to the 
second. 
2. 
IC is to be stored at room temperature. 
3. 
IC is packaged in 5 mL single dose ampules in type 1 brown glass and will be 
provided in a box of 10 ampules.  Each subject will be assigned 1 ampule. 
4. 
The investigator or designee must confirm appropriate temperature conditions have 
been maintained during transit for all study drug received and any discrepancies are 
reported and resolved before use of the study drug. 
5. 
Only subjects enrolled in the study may receive study drug and only authorized site 
staff may supply or administer study drug. All study drug must be stored in a secure, 
environmentally controlled, and monitored (manual or automated) area in accordance 
with the labeled storage conditions with access limited to the investigator and 
authorized site staff. 
6. 
The investigator, institution, or the head of the medical institution (where applicable) 
is responsible for study drug accountability, reconciliation, and record maintenance 
(i.e., receipt, reconciliation, and final disposition records). 
7. 
Further guidance and information for the final disposition of unused study drugs are 
provided in the Study Reference Manual. 
 
6.3. 
Measures to Minimize Bias: Randomization and Blinding 
 
IVRS/IWRS 
All subjects will be centrally assigned to randomized study drug using an Interactive Voice/Web 
Response System (IVRS/IWRS). Before the study is initiated, each site will be provided access and 
training on the enrollment and randomization procedures. 
Study drug will be dispensed during the procedure visit. One vial should be used per subject. 
Blinded IC 
dose using an 
unblinded 
administrator  
Subjects will be randomly assigned in a [1:1] ratio to the IC dose. Investigators will remain blinded to 
each subject’s assigned IC dose throughout the course of the study.  
In order to maintain this blind, an unblinded administrator will draw up and administer the IC.  Only 
the dose of IC is blinded as all subjects receive both 0.9% saline and IC. An unblinded witness will 
confirm the dose administered. 
There will be no dose unblinding permitted at the site level as all subjects will receive both 
0.9% saline and IC. 
In the event of a Quality Assurance audit, the auditor(s) will be allowed access to unblinded study 
drug records at the site(s) to verify that randomization/dispensing has been done accurately. 

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6.4. 
Study Drug Compliance 
The study drug will be administered during the surgical procedure; therefore, the subjects will 
receive study drug directly from the unblinded administrator, under medical supervision.  The 
date and time of each dose (a dose of saline followed by a dose of IC treatment) administered in 
the clinic will be recorded in the source documents and recorded in the CRF.  The randomized 
dose of IC treatment will be provided by IVRS/IWRS to the designated unblinded site staff (i.e. 
pharmacist, coordinator, anesthesiologist, and or operating room/procedure nurse). 
 
6.5. 
Concomitant Therapy 
Any medication or vaccine (including over the counter or prescription medicines, vitamins, 
and/or herbal supplements) that the subject is receiving at the time of enrollment or receives 
during the study must be recorded along with: 
• 
Reason for use 
• 
Dates of administration including start and end dates 
• 
Total Daily Dosage information 
The Medical Monitor should be contacted if there are any questions regarding concomitant or 
prior therapy.  Prior therapy includes medications a subject received within 30 days of study 
enrollment. 
 
6.5 
Prohibited Concomitant Medications/Foods/Supplements 
Prohibited medications, foods, and/or supplements would be anything that could discolor urine 
for example: 
Blue/Green 
Red 
Orange/Bright Yellow 
Brown/Black 
methylene blue 
warfarin 
multivitamins 
metronidazole 
amitriptyline 
rifampin 
isoniazid 
nitrofurantoin 
cimetidine 
phenazopyridine 
sulfasalazine 
chloroquine 
indomethacin 
ibuprofen 
riboflavin (vitamin B2) 
primaquine 
zaleplon 
dantron 
 
furazolidone 
methocarbamol 
pheninidione 
 
cascara 
metoclopramide 
nefopam 
 
levadopa 
triamterene 
clofazimine 
 
phenytoin 

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7. 
DISCONTINUATION OF STUDY DRUG AND SUBJECT 
DISCONTINUATION/WITHDRAWAL 
 
7.1. 
Subject Discontinuation/Withdrawal from the Study 
• 
A subject may withdraw from the study at any time at his/her own request or may be 
withdrawn at any time at the discretion of the investigator for safety, behavioral, 
compliance, or administrative reasons. This is expected to be uncommon for this 
study. 
 
• 
If a subject is discontinued from the study after the subject is randomized but before 
receiving the study drug, the subject will be classified as randomized not treated; 
reason for discontinuation will be collected and no other study procedures will be 
performed. 
 
• 
If a subject is discontinued after the subject has received study drug, at the time of 
discontinuing from the study, if possible, an early discontinuation visit should be 
conducted, as shown in the Schedule of Assessments (SoA). See SoA for data to be 
collected at the time of study discontinuation and follow-up and for any further 
evaluations that need to be completed. 
 
• 
If the subject withdraws consent for disclosure of future information, the sponsor may 
retain and continue to use any data collected before such a withdrawal of consent. 
 
• 
If a subject withdraws from the study, he/she may request destruction of any samples 
taken and not tested, and the investigator must document this in the site study records. 
 
7.2. 
Lost to Follow up 
A subject will be considered lost to follow-up if he or she repeatedly fails to return for scheduled 
follow-up visits and is unable to be contacted by the study site. 
 
The following actions must be taken if a subject fails to return to the clinic for a required study 
visit: 
• 
The site must attempt to contact the subject and reschedule the missed visit as soon as 
possible and counsel the subject on the importance of maintaining the assigned visit 
schedule and ascertain whether or not the subject wishes to and/or should continue in 
the study. 
• 
Before a subject is deemed lost to follow up, the investigator or designee must make 
every effort to regain contact with the subject (where possible, 3 telephone calls and, 
if necessary, a certified letter to the subject’s last known mailing address or local 

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equivalent methods). These contact attempts should be documented in the subject’s 
medical record. 
• 
Should the subject continue to be unreachable, he/she will be considered to have 
withdrawn from the study.  
Discontinuation of specific sites or of the study as a whole are handled as part of Section 10.1.9. 

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8. 
STUDY ASSESSMENTS AND PROCEDURES 
 
The study is set up in 3 phases.  There is a subset of subjects who will participate in the PK 
portion of the study, which will be described in Section 8.4. 
• 
Screening (up to 30 days before the procedure date) 
o Any procedure that is completed as part of the standard of care for the surgery 
is not required to be repeated during screening for participation in the study.   
o These procedures may pre-date the Informed Consent Process. 
o Once consented, the subject will be entered into IVRS/IWRS 
• 
Randomization/Treatment (Day 1/Day of Surgery/Treatment) 
o After a subject is confirmed to be eligible, the subject will be randomized via 
IVRS/IWRS. Randomization will be 1:1 ratio to be assigned to low dose (2.5 
mL IC) or high dose (5 mL IC).    
o Videography will occur by cystoscopy, robotically, or, if an open surgical 
procedure, by use of a video camera.  Recording will occur from immediately 
prior to the start of the 0.9% saline injection until 10 minutes after the 
completion of the IC injection or until adequate visualization/unequivocal has 
occurred for both ureters, whichever comes first. See Section 8.1.3 for 
additional details. 
o Dosing Step by step 
1. The subject will be injected with 5 mL of 0.9% saline for injection 
intravenously over 1 minute, noting the time of injection, and the surgeon will 
assess the patency of the ureters by identifying the efflux of urine from the 
ureteral orifices for up to 10 minutes for each injection.   
a. If both ureteral jets  cannot be assessed on camera simultaneously, then 
the camera will be switched between ureteral orifices every 15-30 seconds 
for the 10-minute observation period or until adequate/unequivocal 
visualization is observed.  Then camera may remain focused on the other 
ureteral orifice until adequate/unequivocal visualization is seen or the 
completion of the 10-minute observation period has lapsed. 
b. If visualization of the ureter is adequate/unequivocal and the time is less 
than 10 minutes, then the time of visualization is to be noted for each 
ureter and the video may be stopped. 
2. After the normal saline observation period of up to 10 minutes, IC (2.5 or 5 
mL, based on randomization) will be administered intravenously over 1 
minute, noting the exact time of injection.   

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3. Again, the surgeon will assess the patency of the ureters by identifying the 
efflux of blue urine from each ureteral orifice for up to 10 minutes.  If /when 
visualization of each ureter is adequate/unequivocal then the time of 
visualization is to be noted for each ureter and the procedure may continue.  
4. The surgeon will document the UOVS score with the use of IC in the 
assessment of the patency of the ureters.  
5. Pharmacokinetic samples will be collected in a subgroup of subjects at each 
dose level at pre-determined time points during the procedure after the 
administration of the IC and stool will be collected at specified time points in 
this same subgroup. 
6. The surgeon will document his/her overall satisfaction by completing  one 
Physician Satisfaction Assessment for each subject. 
7. Subjects will be followed for 30 days (± 2 days) for adverse events. 
 
• 
Study procedures and their timing are summarized in the SoA. Protocol waivers or 
exemptions are not allowed. 
• 
Immediate safety concerns should be discussed with the sponsor immediately upon 
occurrence or awareness to determine if the subject should continue or discontinue 
study drug. 
• 
Adherence to the study design requirements, including those specified in the SoA is 
essential and required for study conduct. 
• 
All screening evaluations must be completed and reviewed to confirm that potential 
subjects meet all eligibility criteria. The investigator will maintain a screening log to 
record details of all subjects screened and to confirm eligibility or record reasons for 
screening failure, as applicable. 
• 
Procedures conducted as part of the subject’s routine clinical management (e.g., blood 
counts) and obtained before signing of 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 defined. 
 
8.1. 
Efficacy Assessments 
8.1.1. 
Ureter Assessment 
Visualization and video recording of the ureteral orifices or the jet flow will be captured through 
time until flow is observed or for 10 minutes post-completion of each injection.  To capture this 
information the following will be recorded: 

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• 
Type of surgical procedure/visualization  
o Cystoscopic 
o Robotic 
o Open  
• 
Time each injection started 
• 
Time each injection completed 
• 
Time of visualization of the ureter jet flow at the ureteral orifice. 
• 
For any procedure requiring that fluid be instilled into the bladder, the amount of and 
type will be captured. 
• 
Type, amount, and timing of hydration and use of any diuretics during the procedure 
will be recorded including dosing times. 
• 
Each ureteral orifice will be assessed independently using UOVS  after each 
injection, once the urine jet flow through the ureter or at the ureteral orifice is 
observed, or for 10 minutes, whichever is shorter, following the completion of the 
injection. 
o 3-point UOVS 
1 = Not visualized – I cannot see the ureteral jet flow  
2 = Inadequately visualized or equivocal – I am less than completely confident 
that the ureter is patent  
3 = Adequately visualized or unequivocal – I am completely confident that the 
ureter is patent  
8.1.2. 
Physician Satisfaction Agreement Scale (PSAS) 
 
After the completion of the procedure, the surgeon will be asked to rate the experience of using 
IC for each patient using the PSAS: 
“Compared to the use of saline treatment, my ability to assess ureteral patency was improved 
after the addition of IC” 
1 = Strongly Agree 
2 = Agree 
3 = Neither Agree nor Disagree 
4 = Disagree 
5 = Strongly disagree 

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The surgeon is considered satisfied with the IC treatment if his/her rating is either a 1 (Strongly 
Agree) or a 2 (Agree); otherwise, the surgeon is considered unsatisfied with the IC treatment. 
8.1.3. 
Videography specifics 
Video recording will be conducted via cystoscopy, robotically or directly with a video camera 
for an open procedure such as open radical prostatectomy. Acquisition parameters will be 
provided to the study sites that detail the video equipment requirements, procedure and views. 
Sites will also receive training materials to assure alignment and consistency of the video 
acquisition.  
 Recording will begin prior to the saline injection and continue uninterrupted until 10 minutes 
after the IC injection or until both ureters have been adequately visualized, whichever comes 
first. If both ureteral orifices cannot be recorded simultaneously in the same field of view, the 
camera will alternate between the ureters approximately every 15-30 seconds until both have 
been adequately visualized. Once adequate visualization has been confirmed with one ureter, the 
camera may remain fixed on the other ureter until patency is verified or the 10-minute 
observation period is complete. 
The videos will be submitted electronically to a central imaging group, for anonymization and to 
confirm adequate image quality and adherence to the acquisition parameters. De-identified 
videos will be provided to a blinded central reviewer for patency assessment using the same 3-
point UOVS used by the surgeon. The TTV and response to the 5-point PSAS will not be 
included in the central reviewer assessments. Central reviewer data will be compiled and 
transferred at pre-defined intervals for evaluation of concordance with the surgeon’s assessment. 
 
8.2. 
Safety Assessments 
Planned time points for all safety assessments are provided in the SoA. 
8.2.1. 
Physical Examinations 
A complete physical examination will include, at a minimum, assessments of the Cardiovascular, 
Respiratory, Gastrointestinal, Urological/Gynecological (as appropriate) and Neurological 
systems. Height and weight will also be measured and recorded.  These will be repeated at the 
follow-up visit except for height.  Investigators should pay special attention to clinical signs 
related to previous serious illnesses. 
8.2.2. 
Vital Signs 
During screening and follow-up visits body temperature, pulse rate, respiratory rate, and blood 
pressure will be assessed. Blood pressure and pulse measurements should be preceded by at least 
5 minutes of rest for the subject in a quiet setting without distractions (e.g., television, cell 
phones). 
Continuous monitoring, including heart rate and rhythm, will be required during the procedure 
and in the immediate post-operative period, vital signs will be collected for the study at the 

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following specific time points: immediately prior to each injection and 5 minutes after each 
injection and thereafter approximately every 15 minutes through 1 hour following the procedure. 
Any clinically significant changes to vital signs or heart rhythm will be reported as an adverse 
event. 
Clinically significant vital sign changes are those which are not associated with the underlying 
disease, unless judged by the investigator to be more severe than expected for the subject's 
condition. 
8.2.3. 
Electrocardiograms 
12-lead ECG will be obtained using an ECG machine that automatically calculates the heart rate 
and measures PR, QRS, QT, and QTc intervals as part of the screening and follow-up visit. 
Initial ECGs will be assessed by an investigator, addition to the measurements, as normal or 
abnormal finding.  Abnormal findings will be rated as clinically significant (CS) or not clinically 
significant (NCS).  Follow-up ECGs will be compared to the original ECG for changes and if the 
changes are CS or NCS.   
Any clinically significant rhythm changes during surgery will require ECG follow-up or rhythm 
strip for self-limiting episodes during surgery.  Clinically significant changes in ECG or heart 
rhythm will be captured as an adverse event. 
8.2.4. 
Clinical Safety Laboratory Assessments 
Laboratory assessments will be done at the local level. The follow-up labs are to be done at the 
same laboratory as the screening labs. 
The following safety labs are to be obtained: 
• 
Hematology: white blood cell count (WBC), red blood cell count (RBC), hemoglobin, 
hematocrit, platelets, differential blood count (neutrophils, lymphocytes, monocytes, 
eosinophils, basophils).  
• 
Serum chemistry: total bilirubin, AST, ALT, alkaline phosphatase (ALP), creatinine, 
blood urea nitrogen, sodium, potassium, chloride, bicarbonate, phosphorus, calcium, 
glucose, albumin. 
The investigator must review the laboratory report, document this review, and record any 
clinically relevant changes occurring during the study in the AE section of the CRF. The 
laboratory reports must be filed with the source documents. Clinically significant abnormal 
laboratory findings are those which are not associated with the underlying disease, unless judged 
by the investigator to be more severe than expected for the subject's condition. 
All laboratory tests with values considered clinically significantly abnormal during participation 
in the study or within 30 days after the last dose of study drug should be repeated until the values 
return to normal or baseline or are no longer considered clinically significant by the investigator 
or medical monitor. 

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• 
If such values do not return to normal/baseline within a period of time judged 
reasonable by the investigator, the etiology should be identified, and the sponsor 
notified. 
• 
If laboratory values from non-protocol specified laboratory assessments performed at 
the institution’s local laboratory require a change in subject management or are 
considered clinically significant by the investigator (e.g., SAE or AE or dose 
modification), then the results must be recorded in the CRF. 
 
8.2.5 
Follow-up for Subjects in whom Ureteral Patency was Not Visualized or Poorly 
Visualized 
Subjects in whom ureteral patency was not visualized or was poorly visualized will be treated 
with appropriate follow-up as per the discretion of the surgeon.  The information regarding 
follow-up will be collected in the eCRF and narratives will be provided in the clinical study 
report (CSR). 
 
8.3. 
Adverse Events and Serious Adverse Events 
AEs will be reported by the subject (or, when appropriate, by a caregiver, surrogate, or the 
subject's legally authorized representative).  See Section 10.2 for Adverse Events: Definitions 
and Reporting. 
Adverse events that occur during the surgical procedure will be reported by the investigator or 
surgical staff designee. 
The investigator and any qualified designees are responsible for detecting, documenting, and 
recording events that meet the definition of an AE or SAE and remain responsible for following 
up AEs that are serious, or considered related to the study drug or study procedures. 
8.3.1. 
Time Period and Frequency for Collecting AE and SAE Information 
All SAEs will be collected from the start of the saline injection until the follow-up visit. 
Medical occurrences that begin before the start of study drug but after obtaining informed 
consent will be recorded on the Medical History/Current Medical Conditions section of the case 
report form (CRF) not the AE section. 
All SAEs will be recorded and reported to the sponsor or designee immediately and under no 
circumstance should this exceed 24 hours, as indicated in Section 10.2.3. The investigator will 
submit any updated SAE data to the sponsor within 24 hours of it being available. 
Investigators are not obligated to actively seek AEs or SAEs after conclusion of the study 
participation. However, if the investigator learns of any SAE, including a death, at any time after 
a subject has been discharged from the study, and he/she considers the event to be reasonably 
related to the study drug or study participation, the investigator must promptly notify the 
sponsor. 

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8.3.2. 
Method of Detecting AEs and SAEs 
The method of recording, evaluating, and assessing causality of AE and SAE and the procedures 
for completing and transmitting SAE reports are provided in Section 10.2. 
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. 
8.3.3. 
Follow-up of AEs and SAEs 
After the initial AE/SAE report, the investigator is required to proactively follow each subject at 
subsequent visits/contacts. All SAEs will be followed until resolution, stabilization, the event is 
otherwise explained, or the subject is lost to follow-up (as defined in Section 7.2). Further 
information on follow-up procedures is provided in Section 10.2. 
8.3.4. 
Regulatory Reporting Requirements for SAEs 
Prompt notification by the investigator to the sponsor of a SAE is essential so that legal 
obligations and ethical responsibilities towards the safety of subjects and the safety of a study 
drug under clinical investigation are met. 
The sponsor has a legal responsibility to notify both the local regulatory authority and other 
regulatory agencies about the safety of a study drug under clinical investigation. The sponsor 
will comply with country-specific regulatory requirements relating to safety reporting to the 
regulatory authority, Institutional Review Boards (IRB)/Independent Ethics Committees (IEC), 
and investigators. 
For all studies, safety reports must be prepared for suspected unexpected serious adverse 
reactions (SUSAR) according to local regulatory requirements and sponsor policy and forwarded 
to investigators as necessary. 
An investigator who receives an investigator safety report describing a SAE or other specific 
safety information (e.g., summary or listing of SAEs) from the sponsor will review and then file 
it in the Site Regulatory binder and will notify the IRB, if appropriate according to local 
requirements. 
8.3.5. 
Pregnancy 
Details of all pregnancies in female subjects will be collected after the start of study drug and 
until study end visit/follow-up telephone call. 
If a pregnancy is reported, the investigator should inform the sponsor within 24 hours of learning 
of the pregnancy. 
Abnormal pregnancy outcomes (e.g., spontaneous abortion, fetal death, stillbirth, congenital 
anomalies, ectopic pregnancy) are considered SAEs. 
 

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8.4. 
Pharmacokinetics 
PK samples of plasma, urine, and stool will be collected from 16 subjects at 2 participating sites 
who have agreed to participate and have been trained in the specific requirements for the study. 
8.4.1. 
Plasma samples  
• 
Approximately 2 mL will be collected for each measurement of plasma 
concentrations of indigo carmine prior to and post injection at 2, 5, 7, 10, 15, 20, 30, 
and 40 mins, then at 1, 2, 3, and 4 hours 
• 
Samples will be used to evaluate the PK of IC and its breakdown products. Each 
plasma sample will be divided into 2 aliquots (1 each for PK and a back-up). Samples 
collected for analyses of IC (plasma) concentration may also be used to evaluate 
safety or efficacy aspects related to concerns arising during or after the study. 
8.4.2 
Urine samples 
• 
A voided urine sample will be collected within 1 hour prior to surgery. 
• 
Post IC urine collection will include all urine voided/drained (if using catheter): 
o 
0-2 hours (this includes all urine drained during surgery) 
o 
2-6 hours 
o 
6-12 hours 
• 
Samples will be used to evaluate the PK of IC and its breakdown products. 
8.4.3 
Stool Samples 
• 
During the evening prior to surgery, the subject will undergo a bowel prep. 
• 
The first post-op stool will be collected and sent for analysis for IC and/or its 
breakdown products; stool will be collected for 24 hours 
Individual PK results will not be reported to investigative sites. 
The pharmacokinetic (PK) analysis will include at least the following parameters:  
1. 
AUC0-t, AUC0-t(90%subj), AUC0-∞  
2. 
AUCextr, AUC%extr  
3. 
Cmax, Clast, Tmax, and Tlast  
4. 
λz: the apparent plasma terminal phase rate constant. 
5. 
t½z: the terminal half-life, where possible, calculated as 0.693/ λz. 
6. 
Total excretion in urine and in stool   
PK samples will be prepared to analyze for plasma concentration of IC and any major 
metabolites.  Non-compartmental Analysis will be used to calculate PK parameters using the 

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observed concentrations and actual sampling times.  All bioanalytical analyses will be completed 
to GLP standards using validated assay methods.  The Bioanalytical Report and Pharmacokinetic 
Analysis Report will be provided as appendices to the final study report. 

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9. 
 STATISTICAL CONSIDERATIONS 
 
9.1. 
Statistical Hypotheses 
It is expected that the IC treatment will improve the ureter visualization with effect size greater 
or equal to 0.10 comparing to the normal saline using the 3-point UOVS measurement. 
 
9.2. 
Sample Size Determination 
The following empirical distribution table (Table 1) is created to illustrate this effect size; sample 
size is determined using the two-group chi-square test comparing the difference in proportions in 
3 categories (Table 2).     
9.2.1. Table 1  Empirical Distribution of Proportion of Subjects in Each UOVS 
Categories and Effect Size between the IC Dose Group vs Saline      
 
% subjects by Ureteral Orifice Visualization Scale [1] 
 
1 
2 
3 
Effect Size ∆² 
Saline (π1) 
0.30 
0.30 
0.40 
 
IC Low Dose (π2) 
0.10 
0.20 
0.70 
0.1009 [2] 
IC High Dose (π2) 
0.05 
0.10 
0.85 
0.2203 [3] 
[1] Score 1 = Not visualized; 2 = Inadequately visualized or equivocal; 3 = Adequately visualized or unequivocal. 
[2] Effect size between IC Low dose and Saline 
[3] Effect size between IC High dose and Saline  
Effect size, ∆² = Σ(π2j-π1j)²/[2(π2j+π1j)] 
9.2.2. Table 2  Number of Subjects Per Group Using Two-group Chi-square Test 
Comparing Proportions in 3 Categories [1, 2] 
 
 
 
 
 
 
[1] nQuery Advisor (version 7) 
[2] Sample size estimates did not account for the repeated measures from each subject. Hence, the actual study 
power could be greater than 80% since each subject is expecting to have 2 measurements under the same treatment 
and each subject is also serving as his/her own control. 
 
The study enrollment target is to have a total of 96 subjects who are randomized and treated with 
study drug: 48 subjects will be randomly assigned to 2.5 mL IC and 48 subjects assigned to 5mL 
Scenario 
1 
2 
Test significance level, α 
0.0500 
0.0500 
Number of categories, C 
3 
3 
Effect size, ∆² = Σ(π2j-π1j)²/[2(π2j+π1j)] 
0.2203 
0.1009 
Power (%) 
80.00 
80.00 
n per group 
22 
48 

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IC. This sample size calculation was based on two-group Chi-square test comparing proportions 
in 3 categories at the 0.05 significance level. The sample size does not account for dropouts, 
protocol deviations, withdrawal of consent, etc. Up to an additional 20% (20) subjects may be 
enrolled to account for protocol deviations, withdrawal of consent, etc.  
A subgroup of subjects at 2 sites (total N=16, 8 from each IC dose level) will be evaluated for 
pharmacokinetics/metabolites. 
Note: "Enrolled" means a subject's, or their legally acceptable representative’s, agreement to 
participate in a clinical study following completion of the informed consent process and the 
subject has been randomized to receive study drug. Potential subjects who are screened for the 
purpose of determining eligibility for the study, but failed to meet all inclusion/exclusion criteria 
to participate in the study, or who are randomized, but did not have a surgical procedure to assess 
ureteral patency are considered screen failures, unless otherwise specified by the protocol.  
 
9.3. 
Populations for Analyses 
The following populations are defined: 
Population 
Description 
Enrolled  
All subjects who sign the ICF 
Intend-to-Treat (ITT) 
Analysis Sets 
All randomized subjects will be included in the ITT Analysis set.  
The ITT set will include subjects randomized but not treated.   
Efficacy Analysis Set 
The efficacy analysis set includes all randomized subjects who 
have a surgical procedure to assess ureteral patency and who 
have received study drug (saline and IC dose).  This will be the 
primary population for efficacy assessments.  
Per Protocol Analysis Set 
All subjects from the Efficacy Analysis Set who did not have any 
major protocol deviation that may confound the interpretation of 
the efficacy assessment. This Analysis Set will serve as a 
confirmation analysis set for efficacy analysis.  who   
Safety Analysis Set 
All randomized subjects who received at least 1 dose of study 
drug. Subjects will be analyzed according to the drug they 
actually received should the randomized IC dose is different 
from the actual received IC dose. 
Pharmacokinetic Analysis 
Set 
All subjects who received at least one dose of study drug, and for 
whom at least one PK parameter value is reported. 

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9.4. 
Statistical Analyses 
The statistical analysis plan will be finalized prior to First Patient First Visit (FPFV) and it will 
include a more technical and detailed description of the statistical analyses. This section is a 
summary of the key elements of the planned statistical analyses. 
9.4.1. 
Type 1 Error Control for the Primary Efficacy Endpoint 
There are two null hypotheses for the primary efficacy endpoint UOVS. 
1. 
there is no difference between the IC high dose and the normal saline 
2. 
there is no difference between the IC low dose and the normal saline 
Multiplicity due to the two null hypotheses will be controlled by Hochberg method.  That is, to 
control family-wide Type I error to be less than or equal to 0.05, when both nominal p-values are 
less or equal to 0.05, both null hypotheses will be rejected and one will conclude that both IC 
dose groups are statistically different from the saline group in the examined parameter.  When 
one of the two nominal p-values is greater than 0.05 but the second nominal p-value is less or 
equal to 0.025, the null hypothesis associated with the first nominal p-value is accepted and the 
null hypothesis associated with the second nominal p-value will be rejected.   If the second 
nominal p-value is greater 0.025, the null hypothesis associated with the second nominal p-value 
is also accepted.  The Overall Type I error control will apply to the primary endpoint UOVS 
only. 
9.4.2. 
Treatment Effect Based on the Operating Surgeon’s UOVS Scores  
In this study each subject will serve as his/her own control and each subject will have repeated 
measures from the same treatment (1 from the left and 1 from the right ureter); therefore, each 
subject is expecting to contribute 4 observations for UOVS endpoint.  The UOVS score provided 
by the surgeon will be the primary efficacy endpoint whereas the score provided by the blinded 
central reviewer will serve only as an estimate of concordance.   
The treatment effect on the UOVS score will be evaluated using a Generalized Estimating 
Equation (GEE) for Repeated Measures to control for the intra-subject correlation.  A full model 
will provide the chi-square tests for the effects from drug (IC high dose, IC low dose, Saline), 
site (left side ureter vs right side ureter), and drug*site interaction on the response based on 
multi-normal distribution for ordinal response.  The model estimates the cumulative logits of 
better outcome (score=3) to the poor outcome (score=1).  Since it is expected that the interaction 
effect will not statistically significant (p >0.10), a reduced model will also be used in which the 
interaction effect will be dropped.  The goodness of fit of the models will be evaluated.  A 
REPEATED statement will be used to control for the repeated measures within a subject.  The 
difference between the IC high dose vs saline, the IC low dose vs saline will be presented in the 
summary along with the estimates and 95% confidence intervals of the estimates.  Effect of a 
source (drug, site, drug*site interaction in full model) will be determined using the Score 
Statistics for Type 3 GEE Analysis.    

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An exploratory comparison will be done on the difference between IC high dose vs IC low dose 
and will be presented in the summary along with the estimates and 95% confidence intervals of 
the estimates.  Effect of a source (drug, site, drug*site interaction in full model) will be 
determined using the Score Statistics for Type 3 GEE Analysis.    
9.4.3. 
Treatment Effect on UOVS Based on Central Rater’s Assessment 
The UOVS score provided by the blinded central reviewer will be analyzed using the same GEE 
approach.  One way to assess the consistency between the surgeon and central rater is to evaluate 
the effect of raters (surgeon or blinded central rater) on the response using covariate approach.  A 
new covariate indicating evaluator (surgeon vs blinded central reviewer) will be added to the 
GEE model. 
Another way to assess the consistency between the raters is to derive the difference in UOVS 
score between the surgeon and the central rater.  This difference will have a value of (-2, -1, 0, 
+1, +2) for each of the 4 readings per subject, with 0 indicating a perfected agreement.  The 
association of the difference in score with treatment and site will also be evaluated using the 
GEE model.         
9.4.4. 
Treatment Effect on Time to Visualization  
Time (minutes) to visualization (TTV) will be evaluated using the Kaplan-Meier Survival 
Analysis approach; time to event percent-tiles (25%, 50%, and 75%) and 95% confidence 
intervals will be estimated along with the log-rank test for equal survival between the groups (IC 
vs Saline); Cox Proportional hazards model approach may be used to estimate hazards ratios if 
data warrant. In this analysis the potential intra-subject correction will not be controlled.  That is, 
each time to event observation will be treated as an independent observation.  Potential 
covariates (such as randomization strata and site will also be evaluated) if data warrants.  
Similarly, the difference in time to visualization between the IC high dose and the IC low dose 
will be evaluated using the log-rank test and Cox PH model. 
Treated subjects with missing data will be censored at 10 minutes.  
9.4.5. 
Assessment of PSAS with IC Treatment 
Ratings in the PSAS with the IC treatment will be tabulated by treatment (IC low dose, IC high 
dose, and study overall); 2-sided 95% confidence intervals on proportion of physicians who are 
satisfied (with ratings 1 or 2) will be derived using normal approximation.  A binominal 1-way 
proportion approach will be used to examine that at least 50% of the physicians are satisfied with 
the IC treatment as an aid for the assessment of ureter patency comparing to the saline treatment. 
Difference in PSAS with the use of IC high dose vs the IC low dose will be compared via a 
CMH test for mean row score.   
      

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9.4.6. 
Assessment of Covariates 
The effect of procedure on the response (randomization stratum) will be assessed via covariate 
analysis.  An appropriate model will be chosen for specific parameter.  
9.4.7. 
Safety Analysis 
Adverse events (AE) occurring on Day 1 post injection of the first dose of study drug and up to 
32 days after the procedure are considered treatment emergent.  The Medical Dictionary for 
Regulatory Activities (MEDRA) (Version 21 or higher) will be used to classify all AEs with 
respect to system organ class and preferred term.  AEs will be summarized by treatment groups.  
Proportion of subjects with clinically important changes in clinical laboratory tests, vital signs, 
and 12-lead ECG before (Screening visit, baseline) and after (follow-up visit, post baseline) 
procedure will be tabulated by treatment and study overall. AEs occurring during continuous 
respiratory and cardiac monitoring during surgery (from the time of the first dose of study drug 
to the discontinuation of monitoring will be recorded.  No formal inferential statistics will be 
performed for the safety parameters.  
9.4.8. 
PK Analysis  
PK parameters will be estimated using Non-compartmental analysis based on the observed 
concentration; descriptive statistics for exposure, excretion in urine, and in stool by IC dose (high 
dose vs low dose) will be provided without inferential statistics.  If data warrants difference in 
exposure and excretion between the IC low dose and high dose will be evaluated. 

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10. 
SUPPORTING DOCUMENTATION AND OPERATIONAL 
CONSIDERATIONS 
 
10.1. 
Regulatory, Ethical, and Study Oversight Considerations 
10.1.1. 
Regulatory and Ethical Considerations 
This study will be conducted in accordance with the protocol and with the following: 
• 
Consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical 
Sciences (CIOMS) International Ethical Guidelines 
• 
Applicable ICH Good Clinical Practice (GCP) Guidelines 
• 
Applicable laws and regulations 
The protocol, protocol amendments, ICF, Investigator Brochure, and other relevant documents 
(e.g., advertisements) must be submitted to an IRB by the investigator and reviewed and 
approved by the IRB before the study is initiated. 
Any amendments to the protocol will require IRB approval before implementation of changes 
made to the study design, except for changes necessary to eliminate an immediate hazard to 
study subjects. 
The investigator will be responsible for the following: 
• 
Providing written summaries of the status of the study to the IRB annually or more 
frequently in accordance with the requirements, policies, and procedures established 
by the IRB 
• 
Notifying the IRB of SAEs or other significant safety findings as required by IRB 
procedures 
• 
Providing oversight of the conduct of the study at the site and adherence to 
requirements of 21 CFR, ICH guidelines, the IRB, European regulation 536/2014 for 
clinical studies (if applicable), and all other applicable local regulations 
10.1.2. 
Financial Disclosure 
Investigators and sub-investigators 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 

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10.1.3. 
Informed Consent Process 
The investigator or his/her representative will explain the nature of the study to the subject or 
his/her legally authorized representative and answer all questions regarding the study. 
Subjects must be informed that their participation is voluntary. Subjects or their legally 
authorized representative will be required to sign a statement of informed consent that meets the 
requirements of 21 CFR 50, local regulations, ICH guidelines, Health Insurance Portability and 
Accountability Act (HIPAA) requirements, where applicable, and the IRB or study center. 
The medical record must include a statement that written informed consent was obtained before 
the subject was enrolled in the study and the date the written consent was obtained. The 
authorized person obtaining the informed consent must also sign the ICF. 
Subjects must be re-consented to the most current version of the ICF(s) during their participation 
in the study. 
A copy of the ICF(s) must be provided to the subject or the subject’s legally authorized 
representative. 
10.1.4. 
Institutional Review Board/Independent Ethics Committee (IRB) 
As required by local regulation, the Investigator or the Sponsor must submit this clinical trial 
protocol to the appropriate IRB and is required to forward to the respective other party a copy of 
the written and dated approval/favorable opinion signed by the Chairman with IRB composition. 
The clinical trial (study number, clinical trial protocol title and version number), the documents 
reviewed (clinical trial protocol, informed consent form, Investigator’s Brochure, Investigator’s 
curriculum vitae [CV], etc.) and the date of the review should be clearly stated on the written 
(IRB) approval/favorable opinion. 
IMP will not be released at the study site and the Investigator will not start the study before the 
written and dated approval/favorable opinion is received by the Investigator and the Sponsor. 
During the clinical trial, any amendment or modification to the clinical trial protocol should be 
submitted to the IRB before implementation, unless the change is necessary to eliminate an 
immediate hazard to the patients, in which case the IRB should be informed as soon as possible. 
It should also be informed of any event likely to affect the safety of patients or the continued 
conduct of the clinical trial, in particular any change in safety. All updates to the Investigator’s 
Brochure will be sent to the IRB. 
A progress report is sent to the IRB at least annually and a summary of the clinical trial’s 
outcome at the end of the clinical trial 
 
 

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10.1.5. 
Data Protection 
Subjects will be assigned a unique identifier by the sponsor. Any subject records or datasets that 
are transferred to the sponsor will contain the identifier only; subject names or any information 
which would make the subject identifiable will not be transferred. 
The subject must be informed that his/her personal study-related data will be used by the sponsor 
in accordance with local data protection law. The level of disclosure must also be explained to 
the subject who will be required to give consent for their data to be used as described in the 
informed consent  
The Investigator should keep a separate log (Patient Master List) of patient’s codes (assigned 
patient number), names, addresses, telephone numbers and hospital numbers (if applicable). 
Documents not for submission should be maintained by the Investigator in strict confidence. 
The subject must be informed that his/her medical records may be examined by Study Monitors, 
Clinical Quality Assurance auditors or other authorized personnel appointed by the sponsor, by 
appropriate IRB members, and by inspectors from regulatory authorities. 
Subject race or ethnicity will be collected in this study because these data are required by several 
regulatory authorities (eg, on afro American population for FDA) 
10.1.6. 
Dissemination of Clinical Study Data 
The Sponsor will be responsible for preparing a CSR and to provide a summary of study results 
to the Investigator. 
The Investigator undertakes not to make any publication or release pertaining to the study and/or 
results of the study prior to the Sponsor’s written consent, being understood that the Sponsor will 
not unreasonably withhold its approval. 
As the study is being conducted at multiple sites, the Sponsor agrees that, consistent with 
scientific standards, a primary presentation or publication of the study results based on global 
study outcomes shall be sought. However, if no multicenter publication is submitted, underway 
or planned within twelve (12) months of the completion of this study at all sites, the Investigator 
shall have the right to publish or present independently the results of this study in agreement with 
other Investigators and stakeholders. The Investigator shall provide the Sponsor with a copy of 
any such presentation or publication for review and comment at least 30 days in advance of any 
presentation or submission for publication. In addition, if requested by the Sponsor, any 
presentation or submission for publication shall be delayed for a limited time, not to exceed 90 
days, to allow for filing of a patent application or such other justified measures as the Sponsor 
deems appropriate to establish and preserve its proprietary rights. 
The Investigator shall not use the name(s) of the Sponsor and/or its employees in advertising or 
promotional material or publication without the prior written consent of the Sponsor. The 
Sponsor shall not use the name(s) of the Investigator and/or the collaborators in advertising or 
promotional material or publication without having received his/her and/or their prior written 
consent(s). 

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The Sponsor has the right at any time to publish the results of the study. 
10.1.7. 
Data Quality Assurance 
All subject data relating to the study will be recorded on printed or electronic CRF unless 
transmitted to the sponsor or designee electronically (e.g., laboratory data). The investigator is 
responsible for verifying that data entries are accurate and correct by physically or electronically 
signing the CRF. 
The investigator must maintain accurate documentation (source data) that supports the 
information entered in the CRF. 
The investigator must permit study-related monitoring, audits, IRB review, and regulatory 
agency inspections and provide direct access to source data documents. 
Monitoring details describing strategy (e.g., risk-based initiatives in operations and quality such 
as Risk Management and Mitigation Strategies and Analytical Risk-Based Monitoring), methods, 
responsibilities and requirements, including handling of noncompliance issues and monitoring 
techniques (central, remote, or onsite monitoring) are provided in the Monitoring Plan. 
The sponsor or designee is responsible for the data management of this study including quality 
checking of the data. 
The sponsor assumes accountability for actions delegated to other individuals (e.g., Contract 
Research Organizations). 
Study monitors will perform ongoing source data verification to confirm that data entered into 
the CRF by authorized site personnel are accurate, complete, and verifiable from source 
documents; that the safety and rights of subjects are being protected; and that the study is being 
conducted in accordance with the currently approved protocol and any other study agreements, 
ICH GCP, and all applicable regulatory requirements. 
Records and documents, including signed ICFs, pertaining to the conduct of this study must be 
retained by the investigator for 15 years after study completion unless local regulations or 
institutional policies require a longer retention period. No records may be destroyed during the 
retention period without the written approval of the sponsor. No records may be transferred to 
another location or party without written notification to the sponsor. 
10.1.8. 
Source Documents 
Source documents provide evidence for the existence of the subject and substantiate the integrity 
of the data collected. Source documents are filed at the investigator’s site. 
Data reported on the CRF or entered in the eCRF that are transcribed from source documents 
must be consistent with the source documents or the discrepancies must be explained. The 
investigator may need to request previous medical records or transfer records, depending on the 
study. Also, current medical records must be available. 

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Definition of what constitutes source data includes: Original documents, data, and records (e.g., 
hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or 
evaluation checklists, pharmacy dispensing records, recorded data from automated instruments, 
copies or transcriptions certified after verification as being accurate copies, microfiches, 
photographic negatives, microfilm or magnetic media, x-rays, subject files, and records kept at 
the pharmacy, at the laboratories and at medico-technical departments involved in the clinical 
trial. 
10.1.9. 
Study and Site Start and Closure 
The study start date is the date on which the clinical study will be open for recruitment of 
subjects. 
The first act of recruitment is the GO-LIVE date defined as the first site having their initiation 
visit and all systems (IVRS/IWRS, eCRF) available for data entry will be the study start date. 
The sponsor designee reserves the right to close the study site or terminate the study at any time 
for any reason at the sole discretion of the sponsor. Study sites will be closed upon study 
completion. A study site is considered closed when all required documents and study supplies 
have been collected and a study-site closure visit has been performed. 
The investigator may initiate study-site closure at any time, provided there is reasonable cause 
and sufficient notice is given in advance of the intended termination. 
Reasons for the early closure of a study site by the sponsor or investigator may include but are 
not limited to: 
• 
Failure of the investigator to comply with the protocol, the requirements of the IRB or 
local health authorities, the sponsor's procedures, or GCP guidelines 
• 
Inadequate recruitment of subjects by the investigator 
• 
Discontinuation of further study drug development 
If the study is prematurely terminated or suspended, the sponsor shall promptly inform the 
Investigators, the IECs/IRBs, the regulatory authorities, and any contract research 
organization(s) used in the study of the reason for termination or suspension, as specified by the 
applicable regulatory requirements. The Investigator shall promptly inform the subject and 
should assure appropriate subject therapy and/or follow-up 
10.1.10. 
Publication Policy 
Any manuscript, abstract or other publication or presentation of results or information arising in 
connection with the study (including any ancillary studies involving trial patients) must be 
prepared in conjunction with the sponsor and must be submitted to the sponsor for review and 
comment at least 8 weeks prior to submission for publication or presentation. 
 

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11. 
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